bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Phosphodiesterase 4 Inhibition on Gene Expression Changes Produced by Long Acting β2‐adrenoceptor Agonists in BEAS‐2B Airway Epithelial Cells
Authors: Joshi Radhika , Yan Dong , Hamed Omar , Giembycz Mark A
Published: June 2021

Phosphodiesterase 4 (PDE4) inhibitors are prescribed as add‐on therapy to a subset of COPD patients that suffer from exacerbations despite treatment with a combination of long‐acting β2‐adrenoceptor agonists (LABA), long‐acting muscarinic receptor antagonist and inhaled corticosteroids. The molecular mechanisms of their beneficial effects in COPD are ill‐defined. We have previously shown that LABAs, through their effect on the canonical Gs‐cAMP pathway, promote genomic effects in airway epithelial cells. Herein, we hypothesized that PDE4 inhibitors, by inhibiting cAMP degradation, promote similar genomic effects, and further potentiate the effects of LABAs in a human bronchial epithelial cell line. Additionally, we investigated whether this interaction between PDE4 inhibitors and LABAs occurred in an additive or a synergistic manner.

Gene expression changes were determined by population‐based RNA‐sequencing in BEAS‐2B cells treated with the PDE4 inhibitor, roflumilast N‐oxide (RNO), alone and in combination with LABA, salmeterol. RNO showed a weak stimulation of gene expression by itself but showed a strong rank‐order correlation with salmeterol‐induced gene expression changes, suggesting a common mechanism of action. RNO augmented the expression of all salmeterol‐regulated genes, but the magnitude of augmentation varied between genes. This variation can be explained by an estimated 7.5‐fold difference in sensitivities of genes to salmeterol. Deming regression analysis of the gene expression changes produced by the combination of salmeterol and RNO versus the sum of their individual effects showed a deviation away from unity slope that may be explained by a synergistic effect of RNO at some DEGs. Consistent with these results, PDE4 inhibitors, GSK 256066, and roflumilast potentiated the effects of LABAs, indacaterol, and formoterol, on a panel of randomly‐selected genes as measured by RT‐PCR.

Collectively, these data suggest that PDE4 inhibition may synergistically augment LABA‐induced gene expression changes in airway epithelial cells. This interaction between LABAs and PDE4 inhibitors may contribute to the therapeutic benefits of their combination in COPD.

Support or Funding Information

Canadian Institutes for Health Research (PJT 152904)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The physiological hypertrophic agonist, norepinephrine, is able to induce PLC‐mediated PI4P hydrolysis in cardiac myocytes via a pool of internal β‐adrenergic receptors
Authors: Nash Craig , Wei Wenhui , Smrcka Alan
Published: June 2021

Previously, we have shown that PLC∑‐mediated phosphatidylinositol‐4‐phosphate (PI4P) can be activated by raising cAMP in cardiac myocytes, however, activation by Isoproterenol was unable to induce PI4P hydrolysis. In the accompanying abstract by Wei et al, we have demonstrated that the cell permeant β‐agonist, Dobutamine is able to induce PI4P hydrolysis. Here, we seek to determine if the physiological agonist, norepinephrine is able to induce PI4P hydrolysis, despite being membrane impermeant. In contrast to Iso, norepinephrine induced sustained PI4P hydrolysis that was blocked by the cell permeant β‐adrenergic antagonist, metoprolol (100μM). The cell impermeant βAR antagonist, sotalol (5mM) could not block norepinephrine effects, suggesting that a pool of internal β‐adrenergic receptors is responsible for NE‐mediated PI4P hydrolysis. To directly determine if internal β1‐ARs in NRVMs can be activated by norepinephrine, we monitored translocation of a YFP‐tagged mini Gs protein in β1AR transfected NRVMs2. Norepinephrine induced robust and rapid recruitment of mini Gs to both the plasma membrane, and the perinuclear region corresponding to the Golgi apparatus. Golgi localization could be reversed by Metoprolol but not Sotalol. Isoproterenol, on the other hand, induced a strong recruitment to the plasma membrane but not to the Golgi. NE‐mediated PI4P hydrolysis is also abrogated by an inhibitor of a cation transporter Oct3, Corticosterone (100μM). Oct3 has been recently shown to be required for epinephrine‐mediated β1‐AR stimulation at the Golgi envelope in HELA cells3. Taken together, these data suggest that norepinephrine can activate β‐adrenergic receptors in internal membranes, potentially at the Golgi apparatus, stimulating PI4P hydrolysis. These internal GPCRs may be responsible for cardiac hypertrophy and heart failure and targeting them may allow for the development of selective therapies which can treat heart failure with little to no effect on other cardiac functions.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Validation of a Manual Cell Counting Assay for Assessing the Kinetics of Cell Unclumping Reagents in Drug Discovery
Authors: Zem Gregory , Nahapetyan V , Crocker K , Tobar D , Santos S Delos , Nazarian D , Hovannes J , Beltran G , Dubin R , Reque L , Singh P , Sarkisyan L , Cardona B , Bachinela G , Oppenheimer S B
Published: June 2021

