Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

PTEN inhibitor bisperoxovanadium protects against noise-induced hearing loss

Studies have shown that phosphatase and tensin homolog deleted on chromosome ten(PTEN)participates in the regulation of cochlear hair cell survival.Bisperoxovanadium protects against neurodegeneration by inhibiting PTEN expression.However,whether bisperoxovanadium can protect against noise-induced hearing loss and the underlying mechanism remains unclear.In this study,we established a mouse model of noise-induced hearing loss by exposure to 105 dB sound for 2 hours.We found that PTEN expression was increased in the organ of Corti,including outer hair cells,inner hair cells,and lateral wall tissues.Intraperitoneal administration of bisperoxovanadium decreased the auditory threshold and the loss of cochlear hair cells and inner hair cell ribbons.In addition,noise exposure decreased p-PI3K and p-Akt levels.Bisperoxovanadium preconditioning or PTEN knockdown upregulated the activity of PI3K-Akt.Bisperoxovanadium also prevented H_(2)O_(2)-induced hair cell death by reducing mitochondrial reactive oxygen species generation in cochlear explants.These findings suggest that bisperoxovanadium reduces noise-induced hearing injury and reduces cochlear hair cell loss.

Poly(A)-specific ribonuclease protein promotes the proliferation,invasion and migration of esophageal cancer cells

BACKGROUND Bioinformatics analysis showed that the expression of the poly(A)-specific ribonuclease(PARN)gene in gastric cancer,head and neck squamous cell carcinoma,melanoma,cervical cancer and lung squamous cell carcinoma tissues was significantly higher than that in normal tissues and was associated with high stage and poor prognosis.The expression of the PARN gene in esophageal cancer(EC)tissue is also significantly higher than that in normal tissues,but the effect of PARN on the proliferation,migration and invasion of EC cells remains unclear.AIM To investigate the relationship between PARN and the proliferation,migration and invasion of EC cells.METHODS The EC tissues of 91 patients after EC surgery and 63 paired precancerous healthy tissues were collected.PARN mRNA levels were measured using a tissue microarray,and the PARN expression level was evaluated using immunohistochemistry to analyze the relationship between PARN expression and clinicopathologic features as well as the survival and prognosis of patients.In addition,the effects of PARN gene knockout on tumor cell proliferation,invasion and migration were studied by using shRNA during the in vitro culture of EC cell lines Eca-109 and TE-1,and the effects of the PARN gene on tumor growth in vivo were verified by a xenotransplantation nude mice model.RESULTS The expression of PARN in EC tissues was higher than that in adjacent normal tissues,and the level of PARN expression was significantly positively correlated with lymphatic metastasis.Patients with high PARN levels had poor overall survival.BIM,IGFBP-5 and p21 levels were significantly increased in the PARN knockout group,while the expression levels of the antiapoptotic proteins Survivin and sTNF-R1 were significantly decreased in the apoptotic antibody array data.In addition,the expression levels of Akt,p-Akt,PIK3CA and CCND1 in the downstream signaling pathway regulating EC progression were significantly decreased.The culture of EC cell lines confirmed that the apoptosis rate of EC cells was significantly increased,the growth and proliferation of tumor cells were significantly inhibited,and the invasion and migration ability of tumor cells were significantly decreased after PARN gene knockout.In vivo experiments of BALB/c nude mice transfected with Eca-109 cells expressing control shRNA(sh-NC)and PARN shRNA(sh-PARN)showed that the tumor volume and weight of nude mice treated with sh-PARN were significantly decreased compared with those of nude mice treated with sh-NC,indicating that PARN knockdown significantly inhibited tumor growth in vivo.CONCLUSION PARN has antiapoptotic effects on EC cells and promotes their proliferation,invasion and migration,which is associated with the development of EC and poor patient prognosis.PARN may become a potential target for the diagnosis,prognosis prediction and treatment of EC.

