# Associated Targets of the Antioxidant Cardioprotection ofGanoderma lucidum in Diabetic Cardiomyopathy by Using Open Targets Platform: A Systematic Review

Authors: Fahmi Shaher; Hongbin Qiu; Shuqiu Wang; Yu Hu; Weiqun Wang; Yu Zhang; Yao Wei; Hisham AL-ward; Mahfoudh A. M. Abdulghani; Sattam Khulaif Alenezi; Salem Baldi; Shaobo Zhou
Journal: BioMed Research International
 (2020)

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## Abstract

Even with substantial advances in cardiovascular therapy, the morbidity and mortality rates of diabetic cardiomyopathy (DCM) continually increase. Hence, a feasible therapeutic approach is urgently needed.Objectives. This work is aimed at systemically reviewing literature and addressing cell targets in DCM through the possible cardioprotection of G. lucidum through its antioxidant effects by using the Open Targets Platform (OTP) website. Methods. The OTP website version of 19.11 was accessed in December 2019 to identify the studies in DCM involving G. lucidum. Results. Among the 157 cell targets associated with DCM, the mammalian target of rapamycin (mTOR) was shared by all evidence, drug, and text mining data with 0.08 score association. mTOR also had the highest score association 0.1 with autophagy in DCM. Among the 1731 studies of indexed PubMed articles on G. lucidum published between 1985 and 2019, 33 addressed the antioxidant effects of G. lucidum and its molecular signal pathways involving oxidative stress and therefore were included in the current work. Conclusion. mTOR is one of the targets by DCM and can be inhibited by the antioxidative properties of G. lucidum directly via scavenging radicals and indirectly via modulating mTOR signal pathways such as Wnt signaling pathway, Erk1/2 signaling, and NF-κB pathways.


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## Body

## 1. Introduction

Cardiovascular complications are associated with diabetes and lead to high mortality [1, 2]. Diabetic cardiomyopathy (DCM) is one of the main causes of heart injury and death in patients with diabetes. A total of 1.6 million deaths worldwide are directly attributed to diabetes every year [3]. Independent of coronary artery disease, DCM has increased prevalence during the last two decades and is experienced by 55% of patients with diabetes [4]. With diabetes being a global epidemic, the number of patients with DCM has increased. For the last two decades, the number of people with diabetes worldwide has increased from 151 million in 2000 to 425 million in 2017 and is estimated to increase to 629 million by 2045 [5]. The risk of developing DCM is higher for patients with diabetes than that for those without diabetes [6] and increases 2 to 4 times for those with more than a 10-year span of diabetes [7, 8]. Once DCM has developed, reducing its morbidity and mortality is difficult even with pharmacological improvement in terms of regulating blood glucose and insulin sensitivity. Clinical and preclinical investigations have examined the complexity of the pathophysiological consequences of DCM.Clinical studies in patients with DCM reported that the pathological remodeling of the heart, which is characterized by left ventricular concentric hypertrophy and perivascular and interstitial fibrosis commencing to diastolic dysfunction and extended contraction and relaxation [9, 10], shortens ventricular ejection and increases wall stiffness [11, 12]. The influence of the diabetic condition on heart and cardiomyocyte function has been experimentally evaluated.DCM and cardiac dysfunction are initiated in diabetic-induced experimental animals from 2 to 12 weeks [13]. Streptozotocin-induced diabetes in mice leads to the morphological changes of heart tissues, interstitial collagen deposition, cardiac hypertrophy, fibrosis, and remarkable elevation of paracrine of angiotensin II level in myocardium and NADPH oxidase activities, which are considered the primary source of free radicals in the cardiomyocytes of diabetic heart [14]. Connective tissue growth factor mediates cardiac fibrosis in diabetes [13, 15]. In diabetic mice with cardiomyopathy, the expression of sarcoplasmic reticulum calcium ATPase and [Ca+2] ion transient is reduced [16]. Sarcoplasmic reticulum calcium ATPase is a primary cardiac isoform of calcium pump transporting calcium from cytoplasm to sarcoplasmic reticulum during diastolic relaxation [17].Even with substantial advances in cardiovascular therapy, diabetic morbidity and mortality rate are continually increasing, and a feasible therapeutic approach for DCM is still lacking. Exploring the medication targets for DCM may further identify novel drugs and improve specific therapies for DCM. Therapeutic targets for DCM with natural resources are considered as one of the main reservoirs for drug discovery. Therefore, novel therapeutics for a range of targets must be developed to prevent DCM progression. This study identifies molecular target involvement and its association with DCM by using the Open Targets Platform (OTP) website established by Biogen, EMBL European Bioinformatics Institute, GlaxoSmithKline, and Wellcome Trust Sanger Institute. The OTP provides comprehensive and up-to-date data for drug molecular targets associated with relative diseases. Oxidative stress (OS) may be a key factor in the molecular and cellular mechanisms of diabetes-induced DCM [18]. Hence, targeting OS-related processes could be a promising therapeutic strategy for DCM.Ganoderma (G.) lucidum, which is known in Chinese as “Lingzhi,” is a medicinal mushroom commonly used as a Chinese herbal medicine and the main ingredient in many conventional combinations or dietary supplements [19]. This name has been proposed by Petter Adolf Karsten from England in the late 19th century and has been applied in various places such as Asia, Africa, Oceania, and Europe [20]. Lingzhi has been widely cultivated in China and has a long history as a traditional Chinese medicine. Chinese G. lucidum exhibits high variability of basidioma morphology and more or less consistency in its microscopic characters, e.g., short clavate cutis elements, Bovista-type ligative hyphae, and strongly echinulated basidiospores [21]. G. lucidum also contains various bioactive compounds, such as flavonoids, ganoderic acid, phenolics, and polysaccharides [21], that can treat many chronic diseases including diabetes and its complications by counteracting OS. Preclinical studies reported the beneficial effects of G. lucidum against OS-induced diseases, its liver protection against CCl4-induced OS [22], skin protection against croton oil-induced lipid peroxidation in mice [23], and thymus and spleen protection against 5-fluorouracil-induced OS in mice [21]. This systematic review is aimed at discussing the potential cell targets and cardioprotective pathway of G. lucidum based on preclinical and clinical investigations.


## 2. Methods

The OTP website version 19.11 (OTP V 19.11) was used to prioritize and identify the targets associated with DCM. The OTP provides score and rank target-disease associations and integrates evidence from six resources, including genetics, genomics, transcriptomics, drugs, animal models, and scientific literature [24, 25]. Two main steps of searching were performed in December 2019. In the first step, the term “diabetic cardiomyopathy” was used, and all the targets associated with DCM were listed according to available evidence recorded through bioinformatic processing, including data evidence of drug, text mining, genetic association, somatic mutation, pathways and signals, RNA signal, and animal model. The resulting targets with the highest association with DCM from the first step were used to further search for evidence on G. lucidum cardioprotection.This systematic review on the antioxidant activity ofG. lucidum was described as follows. Abstracts published from 1985 to July 2019 were reported as guided by the Preferred Reporting Items for Systematic Reviews and Meta-Analyses [26] (Figure 1). The key terms used were G. lucidum and spore of G. lucidum. In this step, the studies were divided into seven 5-year periods to easily read and select related abstracts. The search was limited to studies published in English and Chinese languages. Inclusion criteria were as follows: studies must focus on (1) G. lucidum and its (2) antioxidant, antidiabetic, and cardioprotective activities. Exclusion criteria were as follows: studies focusing on (1) mushrooms other than G. lucidum and (2) not related to its antioxidant activities such as the botanical and genetic studies of G. lucidum.Figure 1
A PRISMA flow diagram summarising the study selection process. Antioxidant ofG. Lucidum; PRISMA: Preferred Reporting Items for Systematic Reviews and Meta-Analyses. ∗After exclusion of other antioxidant activity studies of G. Lucidum.


