基于UHPLC-MS技术及体内外实验探讨参附注射液对慢性心力衰竭心阳虚证大鼠微管蛋白翻译后修饰表达的影响OA

Objective To screen the core active components of Shenfu Injection(参附注射液,SFI)using UHPLC-MS and molecular docking techniques,and to verify the effects of SFI on expressions of post-translational modifications of tubulin in chronic heart failure of heart-yang deficiency syndrome through in vivo and in vitro experiments.Methods UHPLC-MS was applied to identify the main components of SFI,and molecular docking was performed to analyze their interactions with tubulin-related targets.SD rats were randomly divided into control group,model group,colchicine(0.1 mg/kg)group,SFI low-and high-dose(3,6 mL/kg)groups,with eight rats in each group.The CHF heart-yang deficiency model was replicated using sc isoproterenol,and drug intervention was given for two weeks.Echocardiography was used to detect cardiac function.ELISA was used to detect N-terminal pro-B-type natriuretic peptide(NT-proBNP)level in serum.Hematoxylin-eosin(HE)staining was used to detect pathological changes in myocardial tissue.Immunofluorescence was used to detect the expressions of acetyl α-tubulin,polyglut α-tubulin and detyr α-tubulin in myocardial tissue.Immunohistochemistry was used to detect the expressions of tubulin tyrosine ligase(TTL)and histone deacetylase 6(HDAC6)in myocardial tissue.H9c2 cardiomyocytes were treated with isoproterenol to establish a cell injury model,and after intervention with SFI,the level of microtubule protein modification was detected.Results A total of 12 main active ingredients in SFI were obtained through UHPLC-MS screening,and molecular docking results showed strong binding affinity with tubulin and modification related proteins.The animal experiment results showed that compared with model group,SFI could significantly improve cardiac function of rats(P<0.01),reduce the level of NT-proBNP in serum(P<0.01),alleviate the degree of myocardial interstitial fibrosis,reduce the expressions of microtubule protein α-tubulin and microtubule density in myocardial tissue(P<0.05,0.01),reduce the expressions of microtubule protein tyrosine and glutamate,increase acetylation expression,down-regulate HDAC6 positive expression(P<0.01),and up-regulate TTL positive expression(P<0.01).The results of cell experiments were consistent with animal experiments,further verifying that SFI could improve post-translational modifications of microtubule proteins.Conclusion SFI could effectively reduce the levels of tyrosinization and glutamatzation of microtubules,increase acetylation levels,correct the imbalance of post-translational modifications of microtubules,thereby improving the abnormal microtubule network in cardiac myocytes of rats with CHF heart-yang deficiency,enhancing microtubule stability and improving heart function.This provides experimental evidence for the molecular mechanism of SFI treatment for CHF heart-yang deficiency.