This laboratory has developed and used a manual (non‐automated) cell counting assay for examining the kinetics of cell interactions in a yeast model (FASEB J 32:113; 31: 539; 30: 44; 30:49; 29: 52; 29: 65; Acta Histochem 116: 1514; 108: 311). Manual cell counting is often considered as a gold standard in some applications because of direct observation of results (Comparison of Cell Counting Methods…Inhal Toxicol 28(9): 410–429). For many years this assay has been used to evaluate the efficacy of reagents to unclump yeast cell clumps, with possible human applications in unclumping cancer cell clumps, unclumping thrombocytic blockages and reducing biofilms. In this study the assay is further validated by not only counting single cells as done in the past, but also counting cell clumps and number of cells per clump. To accomplish this, in over 400 trials by 14 investigators, sodium citrate dihydrate and magnesium sulfate were tested at 1, 2 and 3 mg per ml deionized water for their effects on fixed (Prefer fixative, Anatech Ltd, Battle Creek, MI) yeast (Saccharomyces cerevisae) over 60 min (with agitation every 20 min) on glass microscope slides and plotting all data with standard error and calculating p values comparing experimentals and controls in each category of results. It was generally observed that when there was no significant change in percentage of single cells in experimentals and controls (no reagent) there was no significant change in number of clumps and number of cells per clump. When there was a significant increase in percent single cells (suggesting that the reagent caused cell unclumping), there was a reduction in clump number and number of cells per clump. A specific example of this finding in 60 trials was that 3 mg sodium citrate dihydrate per ml deionized water caused a 35% increase in single cells by 60 min. The number of clumps decreased by 56% and the number of cells per clump decreased by 39%. Trends for magnesium sulfate were generally similar to those for sodium citrate dihydrate. In summary, this manual assay was further validated here by assessing cell clumps as well as the usual single cells. This assay is considered to be a gold standard for precise assessment of cell unclumping reagents in drug discovery.

Support or Funding Information

Supported by California State University, Northridge Biology Department and Center for Cancer and Developmental Biology Foundation and Corporation accounts including U.S. Presidential Award contribution.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of AMPK‐FoxO3a pathway mediated autophagy inhibits fibroblast‐myofibroblast differentiation in pulmonary fibrosis
Authors: Gao Jian
Published: June 2021

Aim

to investigate the mechanism of AMPK‐FoxO3a pathway mediated autophagy during fibroblast‐myofibroblast differentiation in PF.

Methods

we used BLM to induce PF in SD rats, and treatment with an AMPK activator(metformin) in model group. The pathological change and collagen deposition of lung tissue was observed by H&E and Masson staining. Meanwhile, we detected related proteins by immunohistochemistry and western blot. Meanwhile, human lung fibroblast were stimulated with TGF‐β1, and the cells were treated with AMPK activator(Metformin and AICAR), and activator and inhibitor of autophagy. Furthermore, the FoxO3a small RNA was used to interference cells. The related proteins and autophagy were detected by western blot.

Results

In vivo, AMPK could reduce the destruction of alveolar structure and collagen deposition. In addition, AMPK could increase the expression of FoxO3a and autophagy level. In vitro, the protect effect of AMPK was abolished when FoxO3a was silenced by small RNA. Meanwhile, activation of autophagy by starvation and rapamycin could decrease TGF‐β1 induced fibroblast differentiation. However, when autophagy was inhibited by 3‐MA or chloroquine, the TGF‐β1 induced FMD was aggravated again under the pre‐treatment with metformin.

Conclusion

AMPK‐FoxO3a pathway could attenuate FMD by increasing autophagy in PF

Support or Funding Information

National Natural Science Foundation of China No. 81473267, 81274172.

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This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Maternal Electronic Cigarette Use Can Enhance Offspring Susceptibility to Hypoxic‐Ischemic Brain Injury
Authors: Sifat Ali Ehsan , Nozohouri Saeideh , Villalba Heidi , Vaidya Bhuvaneshwar , Abbruscato Thomas
Published: June 2021

Prenatal exposure to tobacco smoke and nicotine is believed to interfere with fetal brain development predisposing offspring to different neurobehavioral and neuropsychological disorders. Included in this is increased neonatal vulnerability to hypoxic‐ischemic encephalopathy (HIE) which is a major cause of neonatal death and child disability in the US. These effects could be, in part, mediated by fetal nicotine exposure. Use of electronic cigarettes (e‐Cigs), commonly known as vaping, has been rapidly increasing in recent times in the general population, including women of reproductive age. E‐Cig use during pregnancy is also increasing because of the addictive properties of nicotine along with the perception of the relative safety of e‐Cigs. In this study, we aimed to investigate the effects of maternal e‐Cig use on neonatal brain development and HIE utilizing a combination of in vitro and in vivo models. Pregnant CD1 mice were exposed to e‐Cig vapor (2.4% nicotine). Primary cortical neurons were isolated from e‐Cig exposed fetus and cultured for seven days with subsequent exposure to oxygen‐glucose deprivation followed by reoxygenation (OGD/R) to mimic ischemia‐reperfusion injury. Hypoxic‐ischemic brain injury was induced in 8–9 days old mouse pups by a combination of left common carotid artery ligation and 15 minutes exposure to 8% oxygen. We found that e‐Cig exposed cortical neurons demonstrated decreased cell viability and increased caspase‐3 expression in OGD/R condition. These effects were accompanied by a decrease in neurite formation, mitochondrial dysfunctions, and decreased glucose uptake & glucose transporter expression in OGD/R condition. Our preliminary data also indicate increased sensitivity to HI brain injury in prenatally e‐Cig exposed mouse pups. Additionally, in utero e‐Cig exposed mice offspring displayed a significantly increased level of hyperactivity at postnatal day 45 in open field test. These results indicate that maternal e‐Cig exposure could lead to offspring behavioral abnormalities and enhance HI brain injury. Further studies are needed to demonstrate the long‐term anatomical and behavioral effects of HI brain injury in e‐Cig exposed offspring pups. This study is instrumental in elucidating the possible deleterious effects of maternal e‐Cig use in the general population.

