The Study On The Effect And Mechanism Of Qinggan Yishen Qufeng Formula On Hypertensive Kidney Injury

Objectives:The study aims to explore the relationship between hypertensive renal injury and TCM syndrome types.The dominant effect and possible mechanism of Qinggan Yishen Qufeng formula(QYQ)in regulating blood pressure,preventing and treating renal injury were explored by renal histopathology,immunopathology and molecular biology in angiotensin Ⅱ(Ang Ⅱ)-induced hypertensive mice model.The aim of study is to provide objective theoretical basis and experimental data support for the clinical application and promotion of QYQ.Methods:1.Exploring the relationship between renal injury in hypertension patients and TCM syndromes(1)Cross-sectional study method was used in this part.We designed questionnaire according to international standards.Furthermore,the formal questionnaire was constructed and corrected by cardiology and statistics experts after feedback from the former questionnaire.General characteristics of patients,clinical diagnosis,treatment history,physical examination,symptom score and syndrome type determination,etc.were included in final version.(2)Patients with essential hypertension were recruited from the community hospital under the jurisdiction of Shanghai Pudong Wanggang Community Health Service Center,and the TCM syndrome types were diagnosed according to the contents of the questionnaire.(3)The relevant physical examination results of community patients were derived from the examination system,and the results were entered into SPSS in the form of EXCEL.(4)The levels of plasma circulating Creactive protein(CRP),neutrophil gelatinase-associated lipocalin(NGAL),retinol binding protein(RBP),cystatin-C(Cys-C),nitric oxide(NO)and oxidative stress markers were detected by enzyme-linked immunosorbent assay(ELISA).2.Study on the effect and mechanism of QYQ on hypertensive renal injury mice model(1)Micro-osmotic pump loaded with Ang Ⅱ was implanted into C57BL/6J mice subcutaneously to form hypertension model.Then the mice were divided into Sham group,Model(Ang Ⅱ)group,QYQ low dose(QYQ-L)group and QYQ high dose(QYQ-H)group.(2)The renal pathological changes of hypertensive mice were collected by HE staining and periodic acid-Schiff staining(PAS).(3)Real time RT-PCR method was used to detect the effects of Ang Ⅱ and different doses of QYQ on the gene expression levels of monocyte chemoattractant protein-1(MCP-1)and α-smooth muscle actin(α-SMA)in kidney.(4)the level of microalbumin in urine of mice was detected by ELISA method.(5)Immunohistochemistry and western blotting were used to detect the level and distribution of sirtuin3(Sirt3),superoxide dismutase2(SOD2)and acetylation-SOD2(AcSOD2)in kidney tissue of mice.Results:1.The relationship between renal injury in hypertension patients and TCM syndromes(1)Among all the patients,the proportion of each syndrome type from large to small is as follows:liver and kidney deficiency syndrome(35.9%)>liver fire hyperactivity syndrome(27.4%)>yin deficiency and yang hyperactivity syndrome(16.0%)>phlegm and blood stasis syndrome(13.1%).(2)Among all the patients,the median age of each syndrome type was as follows:liver fire hyperactivity syndrome(67 years old)<yin deficiency and yang hyperactivity syndrome(69 years old)<liver and kidney deficiency syndrome(79.5 years old)<phlegm and blood stasis syndrome(81 years old).(3)The level of diastolic blood pressure in hypertensive patients with liver fire hyperactivity syndrome and yin deficiency yang hyperactivity syndrome was significantly higher than that in other syndrome types(P<0.05).(4)There was no significant difference in the levels of circulating NO,CRP,protein carbonyl(PC),malondialdehyde(MDA),3-nitrotyrosine(3-NT),RBP and Cys-C among patients with different syndrome types of hypertension(P>0.05).(5)The level of circulating reactive oxygen species in hypertensive patients with yin deficiency and yang hyperactivity syndrome was significantly higher than that in other syndrome types(P<0.05).(6)The level of circulating NGAL in hypertensive patients with hyperactivity of liver fire was significantly higher than that in other syndrome types(P<0.05).2.The effect of QYQ on renal injury and inflammation in hypertensive model mice(1)The level of blood pressure in Ang Ⅱ group was significantly higher than that in Sham group(P<0.05).Compared with Ang Ⅱ group,QYQ could significantly reduce the level of blood pressure in hypertensive mice(P<0.05),and the antihypertensive effect was positively correlated with dose and time.