The Protective Mechanism Of Qianyang Yuyin Granules In Hypertensive Nephropathy Based On Heat Shock Protein 90? Related Signal Network

ObjectiveHypertensive nephropathy is the second major cause of ESRD after diabetes mellitus.It is of great significance to explore how to prevent and treat renal damage caused by hypertension.HSP90?,as a protein chaperone helping the refolding of related kinases and transcription factors to maintain their activity,has attracted much attention in promoting the development of inflammatory and fibrotic diseases in recent years.Qianyang Yuyin Granule(QYYY)is a characteristic preparation for the prevention and treatment of hypertensive nephropathy in our hospital.To further reveal the specific mechanism of QYYY in renal protection,we conducted this study.Methods1.In vivo experiment1)Effects of QYYY on renal damage from SHR.First,the rats were fed to 14 weeks of age,and the blood pressure and urine indexes were detected in each group.SHR was then randomly divided into five groups,model group,QYYY high-dose group(QYYY-H,6.4g/kg/d),QYYY medium-dose group(QYYY-M,3.2g/kg/d),QYYY low-dose group(QYYY-L,1.6g/kg/d),and Valsartan group(8.5mg/kg/d),with WKY as control.After 8 weeks of intervention,blood pressure,heart rate,urine and other related indexes were detected,and renal pathology of rats in each group was observed by HE staining and Masson staining.2)Expression of Hsp90? in SHR kidney and the effect of QYYY.WB and immunofluorescence(IF)were used to detect the expression of Hsp90? in kidney of rats in each group.3)Effect of QYYY on EMT-related proteins in kidney from SHR.WB and IF were used to detect the expression of EMT-related phenotypic proteins E-cadherin and Vimentin.WB was used to detect the expression of Twistl,an important transcription factor regulating EMT.4)Effect of QYYY on SHR renal inflammation and inflammation-related signaling pathway.The expression of IL-1? and IL-6 inflammatory cytokines were detected by WB and IF,cytoplasmic proteins and nuclear proteins were isolated from renal tissues,and the nuclear transcription of p65 and p38 were detected.The phosphorylation of upstream kinase IKK of p65 was detected by WB.2.In vitro experiment1)The effect of Ang ? on the viability of NRK-52E,and the protective effect of QYYY containing serum.MTT was used to screen optimal concentration of Ang ? for NRK-52E,then the protective effect of QYYY containing serum was detected by MTT to choose two effective concentration(QYYYS-L and QYYYS-H).The influence of Ang II on the expression of HSP90? of NRK-52E was dected by qPCR,WB and IF.2)The effect of QYYY containing serum on Ang II induced EMT of NRK-52E.WB and IF were used to detect the expression of EMT-related phenotypic proteins E-cadherin and Vimentin.Nuclear transcription of Twist1 was observed by WB and IF,and the interaction between Hsp90? and Twistl was detected by Co-IP.3)The effect of QYYY containing serum on Ang ? induced inflamation of NRK-52E.The effect of QYYY containing serum on Ang II induced inflamation was detected by WB.The nuclear transcription of p65 was dected by WB and IF.The phosphorylation of IKK was dected by WB.The laser confocal and CO-IP was used to dected the interaction of HSP90? and IKK.4)The effective component of QYYY inhibits the associated inflammatory fibrosis pathway by binding with HSP90?.Screen the effective component of QYYY,and looking fo the inhibitory residues of HSP90?.Blind docking was performed of effective component over HSP90? to observe whether the active ingredient could bind to the residues of specific inhibition site of Hsp90?.Resunlts1.In vivo experiment1)The blood pressure of SHR at 14 weeks of age was significantly higher than.that of WKY,and the urine mALB,UTP,and NAG of SHR were significantly higher than that of WKY,which confirmed the successful establishment of hypertensive renal damage model.After the intervention of QYYY for 8 weeks,it was found that QYYY could significantly improve the blood pressure,urine UTP and NAG of SHR.HE and Masson staining showed that QYYY could significantly improve the renal pathological structure of SHR and reduce collagen fiber deposition.2)The expression level of HSP90? protein in SHR kidney was significantly higher than that of WKY,but QYYY intervention did not significantly reduce the expression level of HSP90?protein.3)QYYY can inhibit the occurrence of renal EMT in SHR to a certain extent,and at the same time,they also have a certain inhibitory effect on Twistl,an important transcription factor regulating EMT.4)QYYY significantly lower the SHR kidney IL-1? and IL-6,plasma protein separation and cells nucleus protein of tissues indicated that QYYY can significantly reduce the nuclear transcription of p65,has certain influence to the p38 lightning,but the difference did not reach statistical significance,and then the NF-?B upstream kinase IKK testing found that QYYY significantly inhibited the phosphorylation of IKK.2.In vitro experiment1)The damage of 10-6M Ang ? on NRK-52E was found by MTT,and 1.25%and 2.5%of QYYY containing serum could inhibit the injury.The increased expression of HSP90? by Ang ? was dected by qPCR,WB,and IF.2)QYYY containing serum inhibited Ang ? induced EMT,and the decreased expression of Twitlwas dected by WB and IF.At the same time,QYYY containing serum inhibited the nuclear transcription of Twitl,and didn't change the mRNA level of Twist1.Results of CO-IP suggested HSP90? and Twist1 exist interaction relations,the QYYY containing serum inhibits the combination of HSP90? and Twist1.3)QYYY containing serum inhibited Ang ? induced the expression of inflammatory cytokines.WB and IF found the increased nuclear transcription of p65 by Ang ?,and QYYY containing serum inhibited it.Then the inhibition of phosphorylation of IKK was detected by WB.Confocal and CO-IP found that IKK and HSP90? had strong interaction,and the QYYY containing serum inhibits the combination of IKK and HSP90?.4)Molecular docking results show that two main components of QYYY could be combined with the inhibitory residues of HSP90?.0.5?M NVP-BEP800 could significantly inhibit the combination of HSP90? protein with client protein,and the inhibitory effect is greater than QYYY containing serum.However,in terms of improving inflammation fibrosis phenotype both QYYY containing serum and NVP-BEP-800 showed similar effect.ConclusionQYYY could reduce blood pressure of SHR,protect renal function,and improve local inflammatory infiltration and fibrosis collagen deposition in the kidney.At the mechanistic level,it was found that QYYY could inhibit the interaction of HSP90? between IKK and Twistl,leading to the inactivation of related client proteins,thus improving the inflammatory infiltration and EMT in the kidney.However,the effect of QYYY on the inhibition of inflammation and fibrosis may not be specifically derived from its inhibition of HSPO?.Maybe QYYY play a role of kidney protection through multi-targets.