## 3. Results

### 3.1. Targets Associated with DCM in Diabetes Integrated by OTP

#### 3.1.1. DCM and Its Associated Targets

A total of 309 targets were associated with DCM based on evidence from drug and text mining data with overall association scores from 0.004 to 0.177 (Table1, supplementary file (available here)). Among the selected drug data, only two targets, namely, carnitine palmitoyltransferase 1B (CPT1B) and 2 (CPT2) were associated with DCM with 0.1 score association. A total of 306 targets were identified from text mining. Only the mechanistic target of rapamycin kinase (mTOR) was common in both types of data. A total of 309 targets were expressed in 32 tissue organs including the heart and were involved in 19 pathway types (Table 2, supplementary file (available here)). Among these 309 targets, 155 were expressed in the heart tissues with overall association scores ranging from 0.007 to 0.177 (Table 1). Among the 19 pathways, 4 targets were included in autophagy (Table 2), namely, mTOR, beclin 1 (BECN1), parkin RBR E3 ubiquitin protein ligase (PRKN), and voltage-dependent anion channel 1 (VDAC1) with scores of 0.1, 0.06, 0.05, and 0.03, respectively.Table 1
Association sore of 155 targets associated with diabetic cardiomyopathy in heart tissue.
Target nameTarget symbolAssociation scoreData typesKnown drugData typesLiteratureOverall1Tripartite motif containing 55TRIM5500.1770.1772Peroxisome proliferator-activated receptor alphaPPARA00.1170.1173Mechanistic target of rapamycin kinaseMTOR0.10.0540.1134Interleukin 6IL-600.1130.1135Carnitine palmitoyltransferase 1BCPT1B0.10.0000.1006Carnitine palmitoyltransferase 2CPT20.10.0000.1007Tripartite motif containing 54TRIM5400.0810.0818Nuclear factor, erythroid 2 like 2NFE2L200.0720.0729Hydroxysteroid 11-beta dehydrogenase 1HSD11B100.0700.07010Fibroblast growth factor 1FGF100.0700.07011Colony-stimulating factor 3CSF300.0620.06212Beclin 1BECN100.0620.06213Cytochrome P450 family 2 subfamily J member 2CYP2J200.0610.06114Angiotensin I-converting enzyme 2ACE200.0600.06015Aldehyde dehydrogenase 2 family memberALDH200.0590.05916Glycogen synthase kinase 3 betaGSK3B00.0570.05717GelsolinGSN00.0550.05518Toll-like receptor 2TLR200.0540.05419Parkin RBR E3 ubiquitin protein ligasePRKN00.0540.05420ApelinAPLN00.0530.05321ST3 beta-galactoside alpha-2,3-sialyltransferase 4ST3GAL400.0520.05222Peroxisome proliferator activated receptor gammaPPARG00.0520.05223Corin, serine peptidaseCORIN00.0520.05224TitinTTN00.0490.04925AngiogeninANG00.0490.04926Protein kinase D1PRKD100.0490.04927PPARG coactivator 1 alphaPPARGC1A00.0480.04828Vascular endothelial growth factor AVEGFA00.0480.04829Insulin-like growth factor 1IGF100.0470.04730CD36 moleculeCD3600.0470.04731Nitric oxide synthase 3NOS300.0460.04632Apolipoprotein A1APOA100.0440.04433Gap junction protein alpha 1GJA100.0410.04134Calsequestrin 2CASQ200.0410.04135DecorinDCN00.0400.04036UrocortinUCN00.0400.04037Cellular communication network factor 2CCN200.0400.04038Matrix metallopeptidase 2MMP200.0400.04039PeriostinPOSTN00.0390.03940Fibroblast growth factor 2FGF200.0390.03941BCL6 transcription repressorBCL600.0390.03942Tax1-binding protein 1TAX1BP100.0380.03843Solute carrier family 2 member 4SLC2A400.0380.03844Rho-associated coiled-coil containing protein kinase 2ROCK200.0370.03745NADPH oxidase 4NOX400.0360.03646Mitogen-activated protein kinase 9MAPK900.0360.03647Insulin-like growth factor 2IGF200.0360.03648Angiotensin II receptor type 2AGTR200.0360.03649Lipoprotein lipaseLPL00.0360.03650Insulin receptorINSR00.0350.03551Angiopoietin 1ANGPT100.0350.03552Interleukin 33IL3300.0350.03553Caveolin 3CAV300.0340.03454Angiotensin I-converting enzymeACE00.0340.03455Patatin-like phospholipase domain containing 2PNPLA200.0340.03456ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2ATP2A200.0330.03357GlucokinaseGCK00.0320.03258Dimethylarginine dimethylaminohydrolase 2DDAH200.0320.03259Xenotropic and polytropic retrovirus receptor 1XPR100.0320.03260Vascular endothelial growth factor BVEGFB00.0320.03261Phosphodiesterase 5APDE5A00.0310.03162MAPK-activated protein kinase 2MAPKAPK200.0310.03163Heat shock protein family E (Hsp10) member 1HSPE100.0310.03164Sirtuin 2SIRT200.0310.03165DIRAS family GTPase 3DIRAS300.0300.03066SMAD family member 3SMAD300.0300.03067Dual specificity phosphatase 5DUSP500.0300.03068Kruppel-like factor 4KLF400.0300.03069Ryanodine receptor 2RYR200.0290.02970ProhibitinPHB00.0290.02971Estrogen related receptor gammaESRRG00.0280.02872NebulinNEB00.0280.02873Peroxiredoxin 3PRDX300.0280.02874Adrenoceptor beta 2ADRB200.0280.02875Solute carrier family 9 member A1SLC9A100.0280.02876Transglutaminase 2TGM200.0270.02777Poly(ADP-ribose) polymerase 1PARP100.0270.02778Insulin receptor substrate 1IRS100.0270.02779Voltage dependent anion channel 1VDAC100.0260.02680AKT serine/threonine kinase 1AKT100.0250.02581Myocyte enhancer factor 2AMEF2A00.0250.02582Dual specificity phosphatase 1DUSP100.0250.02583MusculinMSC00.0250.02584Diacylglycerol kinase zetaDGKZ00.0240.02485Death associated protein kinase 2DAPK200.0240.02486Solute carrier family 25 member 4SLC25A400.0230.02387SMAD family member 7SMAD700.0230.02388Natriuretic peptide ANPPA00.0230.02389Coiled-coil domain containing 47CCDC4700.0220.02290Lipase E, hormone sensitive typeLIPE00.0220.02291LeptinLEP00.0220.02292Arylsulfatase AARSA00.0210.02193Nitric oxide synthase 2NOS200.0210.02194Nuclear receptor subfamily 3 group C member 2NR3C200.0210.02195Sirtuin 3SIRT300.0210.02196PlasminogenPLG00.0200.02097Spindlin 1SPIN100.0200.02098Serpin family E member 1SERPINE100.0200.02099Tachykinin receptor 1TACR100.0200.020100RNA binding fox-1 homolog 2RBFOX200.0200.020101Fatty acid binding protein 4FABP400.0190.019102Potassium voltage-gated channel subfamily H member 2KCNH200.0190.019103Cell adhesion molecule 1CADM100.0190.019104ProlylcarboxypeptidasePRCP00.0180.018105Nucleotide-binding oligomerization domain containing 1NOD100.0180.018106Activating transcription factor 3ATF300.0180.018107Vasoactive intestinal peptideVIP00.0180.018108Egl-9 family hypoxia inducible factor 3EGLN300.0180.018109Fibronectin 1FN100.0180.018110Endothelin 1EDN100.0180.018111C-C motif chemokine ligand 2CCL200.0180.018112Solute carrier family 5 member 1SLC5A100.0180.018113Fibrinogen-like 2FGL200.0170.017114Monoamine oxidase AMAOA00.0170.017115Sphingosine-1-phosphate receptor 1S1PR100.0170.017116Signal transducer and activator of transcription 3STAT300.0170.017117Toll-like receptor 3TLR300.0170.017118Tripartite motif containing 63TRIM6300.0170.017119TIMP metallopeptidase inhibitor 2TIMP200.0170.017120Nerve growth factorNGF00.0170.017121Natriuretic peptide receptor 2NPR200.0160.016122Cyclin-dependent kinase inhibitor 1ACDKN1A00.0160.016123Cathepsin DCTSD00.0160.016124Thrombospondin 1THBS100.0150.015125Kinase insert domain receptorKDR00.0150.015126Serine/threonine kinase 11STK1100.0150.015127Enolase 3ENO300.0150.015128Gasdermin DGSDMD00.0150.015129Cytochrome c, somaticCYCS00.0150.015130Kallikrein B1KLKB100.0150.015131TIMP metallopeptidase inhibitor 4TIMP400.0150.015132Transforming growth factor beta 3TGFB300.0150.015133Zinc finger and BTB domain containing 16ZBTB1600.0150.015134Collagen type I alpha 1 chainCOL1A100.0150.015135Endothelin receptor type AEDNRA00.0140.014136Cellular communication network factor 1CCN100.0140.014137Secreted protein acidic and cysteine richSPARC00.0140.014138Glucagon like peptide 1 receptorGLP1R00.0140.014139Cystatin CCST300.0140.014140Intercellular adhesion molecule 1ICAM100.0140.014141ElastinELN00.0140.014142Tenascin CTNC00.0140.014143PTEN-induced kinase 1PINK100.0140.014144CalpastatinCAST00.0140.014145CCAAT enhancer binding protein betaCEBPB00.0120.012146Acyl-coA thioesterase 1ACOT100.0120.012147G protein-coupled bile acid receptor 1GPBAR100.0100.010148Annexin A1ANXA100.0100.010149Apolipoprotein L2APOL200.0080.008150Natriuretic peptide BNPPB00.0080.008151Leptin receptorLEPR00.0080.008152Serum response factorSRF00.0080.008153Heat shock protein family B (small) member 3HSPB300.0070.007154Angiotensin II receptor type 1AGTR100.0070.007155Protein phosphatase 5 catalytic subunitPPP5C00.0070.007Table 2
Nineteen pathway types involved in the heart tissues of 155 targets expressed.
No.Pathway (No. of targets)1.Signal transduction (63)2.Immune system (47)3.Metabolism of proteins (39)4.Metabolism (31)5.Gene expression (transcription) (25)6.Hemostasis (23)7.Disease (22)8.Developmental biology (20)9.Extracellular matrix organization (18)10.Cellular responses to external stimuli (14)11.Transport of small molecules (11)12.Muscle contraction (11)13.Vesicle-mediated transport (10)14.Organelle biogenesis and maintenance (4)15.Programmed cell death (4)16.Autophagy (4)17.Neuronal system (3)18.Cell cycle (3)19.Circadian clock (3)mTOR was further investigated, and its association with heart diseases ranged from 0.0004 to 0.8588, which is the overall association score for 49 subtypes of heart diseases. mTOR had 0.1 and 0.8 overall association scores with DCM and hypertrophic cardiomyopathy, respectively (Table3).Table 3
mTOR score association with 49 heart diseases.
No.Heart diseaseAssociation scoreData typesGeneticData typesKnown drugData typesLiteratureData typesAnimal modelOverall1Heart disease0.000410.795500.141610.190280.85882Cardiomyopathy0.000000.778470.119300.190280.83933Hypertrophic cardiomyopathy0.000000.772220.102140.000000.79784Heart failure0.000000.250000.052350.000000.26315Dilated cardiomyopathy0.000000.000000.075680.190280.20926Congestive heart failure0.000000.200000.026360.000000.20667Diastolic heart failure0.000000.200000.000000.000000.20008Barth syndrome0.000000.000000.000000.190280.19039Coronary heart disease0.000000.000000.120090.000000.120110Diabetic cardiomyopathy0.000000.100000.053910.000000.113511Coronary artery disease0.000000.000000.109610.000000.109612Systemic scleroderma0.000000.000000.099140.000000.099113Cardiotoxicity0.000000.000000.090160.000000.090214Glycogen storage disease due to acid maltase deficiency0.000000.000000.083800.000000.083815Myocardial infarction0.000000.000000.064670.000000.064716Persistent truncus arteriosus0.000000.000000.061440.000000.061417Heart neoplasm0.000000.000000.061260.000000.061318Emery-Dreifuss muscular dystrophy0.000000.000000.057800.000000.057819Ischemia reperfusion injury0.000000.000000.057020.000000.057020Myocardial ischemia0.000000.000000.056580.000000.056621Carney complex0.000000.000000.054940.000000.054922Down syndrome0.000000.000000.054880.000000.054923Cardiac rhabdomyoma0.000000.000000.054750.000000.054724Autosomal dominant Emery-Dreifuss muscular dystrophy0.000000.000000.052800.000000.052825Polyarteritis nodosa0.000000.000000.043430.000000.043426Steinert myotonic dystrophy0.000000.000000.042730.000000.042727Acute myocardial infarction0.000000.000000.037980.000000.038028Cardiac arrhythmia0.000410.000000.037210.000000.037329Myocarditis0.000000.000000.032630.000000.032630Duchenne muscular dystrophy0.000000.000000.032530.000000.032531Gaucher disease0.000000.000000.032300.000000.032332Cardiac arrest0.000000.000000.028470.000000.028533Atrial fibrillation0.000000.000000.027200.000000.027234Aortic stenosis0.000000.000000.019100.000000.019135Acute coronary syndrome0.000000.000000.019000.000000.019036Sleep disorder0.000000.000000.018400.000000.018437Williams syndrome0.000000.000000.016400.000000.016438Supravalvular aortic stenosis0.000000.000000.016400.000000.016439Autoimmune myocarditis0.000000.000000.015600.000000.015640Friedreich ataxia0.000000.000000.014800.000000.014841Obstructive sleep apnea0.000000.000000.014800.000000.014842PHACE syndrome0.000000.000000.014400.000000.014443Glycogen storage disease due to LAMP-2 deficiency0.000000.000000.014400.000000.014444Idiopathic pulmonary arterial hypertension0.000000.000000.014000.000000.014045Fabry disease0.000000.000000.013400.000000.013446Becker muscular dystrophy0.000000.000000.008400.000000.008447Hemopericardium0.000000.000000.007200.000000.007248Aortic coarctation0.000000.000000.006800.000000.006849Atrial flutter0.000410.000000.000000.000000.0004mTOR is a serine/threonine-protein kinase playing as a central regulator of cellular metabolism, growth, and survival in response to hormone growth factor [27], nutrients, energy, and stress signals [28, 29]. According to UniPort, mTOR can be found in different subcellular locations including the membranes of endoplasmic reticulum, Golgi apparatus, outer mitochondrion, microsome, and lysosome; lysosome, cytoplasm, nucleus, and PML nuclear body. The RNA and protein expression levels of mTOR are present in several organs including the heart, e.g., the medium RNA and high protein levels of mTOR are expressed in the left ventricle, atrium, and coronary artery but not in the heart muscles (Figure 2).Figure 2
mRNA and protein baseline expression of mTOR in the heart.


