| Objective: To study the effect and mechanism of Erdingerxian Decoction and Icariin on preventing and treating renal interstitial fibrosis in vivo.Methods: Ten-week-old male C57BL/6 mice were randomly divided into seven groups:sham-operated group(SHAM),model group(UUO),model + Erdingerxian Decoction group(EDEX + UUO),model + estradiol group(E2 + UUO),model + icariin group(ICA + UUO),model + GPR30 antagonist group(G-15 + UUO),model + GPR30 antagonist + icariin group(G-15 +ICA+ UUO).Unilateral Ureteral Obstruction(UUO)model was made.After opening the abdominal cavity layer by layer,the left ureter was free.The model was ligated with 4-0 silk thread,the lower level of the left kidney was ligated,the abdominal cavity was closed and sutured layer by layer.In this model,the kidney drainage system was obstructed by surgical ligation of one side of the ureter,which resulted in chronic renal injury and renal interstitial fibrosis.G-15 were subcutaneously implanted with a microosmotic pump(Alzet Minipump model 1007D)at a rate of 200 ug/kg/d for 7 days.After 7 days of treatment,the mice in each group were perfused with heart,blood was taken from heart,serum was extracted,estradiol level was detected,kidneys were removed on both sides,weight was weighed,Masson's,HE and PAS staining were performed after fixation,and pathological changes of kidneys were observed under light microscope.Fluorescence quantitative PCR was used to detect the expression of c-Myc,FN,collagen type I and GPR30.Western Blot method was used to detect the expression of total beta-catenin,phosphorylated beta-catenin(ser-552),GPR30 and c-Myc protein.Immunofluorescence was used to detect a-SMA,type I collagen,immunohistochemistry was used to detect GPR30.Result:(1)There was no significant difference in the weight of mice before and after the experiment(P > 0.05).Compared with G-15+UUO group,serum estradiol level increased in ICA+UUO group(P < 0.05).Compared with G-15+ICA+UUO group,serum estradiol level increased in G-15+ICA+UUO group(P < 0.05).(2)Kidney histopathological staining and Masson's staining showed that compared with sham-operated group,the degree of interstitial fibrosis in model group was obvious,the area of interstitial blue collagen increased significantly,a large number of renal tubules atrophied and dilated under light microscopy,accompanied by a large number of monocytes infiltration in interstitium,partial compensatory enlargement of glomerulus,Erdingerxian Decoction group and icariin group and model group were similar.Compared with the model group,the degree of renal interstitial fibrosis was improved in varying degrees.Generally speaking,the boundary of cortex and medulla was clearer than that of the model group.In addition,some epithelial cells of tubules remained relatively intact.The degree of tubular dilation was lighter than that of the model group,and the area of blue collagen in the interstitium and the infiltration of monocytes were better than that of the model group.PAS staining showed that there were protein tubular formation,renal tubular dilatation,brush edge shedding,interstitial fibrosis,glomerulosclerosis,EREX + UUO and ICA + UUO groups,and renal pathology improved significantly.Renal pathological damage score also showed that the degree of tubular and interstitial injury,arteriosclerosis and glomerulosclerosis in UUO group was higher than that in EREX + UUO and ICA + UUO group.HE staining showed that a large number of inflammatory cells infiltrated in the model group,and the degree of EREX + UUO and ICA + UUO groups was relatively mild.(3)Immunofluorescence showed that in sham-operated group,the expression of alpha-SMA was only in blood vessels,but in UUO group,G-15+UUO group and G-15+ICA+UUO group,the expression of interstitial was increased.Immunofluorescence of a-SMA and collagen I also showed that the fibrosis was aggravated after G-15 intervention with UUO.The degree of renal fibrosis in Erdingerxian Decoction group and icariin group was lighter than that in UUO group.(4)Immunohistochemical staining of GPR30 in kidney tissue showed brown-yellow staining.Compared with SHAM group,the expression of GPR30 in UUO group decreased significantly.(5)Real-time quantitative PCR and Western Blot showed that the expression of FN,c-Myc,total beta-catenin and phosphorylated beta-catenin(ser-552)in UUO groupwas significantly higher than that in SHAM group.The expression of FN,collagen type I,c-Myc,total beta-catenin and phosphorylated beta-catenin(ser-552)increased in GPR30 blocker group.Compared with UUO group,ICA +UUO,EDEX +UUO group decreased the expression of fibrosis in mice.Meanwhile,the expression of GPR30 in EDEX + UUO group was higher than that in ICA + UUO group and UUO group.(6)Immunohistochemical staining of GPR30 showed that in sham-operated group,GPR30 was mainly expressed in renal tubular epithelial cells.In the case of renal obstruction,the expression of GPR30 in renal tubular epithelial cells was significantly decreased after the renal tubule was hit,and the use of G-15(GPR30 antagonist)could aggravate the fibrosis of UUO.At the same time,immunofluorescence staining of renal tissue with alpha-SMA and type I collagen also indicated that the fibrosis was aggravated after G15 intervention with UUO.EDEX+UUO group and ICA+UUO group could improve renal fibrosis,while q PCR results also indicated that FN expression could be significantly increased in G15.Conclusion: 1.Erdianxian Decoction and icariin can increase the estradiol level of UUO renal fibrosis model mice,reduce the accumulation of fibrosis components such as FN,type I collagen,a-SMA,cmyc,and reduce the degree of renal pathological damage,thereby protecting renal function,increasing the expression of GPR30,regulating Wnt/beta-catenin signaling pathway,and delaying the process of renal fibrosis.2.GPR30 antagonists can synergistically aggravate the progress of fibrosis,suggesting that GPR30 may have an important protective effect on renal fibrosis,and blocking its expression may lead to aggravation of fibrosis.Meanwhile,icariin has a protective effect on renal fibrosis,possibly from GPR30,and interfering with renal fibrosis.3.EREX+UUO and ICA+UUO groups showed a significant trend of protein immunoblotting in mice,but there was no statistical significance,suggesting that the action time of drugs may be short,and further research is needed. |
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