冬凌草甲素减轻自噬对阿霉素诱导心肌细胞凋亡的保护作用

Protective effects of oridonin on adriamycin-induced myocardial apoptosis through inhibiting autophagy

目的探究冬凌草甲素对阿霉素诱导心肌细胞凋亡的作用及机制。方法将细胞随机分为H9C2组、Adriamycin组、Oridonin(5μM)组、Oridonin(10μM)组和Oridonin(20μM)组,除H9C2组外,其余组用阿霉素处理,Oridonin(5,10,20μM)组同时给予相应浓度的冬凌草甲素,试剂盒检测超氧化物歧化酶(SOD)和丙二醛(MDA)浓度,CCK8检测细胞增殖,流式细胞术检测细胞凋亡,Western blot检测自噬相关蛋白Beclin1、P62和LC3的表达,免疫荧光检测LC3表达。结果与H9C2组比较,Adriamycin组SOD浓度明显降低,MDA浓度明显升高;与Adriamycin组比较,Oridonin(5,10,20μM)组SOD浓度明显升高,MDA浓度明显降低;同时,Adriamycin组细胞增殖速度明显低于H9C2组,细胞凋亡率明显增高; Oridonin(5,10,20μM)组细胞增殖速度与Adriamycin组比较明显升高,细胞凋亡率明显降低;此外,阿霉素能显著升高Beclin1表达水平和LC3Ⅱ/LC3Ⅰ的比值,抑制P62表达;冬凌草甲素(5,10,20μM)能显著减弱阿霉素对Beclin1、LC3Ⅱ/LC3Ⅰ和P62表达的调控作用。结论冬凌草甲素能通过抑制自噬减轻阿霉素诱导的心肌细胞凋亡。

Objective To investigate the effects and mechanisms of oridonin on adrimycin-induced myocardial apoptosis. Methods Cells were divided into H9 C2,adriamycin,oridonin（ 5 μM）,oridonin（ 10 μM） and oridonin（ 20 μM） group. Cells were treated with adriamycin except H9 C2 group and cells in oridonin（ 5,10,20 μM） group were treated with oridonin at the same time. The concentrations of superoxide dismutase（ SOD） and malondialdehyde（ MDA） were detected and the cell proliferation was detected by CCK8 assay. Cell apoptosis was determined by flow cytometry. The expressions of autophagy-related proteins（ Beclin1,P62 and LC3） were measured by western blot. And the expression of LC3 also detected by immunofluoresence. Results Compared with H9 C2 group,the concentration of SOD decreased and MDA increased in adriamycin group; compared with adriamycin group,SOD increased but MDA decreased in oridonin（ 5,10,20 μM） group significantly. Meanwhile,cell proliferation was inhibited and apoptosis was induced in adriamycin group compared with H9 C2 group; but cell proliferation rate was increased and apoptosis rate was decreased in oridonin（ 5,10,20 μM） group compared with adriamycin group. In addition,adriamycin up-regulated the protein level of Beclin1 and ratio of LC3Ⅱ/LC3Ⅰ,and inhibited the expression of P62; oridonin（ 5,10,20 μM）attenuated the effects of adriamycin on Beclin1,ratio of LC3Ⅱ/LC3Ⅰ and P62 notably. Conclusion Oridonin alleviated adriamycin-indued myocardial apoptosis by inhibiting autophagy.