Toll-like receptor 4-mediated signaling participates in apoptosis of hippocampal neurons

The phosphatidylinositol 3 kinase （PI3K）/protein kinase B （AKT） signaling pathway is considered important for cell survival and has been shown to mediate various anti-apoptotic biological effects. This study explored the role of the Toll-like receptor 4 （TLR4）-mediated PI3K/AKT-glycogen syn-thase kinase 3β （GSK-3β） signaling pathways in lipopolysaccharide-induced apoptosis in a primary culture of hippocampal neurons. Results demonstrated that the apoptotic ratio of hippocampal neurons stimulated by lipopolysaccharide was significantly higher compared with the control group. Both the expression of P-AKTser473 and P-GSK-3βSserg in hippocampal neurons stimulated by lipopolysaccharide decreased compared with the control, while the level of active Caspase-3 and the ratio of Bax/Bcl-2 were significantly increased. The level of active Caspase-3 and the ratio of Bax/Bcl-2 in hippocampal neurons treated with TLR4 antibody or the GSK-3β inhibitor, LiCl, de-creased before intervention with lipopolysaccharide, but increased after treatment with the AKT in-hibitor, LY294002. These findings suggest that the TLR4-PI3K/AKT-GSKβ signaling pathway may be involved in lipopolysaccharide-induced apoptosis of hippocampal neurons.

Insulin-like growth factor-I receptor in proliferation and motility of pancreatic cancer

AIM:To develop a molecular therapy for pancreatic cancer, the insulin-like growth factor-I (IGF-I) signaling pathway was analyzed.METHODS: Pancreatic cancer cell lines (MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4) were cultured in media with 10 mL/L fetal bovine serum. Western blotting analysis was performed to clarify the expression of IGF-I receptor (IGF-IR). Picropodophyllin (PPP), a specific inhibitor of IGF-IR, LY294002, a specific inhibitor of phosphatidylinositol3 kinase (PI3K), and PD98059, a specific inhibitor of mitogen-activated protein kinase, were added to the media. After 72 h, a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) assay was performed to analyze cell proliferation. A wound assay was performed to analyze cell motility with hematoxylin and eosin (HE) staining 48 h after addition of each inhibitor. RESULTS: All cell lines clearly expressed not only IGF-IR but also phosphorylated IGF-IR. PPP significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 36.9% ± 2.4% (mean ± SD), 30.9% ± 5.5%, 23.8% ± 3.9%, 37.1% ± 5.3%, 10.4% ± 4.5%, 52.5% ± 4.5% and 22.6% ± 0.4%, at 2 μmol/L, respectively (P < 0.05). LY294002 significantly suppressed proliferation of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 44.4% ± 7.6%, 32.9% ± 8.2%, 53.9% ± 8.0%, 52.8% ± 4.0%, 32.3% ± 4.2%, 51.8% ± 4.5%, and 30.6% ± 9.4%, at 50 μmol/L, respectively (P < 0.05). PD98059 did not significantly suppress cell proliferation. PPP at 2 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 cells to 3.0% ± 0.2%, 0%, 0%, 2.0% ± 0.1%, 5.0% ± 0.2%, 3.0% ± 0.1%, and 5.0% ± 0.2%, respectively (P < 0.05). LY294002 at 50 μmol/L suppressed motility of MIA-Paca2, NOR-P1, PANC-1, PK-45H, PK-1, PK-59 and KP-4 to 3.0% ± 0.2%, 0%, 3.0% ± 0.2%, 0%, 0%, 0% and 3% ± 0.1%, respectively (P < 0.05). PD980509 at 20 μmol/L did not suppress motility. Cells were observed by microscopy to analyze the morphological changes induced by the inhibitors. Cells in medium treated with 2 μmol/L PPP or 50 μmol/L LY294002 had pyknotic nuclei, whereas those in medium with 20 μmol/L PD98059 did not show apoptosis.CONCLUSION: IGF-IR and PI3K are good candidates for molecular therapy of pancreatic cancer.