#### 3.1.2. Evidence on the Cardioprotection ofG. lucidum

A total of 1731 articles were identified (Figure3) and further divided into seven 5-year time periods. The first period ranged from 1985 to 1989, and the last period ranged from August 2018 to August 2019 (Figure 1). These articles were reviewed in the following three phases. First, 1571 articles remained after the duplicated ones were removed. Second, articles that did not satisfy the inclusion criteria based solely on their titles (remaining 1399 articles) and abstracts (remaining 59 articles) were excluded. Lastly, the remaining articles were scanned, and those that did not meet our inclusion criteria were excluded. After the initial screening of titles and abstracts, the 59 remaining articles were screened for the second time by two individual reviewers. Inclusion of full articles was agreed upon by two reviewers prior to data extraction. Finally, 33 studies were considered eligible for the review (Figure 2). In this section, the collected pieces of evidence were divided into two main parts, namely, the in vivo antioxidant of G. lucidum (14 studies, Table 4), in which the in vivo effect of antioxidant on the parameters related to OS was discussed, and the in vitro antioxidant of G. lucidum (19 studies, Table 5), in which the in vitro effect of antioxidant activities and possible molecular mechanisms was elaborated.Figure 3
Number of studies onG. lucidum during 1985-2019.Table 4
In vivo studies of G. lucidum.
No.AnimalFormDosage (mg/kg)Antioxidant parametersBiological activityPathwayReferences1CCl4-induced acute liver injury miceGLPS100 - 150NOS CYP2E1 MDA, GSHSuppressing free radical lipid peroxidationDecreasing of the protein expression levels of NLRP3, ASC, and caspase-1 in acute liver injury.ASC (apoptosis-associated speck-like protein)NLRP3 (NOD-like receptor 3)Caspase-1GAPDH (glyceraldehyde-3-phosphate hydrogenase[22]2Croton oil applied skin edema in ratsEthanol extract of sporocarps500 and 1000 mg/kgAntiperoxidative, anti-inflammatory, and antimutagenic activitiesDirect anti-inflammatory and free radical scavenging properties of the extract[23]3Photoreceptor cell lesions induced by N-methyl-N-nitrosourea (MNU) in female SD artsGanoderma spore lipid (GSL)500, 1000, 2000, and 4000 mg/kgExpressions of Bax, Bcl-xl, and caspase-3Improve A-wave amplitude (μv) decreased apoptosis levelsRegulate the expressions of Bax, Bcl-xl, and caspases-3, inhibiting MNU-induced rat, photoreceptor cell apoptosis, and protecting retinal function[30]4A carotid-artery-ligation mouse modelGanoderma triterpenoid (GT)300 mg/kg/dayIntimal hyperplasia structural changes VCAM-1, TNF-α, and IL-6Atheroprotective propertiesEndothelin-1, von Willebrand factor, and monocyte chemoattractant protein-1[33]5Swimming-induced oxidative stress in skeletal muscle miceGLPS50, 100, and 200 mg/kgSOD, GPX, and CAT activities as well as by the MDA levelsAttenuates exercise-induced oxidative stress in skeletal muscleIncreasing antioxidant enzyme activities and decrease the MDA levels. Protective effects against exhaustive exercise-induced oxidative stress[32]6Rat gastric cancer modelGLPS400-800 mg/kg for 20 weeksSOD, CAT, and GSH-PxAntioxidantInduced the levels of serum IL-6 and TNF-α levels and increased the levels of serum IL-2, IL-4, and IL-10 in GLP-treated rats compared to gastric cancer model rats[37]7BALB/c female miceGLPS i.p. daily50 mg/kg, 100 mg/kg, and 200 mg/kgSOD and GSH-PxAntioxidantImproved immunity in mice. Increased thymus and spleen index; improved SOD and GSH-Px contents in the mice body[31]8T2DM ratsGLPS200, 400, and 800 mg × kg-1 for 16 weeksNO, SOD, MDA, GSH-Px, and CAT MDA in cardiac tissueAntioxidation in cardiac tissue of T2DM ratsReduce MDA in cardiac tissue and improve the myocardial ultrastructure[34]9Male BALB/c mice (age19-21 months) (aged mice)Ethanolic extract ofG. lucidum50 and 250 mg/kg, once daily for 15 daysGSH Mn-SOD, GPx, and GSTAntioxidant in heart tissuesElevated the levels of GSH as well as activities of MnSOD, GPx, and GST and decreased significantly the levels of lipid peroxidation, AOPP, and ROS. Improve the age-related decline of antioxidant status which was partly ascribed to free radical scavenging activity[38]10B16 mouse melanomaMethanol extract containing total terpenoids (GLme) and a purified methanol extract containing mainly acidic terpenoids (GLpme)A daily i.p. injection of 100 mg/kg body weight (b.w.)Production of oxygen radical caspase-dependent apoptotic cell death-mediated production of reactive oxygen speciesAnticancerThe mechanism of antitumor activity of GLme comprised inhibition of cell proliferation and induction of caspase-dependent apoptotic cell death mediated by upregulated p53 and inhibited Bcl-2 expression[86]11With non-insulin-dependent diabetes mellitus (NIDDM)Ganodermalucidum spores250 mg/kg × d, for 10Xanthine oxidase (XOD), myeloperoxidase (MPO), and mitochondrial succinate dehydrogenase (SDH) in the testisReducing free radical-induceddamage to the testicular tissueProtect the testis of diabetic rats by reducing free radical-induced damage to the testicular tissue and enhancing the activity of SDH[35]12Epididymal cells of type 2 diabetes ratsGanoderma lucidum spores (GLS)250 mg/kg × d, for 10 weeksContents of mitochondrial calcium & cytochrome CAntipoptosis induced by DMProtect epididymal cells and counteract their apoptosis in diabetic condition[36]13Liver tissue of ratsGanoderma lucidum peptide27.1μg/mLMalondialdehyde levelAntioxidantSubstantial antioxidant activity in the rat liver tissue homogenates and mitochondrial membrane peroxidation systems[87]14Lupus miceGanoderma tsugae0.5 mg/kg/dayDecreased proteinuria, decreased serum levels of antidsDNA autoantibodyPrevention of autoantibodyPrevention of autoantibody formation[88]Table 5
In vitro studies of G. lucidum.
No.FormConc.Chemical antioxidant testsBiological text of in vitroExp. parametersBiological activityPathwayReferences1GLP0.5-3.0 mg/mLRSFR=Scavenging of free radicals and reducing powerAntioxidantNM[89]2G. lucidum and Egyptian Chlorella vulgarisCVE (63.5μg/mL) was mixed with GLE (4.1 μg/mL)RSFRAPOther testsLipopolysaccharide-stimulated white blood cellsNitric oxide, tumor necrosis factor- (TNF-)αAntioxidant and anti-inflammatoryDownregulate NF-κB[39]3Polysaccharides inG. lucidum2 mg/mLRSFRAPOther testsNMRadical scavenging reducing powerAntioxidantNM[90]4G. lucidum extract50 mgRSFRAPOther testsNMReducing powerAntimicrobial and antioxidantNM[40]5Ganoderma lucidum G20.32 mgRSFRAPOther testsDNA protectionRadical scavenging reducing powerAntimicrobial and antioxidantNM[41]6Protein extracts2–13μg protein/mLAPOther testsDNA protectionRadical scavenging reducing powerAntioxidant, antibacterialNM[42]7Polysaccharides extraction=FRAPOther testsMCF-7 breast cancer cell line and HeLa cellsRadical scavengingAntioxidantAnticancerNM[43]8G. lucidum and G. resinaceum0.1–1 &0.64±0.040–2.25 mg/mLFRAPOther testsIn vitro cell lineRadical-scavenging chelating lipoxygenase assayAntiproliferative & antioxidantNM[44]9Diff, organic solvent oG. lucidum1-200μg/mLFRAPOther testsNMRadical scavenging, chelating lipid peroxidationAntioxidantAnticholinesteraseNM[45]10Both aqueous and methanolic extracts0.2–30 mg/mL of extractionFRAPOther testsNMRadical scavenging, chelating lipid peroxidationAntioxidantNM[46]11Low-molecular-weightβ-1,3-glucan0–200μg/mLAPOther testsMouse monocyte-macrophage cell line, RAW 264.7H2O2-induced apoptosisAntioxidantAttenuating intracellular reactive oxygen species (ROS) and inhibiting sphingomyelinase (SMase) activity[51]12Polysaccharides0.16-10 mg/mLFRAPOther testsNMRadical scavenging, chelating reducing powerAntioxidantNM[47]13G. lucidumwater-soluble and water-insoluble80-1100μg/mlFRAPOther testsHuman uroepithelial cell (HUC-PC) cellsRadical scavenging, chelating reducing powerAntioxidantOxidative DNA damage. Lingzhi-induced apoptosis in bladder chemoprevention[48]14Ganoderma lucidum polysaccharides0.1-0.6 mg/mlRSCCl-induced injury hepatocytes DNA protectionMDA, SOD, CYP3A, caspase-3, andcaspase-8Suppressing inflammatory responsesReduction of NF-κB activation inhibition of caspase-3, caspase-6, and caspase-9, indicating and suppression extrinsic-induced apoptosis[52]15Ganoderic acid A10-80 lM/mLNMPancreatic cellsRadical scavengingAntiproliferativeAntioxidantAnticancerβ-Catenin in Wnt signaling pathway[54]16Aqueous extract ofG. lucidum5-20μLNMDNA protectionRadical scavengingAntioxidantDNA repairEnhancing reactivity of apurinic/apyrimidinic endonucleases (APE1) a major enzyme of base excision repair (BER)[91]17Methanolic extract ofG. lucidum65 & 130μg/mLNMHuman gastric tumor cellsIncreased the formation of autophagosomesInduces autophagyIncreasing of the cellular levels of LC3-II and decreasing p62 (autophagy-related protein)[92]18G. lucidum (GLPS) and G. sinense (GSPS)19–300μg/mLNMRAW 264.7 mouse macrophage cellsNitric oxide secretion of cytokinesImmunomodulatoryPromoting macrophage phagocytosis, increasing their release of nitric oxide and cytokines interleukin- (IL-) 1a, IL-6, IL-10, and tumor necrosis factor-α[56]19Proteopolysaccharide fromG. lucidum2 - 10μg/mLNMRAW264.7, a mouse macrophage cell lineNitrite productionExpression levels of cytokinesActivation the immune system by modulating cytokine production.NM[57]NM = not mention; RS = radical scavenging; FR = ferric reducing; AP = antilipid peroxidation.


### 3.2.In Vivo Antioxidant Activity and Protective Effect of G. lucidum

According to 10 in vivo experimental studies,G. lucidum has antioxidant activities and protects against OS through four main factors in different tissues, such as the heart, liver, thymus, spleen, eyes, and skeletal muscles, and by regulating chemical-level OS parameters in blood circulation (Table 4). G. lucidum exhibits its antioxidant effects by increasing the antioxidant enzymes and inhibiting the enzymes involved in OS. G. lucidum also increases the activities of superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), mitochondrial succinate dehydrogenase (SDH), and Mn-SOD and reduces glutathione (GSH) levels. By contrast, G. lucidum decreases the activities of nitric oxide synthase (NOS), cytochrome P450 2E1 (CYP2E1), xanthine oxidase (XOD), and myeloperoxidase (MPO). G. lucidum also significantly decreases lipid peroxidation levels, advanced oxidation protein products (AOPPs), and malondialdehyde (MDA) levels.The first factor is the four toxic substances, including CCl4-induced oxidative stress (OS) in the liver, croton oil produced OS in the skin through inflammation, N-methyl-N-nitrosourea (MNU) causing retinal photoreceptor cell lesions in the eyes, and 5-fluorouracil-induced OS in the thymus and spleen of mice. Oral administration of G. lucidum polysaccharides (GLPs) represses free radical lipid peroxidation induced by CCl4 to reduce the enzyme activities of NOS and CYP2E1. Significant inhibition of NOS and CYP2E1 activities and MDA and IL-1β levels was noted in liver tissues, and depleted levels of interleukin- (IL-) 1β, IL-18, IL-6, and tumor necrosis factor-α were found in serum. In CCl4-induced liver damage, highly reactive trichloromethyl free radicals are generated by the cytochrome P450 isozymes (P450s) of the endoplasmic reticulum [22]. Topical administration of G. lucidum ethanol extract inhibits the croton oil-induced lipid peroxidation in the skin of mice [23]. Ganoderma spore lipid (GSL) shows a protective effect on MNU-induced retina injury by inhibiting the related apoptosis to modulate the expression levels of Bax, Bcl-xl, and caspase-3 [30]. GLPs also exhibit an antioxidant effect in 5-fluorouracil-induced OS and improve SOD, an intracellular compound that protects against oxidative processes initiated by superoxide anion and GPx contents in the spleen and thymus of mice [31].The second factor creates conditions in biological systems that can induce OS, such as exercise-like exhaustive swimming, which is OS induced in skeletal muscles, and a carotid artery ligation, which disturbs the flow-induced OS level of manganese-dependent superoxide dismutase (Mn-SOD) in blood vessels. GLPs show protective effects against comprehensive swimming-induced OS by improving the activities of antioxidant enzymes (SOD, GPx, and CAT) and decreasing the MDA levels in the skeletal muscle of mice [32]. Oral ganoderma triterpenoids (GTs) protect against disturbed flow-induced OS through carotid artery ligation, which leads to chronic OS and inflammation that are features of early atherogenesis in mice, and by preventing neointimal thickening 2 weeks after ligation. Early atherogenesis includes neointimal hyperplasia and endothelial dysfunction due to flow turbulence in the ligated artery as induced by OS. GTs alleviate OS and restore the atheroresistent status of endothelium by inhibiting endothelin-1 induction, von Willebrand factor, and monocyte chemoattractant protein-1 after 3-day ligation as atherogenic factors [33]. Inflammatory cytokines, OS-induced endothelial dysfunction, and chronic OS contribute to endothelial impairment and induces atherogenesis.The third factor in OS includes diseases such as type II diabetes mellitus (DM) and cancer. In type II DM, the beneficial effects ofG. lucidum on abnormal heart and testis and epididymal cells of rats with streptozotocin-induced type II DM were evaluated. GLPs improve the myocardial ultrastructure by reducing MDA, activating antioxidant enzymes (GSH-Px, CAT, SOD, and NO) in cardiac tissues, and reducing lipid peroxidation in type II DM rats [34]. G. lucidum spores protect the testis of rats with type II DM by substantially increasing the mitochondrial SDH and decreasing the activities of XOD and MPO [35]. G. lucidum spores protect epididymal cells and counteract their apoptosis that damages the mitochondria and disequilibrium of calcium homeostasis by reducing the amount of mitochondrial cytoplasm cytochrome C in type II DM rats [36]. GLP administration enhances the immunity and antioxidant activities in N-methyl-N9-nitro-nitrosoguanidine-induced gastric cancer in Wistar rats. GLP remarkably reduces the levels of serum IL-6 and TNF-α and increases the levels of serum IL-2, IL-4, and IL-10. In addition, GLP improves the levels of SOD, CAT, and GSH-Px in serum and gastric tissues [37].The fourth factor involved in OS is aging.G. lucidum administration ameliorates the age-related decline of antioxidant status in aged mice, substantially elevates the activities of GST, Mn-SOD, GPx, and CAT, and reduces GSH. By contrast, lipid peroxidation, AOPP, and reactive oxygen species (ROS) are reduced [38] (Table 4).


### 3.3.In Vitro Antioxidant of G. lucidum and Its Possible Pathway

Chemical antioxidant tests consistently revealed the free radical scavenging activity ofG. lucidum. Twelve studies reported the scavenging activity of G. lucidum for different free radicals including 2,2-diphenylpicrylhydrazyl radical (DPPH⋅), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS⋅+), hydroxyl radical (HO⋅), and hydrogen peroxide radicals (H2O2) [39–49] (Table 5). G. lucidum also inhibits lipid peroxidation [23, 49]. In some studies, G. lucidum protects against DNA damage [41, 42, 50]. The results of chemical antioxidant tests regarding the antioxidant properties of G. lucidum are also in agreement with the cell-based antioxidant assays. G. lucidum shows free radical scavenging activity for H2O2 in RAW264.7 cells incubated with G. lucidum lipopolysaccharide and protects against H2O2-induced cell death [48]. G. lucidum also hinders sphingomyelinase activity in incubated RAW264.7 cells with lipopolysaccharide [51]. In addition, G. lucidum prevents lipid peroxidation in two cell models, namely, WBCs incubated with lipopolysaccharide to induce OS [39] and hepatocytes incubated with CCl4 to induce OS [52]. In both cell models, G. lucidum showed protection by elevating the antioxidant enzyme activity (SOD, GPx, and GR) and improving the GSH level. Moreover, G. lucidum protects macrophages in human monocytic cells incubated with lipopolysaccharide to stimulate NO production [53].Wnt, Erk1/2, and NF-κB are the possible signaling pathways of G. lucidum that support its antioxidant and protective effects. A pancreatic cell study suggested β-catenin in the Wnt signaling pathway as a target of ganoderic acid A, thus leading to cell protection and effective scavenging of ROS [54]. The Wnt signaling pathways transfer the signals from extracellular to intercellular and are stimulated by the Wnt protein binding to the cytoplasmic family receptor, which occurs in downstream cell signaling and controls the transcription of genes. In the canonical Wnt pathway, β-catenin accumulates in the cytoplasm and is further translocated into the nucleus, and this phenomenon is widely recognized as a regulation marker of fat and glucose metabolism and β-catenin/Wnt signaling involved in insulin secretion [54]. In 2006, Thyagarajan and his colleagues mentioned that G. lucidum modulates Erk1/2 signaling and transcription factors AP-1 and NF-κB and downregulates c-Fos, whose expression can be induced by OS as the result of the inhibited OS-induced invasive behavior of breast cancer cells. A high H2O2 concentration (5 mM) can stimulate Erk1/2 signaling in MCF-7 cells [55].In addition to its antioxidant activities,G. lucidum also exhibits an anti-inflammatory property and modulates the immune system. It can reverse LPS-induced inflammation by downregulating inflammatory mediators such as NF-κB, thus substantially inhibiting NOS and reducing NO level [39]. G. lucidum also modulates the immune system byregulating cytokine production in RAW264.7 macrophages [56, 57]. Moreover, it increases the formation of autophagosomes and controls proteins (Vps34, beclin 1, LC3-I, LC3-II, and p62) that induce autophagy in a gastric adenocarcinoma cell line. G. lucidum increases the cellular levels of LC3-II and decreases the cellular levels of p62 (Table 5).