Support or Funding Information

R01 DA029121

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Carbamate Insecticides Modulate G Protein‐Dependent Signaling in Cells Expressing Melatonin Receptors
Authors: Jones Anthony Jordan , Mastrandrea Lucy D. , Rajnarayanan Rajendram R. , Dubocovich Margarita L.
Published: June 2021

Melatonin targets MT1 and MT2 melatonin receptors that are expressed in brain as well as peripheral tissues in order to signal “time‐of‐day” messages for the synchronization of circadian rhythms. In mammals, the homeostatic balance of glucose metabolism and insulin release is modulated by Gαi protein‐dependent melatonin receptor (MTR) signaling in pancreatic β‐cells (Kemp et al. 2002). In‐silico screening paired with competition binding has demonstrated that carbamate insecticides with similar pharmacophores to melatonin have affinity for the mammalian MT1 and MT2 melatonin receptors (Jones et al. 2015 EB Abs.; Popovska‐Gorevski et al. 2017; Glatfelter et al. 2017 EB Abs.). Therefore we hypothesize that exposure to these environmental chemicals can disrupt melatonin receptor signaling. The objective of this study was to examine the effect of carbamate insecticides on G protein dependent‐signaling pathways on human MT1 and MT2 melatonin receptors stably expressed in CHO cells and in rat INS‐1 β‐cells expressing MT1. The apparent efficacy of carbamates (ie., carbaryl, fenobucarb, bendiocarb, and carbofuran) assessed by affinity ratios derived from competition with 2‐[125I]‐iodomelatonin binding, with or without G protein inactivation by GTP, suggested antagonist efficacy on hMT1 receptors (no affinity shift with GTP; KiGTP/KiControl:0.59 – 2.3) and agonist efficacy on hMT2 receptors (rightward affinity shift with GTP; KiGTP/KiControl: 4.8 – 11). The same insecticides tested at 10 μM did not modify forskolin‐stimulated cAMP levels in CHO‐hMT1 cells but inhibited stimulated cAMP formation in CHO‐hMT2 cells (47%–72% of melatonin effect on stimulation). Carbaryl (10 pM – 100 μM) acted as a partial agonist at both hMT1 (log IC50= −5.8 ± 0.3 M; Emax= 55% melatonin effect) and hMT2 (log IC50 = −10.0 ± 0.4 M; Emax= 67% melatonin effect) receptors with over 10,000 times higher potency for hMT2 compared to hMT1. Next, carbaryl at 10 μM, but not at 1 μM, inhibited forskolin‐stimulated insulin release from INS‐1 pancreatic β‐cells by 25% (96% melatonin effect) as determined by radioimmunoassay. These data suggest that carbamate insecticides with similar structures to melatonin can alter G protein‐dependent signaling in tissues that express melatonin receptors. Exposure to these chemicals could potentially contribute to the development of diabetes or other metabolic disorders as disruption of β‐cell melatonin receptor signaling and insulin release are linked to increased risk of disease (Tuomi et al. 2016).

Support or Funding Information

Supported by ES 023684 to MLD and RVR.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacokinetics and pharmacodynamics of curcumin in regulating antioxidant and epigenetic gene expression in healthy human volunteers
Authors: Cheng David , Li Wenji , Wang Lujing , Brunetti Luigi , Kong Ah‐Ng
Published: June 2021

Curcumin is a major component of the spice, turmeric (Curcuma longa) often used in food or as a dietary supplement. Many preclinical studies on curcumin suggests benefit in many diseases due to its antioxidant and epigenetic effects. The few human studies and curcumin's unfavorable pharmacokinetics (PK) have limited its potential, leading researchers to study and develop formulations to improve its PK. The purpose of this clinical study is to describe the pharmacokinetics and pharmacodynamics (PK/PD) of commercially marketed curcumin in normal, healthy human volunteers. Twelve volunteers received 4 g of curcumin capsules with standard breakfast. Plasma samples were collected at specified timepoints and analyzed for curcumin levels. RNA was extracted and analyzed for expression of select antioxidant and epigenetic histone deacetylase (HDAC) genes. Plasma levels of parent curcumin were not detected by HPLC‐ITMS/MS/MS. However, curcumin glucuronide, a major metabolite of curcumin, was detected as soon as 30 min. These observations of little to no curcumin and low levels of metabolite are in line with previous studies. Antioxidant genes Nrf2, HO‐1, NQO1, and HDAC1, HDAC2, HDAC3, and HDAC4 were quantified by qPCR. Curcumin glucuronide pharmacokinetics are well‐described by a one‐compartment model and the PK/PD of curcumin glucuronide and its effect on antioxidant and epigenetic gene expression are explained by indirect response model (IDR). Increasing AUC or exposure to curcumin glucuronide was correlated with overall PD response. Physiologically based pharmacokinetic modeling (PBPK) and simulation using Simcyp correlated well with our observed data in the human volunteers. In vitro experiments on curcumin glucuronide in HepG2C8 cells also show that Nrf2‐ARE luciferase activity increases after 24‐hour treatment. These results show that poor bioavailability of curcumin remains a challenge but that oral administration of curcumin delivers detectable levels of curcumin glucuronide and may mediate the antioxidant and epigenetic effects of curcumin.

