Study On The Protective Effects And Mechanisms Of The Compatibility Of Hypaconitine And Glycyrrhetinic Acid On Cardiac Ischemic And Heart Failure

Objective Study on the protective effects and related mechanisms of the compatibility of hypaconitine(HA)and glycyrrhetinic acid(GA)on H9c2 cells under oxygen glucose deprivation(OGD),and HA is one of the major active ingredients of Fuzi,while GA is one of the major effective constituents of Gancao.Besides,the chronic heart failure model was built to observe the relevant effects and mechanisms of the compatibility of HA and GA on it.This study can provide the references of the basic theory to the compatibility of Fuzi and Gancao,and also be the experiment references for their treatments on cardiac ischemia and chronic heart failure.Methods(1)Study on the most suitable concentrations of HA、GA and digoxin on H9c2 cells:First,choosing the concentrations inside the drug safety scales;then,testing the OD levels under MTT methods,and getting the noncytotoxicity concentration scales from the the cell viability formula.(2)Research on the best compatibility ratio of HA and GA on the anti-inflammatory and anti-cell apoptotic effects of H9c2 cells under OGD:Building the OGD model,and grouping them to control group,model group,digoxin group(80μmol·L-1),HA group,and the compatibility groups of HA+GA:1:0.5 group,1:1 group,1:2 group(HA is120μmol·L-1,GA is 60、120、240μmol·L-1),effecting for 4h;Observing the changes of cells morphology;besides,testing the contents of TNF-α,IL-1β and CK in the cell culture supernatant in ELISA methods,and also using the Western Blot method to survey the expressions of caspase-3,Fas and Fas-L proteins.(3)Study on the anti-apoptotic effects and mechanisms of the best compatibility ratio of HA and GA on H9c2 cells under OGD:Grouping the cells into model group,HA group,GA group,HA+GA group,LY294002(LY)group and HA+GA+LY group(HA is 120pmol L-1,GA is 120μmol·L-1,LY is 10μmol·L-1),effecting for 4h.Using the Hoechst33342 staining method to examine the nuclei morphology changes,and the transmission electron microscopy to observe the ultrastructures changes of cells,and also testing the changes of LDH,CK-MB and AST in the cell culture supernatant in ELISA methods,besides the early apoptotic rates by flow cytometry,and also the expressions of Akt,p-Akt,caspase-9,Bax and Bcl-2 proteins by Western Blot methods.(4)Study on the apoptotic pathway effects of the compatibility of HA and GA on chronic heart failure rats:Establishing the chronic heart failure model on rats,and grouping them into Sham group,Model group,Digoxin group,HA group,GA group,HA+GA group(HA is 2.07mg/kg,GA is 25mg/kg,Digoxin is lmg/kg).After the oral administrated once a day for one week,observing the common conditions of rats,and calculating the death rate;and using the ultrasound to test the EF,FS and some other indexes of the heart,and weighing rats and hearts to get the H/B ratios;besides,using ELISA methods to inspect the changes of BNP and cTnI in serum;and also using both the immunohistochemistry staining and Western Blot methods to get the expressions of Bax,Bcl-2 and caspase-3 proteins in heart tissues.Results(1)The results of MTT showed:the noncytotoxicity concentration scale of HA was 0-120μmol·L-1,and GA was 0-240μmol·L-1,besides,digoxin was 0-80pμmol·L-1.(2)The effects of different compatibility ratios of HA and GA on H9c2 cells showed:the(1:1)compatibility improved most in the morphology,and the releases of TNF-α、IL-1β and CK decreased greatest,and also the protein expressions of caspase-3、Fas and Fas-L declined sharply.(3)The protective effects of the compatibility of HA and GA(1:1)reveled as follows:the nuclei morphology and condensations of chromatin became better than other groups;and also improved the situations of membrane,mitochondrial swelling and condensations of nuclei and chromatin;and the releases of LDH、CK-MB and AST declined apparently;and the early apoptotic rate reduced greatly.The Western Blot methods displayed that p-Akt/Akt ratio reduced distinctly in the HA+GA+LY group than HA+GA group,and the expressions of caspase-9 and Bax elevated obviously,oppositely,the Bcl-2 expressions showed a decline notably.(4)The common conditions of rats in HA+GA group much improved than other groups,in the whole process,the mortality of rats was 15.56%.And the ratio of H/B showed a significant decline in HA+GA group than Model group.The cardiac ultrasound reveled that HA+GA group ameliorated the left ventricular remodeling obviously.The levels of BNP and cTnI in the serum reduced greatly in the HA+GA group,while both the immunohistochemistry staining and Western Blot methods showed a decline in the expressions of Bax and caspase-9,and elevated the expression of Bcl-2 and Bcl-2/Bax ratios.Conclusion(1)The best compatibility ratio on H9c2 cells of HA and GA,which are from the major active ingredients of Fuzi and Gancao,is(1:1).(2)The protective effects on H9c2 under OGD of HA and GA,which are from the major active ingredients of Fuzi and Gancao,may be relevant with anti-inflammatory and anti-apoptotic.(3)The protective effects and mechanisms on H9c2 under OGD of HA and GA,which are from the major active ingredients of Fuzi and Gancao,are related with PI3K/Akt signal pathway.(4)The compatibility of HA and GA,which are from the major active ingredients of Fuzi and Gancao,can ameliorate the situations of cardiac hypertrophy,left ventricular remodeling and decreased cardiac function effectively,and the mechanism may be related with inhibiting the apoptosis of myocardial cells.