## 3.1. Targets Associated with DCM in Diabetes Integrated by OTP

### 3.1.1. DCM and Its Associated Targets

A total of 309 targets were associated with DCM based on evidence from drug and text mining data with overall association scores from 0.004 to 0.177 (Table1, supplementary file (available here)). Among the selected drug data, only two targets, namely, carnitine palmitoyltransferase 1B (CPT1B) and 2 (CPT2) were associated with DCM with 0.1 score association. A total of 306 targets were identified from text mining. Only the mechanistic target of rapamycin kinase (mTOR) was common in both types of data. A total of 309 targets were expressed in 32 tissue organs including the heart and were involved in 19 pathway types (Table 2, supplementary file (available here)). Among these 309 targets, 155 were expressed in the heart tissues with overall association scores ranging from 0.007 to 0.177 (Table 1). Among the 19 pathways, 4 targets were included in autophagy (Table 2), namely, mTOR, beclin 1 (BECN1), parkin RBR E3 ubiquitin protein ligase (PRKN), and voltage-dependent anion channel 1 (VDAC1) with scores of 0.1, 0.06, 0.05, and 0.03, respectively.Table 1
Association sore of 155 targets associated with diabetic cardiomyopathy in heart tissue.
Target nameTarget symbolAssociation scoreData typesKnown drugData typesLiteratureOverall1Tripartite motif containing 55TRIM5500.1770.1772Peroxisome proliferator-activated receptor alphaPPARA00.1170.1173Mechanistic target of rapamycin kinaseMTOR0.10.0540.1134Interleukin 6IL-600.1130.1135Carnitine palmitoyltransferase 1BCPT1B0.10.0000.1006Carnitine palmitoyltransferase 2CPT20.10.0000.1007Tripartite motif containing 54TRIM5400.0810.0818Nuclear factor, erythroid 2 like 2NFE2L200.0720.0729Hydroxysteroid 11-beta dehydrogenase 1HSD11B100.0700.07010Fibroblast growth factor 1FGF100.0700.07011Colony-stimulating factor 3CSF300.0620.06212Beclin 1BECN100.0620.06213Cytochrome P450 family 2 subfamily J member 2CYP2J200.0610.06114Angiotensin I-converting enzyme 2ACE200.0600.06015Aldehyde dehydrogenase 2 family memberALDH200.0590.05916Glycogen synthase kinase 3 betaGSK3B00.0570.05717GelsolinGSN00.0550.05518Toll-like receptor 2TLR200.0540.05419Parkin RBR E3 ubiquitin protein ligasePRKN00.0540.05420ApelinAPLN00.0530.05321ST3 beta-galactoside alpha-2,3-sialyltransferase 4ST3GAL400.0520.05222Peroxisome proliferator activated receptor gammaPPARG00.0520.05223Corin, serine peptidaseCORIN00.0520.05224TitinTTN00.0490.04925AngiogeninANG00.0490.04926Protein kinase D1PRKD100.0490.04927PPARG coactivator 1 alphaPPARGC1A00.0480.04828Vascular endothelial growth factor AVEGFA00.0480.04829Insulin-like growth factor 1IGF100.0470.04730CD36 moleculeCD3600.0470.04731Nitric oxide synthase 3NOS300.0460.04632Apolipoprotein A1APOA100.0440.04433Gap junction protein alpha 1GJA100.0410.04134Calsequestrin 2CASQ200.0410.04135DecorinDCN00.0400.04036UrocortinUCN00.0400.04037Cellular communication network factor 2CCN200.0400.04038Matrix metallopeptidase 2MMP200.0400.04039PeriostinPOSTN00.0390.03940Fibroblast growth factor 2FGF200.0390.03941BCL6 transcription repressorBCL600.0390.03942Tax1-binding protein 1TAX1BP100.0380.03843Solute carrier family 2 member 4SLC2A400.0380.03844Rho-associated coiled-coil containing protein kinase 2ROCK200.0370.03745NADPH oxidase 4NOX400.0360.03646Mitogen-activated protein kinase 9MAPK900.0360.03647Insulin-like growth factor 2IGF200.0360.03648Angiotensin II receptor type 2AGTR200.0360.03649Lipoprotein lipaseLPL00.0360.03650Insulin receptorINSR00.0350.03551Angiopoietin 1ANGPT100.0350.03552Interleukin 33IL3300.0350.03553Caveolin 3CAV300.0340.03454Angiotensin I-converting enzymeACE00.0340.03455Patatin-like phospholipase domain containing 2PNPLA200.0340.03456ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2ATP2A200.0330.03357GlucokinaseGCK00.0320.03258Dimethylarginine dimethylaminohydrolase 2DDAH200.0320.03259Xenotropic and polytropic retrovirus receptor 1XPR100.0320.03260Vascular endothelial growth factor BVEGFB00.0320.03261Phosphodiesterase 5APDE5A00.0310.03162MAPK-activated protein kinase 2MAPKAPK200.0310.03163Heat shock protein family E (Hsp10) member 1HSPE100.0310.03164Sirtuin 2SIRT200.0310.03165DIRAS family GTPase 3DIRAS300.0300.03066SMAD family member 3SMAD300.0300.03067Dual specificity phosphatase 5DUSP500.0300.03068Kruppel-like factor 4KLF400.0300.03069Ryanodine receptor 2RYR200.0290.02970ProhibitinPHB00.0290.02971Estrogen related receptor gammaESRRG00.0280.02872NebulinNEB00.0280.02873Peroxiredoxin 3PRDX300.0280.02874Adrenoceptor beta 2ADRB200.0280.02875Solute carrier family 9 member A1SLC9A100.0280.02876Transglutaminase 2TGM200.0270.02777Poly(ADP-ribose) polymerase 1PARP100.0270.02778Insulin receptor substrate 1IRS100.0270.02779Voltage dependent anion channel 1VDAC100.0260.02680AKT serine/threonine kinase 1AKT100.0250.02581Myocyte enhancer factor 2AMEF2A00.0250.02582Dual specificity phosphatase 1DUSP100.0250.02583MusculinMSC00.0250.02584Diacylglycerol kinase zetaDGKZ00.0240.02485Death associated protein kinase 2DAPK200.0240.02486Solute carrier family 25 member 4SLC25A400.0230.02387SMAD family member 7SMAD700.0230.02388Natriuretic peptide ANPPA00.0230.02389Coiled-coil domain containing 47CCDC4700.0220.02290Lipase E, hormone sensitive typeLIPE00.0220.02291LeptinLEP00.0220.02292Arylsulfatase AARSA00.0210.02193Nitric oxide synthase 2NOS200.0210.02194Nuclear receptor subfamily 3 group C member 2NR3C200.0210.02195Sirtuin 3SIRT300.0210.02196PlasminogenPLG00.0200.02097Spindlin 1SPIN100.0200.02098Serpin family E member 1SERPINE100.0200.02099Tachykinin receptor 1TACR100.0200.020100RNA binding fox-1 homolog 2RBFOX200.0200.020101Fatty acid binding protein 4FABP400.0190.019102Potassium voltage-gated channel subfamily H member 2KCNH200.0190.019103Cell adhesion molecule 1CADM100.0190.019104ProlylcarboxypeptidasePRCP00.0180.018105Nucleotide-binding oligomerization domain containing 1NOD100.0180.018106Activating transcription factor 3ATF300.0180.018107Vasoactive intestinal peptideVIP00.0180.018108Egl-9 family hypoxia inducible factor 3EGLN300.0180.018109Fibronectin 1FN100.0180.018110Endothelin 1EDN100.0180.018111C-C motif chemokine ligand 2CCL200.0180.018112Solute carrier family 5 member 1SLC5A100.0180.018113Fibrinogen-like 2FGL200.0170.017114Monoamine oxidase AMAOA00.0170.017115Sphingosine-1-phosphate receptor 1S1PR100.0170.017116Signal transducer and activator of transcription 3STAT300.0170.017117Toll-like receptor 3TLR300.0170.017118Tripartite motif containing 63TRIM6300.0170.017119TIMP metallopeptidase inhibitor 2TIMP200.0170.017120Nerve growth factorNGF00.0170.017121Natriuretic peptide receptor 2NPR200.0160.016122Cyclin-dependent kinase inhibitor 1ACDKN1A00.0160.016123Cathepsin DCTSD00.0160.016124Thrombospondin 1THBS100.0150.015125Kinase insert domain receptorKDR00.0150.015126Serine/threonine kinase 11STK1100.0150.015127Enolase 3ENO300.0150.015128Gasdermin DGSDMD00.0150.015129Cytochrome c, somaticCYCS00.0150.015130Kallikrein B1KLKB100.0150.015131TIMP metallopeptidase inhibitor 4TIMP400.0150.015132Transforming growth factor beta 3TGFB300.0150.015133Zinc finger and BTB domain containing 16ZBTB1600.0150.015134Collagen type I alpha 1 chainCOL1A100.0150.015135Endothelin receptor type AEDNRA00.0140.014136Cellular communication network factor 1CCN100.0140.014137Secreted protein acidic and cysteine richSPARC00.0140.014138Glucagon like peptide 1 receptorGLP1R00.0140.014139Cystatin CCST300.0140.014140Intercellular adhesion molecule 1ICAM100.0140.014141ElastinELN00.0140.014142Tenascin CTNC00.0140.014143PTEN-induced kinase 1PINK100.0140.014144CalpastatinCAST00.0140.014145CCAAT enhancer binding protein betaCEBPB00.0120.012146Acyl-coA thioesterase 1ACOT100.0120.012147G protein-coupled bile acid receptor 1GPBAR100.0100.010148Annexin A1ANXA100.0100.010149Apolipoprotein L2APOL200.0080.008150Natriuretic peptide BNPPB00.0080.008151Leptin receptorLEPR00.0080.008152Serum response factorSRF00.0080.008153Heat shock protein family B (small) member 3HSPB300.0070.007154Angiotensin II receptor type 1AGTR100.0070.007155Protein phosphatase 5 catalytic subunitPPP5C00.0070.007Table 2
Nineteen pathway types involved in the heart tissues of 155 targets expressed.
No.Pathway (No. of targets)1.Signal transduction (63)2.Immune system (47)3.Metabolism of proteins (39)4.Metabolism (31)5.Gene expression (transcription) (25)6.Hemostasis (23)7.Disease (22)8.Developmental biology (20)9.Extracellular matrix organization (18)10.Cellular responses to external stimuli (14)11.Transport of small molecules (11)12.Muscle contraction (11)13.Vesicle-mediated transport (10)14.Organelle biogenesis and maintenance (4)15.Programmed cell death (4)16.Autophagy (4)17.Neuronal system (3)18.Cell cycle (3)19.Circadian clock (3)mTOR was further investigated, and its association with heart diseases ranged from 0.0004 to 0.8588, which is the overall association score for 49 subtypes of heart diseases. mTOR had 0.1 and 0.8 overall association scores with DCM and hypertrophic cardiomyopathy, respectively (Table3).Table 3
mTOR score association with 49 heart diseases.
No.Heart diseaseAssociation scoreData typesGeneticData typesKnown drugData typesLiteratureData typesAnimal modelOverall1Heart disease0.000410.795500.141610.190280.85882Cardiomyopathy0.000000.778470.119300.190280.83933Hypertrophic cardiomyopathy0.000000.772220.102140.000000.79784Heart failure0.000000.250000.052350.000000.26315Dilated cardiomyopathy0.000000.000000.075680.190280.20926Congestive heart failure0.000000.200000.026360.000000.20667Diastolic heart failure0.000000.200000.000000.000000.20008Barth syndrome0.000000.000000.000000.190280.19039Coronary heart disease0.000000.000000.120090.000000.120110Diabetic cardiomyopathy0.000000.100000.053910.000000.113511Coronary artery disease0.000000.000000.109610.000000.109612Systemic scleroderma0.000000.000000.099140.000000.099113Cardiotoxicity0.000000.000000.090160.000000.090214Glycogen storage disease due to acid maltase deficiency0.000000.000000.083800.000000.083815Myocardial infarction0.000000.000000.064670.000000.064716Persistent truncus arteriosus0.000000.000000.061440.000000.061417Heart neoplasm0.000000.000000.061260.000000.061318Emery-Dreifuss muscular dystrophy0.000000.000000.057800.000000.057819Ischemia reperfusion injury0.000000.000000.057020.000000.057020Myocardial ischemia0.000000.000000.056580.000000.056621Carney complex0.000000.000000.054940.000000.054922Down syndrome0.000000.000000.054880.000000.054923Cardiac rhabdomyoma0.000000.000000.054750.000000.054724Autosomal dominant Emery-Dreifuss muscular dystrophy0.000000.000000.052800.000000.052825Polyarteritis nodosa0.000000.000000.043430.000000.043426Steinert myotonic dystrophy0.000000.000000.042730.000000.042727Acute myocardial infarction0.000000.000000.037980.000000.038028Cardiac arrhythmia0.000410.000000.037210.000000.037329Myocarditis0.000000.000000.032630.000000.032630Duchenne muscular dystrophy0.000000.000000.032530.000000.032531Gaucher disease0.000000.000000.032300.000000.032332Cardiac arrest0.000000.000000.028470.000000.028533Atrial fibrillation0.000000.000000.027200.000000.027234Aortic stenosis0.000000.000000.019100.000000.019135Acute coronary syndrome0.000000.000000.019000.000000.019036Sleep disorder0.000000.000000.018400.000000.018437Williams syndrome0.000000.000000.016400.000000.016438Supravalvular aortic stenosis0.000000.000000.016400.000000.016439Autoimmune myocarditis0.000000.000000.015600.000000.015640Friedreich ataxia0.000000.000000.014800.000000.014841Obstructive sleep apnea0.000000.000000.014800.000000.014842PHACE syndrome0.000000.000000.014400.000000.014443Glycogen storage disease due to LAMP-2 deficiency0.000000.000000.014400.000000.014444Idiopathic pulmonary arterial hypertension0.000000.000000.014000.000000.014045Fabry disease0.000000.000000.013400.000000.013446Becker muscular dystrophy0.000000.000000.008400.000000.008447Hemopericardium0.000000.000000.007200.000000.007248Aortic coarctation0.000000.000000.006800.000000.006849Atrial flutter0.000410.000000.000000.000000.0004mTOR is a serine/threonine-protein kinase playing as a central regulator of cellular metabolism, growth, and survival in response to hormone growth factor [27], nutrients, energy, and stress signals [28, 29]. According to UniPort, mTOR can be found in different subcellular locations including the membranes of endoplasmic reticulum, Golgi apparatus, outer mitochondrion, microsome, and lysosome; lysosome, cytoplasm, nucleus, and PML nuclear body. The RNA and protein expression levels of mTOR are present in several organs including the heart, e.g., the medium RNA and high protein levels of mTOR are expressed in the left ventricle, atrium, and coronary artery but not in the heart muscles (Figure 2).Figure 2
mRNA and protein baseline expression of mTOR in the heart.


