# Network Pharmacology Analysis on the Mechanism of Huangqi Sijunzi Decoction in Treating Cancer-Related Fatigue

Authors: Yixin Cui; Haiming Wang; Decai Wang; Jiwei Mi; Gege Chen; Fagen Li; Yujia Wang; Yin Zhang
Journal: Journal of Healthcare Engineering
 (2021)

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## Abstract

Objective. This study aimed to determine the active ingredients of Huangqi Sijunzi Decoction (HQSJZD) and the targets in treating cancer-related fatigue (CRF) so as to investigate the treatment mechanism of HQSJZD for CRF. Methods. This study adopted the method of network pharmacology. The active ingredients and targets of HQSJZD were retrieved, and the targets of HQSJZD in treating CRF were obtained using a Venn diagram. Next, a protein-protein interaction (PPI) network was constructed using the String database. The core targets of HQSJZD in treating CRF were identified through topological analysis, and functional annotation analysis and pathway enrichment analysis were carried out. Subsequently, a compound-disease-target regulatory network was constructed using Cystoscape 3.8.0 software. Results. A total of 250 targets of HQSJZD ingredients, 1447 CRF-related genes, and 144 common targets were obtained. Through topological analysis, 61 core targets were screened. Bioinformatics annotation of these genes identified 2366 gene ontology (GO) terms and 172 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Conclusion. The active ingredients in HQSJZD, that is, quercetin, luteolin, kaempferol, and naringenin, may act on AKT1, IL-6, VEGFA, MAPK3, CASP3, JUN, and EGFR to regulate the PI3K-Akt, TNF, and IL-17 signaling pathways, thereby suppressing inflammatory response, tumor gene expression, and tumor angiogenesis to treat CRF. This study investigated the pharmacological basis and mechanism of HQSJZD in the treatment of CRF using systematic pharmacology, which provides an important reference for further elucidation of the anti-CRF mechanism and clinical applications of HQSJZD, and also provides a method protocol for similar studies in the future.


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## Body

## 1. Introduction

Cancer-related fatigue (CRF) refers to the painful, continuous, and subjective feeling of fatigue or exhaustion resulting from malignant tumor or tumor-related treatments, which is accompanied by symptoms such as limb heaviness, insomnia or somnolence, difficulty in activities, and abnormal emotional responses, but is not related to the amount of exercise [1]. CRF has an incidence rate in tumor patients of as high as 75% [2], and patients with CRF demonstrate higher degrees of fatigue than ordinary people, a longer course of disease, no remission after resting, and a greater propensity for negative emotions, which seriously affect the quality of life of the patients. It is currently well known by scholars worldwide that the production of cytokines, the abnormal accumulation of muscle metabolites, and mitochondrial dysfunction are involved in CRF [3]. Cytokines released by tumor cells, such as IL-2, IL-6, and tumor necrosis factor (TNF), not only affect the metabolism of normal cells but also promote tumor cell growth, resulting in impaired body functions and the occurrence of fatigue symptoms [4]. Peripheral inflammatory cytokines can enter the brain via various paths, and cytokines like IL-6 and TNF may affect the hypothalamic-pituitary-adrenal (HPA) axis and suppress cortisol synthesis and release, thereby aggravating fatigue symptoms [5]. After chemotherapy or radiotherapy, the mitochondrial structure and function are destroyed, which can also aggravate fatigue [6]. According to the NCCN Cancer-Related Fatigue (CRF) guidelines, CRF is mainly treated with drugs such as psychostimulants or those for improving sleep dysfunction; nondrug treatments like social-psychic intervention and acupuncture, however, do not provide satisfactory effects.According to traditional Chinese medicine (TCM), CRF belongs to the “consumptive disease” category. When cancer toxins have invaded the body for a prolonged period, the blood and Qi of the patients are impaired, which may lead to the impairment of kidney functions and the failure of the five organs, resulting in the occurrence of CRF. Moreover, surgery, radiotherapy, and chemotherapy aggravate blood and Qi impairment, thereby exacerbating fatigue symptoms. Therefore, the pathogenesis of CRF in TCM involves the impairment of the five organs and Qi/blood deficiency. In this regard, CRF is mainly treated by strengthening the body’s resistance and tonifying the body to compensate for the deficiency.In recent studies, the therapeutic effects of Huangqi Sijunzi Decoction (HQSJZD) on treating CRF have been recognized by numerous doctors. HQSJZD is modified from Sijunzi Decoction, with the addition of Astragalus, which has Qi-tonifying, blood-replenishing, body-resistance-enhancing, and deficiency-tonifying effects. The Sijunzi Decoction consists of ginseng (which is often replaced byCodonopsis pilosula owing to the rareness of ginseng), Atractylodes, poria cocos, and radix glycyrrhizae. This is a classical and representative TCM preparation for Qi tonifying. The addition of Astragalus further enhances the Qi- and spleen-tonifying, body-resistance-strengthening, and consolidating effects. Pharmacological research [7] has confirmed that Sijunzi Decoction can accelerate the recovery of NK cell and K cell activities that are suppressed after chemotherapy and enhance granulocyte, monocyte, and bone marrow hematopoietic functions. Moreover, the addition of Astragalus to the Sijunzi Decoction has been found to improve RNA synthesis in the liver and enhance the energy metabolic rate of mice with spleen deficiency and to reduce mitochondrial injury and improve energy metabolic disorder [8]. HQSJZD has been proved in clinical practice to correct anemia, improve immune function, and mitigate energy metabolic disorder, thus relieving CRF symptoms [8]. However, the molecular mechanism of HQSJZD in CRF treatment remains unclear, which limits its promotion and development.Network pharmacology can systematically analyze the interaction network of drug components, protein targets, diseases, and genes and is effective for establishing a “compound-protein/gene-disease” network in a high-throughput manner [9, 10]. This approach is powerful for the analysis of TCM preparations. A number of researchers have applied network pharmacology to study the mechanism of TCM in various diseases [11–14]. This study screened the active ingredients of HQSJZD and the human targets of these ingredients as well as CRF-related genes. Network pharmacology was used to explore the relationship between the active ingredients of HQSJZD and CRF targets and the mechanism of HQSJZD treatment of CRF to provide reference for further elucidation of the anti-CRF mechanism and clinical applications of HQSJZD.


