| Objective :To investigate the effects of quercetin(Que)on the protein expression of phosphorylated adenylate activated protein kinase(p-AMPKα)and the signaling pathway of TGF-β1/Smad3 in renal tissues of rats with unilateral ureteral obstruction(UUO),and to explore the possible mechanisms of quercetin in the process of anti renal fibrosis.Methods :(1)72 male Wistar rats weighing approximately 200 ± 10 g were selected and randomly divided into sham operated group(Sham group),model group(UUO group),quercetin treated group(Que group)with 24 rats in each group.(2)Rats in Sham,UUO,and Que groups were anesthetized by intraperitoneal injection(10% chloral hydrate 0.3ml/100g),then holding forceps clamped with iodophos cotton balls for surgical mouth disinfection,covered sterile hole towels,and cut erector spinally using a scalpel at the surgical site.In the first step,the skin was first incised,followed by the subcutaneous tissue,then blunt separation of the muscle layers,and then each layer of the parietal peritoneum and visceral peritoneum were cut to free the left ureter:(1)rats in the Sham group only had their ureters stripped without ligation;(2)rats in the UUO and Que groups were ligated to the left ureter.(3)The Que group was treated with quercetin(dissolved in DMSO)at 100 mg / kg / D by gavage 1 day before the model operation,and once / D,the Sham and UUO groups were administered an equal volume of 0.9% saline.(4)In each group,eight rats from each group were sacrificed in random batches at the end of days 3,7,and 14 after modeling,and the obstructed kidney tissues were removed,fixed in neutral formaldehyde solution,dehydrated,embedded in paraffin,sectioned,and subjected to he and Masson staining,followed by observation with a 400 X light microscope,immunohistochemical detection(p-AMPKα,TGF-β1,Smad3),and visualization by image pro The average optical density value(AOD)was calculated by Plus6.0 software,and spss25.0statistical software was used for data analysis again.Results :1.HE staining results : No significant pathological changes were observed in the renal tissues of Sham group,and there was no significant difference among groups at 3,7,or 14 days(P>0.05).The UUO group showed a slightly higher tis than Sham group at each corresponding time point,which was statistically significant(P<0.05).In the UUO group,on the 3rd day of obstruction,the morphology of obstructed kidneys changed,and the renal tubules slowly appeared damaged,and on the 7th and 14 th days of obstruction,the renal tubules gradually dilated,and some The tubules appeared atrophy destruction,inflammatory cell infiltration,and the tubulointerstitial injury score(TIS)gradually increased,which was statistically significant(P<0.05)at 3 days,7 days,and 14 days within the group.At each corresponding time point,the Que group showed a little decrease in tis compared with the UUO group,which was statistically significant(P<0.05).2.Masson staining results : The green stained area of interstitial collagen fibrils in Sham group was small,and the difference was not statistically significant at 3,7,or 14 days within groups(P>0.05).At each corresponding time point,the renal interstitial collagen fibril green stained slightly higher in UUO group than sham group,and the difference was statistically significant(P<0.05).At day 3 of infarction,interstitial fibrils began to appear,and at days 7,14 of obstruction,renal interstitial collagen fibril green stained Area increased stepwise,and the RIF index increased gradually,with statistically significant differences at 3 days,7 days,and 14 days within the group(P<0.05).At each corresponding time point,the Que group had a lower RIF index than the UUO group,with statistically significant differences(P<0.05).3.Immunohistochemical results:(1)The p-AMPKα protein was mainly expressed in the cytoplasm and nucleus of tubular epithelial cells,while the p-AMPKα protein was abundantly expressed in the Sham group,and the differences were not statistically significant at 3 days,7days,and 14 days within the group(P>0.05).At each corresponding time point,the expression of p-AMPKα protein was reduced,and the differences were statistically significant(P<0.05)in the UUO group compared with the Sham group;UUO Group rats showed a gradual decrease in the expression of p-AMPKα protein with the extension of obstruction time.The differences were statistically significant at 3 days,7 days,and 14 days in the UUO group(P<0.05).At each corresponding time point,the expression of p-AMPKα protein increased in the Que group compared with the UUO group,and the differences were statistically significant(P<0.05).(2)TGF-β1 protein expression was mainly in tubular epithelial cells,Smad3 protein expression was mainly in the cytoplasm and nucleus of tubular epithelial cells,TGF-β1 and Smad3 proteins were less expressed in the Sham group,and there were no significant differences among groups at 3 days,7 days,and 14 days(P>0.05).At each corresponding time point,UUO The expression of TGF-β1 and Smad3 proteins increased in the UUO group compared with the Sham group,with statistically significant differences(P<0.05).The expression of TGF-β1 and Smad3 proteins increased gradually in the UUO group as the obstruction time extended.The UUO group differed from the UUO group at 3,7,and 14 days(P<0.05).At each corresponding time point,the Que group differed from the UUO group The protein expressions of TGF-β1 and Smad3 were reduced compared with the other groups,and the differences were statistically significant(P<0.05).Conclusions :1.The decrease of p-AMPKα protein expression and the increase of TGF-β1 and Smad3 protein expression suggest that AMPK and TGF-β1/Smad3 signaling pathway are involved in the process of renal interstitial fibrosis.2.After treatment with quercetin,the expression of p-AMPKα protein increased,while the expression of TGF-β1 and Smad3 protein decreased,indicating that quercetin may activate AMPK,act on TGF-β1 signaling pathway,inhibit Smad3 phosphorylation,resulting in the decrease of Smad3 expression,thereby alleviating renal fibrosis. |
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