黄芪总黄酮通过网腔钙结合蛋白调控线粒体钙循环及内质网应激介导CVB3感染心肌细胞保护作用研究

The protective effect of total flavonoids of Astragalus on the mitochondrial calcium circu-lation and ER stress caused by CVB3 injured cardiomyocytes through Calumenin protein

目的研究黄芪总黄酮对CVB3感染心肌细胞凋亡、网腔钙结合蛋白(calumenin)、GRP78、chop表达、线粒体膜电位(ΔΦm)及线粒体通透性转换孔(MPTP)作用。方法乳鼠心肌细胞分离后分为空白组、模型组和黄芪总黄酮治疗组,每组设计3个复孔。模型组和治疗分别以108PFU/ml CVB3感染细胞。同时治疗组加入20mg·L^-1 TFA处理细胞3天.采用Western blot检测各组心肌细胞calumenin、GRP78、chop表达,采用钙黄绿素(calcein)-AM绿色荧光染色测定各组心肌细胞线粒体通透性转换孔(MPTP)开放情况,采用Rho123检测各组心肌细胞线粒体膜电位(ΔΦm),采用TUNEL检测各组心肌细胞凋亡。结果与CVB3感染心肌细胞比较,黄芪总黄酮组心肌细胞ΔΦm及MPTP发生明显改变(P<0.01);同时,calumenin表达增加,GRP78、chop表达减少;细胞凋亡减少。结论黄芪总黄酮明显减少CVB3感染心肌细胞凋亡,该作用可能通过增加calumenin表达,改善线粒体钙循环及缓解内质网应激所介导的。

Objective To research the effect of total flavonoids of Astragalus on apoptosis, the expression of calumenin, GRP78 and chop, mitochondrial membrane potential(ΔΦm) and mitochondrial permeability transition pore(MPTP) in CVB3 infected cardiomyocytes. Methods Cardiomyocyte was isolated from neonatal mice, and divided three group: control, model and treatment group. Each group was designed with three compound experiment. The model group and treatment group were infected with 108 PFU/ml CVB3. Then the cardiomyocytes from treatment group were treatment with 20 mg·L-1 TFA for 24 h. The expression of calumenin,GRP78 and chop was detected by western blotting, MPTP was analyzed by calcein-AM green fluorescence staining and ΔΦm was identified by Rho123 staining. Results Compared with model group, ΔΦm and MPTP was declined(P<0.01), the expression of calumenin was up-regulated and GRP78 and chop was down-regulated, and apoptosis was decreased in cardiomyocytes treatment with total flavonoids of Astragalus. Conclusion Total flavonoids of Astragalus could significantly reduce apoptosis by CVB3, the mechanism may improve mitochondrial calcium circulation and ER stress by calumenin protein.