## 2. Methods

The study strategy is illustrated in Figure1.Figure 1
Illustration of the study strategy.


### 2.1. Screening for the Targets of HQSJZD Ingredients

The components of HQSJZD, Atractylodes,Codonopsis pilosula, Poria cocis, radix glycyrrhizae, and Astragalus were searched in the TCMSP database [15] (https://tcmspw.com/tcmsp.php), with oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18 used as screening criteria to screen for targets of corresponding ingredients. Next, the uniprot database (https://www.uniprot.org/) [16] was used for target correction, with species restricted to “Homo sapiens,” and the target proteins were transformed into corresponding genes.


### 2.2. Screening of CRF-Related Genes

Using “cancer-related fatigue” as keyword, human genes were screened in the GeneCards database [17] (https://www.genecards.org), NCBI database [18] (https://www.ncbi.nlm.nih.gov), and OMIM database [19] (https://www.omim.org) for CRD-related genes. Targets in the GeneCards database were screened according to the median value, so more related targets were obtained.


### 2.3. Prediction of Potential Targets of HQSJZD in Treating CRF

The Venn diagram generation software Venny 2.1 was used to obtain the potential targets of HQSJZD in treating CRF from targets corresponding to the active ingredients of HQSJZD and the CRF-related targets.


### 2.4. Construction of the PPI Network and Topological Analysis

The PPI network was constructed using the String database [20] (https://string-db.org/cgi/input.pl), with the common drug-disease targets screened. The biological species was set to “Homo sapiens,” and the minimum interaction score was set at medium confidence (0.400). The NetworkAnalyzer tool of the Cystoscape 3.8.0 software [21] was used for topological analysis of the PPI network. Genes were sorted based on degree values, and genes with scores greater than the mean value were selected as the core targets.


### 2.5. Screening of Key Active Ingredients

To better understand the complicated interactions among drug components, disease, and corresponding targets, a component-disease-target network was constructed based on the screened drug ingredients, disease, and targets using the Cystoscape 3.8.0 software for topological analysis. Drug ingredients were sorted according to the degree values; the higher the degree value, the more important the ingredient.


### 2.6. GO Functional Enrichment and KEGG Pathway Enrichment Analyses

GO enrichment analysis was performed on the common drug-disease targets. Using the String database, terms with a corrected P value <0.05 were selected. Next, the clusterProfiler, enrichplot, and ggplot2 packages of the R 4.0.3 software were employed to plot the bubble chart. The common drug-disease targets were also applied to KEGG pathway enrichment analysis, pathways with corrected P value <0.05 were selected, and the top 20 pathways were visualized.


### 2.7. Construction of the Component-Disease Pathway-Target Network

Drug components, the top 20 KEGG pathways, and core targets were imported into the Cytoscape 3.8.0 software to construct the component-disease pathway-target network.


## 2.1. Screening for the Targets of HQSJZD Ingredients

The components of HQSJZD, Atractylodes,Codonopsis pilosula, Poria cocis, radix glycyrrhizae, and Astragalus were searched in the TCMSP database [15] (https://tcmspw.com/tcmsp.php), with oral bioavailability (OB) ≥30% and drug likeness (DL) ≥0.18 used as screening criteria to screen for targets of corresponding ingredients. Next, the uniprot database (https://www.uniprot.org/) [16] was used for target correction, with species restricted to “Homo sapiens,” and the target proteins were transformed into corresponding genes.


