Effects of Zhigancao Decoction on atrial remodeling in atrial fibrillation rat model via modulating the miR-26b-5p/SMAD4 pathway

LIU Kuizhi; XUAN Xuexi; ZHOU Peng; YUAN Xiaowei; ZHU Ziqiang

doi:10.11958/20252534

Abstract：Objective To explore the effect of Zhigancao Decoction on atrial remodeling and the microrNA(miR)-26b-5p/maternal DPP homologous product(SMAD)4 pathway in rat model of atrial fibrillation(AF).Methods The AF rat model was constructed.The successfully modeled rats were randomly divided into the AF group,the verapamil group,the low-dose Zhigancao Decoction(Zhigancao Decoction-L)group,the high-dose Zhigancao Decoction(Zhigancao Decoction-H)group,the Zhigancao Decoction-H+anti-NC group and the Zhigancao Decoction-H+miR-26b-5p inhibitor(anti-miR-26b-5p)group,with 18 rats in each group.Another 18 healthy rats were taken as the control group.Cardiac function of rats and induction rate of AF in vitro were detected in each group.Hematoxylin-eosin(HE)staining and Masson staining were used to observe the pathological changes of atrial tissue.TdT-mediated dUTP nick-end labeling(TUNEL)staining was used to observe cardiomyocyte apoptosis in atrial tissue.Immunohistochemistry was used to detect expressions of type Ⅰ collagen(ColⅠ)and α-smooth muscle actin(α-SMA)in atrial tissue.qRT-PCR was used to explore the relative expression level of miR-26b-5p.Western blot assay was used to detect the expression levels of SMAD4,BCL-2 associated X protein(BAX)and activated caspase-3(C-caspase3).Dual-luciferase assay was performed to verify the targeting relationship between miR-26b-5p and SMAD4.Results Compared with the control group,the atrial tissue in the AF group exhibited severe pathological damage,including abnormal myocardial cell structure,swelling,sparse and disordered arrangement,and significant blue collagen deposition(P＜0.05).In cardiac function indexes of AF group,left ventricular end-systolic diameter(LVESD)and left ventricular end-diastolic diameter(LVEDD)were increased,while left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)were decreased(P＜0.05).Furthermore,the induction rate of AF,collagen area percentage and cell apoptosis rate of atrial fibrillation were increased.The protein expression levels of BAX,C-caspase3,ColⅠ,α-SMA and SMAD4 were enhanced,and the level of miR-26b-5p was decreased(P＜0.05).Compared with the AF group,treatment with verapamil and Zhigancao Decoction reversed the trends of the above indicators,alleviated the pathological damage in atrial tissue and reduced blue collagen deposition.Zhigancao Decoction-H+anti-miR-26b-5p reversed the ameliorative effects of Zhigancao Decoction-H on atrial remodeling in the AF rat model.Starbase website and the dual-luciferase gene report indicated a targeting relationship between miR-26b-5p and SMAD4.Conclusion Zhigancao Decoction may improve atrial remodeling in AF rats by up-regulating miR-26b-5p and thereby inhibiting the expression of SMAD4.