### 3.1.2. Evidence on the Cardioprotection ofG. lucidum

A total of 1731 articles were identified (Figure3) and further divided into seven 5-year time periods. The first period ranged from 1985 to 1989, and the last period ranged from August 2018 to August 2019 (Figure 1). These articles were reviewed in the following three phases. First, 1571 articles remained after the duplicated ones were removed. Second, articles that did not satisfy the inclusion criteria based solely on their titles (remaining 1399 articles) and abstracts (remaining 59 articles) were excluded. Lastly, the remaining articles were scanned, and those that did not meet our inclusion criteria were excluded. After the initial screening of titles and abstracts, the 59 remaining articles were screened for the second time by two individual reviewers. Inclusion of full articles was agreed upon by two reviewers prior to data extraction. Finally, 33 studies were considered eligible for the review (Figure 2). In this section, the collected pieces of evidence were divided into two main parts, namely, the in vivo antioxidant of G. lucidum (14 studies, Table 4), in which the in vivo effect of antioxidant on the parameters related to OS was discussed, and the in vitro antioxidant of G. lucidum (19 studies, Table 5), in which the in vitro effect of antioxidant activities and possible molecular mechanisms was elaborated.Figure 3
Number of studies onG. lucidum during 1985-2019.Table 4
In vivo studies of G. lucidum.
No.AnimalFormDosage (mg/kg)Antioxidant parametersBiological activityPathwayReferences1CCl4-induced acute liver injury miceGLPS100 - 150NOS CYP2E1 MDA, GSHSuppressing free radical lipid peroxidationDecreasing of the protein expression levels of NLRP3, ASC, and caspase-1 in acute liver injury.ASC (apoptosis-associated speck-like protein)NLRP3 (NOD-like receptor 3)Caspase-1GAPDH (glyceraldehyde-3-phosphate hydrogenase[22]2Croton oil applied skin edema in ratsEthanol extract of sporocarps500 and 1000 mg/kgAntiperoxidative, anti-inflammatory, and antimutagenic activitiesDirect anti-inflammatory and free radical scavenging properties of the extract[23]3Photoreceptor cell lesions induced by N-methyl-N-nitrosourea (MNU) in female SD artsGanoderma spore lipid (GSL)500, 1000, 2000, and 4000 mg/kgExpressions of Bax, Bcl-xl, and caspase-3Improve A-wave amplitude (μv) decreased apoptosis levelsRegulate the expressions of Bax, Bcl-xl, and caspases-3, inhibiting MNU-induced rat, photoreceptor cell apoptosis, and protecting retinal function[30]4A carotid-artery-ligation mouse modelGanoderma triterpenoid (GT)300 mg/kg/dayIntimal hyperplasia structural changes VCAM-1, TNF-α, and IL-6Atheroprotective propertiesEndothelin-1, von Willebrand factor, and monocyte chemoattractant protein-1[33]5Swimming-induced oxidative stress in skeletal muscle miceGLPS50, 100, and 200 mg/kgSOD, GPX, and CAT activities as well as by the MDA levelsAttenuates exercise-induced oxidative stress in skeletal muscleIncreasing antioxidant enzyme activities and decrease the MDA levels. Protective effects against exhaustive exercise-induced oxidative stress[32]6Rat gastric cancer modelGLPS400-800 mg/kg for 20 weeksSOD, CAT, and GSH-PxAntioxidantInduced the levels of serum IL-6 and TNF-α levels and increased the levels of serum IL-2, IL-4, and IL-10 in GLP-treated rats compared to gastric cancer model rats[37]7BALB/c female miceGLPS i.p. daily50 mg/kg, 100 mg/kg, and 200 mg/kgSOD and GSH-PxAntioxidantImproved immunity in mice. Increased thymus and spleen index; improved SOD and GSH-Px contents in the mice body[31]8T2DM ratsGLPS200, 400, and 800 mg × kg-1 for 16 weeksNO, SOD, MDA, GSH-Px, and CAT MDA in cardiac tissueAntioxidation in cardiac tissue of T2DM ratsReduce MDA in cardiac tissue and improve the myocardial ultrastructure[34]9Male BALB/c mice (age19-21 months) (aged mice)Ethanolic extract ofG. lucidum50 and 250 mg/kg, once daily for 15 daysGSH Mn-SOD, GPx, and GSTAntioxidant in heart tissuesElevated the levels of GSH as well as activities of MnSOD, GPx, and GST and decreased significantly the levels of lipid peroxidation, AOPP, and ROS. Improve the age-related decline of antioxidant status which was partly ascribed to free radical scavenging activity[38]10B16 mouse melanomaMethanol extract containing total terpenoids (GLme) and a purified methanol extract containing mainly acidic terpenoids (GLpme)A daily i.p. injection of 100 mg/kg body weight (b.w.)Production of oxygen radical caspase-dependent apoptotic cell death-mediated production of reactive oxygen speciesAnticancerThe mechanism of antitumor activity of GLme comprised inhibition of cell proliferation and induction of caspase-dependent apoptotic cell death mediated by upregulated p53 and inhibited Bcl-2 expression[86]11With non-insulin-dependent diabetes mellitus (NIDDM)Ganodermalucidum spores250 mg/kg × d, for 10Xanthine oxidase (XOD), myeloperoxidase (MPO), and mitochondrial succinate dehydrogenase (SDH) in the testisReducing free radical-induceddamage to the testicular tissueProtect the testis of diabetic rats by reducing free radical-induced damage to the testicular tissue and enhancing the activity of SDH[35]12Epididymal cells of type 2 diabetes ratsGanoderma lucidum spores (GLS)250 mg/kg × d, for 10 weeksContents of mitochondrial calcium & cytochrome CAntipoptosis induced by DMProtect epididymal cells and counteract their apoptosis in diabetic condition[36]13Liver tissue of ratsGanoderma lucidum peptide27.1μg/mLMalondialdehyde levelAntioxidantSubstantial antioxidant activity in the rat liver tissue homogenates and mitochondrial membrane peroxidation systems[87]14Lupus miceGanoderma tsugae0.5 mg/kg/dayDecreased proteinuria, decreased serum levels of antidsDNA autoantibodyPrevention of autoantibodyPrevention of autoantibody formation[88]Table 5
In vitro studies of G. lucidum.
No.FormConc.Chemical antioxidant testsBiological text of in vitroExp. parametersBiological activityPathwayReferences1GLP0.5-3.0 mg/mLRSFR=Scavenging of free radicals and reducing powerAntioxidantNM[89]2G. lucidum and Egyptian Chlorella vulgarisCVE (63.5μg/mL) was mixed with GLE (4.1 μg/mL)RSFRAPOther testsLipopolysaccharide-stimulated white blood cellsNitric oxide, tumor necrosis factor- (TNF-)αAntioxidant and anti-inflammatoryDownregulate NF-κB[39]3Polysaccharides inG. lucidum2 mg/mLRSFRAPOther testsNMRadical scavenging reducing powerAntioxidantNM[90]4G. lucidum extract50 mgRSFRAPOther testsNMReducing powerAntimicrobial and antioxidantNM[40]5Ganoderma lucidum G20.32 mgRSFRAPOther testsDNA protectionRadical scavenging reducing powerAntimicrobial and antioxidantNM[41]6Protein extracts2–13μg protein/mLAPOther testsDNA protectionRadical scavenging reducing powerAntioxidant, antibacterialNM[42]7Polysaccharides extraction=FRAPOther testsMCF-7 breast cancer cell line and HeLa cellsRadical scavengingAntioxidantAnticancerNM[43]8G. lucidum and G. resinaceum0.1–1 &0.64±0.040–2.25 mg/mLFRAPOther testsIn vitro cell lineRadical-scavenging chelating lipoxygenase assayAntiproliferative & antioxidantNM[44]9Diff, organic solvent oG. lucidum1-200μg/mLFRAPOther testsNMRadical scavenging, chelating lipid peroxidationAntioxidantAnticholinesteraseNM[45]10Both aqueous and methanolic extracts0.2–30 mg/mL of extractionFRAPOther testsNMRadical scavenging, chelating lipid peroxidationAntioxidantNM[46]11Low-molecular-weightβ-1,3-glucan0–200μg/mLAPOther testsMouse monocyte-macrophage cell line, RAW 264.7H2O2-induced apoptosisAntioxidantAttenuating intracellular reactive oxygen species (ROS) and inhibiting sphingomyelinase (SMase) activity[51]12Polysaccharides0.16-10 mg/mLFRAPOther testsNMRadical scavenging, chelating reducing powerAntioxidantNM[47]13G. lucidumwater-soluble and water-insoluble80-1100μg/mlFRAPOther testsHuman uroepithelial cell (HUC-PC) cellsRadical scavenging, chelating reducing powerAntioxidantOxidative DNA damage. Lingzhi-induced apoptosis in bladder chemoprevention[48]14Ganoderma lucidum polysaccharides0.1-0.6 mg/mlRSCCl-induced injury hepatocytes DNA protectionMDA, SOD, CYP3A, caspase-3, andcaspase-8Suppressing inflammatory responsesReduction of NF-κB activation inhibition of caspase-3, caspase-6, and caspase-9, indicating and suppression extrinsic-induced apoptosis[52]15Ganoderic acid A10-80 lM/mLNMPancreatic cellsRadical scavengingAntiproliferativeAntioxidantAnticancerβ-Catenin in Wnt signaling pathway[54]16Aqueous extract ofG. lucidum5-20μLNMDNA protectionRadical scavengingAntioxidantDNA repairEnhancing reactivity of apurinic/apyrimidinic endonucleases (APE1) a major enzyme of base excision repair (BER)[91]17Methanolic extract ofG. lucidum65 & 130μg/mLNMHuman gastric tumor cellsIncreased the formation of autophagosomesInduces autophagyIncreasing of the cellular levels of LC3-II and decreasing p62 (autophagy-related protein)[92]18G. lucidum (GLPS) and G. sinense (GSPS)19–300μg/mLNMRAW 264.7 mouse macrophage cellsNitric oxide secretion of cytokinesImmunomodulatoryPromoting macrophage phagocytosis, increasing their release of nitric oxide and cytokines interleukin- (IL-) 1a, IL-6, IL-10, and tumor necrosis factor-α[56]19Proteopolysaccharide fromG. lucidum2 - 10μg/mLNMRAW264.7, a mouse macrophage cell lineNitrite productionExpression levels of cytokinesActivation the immune system by modulating cytokine production.NM[57]NM = not mention; RS = radical scavenging; FR = ferric reducing; AP = antilipid peroxidation.


