Improvement of Myocardial Injury by Hydroxy-α-Sanshool from Extracts of ZANTHOXYLI PERICARPIUM via Regulating Nrf2/HO-1 Pathway
Objective:To investigate the protective effect and mechanism of hydroxy-α-sanshool(HAS)from extracts of ZANTHOXYLI PERI-CARPIUM against rats with isoprenaline(ISO)-induced myocardial ischemia(MI)and oxidative damage in myocardial cells of rats(H9c2)induced by hydrogen peroxide(H2O2).Methods:A total of 36 SD male rats were randomly divided into a normal control group,a model con-trol group,diltiazem hydrochloride group of 20 mg/kg,and HAS groups of 5,10,and 20 mg/kg.Rats were administered for 14 days,and ISO of 40 mg/kg was injected intraperitoneally on the 13th and 14th day to induce the MI model.Echocardiography was used to evaluate the car-diac function of rats in each group.The malondialdehyde(MDA),glutathione(GSH),superoxide dismutase(SOD),cardiac troponin Ⅰ(cT-nⅠ),lactate dehydrogenase(LDH),and creatine kinase isoenzyme(CK-MB)were detected by kits.The contents of tumor necrosis factor-α(Tnf-α),interleukin-1β(Il-1β),and Il-6 in the myocardial tissue were detected by real-time fluorescence quantitative qRT-PCR.Hematoxy-lin-Eosin(HE)staining and Masson staining were used to observe the pathological changes in the myocardial tissue.H9c2 was cultured in vitro,and oxidative damage was induced by treating H9c2 with H2O2 of 600 μmol/L for six hours to establish an in vitro cell model.The cells were randomly divided into a normal control group,model control group,and HAS groups of 2.5,5,and 10 µg/mL.The optimal drug concen-tration of HAS was selected using the cell counting kit(CCK-8).Cell apoptosis was observed by using flow cytometry and laser confocal mi-croscopy.The reactive oxygen species(ROS)concentration of the cells was measured using flow cytometry.The contents or activities of an-tioxidant enzymes MDA,GSH-Px,and SOD were determined using the enzyme-linked immunosorbent assay(ELISA).The expression levels of Nrf2,Bax,HO-1,cleaved-Caspase-3,and Bcl-2 were detected by Western blot(WB).Results:Compared with those in the normal control group,the left ventricular fraction shortening(LVFS)and left ventricular ejection fractions(LVEF)were significantly decreased in the model control group(P＜0.01),and the contents of MDA,cTnl,LDH,and CK-MB were significantly increased(P＜0.01).The activities of GSH and SOD were significantly decreased(P＜0.01),and the relative mRNA expressions of Il-1β,Il-6,and Tnf-α in the myocardial tissue were significantly up-regulated(P＜0.05).Compared with the model group,the LVFS and LVEF in the HAS groups were significantly increased(P＜0.05),and the contents of MDA,cTnl,LDH,and CK-MB in the HAS group of 20 mg/kg were significantly decreased(P＜0.05).The activities of GSH and SOD were significantly increased(P＜0.05 and P＜0.01),and the relative mRNA expressions of Il-1β,Il-6,and Tnf-αwere significantly down-regulated(P＜0.05).The damage to the myocardial tissue in the HAS groups was alleviated,and the collagen deposi-tion of myocardial cells was reduced.Compared with the normal control group,the viability and mitochondrial membrane potential of H9c2 in the model control group with H2O2 was decreased,and the cell apoptosis rate,ROS level,and the contents of MDA,TNF-α,IL-1β,and IL-6 were increased.GSH-Px and SOD levels were decreased(P＜0.01).After pre-treatment with HAS,the viability of H9c2 was significantly in-creased,and the mitochondrial membrane potential was enhanced.The cell apoptosis rate,as well as the levels of ROS,MDA,IL-1β,and IL-6 was decreased in the HAS groups of 5 and 10 μg/mL,and the GSH-Px and SOD activities were significantly increased(P＜0.01).In the HAS group of 10 μg/mL,the TNF-α level was decreased significantly(P＜0.01).After HAS pre-treatment,the protein expression levels of Nrf2(nuclear),HO-1,and Bcl-2 were up-regulated,while the protein expression levels of Nrf2(cytoplasmic),Bax,and cleaved-caspase3 were down-regulated(P＜0.01).Conclusion:HAS could improve cardiac function,myocardial injury,and fibrosis in ISO-induced rats with MI,protect against H2O2-induced oxidative damage in H9c2,and improve apoptosis in H9c2.Its mechanism may be related to the inhibition of oxidative stress,release of inflammatory factors,and activation of the Nrf2/HO-1 signaling pathway.
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维普