Support or Funding Information

Grant: This work was supported in part by institutional funds and R01AT007065 from the National Center for Complementary and Alternative Medicines (NCCAM) and the Office of Dietary Supplements (ODS).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative Proteomics‐based Bottom‐up PBK Modeling: A Case Study of 3 Statins
Authors: Chan James Chun Yip , Tan Shawn Pei Feng , Chan Eric Chun Yong
Published: June 2021

In the consumer care, cosmetics and chemical industries, there is a growing need for alternatives to animal testing to derive biokinetic data to evaluate both efficacy and safety of chemicals. One promising alternative is bottom‐up physiologically‐based biokinetic (PBK) modeling, which utilizes in vitro‐to‐in vivo extrapolation (IVIVE) for prediction of biokinetic parameters. The main challenges of IVIVE lie in suboptimal biokinetic predictions, particularly when using in vitro transporter data which often requires empirical fitting to human biokinetic data. As part of an Organization for Economic Cooperation and Development (OECD) case study, the primary objective of this work is to develop and validate quantitative proteomics‐based bottom‐up PBK models using a set of chemicals rich with in vitro and human in vivo data. Three HMG‐CoA reductase inhibitors that undergo differential elimination processes were chosen: rosuvastatin (transporter‐dependent), fluvastatin (metabolism‐dependent) and pitavastatin (mixed‐mode). PBK models were built using the Simcyp® Simulator by incorporating: (1) in vitro transporter and metabolism data (Vmax, Jmax, Km and CLint) and (2) animal tissue distribution data from literature to inform tissue‐to‐plasma equilibrium distribution ratio (Kp). Simulations were performed for single intravenous, single oral and multiple oral dosing of these chemicals. The successful prediction was based on a two‐fold criterion when compared against human biokinetic parameters. Our results showed that predicted systemic exposure (AUC0‐∞h), maximum plasma concentration (Cmax), plasma clearance (CL) and time to reach Cmax (Tmax) were within two‐fold of the observed data, with the exception of parameters associated with multiple oral pitavastatin dosing and Tmax of single oral fluvastatin dosing. The use of animal Kp data improved the predicted plasma‐concentration time profiles but did not significantly alter predicted biokinetic parameters (Figure 1). Our study demonstrated that quantitative proteomics‐based mechanistic IVIVE could account for differences in transporter and metabolic enzyme expression levels between in vitro systems and in vivo organs, allowing the prediction of whole organ clearances without any empirical scaling. We conclude that bottom‐up PBK modeling incorporating mechanistic IVIVE could be a viable alternative to animal testing in obtaining predictions of human biokinetics of chemicals.

Support or Funding Information

This work was supported by the Innovations in Food and Chemical Safety Programme [Grant number H18/01/a0/C14] and NUS Department of Pharmacy [Grant number C‐148‐000‐003‐001]. Travel funding for this conference was provided by the Innovations in Food and Chemical Safety Programme and the Skin Research Institute of Singapore.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hyperglycemia increases breast cancer cell and normal breast epithelial cell proliferation through polyamine deregulation
Authors: Capellen Caleb , Ortega Jose , Dunworth Matthew , Casero Robert A. , Chandra Surabhi
Published: June 2021

Polyamines (spermine, spermidine, and putrescine) are involved in basic cellular processes such as cell growth, replication, and transcription. Upregulation of polyamines can thus develop tumors due to hyper‐proliferation of cells as observed in cancers of the colon, skin, prostate and breast. Inhibitors of polyamine pathway prevent tumor growth as shown in animal models of colon and skin cancer. Diabetic conditions further accelerate the growth and metastasis of cancers such as pancreas, breast, liver, and colorectum, however the role of polyamines in such states has not been investigated. We hypothesized that polyamine pathway rapidly affects proliferation of breast cancer cells and normal breast epithelial cells in diabetic conditions. Breast cancer cells (MDA‐MB‐231) and normal breast epithelial cells (MCF‐10A) were treated with low glucose (LG, 5mM) or high glucose (HG, 25 mM) in the presence/absence of polyamine pathway inhibitors. There was a time dependent increase in cell proliferation with HG from 48–72 hr compared to LG. This was correlated with an increase in polyamines (putrescine, spermidine) though spermine levels were not changed considerably. In addition, gene expression and activity of catabolic enzymes in the polyamine pathway, spermine‐spermidine acetyltransferase (SSAT) and spermine oxidase (SMOX) were decreased with HG treatment, further suggesting a build‐up of polyamines. Simultaneous treatment with an ornithine decarboxylase (polyamine synthesis enzyme) inhibitor, difluoromethylornithine (DFMO) could prevent cell proliferation, restore polyamines, and normalize polyamine catabolic enzymes with high glucose exposure. In conclusion, polyamine pathway is responsible for proliferation of breast cancer cells and normal breast epithelial cells in diabetic states, which can be prevented using inhibitors of polyamine synthesis.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Heterozygous knockdown insulin‐like growth factor‐1 receptor (IGF‐1R) regulates mitochondrial functions and prevents colitis and colorectal cancer
Authors: Wang Shu Qing , Cui Shu Xiang
Published: June 2021