## 3.1.1. DCM and Its Associated Targets

A total of 309 targets were associated with DCM based on evidence from drug and text mining data with overall association scores from 0.004 to 0.177 (Table1, supplementary file (available here)). Among the selected drug data, only two targets, namely, carnitine palmitoyltransferase 1B (CPT1B) and 2 (CPT2) were associated with DCM with 0.1 score association. A total of 306 targets were identified from text mining. Only the mechanistic target of rapamycin kinase (mTOR) was common in both types of data. A total of 309 targets were expressed in 32 tissue organs including the heart and were involved in 19 pathway types (Table 2, supplementary file (available here)). Among these 309 targets, 155 were expressed in the heart tissues with overall association scores ranging from 0.007 to 0.177 (Table 1). Among the 19 pathways, 4 targets were included in autophagy (Table 2), namely, mTOR, beclin 1 (BECN1), parkin RBR E3 ubiquitin protein ligase (PRKN), and voltage-dependent anion channel 1 (VDAC1) with scores of 0.1, 0.06, 0.05, and 0.03, respectively.Table 1
Association sore of 155 targets associated with diabetic cardiomyopathy in heart tissue.
Target nameTarget symbolAssociation scoreData typesKnown drugData typesLiteratureOverall1Tripartite motif containing 55TRIM5500.1770.1772Peroxisome proliferator-activated receptor alphaPPARA00.1170.1173Mechanistic target of rapamycin kinaseMTOR0.10.0540.1134Interleukin 6IL-600.1130.1135Carnitine palmitoyltransferase 1BCPT1B0.10.0000.1006Carnitine palmitoyltransferase 2CPT20.10.0000.1007Tripartite motif containing 54TRIM5400.0810.0818Nuclear factor, erythroid 2 like 2NFE2L200.0720.0729Hydroxysteroid 11-beta dehydrogenase 1HSD11B100.0700.07010Fibroblast growth factor 1FGF100.0700.07011Colony-stimulating factor 3CSF300.0620.06212Beclin 1BECN100.0620.06213Cytochrome P450 family 2 subfamily J member 2CYP2J200.0610.06114Angiotensin I-converting enzyme 2ACE200.0600.06015Aldehyde dehydrogenase 2 family memberALDH200.0590.05916Glycogen synthase kinase 3 betaGSK3B00.0570.05717GelsolinGSN00.0550.05518Toll-like receptor 2TLR200.0540.05419Parkin RBR E3 ubiquitin protein ligasePRKN00.0540.05420ApelinAPLN00.0530.05321ST3 beta-galactoside alpha-2,3-sialyltransferase 4ST3GAL400.0520.05222Peroxisome proliferator activated receptor gammaPPARG00.0520.05223Corin, serine peptidaseCORIN00.0520.05224TitinTTN00.0490.04925AngiogeninANG00.0490.04926Protein kinase D1PRKD100.0490.04927PPARG coactivator 1 alphaPPARGC1A00.0480.04828Vascular endothelial growth factor AVEGFA00.0480.04829Insulin-like growth factor 1IGF100.0470.04730CD36 moleculeCD3600.0470.04731Nitric oxide synthase 3NOS300.0460.04632Apolipoprotein A1APOA100.0440.04433Gap junction protein alpha 1GJA100.0410.04134Calsequestrin 2CASQ200.0410.04135DecorinDCN00.0400.04036UrocortinUCN00.0400.04037Cellular communication network factor 2CCN200.0400.04038Matrix metallopeptidase 2MMP200.0400.04039PeriostinPOSTN00.0390.03940Fibroblast growth factor 2FGF200.0390.03941BCL6 transcription repressorBCL600.0390.03942Tax1-binding protein 1TAX1BP100.0380.03843Solute carrier family 2 member 4SLC2A400.0380.03844Rho-associated coiled-coil containing protein kinase 2ROCK200.0370.03745NADPH oxidase 4NOX400.0360.03646Mitogen-activated protein kinase 9MAPK900.0360.03647Insulin-like growth factor 2IGF200.0360.03648Angiotensin II receptor type 2AGTR200.0360.03649Lipoprotein lipaseLPL00.0360.03650Insulin receptorINSR00.0350.03551Angiopoietin 1ANGPT100.0350.03552Interleukin 33IL3300.0350.03553Caveolin 3CAV300.0340.03454Angiotensin I-converting enzymeACE00.0340.03455Patatin-like phospholipase domain containing 2PNPLA200.0340.03456ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2ATP2A200.0330.03357GlucokinaseGCK00.0320.03258Dimethylarginine dimethylaminohydrolase 2DDAH200.0320.03259Xenotropic and polytropic retrovirus receptor 1XPR100.0320.03260Vascular endothelial growth factor BVEGFB00.0320.03261Phosphodiesterase 5APDE5A00.0310.03162MAPK-activated protein kinase 2MAPKAPK200.0310.03163Heat shock protein family E (Hsp10) member 1HSPE100.0310.03164Sirtuin 2SIRT200.0310.03165DIRAS family GTPase 3DIRAS300.0300.03066SMAD family member 3SMAD300.0300.03067Dual specificity phosphatase 5DUSP500.0300.03068Kruppel-like factor 4KLF400.0300.03069Ryanodine receptor 2RYR200.0290.02970ProhibitinPHB00.0290.02971Estrogen related receptor gammaESRRG00.0280.02872NebulinNEB00.0280.02873Peroxiredoxin 3PRDX300.0280.02874Adrenoceptor beta 2ADRB200.0280.02875Solute carrier family 9 member A1SLC9A100.0280.02876Transglutaminase 2TGM200.0270.02777Poly(ADP-ribose) polymerase 1PARP100.0270.02778Insulin receptor substrate 1IRS100.0270.02779Voltage dependent anion channel 1VDAC100.0260.02680AKT serine/threonine kinase 1AKT100.0250.02581Myocyte enhancer factor 2AMEF2A00.0250.02582Dual specificity phosphatase 1DUSP100.0250.02583MusculinMSC00.0250.02584Diacylglycerol kinase zetaDGKZ00.0240.02485Death associated protein kinase 2DAPK200.0240.02486Solute carrier family 25 member 4SLC25A400.0230.02387SMAD family member 7SMAD700.0230.02388Natriuretic peptide ANPPA00.0230.02389Coiled-coil domain containing 47CCDC4700.0220.02290Lipase E, hormone sensitive typeLIPE00.0220.02291LeptinLEP00.0220.02292Arylsulfatase AARSA00.0210.02193Nitric oxide synthase 2NOS200.0210.02194Nuclear receptor subfamily 3 group C member 2NR3C200.0210.02195Sirtuin 3SIRT300.0210.02196PlasminogenPLG00.0200.02097Spindlin 1SPIN100.0200.02098Serpin family E member 1SERPINE100.0200.02099Tachykinin receptor 1TACR100.0200.020100RNA binding fox-1 homolog 2RBFOX200.0200.020101Fatty acid binding protein 4FABP400.0190.019102Potassium voltage-gated channel subfamily H member 2KCNH200.0190.019103Cell adhesion molecule 1CADM100.0190.019104ProlylcarboxypeptidasePRCP00.0180.018105Nucleotide-binding oligomerization domain containing 1NOD100.0180.018106Activating transcription factor 3ATF300.0180.018107Vasoactive intestinal peptideVIP00.0180.018108Egl-9 family hypoxia inducible factor 3EGLN300.0180.018109Fibronectin 1FN100.0180.018110Endothelin 1EDN100.0180.018111C-C motif chemokine ligand 2CCL200.0180.018112Solute carrier family 5 member 1SLC5A100.0180.018113Fibrinogen-like 2FGL200.0170.017114Monoamine oxidase AMAOA00.0170.017115Sphingosine-1-phosphate receptor 1S1PR100.0170.017116Signal transducer and activator of transcription 3STAT300.0170.017117Toll-like receptor 3TLR300.0170.017118Tripartite motif containing 63TRIM6300.0170.017119TIMP metallopeptidase inhibitor 2TIMP200.0170.017120Nerve growth factorNGF00.0170.017121Natriuretic peptide receptor 2NPR200.0160.016122Cyclin-dependent kinase inhibitor 1ACDKN1A00.0160.016123Cathepsin DCTSD00.0160.016124Thrombospondin 1THBS100.0150.015125Kinase insert domain receptorKDR00.0150.015126Serine/threonine kinase 11STK1100.0150.015127Enolase 3ENO300.0150.015128Gasdermin DGSDMD00.0150.015129Cytochrome c, somaticCYCS00.0150.015130Kallikrein B1KLKB100.0150.015131TIMP metallopeptidase inhibitor 4TIMP400.0150.015132Transforming growth factor beta 3TGFB300.0150.015133Zinc finger and BTB domain containing 16ZBTB1600.0150.015134Collagen type I alpha 1 chainCOL1A100.0150.015135Endothelin receptor type AEDNRA00.0140.014136Cellular communication network factor 1CCN100.0140.014137Secreted protein acidic and cysteine richSPARC00.0140.014138Glucagon like peptide 1 receptorGLP1R00.0140.014139Cystatin CCST300.0140.014140Intercellular adhesion molecule 1ICAM100.0140.014141ElastinELN00.0140.014142Tenascin CTNC00.0140.014143PTEN-induced kinase 1PINK100.0140.014144CalpastatinCAST00.0140.014145CCAAT enhancer binding protein betaCEBPB00.0120.012146Acyl-coA thioesterase 1ACOT100.0120.012147G protein-coupled bile acid receptor 1GPBAR100.0100.010148Annexin A1ANXA100.0100.010149Apolipoprotein L2APOL200.0080.008150Natriuretic peptide BNPPB00.0080.008151Leptin receptorLEPR00.0080.008152Serum response factorSRF00.0080.008153Heat shock protein family B (small) member 3HSPB300.0070.007154Angiotensin II receptor type 1AGTR100.0070.007155Protein phosphatase 5 catalytic subunitPPP5C00.0070.007Table 2
Nineteen pathway types involved in the heart tissues of 155 targets expressed.
No.Pathway (No. of targets)1.Signal transduction (63)2.Immune system (47)3.Metabolism of proteins (39)4.Metabolism (31)5.Gene expression (transcription) (25)6.Hemostasis (23)7.Disease (22)8.Developmental biology (20)9.Extracellular matrix organization (18)10.Cellular responses to external stimuli (14)11.Transport of small molecules (11)12.Muscle contraction (11)13.Vesicle-mediated transport (10)14.Organelle biogenesis and maintenance (4)15.Programmed cell death (4)16.Autophagy (4)17.Neuronal system (3)18.Cell cycle (3)19.Circadian clock (3)mTOR was further investigated, and its association with heart diseases ranged from 0.0004 to 0.8588, which is the overall association score for 49 subtypes of heart diseases. mTOR had 0.1 and 0.8 overall association scores with DCM and hypertrophic cardiomyopathy, respectively (Table3).Table 3
mTOR score association with 49 heart diseases.
No.Heart diseaseAssociation scoreData typesGeneticData typesKnown drugData typesLiteratureData typesAnimal modelOverall1Heart disease0.000410.795500.141610.190280.85882Cardiomyopathy0.000000.778470.119300.190280.83933Hypertrophic cardiomyopathy0.000000.772220.102140.000000.79784Heart failure0.000000.250000.052350.000000.26315Dilated cardiomyopathy0.000000.000000.075680.190280.20926Congestive heart failure0.000000.200000.026360.000000.20667Diastolic heart failure0.000000.200000.000000.000000.20008Barth syndrome0.000000.000000.000000.190280.19039Coronary heart disease0.000000.000000.120090.000000.120110Diabetic cardiomyopathy0.000000.100000.053910.000000.113511Coronary artery disease0.000000.000000.109610.000000.109612Systemic scleroderma0.000000.000000.099140.000000.099113Cardiotoxicity0.000000.000000.090160.000000.090214Glycogen storage disease due to acid maltase deficiency0.000000.000000.083800.000000.083815Myocardial infarction0.000000.000000.064670.000000.064716Persistent truncus arteriosus0.000000.000000.061440.000000.061417Heart neoplasm0.000000.000000.061260.000000.061318Emery-Dreifuss muscular dystrophy0.000000.000000.057800.000000.057819Ischemia reperfusion injury0.000000.000000.057020.000000.057020Myocardial ischemia0.000000.000000.056580.000000.056621Carney complex0.000000.000000.054940.000000.054922Down syndrome0.000000.000000.054880.000000.054923Cardiac rhabdomyoma0.000000.000000.054750.000000.054724Autosomal dominant Emery-Dreifuss muscular dystrophy0.000000.000000.052800.000000.052825Polyarteritis nodosa0.000000.000000.043430.000000.043426Steinert myotonic dystrophy0.000000.000000.042730.000000.042727Acute myocardial infarction0.000000.000000.037980.000000.038028Cardiac arrhythmia0.000410.000000.037210.000000.037329Myocarditis0.000000.000000.032630.000000.032630Duchenne muscular dystrophy0.000000.000000.032530.000000.032531Gaucher disease0.000000.000000.032300.000000.032332Cardiac arrest0.000000.000000.028470.000000.028533Atrial fibrillation0.000000.000000.027200.000000.027234Aortic stenosis0.000000.000000.019100.000000.019135Acute coronary syndrome0.000000.000000.019000.000000.019036Sleep disorder0.000000.000000.018400.000000.018437Williams syndrome0.000000.000000.016400.000000.016438Supravalvular aortic stenosis0.000000.000000.016400.000000.016439Autoimmune myocarditis0.000000.000000.015600.000000.015640Friedreich ataxia0.000000.000000.014800.000000.014841Obstructive sleep apnea0.000000.000000.014800.000000.014842PHACE syndrome0.000000.000000.014400.000000.014443Glycogen storage disease due to LAMP-2 deficiency0.000000.000000.014400.000000.014444Idiopathic pulmonary arterial hypertension0.000000.000000.014000.000000.014045Fabry disease0.000000.000000.013400.000000.013446Becker muscular dystrophy0.000000.000000.008400.000000.008447Hemopericardium0.000000.000000.007200.000000.007248Aortic coarctation0.000000.000000.006800.000000.006849Atrial flutter0.000410.000000.000000.000000.0004mTOR is a serine/threonine-protein kinase playing as a central regulator of cellular metabolism, growth, and survival in response to hormone growth factor [27], nutrients, energy, and stress signals [28, 29]. According to UniPort, mTOR can be found in different subcellular locations including the membranes of endoplasmic reticulum, Golgi apparatus, outer mitochondrion, microsome, and lysosome; lysosome, cytoplasm, nucleus, and PML nuclear body. The RNA and protein expression levels of mTOR are present in several organs including the heart, e.g., the medium RNA and high protein levels of mTOR are expressed in the left ventricle, atrium, and coronary artery but not in the heart muscles (Figure 2).Figure 2
mRNA and protein baseline expression of mTOR in the heart.


