| ObjectiveOn the basis of previous studies,this research aimed to further explore the mechanism of the anti renal fibrosis of Qing Shen granule with Wnt/ beta-catenin signal pathway as the breakthrough point.To observe the expression of type I collagen(Col-Ⅰ),fibronectin(FN),alpha smooth muscle actin(α-SMA),E-cadherin,Wnt1,P-GSK-3beta,beta-catenin in renal tissue of unilateral ureteral obstruction(UUO)model rats,and the intervention effect of Qing Shen granule on the expression of these proteins.To investigate the role of Wnt/beta-catenin signaling pathway in renal fibrosis,and observe whether Qing Shen granule can inhibit the activity of this signaling pathway,reduce tubular epithelial-mesenchymal transformation(EMT),reduce the accumulation of extracellular matrix,play a role in anti renal fibrosis,to further clarify the mechanism on anti renal fibrosis of Qing Shen granule.This study provides new experimental evidence for a new therapeutic target of prevention and treatment on renal fibrosis.Meanwhile,to observe the pathological changes of the non obstructive kidneyt in UUO rats and the intervention effect of Qing Shen granule.From the perspective of holistic view of traditional Chinese Medicine,to explore the effect of damp heat and blood stasis syndrome in the non obstructive kidneyt of UUO rats,and provides experimental evidence for "Preventive Treatment of Disea se" by clearing damp-heat and dispelling blood stasis method.Methods fter closed management and adaptive feeding for a week,All 60 healthy two month old male Sprague-Dawley(SD)rats of clean grade,weight 180 ~ 220 g,which purchased from the experimental animal center of Medical University Of Anhui,according to the method of random sampling,the rats were divided into 12 groups:normal group,clear kidney granule group,sham operation group,Benner Pury group and model group.All rats weighed before the experiment,and the urine was collected for 24 hours.The blood samples were collected from the tail vein to detect the blood urea nitrogen(BUN)and the serum creatinine(Scr).In the model group,the rats were treated with Qingshen granule group and the candecartan losartan group,while the sham operation group was separated by the same operation route without ligation of the ureter in the model group(UUO).Qing Shen Granule group with renal clearance granule dissolved in warm water by 0.4g/ml was gavaged with the dose of 1ml/100 g.Benazepril group with 0.16g/ml benazepril dissolved in warm water was gavaged with the dose of 1ml/100 g.Model group,sham operation group and normal group were all gavaged by warm water with the dose of 1ml/100 g.Rats in each group were given once a day for 4 weeks.After the end of the experiment,the blood was taken out of the abdominal aorta and bilateral kidneys were taken out and weighed.Before the operation,the rats were weighed and 24 hours urine was collected.The serum levels of BUN,Scr and 24 hours urinary protein were measured by biochemical method before and after treatment in all groups.HE and Masson staining were used to observe the morphological changes of bilateral kidneys.Immunohistochemical method and Western blot method were usd to detect in semi quantitative the expression of type I collagen,fibronectin(FN)and alpha-SMA,E-cadherin in obstructive renal tubulointerstitial.Real time fluorescence PCR and Western blot were used to detect the m RNA and protein expression of Wnt1,P-GSK-3βandβ-catenin.Result1.Comparison of body weight changes in rats:There was no significant difference in the body weight of rats before treatment(P>0.05).After treatment,the weight of rats in each group increased,the weight gain of the normal group and the sham operation group was the most obvious.The weight of benazepril group and Qing Shen granule group were both significantly higher than the model group,while Qing Shen granule group weight increased was significantly higher than benazepril group.2.Comparison of BUN and Scr in serum of rats in each group:Before treatment,there was no significant difference between BUN and Scr in each group(P >0.05).After treatment: compared with the sham operation group,the BUN and Scr of Qing Shen granule group,benazepril group and the model group were significantly higher than those of the sham operation group.BUN and Scr of Qing Shen granule group and benazepril group were both lower than the model group.BUN and Scr of Qing Shen granule group were lower than benazepril group,and the differences were significant(P<0.05).3.Quantitative comparison of 24 hours urine protein of rats in each group:There was no significant difference between 24 hours urine protein in each group before treatment.After treatment,compared with the sham operation group,24 hours urine protein quantitation in each model group was significantly higher than that in the sham operation