## 2.2. Screening of CRF-Related Genes

Using “cancer-related fatigue” as keyword, human genes were screened in the GeneCards database [17] (https://www.genecards.org), NCBI database [18] (https://www.ncbi.nlm.nih.gov), and OMIM database [19] (https://www.omim.org) for CRD-related genes. Targets in the GeneCards database were screened according to the median value, so more related targets were obtained.


## 2.3. Prediction of Potential Targets of HQSJZD in Treating CRF

The Venn diagram generation software Venny 2.1 was used to obtain the potential targets of HQSJZD in treating CRF from targets corresponding to the active ingredients of HQSJZD and the CRF-related targets.


## 2.4. Construction of the PPI Network and Topological Analysis

The PPI network was constructed using the String database [20] (https://string-db.org/cgi/input.pl), with the common drug-disease targets screened. The biological species was set to “Homo sapiens,” and the minimum interaction score was set at medium confidence (0.400). The NetworkAnalyzer tool of the Cystoscape 3.8.0 software [21] was used for topological analysis of the PPI network. Genes were sorted based on degree values, and genes with scores greater than the mean value were selected as the core targets.


## 2.5. Screening of Key Active Ingredients

To better understand the complicated interactions among drug components, disease, and corresponding targets, a component-disease-target network was constructed based on the screened drug ingredients, disease, and targets using the Cystoscape 3.8.0 software for topological analysis. Drug ingredients were sorted according to the degree values; the higher the degree value, the more important the ingredient.


## 2.6. GO Functional Enrichment and KEGG Pathway Enrichment Analyses

GO enrichment analysis was performed on the common drug-disease targets. Using the String database, terms with a corrected P value <0.05 were selected. Next, the clusterProfiler, enrichplot, and ggplot2 packages of the R 4.0.3 software were employed to plot the bubble chart. The common drug-disease targets were also applied to KEGG pathway enrichment analysis, pathways with corrected P value <0.05 were selected, and the top 20 pathways were visualized.


## 2.7. Construction of the Component-Disease Pathway-Target Network

Drug components, the top 20 KEGG pathways, and core targets were imported into the Cytoscape 3.8.0 software to construct the component-disease pathway-target network.


## 3. Results

### 3.1. Screening of the Potential Targets of HQSJZD in Treating CRF

The active ingredients of HQSJZD were retrieved from the TCMSP database, including 20 Astragalus ingredients, 21 Codonopsis pilosula ingredients, 7 Atractylodes ingredients, 15 Poria cocis ingredients, and 93 radix glycyrrhizae ingredients. Moreover, the targets of these active ingredients were also obtained, including 20 targets of Astragalus ingredients, 106 targets ofCodonopsis pilosula ingredients, 16 targets of Atractylodes ingredients, 23 targets of Poria cocis ingredients, and 225 targets of radix glycyrrhizae ingredients. Finally, a total of 250 targets were obtained after eliminating duplicates.A total of 1446 CRF-related genes were obtained after searching the GeneCards database, 13 were obtained from the NCBI database, and 2 were obtained from the OMIM database. After eliminating duplicated genes, 1447 CRF-related genes were retrieved. The screened drug targets and disease targets were intersected to obtain 144 common targets between HQSJZD and CRF (Figure2), which were used as the potential targets of HQSJZD in treating CRF in subsequent network construction and pathway enrichment analysis.Figure 2
Venn diagram of the targets of the active ingredients of HQSJZD and the CRF-related targets.


### 3.2. Core Targets of HQSJZD in Treating CRF

In the PPI network constructed using the 144 common targets (Figure3), the color and size of the nodes were plotted based on the degree value; a bigger node and deeper color indicate a greater degree value. A total of 3074 edges, representing protein interactions, were established. The edge thickness represents the edge betweenness, and a thicker line indicates a stronger relationship between the two targets. The average degree value of the PPI network is 42.7. Topological analysis of the PPI networks identified 61 genes with scores greater than the average as the core targets. The top 20 genes were plotted (Figure 4), with the X-coordinate being the degree of each gene; the greater the value, the more important the role the gene plays in the network. Therefore, the AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, EGFR, MAPK1, and MYC genes may play the most important roles in the HQSJZD treatment of CRF.Figure 3
PPI network of the common targets of HQSJZD and CRF.Figure 4
20 key targets determined in the PPI network.