## 3.1.2. Evidence on the Cardioprotection ofG. lucidum

A total of 1731 articles were identified (Figure3) and further divided into seven 5-year time periods. The first period ranged from 1985 to 1989, and the last period ranged from August 2018 to August 2019 (Figure 1). These articles were reviewed in the following three phases. First, 1571 articles remained after the duplicated ones were removed. Second, articles that did not satisfy the inclusion criteria based solely on their titles (remaining 1399 articles) and abstracts (remaining 59 articles) were excluded. Lastly, the remaining articles were scanned, and those that did not meet our inclusion criteria were excluded. After the initial screening of titles and abstracts, the 59 remaining articles were screened for the second time by two individual reviewers. Inclusion of full articles was agreed upon by two reviewers prior to data extraction. Finally, 33 studies were considered eligible for the review (Figure 2). In this section, the collected pieces of evidence were divided into two main parts, namely, the in vivo antioxidant of G. lucidum (14 studies, Table 4), in which the in vivo effect of antioxidant on the parameters related to OS was discussed, and the in vitro antioxidant of G. lucidum (19 studies, Table 5), in which the in vitro effect of antioxidant activities and possible molecular mechanisms was elaborated.Figure 3
Number of studies onG. lucidum during 1985-2019.Table 4
In vivo studies of G. lucidum.
No.AnimalFormDosage (mg/kg)Antioxidant parametersBiological activityPathwayReferences1CCl4-induced acute liver injury miceGLPS100 - 150NOS CYP2E1 MDA, GSHSuppressing free radical lipid peroxidationDecreasing of the protein expression levels of NLRP3, ASC, and caspase-1 in acute liver injury.ASC (apoptosis-associated speck-like protein)NLRP3 (NOD-like receptor 3)Caspase-1GAPDH (glyceraldehyde-3-phosphate hydrogenase[22]2Croton oil applied skin edema in ratsEthanol extract of sporocarps500 and 1000 mg/kgAntiperoxidative, anti-inflammatory, and antimutagenic activitiesDirect anti-inflammatory and free radical scavenging properties of the extract[23]3Photoreceptor cell lesions induced by N-methyl-N-nitrosourea (MNU) in female SD artsGanoderma spore lipid (GSL)500, 1000, 2000, and 4000 mg/kgExpressions of Bax, Bcl-xl, and caspase-3Improve A-wave amplitude (μv) decreased apoptosis levelsRegulate the expressions of Bax, Bcl-xl, and caspases-3, inhibiting MNU-induced rat, photoreceptor cell apoptosis, and protecting retinal function[30]4A carotid-artery-ligation mouse modelGanoderma triterpenoid (GT)300 mg/kg/dayIntimal hyperplasia structural changes VCAM-1, TNF-α, and IL-6Atheroprotective propertiesEndothelin-1, von Willebrand factor, and monocyte chemoattractant protein-1[33]5Swimming-induced oxidative stress in skeletal muscle miceGLPS50, 100, and 200 mg/kgSOD, GPX, and CAT activities as well as by the MDA levelsAttenuates exercise-induced oxidative stress in skeletal muscleIncreasing antioxidant enzyme activities and decrease the MDA levels. Protective effects against exhaustive exercise-induced oxidative stress[32]6Rat gastric cancer modelGLPS400-800 mg/kg for 20 weeksSOD, CAT, and GSH-PxAntioxidantInduced the levels of serum IL-6 and TNF-α levels and increased the levels of serum IL-2, IL-4, and IL-10 in GLP-treated rats compared to gastric cancer model rats[37]7BALB/c female miceGLPS i.p. daily50 mg/kg, 100 mg/kg, and 200 mg/kgSOD and GSH-PxAntioxidantImproved immunity in mice. Increased thymus and spleen index; improved SOD and GSH-Px contents in the mice body[31]8T2DM ratsGLPS200, 400, and 800 mg × kg-1 for 16 weeksNO, SOD, MDA, GSH-Px, and CAT MDA in cardiac tissueAntioxidation in cardiac tissue of T2DM ratsReduce MDA in cardiac tissue and improve the myocardial ultrastructure[34]9Male BALB/c mice (age19-21 months) (aged mice)Ethanolic extract ofG. lucidum50 and 250 mg/kg, once daily for 15 daysGSH Mn-SOD, GPx, and GSTAntioxidant in heart tissuesElevated the levels of GSH as well as activities of MnSOD, GPx, and GST and decreased significantly the levels of lipid peroxidation, AOPP, and ROS. Improve the age-related decline of antioxidant status which was partly ascribed to free radical scavenging activity[38]10B16 mouse melanomaMethanol extract containing total terpenoids (GLme) and a purified methanol extract containing mainly acidic terpenoids (GLpme)A daily i.p. injection of 100 mg/kg body weight (b.w.)Production of oxygen radical caspase-dependent apoptotic cell death-mediated production of reactive oxygen speciesAnticancerThe mechanism of antitumor activity of GLme comprised inhibition of cell proliferation and induction of caspase-dependent apoptotic cell death mediated by upregulated p53 and inhibited Bcl-2 expression[86]11With non-insulin-dependent diabetes mellitus (NIDDM)Ganodermalucidum spores250 mg/kg × d, for 10Xanthine oxidase (XOD), myeloperoxidase (MPO), and mitochondrial succinate dehydrogenase (SDH) in the testisReducing free radical-induceddamage to the testicular tissueProtect the testis of diabetic rats by reducing free radical-induced damage to the testicular tissue and enhancing the activity of SDH[35]12Epididymal cells of type 2 diabetes ratsGanoderma lucidum spores (GLS)250 mg/kg × d, for 10 weeksContents of mitochondrial calcium & cytochrome CAntipoptosis induced by DMProtect epididymal cells and counteract their apoptosis in diabetic condition[36]13Liver tissue of ratsGanoderma lucidum peptide27.1μg/mLMalondialdehyde levelAntioxidantSubstantial antioxidant activity in the rat liver tissue homogenates and mitochondrial membrane peroxidation systems[87]14Lupus miceGanoderma tsugae0.5 mg/kg/dayDecreased proteinuria, decreased serum levels of antidsDNA autoantibodyPrevention of autoantibodyPrevention of autoantibody formation[88]Table 5
In vitro studies of G. lucidum.
No.FormConc.Chemical antioxidant testsBiological text of in vitroExp. parametersBiological activityPathwayReferences1GLP0.5-3.0 mg/mLRSFR=Scavenging of free radicals and reducing powerAntioxidantNM[89]2G. lucidum and Egyptian Chlorella vulgarisCVE (63.5μg/mL) was mixed with GLE (4.1 μg/mL)RSFRAPOther testsLipopolysaccharide-stimulated white blood cellsNitric oxide, tumor necrosis factor- (TNF-)αAntioxidant and anti-inflammatoryDownregulate NF-κB[39]3Polysaccharides inG. lucidum2 mg/mLRSFRAPOther testsNMRadical scavenging reducing powerAntioxidantNM[90]4G. lucidum extract50 mgRSFRAPOther testsNMReducing powerAntimicrobial and antioxidantNM[40]5Ganoderma lucidum G20.32 mgRSFRAPOther testsDNA protectionRadical scavenging reducing powerAntimicrobial and antioxidantNM[41]6Protein extracts2–13μg protein/mLAPOther testsDNA protectionRadical scavenging reducing powerAntioxidant, antibacterialNM[42]7Polysaccharides extraction=FRAPOther testsMCF-7 breast cancer cell line and HeLa cellsRadical scavengingAntioxidantAnticancerNM[43]8G. lucidum and G. resinaceum0.1–1 &0.64±0.040–2.25 mg/mLFRAPOther testsIn vitro cell lineRadical-scavenging chelating lipoxygenase assayAntiproliferative & antioxidantNM[44]9Diff, organic solvent oG. lucidum1-200μg/mLFRAPOther testsNMRadical scavenging, chelating lipid peroxidationAntioxidantAnticholinesteraseNM[45]10Both aqueous and methanolic extracts0.2–30 mg/mL of extractionFRAPOther testsNMRadical scavenging, chelating lipid peroxidationAntioxidantNM[46]11Low-molecular-weightβ-1,3-glucan0–200μg/mLAPOther testsMouse monocyte-macrophage cell line, RAW 264.7H2O2-induced apoptosisAntioxidantAttenuating intracellular reactive oxygen species (ROS) and inhibiting sphingomyelinase (SMase) activity[51]12Polysaccharides0.16-10 mg/mLFRAPOther testsNMRadical scavenging, chelating reducing powerAntioxidantNM[47]13G. lucidumwater-soluble and water-insoluble80-1100μg/mlFRAPOther testsHuman uroepithelial cell (HUC-PC) cellsRadical scavenging, chelating reducing powerAntioxidantOxidative DNA damage. Lingzhi-induced apoptosis in bladder chemoprevention[48]14Ganoderma lucidum polysaccharides0.1-0.6 mg/mlRSCCl-induced injury hepatocytes DNA protectionMDA, SOD, CYP3A, caspase-3, andcaspase-8Suppressing inflammatory responsesReduction of NF-κB activation inhibition of caspase-3, caspase-6, and caspase-9, indicating and suppression extrinsic-induced apoptosis[52]15Ganoderic acid A10-80 lM/mLNMPancreatic cellsRadical scavengingAntiproliferativeAntioxidantAnticancerβ-Catenin in Wnt signaling pathway[54]16Aqueous extract ofG. lucidum5-20μLNMDNA protectionRadical scavengingAntioxidantDNA repairEnhancing reactivity of apurinic/apyrimidinic endonucleases (APE1) a major enzyme of base excision repair (BER)[91]17Methanolic extract ofG. lucidum65 & 130μg/mLNMHuman gastric tumor cellsIncreased the formation of autophagosomesInduces autophagyIncreasing of the cellular levels of LC3-II and decreasing p62 (autophagy-related protein)[92]18G. lucidum (GLPS) and G. sinense (GSPS)19–300μg/mLNMRAW 264.7 mouse macrophage cellsNitric oxide secretion of cytokinesImmunomodulatoryPromoting macrophage phagocytosis, increasing their release of nitric oxide and cytokines interleukin- (IL-) 1a, IL-6, IL-10, and tumor necrosis factor-α[56]19Proteopolysaccharide fromG. lucidum2 - 10μg/mLNMRAW264.7, a mouse macrophage cell lineNitrite productionExpression levels of cytokinesActivation the immune system by modulating cytokine production.NM[57]NM = not mention; RS = radical scavenging; FR = ferric reducing; AP = antilipid peroxidation.


## 3.2.In Vivo Antioxidant Activity and Protective Effect of G. lucidum

According to 10 in vivo experimental studies,G. lucidum has antioxidant activities and protects against OS through four main factors in different tissues, such as the heart, liver, thymus, spleen, eyes, and skeletal muscles, and by regulating chemical-level OS parameters in blood circulation (Table 4). G. lucidum exhibits its antioxidant effects by increasing the antioxidant enzymes and inhibiting the enzymes involved in OS. G. lucidum also increases the activities of superoxide dismutase (SOD), glutathione-S-transferase (GST), glutathione peroxidase (GPx), catalase (CAT), mitochondrial succinate dehydrogenase (SDH), and Mn-SOD and reduces glutathione (GSH) levels. By contrast, G. lucidum decreases the activities of nitric oxide synthase (NOS), cytochrome P450 2E1 (CYP2E1), xanthine oxidase (XOD), and myeloperoxidase (MPO). G. lucidum also significantly decreases lipid peroxidation levels, advanced oxidation protein products (AOPPs), and malondialdehyde (MDA) levels.The first factor is the four toxic substances, including CCl4-induced oxidative stress (OS) in the liver, croton oil produced OS in the skin through inflammation, N-methyl-N-nitrosourea (MNU) causing retinal photoreceptor cell lesions in the eyes, and 5-fluorouracil-induced OS in the thymus and spleen of mice. Oral administration of G. lucidum polysaccharides (GLPs) represses free radical lipid peroxidation induced by CCl4 to reduce the enzyme activities of NOS and CYP2E1. Significant inhibition of NOS and CYP2E1 activities and MDA and IL-1β levels was noted in liver tissues, and depleted levels of interleukin- (IL-) 1β, IL-18, IL-6, and tumor necrosis factor-α were found in serum. In CCl4-induced liver damage, highly reactive trichloromethyl free radicals are generated by the cytochrome P450 isozymes (P450s) of the endoplasmic reticulum [22]. Topical administration of G. lucidum ethanol extract inhibits the croton oil-induced lipid peroxidation in the skin of mice [23]. Ganoderma spore lipid (GSL) shows a protective effect on MNU-induced retina injury by inhibiting the related apoptosis to modulate the expression levels of Bax, Bcl-xl, and caspase-3 [30]. GLPs also exhibit an antioxidant effect in 5-fluorouracil-induced OS and improve SOD, an intracellular compound that protects against oxidative processes initiated by superoxide anion and GPx contents in the spleen and thymus of mice [31].The second factor creates conditions in biological systems that can induce OS, such as exercise-like exhaustive swimming, which is OS induced in skeletal muscles, and a carotid artery ligation, which disturbs the flow-induced OS level of manganese-dependent superoxide dismutase (Mn-SOD) in blood vessels. GLPs show protective effects against comprehensive swimming-induced OS by improving the activities of antioxidant enzymes (SOD, GPx, and CAT) and decreasing the MDA levels in the skeletal muscle of mice [32]. Oral ganoderma triterpenoids (GTs) protect against disturbed flow-induced OS through carotid artery ligation, which leads to chronic OS and inflammation that are features of early atherogenesis in mice, and by preventing neointimal thickening 2 weeks after ligation. Early atherogenesis includes neointimal hyperplasia and endothelial dysfunction due to flow turbulence in the ligated artery as induced by OS. GTs alleviate OS and restore the atheroresistent status of endothelium by inhibiting endothelin-1 induction, von Willebrand factor, and monocyte chemoattractant protein-1 after 3-day ligation as atherogenic factors [33]. Inflammatory cytokines, OS-induced endothelial dysfunction, and chronic OS contribute to endothelial impairment and induces atherogenesis.The third factor in OS includes diseases such as type II diabetes mellitus (DM) and cancer. In type II DM, the beneficial effects ofG. lucidum on abnormal heart and testis and epididymal cells of rats with streptozotocin-induced type II DM were evaluated. GLPs improve the myocardial ultrastructure by reducing MDA, activating antioxidant enzymes (GSH-Px, CAT, SOD, and NO) in cardiac tissues, and reducing lipid peroxidation in type II DM rats [34]. G. lucidum spores protect the testis of rats with type II DM by substantially increasing the mitochondrial SDH and decreasing the activities of XOD and MPO [35]. G. lucidum spores protect epididymal cells and counteract their apoptosis that damages the mitochondria and disequilibrium of calcium homeostasis by reducing the amount of mitochondrial cytoplasm cytochrome C in type II DM rats [36]. GLP administration enhances the immunity and antioxidant activities in N-methyl-N9-nitro-nitrosoguanidine-induced gastric cancer in Wistar rats. GLP remarkably reduces the levels of serum IL-6 and TNF-α and increases the levels of serum IL-2, IL-4, and IL-10. In addition, GLP improves the levels of SOD, CAT, and GSH-Px in serum and gastric tissues [37].The fourth factor involved in OS is aging.G. lucidum administration ameliorates the age-related decline of antioxidant status in aged mice, substantially elevates the activities of GST, Mn-SOD, GPx, and CAT, and reduces GSH. By contrast, lipid peroxidation, AOPP, and reactive oxygen species (ROS) are reduced [38] (Table 4).


