参附注射液对异丙肾上腺素诱导的心肌细胞损伤模型中SIRT1去乙酰化修饰调控HMGB1/TLR4/NF-κB通路的影响  

Effect of Shenfu Injection (参附注射液) on SIRT1 Deacetylation-modified Regulation of HMGB1/TLR4/NF-κB Pathway in Isoproterenol-induced Cardiomyocyte Injury Model

作　　者：黄淑敏 廖晓倩 范星宇 王梓仪 胡志希[2] HUANG Shumin;LIAO Xiaoqian;FAN Xingyu;WANG Ziyi;HU Zhixi(The Second People's Hospital of Fujian University of Traditional Chinese Medicine,Fuzhou,350003;Hunan University of Chinese Medicine;Guang'anmen Hospital,China Academy of Chinese Medical Sciences)

机构地区：[1]福建中医药大学附属第二人民医院,福建省福州市350003 [2]湖南中医药大学 [3]中国中医科学院广安门医院

出　　处：《中医杂志》2024年第14期1488-1495,共8页Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(82274412,81774208);湖南省自然科学基金(2020JJ4062,2020JJ5408);广东省重点领域研发计划项目(2020B1111100001);湖南省教育厅科学研究项目(21A0230,21B0361);湖南省学位与研究生教育改革研究项目(2020JGZX012)。

摘　　要：目的 从沉默信息调节因子1 (SIRT1)去乙酰化修饰调控高迁移率族蛋白B1 (HMGB1)/Toll样受体4 (TLR4)/核因子κB (NF-κB)通路探讨参附注射液对异丙肾上腺素诱导的大鼠心肌细胞损伤的影响及可能作用机制。方法 通过CCK-8法筛选盐酸异丙肾上腺素最佳干预浓度及干预时间、参附注射液药物最佳干预浓度。取对数生长的H9c2大鼠心肌细胞以5×104个细胞/孔,分为正常组、模型组、参附注射液组、SIRT1抑制剂组,每组3个复孔。除正常组外其余各组细胞采用盐酸异丙肾上腺素诱导建立慢性心力衰竭细胞模型。造模后参附注射液组再加最佳干预浓度的参附注射液处理,SIRT1抑制剂组造模后加1μmol/L的SIRT1抑制剂EX-527处理,时间为筛选的最佳干预时间。采用CCK-8法检测各组细胞活性及计算抑制率;ELISA法检测心肌细胞中烟酰胺腺嘌呤二核苷酸氧化态/烟酰胺腺嘌呤二核苷酸还原态(NAD+/NADH)值;免疫荧光法检测心肌细胞HMGB1及SIRT1免疫荧光定位情况;蛋白免疫印迹法检测心肌细胞乙酰化HMGB1蛋白表达,细胞核、细胞质中HMGB1蛋白表达,心肌细胞中SIRT1、 TLR4、髓样细胞分化因子88(MYD88)、NF-κB p65蛋白表达;RT-qPCR法检测心肌细胞SIRT1、HMGB1、TLR4、MYD88、NF-κB p65mRNA表达。结果 最终选择盐酸异丙肾上腺素最佳干预浓度为300μmol/L,干预时间为48 h;8%为参附注射液最佳干预浓度。与正常组比较,模型组心肌细胞活性、NAD+/NADH值、细胞核HMGB1蛋白表达、心肌细胞SIRT1蛋白及mRNA表达均降低;细胞抑制率、心肌细胞乙酰化HMGB1蛋白、细胞质HMGB1蛋白表达,心肌细胞TLR4、MYD88、NF-κB p65蛋白及mRNA表达,HMGB1 mRNA表达均升高(P<0.05);荧光定位显示,模型组心肌细胞HMGB1含量增加并且定位在细胞核与细胞质中均有体现。与模型组和SIRT1抑制剂组比较,参附注射液组上述各指标均显著改善(P<0.05);荧光定位显示,参附注射液组SObjective To investigate the effect and possible mechanism of Shenfu Injection(参附注射液)on rat cardiomyocyte injury induced by isoproterenol from the perspective of regulating the high mobility group protein B1(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factorκB(NF-κB)pathway through the deacetylation modification of silent information regulator 1(SIRT1).Methods The optimal concentration and intervention duration of isoproterenol hydrochloride and the optimal intervention concentration of Shenfu Injection were screened out by CCK-8 method.Logarithmically growing H9c2 rat cardiomyocytes were taken at 5×104 cells/well and divided into normal group,model group,Shenfu Injection group,and SIRT1 inhibitor group,with 3 replicates in each group.Except for the normal group,the cells in the other groups were induced by isoproterenol hydrochloride to establish a chronic heart failure cell model.After modeling,the Shenfu Injection group was given Shenfu Injection at the optimal intervention concentration,and the SIRT1 inhibitor group was given 1μmol/L of SIRT1 inhibitor EX-527,for optimal intervention duration.CCK-8 assay was used to detect the cell activity and calculate the inhibitory rate.ELISA assay was used to detect the nicotinamide adenine dinucleotide oxidation state/nicotinamide adenine dinucleotide reduction state(NAD+/NADH)in cardiomyocytes.Immunofluorescence was used to detect the immunofluorescence localization of HMGB1 and SIRT1 in cardiomyocytes.Western blotting was used to detect the protein expression of acetylated HMGB1 in cardiomyocytes,HMGB1 in the nucleus and cytoplasm,and SIRT1,TLR4,myeloid differentiation factor 88(MYD88)and NF-κB p65 in cardiomyocytes.RT-qPCR was used to detect the mRNA expression of SIRT1,HMGB1,TLR4,MYD88 and NF-κB p65 in cardiomyocytes.Results The optimal intervention concentration of isoproterenol hydrochloride was 300μmol/L,and the intervention duration was 48 hours;8%was the optimal intervention concentration of Shenfu Injection.Compared to those in the normal group,

关 键 词：慢性心力衰竭 心肌细胞 参附注射液 沉默信息调节因子1 去乙酰化 高迁移率族蛋白B1 

分 类 号：R285.5[医药卫生—中药学]