### 3.3. Screening of Key Components

Topological analysis of the component-disease-target network constructed using the Cystoscape 3.8.0 software identified the key components of HQSHZD and ranked them based on degree values; a higher value represents greater importance. Components with a value greater than 10 were chosen for subsequent analysis (Table1). The component with the highest value was quercetin, followed by luteolin, kaempferol, naringenin, formononetin, and isorhamnetin.Table 1
Top active ingredients of HQSJZD identified in the component-target network.
MOL IDNameDegreeMOL000098Quercetin106MOL000006Luteolin46MOL000422Kaempferol36MOL004328Naringenin24MOL000392Formononetin21MOL000354Isorhamnetin20MOL0038967-Methoxy-2-methyl isoflavone20MOL000497Licochalcone19


### 3.4. GO Enrichment Analysis

The 144 HQSJZD-CRF common targets were subjected to GO enrichment analysis. A total of 2332 BP terms (P<0.01), 6 CC terms, and 27 MF terms were retrieved. The top 10 GO terms of each category were plotted in the bubble chart (Figure 5). The top BP terms are mainly involved in oxidative stress response, cellular response to chemical stress, response to bacterial molecules, cellular response to oxidative stress, reactive oxygen species metabolic process, regulation of the apoptotic signaling pathway, epithelial cell proliferation, regulation of inflammatory response, regulation of DAN binding transcription factor activity, response to hypoxia, and negative regulation of the apoptotic signaling pathway. The common targets were mainly enriched in six CC terms, including membrane raft, membrane microdomain, membrane region-protein kinase complex, cyclin-dependent protein kinase holoenzyme complex, serine/threonine protein kinase complex, and vesicle lumen. The enriched MF terms mainly include DNA binding transcription factor binding, RNA-polymerase-specific DNA binding transcription factor binding, nuclear receptor activity, ligand activation, transcription factor activity, phosphatase binding-cytokine receptor binding, heme binding tetrapyrrole binding, cytokine activity, and protein phosphatase binding.Figure 5
Top GO enriched terms of HQSJZD in treating CRF. TheX-coordinate indicates the number of enriched genes, and the color of the dot represents the P value of the corresponding term. A bigger dot indicates that more genes are enriched.


### 3.5. KEGG Pathway Analysis

The 144 common targets were significantly enriched into 172 pathways (P<0.05). The top 20 pathways are shown in Figure 6. Pathways not related to CRF, such as the “AGE-RAGE signaling pathway in diabetic complications,” were excluded. Finally, 9 pathways with the smallest P values were selected: phosphatidylinositol 3 kinase/protein kinase B (PI3K-Akt) signaling pathways, prostate cancer, bladder cancer, pancreatic cancer, IL-17 signaling pathway, non-small cell lung cancer, TNF signaling pathway, and small cell lung cancer.Figure 6
Top KEGG enriched pathways of HQSJZD in treating CRF. A bigger dot indicates that more genes are enriched in that pathway, and a dot with a darker red color represents a smallerP value.


### 3.6. Construction of the Ingredient-Disease Pathway-Target Network

The ingredient-disease pathway-target network was constructed using Cytoscape3.8.0 software (Figure7). The network contains 5 drugs, 114 active ingredients (ingredients not directly connected to targets were eliminated), 144 CRF-related disease targets (free protein spots were eliminated), and 20 CRF target-related pathways. Among the active ingredients, quercetin had the highest number of connected edges, followed by luteolin, kaempferol, naringenin, formononetin, and isorhamnetin. Among the potential targets, AKT1, IL6, VEGFA, and MAPK3 had relatively more connected edges. Among the pathways, the PI3K-Akt signaling pathway, prostate cancer, bladder cancer, pancreatic cancer, IL-17 signaling pathway, non-small cell lung cancer, TNF signaling pathway, and small cell lung cancer showed more connections with the potential targets.Figure 7
Drug component-target-pathway network.


## 3.1. Screening of the Potential Targets of HQSJZD in Treating CRF

The active ingredients of HQSJZD were retrieved from the TCMSP database, including 20 Astragalus ingredients, 21 Codonopsis pilosula ingredients, 7 Atractylodes ingredients, 15 Poria cocis ingredients, and 93 radix glycyrrhizae ingredients. Moreover, the targets of these active ingredients were also obtained, including 20 targets of Astragalus ingredients, 106 targets ofCodonopsis pilosula ingredients, 16 targets of Atractylodes ingredients, 23 targets of Poria cocis ingredients, and 225 targets of radix glycyrrhizae ingredients. Finally, a total of 250 targets were obtained after eliminating duplicates.A total of 1446 CRF-related genes were obtained after searching the GeneCards database, 13 were obtained from the NCBI database, and 2 were obtained from the OMIM database. After eliminating duplicated genes, 1447 CRF-related genes were retrieved. The screened drug targets and disease targets were intersected to obtain 144 common targets between HQSJZD and CRF (Figure2), which were used as the potential targets of HQSJZD in treating CRF in subsequent network construction and pathway enrichment analysis.Figure 2
Venn diagram of the targets of the active ingredients of HQSJZD and the CRF-related targets.


