| Objective:1.To observe the ameliorating effect of nebivolol on tubulointerstitial fibrosis(TIF)in rats with unilateral ureteral obstruction(UUO).2.Based on proteomic bioinformatics analysis,the mechanism of the improvement of TIF by nebivolol was preliminarily discussed,so as to provide theoretical basis for its clinical application.Methods:Male SD rats at about 220 g were selected.After anesthesia,abdomens were opened and left ureter was ligated twice to prepare the TIF model of UUO rats.The left ureter of rats in the sham operation group was only separated but not ligated.The following experiments were carried out:1.Effects of different durations of administration of nebivolol(10 mg/kg/day)on kidney function,structure and α-SMA protein expression in UUO ratsThe modeling duration of UUO rats was divided into 7 d,14 d,21 d and 28 d,and each time period was divided into the following groups:(1)sham operation group(constant volume of distilled water,ig);(2)UUO group(constant volume distilled water,ig);(3)UUO + nebivolol group(nebivolol 10 mg/kg/day,ig).The 24 h urine of rats in each group was collected 1 d before the end of the experiment to detect urinary creatinine(Ucr)and urinary protein.Then the rats were anesthetized and blood samples were collected from the abdominal aorta to detect serum creatinine(Scr)and blood urea nitrogen(BUN),and creatinine clearance rate(Ccr)was calculated.The obstructed kidney were separated,photographed and weighed,and part of the kidney were embedded in paraffin wax with 4%paraformaldehyde: the kidney structure was observed by HE staining,and kidney fibrosis was observed by Masson staining;The other part of kidney was frozen in liquid nitrogen and stored at-80℃: the expression of α-SMA protein was detected by Western blot assay.2.Effects of different concentrations of nebivolol on kidney function,structure andα-SMA protein expression in UUO rats after 21 d administrationThe modeling time of UUO rats was 21 d,and the rats were divided into the following groups:(1)sham operation group(constant volume distilled water,ig);(2)sham operation +nebivolol group(nebivolol 10 mg/kg/day,ig);(3)UUO group(constant volume distilled water,ig);(4)UUO + low concentration nebivolol group(nebivolol 1 mg/kg/day,ig);(5)UUO + medium concentration nebivolol group(nebivolol 3 mg/kg/day,ig);(6)UUO +high concentration nebivolol group(nebivolol 10 mg/kg/day,ig).The 24 h urine of rats in each group was collected 1 d before the end of the experiment to detect Ucr and urinary protein.Then the rats were anesthetized and blood samples were collected from the abdominal aorta to detect Scr and BUN,and Ccr was calculated.The obstructed kidney were separated,photographed and weighed,and part of the kidney were embedded in paraffin wax with 4% paraformaldehyde: the kidney structure was observed by HE staining,and kidney fibrosis was observed by Masson staining;The other part of kidney was frozen in liquid nitrogen and stored at-80℃: the expression of α-SMA protein was detected by Western blot assay.3.Nebivolol improved the TIF mechanism in UUO ratsAccording to the above experimental results,the rats in the sham operation group,the UUO group and the UUO + high concentration nebivolol(10 mg/kg/day)group were selected for further TMT proteomics quantitative analysis of the kidney of the rats on the obstruction side.Results:1.Effects of different administration time of nebivolol(10 mg/kg/day)on kidney function,structure and α-SMA protein expression in UUO rats(1)Compared with sham operation group at the same period,the body weight of rats in UUO group decreased significantly from 14 d with the prolongation of ureteral obstruction(p <0.05),and nebivolol(10 mg/kg/day)had no significant effect.(2)Compared with the sham operation group at the same period,Scr of UUO rats at different modeling time had a tendency to increase,but the difference was not statistically significant,while Ccr decreased significantly and BUN increased significantly after 14 d of modeling(p <0.05),and there was no significant change in urinary protein content at different time points.Nebivolol(10 mg/kg/day)for 14 d significantly decreased Scr and increased Ccr(p <0.05),but other administration time had no significant effect on kidney function in UUO rats.(3)Compared with the sham operation group at the same period,the kidney organ index of the obstructed side of UUO rats was significantly increased at 7 d after modeling(p <0.05),and then gradually decreased.With the prolongation of ureteral obstruction time,the kidney cortex of UUO rats gradually thinned and a large number of kidney tubule atrophy disappeared(HE staining);The tubulointerstitial fibrosis index was significantly increased from 7 d(p <0.05,Masson staining);The expression of α-SMA protein in kidney was also significantly increased from 7 d(p <0.05,Western blot test).Nebivolol(10mg/kg/day)reduced kidney organ index in the obstructed side of UUO rats only after 14 d of administration(p <0.05),the increase of tubulointerstitial fibrosis index and α-SMA protein expression in UUO rats were significantly inhibited after 21 d and 28 d administration,especially at 21 d(p <0.05).2.Effects of different concentrations of nebivolol on kidney function,structure andα-SMA protein expression in UUO rats after 21 d administration(1)Nebivolol 10 mg/kg/day had no significant effect on the body weight of rats in the sham operation group.The body weight of UUO rats decreased after 21 d modeling(p<0.05),there was no significant effect of different concentrations of nebivolol(1,3,10mg/kg/day)on 21 d.(2)Nebivolol 10 mg/kg/day had no significant effect on Scr,Ccr and BUN of rats in the sham operation group.After 21 d of modeling,Scr of UUO rats increased,Ccr decreased significantly,BUN increased significantly(p <0.05),and urinary protein content had no significant change.The above indexes(Scr,Ccr,BUN)were not significantly affected by different concentrations of nebivolol(1,3,10 mg/kg/day).(3)Nebivolol 10 mg/kg/day had no significant effect on kidney organ index and kidney structure of rats in sham operation group.Compared with the sham operation group,there was no significant change in kidney organ index of UUO rats after 21 d of modeling;The kidney cortex was thinned and tubules were atrophied(HE staining);The tubulointerstitial fibrosis index was significantly increased(p <0.05,Masson staining);α-SMA protein expression was also significantly increased(p <0.05,Western blot test).The expression of α-SMA protein in UUO rats was decreased by different concentrations of nebivolol(1,3,10 mg/kg/day),and the high concentration of nebivolol(10 mg/kg/day)had the most significant effect(p <0.05),and only high concentration of nebivolol(10mg/kg/day)significantly reduced tubulointerstitial fibrosis index in UUO rats(p <0.05).3.Nebivolol improved the TIF mechanism in UUO ratsCompared with the sham operation group,a total of 3030 proteins were significantly changed in the UUO group.Compared with 21 d of UUO modeling,a total of 479 proteins were significantly changed in the 10 mg/kg/day administration group of nebivolol.A total of 179 cross-regulated and reverse regulated proteins were screened among the three groups,nebviolol(10 mg/kg/day)was significantly up-regulated by 44 proteins and down-regulated by 135 proteins after 21 d of administration,among which Col-IIIα1,FN1,CRIP1 and HSPA5 may be related to its anti-TIF.Conclusion:Nebivolol(10 mg/kg/day)could significantly improve TIF in UUO rats after 21 d administration,and its effects involved multiple targets,possibly related to Col-IIIα1,FN1,CRIP1,HSPA5 and other proteins. |
PDF Full Text Request