目的：在细胞水平研究中药提取物小檗碱(BBR)对多柔比星(DOX)引起的心肌细胞损伤的影响,并探索该过程中可能的机制。方法：培养H9C2大鼠心肌细胞,将DOX分为不同的浓度,确定DOX的造模浓度,再分组为正常对照组、DOX模型组、治疗组(低剂量BBR、中剂量BBR、高剂量BBR),来处理H9C2细胞。通过倒置显微镜观察细胞生长趋势和细胞形态,使用细胞计数盒(CCK8)法测定心肌细胞活力,用流式细胞仪检测细胞凋亡,蛋白免疫印迹法(Western-blot)测定不同组凋亡、自噬相关蛋白Bax、Bcl-2、Beclin1、P62的相对表达水平。结果：显微镜下发现随着DOX的浓度增大,心肌细胞损伤程度依次增加。通过CCK8法选取1 μmol/L DOX为心肌损伤的最适造模浓度,与对照组相比,治疗组不同浓度BBR均可提高DOX诱导的心肌损伤细胞活力;其中治疗组的中剂量促凋亡蛋白Bax和自噬相关蛋白Beclin1的蛋白表达量明显下降,抗凋亡蛋白Bcl-2和自噬相关蛋白P62的蛋白表达量升高(P＜0.05)。结论：小檗碱可以通过减少细胞凋亡保护DOX诱导的心肌细胞损伤,这种保护机制和细胞自噬有相关性。
Objective: To investigate the effect of berberine (BBR),a traditional Chinese medicine extract,on doxorubicin (DOX) induced myocardial cell damage at the cellular level,and explore the possible mechanisms involved in this process. Methods: H9C2 rat cardiomyocytes were cultured,and DOX was divided into different concentrations to determine the concentration of DOX.Then they were divided into normal control group,DOX model group and treatment group (low-dose BBR,medium-dose BBR,high-dose BBR) to treat H9C2 cells. The cell growth trend and cell morphology were observed by inverted microscope. The cell viability was determined by cell counting kit (CCK8) method. The apoptosis was detected by flow cytometry. The relative expression levels of apoptosis and autophagy related proteins Bax,Bcl-2,Beclin1 and P62 in different groups were determined by Western-blot. Results: Under the microscope,it was found that with the increase of DOX concentration,the degree of myocardial cell injury increased in turn. CCK8 method was used to select 1 μmol/L DOX as the optimal modeling concentration for myocardial injury,compared with the control group,different concentrations of BBR in the treatment group could increase the viability of DOX-induced myocardial injury cells. The expression of pro-apoptotic protein Bax and autophagy-related protein Beclin1 in the middle dose of the treatment group was significantly decreased,and the expression of anti-apoptotic protein Bcl-2 and autophagy-related protein P62 was increased (P＜0.05). Conclusion: Berberine can protect DOX-induced cardiomyocyte injury by reducing apoptosis,and this protective mechanism is related to autophagy.
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