The role of mechanical stretch and TGF-β2 in epithelial-mesenchymal transition of retinal pigment epithelial cells

AIM: To explore the effects and mechanisms of mechanical stress and transforming growth factor-beta2(TGF-β2) on epithelial-mesenchymal transition(EMT) in cultured human retinal pigment epithelial(RPE) cells. METHODS: Human RPE cells were inoculated on BioF ex 6-well plates and RPE cells received 0, 1, 2, 3, or 4 mild stretch injuries delivered 3h apart after 24h of culture. The device of mechanical stress parameters were set to sine wave, frequency 1 Hz, stretch strength 20%. For treatment with TGF-β2, when the inoculated RPE cells in 6-well plates were around 60% confluent, serum was reduced to 0 for 12h and recombinant human TGF-β2(0, 1, 5, 10 ng/mL)was added for 48h. α-SMA, Vimentin and N-Cadherin, fibronectin proteins expressions were detected by Western blotting, confocal cell immunofluorescence and quantitative real-time polymerase chain reaction(q RT-PCR). Then we detected the change of mi RNA-29b and ascertained the changes of phosphatidylinositol 3-kinase-serine threonine protein kinase(PI3K/Akt) pathway after RPE cells were stretched by the device of mechanical stress and induced by TGF-β2 by Western blotting, confocal cell immunofluorescence and qR T-PCR. RESULTS: Mechanical stress induce EMT and activate the PI3K/Akt pathway in ways that lead to the EMT process. TGF-β2 induce RPE cells EMT and in a certain range and TGF-β2 decrease the miR NA-29b expression in RPE cells, and the inhibitory effect is more obvious with the increase of TGF-β2 concentration. CONCLUSION: Our findings are crucial steps in determining the critical roles of the PI3K/Akt signaling pathway and mi RNA-29b in pathogenesis of proliferative vitreoretinopathy(PVR) which may be a potential target for preventing or treating PVR.

Clean-DM1, a Korean Polyherbal Formula, Improves High Fat Diet-Induced Diabetic Symptoms in Mice by Regulating IRS/PI3K/AKT and AMPK Expressionsin Pancreas and Liver Tissues

Objective:To investigate the effects of Clean-DM1(C-DM1),a polyherbal formulation of Radix Scrophulariae,Radix Astragali,Rhizoma Atractylodis,and Radix Salviae Miltiorrhizae,on high-fat diet(HFD)-induced diabetes mice.Methods:The information about active components of C-DM1 extract and molecular mechanism was obtained from network pharmacology analysis.Main compounds of C-DM1 extract by high performance liquid chromatography-mass spectrometry(HPLC-MS)analysis were conducted for quality control.For in vivo study,mice were induced diabetes by HFD for 12 weeks.The mice in the normal group(Nor)were maintained with a regular diet and treated with saline by gavage.The HFD model mice were randomly divided into 3 groups,including a HFD diabetic model group,a C-DM1 extract-administered group(C-DM1,500 mg/kg),and metformin-administered groups(Met,500 mg/kg),8 mice in each group.Food intake,body weight(BW),and fasting blood glucose(FBG)levels were recorded weekly for 4 weeks.After 4 weeks of treatment,alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood glucose,low-density lipoprotein cholesterol(LDL-C)were determined using an automated clinical chemistry analyzer,and homeostatic model for assessing insulin resistance(HOMA-IR)levels and oral glucose tolerance test(OGTT)were detected.The histopathological changes of liver and pancreatic tissues were observed by hematoxylin-eosin staining.Insulin receptor substrate(IRS)/phosphatidylinositol 3 kinase(PI3K)/protein kinase B(AKT)and adenosine 5'-monophosphate-activated protein kinase(AMPK)expressions in liver and pancreas tissues were detected by Western blot analysis.Results:HPLC-MS identified dihydroisotanshinone,dihydroisotanshinone I,cryptotanshinone,harpagoside,and atractyloside A in C-DM1 extract.The administration of C-DM1 extract significantly decreased body weight,calorie intake,and the levels of blood glucose and insulin in the diabetic mice(P<0.05 or P<0.01).The C-DM1 extract administration improved the impaired glucose tolerance and insulin resistance in the diabetic mice and significantly decreased the levels of LDL-C,ALT and AST(P<0.01).The C-DM1 extract inhibited the histopathological changes of fatty liver and hyperplasia of pancreatic islets in the diabetic mice.The C-DM1 extract significantly increased the phosphorylation of IRS,AKT,and AMPK and the expression of PI3K in pancreas and liver tissues(P<0.05 or P<0.01),which was consistent with the analysis results of network pharmacology.Conclusion:C-DM1 extract improved diabetes symptoms in longterm HFD-induced mice by regulation of IRS/PI3K/AKT and AMPK expressions in pancreas and liver tissues,suggesting that C-DM1 formulation may help prevent the progression of T2DM.