## 3.2. Core Targets of HQSJZD in Treating CRF

In the PPI network constructed using the 144 common targets (Figure3), the color and size of the nodes were plotted based on the degree value; a bigger node and deeper color indicate a greater degree value. A total of 3074 edges, representing protein interactions, were established. The edge thickness represents the edge betweenness, and a thicker line indicates a stronger relationship between the two targets. The average degree value of the PPI network is 42.7. Topological analysis of the PPI networks identified 61 genes with scores greater than the average as the core targets. The top 20 genes were plotted (Figure 4), with the X-coordinate being the degree of each gene; the greater the value, the more important the role the gene plays in the network. Therefore, the AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, EGFR, MAPK1, and MYC genes may play the most important roles in the HQSJZD treatment of CRF.Figure 3
PPI network of the common targets of HQSJZD and CRF.Figure 4
20 key targets determined in the PPI network.


## 3.3. Screening of Key Components

Topological analysis of the component-disease-target network constructed using the Cystoscape 3.8.0 software identified the key components of HQSHZD and ranked them based on degree values; a higher value represents greater importance. Components with a value greater than 10 were chosen for subsequent analysis (Table1). The component with the highest value was quercetin, followed by luteolin, kaempferol, naringenin, formononetin, and isorhamnetin.Table 1
Top active ingredients of HQSJZD identified in the component-target network.
MOL IDNameDegreeMOL000098Quercetin106MOL000006Luteolin46MOL000422Kaempferol36MOL004328Naringenin24MOL000392Formononetin21MOL000354Isorhamnetin20MOL0038967-Methoxy-2-methyl isoflavone20MOL000497Licochalcone19


## 3.4. GO Enrichment Analysis

The 144 HQSJZD-CRF common targets were subjected to GO enrichment analysis. A total of 2332 BP terms (P<0.01), 6 CC terms, and 27 MF terms were retrieved. The top 10 GO terms of each category were plotted in the bubble chart (Figure 5). The top BP terms are mainly involved in oxidative stress response, cellular response to chemical stress, response to bacterial molecules, cellular response to oxidative stress, reactive oxygen species metabolic process, regulation of the apoptotic signaling pathway, epithelial cell proliferation, regulation of inflammatory response, regulation of DAN binding transcription factor activity, response to hypoxia, and negative regulation of the apoptotic signaling pathway. The common targets were mainly enriched in six CC terms, including membrane raft, membrane microdomain, membrane region-protein kinase complex, cyclin-dependent protein kinase holoenzyme complex, serine/threonine protein kinase complex, and vesicle lumen. The enriched MF terms mainly include DNA binding transcription factor binding, RNA-polymerase-specific DNA binding transcription factor binding, nuclear receptor activity, ligand activation, transcription factor activity, phosphatase binding-cytokine receptor binding, heme binding tetrapyrrole binding, cytokine activity, and protein phosphatase binding.Figure 5
Top GO enriched terms of HQSJZD in treating CRF. TheX-coordinate indicates the number of enriched genes, and the color of the dot represents the P value of the corresponding term. A bigger dot indicates that more genes are enriched.


## 3.5. KEGG Pathway Analysis

The 144 common targets were significantly enriched into 172 pathways (P<0.05). The top 20 pathways are shown in Figure 6. Pathways not related to CRF, such as the “AGE-RAGE signaling pathway in diabetic complications,” were excluded. Finally, 9 pathways with the smallest P values were selected: phosphatidylinositol 3 kinase/protein kinase B (PI3K-Akt) signaling pathways, prostate cancer, bladder cancer, pancreatic cancer, IL-17 signaling pathway, non-small cell lung cancer, TNF signaling pathway, and small cell lung cancer.Figure 6
Top KEGG enriched pathways of HQSJZD in treating CRF. A bigger dot indicates that more genes are enriched in that pathway, and a dot with a darker red color represents a smallerP value.


## 3.6. Construction of the Ingredient-Disease Pathway-Target Network

The ingredient-disease pathway-target network was constructed using Cytoscape3.8.0 software (Figure7). The network contains 5 drugs, 114 active ingredients (ingredients not directly connected to targets were eliminated), 144 CRF-related disease targets (free protein spots were eliminated), and 20 CRF target-related pathways. Among the active ingredients, quercetin had the highest number of connected edges, followed by luteolin, kaempferol, naringenin, formononetin, and isorhamnetin. Among the potential targets, AKT1, IL6, VEGFA, and MAPK3 had relatively more connected edges. Among the pathways, the PI3K-Akt signaling pathway, prostate cancer, bladder cancer, pancreatic cancer, IL-17 signaling pathway, non-small cell lung cancer, TNF signaling pathway, and small cell lung cancer showed more connections with the potential targets.Figure 7
Drug component-target-pathway network.


