Study On The Mechanism Of YiShen HuanShuai Recipe In The Treatment Of Atherosclerotic Renal Disease Through Regulating IS/AhR/NOX4 Pathway

BackgroundAtherosclerotic renal disease(ARD)is a kind of renal injury caused by atherosclerotic renal artery stenosis and decreased blood flow,which is the main cause of end stage renal disease(ESRD)in the elderly.With the continuous decrease of renal perfusion,the microvascular endothelium around the renal tubules is gradually damaged,showing thinning,and the pathological chain of "microvascular waste and tubulointerstitial fibrosis" is gradually formed,which is the key pathological link in the development of ARD.At present,the treatment of ARD mainly includes drug therapy and revascularization to control blood pressure,blood lipid,reduce the probability of cardiovascular events and protect renal function However,the effect of treatment is not ideal,and the end point of renal disease has not been improved.This also suggests that clinical workers should actively seek new intervention measures and theraputic targets in the treatment of ARD,such as choosing tubulointerstitial fibrosis,which can determine the prognosis of patients with ARD.It is found that tubulointerstitial fibrosis can be accelerated obviously when tubulointerstitial microvascular is damaged.Therefore,it is of great significance to explore measures to delay tubulointerstitial fibrosis from the perspective of protecting tubulointerstitial microvascular endothelium in ARD.Indoxyl sulfate(IS),as an gut-derived uremic toxin,is produced after tryptophan is decomposed by intestinal microflora and transformed by the liver,which is mainly eliminated by the kidneys.When renal function declined,IS accumulated significantly in the body which is not easy to be removed by dialysis as it is easy to bind to protein,IS can activate aryl hydrocarbon receptor(AhR)and nicotinamide adenine dinucleotide phosphatase oxidant-4(NOX4)was further activated to induce oxidative stress and inflammatory response,leading to endothelial dysfunction,microvascular waste and tubulointerstitial fibrosis.Therefore,under the stimulation of IS,AhR/NOX4 pathway can be activated and then mediate oxidative stress and inflammatory response,which may be the main mechanism of tubulointerstitial microvascular damage,thus aggravating tubulointerstitial fibrosis in the progression of ARD.Based on the previous syndrome research and clinical experience,the basic pathogenesis of ARD is based on the deficiency of qi in spleen and kidney,and the miture of dampness,turbidity,stasis and toxin.Professor Dai Xiwen,a well-known nephrologist,proposed YiShen HuanShuai Recipe(YSHSR)with the method of invigorating qi,activating blood circulation and removing dampness,turbidity and toxin combined with her many years of clinical experience in the treatment of chronic kidney disease.The treatment principle of the prescription is consistent with the pathogenesis of ARD,which has a certain clinical effect in delaying the progress of renal function of ARD.The basic research of YSHSR suggested that it played an important role in protecting vascular endothelium and improving tubulointerstitial fibrosis.In view of the similar pathogenic characteristics between IS and "turbid toxin" of traditional Chinese medicine,and IS can damage the vascular endothelium by activating AhR and NOX4,so it can be considered that the protective effect of YSHSR on tubulointerstitial microvascular may be related to its inhibition of is toxicity.And the hypothesis that "YSHSR may improve microvascular waste and alleviate the tubulointerstitial fibrosis by regulating the AhR/NOX4 pathway mediated by IS,thus delaying the progress of ARD" was put forward.Based on this hypothesis,this study firstly constructed ARD model with ApoE gene knockout mice fed with high fat diet and 5/6 nephrectomy,and observed the effect of YSHSR on the level of serum IS,tubulointerstitial microvascular damage,tubulointerstitial fibrosis,molecular expression level related to AhR/NOX4 pathway,and the oxidative stress and inflammatory molecular changes in its downstream in ARD mice.Secondly,human umbilical vein endothelial cells(HUVECs)stimulated by IS were used to construct a cell model by toxic injury,and to observe the effect of serum containing YSHSR on the viability,aging,oxidative stress and AhR/NOX4 pathway of HUVECs induced by IS.The purpose of this study was to elaborate the protective effect and mechanism of YSHSR on the interstitial microvascular of ARD from the perspective of IS,so as to provide a theoretical basis for this prescription to delay the interstitial fibrosis of ARD.Objective1.To observe the effect of YSHSR on the change of the level of serum IS,tubulointerstitial fibrosis and tubulointerstitial microvascular injury in ARD mice,and the protective effect of YSHSR on the damage of vascular endothelial cells induced by IS.2.To reveal the protective effect and the mechanism of YSHSR on