| ObjectiveRenal Fibrosis,especially Renal interstitial Fibrosis,is the common outcome of almost all progressive chronic kidney diseases.At present,the mechanism of renal fibrosis in modern medicine is still not fully understood,and there is no effective treatment.Wnt/?-catenin signaling is considered to be one of the most important signaling pathways mediating the occurrence of renal fibrosis.Xiezhuo Tongluo Decoction(XZTLD),the experience formula of professor Shuifu Tang,has been used in clinical for many years to treat chronic kidney disease,which can effectively delay the progression of chronic kidney disease.Based on clinical observation and combined with the etiology and pathogenesis of renal fibrosis,this study explored the inhibitory effect of XZTLD on renal fibrosis through experimental,and elucidated the specific mechanisms underlying the therapeutic benefits based on the Wnt/?-catenin signal pathway.So as to provide experimental support for the clinical application of XZTLD.MethodsIn this study,A total of 70 male SD rats(180?200 g)were used.Rats were acclimatized for 5 days prior to experiments then 8 rats were randomly selected as the normal control group and the other 8 rats as the sham operation group.The remaining 54 rats underwent 5/6 nephrectomy and the sham operation group rats were operated simultaneously.Two weeks after the second stage surgery,the 51 rats in the 5/6nephrectomy group that survived were randomly divided into the model group(n=11),the low-dose group(n=10),the median-dose group(n=10),the high-dose group(n=10),the positive control group(n=10).Blood was collected from the inner canthus of rats in each group and 24h-urine samples were collected to determine the success of the model.Subsequently,drug intervention was initiated.The rats of the low-,median-,high-dose group were administered with corresponding dose of XZTLD liquid by gavage(equivalent to 11.4 g,22.7 g and 45.5 g per kilogram of body weight)and the positive group was given losartan solution by gavage(equivalent to5.1mg/kg body weight).The normal group,the sham operation group,and the model group were given the corresponding volume of distilled water intragastrically.The rats in each group were given gavage for 8 weeks continuously.The body weight of all rats was measured at regular intervals every week,and the dosage was adjusted according to the body weight once a week.After 4 weeks of intragastric administration,the rats in each group took blood from the inner canthus again and collect 24h-urine for examination.After 8 weeks of intragastric administration,24h-urine was taken from all the rats one day before the death,and then all the rats were killed to obtain serum and kidney for examinationThe blood samples were used to detect serum creatinine,urea nitrogen,AST and ALT.Urine samples were used to detect 24 h protein and urine MMP-7(ELISA method).HE and Masson staining were used to observing the pathological changes of renal tissue.The expression of ?-catenin and Ecadherin in renal tissue was observed by immunohistochemistry and the expression of wnt1,?-catenin,MMP-7,Fn,collagen I,E-cadherin and ?-SMA protein in renal tissue was detected by Western-blotting.The mRNA expression of ?-catenin,MMP-7,and Snail1 in renal tissue was detected by q PCR.Results1.Comparison of rats' weight in each groupBefore grouping,there was no significant difference in rats' weight between the normal group,the sham operation goup and the 5/6 nephrectomy group.This indicated that the baseline of rats' weight in each group was relatively uniform.Two weeks after the second stage operation,compared with the normal group,the rats in the 5/6 nephrectomy group showed significant weight loss(P<0.01).After 5/6 nephrectomy rats were divided into groups(before intervention),there was no significant difference in rats' weight among the model group,low-dose group,median-dose group,high-dose group and positive control group.After 8 weeks of intervention,the weight of rats in the model group,the low-dose group,the median-dose group,the high-dose group and the positive control group was still significantly lower than that of in the normal group(P<0.05).There was no significant difference in rats' weight between the model group,lowdose group,median-dose group,high-dose group,positive control group and the sham operation group(P>0.05).However,the mean weight of the model group was the lowest among all groups.2.The changes of 24 h protein in the urine of rats in each groupTwo weeks after the second stage operation,the 24 h urinary protein in5/6 nephrectomy group was significantly higher than that in the normal group(P<0.05)?After 4 weeks of intervention,the 24 h urine protein of the model group was significantly higher than that of the normal group and the sham operation goup(P<0.01).Compared with the model group,The 24 h urine protein of rats in the low-,median-and high-dose groups and the positive control group decreased and the difference between the positive control group and the model group was significant(P<0.01).After the intervention for 8 weeks,the 24 h urine protein of rats in the model group continued to increase,while the 24 h urine protein of rats in the high-dose group of XZTLD and the positive control group decreased significantly compared with the model group(P<0.01).3.The changes of serum creatinine and urea nitrogen in each groupTwo weeks after the second stage operation,the serum creatinine and urea nitrogen in the model group were significantly higher than those in the normal group.There were no statistically significant differences in serum creatinine and urea nitrogen between the model