Mechanism Of Danzhi Jiangtang Capsule Inhibiting Apoptosis Of DCM Cardiomyocytes By Regulating PI3K/AKT/GSK-3β Pathway

The first part:To study the material basis and mechanism of Danzhi Jiangtang capsule in treating diabetic cardiomyopathy based on network pharmacologyObjective: To study the substance basis and mechanism of Danzhi Jiangtang Capsule(DJC)in the treatment of Diabetic cardiomypathy(DCM)based on network pharmacology.Methods: The main active components of DJC were obtained by Traditional Chinese Medicine Systems Pharmacology Database(TCMSP).The chemical components of Radix rehmanniae recen and Hirudo not included in TCMSP database were collected from CNKI,Wanfang and Pubmed databases,and the Swiss Target Prediction platform was used to predict the potential targets of the active ingredients.The target of DCM was retrieved in GEO,Pharm Gkb,OMIM,Disgenet and NCBI Gene database by means of "Diabetic Cardiomyopathy".Protein-protein interaction network(PPI)was constructed using String database and Cytoscape 3.7.2 software.GO and KEGG analysis of DJC therapeutic targets for DCM was performed using R language.Auto Dock Tools1.5.6 was used to conduct molecular docking between the active components of DJC and important target proteins.Results:(1)52 active components were screened from DJC,including 12 active components and 129 targets in Radix pseudostellariae,12 active components and206 targets in Dodder,7 active components and 20 targets in Alisma orientalis,9act ive components and 230 targets in Cortex moutan,7 active components and57 targets in Rehmannia glutinosa,11 active components and 242 targets in Leeches.There were 486 targets corresponding to the active components of DJC after removing the repeated components.(2)282,76,15,220 and 560 DCM targets were retrieved from GSE3585 dataset,Pharm Gkb database,OMIM database,Disgenet database and NCBI Gene database respectively.A total of 863 DCM targets were obtained after removing repeatedtargets.(3)DJC has 128 therapeutic targets for DCM,12 key pharmacophore molecules and 17 core targets were screened out by topology analysis.The 12 key chemical constituents were quercetin,luteolin,kamanol,crocin,paeoniflorin,ursolic acid,isorhamnetin,β sitosterol,genipinin,Gardnerilin A,robinin,hyperin.The 17 core targets were AKT1,TNF,IL6,IL1 B,VEGFA,TP53,CASP3,JUN,EGFR,STAT3,FN1,PPARG,MYC,MMP9,PTGS2,CXCL8 and CCL2.(4)According to GO enrichment analysis,these targets are related to biological functions such as regulating gene transcription,cell proliferation,cell differentiation,cell apoptosis,oxidative stress and inflammatory response.Enrichment analysis of KEGG pathway showed that PI3K-Akt,TNF,TLR,NLRP,HIF-1,Fox O,VEGF,MAPK,IL-17,Th17,JAK-STAT and other signaling pathways play a role.(5)According to the information of the top ten core target proteins with DC value obtained from PDB database,the minimum binding energy between most of the allocators and the active ingredient molecule was≤-5.0kal/mol,indicating that the target protein and the active ingredient had a good affinity.Conclusion:DJC treatment of DCM is characterized by multiple components,multiple targets and multiple pathways,and its mechanism may be related to inhibiting inflammatory response,reducing oxidative stress level,regulating apoptosis and regulating immune response.The second part:Danzhi Jiangtang capsule regulates PI3K/AKT/GSK-3β signaling pathway to inhibit myocardial cell apoptosis in diabetic cardiomyopathy rats Objective: To observe the effects of Danzhi Jiangtang capsule on cardiac function and myocardial cell apoptosis in diabetic cardiomyopathy rats based on PI3K/AKT/GSK-3βsignaling pathway.Methods: Ten of the 90 male SD rats were randomly selected as the normal control group(NC),and the remaining 80 rats were given a single intraperitoneal injection of streptozotocin(STZ)55mg/kg combined with high sugar and high fat diet to establish type 2 diabetic cardiomyopathy model.At last,70 rats were successfully established.They were randomly divided into Danzhi Jiangtang capsule(DJC)low-dose,medium-dose and high-dose groups,Metformin group and model control(MC)group.DJC low-dose,medium-dose and high-dose groups were given 0.5,1,2 times(270,540,1080mg/kg)of the equivalent dose of 6g/d for adults by gavage.Metformin group was given Metformin at 150mg/(kg.d).Model control group(MC)and NC group were given equal volume distilled water intragastric treatment with 14 rats in each group.Body weight,water intake,food intake and random blood glucose levels were monitored at 0,2,4,6 and 8w after drug intervention respectively in each group.After 8 weeks of intervention,samples were collected under anesthesia.Serum and myocardial tissue were collected to detect serum TC,TG,LDL-C and HDL-C of rats.Histopathological changes of myocardium were observed by HE staining and Masson staining.Cardiac function of rats in each group was detected by echocardiography.The expression levels of key proteins in PI3K/AKT/GSK-3β signaling pathway were detected by western blotting.Apoptosis of myocardial cells was detected by TUNEL immunofluorescence staining.Results :(1)DCM model rats presented typical symptoms of "polydipsia,polyophagia,polyuria,loss-weight ",lethargy,clumsy movement,coarse hair;DJC and metformin can significantly increase body weight,reduce water intake and random blood glucose level,and improve the general condition of DCM rats.(2)Compared with NC group,the contents of TC,TG and LDL-C in serum of MC group were significantly increased,while the content of HDL-C was significantly decreased(P<0.01);Compared with MC group,the contents of TC,TG and LDL-C in serum of Metformin group and DJC groups were significantly decreased,while the content of HDL-C was significantly increased(P<0.05 or P<0.01).