the tubulointerstitial microvascular of ARD from the point that IS can activate"AhR/NOX4" pathway and then inducing oxidative stress and inflammatory reaction,which provided a theoretical basis for YSHSR recipe to delay the tubulointerstitial fibrosis of ARD.Methods1.In vivo part:the ARD model was established by feeding ApoE gene knockout(ApoE-/-)mice with high fat diet and 5/6 nephrectomy.The experiment was divided into control group,sham operation group,model group,low dose YSHSR group(16.76g/kg.day),medium dose YSHSR group(33.52g/kg.day),high dose YSHSR group(67.04g/kg.day)and AXT group(0.4g/kg.D)for 20 weeks.The general conditions of mice were observed,and the changes of renal function(Scr,BUN)and the level of blood lipid(TC,TG,LDL-C)were tested automatic biochemical instrument.The level of serum IS was tested by the method of high-effective liquid chromatography fluorescence,and the indexes of oxidative stress(SOD,MDA)and the level of ADMA were measured with ELISA method.The degree of renal artery atherosclerosis was judged by HE staining and VG staining,the histomorphology and interstitial collagen deposition of renal tissues were judged by HE staining,PAS staining and Masson staining.Ultrastructural observation of renal tissues were judged by electron microscopy.The indexes(Collagen ?,?-SMA,TGF-?1)related to interstitial fibrosis and the expression level of AhR protein and the microvascular endothelial protection index(VWF)were detected by immunohistochemistry.The mRNA expression levels of some molecules(AR,NOX4,MCP-1)related to AhR/NOX4 pathway and TGF-?1,an index related to interstitial fibrosis were detected by real time PCR.2.In vitro part:HUVECs were stimulated by 500?mol/L IS for 24 hours to establish the toxin model of endothelial injury.First of all,the changes of HUVECs viability after the stimulation of different concentrations of IS(0?mol/L,250umol/L,500?mol/L,1000?mol/L)and the effects of different concentrations of the serum containing YSHSR(5%,10%,20%)on HUVECs viability under the stimulation of IS were detected by CCK-8 method.At the same time,the modeling concentration of IS and the intervent concentration of the containing serum were determined.Secondly,the experiment was divided into four groups:blank control group,control group with serum containing drug,toxin model group and experimental group with serum containing drug.The cell aging degree of each group was detected by SA-?galactosidase method,and ROS production of each group was detected by chemical fluorescence microplate method.Finally,the AhR inhibitor group(CH223191)and AhR inhibitor+serum containing group were added,and the cell viability,cell aging and ROS production of each group were further detected.The protein expression of AhR and NOX4 was detected by Western blot,and the mRNA expression of AhR,NOX4 and inflammatory factor MCP-1 was detected by real time PCRResults1.In vivo part:(1)compared with the control group,the weight of mice in the sham operation group increased,but there was no statistical significance(P>0.05);the level of TC?TG?LDL-c increased significantly(P<0.05),the activity of serum SOD decreased significantly(P<0.05),but the serum level of Scr,BUN,IS,MDA and ADMA did not increase significantly(P>0.05);The intima of renal artery was thickened,the lumen was narrowed and the renal tubules were dilated,but the ultrastructural changes of renal tubules were not obvious and the deposition of collagen fibers in renal interstitium was not obvious(P>0.05);The expression of TGF-?1,collagen ?,?-SMA and AhR in renal interstitium were not significantly increased(P>0.05),but the expression of vWF in renal interstitium was significantly decreased(P<0.05);There was no significant difference in mRNA expression level of AhR and TGF-?1(P>0.05),but there was significant difference in the expression level of NOX4 and MCP-1(P<0.05).(2)Compared with the control group,there was no significant difference in body weight in the model group(P>0.05);the serum levels of Scr?TC?TG?LDL-c were significantly increased(P<0.05);serum SOD activity decreased while the content of MDA and ADMA increased(P<0.05);The intima of renal artery thickened and the lumen became narrow,and there were obvious glomerulosclerosis,vacuolar degeneration of renal tubules and tubulointerstitial fibrosis with the thinning and loss of microvilli in lumen;The deposition of collagen fibers in renal interstitium increased significantly(P<0.05);The protein expression level of collagen ?,?-SMA,TGF-?1 and AhR increased(P<0.05).(3)Compared with sham operation group,the average body weight of mice in model group was lower than that in sham operation group,but there was statistical difference only at 8 weeks of diet intervention(P<0.05);the levels of Scr,IS,MDA and ADMA were higher(P<0.05),but there was no significant difference in blood lipid level and SOD activity(P>0.05);the intimal thickening and lumen stenosis of renal artery were more obvious,and the glomerulosclerosis was more obvious with the thinning and loss of microvilli in lumen.The deposition of collagen fibers in renal interstitium was more obvious(P<0.05).The expression levels of collagen IV,?