Although insulin‐like growth factor‐1 receptor (IGF‐1R) has been accepted as a major determinant of tumorigenesis, the mechanisms of its roles in the pathogenesis of cancers have remained elusive. Herein, we demonstrate that IGF‐1R plays pivotal roles in regulation of mitochondrial functions and resistance to oxidative stress in colitis and colorectal tumorigenesis. Heterozygous knockdown IGF‐1R attenuated azoxymethane (AOM)/dextran sulfate sodium (DSS)‐induced colitis and colitis associated cancer (CAC) in Igf1r+/− mice. Knockdown of IGF‐1R confers the resistance to oxidative stress‐induced damage on colorectal epithelium through protection the mitochondrial dynamics and structures. Knockdown of IGF‐1R develops the function of mitochondrial fusion under oxidative stress. Mechanically, an increase of mitochondrial coupling index RCI and oxidative phosphorylation index ADP/O was seen in the epithelial cells of Igf1r+/− mice. Seahorse XFe‐24 analyzer analysis of mitochondrial bioenergetics showed an increase of oxygen consumption rate (OCR) and a decrease of extracellular acidification rate (ECAR) in Igf1r+/− cells. Further analysis suggests the protection mechanisms of colorectal epithelial cells from oxidative stress through activating mitochondrial respiratory chain and the LKB1/AMPK pathways. These results support the roles of IGF‐1R at the nexus between oxidative damage and mitochondrial function and connect colitis and colorectal tumorigenesis.

Support or Funding Information

This work was supported by Natural Science Foundation of China (81673449, 91629303).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sphingosine kinase inhibitor regulates pro‐inflammatory cytokines to subdue the neoplastic lesions in hepatocellular carcinoma
Authors: CHATTEERJEE NABANITA , Das Subhadip , Bose Dipayan , Banerjee Somenath , Saha Krishna Das
Published: June 2021

Hepatocellular carcinoma (HCC), is the most leading cause of cancer‐related death in all over the world (seems to more than 80% of death cases). It is frequently linked with continuous hepatocytes death, inflammatory cell infiltration, and compensatory liver regeneration with higher angiogenic rate. Considering the different signalling process during this tumorogenesis with the involvement of new ‘lead’ identification of different sources, desire to be less toxic and higher bioavailability. The use of live, attenuated or genetically compensated microbes or its cellular component(s) or metabolites has begun to emerge as a potential new approach in medicinal research to deliver bioactive entities. Thus, advancing our knowledge of such microbes‐mediated therapy may suggest new avenues for therapeutic intervention in many fatal diseases.

Sphingosine kinase inhibitor (SKI) II has been reported as a dual inhibitor of sphingosine kinases (SKs) 1 and 2 and has been extensively used to prove the involvement of SKs and sphingosine‐1‐phosphate (S1P) in cellular processes and able to induce apoptosis in human HepG2 (Hepatocellular carcinoma cell, human) cells, as characterized by phosphatidylserine serine exposure, activation of series of caspases and cleaved PARP. SKI also modulates the various transcription factors like NF‐kB‐65, p53 through regulation of cell proliferation exerted the death cause. Moreover, this study also focused that SKI effectively suppressed diethylnitrosamine (DEN) induced liver inflammation and hyperplasia in murine, also regulated the lipid peroxidation and enzymic antioxidants (SOD, CAT, GPx, GR and GST) status with an improvement of their survival rate. It reduced the elevated level of cytokines including tumor necrosis factors, interleukins, interferon (alpha and gamma), and transforming growth factors in hepatocytes from liver fibrosis bearing mice. As evident from an immunochemical analysis, DEN‐induced neoplastic lesions may up‐regulate the various growth factors including EGF, HGF, endothelin‐1 regulation with promoting angiogenic activity via modulation of CD34, VEGF, HIF‐1α expression and we found that the alteration of these changes were balanced by SKI treatment in vivo system. So taken together, these finding indicated that, SKI may recover in hyperproliferating cancer cells with tumor growth regulation in carcinogen‐exposed rodent system. In future direction, we will check the p53−/− and cmyc−/− mice that hoe it could regulate cancer metastasis with this SKI. and the involvement of od tumor microenvironment.