group,and the differences were significant(P<0.01).24 hours of urinary protein in the drug intervention group were lower than those in the model group.24 hours urine protein of Qing Shen granule group was significant than benazepril group,(P<0.05).4.Pathological changes of kidney of rats in each group:The histopathological results showed that renal tubules and glomeruli were both normal in normal group and the sham operation group.The renal tubular expansion,renal tubular epithelial cell shedding,vacuolar degeneration of renal tubular interstitial infiltration of inflammatory cells,and renal interstitial blue collagen deposition were significant in each model group.The renal interstitial injury index and the relative area of collagen increased.The above-mentioned pathological changes of Qing Shen granule group were significantly reduced than the model group.The pathological changes of benazepril group were between the model group and Qingshen granule group.5.Immunohistochemical detection of renal tissue in rats in each group:Compared with the sham group,the coloring semi quantitative scores of renal tubular model rats and interstitial collagen, fibronectin(FN),alpha smooth muscle actin(alpha-SMA)in each model group were higher.The protein semi quantitative score of Qing Shen granule group and benazepril group were lower than that of model group(P<0.01).Compared with benazepril group,the protein semi quantitative score of Qing Shen granule group was lower than that of benazepril group,(P<0.05).Compared with the sham operation group,the semi quantitative scores of E-cadherin staining in the renal tubules was lower in each model group.E-cadherin staining semi quantitative scores of Qingshen group and benazepril group were both higher than that of the model group(P<0.01).Coloring semi quantitative score of E-cadherin in Qing Shen granule group was higher than that of benazepril group(P<0.05).6.Detection of renal tissue of rats in each group by Western blot method:Expression of Col-I,FN,alpha-SMA,Wnt1,P-GSK-3 and-catenin in the renal tissue of each model group was higher than sham operation group.Protein expression of Qing Shen granule group and benazepril group were both lower than the model group(P<0.01).Compared with the benazepril group,the protein expression of Qing Shen granule group was significantly lower than the benazepril group(P<0.05).Compared with sham operation group,the expression of E-cadherin in renal tissue of each model group was lower(P<0.01).E-cadherin was higher in Qing Shen granule than that of benazepril group(P<0.05).7.Real time fluorescence PCR detection of renal tissue in rats in each group:The m RNA expression of Wnt1,P-GSK-3 and beta-catenin in each model group rats renal tissue was higher.The m RNA protein expression in Qing Shen granule group and benazepril group were lower than that in the model group(P<0.01).Compared with the benazepril group,the m RNA expression in the granule group was lower.8.Pathological changes of non obstructive renal in rats:Compared with the UUO model group,the renal weight / body weight ratio was significantly lower in the model group;HE staining and Masson staining of non obstructive kidney show : In Qing Shen Granule group,the renal glomerular congestion,mild mesangial proliferation,a small number of renal tubules in the renal medulla were dilated,and necrotic and exfoliated epithelial cells were seen in the lumen.The overall pathological changes were reduced compared with the model group,and the collagen fiber deposition in the mesangial area and the renal interstitium was significantly lower than that in the model group.The renal interstitial injury index and the relative area of collagen were significantly lower than those in model group,The results indicated that Qing Shen Granule could improve the pathological damage of non obstructive kidney in UUO rats,Compared with benazepril,there was no significant difference between the two groups.ConclusionActivation of Wnt/ beta-catenin signaling pathway is involved in the pathogenesis of renal fibrosis in UUO rats.Qing Shen Granule can improve renal function in rat model of UUO,decrease urine protein,inhibiting fibrosis.The mechanism may be through down-regulation of Wnt1,P-GSK-3 beta,beta-catenin expression,inhibition of Wnt/ beta-catenin signal pathway was activated to upregulate the expression of E-cadherin,reduce the expression of alpha-SMA,inhibit renal tubular EMT,reduce the generation of extracellular matrix include FN 、Col-1,playing the role of anti renal fibrosis.At the same time,Qing Shen Granule can reduce the renal weight / body weight ratio in the non obstructive side of UUO rats,and improve the pathological damage of non obstructive kidney.The specific mechanism needs further study. |
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