## 3.3.In Vitro Antioxidant of G. lucidum and Its Possible Pathway

Chemical antioxidant tests consistently revealed the free radical scavenging activity ofG. lucidum. Twelve studies reported the scavenging activity of G. lucidum for different free radicals including 2,2-diphenylpicrylhydrazyl radical (DPPH⋅), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) radical (ABTS⋅+), hydroxyl radical (HO⋅), and hydrogen peroxide radicals (H2O2) [39–49] (Table 5). G. lucidum also inhibits lipid peroxidation [23, 49]. In some studies, G. lucidum protects against DNA damage [41, 42, 50]. The results of chemical antioxidant tests regarding the antioxidant properties of G. lucidum are also in agreement with the cell-based antioxidant assays. G. lucidum shows free radical scavenging activity for H2O2 in RAW264.7 cells incubated with G. lucidum lipopolysaccharide and protects against H2O2-induced cell death [48]. G. lucidum also hinders sphingomyelinase activity in incubated RAW264.7 cells with lipopolysaccharide [51]. In addition, G. lucidum prevents lipid peroxidation in two cell models, namely, WBCs incubated with lipopolysaccharide to induce OS [39] and hepatocytes incubated with CCl4 to induce OS [52]. In both cell models, G. lucidum showed protection by elevating the antioxidant enzyme activity (SOD, GPx, and GR) and improving the GSH level. Moreover, G. lucidum protects macrophages in human monocytic cells incubated with lipopolysaccharide to stimulate NO production [53].Wnt, Erk1/2, and NF-κB are the possible signaling pathways of G. lucidum that support its antioxidant and protective effects. A pancreatic cell study suggested β-catenin in the Wnt signaling pathway as a target of ganoderic acid A, thus leading to cell protection and effective scavenging of ROS [54]. The Wnt signaling pathways transfer the signals from extracellular to intercellular and are stimulated by the Wnt protein binding to the cytoplasmic family receptor, which occurs in downstream cell signaling and controls the transcription of genes. In the canonical Wnt pathway, β-catenin accumulates in the cytoplasm and is further translocated into the nucleus, and this phenomenon is widely recognized as a regulation marker of fat and glucose metabolism and β-catenin/Wnt signaling involved in insulin secretion [54]. In 2006, Thyagarajan and his colleagues mentioned that G. lucidum modulates Erk1/2 signaling and transcription factors AP-1 and NF-κB and downregulates c-Fos, whose expression can be induced by OS as the result of the inhibited OS-induced invasive behavior of breast cancer cells. A high H2O2 concentration (5 mM) can stimulate Erk1/2 signaling in MCF-7 cells [55].In addition to its antioxidant activities,G. lucidum also exhibits an anti-inflammatory property and modulates the immune system. It can reverse LPS-induced inflammation by downregulating inflammatory mediators such as NF-κB, thus substantially inhibiting NOS and reducing NO level [39]. G. lucidum also modulates the immune system byregulating cytokine production in RAW264.7 macrophages [56, 57]. Moreover, it increases the formation of autophagosomes and controls proteins (Vps34, beclin 1, LC3-I, LC3-II, and p62) that induce autophagy in a gastric adenocarcinoma cell line. G. lucidum increases the cellular levels of LC3-II and decreases the cellular levels of p62 (Table 5).


## 4. Discussion

Among the 155 targets associated with DCM, mTOR, CPT1B, and CPT2 have the highest association. mTOR acts as a core regulator of cellular metabolism, growth, and survival in response to hormone growth factors, nutrients, energy, and stress signals. An animal study confirmed that streptozotocin-induced diabetes increases mTOR levels in rats [58]. mTOR can be found in different cellular locations including membrane, cytoplasm, and nucleus and different cellular organs (mitochondria, Golgi, and endoplasmic reticulum) and therefore is involved directly or indirectly in regulating the phosphorylation of at least 800 proteins (OPT.V19.11). mTOR functions through two distinct signaling complexes of mTORC1 and mTORC2 [59]. When activated, mTORC1 upregulates protein synthesis by phosphorylating the key regulators of mRNA translation and ribosome synthesis. mTORC1 also regulates protein synthesis [29], lipid synthesis [60], and mitochondrial biogenesis and stimulates the pyrimidine biosynthesis pathway through acute and delayed regulations. In acute regulation, mTORC1 stimulates pyrimidine biosynthesis through the ribosomal protein S6 kinase B1-mediated phosphorylation of biosynthetic enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase; these enzymes catalyze the first three steps in de novo pyrimidine synthesis [61]. In delayed regulation, mTORC1 stimulates pyrimidine biosynthesis through the transcriptional enhancement of the pentose phosphate pathway, which produces 5-phosphoribosyl-1-pyrophosphate, an allosteric activator of pyrimidine biosynthesis enzyme at a later step in the synthesis. In addition, mTORC1 regulates ribosome synthesis by activating RNA polymerase III-dependent transcription through the phosphorylation and inhibition of MAF1 protein, a RNA polymerase III-repressor. When nutrients are available and mTOR kinase is active, MAF1 is hyperphosphorylated, and RNA polymerase III is engaged in the transcription [62]. Stress-induced MAF1 dephosphorylation resulted in nuclear localization, increased targeting of gene-bound RNA polymerase III, and decreased transcriptional readout [63, 64]. Moreover, mTORC1 is involved in the negative feedback regulation of autophagy on upstream growth factor signaling during microtubule regulation [64–66].mTORC2 regulates other cellular processes such as survival and organization of cytoskeleton, actin cytoskeleton [67], osteoclastogenesis, and circadian clock function. In a pressure-overloaded male mouse heart, mTORC2 maintains a contractile function [68]. In brown adipose tissues, mTOR complex 2 has a role in β3-adrenoceptor-stimulated glucose uptake by stimulating the translocation of newly synthesized GLUT1 to the plasma membrane, thereby increasing the glucose uptake [69]. mTOR complex 2 regulates the proper turnover of insulin receptor substrate-1 [70].G. lucidum exhibits cardiac protection via its antioxidant properties through OS modulation. This systemic review of 33 studies has documented its antioxidant activities. At the molecular and cellular levels, OS is a key in diabetes-induced DCM [18]. The antioxidant effects of G. lucidum are facilitated by increasing the antioxidant enzymes and inhibiting the enzymes involved in OS [33–35, 38]. G. lucidum consistently shows free radical scavenging activity against several free radicals including DPPH⋅, ABTS⋅+, HO⋅, and H2O2. As confirmed by the in vitro (chemical and cell-based) antioxidant tests, G. lucidum inhibits lipid peroxidation and protects against DNA damage.G. lucidum modulates several signal pathways including Erk1/2, NF-κB, and Wnt. Its antioxidant activity protects against inflammation and directly modulates immunity through scavenging radicals and through the oxidative signal pathways, thereby protecting the cells. These effects of G. lucidum may contribute to its positive influence on DCM.DM is a state of persistent inflammation that upregulates mTOR at different levels of the myocardium, thereby influencing several signal pathways. The elevation of cellular cAMP levels disrupts phosphodiesterase-Rheb interaction, increases Rheb-mTOR interaction, and consequently leads to mTOR1 activation. Phosphodiesterase binds with Rheb and thereby inhibits the latter’s ability to activate mTOR [71]. Heart myocardium responds to high blood glucose by adapting its energy metabolism and using only fatty acids as a substrate, thus increasing OS through the upregulation of NADPH-oxidases, NO synthases [72], and reversible oxidative modifications for myocardial titin elastic protein [73]. mTOR upregulation and oxidative modification alter titin-based stiffness and titin isoform composition, thereby impairing myocardium contractility. The PI3K-Akt-mTOR kinase axis regulates the composition of titin isoform [73]. OS decreases NO levels, leading to the impairment of the NO-soluble guanylate cyclase- (sGC-) cyclic guanosine monophosphate- (cGMP-) protein kinase G (PKG) pathway, an important regulator of cardiac contractility [72]. Chronic intrude accumulation to high free fatty acids downregulates PPAR-α and impairs mTOR-PPAR-α, thereby causing mitochondrial dysfunction in rodent cardiomyocytes and further deteriorating cardiac function through the inhibition of fatty acid oxidation and increase in intracellular fat accumulation. PPAR-α is involved in the upregulation of carnitine palmitoyltransferase I, which increases the uptake of long-chain fatty acid in the mitochondria and facilitates the beta-oxidation of fatty acids. mTOR-PPAR-α axis modification can lead to inflammation [74] and immune dysfunction [75]. mTOR upregulation leads to the impaired response to adrenergic stimulation in DCM mice and further reduces heart contractility [58]. mTOR inhibition improves contractility via the chronic administration of PDE inhibitor in animals and patients with diabetes [76] and restores the impaired response to adrenergic stimulation in DCM mice [58]. G. lucidum shows its effects via several signal pathways such as Wnt, Erk1/2, and NF-κB pathway and consequently reduces the upregulated mTOR and its effects. mTOR is the main target of G. lucidum, and this finding supports its antioxidant and cardioprotective effects. G. lucidum inhibits the Wnt pathway [54] and may decrease the activity of mTOR via the Wnt/GSk/mTOR signal pathway. A pathologically stressed heart reactivates the Wnt signal pathway, which is modulated during left ventricular remodeling [77]. In heart cells, the Wnt pathway plays a role in the release of intracellular Ca2+ whose accumulation activates several Ca+2-sensitive proteins, fat and glucose metabolism, and cell fate decisions, such as renewal, differentiation, and apoptosis. Wnt dysregulation has an important role in cardiac diseases such as hypertrophy and fibrosis [78]. The Wnt pathway is important in the response to heart injuries leading to adverse effects on the heart [79] and is integrated with bioenergetic status to control mTOR activity [80]. Wnt is activated in late-stage inflammation of heart tissue [81]. G. lucidum suppresses Erk1/2 signaling [55] and consequently reduces the mTOR level. Erk1/2 signaling inhibits the TSC1/2 complex, which is the downregulator of mTOR, and thus activates mTOR [82]. The antioxidant properties of G. lucidum abolish the activation of the Erk pathway by OS. NADPH oxidase 2 is involved in Erk activation [83], and the inhibition of Erk/mTOR by G. lucidum also prevents NF-κB. mTOR activates NF-κB by phosphorylating the NF-κB p65 subunit, increasing p65 nuclear translocation, and activating gene transcription. With its anti-inflammatory effect, G. lucidum inhibits NF-κB via decreasing inflammatory mediators and cytokines such as TNF or IL-1, and innate immune response effectors activate NF-κB via the IKK complex through IκB protein phosphorylation with subsequent ubiquitination and degradation [84]. Inhibiting mTOR and NF-κB may improve the contractility of the heart, abolish the angiotensin II-induced hypertrophic response of cardiomyocytes [83], and prevent heart failure. A prolonged NF-κB activation promotes heart failure by evoking signals that induce chronic inflammation through the enhancement of cytokines including tumor necrosis factor, IL-1, and IL-6, commencing to endoplasmic reticulum stress responses and cell death [85].Our results concluded that the antioxidant properties ofG. lucidum and the cardioprotection of its polysaccharides may have a direct effect. Its free radical scavenging ability reduces OS and upregulates mTOR via several pathways including Wnt, Erk1/2, and NF-κB/IKK/TOR, thereby improving myocardium contractility (Figure 4). The anti-inflammatory properties may enhance the cAMP/cGMP/mTOR/PPAR pathway and its related protein or/and pathway and mitochondrial function, thus improving myocardium hemostasis. Further study is needed to identify the specific target of GLP in heart tissues.Figure 4
G. lucidum inhibits mTOR via several signal pathways (- inhibit; + stimulate). Red +/- effects of inflammation, glucose starvation and fat metabolism, and oxidative stress on different effectors of different pathways in cardiocytes; black +/- protective effects of G lucidum at different effectors of different pathways in cardiocytes.



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