基于外泌体miR-30转运探讨益肾解毒法对尿毒症小鼠心肌自噬的作用

Study on the Effect of Yishen Jiedu Method on Myocardial Autophagy in Uremic Mice Based on Exosomal miR-30 Transport

【目的】基于循环外泌体(Exos)miR-30转运探讨益肾解毒法对尿毒症小鼠心肌自噬的作用。【方法】(1)建立5/6肾切除尿毒症小鼠模型,采用益肾解毒法中药复方(YSJD,8.0 g·kg^(-1)·d^(-1))灌胃治疗3周后,通过超声系统评估小鼠心脏功能,采用苏木素-伊红(HE)染色法观察心脏组织病理学形态,Masson三相染色法评估心脏纤维化含量,酶联免疫吸附分析(ELISA)检测血清乳酸脱氢酶(LDH)、肌酸激酶同工酶MB(CK-MB)活性,脱氧核糖核苷酸末端转移酶介导的缺口末端标记(TUNEL)法检测心肌细胞凋亡,免疫组织化学法检测心脏组织中LC3蛋白表达情况,透射电镜(TEM)观察心肌超微结构。(2)分离小鼠外周血Exos,与小鼠原代心肌细胞共培养,采用TUNEL法检测心肌细胞凋亡,免疫荧光染色法检测心肌细胞LC3表达,实时定量聚合酶链反应(RT-qPCR)法检测Exos中miR-30表达,采用Western Blot法检测心肌细胞中自噬相关蛋白表达。【结果】益肾解毒法治疗可明显防止尿毒症心肌病(UCM)诱导的心脏功能障碍、心脏形态变化和重塑,降低血清CK-MB和LDH水平,抑制UCM小鼠心肌细胞凋亡,抑制自噬囊泡形成和自噬标记蛋白LC3的表达。从各组外周血中分离Exos,并通过RT-qPCR证实UCM+YSJD组Exos中miR-30表达较UCM组显著下调(P<0.001)。当在UCM+YSJD-Exos与原代心肌细胞共培养系统中向心肌细胞转染miR-30时,心肌细胞的凋亡率、自噬体形成和自噬相关蛋白的表达均显著增加(P<0.05)。【结论】益肾解毒法可通过干预外周血Exos中miR-30转运抑制UCM诱导的心肌细胞凋亡、自噬,发挥心脏保护作用。

Objective To investigate the effect of Yishen Jiedu method on myocardial autophagy in uremic mice based on exosomal miR-30 transport.Methods(1)A 5/6 nephrectomized uremic mouse model was established and treated by gavage for 3 weeks using the Yishen Jiedu(kidney-nourishing and toxins-releasing method)Chinese herbal compound(YSJD,8.0 g·kg^(-1)·d^(-1)).Cardiac function was assessed by ultrasound system,cardiac histopathological morphology was observed by hematoxylin-eosin(HE)staining,cardiac fibrosis content was assessed by Masson’s three-phase staining,enzyme-linked immunosorbent assay(ELISA)was used to detect serum lactate dehydrogenase(LDH)and creatine kinase isoenzyme MB(CK-MB)activities,deoxyribonucleotide terminal transferase-mediated nick end labeling(TUNEL)assay was used to detect apoptosis in cardiac myocytes,immunohistochemistry staining was used to detect LC3 protein expression in cardiac tissue,and myocardial ultrastructure was observed by transmission electron microscopy(TEM).(2)Peripheral blood Exos was isolated from mice and co-cultured with primary mouse cardiomyocytes.Apoptosis of cardiomyocytes was detected by TUNEL,immunofluorescence staining was used to detect LC3 protein expression in cardiac cells,miR-30 expression in Exos was detected by real-time quantitative polymerase chain reaction(RT-qPCR),and autophagyrelated protein expression in cardiomyocytes was detected by Western Blot.Results Treatment with Yishen Jiedu Method significantly prevented uremic cardiomyopathy(UCM)-induced cardiac dysfunction,cardiac morphological changes and remodelling,reduced serum CK-MB and LDH levels,inhibited apoptosis and suppressed autophagic vesicle formation and expression of autophagy marker protein LC3 in cardiac myocytes of UCM mice.Exos was isolated from the peripheral blood of each group and miR-30 expression in Exos was significantly downregulated in the UCM+YSJD group compared to the UCM group(P<0.001)as confirmed by RT-qPCR.When miR-30 was transfected into cardiomyocytes in the UCM+YSJD-Exos co-culture system with primary cardiomyocytes,apoptosis rate,autophagosome formation and expression of autophagy-related proteins were significantly increased in cardiomyocytes(P<0.05).Conclusion Yishen Jiedu Method can inhibit UCM-induced cardiomyocyte apoptosis and autophagy by interfering with miR-30 transport in peripheral blood Exos and exert cardioprotective effects.