Study On The Mechanism Of Yishen Huoxue Decoction In Regulating Renal Tissue Cell Apoptosis And Anti-Renal Fibrosis In UUO Rats

Objective:Renal fibrosis（RF）is an important pathway in the progression of almost all chronic kidney diseases（Chronic kidney disease,CKD）,but the specific mechanism of action of renal fibrosis has not been fully elucidated.In this experiment,we investigated the effect of Yishen Huoxue Decoction on apoptosis in unilateral ureteral obstruction（UUO）induced renal fibrosis model in rats using HE,Masson,TUNEL,Western Blot and ELISA techniques to provide a new perspective on the road to treat chronic kidney disease.Methods:In the experiment,50 male SD rats of clean grade were randomly divided into sham operation group,model group,losartan potassium group,low dose group of Yishen Huoxue Decoction,medium dose group of Yishen Huoxue Decoction,and high dose group of Yishen Huoxue Decoction,with 8 rats in each group.On the first day（d1）after modeling,the sham-operated group and the UUO group were given normal saline by gavage,the Yishen Huoxue Decoction（low,medium and high）dose group were given Yishen Huoxue Decoction（9.03,18.06,36.12）g·kg^-1·d^-1by gavage,and the losartan potassium group were given losartan potassium 50 mg·kg^-1·d^-1by gavage,and each group was given one gavage daily.After 14 days of continuous gavage,the rats were killed and taken.The general conditions（hair,diet,mental and behavioral status,surgical wound infection,body weight,bilateral kidney weight,etc.）of rats in each group were observed.HE staining was used to observe the changes of renal pathological structure.Masson staining was used to detect the degree of renal fibrosis.TUNEL staining was used to observe the scorching of renal tubular epithelial cells in renal tissue.Western Blot was used to detect NLRP3,ASC,Caspase-1 The expression level of GSDMD pyrolytic protein and the content of serum IL-1βwere detected by ELISA.Results:1.General results of rats:rats in the sham operation group had tight and bright hair,normal diet,sensitive response,normal activity,normal size and shape of both kidneys,and fast weight growth.The rats in the UUO model group showed decreased appetite,mental depression,irritable behavior,hunched back,decreased activity,hair loss,slow weight growth,and significantly increased renal coefficient（P<0.05）.After the intervention of Yishen Huoxue Decoction and losartan potassium,the water consumption of rats can be reduced,the activity and body weight can be improved,and the kidney coefficient can be significantly reduced（P<0.05）.2.HE staining results:The renal tissue of rats in the sham-operation group was normal under the microscope,without specific changes,with clear and visible outline,renal tubules arranged in order,lumen without expansion or contraction,interstitial area without inflammatory cell exudation,and no cell necrosis.The renal structure of rats in the model group is irregular,the boundary of glomerulus is unclear,the renal tubules are arranged in disorder,some renal tubules are enlarged,and the renal interstitium is widened.Compared with the sham operation group,a large number of inflammatory cells can be seen.Compared with the model group,the infiltration of interstitial inflammatory cells in losartan potassium group was significantly reduced,and the structure and morphology of glomerulus were improved to a certain extent.The rats in the Yishen Huoxue Decoction（low,medium and high）dose group can see that the infiltration of inflammatory cells in the interstitium and the swelling of the renal interstitium are significantly reduced,and the morphology and structure of the glomerulus,the atrophy of renal tubules or the expansion of renal lumen are significantly improved.3.Masson staining results:In the sham operation group,there was no abnormal change in the cortex and medulla of the renal structure,the morphology and structure of renal tubules and glomeruli were normal,and a small amount of collagen fibers were found in the renal interstitium.Compared with the sham operation group,the renal tissue structure of the model group was significantly changed,the renal tubules were atrophic,the renal interstitium was widened,a large number of blue collagen fibers were gathered,and the area of fibrosis was significantly increased（P<0.05）.Compared with the model group,the arrangement of renal tubular structure in losartan potassium group was significantly improved,the glomerular structure was clear and regular,and the area of interstitial blue collagen fibrosis in renal tissue was significantly reduced（P<0.05）.The morphological and structural changes of glomerulus and renal tubules in rats of Yishen Huoxue Decoction（low,medium and high）dose group were reduced,and the area of inflammatory cells infiltration and fibrosis in renal interstitium was significantly reduced.Compared with losartan potassium group,there was no significant difference in the area of collagen fibrosis in the Yishen Huoxue Decoction（middle and high）dose groups（P>0.05）;Compared with the low-dose group of Yishen Huoxue Decoction,the change of fibrosis area of rats in the low-dose group of Yishen Huoxue Decoction（middle and high）was statistically significant（P<0.05）;Compared with the middle dose group of Yishen Huoxue Decoction,the fibrosis area of rats in the high dose group of Yishen Huoxue Decoction was significantly reduced（P<0.05）.4.TUNEL staining results:In the sham operation group,the nuclei of kidney tissue were large and round,no obvious inflammatory infiltration was found,and a small number of positive cells were visible;Compared with the sham-operated group,the positive rate of renal tissue cells in the model group was significantly higher,and obvious cell death was observed（P<0.05）;Compared with the model group,the inflammatory infiltration in losartan potassium group and Yishen Huoxue Decoction（low,medium and high）dose group was significantly reduced,and partially reversed the occurrence of renal tissue cell death（P<0.05）;Compared with losartan potassium group,the positive rate of Yishen Huoxue Decoction（low and medium）dose group was higher,while the positive rate of Yishen Huoxue Decoction（high dose group）was lower（P<0.05）;Compared with the low dose of Yishen Huoxue Decoction,the positive rate of Yishen Huoxue Decoction（middle and high dose）decreased（P<0.05）;Compared with the middle dose of Yishen Huoxue Decoction,the high dose of Yishen Huoxue Decoction has a better effect on inhibiting cell scorching（P<0.05）.5.Results of Western blot assay:compared with the sham group,the expression levels of NLRP3,Caspase-1,ASC,and GSDMD proteins in the model group were significantly up-regulated（P<0.05）;Compared with the model group,NLRP3,Caspase-1,ASC,and GSDMD protein expressions were down regulated in losartan potassium group and Yishen Huoxue Decoction（low,medium,and high）dosage groups（P<0.05）,in which NLRP3 and GSDMD were down regulated most significantly in Yishen Huoxue Decoction high dosage group（P<0.05）,and most significantly in losartan potassium group（P<0.05）..6.ELISA results:Compared with sham operation group,the expression level of serum IL-1βinflammatory factor in kidney tissue of model group was significantly increased（P<0.05）;Compared with the model group,losartan potassium group and Yishen Huoxue Decoction（low,medium and high）dose groups can down-regulate the expression level of IL-1βinflammatory factors,and the down-regulation effect of Yishen Huoxue Decoction high-dose group is the most significant（P<0.05）.Conclusion（s）:1.Unilateral ureteral obstruction model can induce the overexpression of pyrolytic proteins NLRP3,ASC,Caspase-1,GSDMD in kidney tissue,release IL1βinflammatory factors,and seriously damage the kidney.2.Yishen Huoxue Decoction can reduce the induction of renal tubular epithelial cell scorching by inhibiting NLRP3/Casapse-1/GSDMD pathway,and play an anti-RF role.