(2)The results of HE stains showed that there were no significant changes in the structure of glomeruli and tubules and no significant renal interstitial fibrosis in,Ang Ⅱ group after subcutaneous continuous infusion for 1 wk and 2 wk.(3)The results of PAS showed that the glomerular mesangial matrix was significantly expanded in,Ang Ⅱ group compared with Sham group,and QYQ intervention could significantly inhibit the expansion of glomerular Mesangial matrix compared with Ang Ⅱgroup(P<0.05).QYQ intervention could significantly inhibit the expansion of glomerular mesangial matrix compared with Ang Ⅱ group(P<0.05).(4)The results of ELISA showed that the level of urinary Microalbumin in Ang Ⅱ group was significantly higher than that in Sham group(P<0.05),and QYQ intervention could significantly reduce urinary microalbumin excretion compared with Ang Ⅱ group(P<0.05).(5)The results of immunohistochemistry showed that the infiltration of macrophages and the level of α-SMA in glomeruli and tubules,Ang Ⅱ group were significantly higher than those in Ang Ⅱ group(P<0.05),and the intervention of QYQ could significantly inhibit the infiltration of renal macrophages and the increase of α-SMA compared with Ang Ⅱgroup(P<0.05)(6)The results of real time RT-PCR showed that the mRNA expression of MCP-1 and αSMA in Ang Ⅱ group was significantly higher than that in Sham group(P<0.05),and the mRNA expression level of MCP-1 and α-SMA in QYQ group was significantly lower than that in Ang Ⅱ group(P<0.05).3.The mechanism of QYQ on renal injury in hypertensive mice model mice(1)The production of nitrotyrosine in Ang Ⅱ group was significantly higher than that in Sham group compared with Sham group(P<0.05),especially in renal tubules,and QYQ intervention could significantly inhibit nitrification in renal tubules compared with AngⅡ group(P<0.05).(2)One week after Ang Ⅱ(1000 ng/kg/min)infusion,the level of Sirt3 in kidney tissue,Ang Ⅱ group was significantly higher than that of Sham group(P<0.05).Compared with Ang Ⅱ group,QYQ intervention could significantly inhibit the compensatory increase of Sirt3 level(P<0.05).Two weeks after infusion,the level of Sirt3 in kidney tissue,Ang Ⅱgroup was significantly lower than that of Sham group,and QYQ intervention could significantly inhibit the decrease of Sirt3 level in kidney tissue compared with Ang Ⅱgroup(P<0.05).(3)One week after Ang Ⅱ(1000 ng/kg/min)infusion,there was no significant difference in total SOD2 level in kidney tissue of all groups,but 2 weeks after infusion,the total SOD2 level in Ang Ⅱ group was significantly lower than that in Sham group(P<0.05),and QYQ intervention could significantly inhibit the decrease of total SOD2 level induced by Ang Ⅱ compared with Ang Ⅱ group(P<0.05).(4)One week after Ang Ⅱ(1000 ng/kg/min)infusion,the ratio of AcSOD2/SOD2 in renal tissue of Ang Ⅱ group and QYQ group was higher than that in Sham group,and the increase of AcSOD2/SOD2 ratio in QYQ group was significantly higher than that in Sham group(P<0.05).At 2 weeks after infusion,the ratio of AcSOD2/SOD2 in,Ang Ⅱgroup was higher than that in Sham group,but there was no significant difference between Sham group and Ang Ⅱ group.QYQ intervention could significantly reduce the ratio of AcSOD2/SOD2 compared with Ang Ⅱ group(P<0.05).Conclusion:1.The level of diastolic blood pressure in patients with liver fire hyperactivity syndrome and yin deficiency and yang hyperactivity syndrome was significantly higher than that of other syndromes,in which the level of circulating NGAL of liver fire hyperactivity syndrome was significantly higher than that of other syndromes,and the level of circulating ROS of yin deficiency and yang hyperactivity syndrome was significantly higher than that of other syndromes,which may be related to the mechanism of renal injury in the early stage of hypertension.2.Qinggan Yishen Qufeng formula can significantly inhibit the early renal tissue injury induced by Ang Ⅱ in mice.And its mechanism may be related to the following aspects:(1)QYQ significantly inhibited the phenotypic transformation of glomerular Mesangial cells.(2)QYQ significantly inhibited the inflammatory reaction of renal tissue in the state of hypertension.(3)QYQ significantly up regulated the level of Sirt3 and inhibited the renal injury caused by hypertension by activating Sirt3/SOD2 pathway,which may have a renal protective effect except hemodynamics.