沙库巴曲缬沙坦钠通过Nrf2/HO-1信号通路缓解心力衰竭的机制研究

Mechanistic study of Sacubitril/Valsartan in alleviating heart failure via the Nrf2/HO-1 signaling pathway

摘要:

目的 探究沙库巴曲缬沙坦钠通过Nrf2/HO-1信号通路改善心力衰竭（HF）细胞和动物模型的分子机制。方法 通过盐酸阿霉素（DOX）处理H9C2细胞后制备HF细胞模型，然后分别给予沙库巴曲缬沙坦钠（LCZ696）和Nrf2抑制剂（ML385）治疗。将实验细胞分为对照组、DOX组、DOX+ LCZ696组、DOX+ ML385组、DOX+ LCZ696+ ML385组。用DOX诱导HF模型小鼠，并给予LCZ696和ML385治疗，又分为假手术组、DOX组、DOX+ LCZ696组、DOX+ LCZ696+ ML385组。CCK-8检测细胞活力；流式细胞术检测细胞凋亡；采用酶联免疫吸附试验（ELISA）检测细胞ROS、MDA、SOD水平，以及炎症因子（TNF-α、IL-1β、IL-6）蛋白浓度；Western blotting检测各组细胞和小鼠Bax、Bcl-2、C-Caspase-3、Nrf2和HO-1蛋白相对表达量。观察各组小鼠心脏组织HE、Masson和TUNEL染色。结果 与对照组比较，DOX组细胞活力降低（P <0.05），细胞凋亡率升高（P <0.05），炎症因子、ROS、MDA水平和促凋亡蛋白表达均升高（P <0.05），SOD水平、Nrf2和HO-1蛋白表达均降低（P <0.05）；与DOX组比较，DOX+ LCZ696组抑制了DOX造成的细胞损伤，而DOX + ML385组加重了细胞损伤程度；与DOX+ LCZ696组比较，DOX + LCZ696 + ML385组细胞损伤更为严重。而与DOX+ ML385组比较，DOX+ LCZ696+ ML385组细胞损伤减轻。与假手术组比较，DOX诱导小鼠心脏组织损伤严重，DOX + LCZ696组则缓解了DOX造成的损伤，DOX + LCZ696+ ML385组损伤加重。结论 沙库巴曲缬沙坦钠通过Nrf2/HO-1信号通路可缓解DOX诱导的心肌细胞损伤和小鼠心脏组织损伤。

Abstract:

Objective To explore the molecular mechanism of Sacubitril/Valsartan Sodium improving heart failure cell model and animal model through Nrf2/HO-1 signaling pathway.Methods H9C2 cells were treated with Doxorubicin hydrochloride (DOX) to construct a heart failure cell model, and then treated with Sacubitril/Valsartan Sodium (LCZ696) and Nrf2 inhibitor (ML385), respectively. They were divided into five groups: Control, DOX, DOX + LCZ696, DOX + ML385, DOX + LCZ696 + ML385. Mice model of heart failure was induced by DOX and treated with LCZ696 and ML385, which were divided into sham operation group, DOX group, DOX + LCZ696 group, and DOX + LCZ696 + ML385 group. Cell viability and apoptosis was analyzed by CCK-8 and flow cytometry. ROS, MDA and SOD levels were detected with the kit. The levels of TNF-α, IL-1β and IL-6 were detected by ELISA. Bax, Bcl-2, C-caspase-3, Nrf2 and HO-1 proteins was detected via Western blotting. Heart tissue of mice with heart failure was stained by HE, Masson and TUNEL.Results Compared with control group, DOX-induced cell viability was significantly decreased (P < 0.05); cell apoptosis ratio was increased (P < 0.05); the levels of inflammatory factors, ROS, MDA, and pro-apoptotic proteins were increased (P < 0.05); SOD level, Nrf2, and HO-1 proteins were decreased (P < 0.05). Compared with DOX group, DOX + LCZ696 group significantly inhibited cell damage caused by DOX, while DOX + ML385 group significantly aggravated cell damage. Compared with DOX + LCZ696 group, the cell injury was more severe in DOX + LCZ696 + ML385 group, while the cell injury was reduced in DOX + LCZ696 + ML385 group. Compared with the sham group, the heart tissue damage of mice induced by DOX was serious, the damage caused by DOX was alleviated in DOX + LCZ696 group, and the damage was aggravated in DOX + LCZ696 + ML385 group.Conclusion Sacubitril/Valsartan sodium alleviates DOX-induced myocardial cell injury through Nrf2/HO-1 signaling pathway.