group,the XZTLD group(three groups in total),and the positive control group(P>0.05).After 4weeks of intervention,serum creatinine and blood urea nitrogen of rats in the high-dose group of XZTLD were significantly lower than those in the model group(P<0.05 or P<0.01).Compared with the model group,the mean levels of serum creatinine and blood urea nitrogen in the low-and median-dose groups decreased,but the difference was not statistically significant(P>0.05).After 8 weeks of intervention,compared with the model group,serum creatinine and urea nitrogen were significantly reduced in the highdose group,with statistically significant differences(P<0.01).Serum creatinine and blood urea nitrogen in the positive group were significantly decreased compared with the model(P<0.05 or P<0.01).4.Changes of liver function of rats in each groupAfter 4 weeks of intervention,compared with the normal group,shamoperated group and model group,there was no significant difference in the AST,ALT of each drug intervention group(P>0.05).After 8 weeks of intervention,the AST of rats in the model group,low-dose group,mediandose group,high-dose group and positive control was significantly lower than that in the normal group(P<0.05 or P<0.01),while the ALT of rats in each group was still not significantly different(P>0.05).5.pathological morphological changes of rats in each groupNo significant changes were observed in the pathological morphology of the kidney in the normal group and the sham operation goup.In the model group,the renal tissue structure was disorganized,the glomeruli were sparse,and multiple sclerosed glomeruli could be seen,the renal interstitium was widened significantly,and the Focal infiltration of inflammatory cells,mainly lymphocytes.The tubular structure was destroyed obviously,and occasionally the tubular wall was broken,the renal tubules were dilated,and the epithelial cells were shed and necrotic.Compared with the model group,the low-dose,median-dose and high-dose groups of XZTLD showed less epithelial cell shedding and necrosis,less inflammatory cell infiltration,and the lesion was roughly between the normal group and the model group.Masson staining showed that the blue staining area of the renal interstitium in the model group was significantly increased,while the blue staining area of the renal tissue in the normal group and the sham operation goup was only a little,and the blue staining area of the renal tissue in the low-,median-and high-dose groups of XZTLD was between the model group and the normal group.By relative quantitative analysis,the collagen area of renal tissue in the median-dose group,high-dose group and positive control group was significantly reduced compared with the model group(P<0.05 or P<0.01).6.The expression of MMP-7 in the urine of rats in each groupTwo weeks after the second stage operation,the urinary MMP-7 content in 5/6 nephrectomy group was significantly higher than that in the normal group and the sham operation goup(P<0.01).After eight weeks of intervention,the urine MMP-7 level in the model group continued to increase.Compared with the model group,urine MMP-7 level in the median-and highdose groups of XZTLD and the positive control group were significantly reduced,and the effect was most obvious in the high-dose group(P<0.01).7.Protein expressions of ?-catenin,Wnt1,MMP-7,Collagen I,FN,Ecadherin and ?-SMA in rat kidney tissues in each groupCompared with normal and sham operation goup,the expression of ?-catenin,Wnt1,MMP-7,Collagen I,FN,?-SMA in the model group rats' kidney increased significantly(P<0.01).After the treatment of XZTLD,compared with the model group,the expression of the above proteins decreased,and the effect was most obvious in the high-dose group(P<0.05 or P<0.01).There was no significant difference in the expression of the above proteins between the high-dose group and the positive group.In contrast to the above protein expression model,The expression of E-cadherin in the model group was significantly lower than that in the normal group and the sham operation goup(P<0.01).After 8 weeks of intervention,the expression of E-cadherin in the high-dose group and the positive control group increased significantly compared with the model group(P<0.01).8.mRNA expression of ?-catenin,MMP-7 and Snail1 in rat kidney tissues in each groupThe mRNA expression levels of ?-catenin,MMP-7 and Snail1 in the model group were significantly increased compared with the normal group and the sham operation goup(P<0.01).Compared with the model group,the mRNA expressions of ?-catenin,MMP-7 and Sanil1 in the low-,median-and highdose groups were decreased,and the effect was more obvious in the highdose group(P<0.05 or P<0.01).9.Immunohistochemistry of ?-catenin and E-cadherin in each groupCompared with the normal group and sham operation goup,the expression of E-cadherin expression in the model group was significantly decreased,however,the expression of ?-catenin was significantly increased.After the administration of XZTLD,the expression of E-cadherin was higher and the expression of E-catenin was lower than that in the model group,both effects were significant in the high-dose group.ConclusionAfter 5/6 nephrectomy,the Wnt/?-catenin signal pathway in kidney was continuously activated in the renal tissue of rats.XZTLD could decrease the blood creatinine,blood urea nitrogen and urine protein in rats.In addition,the XZTLD is safe,and the low-,medium-and high-doses of XZTLD have no obvious damage to the liver function of rats.In summary,the XZTLD can inhibit the Wnt/?-catenin signal pathway of kidney and improve renal fibrosis. |
PDF Full Text Request