## 4. Analysis

### 4.1. Active Ingredients of HQSJZD in Treating CRF

In this study, 144 active ingredients of HQSJZD in treating CRF were obtained; the most promising ones were quercetin, luteolin, kaempferol, naringenin, formononetin, and isorhamnetin. All of them are flavonoids with pharmacological activities such as enzyme regulation, blood circulation improvement, apoptosis regulation, and stem cell signal transduction regulation. Therefore, they have anti-inflammation, antioxidation, and antitumor effects [22]. Modern pharmacological studies have found that quercetin can eliminate oxygen free radicals to maintain oxidative balance [23], reduce the secretion of inflammatory factors [24], and enhance body functions. Moreover, quercetin has been shown to suppress the PI3K/Akt/mTOR pathway in breast cancer stem cells [25], induce apoptosis, reduce the phosphorylation of IKKβ and IκB, and suppress the invasion and progression of malignant tumor cells [26]. Therefore, quercetin has antitumor effects through various pathways [27] and is conducive to relieving CRF. Luteolin can modulate cell signaling pathways, suppress macrophage phosphorylation, reduce NF-kB activity, inhibit lipopolysaccharide- (LPS-) induced production of cytokines like IL-6 and TNF-α by macrophages, and inhibit cancer cell invasion by controlling tumor cell growth factor production and regulating kinase activities [28]. In addition, it can arrest cell cycle progression to suppress tumor cell growth [28] and induce apoptosis through inhibiting AKT activation, thereby increasing P53/bax/cleaved-caspase3 expression and decreasing bcl-2 expression [29]. It has been reported that luteolin can induce changes in cancer cell mitochondrial membrane structure to increase the membrane permeability, resulting in the release of cytochrome C and apoptosis-inducing factor (AIF), which promote cancer cell apoptosis [30]. Kaempferol can downregulate the PI3K/Akt/mTOR signaling pathway and induce cell autophagy to suppress the proliferation and induce apoptosis of human colorectal cancer RKO cells [31]. Isorhamnetin can reduce mTOR phosphorylation level to suppress tumor cell proliferation and migration [32]. Naringenin has antioxidative activity to relieve oxidative stress, suppresses the production of inflammatory mediators [33], and reduces Bcl-2 expression to improve mitochondrial function and decrease tumor cell apoptosis, thus achieving antitumor effects [34]. Therefore, we speculate that the active ingredients of HQSJZD in treating CRF are mainly flavonoids, which exert their functions through resisting oxidative stress, suppressing inflammatory cytokines, and inhibiting AKT activation.


### 4.2. Key Targets of HQSJZD in Treating CRF

In our analysis, the degree values of the targets, AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, EGFR, MAPK1, and MYC, were greater than 90, suggesting that they are important core targets with great biological importance and close involvement in the treatment of CRF. AKT1 is a serine/threonine protein kinase and the key member of the PI3K pathway. Activated AKT1 can activate or suppress a series of downstream effectors, thus participating in the regulation of multiple biological processes, such as cell metabolism, proliferation, differentiation, apoptosis, and migration [35]. Research suggests that, during tumor cell metastasis, the lack of original matrix support is disadvantageous to tumor cell survival [36]. AKT1 can enhance the adaptability of tumor cells to the environment and promote tumor cell migration and colonization and thus is closely related to the high invasion of tumor cells [37]. Multiple clinical studies have shown that flavonoid monomers of Chinese traditional herbs can control tumor gene expression, induce apoptosis, promote cell autophagy, inhibit cell proliferation, and suppress tumor angiogenesis through regulating the PI3K/Akt signaling pathway to achieve antitumor effects [38]. Therefore, combined with previous results, AKT1 can be considered as the core target of HQSJZD, with great importance in treating CRF. Mitogen-activated protein kinase (MAPK) is also a serine/threonine protein kinase, and its kinase activity can be activated by cytokines, neurotransmitters, and hormones. Activated MAPK can phosphorylate multiple substrates, including transcription factors, cytoskeletal proteins, and enzymes, to regulate numerous cellular physiological processes. Moreover, it plays an important role in mediating inflammatory response and cytokine production [39]. c-JUN is expressed at a low level in normal cells but is abnormally highly expressed after stimulation, and it forms AP-1 with c-Fos, regulating cell differentiation, proliferation, and apoptosis [40]. VEGF participates in the pathogenesis and progression of numerous angiogenesis-dependent diseases, including cancers, certain inflammatory diseases, and diabetic retinopathy.


