Based On The Theory Of "heart-kidney Correlation" To Explore The Mechanism Of Hypertension, Heart Failure And Renal Fibrosis And The Intervention Study Of Prescription Testing

Objective:1.Based on the theory of heart and kidney related,the pathological mechanism of renal fibrosis caused by hypertension and heart failure was studied,and the "Test and verify with a prescription" method was used to clarify the syndrome nature of hypertension and renal failure.2.To clarify the mechanism and key target of Shenmai injection and Shenfu injection regulating TGF-β/Smad signal transduction pathway in intervention of renal fibrosis in hypertensive heart failure.Method:1.50 Dahl/SS salt-sensitive rats were randomly divided into blank control group(6 rats)and model group(44 rats),and 10 SS-13 BN salt-tolerant rats were salt-sensitive control groups,The blank control group was fed with low salt diet,the salt sensitive control group and the model group were fed with high salt diet for 20 weeks.to establish and validate salt-sensitive hypertensive heart failure rat model.40Dahl/SS rats that had developed a model of hypertension and heart failure were randomly divided into model group,Shenmai injection group,Shenfu injection group and pirfenidone inhibitor group,10 rats in each group,injected intraperitoneally combined with intragastric administration for 15 days.At the end of administration,the EF and FS values of rats in each group were detected by ultrasound,the content of serum NT-pro BNP and TGF-β,the content of Cystatin C and NGAL were measured by ELISA,the 24-hour urine protein quantity and renal function(SCR,BUN and UA)of rats in each group were detected by urine collection,HE staining was used to observe the pathological changes of kidney tissue,Masson staining was used to observe the changes of renal fibrosis,the changes of Collagen type I(Col I)in renal tissue was detected by immunofluorescent techniques,and the expression of TGF-βand the phosphorylation of Smad2 and Smad3 were detected by Western blot,and the m RNA expression of Smad2,Smad3,TGF-β and Col I were detected by RT-q PCR.2.30 Dahl/SS salt sensitive rats were randomly divided into three groups: blank control group,Shenmai injection group and Shenfu injection group.The rats were injected intraperitoneally with 6.0m L/kg of medicine for 7 days,once a day,and blood was taken from abdominal aorta to prepare serum containing drug.Shenmai serum,Shenfu serum,blank serum,TGF-β1 and Pirfenidone were applied to renal tubular epithelial cells of primary rats for 48 hours,and CCK-8 method was used to determine the best concentration(dose effect)and the best intervention time(time effect).The blank control group,TGF-β1 group,Shenmai serum combined with TGF-β1 group,Shenfu serum combined with TGF-β1 group,blank serum combined with TGF-β1 group,pirfenidone combined with TGF-β1 group were set up,CCK-8method was used to detect the cell proliferation inhibition rate of drug-treated cells in each group for 48 hours,Western Blot method was used to detect the degree of Smad2 and Smad3 phosphorylation,and RT-q PCR method was used to detect the relative m RNA expression of Smad2 and Smad3.Results:1.At the end of modeling,compared with the control group(blank control,salt sensitive control),the general situation: the overall state of the model group rats(eating condition,mental state,behavior signs)abnormal,the abdomen is swollen,the blood pressure is significantly increased,and the systolic pressure can reach more than 200 mm Hg;In terms of cardiac function: the level of cardiac function parameters(EF,FS)in the model group was significantly reduced,and the level of serum NT-pro BNP was significantly increased;Renal function: in the model group,urinary protein excretion increased,renal function indexes(Scr,BUN,UA)increased,and renal tissue showed different degrees of renal tubular atrophy,interstitial fibrosis,inflammatory cell infiltration and other pathological changes.The above results indicate that the renal fibrosis model of hypertensive heart failure is successfully established.2.After administration,compared with the Shenfu group,the urinary protein excretion increased,the levels of Scr,BUN and UA in renal function increased,thelevels of TGF-β and Col I in the kidney tissue of rats increased,and the degree of phosphorylation of Smad2 and Smad3 protein in the kidney of the model group increased,which suggested that there was a process of renal tubular epithelial mesenchymal cell transdifferentiation(EMT)in the hypertensive heart failure rats.Compared with the model group,in Shenmai group,the urinary protein excretion decreased,the levels of SCR,BUN and UA decreased,the pathological changes such as glomerulosclerosis,tubular atrophy,interstitial fibrosis and inflammatory cell infiltration decreased,the content of TGF-β1,the phosphorylation of Smad2 and Smad3 protein,the volume fraction of collagen and Col I average fluorescence intensity decreased,the above results suggest that the degree of renal fibrosis is reduced.3.TGF-β1,Shenmai serum,Shenfu serum and blank serum all had different proliferative effects on rat renal tubular epithelial cells,while pirfenidone had inhibitory effect on rat renal tubular epithelial cells,and the dose dependence is not obvious.10% drug serum,10μg/L TGF-β1,1g/L pirfenidone and 48 h were determined as the optimal concentration and intervention time.after each drug combined with TGF-β1,the cells in Shenfu group and blank combination group proliferated further,the growth of cells in Shenmai group and pirfenidone group was significantly inhibited,and the inhibition rate of cells in Shenmai group was significantly higher than other groups.4.After the renal tubular epithelial cells of rats were incubated with the drug containing serum,pirfenidone and TGF-β1,The protein phosphorylation degree of Smad2 and Smad3 and the relative m RNA expression of Smad2 and Smad3 in Shenmai combined group were significantly lower than those in the blank control group,Shenfu combined group,blank combined group and pirfenidone combined group.This suggested that Shenmai injection can reduce renal fibrosis by inhibiting the activation of TGF-β1/Smad signal pathway in renal tissue.Conclusion:1.This study confirmed that the two-way interaction damage of heart andkidney is the main reason for the occurrence and development of renal fibrosis in hypertensive heart failure.The activation of TGF-β1/Smad signal transduction pathway in renal tissue of model rats and its involvement in the process of renal tubular epithelial mesenchymal cell transdifferentiation are important pathological mechanisms of renal fibrosis in hypertensive heart failure.2.Shenmai injection can regulate TGF-β 1/Smad signal transduction pathway and inhibit renal fibrosis,and its mechanism may be related to preventing TGF-β1/Smad signal transduction pathway activation and renal tubular epithelial-stromal cell transdifferentiation.3.In this study,based on the theory of syndrome identification,Shenmai injection is the main treatment,and Shenfu injection is the reference for comparison.It is confirmed that the essence of syndrome in hypertensive heart failure and renal fibrosis rats is deficiency syndrome of Qi-yin both heart and kidney.