| AbstractObjective:The animal model of renal interstitial fibrosis was induced with the method of unilateral ureteral obstruction(UUO),to observe the effects of Shenxiankang granules on the kidney functions,tubule injury index,the degree of renal interstitial fibrosis and wnt/β-catenin signaling pathway with benazepril as positive reference drug.Exploring the therapeutical effects and its possible mechanism on renal interstitial fibrosis.Methods:Sixty male SD rats were randomly divided into sham operated group(SOR group),UUO model group(UUO group),UUO model treated with benazepril group(Lotensin group) and UUO model treated with Shenxiankang Granules group(Shenxiankang group).Every group has fifteen rats.The rats were iduced by unilateral ureteral obstruction except SOR group,which only received pute celiotomy without ureteral ligation.From the first day after operation,Shenxiankang group and Lotensin group rats were fed with Shenxiankang Granules(4.2g/kg?d) and benazepril(10mg/kg?d) respectively through intragastric administration.Both SOR group and UUO group were received equal distilled water through intragastric administration.Five rats in each guoup were randomly selected and killed respectively on the day 7, day 14, day 21 after operation.Recorded the body weight and kidney weight,and calculated the ratio of kidney weight to body weight(KW/BW).Measured the level of 24 hour urinary albumin,serum creatinine(Scr) and blood urea nitrogen(BUN).Observed the pathological changes in kidney by Masson and HE staining,the expression of wnt4,β-catenin and α-SMA protein detected by immunohistochemistry and western blot,the level of wnt4,β-catenin and α-SMA m RNA detected by Real time PCR.Results:1. Body weight and KW/BW:Compared with the SOR group,The KW/BW of all the other groups,which had been received the operation of UUO,was significantly increased at various time points(P<0.05),while the body weight was significantly decreased(P<0.05).Compared with the UUO group,the body weight of both Shenxiankang group and Lotensin group was significantly increased at various time points(P<0.05),and the KW/BW was significantly decreased on day 7 and day 14,while the KW/BW was significantly increased on day 21(P<0.05).There was no significant differences between the Shenxiankang group and Lotensin group on this item(P>0.05).2. 24 hour urinary albumin:To all groups,the difference in 24 hour urinary albumin was not statistically significant,for both intra-group comparison and the comparison among groups(P>0.05).3. Renal function:Compared with the SOR group,the level of both Scr and BUN of all the other groups after the operation of UUO was significantly increased(P<0.05).Compared with the UUO group,The level of both Scr and BUN in both Shenxiankang group and Lotensin group was significantly decreased(P<0.05).There was no significant differences between the Shenxiankang group and Lotensin group on this item(P>0.05).4. Pathological changes in kidney:HE and Masson staining showed that both glomerular and tubular morphology were normal at each time point and there was no pathological changes in SOR group.In the other groups,renal cortical became thin,and showed varying degrees of tubular dilatation,luminal atrophy or occlusion,epithelial cell degeneration and inflammatory cell infiltration,and collagen fibers in interstitial gradually increased until day 21.Compared with the SOR group,the tubulointerstitial damage index and collagen staining area in the other groups were significantly increased(P<0.05).Compared with the UUO group,The tubulointerstitial damage index and collagen staining area in both Shenxian kang group and Lotensin group were significantly decreased(P<0.05).There was no significant differences between the Shenxiankang group and Lotensin group on this item(P>0.05).5 Immunohistochemical staining5.1 α-SMA:A little expression of α-SMA,without significant changes with the prolonging of time(P>0.05),was observed on the vascular smooth muscle cells of glomeruli in SOR group,while expression of α-SMA,with gradually increase with the prolonging of time(P<0.05),was observed in the renal tubular epithelial cell plasm among the groups after the operation of UUO.Compared with UUO group,the expression of α-SMA in both Shenxiankang group and Lotensin group was significantly decreased(P<0.05).There was no significant differences between the Shenxiankang group and Lotensin group on this item(P>0.05).5.2 β-catenin and wnt4:A little expression of β-catenin and wnt4,without significant changes among time points(P>0.05),was observed on the basal part of tubular epithelial cells in SOR group,while the expression of β-catenin and wnt4,with gradually increase with the prolonging of time(P<0.05),was observed in the renal tubular epithelial cells among the groups after the operation of UUO.Compared with UUO group,The expression of β-catenin and wnt4 in both Shenxian kang group and Lotensin group were significantly decreased(P<0.05).There was no significant differences between the Shenxiankang group and Lotensin group on this item(P>0.05).5.3 Correlation analysis:There was a positive correlation between the expression of Wnt4 as well as β-catenin and the expression of α-SMA in renal tubulointerstitial(r = 0. 982,0.934;P<0.05). 6. Western blot detectionA little expression of Wnt4,α-SMA and β-catenin protein was observed in SOR group,while a gradually increased expression with the prolonging of time was observed in the UUO group(P<0.05).Compared with the UUO group,the expression of these proteins in both Shenxiankang group and Lotensin group was significantly decreased(P<0.05).There was no significant differences between the Shenxiankang group and Lotensin group on this item(P>0.05).7. Realtime PCR detectionA little expression of Wnt4,α-SMA and β-catenin m RNA was observed in SOR group,while it gradually increased with the prolonging of time after the operation of UUO(P<0.05). Compared with the UUO group,the expression of these m RNA in both Shenxiankang group and Lotensin group was significantly decreased(P<0.05).There was no significant differences between the Shenxiankang group and Lotensin group on this item(P>0.05).conclusion1. Shenxiankang Granules can reduce Scr,BUN,tubular injury index and collagen fiber area in UUO rats,and the curative effect was approximately equal to benazepril.2. Wnt/β-catenin signaling increased in the kidney of UUO rats,and the expression of its key signaling,Wnt4 and β-catenin,was associated with the degree of renal interstitial fibrosis.3. Shenxiankang Granules can reduce the expression of α-SMA and collagen fibers of kidney in UUO rats and delay the progression of renal interstitial fibrosis,and the possible mechanism may through inhibiting Wnt/β-catenin signaling pathway. |
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