(3)Compared with NC group,LVEF and LVFS in MC group were significantly decreased,while LVDs and LVDd were significantly increased(P<0.01);Compared with MC group,LVEF and LVFS in medium/high dose DJC group and metformin group were significantly increased,while LVDs and LVDd were significantly decreased(P<0.05 or P<0.01).(4)Compared with NC group,HW/BW and LVM/BW of MC group were significantly increased(P< 0.01);Compared with MC group,HW/BW and LVM/BW of DJC groups and metformin group were significantly decreased(P<0.05 or P<0.01).(5)Compared with NC group,MC group showed more disordered cardiac muscle fibers under light microscope,with partial dissolution and fracture,different positions of cell nuclei,unclear boundaries between cells,and fibrocyte proliferation in interstitium.Compared with MC group,the pathological changes of myocardial cells in metformin group and DJC groups were significantly reduced under light microscope.(6)Masson results showed that myocardial fibrosis was significantly increased in MC group compared with NC group.Compared with MC group,the myocardial fibrosis area of DJC group and metformin group was improved.(7)Compared with NC group,protein expression levels of p-PI3K/PI3 K,p AKT/AKT and p-GSK-3β/GSK-3β in myocardium of MC group were decreased significantly(P<0.01);Compared with MC group,protein expression levels of p-PI3K/PI3 K,p-AKT/AKT and p-GSK-3β/GSK-3β in myocardium of rats in DJC groups and metformin groups were increased significantly(P<0.01).(8)compared with NC group,the activities of Caspase3 and Caspase9 in myocardial tissue unit concentration in MC group were significantly increased(P<0.01);Compared with MC group,the activities of Caspase3 and Caspase9 in myocardial tissue unit concentration of rats in DJC groups and metformin groups were significantly decreased(P<0.05 or P<0.01).(9)TUNEL immunofluorescence staining showed that the apoptosis rate of myocardial cells in MC group was significantly higher than that in NC group(P<0.01).Compared with MC group,the apoptosis rate of myocardial cells in all drug intervention groups was significantly decreased(P<0.01).Conclusion: Danzhi Jiangtang capsule may inhibit myocardial apoptosis,improve cardiac function and play a protective role in diabetic cardiomyopathy rats by activating PI3K/AKT/GSK-3β signaling pathway and increasing PI3 K,AKT and GSK-3βphosphorylation.The third part:To study the effect of Danzhi Jiangtang capsule on high glucose induced apoptosis of H9C2 cardiomyocytes based on PI3K/AKT/GSK-3β signaling pathway.Objective:To observe the effect of Danzhi Jiangtang capsule on H9C2 myocardial cell apoptosis induced by high glucose based on PI3K/AKT/GSK-3β signaling pathway.Methods:H9C2 myocardial cell injury model induced by high glucose was established by vitro experiments.MTT assay was used to observe the effect of high glucose on myocardial cell viability.After intervention with PI3 K specific inhibitor LY294002,Metformin and Danzhi Jiangtang capsule containing serum,the changes of key proteins in PI3K/AKT/GSK-3β signaling pathway were detected by WB method,and the apoptosis rate of H9C2 cells in each group was detected by flow cytometry.To elucidate the pathological mechanism of PI3K/AKT/GSK-3β signaling pathway regulating the apoptosis process of cardiomyocytes induced by high glucose,and to reveal the effect of Danzhi Jiangtang capsule on the apoptosis of cardiomyocytes induced by high glucose and its relationship with the regulation of PI3K/AKT/GSK-3βsignaling pathway.Results:(1)Different glucose concentrations(5.5,15,30,45,60mmol/L)and different intervention time(12,24,48h),the survival rate of H9C2 cardiomyocytes showed that compared with the normal control group(5.5mmol/L),the survival rate of cardiomyocytes in high glucose model group(30mmol/L)was significantly decreased for 48h(P<0.01).(2)Compared with normal control group,the relative protein expression of p-PI3K/PI3 K,p-AKT/AKT and p-GSK-3β/GSK-3β in cardiomyocytes of high glucose model group was significantly decreased(P<0.01).The apoptosis rate by flow cytometry and Caspase3 and Caspase9 activities of cardiomyocytes were significantly increased(P<0.01).(3)Compared with the high glucose model group,the relative protein expressions of p-PI3K/PI3 K,p-Akt/AKT and P-GSK-3β/GSK-3β in cardiomyocytes in the high glucose model+Metformin group,high glucose model+Danzhi Jiangtang capsule group,high glucose model+Metformin+LY294002group and high glucose model+Danzhi Jiangtang capsule+LY294002 group were significantly increased.The apoptosis rate of myocardial cells and the activities of Caspase3 and Caspase9 were significantly decreased(P<0.01).(4)Compared with the high-glucose model+ Metformin group,the relative protein expression of p-PI3K/PI3 K,p-AKT/AKT and p-GSK-3β/GSK-3β were significantly decreased in the high-glucose model+ Metformin+LY294002 group.The apoptosis rate of myocardial cells and the activities of Caspase3 and Caspase9 were significantly increased(P<0.01).(5)The relative protein expression of p-PI3K/PI3 K,p-AKT/AKT and p-GSK-3β/GSK-3β in cardiomyocytes in high-glucose model+Danzhi Jiangtang capsule+LY294002 group was significantly lower than that in high-glucose model+Danzhi Jiangtang capsule group.The apoptosis rate of myocardial cells and the activities of Caspase3 and Caspase9 were significantly increased(P<0.01).Conclusion:Danzhi Jiangtang capsule may promote GSK-3β phosphorylation and inhibit H9C2 myocardial cell apoptosis induced by high glucose by regulating PI3K/AKT/GSK-3β signaling pathway.