心康颗粒调节LncRNA GAS5/miR-21抗慢性心力衰竭患者心肌纤维化和自噬及其安全性研究

Research on the Regulation of LncRNA GAS5/miR-21 by Xinkang Granule(心康颗粒)in Anti-fibrosis and Autophagy of Patients with Chronic Heart Failure and Its Safety

目的 探讨心康颗粒对慢性心力衰竭患者心肌纤维化及自噬的作用及其安全性。方法 根据纳入排除标准选取60例慢性心力衰竭患者,分为基础组、阳性对照组、治疗组,每组20例。基础组予西医基础治疗,治疗组在基础治疗上给予心康颗粒口服,阳性对照组在基础治疗上给予芪苈强心胶囊口服,共治疗8周。采用荧光实时定量PCR(quantitative real-time PCR,qRT-PCR)法检测全血长链非编码RNA生长阻滞特异转录物5(LncRNA growth arrest-specific transcript 5,LncRNA GAS5)、微小RNA-21(microRNA-21,miR-21)基因表达,酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法检测血浆B型钠尿肽(brain natriuretic peptide,BNP)、基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)、基质金属蛋白酶抑制剂-1(tissue inhibitor of metalloproteinase-1,TIMP1)、溶酶体关联膜蛋白2(human lysosomal associated membrane protein 2,LAMP2)及微管相关蛋白轻链3Ⅱ(human microtubule-associated protein light chain 3Ⅱ,LC3Ⅱ)水平,超声心动图检测左室射血分数(left ventricular ejection fractions,LVEF),六分钟步行距离(six-minute walk distance,6MWD)评估患者活动耐量,生化法检测安全性指标谷丙转氨酶(alanine aminotransferase,ALT)、肌酐(creatinine,Cre)、心肌肌钙蛋白Ⅰ(cardiac troponin I,cTnI)、肌酸激酶同工酶(creatine kinase isoenzyme,CK-MB)水平。结果 与基础组比较,治疗组能显著降低心力衰竭患者BNP、LAMP2、LC3Ⅱ、MMP-2、ALT、Cre、cTnI、CK-MB水平,增加LncRNA GAS5表达,抑制miR-21表达,提高TIMP1、LVEF、6MWD水平,P<0.05。与对照组比较,治疗组能明显提高TIMP1,降低miR-21表达及BNP水平,P<0.05;但在改善MMP-2、LAMP2、LC3Ⅱ、LVEF、6MWD、ALT、Cre、CK-MB、cTnI水平和LncRNA GAS5表达比较差异无统计学意义,P>0.05。LncRNA GAS5与LAMP2、LC3Ⅱ、MMP-2、TIMP1、BNP呈负相关,与6MWD、LVEF呈正相关。MiR-21与LAMP2、LC3Ⅱ、MMP-2、TIMP1、BNP呈正相关,与6MWD、LVEF呈负相关。结论 心康颗粒可能通过调节LncRNA GAS5/miR-21表达,降低心力衰竭患者自噬反应,调节细胞外基质MMP-2与TIMP1平衡,减轻心肌纤维化且安全有效。

Objective To investigate the effect and safety of Xinkang Granule(心康颗粒)on myocardial fibrosis and autophagy in patients with chronic heart failure.Methods Sixty patients with chronic heart failure were selected according to the inclusion and exclusion criteria and divided into basic group,positive control group,and treatment group,with 20 cases in each group.The basic group received Western medicine basic treatment,the treatment group received oral Xinkang granule on the basis of basic treatment,and the positive control group received oral Qili Qiangxin Capsules(芪苈强心胶囊)on the basis of basic treatment.The treatment lasted for 8 weeks.Quantitative Real-time PCR(qRT-PCR)was used to detect the gene expression of LncRNA growth arrest-specific transcript 5(LncRNA GAS5)and microRNA-21(miR-21)in whole blood.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of plasma brain natriuretic peptide(BNP),matrix metallo proteinase 2(MMP-2),tissue inhibitor of metallo proteinase-1(TIMP1),lysosomal associated membrane protein 2(LAMP2),and microtubule-associated protein light chain 3Ⅱ(LC3Ⅱ).Echocardiography was used to determine the left ventricular ejection fraction(LVEF),and the six-minute walk distance(6MWD)test was used to assess patients'activity tolerance.Biochemical method was used to detect safety indicators such as alanine aminotransferase(ALT),creatinine(Cre),myocardial troponin I(cTnI),and creatine kinase isoenzyme(CK-MB).Results Compared with the basic group,the treatment group could significantly reduce BNP,LAMP2,LC3Ⅱ,MMP-2,ALT,Cre,cTnI,CK-MB,increase LncRNA GAS5,inhibit miR-21,increase TIMP1,LVEF,and 6MWD,P<0.05.Compared with the positive control group,the treatment group could significantly increase TIMP1 and BNP,reduce miR-21,P<0.05,but there was no difference in improving MMP-2,LAMP2,LC3Ⅱ,LVEF,6MWD,LncRNA GAS5,ALT,Cre,CK-MB,cTnI,P>0.05.LncRNA GAS5 showed a negative correlation with LAMP2,LC3Ⅱ,MMP-2,TIMP1,BNP,and a positive correlation with 6MWD and LVEF.MiR-21 showed a positive correlation with LAMP2,LC3Ⅱ,MMP-2,TIMP1,BNP,and a negative correlation with 6MWD and LVEF.Conclusion Xinkang Granule may reduce autophagy response of patients with heart failure,regulate the balance of extracellular matrix MMP-2 and TIMP1,alleviate myocardial fibrosis and be safe and effective by regulating the expression of LncRNA GAS5/miR-21.