### 4.3. Pathways of HQSJZD in Treating CRF

GO functional enrichment analysis and KEGG pathway enrichment analysis identified important functional terms and pathways that are closely related to HQSJZD and CRF targets, and further construction of the ingredient-disease pathway-target network established the connections between drug ingredients and pathways and revealed the molecular mechanisms of HQSJZD in treating CRD. The PI3K-Akt signaling pathway, TNF signaling pathway, and IL-17 signaling pathway were among the top pathways identified in KEGG analysis. The PI3K-Akt pathway plays an important role in tumor cell differentiation and proliferation in prostate cancer and ovarian cancer. Quercetin, the main active ingredient of HQSJZD, can suppress the overexpression of oncogene PTEN, downregulate PIP3 expression, inhibit AKT activity, and block the activation of the PI3K/Akt pathway to regulate tumor progression [41–43] and improve fatigue symptoms. Luteolin, another major ingredient, also downregulates the expression of P-PI3K, p-AKT, and AKT-1 proteins to suppress the tumorigenesis and progression of lung cancer [44]. The persistent activation of the TNF signaling pathway will lead to uncontrolled cell growth, suppress cell apoptosis, and promote tumor metastasis and angiogenesis [45]. The IL-17 signaling pathway is a classical important pathway related to immune responses, apoptosis, and tumorigenesis [46]. Animal experiments have shown that the main active ingredients of HQSJZD, that is, quercetin, luteolin, and kaempferol, can reduce the expression of TNF-α and IL-17 [47], thus improving the tumor microenvironment to effectively relieve CRF. Moreover, it has been revealed by KEGG pathway enrichment analysis that HQSJZD acts on EGFR, MMPS, and ERBB2 to improve bladder cancer-related fatigue and on EGFR, MET, ERBB2, and PI3K-Akt to improve non-small cell lung cancer-related fatigue. Thus, CRF caused by prostate cancer, bladder cancer, pancreatic cancer, and small cell lung cancer can be improved via the PI3K-Akt, TNF, and IL-17 signaling pathways, especially the PI3K-Akt pathway.In conclusion, the active ingredients of HQSJZD, that is, quercetin, luteolin, kaempferol, and naringenin, may act on multiple targets like AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, and EGFR and regulate multiple signaling pathways like PI3K-Akt, TNF, and IL-17 to suppress inflammatory responses, tumor gene expression, and tumor angiogenesis, thereby achieving the treatment of CRF. This study employed the method of network pharmacology and clearly revealed the “multicomponent, multitarget, and multipathway” mechanism of HQSJZD in treating CRF through network construction. Considering the limitation of network pharmacology, experimental studies are required to further analyze and validate the mechanism of HQSJZD in treating CRF identified in this study.


## 4.1. Active Ingredients of HQSJZD in Treating CRF

In this study, 144 active ingredients of HQSJZD in treating CRF were obtained; the most promising ones were quercetin, luteolin, kaempferol, naringenin, formononetin, and isorhamnetin. All of them are flavonoids with pharmacological activities such as enzyme regulation, blood circulation improvement, apoptosis regulation, and stem cell signal transduction regulation. Therefore, they have anti-inflammation, antioxidation, and antitumor effects [22]. Modern pharmacological studies have found that quercetin can eliminate oxygen free radicals to maintain oxidative balance [23], reduce the secretion of inflammatory factors [24], and enhance body functions. Moreover, quercetin has been shown to suppress the PI3K/Akt/mTOR pathway in breast cancer stem cells [25], induce apoptosis, reduce the phosphorylation of IKKβ and IκB, and suppress the invasion and progression of malignant tumor cells [26]. Therefore, quercetin has antitumor effects through various pathways [27] and is conducive to relieving CRF. Luteolin can modulate cell signaling pathways, suppress macrophage phosphorylation, reduce NF-kB activity, inhibit lipopolysaccharide- (LPS-) induced production of cytokines like IL-6 and TNF-α by macrophages, and inhibit cancer cell invasion by controlling tumor cell growth factor production and regulating kinase activities [28]. In addition, it can arrest cell cycle progression to suppress tumor cell growth [28] and induce apoptosis through inhibiting AKT activation, thereby increasing P53/bax/cleaved-caspase3 expression and decreasing bcl-2 expression [29]. It has been reported that luteolin can induce changes in cancer cell mitochondrial membrane structure to increase the membrane permeability, resulting in the release of cytochrome C and apoptosis-inducing factor (AIF), which promote cancer cell apoptosis [30]. Kaempferol can downregulate the PI3K/Akt/mTOR signaling pathway and induce cell autophagy to suppress the proliferation and induce apoptosis of human colorectal cancer RKO cells [31]. Isorhamnetin can reduce mTOR phosphorylation level to suppress tumor cell proliferation and migration [32]. Naringenin has antioxidative activity to relieve oxidative stress, suppresses the production of inflammatory mediators [33], and reduces Bcl-2 expression to improve mitochondrial function and decrease tumor cell apoptosis, thus achieving antitumor effects [34]. Therefore, we speculate that the active ingredients of HQSJZD in treating CRF are mainly flavonoids, which exert their functions through resisting oxidative stress, suppressing inflammatory cytokines, and inhibiting AKT activation.


