Study On The Effect And Mechanism Of Jiawei Shenkang Prescription On Curing Renal Fibrosis Based On Hedgehog Signaling Pathway

Objective:In this study we observed the expression of Hedgehog signaling pathway-related molecules in kidney tissue of unilateral ureteral ligation rats and transforming growth factor beta-induced HK-2 cells cultured in vitro,and explored the Anti-renal fibrosis effect and mechanism of Jiawei Shenkang Prescription,in order to provide new ideas for the prevention and treatment of renal fibrosis by traditional Chinese medicine.Method:Experiment one:Effect of Jiawei Shenkang Prescription on Renal Fibrosis and HH Signaling Pathway Expression in UUO Model Rats126 SPF male SD rats were randomly divided into 7 groups:normal group,sham-operated group,unilateral ureteral ligation model group(UUO model group),western medicine group and low/medium/high concentration group of traditional Chinese medicine.The normal group was not treated.The sham operation group was sutured only after freeing the left ureter and the other groups were treated with left ureteral ligation.From the third day after operation,the low/medium/high concentration group was given low/medium/high concentration Jiawei Shenkang Prescription,the western medicine group was given benazepril hydrochloride,and the normal group,sham operation group and UUO model group were given the same amount of saline.Abdominal aorta blood samples were collected from 7/14/21 days after gastric administration under 10%chloral hydrate anesthesia for the detection of serum creatinine(Scr)and urea nitrogen(BUN),and left kidney tissues were taken for pathological sections.HE staining was used to observe the pathological changes of kidney tissue,Masson staining was used to observe the degree of renal fibrosis,immunohistochemistry was used to detect the expression of Shh,Ptch1,Smo and Glil related molecules of a-SMA and HH signaling pathway,and SPSS24.0 statistical software was used for statistical analysis.Experiment two:Effect of Serum Containing Jiawei Shenkang Prescription on HK-2 CellsSPF male SD rats were selected to prepare Jiawei Shenkangfang drug-containing serum and blank serum.HK-2 cells were cultured and divided into four groups:control group,TGF-?1 induced model group,drug-containing serum group and traditional Chinese medicine treatment group.After 48 hours of cell intervention,the expression of Vimentin,E-Cad,Shh and Glil was detected by RT-PCR.The expression of Vimentin,E-C ad,Shh and Glil was analyzed by SPSS24.0 statistical software.Result:Experiments one:(1)Biochemical test:Compared with sham-operated group,the level of urea nitrogen in UUO model group increased(P<0.05);compared with UUO model group,urea nitrogen in western medicine group and Chinese medicine low/medium/high concentration group decreased(P<0.05).At the 14-day stage,there was no significant difference in the level of urea nitrogen among the groups(P>0.05).At the 21-day stage,compared with the normal group and sham-operated group,the level of urea nitrogen in UUO model group increased(P<0.05).There was no significant difference in creatinine levels between the 7-day and 14-day groups(P>0.05).At the 21-day stage,compared with normal group and sham-operated group,urea nitrogen level in UUO model group increased(P<0.05),while creatinine level in western medicine group decreased(P<0.05).(2)HE staining and Mason staining:There were no obvious pathological changes in the normal group and sham-operated group.Inflammatory cell infiltration,renal tubular epithelial cells and interstitial injury,tubular dilatation,interstitial area enlargement and interstitial collagen fibers deposition were observed in the other groups.The degree of fibrosis in UUO model group was more serious.(3)Immunohistochemical detection:Expression of a-SMA:At 7 days,there was no significant difference in ?-SMA expression between the groups(P>0.05).In the 14-day and 21-day stages,the expression level of the UUO model group was the highest.Compared with the UUO model group,the expression levels of the Western medicine group and the low/medium/high concentration group of Chinese medicine were significantly lower(P<0.05).The expression of Shh:The expression of UUO model group was the highest.Compared with UUO model group,the expression of Western medicine group and Chinese medicine low/medium/high concentration group were decreased(P<0.05).Expression of Ptchl:The expression of UUO model group was the lowest,compared with UUO model group,the expression of Western medicine group and low/medium/high concentration group of traditional Chinese medicine were increased(P<0.05).Smo's expression:The expression of UUO model group was the highest,and the expression of Western medicine group and low/medium/high concentration group of traditional Chinese medicine was lower than that of UUO model group(P<0.05).The expression of Glil:The expression of UUO model group was the highest,and the expression of Western medicine group and low/medium/high concentration group of traditional Chinese medicine was lower than that of UUO model group(P<0.05).Experiment two:(1)Changes of E-cad expression:Compared with the control group,the expression of E-Cad mRNA in the model group induced by TGF-?1 decreased significantly;Compared with the model group induced by TGF-?1,the expression of E-Cad mRNA in the traditional Chinese medicine treatment group decreased significantly(P<0.05).(2)Changes of Vimentin expression:Compared with the control group,the expression of Vimentin mRNA in the model group induced by TGF-?1 was significantly increased;compared with the model group induced by TGF-?1,the expression of Vimentin mRNA in the traditional Chinese medicine group was significantly decreased(P<0.05).(3)Changes of Shh expression:Compared,with the control group,the expression of Shh in the model group induced by TGF-?1 increased significantly,and the expression of Shh in the treatment group decreased significantly compared with the model group induced by TGF-?1(P<0.05).(4)Changes of Gli1 expression:Compared with the control group,the expression of Gli1 in the model group induced by TGF-?1 increased,while that in the treatment group decreased(P<0.05).Conclusion:(1)Jiawei Shenkang Decoction can improve inflammatory cell infiltration and damage of renal tubular epithelial cells and renal interstitium in rats with renal fibrosis,alleviate collagen deposition,up-regulate the ex ression of E-Cad,down-regulate the expression of alpha-SMA and Vimentin,which has a certain therapeutic effect on renal fibrosis.(2)The expression of Shh,Smo and Gli1 in renal tissue of UUO model rats increased significantly,while the expression of Ptch1 decreased,suggesting that the activation of HH signaling pathway may be involved in the occurrence and development of renal fibrosis.(3)Jiawei Shenkang Prescription can down-regulate the expression levels of Shh,Smo and Glil related molecules of HH signaling pathway,and up-regulate the expression of Ptchl to achieve anti-fibrosis effect,suggesting that the mechanism of Jiawei Shenkang Prescription in treating renal fibrosis may be related to the inhibition of HH signaling pathway activity.