Hydroxysafflor Yellow A Promotes HaCaT Cell Proliferation and Migration by Regulating HBEGF/EGFR and PI3K/AKT Pathways and Circ_0084443

Objective:To investigate the effect and possible mechanism of hydroxysafflor yellow A(HSYA) on human immortalized keratinocyte cell proliferation and migration.Methods:HaCaT cells were treated with HSYA.Cell proliferation was detected by the cell counting kit-8 assay,and cell migration was measured using wound healing assay and Transwell migration assay.The mRNA and protein expression levels of heparin-binding epidermal growth factor(EGF)-like growth factor(HBEGF),EGF receptor(EGFR),phosphatidylinositol 3-kinase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(mTOR),and hypoxia-inducible factor-1α(HIF-1α) were detected by quantitative real-time polymerase chain reaction(qRT-PCR) and Western blot,respectively.Circ_0084443-overexpressing HaCaT cells and empty plasmid HaCaT cells were constructed using the lentiviral stable transfection and treated with HSYA.The expression of circ_0084443 was detected by qRT-PCR.Results:HSYA(800 μmol/L) significantly promoted HaCaT cell proliferation and migration(P<0.05or P<0.01).It also increased the mRNA and protein expression levels of HBEGF,EGFR,PI3K,AKT,mTOR and HIF-1α,and increased the phosphorylation levels of PI3K and AKT(P<0.05 or P<0.01).Furthermore,HSYA promoted HaCaT cell proliferation and migration via the HBEGF/EGFR and PI3K/AKT/m TOR signaling pathways(P<0.01).Circ_0084443 attenuated the mRNA expression levels of HBEGF,EGFR,PI3K,AKT,mTOR and HIF-1α(P<0.05).HSYA inhibited the circ_0084443 expression,further antagonized the inhibition of circ_0084443on HBEGF,EGFR,PI3K,AKT,m TOR and HIF-1α,and promoted the proliferation of circ_0084443-overexpressing HaCaT cells(P<0.05 or P<0.01).However,HSYA could not influence the inhibitory effect of circ_0084443 on HaCaT cell migration(P>0.05).Conclusion:HSYA played an accelerative role in HaCaT cell proliferation and migration,which may be attributable to activating HBEGF/EGFR and PI3K/AKT signaling pathways,and had a particular inhibitory effect on the keratinocyte negative regulator circ_0084443.

