Exploring Anti-Myocardial Fibrosis Mechanism of Linggui Qihua Formula(苓桂气化方)Based on miRNA21-5p Regulating TGF-β1/Smads Pathway
Objective To explore the anti-myocardial fibrosis mechanism of Linggui Qihua Formula(芩桂气化方,LGQH)based on miRNA21-5p regulating transforming growth factor-β1(TGF-β1)/Smads signaling pathway.Methods Forty 4-week-old spontaneously hypertensive rats(SHR)were equally divided into the heart failure with preserved ejection fraction(HF-pEF)group,sacubitril/valsartan group(LCZ696,0.018 g·kg-1),low dose LGQH group(LGQH-L,3.87 g·kg-1)and high dose LGQH group(LGQH-H,7.74 g·kg-1).HFpEF rat model was established by feeding with the high-fat-salt-sugar di-et for 16 weeks and intraperitoneal injection of streptozotocin solution for 8 weeks.10 Wistar Kyoto(WKY)rats and 10 SHRs were used as the control group and were given the regular feed until the end of the experiment.After successful molding,the WKY,SHR and HFpEF groups were given the equal dose of saline,and the other three groups followed the pre-specified inter-vention by gavage once a day for 6 weeks.After the intervention,echocardiography was performed to measure left ventricle(LV)end-diastolic anterior wall thickness(LVAWd),LV end-diastolic posterior wall thickness(LVPWd),LV end-diastolic inter-nal diameter(LVIDd),LV ejection fraction(LVEF),diastolic mitral inflow peak velocity(E),late diastolic mitral inflow peak ve-locity(A),early diastolic mitral annular velocity(e')and E/A and E/e'were calculated.ELISA was used to detect serum atrial natriuretic peptide(ANP),B-type brain natriuretic peptide(BNP)and galectin-3(Gal-3).Hematoxylin eosin and Masson staining for pathology was used to visualize myocardial hypertrophy and fibrosis and calculate the LV wall thickness(LVWT),col-lagen volume fraction(CVF)and perivascular fibrosis ratio(PFR).The qPCR method was used to detect the expressions of miR-NA21-5p and mRNA related to TGF-β1/Smads pathway in LV myocardium.Western blot was performed to detect the expres-sion of protein related to TGF-β1/Smads pathway.Results Compared with the control group,the HFpEF group showed signifi-cantly higher levels of LVAWd,LVPWd,LVIDd,E/A,E/e',ANP,BNP,Gal-3,LVWT,CVF and PFR(P＜0.05 or P＜0.01),up-regulated miRNA21-5p expression,mRNA expressions of α-smooth muscle actin(α-SMA),Coll Ⅰ,Coll Ⅲ,TGF-β1,Smad2 and Smad3,and protein expressions of α-SMA,Coll Ⅰ,Coll Ⅲ,TGF-β1 and P-Smad2/3(P＜0.05 or P＜0.01),down-regulated Smad7 mRNA and protein expressions(P＜0.05 or P＜0.01).Compared to the HFpEF group,LGQH dose-dependently reduced the levels of LVAWd,LVPWd,LVIDd,E/A,E/e',ANP,BNP,Gal-3,LVWT,CVF and PFR(P＜0.05 or P＜0.01),down-regulated miRNA21-5p,mRNA expressions of α-SMA,Coll Ⅰ,Coll Ⅲ,TGF-β1,Smad2 and Smad3 and protein expressions of α-SMA,Coll Ⅰ,TGF-β 1 and P-Smad2/3(P＜0.05 or P＜0.01),up-regulated Smad7 mRNA and protein expression(P＜0.05 or P＜0.01).Conclusions LGQH may inhibit myocardial fibrosis by targeting miRNA21-5p to regulate the TGF-β1/Smads signaling pathway,thereby alleviating LV remodeling and diastolic dysfunction in HFpEF rats,and is an effective herbal compound for HFpEF treatment.
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