Support or Funding Information

CSIR and Department of Science and Technology, India

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phloretin ameliorates hyperuricemia‐induced chronic renal dysfunction through inhibiting NLRP3 inflammasome and uric acid reabsorption
Authors: Cui Danli , Liu Shuyun , Zhao Meng , Mao Ruiwen , Wang Chengshi , Li Lan , Chen Younan , Cheng Jingqiu , Lu Yanrong , Liu Jingping
Published: June 2021

Objective

Hyperuricemia is an important risk factor for renal diseases and contributes to renal fibrosis, and it's a big challenge for clinic. Phloretin, a plant‐derived dihydrochalcone, has shown antioxidant/anti‐inflammatory properties, and could inhibit uric acid (UA) uptake in vitro, but its role on renal injury remains unknown. In this study, we aimed to investigate the therapeutic potential of phloretin on hyperuricemia‐induced renal dysfunction and underlying mechanism in mice.

Methods

Mice were induced hyperuricemic by oral gavage of adenine/potassium oxonate mixture, and then treated with phloretin. The effects of phloretin on renal function, inflammation and interstitial fibrosis were evaluated in hyperuricemic mice. The changes of renal mitochondrial oxidative stress and UA metabolism were also analyzed.

Results

Hyperuricemic mice showed renal dysfunction with elevated renal inflammation and fibrosis. By contrast, phloretin significantly improved renal function and morphologic lesions, as indicated by reduced levels of serum blood urea nitrogen (BUN), creatinine and urinary albumin to creatinine ratio (UACR), as well as decreased levels of kidney tubular necrosis, interstitium extracellular matrix (ECM) deposition and interstitial fibroblasts activation in the kidneys of hyperuricemic mice. Furthermore, phloretin significantly suppressed renal mitochondrial reactive oxygen species (ROS) production and NOD‐like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling, and inhibited glucose transporter 9 (GLUT9) and UA reabsorption in hyperuricemic mice.

Conclusion

Phloretin can ameliorate hyperuricemia‐induced chronic renal injury through co‐inhibiting NLRP3 pathway and UA reabsorption, and thus it might be a promising therapy to hyperuricemia‐related renal diseases.

Support or Funding Information

This work was supported by grants from National Natural Science Foundation of China (31200754, 81571808, 31871001) and China Postdoctoral Science Foundation (2012M511931).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Roles of Qishen Granules Recipes, Qingre Jiedu, Wenyang Yiqi and Huo Xue, in the Treatment of Heart Failure
Authors: Gao Sheng , Wang Yong , Tian Chuan , Li Chun , Wu Dingfeng , Jiao Na , Zhu Lixin , Zhu Ruixin , Wang Wei
Published: June 2021

Background

Recipes (Qingre Jiedu (QJ), Wenyang Yiqi (WYYQ) and HuoXue (HX)) in Qishen granules (QSG) are believed to synergistically exert cardio‐protective effects and improve heart function. Further studies, however, are necessary to interpret the underlying pattern of each decomposed recipe in QSG and their synergistic effect in the treatment of heart failure.

Methods

In this study, the efficacy of QJ, WYYQ and HX was evaluated by using heart failure model Sprague‐Dawley rats. Cardiac tissues were harvested for RNA Sequencing. Differentially expressed genes (DEGs) analysis and pathway enrichment analysis were performed with NOIseq and clusterProfiler approach, respectively. Furthermore, an optimized quantitative pathway analysis method that maximally acquires gene expression information was introduced to perform differential pathways analysis between different groups.

Results

QJ, WYYQ and HX are shown to improve left ventricular ejection fraction, indicating their common contribution within QSG to the treatment of heart failure. Compared to model group, there are 858, 124, 1195, 1733 DEGs in QSG, QJ, WYYQ and HX group respectively, which are enriched in all or part of pathways including energy metabolism, inflammation reactions and ventricular remodelling. Quantitative pathway analysis show that decomposed recipes QJ, WYYQ and HX have common but differentiated contributions in the treatment of heart failure. Especially, ATP1A3 gene in thyroid hormone signalling pathway is up‐regulated significantly in both QSG and HX groups. Besides, ADCY1 gene in relaxin signalling pathway is up‐regulated in both QSG, WYYQ and HX groups. Therefore, WYYQ and HX might have complementary effects on energy supply in the treatment of heart failure through activation of thyroid hormone signalling and relaxin signalling pathways.

Conclusion

Our results have indicated that QJ, WYYQ and HX recipes in QSG achieve synergistic therapeutic effects in myocardial energy metabolism. This provides theoretical foundations and evidence for the optimization of QSG formulas. Quantitative pathway analysis is proved as an effective tool to decompose the recipes of Traditional Chinese Medicine.