Apelin protects against cardiomyocyte apoptosis induced by glucose deprivation

Background Apoptosis is a major cause of ischemic heart dysfunction. Apelin, the endogenous ligand for the G-protein-coupled APJ receptor, has been reported to exert cardioprotective effects during myocardial injury. The aim of this study was to investigate the effects of apelin on apoptosis of rat cardiomyocytes induced by glucose deprivation （GD） and study the related signaling pathway. Methods Apelin and APJ mRNA expression were determined by RT-PCR in neonatal rat cardiomyocytes during different durations of GD. Cardiomyocyte apoptosis was detected by annexin V-FITC/propidium iodide （PI） staining after GD for 12 hours with or without apelin-13 （10 and 100 nmol/L） pretreatment. Protein levels of Akt and the mammalian target of rapamycin （mTOR） as well as cell apoptosis were detected in the presence or absence of LY294002 （a phosphatidylinositol 3-kinases （PI3K） inhibitor） or rapamycin （a mTOR inhibitor）. Results Apelin mRNA expression was up-regulated when cardiomyocytes were exposed to GD for 6, 12, 18, and 24 hours compared with the base level （P 〉0.05, P 〈0.01, P 〈0.01, P 〈0.01）. However, when cardiomyocytes were exposed to GD for up to 36 hours, apelin mRNA expression was 17% lower than the base level （P〈0.05）. APJ mRNA expression paralleled that of apelin. Apelin-13 pretreatment at 100 nmol/L significantly inhibited GD-induced cardiomyocyte apoptosis （P 〈0.05） and increased Akt and mTOR phosphorylation （P 〈0.01, P 〈0.01）. At the same time apelin-13 （100 nmol/L） up-regulated Bcl-2 protein expression and down-regulated Bax and cleaved caspase-3 expression （P 〈0.01, P 〈0.05, P 〈0.05）. The anti-apoptotic effect of apelin-13 was blocked by LY294002 （P 〈0.01） but not by rapamycin. Conclusions The endogenous apelin-APJ system is compensatorily up-regulated and ultimately down-regulated following sustained myocardial ischemia. Apelin protects against ischemic cardiomyocyte apoptosis via activation of the PI3K/Akt pathway.

Effects of Panax Quinquefolium Saponin on Phosphatidylinositol 3-Kinase/Serine Threonine Kinase Pathway of Neonatal Rat Myocardial Cells Subjected to Hypoxia

Objective： To explore the effects of Panax Quinquefolium Saponin （PQS） on phosphatidylinositol 3-kinase/serine threonine kinase （P13/Akt） pathway of neonatal rat myocardial cells subjected to hypoxia. Methods： Neonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups： the normal group, the model group, the positive control group （Ciclosporin A, 2 p, mol/L）, the low-dose PQS group （PQSL, 25mg/L）, and the high-dose PQS group （PQSH, 50 mg/L）. Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase （LDH） leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase （GSK）-3β and phospho-Akt as well as cytochrome C were determined by Western blot. Results： LDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group （P〈0.05）. Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group （P〈0.05, P〈0.01）. Compared with the model group （16.41 ± 1.74; 35.28 ± 6.30）, both the integrated optical density of mitochondrial permeability transition pore （MPTP） and the mitochondrial transmembrane potential significantly increased in the PQSH group （42.74± 2.12; 71.36 ± 6.54） and the PQSL group （39.58± 1.49; 66.99± 5.45; P〈0.05, P〈0.01）. However, the protein of cytochrome C outside the mitochonddon decreased in the PQSH group （273.66 ± 14.61） and the PQSL group （259.62 ± 17.31） compared with the model group （502.41 ± 17.76; P〈0.05）. Conclusion： Through activation of the P13K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.

TopoisomeraseⅡalpha promotes gallbladder cancer proliferation and metastasis through activating phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway

