PKC/HSP70通路介导白藜芦醇对心肌细胞缺氧/复氧损伤的保护作用

目的：探讨白藜芦醇（resveratrol,RSVL）对心肌细胞缺氧/复氧损伤（anoxia/reoxygenation injury,ARI）的保护作用及机制。方法：分离、纯化SD乳鼠原代心肌细胞,建立缺氧/复氧损伤模型,采用随机数字量表法分为6组：正常对照（CON）组、ARI组、ARI+不同浓度RSVL（25,50,75 μmol·L^-1）预处理组、ARI+RSVL（75 μmol·L^-1）+PKC抑制剂白屈菜红碱（1 μmol·L^-1）组。倒置相差显微镜观察心肌细胞生长形态,自发搏动频率和节律;Hoechst 33258染色计算细胞凋亡率;比色法测定细胞培养液中LDH、CK漏出量及细胞内Ca²⁺-ATPase活性;荧光分光光度计测定细胞内游离钙离子浓度;Western blot检测心肌细胞PKC、HSP70及Ca²⁺-ATPase蛋白水平。结果：不同浓度RSVL与ARI组相比,心肌细胞折光性好,搏动有力而规则,频率在90~115次/分（P<0.01）;呈剂量依赖性减少细胞凋亡（P<0.01）,降低培养液中LDH和CK漏出量（P<0.05或P<0.01）,提高心肌细胞Ca²⁺-ATPase活性及降低细胞内Ca²⁺]_i量（P<0.05或P<0.01）;心肌细胞PKC、HSP70及Ca²⁺-ATPase蛋白表达增加（P<0.05或P<0.01）。而RSVL的保护作用可被PKC抑制剂白屈菜红碱所取消（P<0.05或P<0.01）。结论： RSVL可能通过PKC/HSP70通路产生对心肌细胞缺氧/复氧损伤的保护作用。

Protective effects of resveratrol on cardiomyocytes against anoxia/reoxygenation injury through a PKC/HSP70 pathway

OBJECTIVE To investigate the effects of resveratrol (RSVL) on cardiomyocytes during anoxia/reoxygenation injury (ARI) and its possible mechanism.METHODS Primary cardiomyocytes were isolated and purified from neonatal SD rats, which randomly divided into control group, ARI group, ARI+ RSVL (25, 50, 75 μmol·L^-1) pretreatment group, ARI +RSVL (75 μmol·L^-1) + chelerythrine (1 μmol·L^-1) pretreatment group. The cell viability was observed by inverted microscope. Apoptotic rates in cardiomyocytes were calculated by Hoechst 33258 staining. The leakage of lactate dehydrogenase (LDH) and creatine kinase (CK) in the cell culture supernatant, and the activity of Ca²⁺-ATPase in cardiomyocytes were measured by colorimetric method. The free calcium in cardiomyocytes was determined by fluorescence spectrophotometer. The protein expression of PKC, HSP70 and Ca²⁺-ATPase in cardiomyocytes were detected by Western blot.RESULTS Compared with ARI group, resveratrol resulted in a dose dependent increase in the cell viability and beating frequency (P<0.01). Resveratrol dose dependently decreased the cardiomyocyte apoptosis (P<0.01), the leakage of LDH and CK in the cell culture supernatant (P<0.05 or P<0.01) and the free calcium in cardiomyocytes (P<0.05 or P<0.01), and increased the activity of Ca²⁺-ATPase in cardiomyocytes (P<0.05 or P<0.01). The above data changes were accompanied with the increase of protein levels of PKC, HSP70 and Ca²⁺-ATPase in cardiomyocytes (P<0.05 or P<0.01). However, the protective effects of resveratrol was abolished by chelerythrine (P<0.05 or P<0.01).CONCLUSION Resveratrol may protect cardiomyocytes against anoxia/reoxygenation injury through a PKC/HSP70 pathway.