温阳通脉方经内质网应激相关通路对H9C2心肌细胞缺氧/复氧损伤的作用

The effect of Wenyangtongmaifang on endoplasmic reticulum stress-related pathway in hypoxia/reoxygenation injury of H9C2 cardiomyocytes

目的:观察温阳通脉方含药血清是否在内质网应激相关通路对心肌细胞缺氧/复氧损伤的作用,并探究体外培养时最佳给药剂量。方法:以20%含药血清处理,用四甲基偶氮唑盐(MTT)法测得CoCl_2烫最佳浓度和各组细胞存活率;比色法检测各组半胱氨酸蛋白酶3(Caspase3)酶活性和上清液中乳酸脱氢酶(LDH)活性;Hoechst 33342荧光染色观察各组细胞凋亡形态变化;流式细胞术检测各组细胞凋亡率;Western Blot法检测各组内质网应激相关蛋白GRP78、Caspase12、CHOP表达以及凋亡相关蛋白Bcl-2、Bax表达情况。结果:与空白对照组血清相比,模型组细胞活性明显降低,Caspase3酶活性和LDH活性明显升高,细胞凋亡数量明显增多,凋亡率明显升高, GRP78、Caspase12、CHOP和Bax表达明显增多,Bcl-2表达明显减少;与模型组相比,3.24、6.48、12.96g/kg组含药血清能够不同程度改善细胞活性,Caspase3酶活性和LDH活性明显减少,凋亡率不同程度降低,GRP78、Caspase12、CHOP和Bax表达明显减少,Bcl-2表达不同程度增多。结论:温阳通脉方能减轻缺氧/复氧后H9C2心肌细胞损伤,其机制可能与温阳通脉方抵抗内质网应激介导的细胞凋亡有关,其抗凋亡程度呈剂量依赖性,以温阳通脉方12.96g/kg组效果最佳。

Objective: To observe the effect of Wenyangtongmaifang and its drug-containing serum on the hypoxia/reoxygenation injury of cardiomyocytes induced by endoplasmic reticulum stress-related pathways,and to explore the optimal dose for in vitro culture. Methods: The cells in different groups were treated with 20% blank serum or 20% drug-containing serum respectively. The optimal concentration of CoC l2 was determined and the cell viability in each group was measured by MTT. Caspase3 enzyme activity and supernatant LDH activity was determined by the colorimetric method. Morphological changes of apoptosis were observed by Hoechst 33342 fluorescence staining. Apoptosis rate was detected by flow cytometry. The expressions of GRP78,Caspase12,CHOP,Bcl-2 and Bax were detected by Western Blot. Results: Compared with the blank control group,the survival rates in the model group was significantly reduced,the levels of Caspase3 and LDH were significantly increased,the number of apoptotic cells was significantly increased and the apoptosis rate was increased. The expressions of GRP78,Caspase12,CHOP and Bax were significantly up-regulated while Bcl-2 was significantly down-regulated. Compared with the model group,the levels of Caspase3 and LDH,and the rates of apoptosis were decreased in all doses of Wenyangtongmaifang groups,the expressions of GRP78,Caspase12,CHOP and Bax were decreased while Bcl-2 was increased. Conclusion: Wenyangtongmaifang can alleviate H9 C2 cells injury after hypoxia/reoxygenation,and its mechanism may be related to endoplasmic reticulum stress-mediated apoptosis. Its anti-apoptotic effect is dose-dependent and the 12. 96 g/kg is suggested the optimal dose.