| Objective: Establishment of a rat model of unilateral ureteral obstruction,To study used Chongcaoyishen decoction observe renal function,renal pathology and the effect of Sonic Hedgehog signaling pathway-related protein Sonic Hedgehog(Shh),membrane receptor protein Patched(Ptch1),G protein coupled receptor protein Smoothened(Smo),glioma-associated oncogene homolog 1(Gli1).To explore the mechanism of renal interstitial fibrosis and the possible mechanism of action of Chongcaoyishen decoction in the process of anti-renal interstitial fibrosis.Methods: One hundred SPF male Sprague-Dawley rats were randomly divided into normal group,sham operation group,model group,Chongcaoyishen group and Irbesartan group,with 20 rats in each group.The renal interstitial fibrosis model was established by the left ureteral obstruction method.On the first day after modeling,the stomach began to be administered,the Chongcaoyishen group was given 6.57g/kg of Chongcaoyishen decoction daily,the Irbesartan group was given a daily dose of 13.5 mg/kg Irbesartan solution,and the normal group,the sham operation group and the model group were given an equal volume of distilled water for gavage once a day.The general state of the rats was observed.On the 6th and 13 th day,10 rats randomly selected from each group were placed in the metabolic cage for 24 hours.The water was fasted,urine was collected,and the urine volume was recorded for 24 hours.Uri ne protein quantification(24hUPQ);On the 7th and 14 th day,the rats were sacrificed and serum was collected for biochemical indicators(serum creatinine,serum urea nitrogen);renal tissue was collected for examined by HE and Masson staining.The kidney tissue was selected on the 14 th day.The expression and distribution of Shh,Ptch1,Smo and Gli1 proteins were detected by immunohistochemistry.The levels of Shh mRNA,Ptch1 mRNA,Smo mRNA and Gli1 mRNA were detected by Real-Time PCR.The expression of Shh,Ptch1,Smo,and Gli1 proteins were detected by Western Blot method.Results: 1.In the model group,the rats were in poor condition,the fur was dull,the reaction was slow,the activity was reduced,and the sputum was reduced.The diet was significantly lower than that of the normal group and the sham-operated group.Chongcaoyishen group could significantly improve t he general state of UUO rats,it was superior to the model group and the Irbesartan group in terms of fur color,mental state,food intake and weight gain.2.24 h urine protein quantification,serum creatinine and serum urea nitrogen in the model group were significantly higher than those in the normal group and the sham operation group(P<0.05);the Chongcaoyishen group and the Irbesartan group compared with the model group,24 h urine protein quantitation,serum creatinine and serum urea nitrogen levels in UUO rats were reduced(P<0.05).3.HE and Masson staining results showed that Chongcaoyishen decoction could improve the pathological changes of kidney in UUO rats,r educe the percentage of renal tissue fibrosis in UUO rats,and reduce the renal organ coefficient in rats.It was statistically significan t compared with the model group(P<0.05).4.Immunohistochemical results showed that the expression of Shh,Smo and Gli1 in the renal tissue of the model group was significantly increased compared with the normal group and the sham operation group at 14 days(P<0.05),and the expression of Ptch1 protein was lower than that of the normal group.In the operation group(P<0.05),the expressions of Shh,Smo and Gli1 in the kidney tissues of the rats with Chongcaoyishen group and Irbesartan group were lower than those in the model group(P<0.05),and the expression of Ptch1 protein was higher than that in the model group(P<0.05).Shh and Ptch1 proteins are expressed in the renal cytoplasm and nucleus of renal cortex,and Smo and Gli1 proteins are expressed in renal cortical renal tubular cytoplasm.5.Real-Time PCR results showed that at 14 days,the transcription levels of Shh mRNA,Smo mRNA and Gli1 mRNA in the renal tissue of the model group were significantly higher than those in the normal group and the sham operation group(P<0.05),and the level of Ptch1 mRNA transcription.It was lower than the normal group and the sham operation group(P<0.05).Compared with the model group,the Chongcaoyishen group and the Irbesartan group could down-regulate the transcription of Shh mRNA,Smo mRNA and Gli1 mRNA(P<0.05),Ptch1 mRNA.The level of transcription was higher than that of th e model group(P<0.05).Compared with the Irbesartan group,the level of Shh mRNA transcription was lower in the Chongcaoyishen group(P<0.05).6.Western Blot results showed that the expression of Shh,Smo and Gli1 in the renal tissue of the model group was significantly higher than that in the normal group and the sham operation group at 14 days(P<0.05),and the Ptch1 protein expression was significantly decreased(P<0.05).Compared to the model group,the expression of Shh,Smo and Gli1 protein was down-regulated(P<0.05),and the expression of Ptch1 protein was up-regulated(P<0.05)in the Chongcaoyishen group and Irbesartan group.Conclusion: 1.Chongcaoyishen decoction can improve the general state of UUO rats and increase the body weight and urine volume of UUO rats.2.Chongcaoyishen decoction can reduce 24 h urine protein quantitation,serum creatinine and serum urea nitrogen levels in UUO rats,and improve renal function in UUO rats.3.Chongcaoyishen decoction can improve renal pathological changes in UUO rats due to ureteral obstruction.4.Chongcaoyishen decoction can delay renal interstitial fibrosis by regulating the gene level of Sonic Hedgehog signaling pathway,which may be related to down-regulation of Shh mRNA,Smo mRNA,Gli1 mRNA transcription and up-regulation of Ptch1 mRNA transcription.5.Chongcaoyishen decoction can down-regulate the expression of Shh and downstream Smo and Gli1 proteins and up-regulate the expression of Ptch1 protein by regulating the protein level of Sonic Hedgehog signaling pathway,thereby delaying renal interstitial fibrosis and protecting k idney function. |
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