The Research On Mechanism Of Myocardial Fibrosis Of Hypertensive Heart Failure Based On TGF-β/Smad Pathway And Intervention According To "Syndrome Detection By Formula"

Objective:This project try to pinpoint the pathological mechanism of myocardial fibrosis of hypertensive heart failure and the syndrome essence of hypertensive heart failure rat model according to "syndrome detection by formula" method,by administering Shen-Mai injection and Shen-Fu injection on D ah1 salt-sensitive rat model of heart failure,comparative analysizing the effect of intervening myocardial fibrosis and improving cardiac function through the regulation of TGF-β/Smad signaling pathway.Methods:1.50 Dahl/SS rats were randomly divided into blank control group(6 rats)and model group(44 rats),another 10 SS-13 BN salt-sensitive control rats were salt-sensitive control group.The blank control group was given low salt,while salt-sensitive control group and model group were fed a high-salt diet for 20 weeks to establish and validate a salt-sensitive hypertensive heart failure rat model.Then 40 Dahl/SS rats with hypertensive heart failure were randomly divided into model group,Shen-Mai injection group,Shen-Fu injection group and pirfenidone group with 10 rats in each group,administered by intraperitoneal injection combined with intragastric administration for 15 days.After the end of the administration,the EF and FS values of the rats were detected by ultrasound,the serum levels of NT-proBNP and TGF-β were detected by ELISA,the pathological changes of myocardial tissue were observed by HE staining,the changes of myocardial fibrosis were observed by Masson staining,and the content of Collagen type I(Col I)was detected by immunofluorescence staining.Western blot assay detected the expression of TGF-β and Smad2/Smad3 phosphorylation,and the relative expression level of mRNA of Smad2,Smad3,TGF-β and Col I were detected by RT-qPCR.2.30 Dahl/SS rats were randomly divided into blank control group,Shen-Mai injection group and Shen-Fu injection group,all rats were given consecutively intraperitoneal injection of 6.0mL/kg drug for 5 days,once a day,then drug-containing serum was prepared by abdominal aorta blood sampling.The primary rat cardiac fibroblasts were treated by 5% / 10% / 20% Shen-Mai serum / Shen-Fu serum / blank serum,2.5μg/L / 5μg/L / 10μg/L TGF-β1,0.25g/L / 0.5g/L / 1g/L Pirfenidone for 48 h,the dose-effect relationship of drug effects were detected by CCK-8 method,and the concentration and intervention time were determined.The rat cardiac fibroblasts were divided into blank control group,TGF-β1 group,Shen-Mai serum combined with TGF-β1 group,Shen-Fu serum combined with TGF-β1 group,blank serum combined with TGF-β1 group and pirfenidone combined with TGF-β1 group,CCK-8 assay detected the cell inhibitory rate,Western Blot assay detected the Smad2/Smad3 phosphorylation and RT-qPCR detected the expression of mRNA of Smad2 and Smad3.Results:1.After the end of modeling,compared with SS-13 BN rats and rats in the blank control group,the rats in the model group showed significant changes in mental state and behavioral signs,blood pressure continued to rise,and the systolic blood pressure reached 212 mmHg.The EF and FS values of the model group were significantly lower than those of the blank control group and the SS control group,which were also significantly lower than those of the pre-modeling values;the serum NT-proBNP was significantly increased.All indexes of model group were significantly higher than those of the blank control group and the SS control group,indicating that the model group of rats with hypertension heart failure model was initially established.2.After administration,compared with the rats in Shen-Fu group,the EF and FS values of Shen-Mai group were significantly increased;the serum levels of NT-proBNP and TGF-β1 of Shen-Mai group were significantly decreased.In the Shen-Mai group,the structure of cardiomyocytes was clear,the muscle fibers were arranged neatly,and the cells were no pathological changes such as edema;myocardial fibrosis was significantly reduced,collagen volume fraction decreased;myocardial collagen fiber content decreased significantly,and Col I average fluorescence intensity decreased significantly.Shen-Mai injection,Shen-Fu injection and pirfenidone could inhibit the phosphorylation and mRNA expression of Smad2 and Smad3 protein,the expression of TGF-β mRNA and protein,and the expression of Col I mRNA in rat myocardial tissue,while all indicators of Shen-Mai group are better than that of Shen-Fu group.3.TGF-β1,Shen-Mai serum,Shen-Fu serum and blank serum had different degrees of proliferation of rat cardiac fibroblasts,while pirfenidone had cell proliferation inhibition,all drugs had no obvious dose-dependent effect.Determined 10% drug-containing serum,10μg/L TGF-β1,1g/L pirfenidone and 48 h were the optimal concentration and intervention time.After combination of each drug with TGF-β1,the cells in the Shen-Fu combination group and the blank combination group were further proliferated,while cell growth of Shen-Mai combined group and pirfenidone combined group was significantly inhibited,and the cell inhibition rate of Shen-Mai combined group was significantly higher than the other groups.4.The Smad2/Smad3 phosphorylation and the relative expression of mRNA of Smad2 and Smad3 in Shen-Mai combined group were significantly reduced compared with those of blank control group,Shen-Fu combination group,blank combination group and pirfenidone combination group.Conclusion:This study preliminarily confirmed the scientificity and feasibility of the rat model of hypertensive heart failure using Dahl/SS salt-sensitive rats.Shen-Mai injection could significantly improve the heart function parameters of hypertensive heart failure rats,inhibit the fibrosis pathway of myocardial tissue and cardiac fibroblasts in rats with hypertensive heart failure through regulating TGF-β/Smad signaling pathway,and the effect of Shen-Mai injection was significantly better than that of Shen-Fu injection.From this it verifies the syndrome essence of “Qi and Yin deficiency syndrome” of hypertensive heart failure rat model according to "syndrome detection by formula" method,and it preliminary reveals the pathological mechanism of myocardial fibrosis of hypertensive heart failure and the key target for Shen-Mai injection combating hypertensive heart failure.