苓桂术甘汤对心肌梗死后小鼠心肌纤维化及Lats1/Yap信号通路的影响

Effect of Linggui Zhugan Decoction on myocardial fibrosis and Lats1/Yap signaling pathway in mice after myocardial infraction

观察苓桂术甘汤对心肌梗死(MI)后小鼠心肌纤维化(MF)及心肌组织大肿瘤抑制激酶1(Lats1)/Yes相关蛋白(Yap)信号通路的影响,探讨其抑制MF的作用和机制。采用冠状动脉左前降支结扎法构建MI后小鼠缺血模型,造模24 h后,连续干预6周。采用小动物超声成像系统检测小鼠左室射血分数(LVEF)、左室短轴缩短率(LVFS)、左室收缩末期内径(LVIDs)、左室舒张末期内径(LVIDd);采用HE及Masson染色观察小鼠心肌组织病理学变化;采用ELISA法检测小鼠血清肌酸激酶同工酶MB(CK-MB)、乳酸脱氢酶(LDH)水平;采用RT-qPCR法检测小鼠心肌组织Lats1、Yap、结缔组织生长因子(CTGF)mRNA水平;采用Western blot法检测小鼠心肌组织α-平滑肌肌动蛋白(α-SMA)、胶原蛋白Ⅰ(ColⅠ)、胶原蛋白Ⅲ(ColⅢ)、基质金属蛋白酶抑制剂1(TIMP1)、基质金属蛋白酶2(MMP2)、Lats1、Yap、磷酸化Yap(phosphorylation-Yap,p-Yap)、细胞核Yap(n-Yap)蛋白表达。与假手术组比较,模型组小鼠LVEF、LVFS水平显著降低,LVIDd、LVIDs水平显著升高(P<0.01);心肌细胞排列紊乱,心肌纤维部分断裂及胶原纤维大量沉积;小鼠血清CK-MB、LDH水平显著升高(P<0.01);心肌组织Lats1 mRNA水平显著降低(P<0.01),Yap、CTGF mRNA水平显著升高(P<0.01);心肌组织α-SMA、ColⅠ、ColⅢ、MMP2、Yap、n-Yap蛋白表达显著升高(P<0.01),Lats1、TIMP1、p-Yap蛋白表达及p-Yap/Yap比值显著降低(P<0.01)。与模型组比较,经高剂量苓桂术甘汤干预后,小鼠LVEF、LVFS水平显著升高,LVIDd、LVIDs水平显著降低(P<0.01);心肌细胞排列较整齐,心肌纤维断裂及胶原纤维沉积明显减少;小鼠血清CK-MB、LDH水平显著降低(P<0.01);心肌组织Lats1 mRNA水平显著升高(P<0.01),Yap、CTGF mRNA水平显著降低(P<0.01);心肌组织α-SMA、ColⅠ、ColⅢ、MMP2、Yap、n-Yap蛋白表达显著降低(P<0.01),Lats1、TIMP1、p-Yap蛋白表达及p-Yap/Yap比值显著升高(P<0.01)。苓桂术甘汤能够抑制MI后模型小鼠MF,改善小鼠心功能,此作用与其激活Lats1/Yap信号通路密切相关。

This study aims to investigate the effects of Linggui Zhugan Decoction(LGZGD)on myocardial fibrosis(MF)and the Lats1/Yap signaling pathway in mice after myocardial infarction(MI),exploring its role and mechanism in inhibiting MF.The MIinduced ischemic mouse model was established by left anterior descending coronary artery ligation,followed by continuous intervention for six weeks.Doppler ultrasound imaging-system of small animals was used to detect left ventricular ejection fraction(LVEF),left ventricular fractional shortening(LVFS),left ventricular internal diameter at end-systole(LVIDs),and left ventricular internal diameter at end-diastole(LVIDd).Pathological changes in myocardial tissue were observed by HE and Masson staining.Serum levels of creatine kinase isoenzyme MB(CK-MB)and lactate dehydrogenase(LDH)were detected by using ELISA.Myocardial tissue mRNA levels of Lats1,Yap,and connective tissue growth factor(CTGF)were determined by RT-qPCR.Protein expression of alpha-smooth muscle actin(α-SMA),collagenⅠ(ColⅠ),collagenⅢ(ColⅢ),tissue inhibitor of metal protease 1(TIMP1),matrix metallopeptidase 2(MMP2),Yap,p-Yap,and n-Yap was determined by Western blot.Compared with the sham group,the model group showed significantly decreased LVEF and LVFS levels,increased LVIDd and LVIDs levels(P<0.01),disordered arrangement of myocardial cells,partial fracture of myocardial fibers,and massive deposition of collagen fibers.Moreover,serum levels of CK-MB and LDH were significantly increased(P<0.01),while myocardial tissue mRNA levels of Lats1 were significantly decreased(P<0.01),and mRNA levels of Yap and CTGF were significantly increased(P<0.01).Protein expression ofα-SMA,ColⅠ,ColⅢ,MMP2,Yap,and n-Yap was significantly increased(P<0.01),while protein expression of Lats1,TIMP1,p-Yap,and the ratio of p-Yap/Yap were significantly decreased(P<0.01).Compared with the model group,after intervention with LGZGD(9.36 g·kg^(-1)),mice showed significantly increased LVEF and LVFS levels,decreased LVIDd and LVIDs levels(P<0.01),more orderly arrangement of myocardial cells,significantly reduced myocardial fiber fracture and collagen fiber deposition.Serum levels of CK-MB and LDH were significantly decreased(P<0.01),while myocardial tissue mRNA levels of Lats1 were significantly increased(P<0.01),and mRNA levels of Yap and CTGF were significantly decreased(P<0.01).Protein expression ofα-SMA,ColⅠ,ColⅢ,MMP2,Yap,and n-Yap was significantly decreased(P<0.01),while protein expression of Lats1,TIMP1,p-Yap,and the ratio of p-Yap/Yap were significantly increased(P<0.01).LGZGD can inhibit MF in mice after MI and improve mouse cardiac function,which is closely related to the activation of the Lats1/Yap signaling pathway.