Support or Funding Information

This work was supported by National Natural Science Foundation of China 81770571 (to LZ), 81774152 (to RZ), 41530105 (to RZ).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Efficacy of Tramadol in Thermoreversibles Gels in a Model of Postoperative Pain
Authors: Tofoli Giovana , Papini Juliana , Cereda Cintia , Napimoga Juliana , Macedo Cristina , de Araujo Daniele
Published: June 2021

Tramadol (TR) is an opioid analgesic with short duration of action, still it may be consider valuable when compared to other opioids, as it produces mild adverse effects. Due to these properties, trmadol was incorporated in thermoreversible poloxamers and the effectiveness of these new drug delivery formulations was evaluated in a rat model of postoperative pain. The poloxamer (PL)‐tramadol systems were prepared by direct dispersion of the drug in solutions with PL 407 (20%) or the association of PL 407 (20%) and PL 188 (10%). Male Wistar rats were submitted to hindpaw incision and after 24 hours treated by subcutaneous route with one of the two formulations or plain TR 2% (10 μg.kg−1), saline or the poloxamers vehicles. Mechanical hypersensitivity was measured using von Frey filaments after 15, 30, 60, 90, 120,180, 240, 300 and 360 minutes from the injections. The protein levels of TNF‐α, IL‐1β, Substance P and CGRP were obtained through ELISA assays after 1, 3 and 6 hours after tramadol injections. All animals presented postincisional hypersensitivity showed by increased withdrawal response when compared to the preoperative period (p<0.05). Poloxamers formulations presented greater and longer analgesic effect than plain tramadol (p<0.05). Also, the new formulations were able to reduce the levels of TNF‐α after 6 hours (p<0.05). One hour far ahead of the formulations' injection CGRP levels with the PL 407 +PL 188 +TR 2% formulation was smaller than this vehicle without TR (p<0.05). The association of PL 407 and PL188 in an injectable formulation of tramadol promoted prolonged and effective postoperative pain relief in a rat model.

Support or Funding Information

FAPESP (#2015/20744‐0)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Glucocorticoids in Gastroprotection Induced by Capsaicin
Authors: Filaretova Ludmila , Podvigina Tatiana , Morozova Olga
Published: June 2021

The pivotal role of capsaicin‐sensitive peptidergic sensory fibers in maintaining of gastric mucosal integrity against injurious interventions has been well established in rats and then in human. Clinical finding suggests that capsaicin‐induced gastric mucosal protection against the injurious effects of ethanol or indomethacin is due to the acute stimulatory effect of the compound. It was concluded that patients taking non‐steroidal anti‐inflammatory drugs (NSAID) regularly can decrease the incidence and severity of gastric ulceration, if they have moderate spicy foods. Opposite effect has been observed in rats desensitized by capsaicin. Functional blockade of gastric sensory nerve endings elicited by systemic capsaicin application was resulted in impaired mucosal protection against various ulcerogenic agents. Our previous findings suggest a pivotal compensatory role of glucocorticoids in maintenance of gastric mucosal integrity in rats desensitized by capsaicin. In this study, we tested if glucocorticoids can participate in capsaicin‐caused gastroprotection during NSAID‐caused gastropathy accompanied by prostaglandin deficiency in rats. Gastric injury was induced by indomethacin (IM). A single IM administration (35 mg/kg, sc) into fasting (24 h) rats caused the formation of gastric erosions 4 h after injection. The effects of small doses (0.1, 0.5, 1.0, 2.0, 3.0, 5.0, 10.0 mg/kg, sc) of capsaicin on the blood corticosterone and glucose levels and IM‐induced gastric injury were tested. A single capsaicin administration was performed one hour before IM administration. To test the participation of corticosterone in the gastroprotective effect of capsaicin, the pretreatment by the inhibitor of glucocorticoid synthesis, metyrapone (30 mg/kg, i.p., 30 min before capsaicin) was used. We found that the pretreatment with capsaicin (0.5–10.0 mg/kg, sc) reduced dose dependently the area of IM‐induced erosions. This protection was accompanied by a gradual increase in blood corticosterone and glucose levels. Metyrapone injected shortly before capsaicin administration (1.0 mg/kg) caused a fast inhibition of capsaicin‐induced corticosterone response and reversed the protective effect of capsaicin on the gastric mucosa against the IM‐produced injury. The present study confirms evidence for the powerful gastroprotective potency of capsaicin during NSAID‐caused gastropathy. The findings obtained demonstrate that capsaicin administration resulted in an increase in plasma corticosterone levels which in turn plays a pivotal role in maintaining of gastric mucosal integrity against injurious intervention of IM.

Support or Funding Information

The study was supported by grants of RSF N 19‐15‐00430 (NSAID‐caused gastropathy with prostaglandin deficiency) and RFBR N 19‐015‐00514a (capsaicin‐induced gastroprotection)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanosensitive Ion Channels in the Development of Ileus
Authors: Uray Karen , Sipos Ádám , Vo Duong Nhu Y , Thi Hai Yen Nguyen , Docsa Tibor
Published: June 2021

Introduction

Approximately 30% of trauma patients develop ileus, a decrease or cessation of intestinal motility resulting in pathological distension of the intestinal wall. Mechanosensitive ion channels play a role in both the physiological and pathological response to mechanical stimuli.

Objective

The objective of this study was to determine if mechanosensitive ion channels play a role in the development of ileus and the response of the intestine to pathological stretch.

Methods

In a rodent model, we induced ileus by a combination of hemodilution and mesenteric venous hypertension. We measured intestinal function using an ex vivo organ bath system. We also measured smooth muscle myosin light chain phosphorylation and intestinal wall fluid content. We collected tissue for microarray analysis.