-SMA,TGF-?1,AhR and vWF in renal interstitium were significantly increased(P<0.05);The expression levels of TGF-?1,AhR,NOX4 and MCP-1 mRNA in renal tissue were significantly increased(P<0.05).(4)Compared with the model group,the low dose and middle dose of YSHSR group had no significant effect on the body weight of mice(P>0.05).The body weight of high dose group and AXT group was significantly lower than that of the model group(P>0.05);The low,middle,high dose of YSHSR group and AXT group could significantly reduce serum creatinine and IS levels of mice(P<0.05),but the change of BUN and blood lipid levels was not significant(P>0.05);the low dose of YSHSR group could significantly increase the activity of serum SOD and decrease the content of serum MDA and ADMA in mice(P<0.05);the middle and high dose groups could significantly reduce the content of serum MDA in mice(P<0.05);the AXT group could significantly increase the activity of serum SOD and reduce the content of serum MDA in mice(P<0.05);the low,middle,high dose of YSHSR group and AXT group all could improve the thickening of renal artery intima,tubular vacuolation to some extent and ultrastructure of renal tubules,but only the low dose group could reduced the deposition of collagen fibers in renal interstitium significantly(P<0.05);The expression of collagen ?,?-SMA,AhR and vWF in renal interstitium were significantly decreased in the low dose group and the middle dose group(P<0.05);the expression of AhR,NOX4,MCP-1 mRNA in renal tissue was significantly decreased in the low dose group and the middle dose group(P<0.05),and the protein expression and mRNA expression of TGF-?1 in renal tissue was significantly decreased in the low dose group(P<0.05).There was no significant difference in protein expression of collagen ?,?-SMA,TGF-?1,AhR and vWF,and mRNA expression of TGF-?1,AhR,NOX4 and MCP-1 both high dose group and AXT group(P>0.05).2.In vitro part:(1)compared with 0?mol/L IS group,the cell viability of HUVECs in 500?mol/L IS group and 1000?mol/L IS group decreased significantly(P<0.05)after 24 hours,48 hours and 72 hours intervention,and the cell viability in 250?mol/L IS group decreased significantly(P<0.05)after 48 hours of intervention However,the number of cells decreased after the intervention of 1000?mol/L IS.(2)Compared with the normal rats serum group with the same concentration,10%and 20%of the drug-containing serum could significantly increase the cell viability under IS stimulation(P<0.05);When there was no IS stimulation,5%and 10%of the drug-containing serum had no significant effect on the cell viability(P>0.05),and 20%of the drug-containing serum can significantly increase the cell viability(P<0.05).(3)Compared with the blank control group,the drug-containing serum control group had no significant effect on cell viability,cell aging,ROS generation and the expression of protein and mRNA of AhR and NOX4(P>0.05).(4)Compared with the blank control group,the viability of cells in the toxin model group decreased and the staining rate of senescent cells increased;the relative production of ROS increased(P<0.05)and the protein expression of AhR and NOX4 in nucleus increased,and the mRNA expression of AhR,NOX4 and MCP-1 increased(P<0.05).(5)Compared with the toxin model group,the drug-containing serum group,AhR inhibitor group and "AhR inhibitor+ drug-containing serum" group all significantly increased the cell viability and decreased the staining rate of senescent cells and the relative production of ROS(P<0.05);the protein expression levels of AhR and NOX4 were decreased,and the mRNA expression levels of AhR,NOX4 and MCP-1 were down-regulated(P<0.05).(6)Compared with AhR inhibition group,"AhR inhibitor+drug containing serum" group had more significant effects in increasing cell viability,reducing the staining rate of senescent cells and the relative production of ROS,as well as down-regulating the protein expression level of AhR and NOX4 and the mRNA expression level of AhR,NOX4 and MCP-1(P<0.05).Conclusions1.YSHSR can improve the renal function and the pathomorphological damage of renal artery and renal tissue,alleviate the interstitial fibrosis of ARD mice,thus delaying the progression of ARD.2.YSHSR can reduce the level of IS in the serum of ARD mice,improve the damage of the vascular endothelium induced by IS and the tubulointerstitial microvascular waste,and then reduce tubulointerstitial fibrosis.3.The mechanism that YSHSR can improve the tubulointerstitial microvascular waste in ARD mice and the injury of human umbilical vein endothelial cells induced by IS may be related to the inhibition of the activation of AhR/NOX4 pathway induced by IS and the improvement of oxidative stress and inflammatory response downstream.