| Objective:In the present research based on the theory of tonifying kidney and harmonizing kidney,Bushen Hemai formula was invented to treat the elderly spontaneously hypertensive rates(SHR);to evaluate the hypotensive and protective effect on the target organ like kidney of the Bushen Hemai granule;to explore the target of renoprotection and the pharmacological mechanism of Bushen Hemai granule on anti-hypertension from the molecular level though observing its effect on angiotensin ? type 1 receptor(AT1)and downstream phosphatidylinositol 3-kinase(PI3K)/AKT,and RhoA/ROCK pathway in the elderly SHR;to evaluate the glomerular perfusion by determining renal artery blood flow and renal hemodynamic parameters of rats before and after drug intervention,including resistance index(RI),pulsatility index(PI)and blood flow velocity by Doppler.We try to provide an objective and macroscopic hemodynamic data to support the thought,treating elderly hypertension from kidney and meridian;to evaluate the degree of glomerular sclerosis and renal fibrosis in SHRs by the observing the changes of the expression level of smooth muscle alpha actin(alpha-SMA),connective tissue growth factor(CTGF),transforming growth factor-?1(TGF-?1),drosophila mothers against decapentaplegic 3(SMAD3),fibroblast-specific protein-1(FSP-1)in kidney tissues.Methods:1.Thirty 16-month old male SHRs were randomly divided into model group,western medicine group and Bushen granule group(n=10),and 10 16-month old male WKY rats and 10 8-week old male WKY rats were taken as control groups.Bushen Hemai granule was administrated to the SHR in the Bushen granule group intragastrically(concentration of the crude herb was 14.22g/kg.d)and valsartan was given to the SHR in the western medicine group(14.4mg/kg.d)and 2mL normal saline was given to the rats of model group and blank control groups once daily for 8 weeks.2.The pharmacodynamic study of the Bus hen Hemai granule.(1)Measurement of blood pressure of rats was determined by tail artery compression.(2)The renal artery blood flow(including the main renal artery and segmental artery)and renal hemodynamic parameters of rats was measuredusing the color Doppler ultrasound diagnostic instrument directly and renal artery blood flow parameters(Vp),the minimum velocity(Vd),mean velocity(Vm)and resistance index(RI),pulsatility index(PI)was recorded.(3)The level of urinary retinol binding protein(RBP)and urine ?2-microglobulin(?2-MG),and urinary cystatin C was determined by enzyme linked immunosorbent assay(ELISA).After 8 weeks,the rats were sacrificed and blood samples were obtained from inferior vena cava,and the serum was isolated.The samples were prepared for routine pathology observationand the serum creatinine and urea nitrogen assay.The serum level of angiotensin II(Ang II),arginine vasopressin(AVP)and endothelin(ET)was detected by ELISA.(4)The rat kidneys and the main renal arterys were isolated and morphological changes were observed using the HE staining and transmission electron microscopy.By the method of immunohistochemical(IHC),the distribution of AT1,TGF-? in kidneys was analyzed.3.To explore the pharmacological mechanism of Bushen Hemai granule:(1)After the mRNA and total protein was extracted from renal cortex,the mRNA expression level of AT1 and its downstream PI3K,AKT,RhoA and ROCK was detected by real-time quantitive PCR(qPCR)respectively,and the protein expression of AKT was determined by western blotting.4.To study the effects of Bushen Hemai granule onhypertensive renal fibrosis:the collagen deposition in extracellular matrix(ECM)and renal fibrosis in rat kidneys was observed by Masson staining.The renal mRNA and total protein was extracted from renal cortex,the mRNA level of a-SMA,CTGF,TGF-,SMAD3 and FSP-1 was detected by qPCR,and the protein expression of CTGF,FSP-1 and TGF-? was detectedby the method of western blotting.Results:1.The weight of SHR rats was significantly lower than that of WKY rats(P<0.05),with the slightly yellow hair color,mental excitement,as well as poor sleep.After the treatment,the general condition of rats was improved by various degrees,and the average weight of rats in the Bushen granule group were increased slightly.2.The pharmacodynamic study of Bushen Hemai granule:(1)After 8 weeks of treatment,both Bushen Hemai granule and valsartan had significantly decreased the level of systolic blood pressure,diastolic blood pressure and mean arterial pressure.The pulse pressure of Bushen Hemai granule group was significantly lower than that in the valsartan group.(2)Color Doppler ultrasound of kidney demonstrated that Bushen Hemai granule and valsartan increased the maximum blood flow velocity and the minimum blood flow velocity of renal artery and decreased the RI and PI values in the SHR(P<0.05).(3)After 8 weeks of treatment,the urine level of RBP,?2-MG and cystain C in Bushen group and valsartan group was significantly lower than those in the model group(P<0.05).(4)Serum creatinine and urea nitrogen level decreased after Bushen Hemai granule treatment(P<0.05).(5)The serum levels of Ang?,AVP and ET were significantly lowered after Bushen Hemai granule treatment(P<0.05).(6)HE staining showed that the glomerular size was significantly normalized,mesangial proliferation was less,mesangial matrix and collagen proliferation was reduced,the degree of capillary expansion was mild,renal interstitial fibroblasts were of mild hyperplasia,accompanied by a small amount of inflammatory cell infiltration in the Bushen group and valsartan group compared with the SHR models.Under the transmission electron microscopy,it was showed that in Bushen Hemai granule group,significantly improved fusion,significantly reduced mesangial cells and stromal hyperplasia,abundant organelles,swelling mitochondria,actived autophagosomes and normal basement membrane structure;in renal artery,there was smooth vascular wall,' cell distribution in order and smooth muscle thickened phenomenon eased after Bushen Hemai granule and valsartan administration.(7)Masson staining showed less collagen deposition in renal tubules,renal interstitium,renal tubular dilatation,mild renal cysts expansion,and eased glomerular fibrosis in Bushen Hemai granule and valsartan group.(8)Immunohistochemical staining showed that the density of AT1 and TGF-? was significantly lower in the Bushen group than that of model group.(P<0.05).It was suggested that the Bushen Hemai granule effectively controlled blood pressure and postponed the hypertensive renal damage.3.The pharmacological study of Bushen Hemai granule:qPCR demonstrated that the mRNA expression level of PI3K,AKT,RhoA,Rock,SMA-?,CTGF,TGF-?,SMAD3 and FSP-1 were significantly lower in Bushen group than that in the model group(P<0.05).The protein expression level of AKT,CTGF,TGF? and FSP-1 in Bushen group were significantly lower than that in model group(P<0.05).Conclusion:Bushen Hemai granule effectively controlled the blood pressure,improved the renal hemodynamic parameters and relieved the process and progress of hypertensive renal fibrosis in SHR.The pharmacological mechanism of Bushen Hemai granule may be close related to blocking AT1 and inhibiting the activation of PI3K/AKT and RhoA/ROCK pathway;and protected the target organ and retarded renal fibrosis by inhibition of TGF-?/CTGF pathway activation. |
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