Results

Intestinal wall fluid content increased significantly, indicating the development of intestinal edema. Edema development resulted in distension of the intestinal wall, decreased intestinal smooth muscle contractile activity, and decreased smooth muscle myosin light chain phosphorylation. We used microarray analysis to compare normal (unstretched) intestinal smooth muscle to edematous (stretched) intestinal smooth muscle to characterize changes in ion channel expression. We identified hyperpolarization‐activated cyclic nucleotide‐gated 2 (HCN2), a cyclic nucleotide gated ion channel, which is also stretch‐activated. HCN2 gene expression decreased in edematous intestinal tissue. In the ex vivo organ bath system, we tested the effects of HCN2 inhibitors on intestinal function. In preliminary experiments, HCN2 inhibition with ZD7288 resulted in decreased response to carbachol, with little change in spontaneous contractile activity or frequency.

Conclusions

Our data suggest that HCN2 downregulation/inhibition can suppress intestinal contractile activity. Thus, HCN2 is a potential therapeutic target for improving intestinal motility. In future experiments, we will determine the role of HCN2 in the intestinal response to stretch.

Support or Funding Information

Hungarian National Research Development and Innovation Office (K120669, EFOP‐3.6.2‐16‐2017‐00006)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Non‐Polarized mpkCCD Cell Model for Aquaporin‐2 Phosphorylation and Membrane Trafficking Study
Authors: CHAN TZU‐HSIEN , YU MING‐JIUN
Published: June 2021

Aquaporin‐2 (AQP2) is a water channel protein expressed in the kidney collecting duct principal cells responsible for water reabsorption. AQP2's ability to regulate water reabsorption depends on its phosphorylation states and trafficking to and from the apical plasma membrane regulated by the peptide hormone vasopressin. The mpkCCD cells have been an instrumental collecting duct cell model for studying AQP2 phosphorylation and trafficking. However, the cells require a time‐consuming polarization process before they can respond to vasopressin. We sought to circumvent this issue by examining AQP2 phosphorylation and trafficking in non‐polarized AQP2‐overexpressing mpkCCD cells treated with forskolin, which elevates intracellular cyclic AMP (cAMP), a second messenger of vasopressin. We found that forskolin induced AQP2 phosphorylation at serine 269 (S269) by 1.6 folds as observed in polarized mpkCCD and isolated rat inner medullary collecting duct cells treated with vasopressin. Before forskolin stimulation, most AQP2 was intracellular and was not phosphorylated at S269 by confocal immunofluorescence microscopy. In response to forskolin, AQP2 was found in the plasma membrane and was phosphorylated at S269, similar to those observed in the polarized mpkCCD cells and native collecting duct cells stimulated with vasopressin. As a proof of principle, we found that small hairpin RNA‐mediated protein kinase A knockdown reduced AQP2 phosphorylation at S269 and membrane localization in the non‐polarized mpkCCD cells treated with forskolin. Thus, non‐polarized AQP2‐overexpressing mpkCCD cells can be used in lieu of polarized mpkCCD cells when time is a limiting factor including transient small hairpin RNA‐mediated gene knockdown experiments.

Support or Funding Information

This work was supported by the Ministry of Science and Technology, TAIWAN (MOST 107‐2320‐B‐002‐057‐MY3).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Complete NEM Modification of Highly Reactive Cysteines Induces Full TRPA1 Activation
Authors: Parks Thomas , Taylor‐Clark Thomas
Published: June 2021

Transient Receptor Potential Ankyrin 1 (TRPA1) is a tetrameric, non‐selective, cationic channel that is expressed in the dorsal root ganglia, trigeminal ganglia, and vagal ganglia and is activated by harmful chemicals including pollutants and endogenous irritants produced during inflammation and oxidative stress. Many irritants activate TRPA1 via electrophilic modification of highly reactive cysteine residues located on the cytosolic side of the channel, however the underlying processes that precede activation are poorly understood. Our lab has identified four highly reactive cysteines on TRPA1 (thus 16 highly reactive cysteines per channel) that rapidly bind electrophiles: C273, C621, C665 and C1085. C621 was the most reactive and point mutation studies show that both C621 and C665 are required for activation. In this study, we use cell‐attached single‐channel patch clamp analyses of transiently expressed human TRPA1 (hTRPA1) in HEK293 cells to determine the relationship between irreversible electrophilic binding and channel activity. We recorded stochastic gating events of single channels in response to N‐ethylmaleimide (NEM) treatment. Our open probability (Po) analysis indicates that NEM evoke two distinct hTRPA1 activation profiles: (1) weak activation (low Po) throughout NEM treatment which does not increase with time; (2) weak activation which then suddenly and spontaneously progresses to full activation (near 100% Po). Open time histogram analyses suggest that the weak activation state evoked by NEM has similar open times to spontaneous hTRPA1 activation in control conditions; whereas the full activation state has significantly longer open times. We conclude that the weak and full hTRPA1 activation states by NEM are due to differences in progressive cysteine modification. Furthermore, we suggest that full activation is not the summation of multiple weak states, but is instead a separate activation state that can only be evoked by a particular complement of cysteine modifications.

Support or Funding Information

This research is funded by the National Heart, Lung and Blood Institute (5R01HL119802‐S1).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.