Background:TopoisomeraseⅡalpha(TOP2A)has been reported to play a crucial role in the tumorigenesis of various cancer types.However,the biological role of TOP2A in gallbladder cancer(GBC)remains unknown.The current study aimed to explore the function and potential mechanism of TOP2A in GBC.Methods:Based on Gene Expression Profiling Interactive Analysis data,we found TOP2A was significantly up-regulated in GBC tissues and resulting in shorter overall survival.Quantitative real-time polymerase chain reaction and immunohistochemistry were conducted to detect the expression of TOP2A in 45 pairs of GBC tissues and adjacent non-tumor tissues.In vitro,cell proliferation,migration,and invasion ability were examined by cell counting kit-8 and transwell assay,respectively.Epithelial-mesenchymal transition(EMT)related and phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway-related markers were measured by Western blotting.Xenograft model assay was performed to evaluate the effect of TOP2A in vivo.Results:TOP2A was found up-regulated in GBC(tumor vs.normal,12.62 vs.0.34)and correlated with the late tumor node metastasis stage(P=0.0032),present of lymph node metastasis(P=0.0273),and poor prognosis in GBC patients(log-rank P=0.028).In vitro and in vivo assays showed that knockdown of TOP2A notably inhibited cell proliferation,migration,invasion,EMT process,and tumor growth in GBC.In addition,TOP2A down-regulation significantly decreased the protein levels of phosphor(p)-PI3K,p-Akt,and p-mTOR.Conclusion:Our study demonstrates that TOP2A was overexpressed in GBC and associated with poor prognosis in GBC patients.TOP2A promotes GBC cell proliferation,migration,invasion,EMT process,and tumor growth through activating PI3K/Akt/mTOR signaling pathway,and may serve as a novel prognostic biomarker and therapeutic target for GBC.

Integration of network and experimental pharmacology to decipher the antidiabetic action of Duranta repens L.

Objective:Duranta repens is reported to contain a wide array of secondary metabolites,including aamylase and a-glucosidase inhibitors,and-has potent antioxidant activity.The present study evaluated the network pharmacology of D.repens(whole plant)with targets related to diabetes mellitus and assessed its outcome by evaluating the effects of the hydroalcoholic extract of D.repens in streptozotocin-nicotinamide-induced diabetes mellitus in rats.Methods:Phytoconstituents of D.repens were retrieved from an open-source database and published literature,and their targets were predicted for diabetes mellitus using Binding DB and the therapeutic target database.Protein-protein interaction was predicted using STRING,and pathways involved in diabetes mellitus were identified using the Kyoto Encyclopedia of Genes and Genomes pathway browser.Druglikeness,ADMET profile(absorption,distribution,metabolism,excretion and toxicity)and cytotoxicity of compounds modulating proteins involved in diabetes were predicted using Mol Soft,admet SAR2.0 and CLC-Pred,respectively.The interaction network among phytoconstituents,proteins and pathways was constructed using Cytoscape,and the docking study was performed using Auto Dock4.0.The hydroalcoholic extract of D.repens was evaluated using streptozotocin-nicotinamide-induced diabetes mellitus animal model for 28 d,followed by an oral glucose tolerance test.At the end of the study,biochemical parameters like glycogen content,hepatic enzymes,antioxidant biomarkers and lipid profiles were quantified.Further,the liver and pancreas were collected for a histopathology study.Results:Thirty-six different secondary metabolites from D.repens were identified to regulate thirty-one targets involved in diabetes mellitus,in which protein-tyrosine phosphatase 1 B(PTP1 B)was primarily targeted.Enrichment analysis of modulated proteins identified 12 different pathways in diabetic pathogenesis in which the phosphatidylinositol 3-kinase-protein kinase B(PI3 K-Akt)signaling pathway was chiefly regulated.The docking study found that durantanin I possessed the highest binding affinity(à8.9 kcal/mol)with PTP1 B.Similarly,ADMET profiling showed that the majority of bioactive constituents from D.repens had higher human intestinal absorptivity and minimal cytotoxicity to normal cell lines,than tumor cell lines.Further,an in vivo animal study reflected the efficacy of the hydroalcoholic extract of D.repens to lower the elevated blood glucose level by stimulating insulin secretion,maintaining pancreatic b cell mass,regulating glycolysis/gluconeogenesis and enhancing the glucose uptake in skeletal muscles.Conclusion:The present study reflected the probable network interaction of bioactive constituents from D.repens,their targets and modulated pathways,which identified the prime regulation of the PI3 K-Akt signaling pathway and PTP1 B protein.Modulation of PTP1 B protein and PI3 K-Akt signaling pathway could contribute to enhancing glucose uptake,insulin production and glycolysis and decreasing gluconeogenesis in diabetes,which was evaluated via the experimental study.