## 4.2. Key Targets of HQSJZD in Treating CRF

In our analysis, the degree values of the targets, AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, EGFR, MAPK1, and MYC, were greater than 90, suggesting that they are important core targets with great biological importance and close involvement in the treatment of CRF. AKT1 is a serine/threonine protein kinase and the key member of the PI3K pathway. Activated AKT1 can activate or suppress a series of downstream effectors, thus participating in the regulation of multiple biological processes, such as cell metabolism, proliferation, differentiation, apoptosis, and migration [35]. Research suggests that, during tumor cell metastasis, the lack of original matrix support is disadvantageous to tumor cell survival [36]. AKT1 can enhance the adaptability of tumor cells to the environment and promote tumor cell migration and colonization and thus is closely related to the high invasion of tumor cells [37]. Multiple clinical studies have shown that flavonoid monomers of Chinese traditional herbs can control tumor gene expression, induce apoptosis, promote cell autophagy, inhibit cell proliferation, and suppress tumor angiogenesis through regulating the PI3K/Akt signaling pathway to achieve antitumor effects [38]. Therefore, combined with previous results, AKT1 can be considered as the core target of HQSJZD, with great importance in treating CRF. Mitogen-activated protein kinase (MAPK) is also a serine/threonine protein kinase, and its kinase activity can be activated by cytokines, neurotransmitters, and hormones. Activated MAPK can phosphorylate multiple substrates, including transcription factors, cytoskeletal proteins, and enzymes, to regulate numerous cellular physiological processes. Moreover, it plays an important role in mediating inflammatory response and cytokine production [39]. c-JUN is expressed at a low level in normal cells but is abnormally highly expressed after stimulation, and it forms AP-1 with c-Fos, regulating cell differentiation, proliferation, and apoptosis [40]. VEGF participates in the pathogenesis and progression of numerous angiogenesis-dependent diseases, including cancers, certain inflammatory diseases, and diabetic retinopathy.


## 4.3. Pathways of HQSJZD in Treating CRF

GO functional enrichment analysis and KEGG pathway enrichment analysis identified important functional terms and pathways that are closely related to HQSJZD and CRF targets, and further construction of the ingredient-disease pathway-target network established the connections between drug ingredients and pathways and revealed the molecular mechanisms of HQSJZD in treating CRD. The PI3K-Akt signaling pathway, TNF signaling pathway, and IL-17 signaling pathway were among the top pathways identified in KEGG analysis. The PI3K-Akt pathway plays an important role in tumor cell differentiation and proliferation in prostate cancer and ovarian cancer. Quercetin, the main active ingredient of HQSJZD, can suppress the overexpression of oncogene PTEN, downregulate PIP3 expression, inhibit AKT activity, and block the activation of the PI3K/Akt pathway to regulate tumor progression [41–43] and improve fatigue symptoms. Luteolin, another major ingredient, also downregulates the expression of P-PI3K, p-AKT, and AKT-1 proteins to suppress the tumorigenesis and progression of lung cancer [44]. The persistent activation of the TNF signaling pathway will lead to uncontrolled cell growth, suppress cell apoptosis, and promote tumor metastasis and angiogenesis [45]. The IL-17 signaling pathway is a classical important pathway related to immune responses, apoptosis, and tumorigenesis [46]. Animal experiments have shown that the main active ingredients of HQSJZD, that is, quercetin, luteolin, and kaempferol, can reduce the expression of TNF-α and IL-17 [47], thus improving the tumor microenvironment to effectively relieve CRF. Moreover, it has been revealed by KEGG pathway enrichment analysis that HQSJZD acts on EGFR, MMPS, and ERBB2 to improve bladder cancer-related fatigue and on EGFR, MET, ERBB2, and PI3K-Akt to improve non-small cell lung cancer-related fatigue. Thus, CRF caused by prostate cancer, bladder cancer, pancreatic cancer, and small cell lung cancer can be improved via the PI3K-Akt, TNF, and IL-17 signaling pathways, especially the PI3K-Akt pathway.In conclusion, the active ingredients of HQSJZD, that is, quercetin, luteolin, kaempferol, and naringenin, may act on multiple targets like AKT1, IL6, VEGFA, MAPK3, CASP3, JUN, and EGFR and regulate multiple signaling pathways like PI3K-Akt, TNF, and IL-17 to suppress inflammatory responses, tumor gene expression, and tumor angiogenesis, thereby achieving the treatment of CRF. This study employed the method of network pharmacology and clearly revealed the “multicomponent, multitarget, and multipathway” mechanism of HQSJZD in treating CRF through network construction. Considering the limitation of network pharmacology, experimental studies are required to further analyze and validate the mechanism of HQSJZD in treating CRF identified in this study.



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*Source: 9780677-2021-11-18.xml*