To Explore The Effect Of Ershen Zhenwu Decoction On Myocardial Fibrosis Of Chronic Heart Failure Rats With Heart-kidney Yang Deficiency Based On TGF-β1/Smads And PI3K/AKT/mTOR Signaling Pathway

Chronic heart failure is a cardiovascular disease with a high incidence in middle-aged and elderly patients.Chronic heart failure with heart-kidney yang deficiency is a type of CHF frequently witnessed by kidney yang deficiency.Ventricular remodeling is the pathological mechanism of chronic heart failure.The basis of ventricular remodeling is myocardial fibrosis.Er Shen Zhen Wu Decoction(ESZWD)is based on the ancient classical formula Zhen Wu Tang with the addition of two herbs,red ginseng and salvia,based on many years of clinical experience by Cheng Xiaoyu,director of the First Affiliated Hospital of Ancient Chinese Medicine.It is clinically effective against CHF-HKYd.Based on the previous studies,the material basis of ESZWD’s efficacy has been clarified,but the mechanism of action is still unclear.In this paper,we investigate the mechanism of ESZWD’s action on CHF-HKYd based on the previous studies.Aim:(1)Investigating the pharmacological efficacy of ESZWD on CHF-HKYd myocardial fibrosis.(2)Investigating the therapeutic effects of ESZWD on cardiac fibrosis cell models based on TGF-β1/Smads and PI3K/AKT/mTOR pathways.(3)To investigate the therapeutic effects of ESZWD on myocardial fibrosis in CHF rats with heart-kidney yang deficiency based on TGF-β1/Smads and PI3K/AKT/mTOR pathways.Methods: For animals,60 rats were divided equally into six groups,normal group,model group,ESZWD low,medium and high dose(3.96,7.92,15.84 g/kg)group and positive drug benazepril(0.5 mg/kg)group,and established by bilateral thyroidectomy combined with Adriamycin(0.02% Adriamycin 1 m L/100 g intraperitoneal injection twice a week)injection CHF-HKYd rat model.The rat’s cardiac function was measured and blood was taken from the eye socket to test the levels of amino-terminal pro brain natriuretic peptide(NT-pro BNP),triiodothyronine(T3)and thyroxine(T4)to validate the model.Four weeks after gavage of ESZWD,the heart tissue was stained with HE,Masson and immunohistochemically labelled with α-SMA to detect the degree of fibrosis,and Western blot to detect the protein levels and m RNA content of collagen I,collagen III and α-SMA in the heart tissue;In terms of cells,primary cardiac fibroblasts are extracted from mammary rats,stained with Taipan Blue for cell activity and immunofluorescence for waveform proteins to verify cell purity,and then used for subsequent experiments using 2 to 3 generations of cells.Cells were stimulated using medium containing different concentrations of Ang II(0.1 μmol/L,1 μmol/L,10μmol/L),and CCK-8 determined the timing and concentration of angiotensin II(Ang II)stimulation in primary cardiac fibroblasts to establish a fibrotic cell model.Thirty rats were divided into three groups and gavaged with saline,ESZWD(15.84 g/kg)and Benadryl(0.5 mg/kg)once a day for 7 days,and blood was taken 1 hour after the last gavage to prepare serum.Cells were divided into four groups,normal group,model group(1μmol/L Ang II stimulation for 24h),ESZWD group and benazepril group.Detection of intracellular collagen I,collagen III,α-SMA protein levels and m RNA content.Finally,TGF-β1/Smads and PI3K/AKT/mTOR pathway-related protein levels and m RNA content were measured in rat heart tissues and primary cardiac fibroblasts,and TGF-β1 and PI3 K expression was detected by immunofluorescence in rat heart tissues and primary cardiac fibroblasts.Results:(1)ESZWD significantly inhibited the decline in cardiac function in rats with chronic heart failure of the heart-kidney-yang deficiency type.(2)ESZWD effectively attenuates cardiac injury in CHF-HKYd rats.(3)ESZWD effectively attenuated myocardial fibrosis in CHF-HKYd rats.(4)ESZWD-containing serum ameliorates Ang II-induced cellular fibrosis.(5)ESZWD can improve CHF-HKYd by inhibiting TGF-β1/Smads and PI3K/AKT/mTOR signaling pathways to attenuate MF.Conclusion: ESZWD can regulate TGF-β1/Smads and PI3K/AKT/mTOR signaling pathways to reduce collagen production,improve cellular autophagy,attenuate myocardial fibrosis and inhibit ventricular remodeling,ultimately improving chronic heart failure in the heart-kidney-yang deficiency type.