| Objective: To observe the expression of marker for renal tubular epithelial cell phenotypic(α-Smooth muscle actin)α-SMA and detect the change of regulation signaling pathways(tumor necrosis factor)TNF-α and(nuclear factor) NF-κB which are related to α-SMA. To Investigate the mechanism and the role of Chinese herbs shenluotong on inhibiting renal tubular epithelial cell phenotype transformation and provide the evidence for the clinical application.Methods: Healthy female Wistar rats 48 weight about 180±20g were used in this study. The experimental rats eat and drink freely, temperature controled in(22±2)℃. After adaptive feeding one week, 48 Wistar rats randomly divided into Sham group, UUO group,EPL group, SLT group, and each group with 12 rats. After anesthesia with 10% chloral hydrate, the rats were sliced through the left middle abdominal skin, separated the left ureter, ligated respectively on ureteral 1/3 and middle 1/3 ligation with silk, cut off the ureter. Then sutured in skin by layer to layer. The rats in Sham group were only separated the left ureter but not ligated and cutted off the ureter. After operation, the rats were treated with 100mg/(kg.d) Eplerenone in fodder in EPL group and 26g/kg in SLT group; others in sham and UUO group administered with the same volume of saline once a day. Continuous intervention was for 10 days. Beheaded after weigh the rats 10 days later, retain blood specimens for detection.Cut down the obstruction kidney, weigh the tissue after remove integument, part of the kidneys were fixed by 4% paraformaldehyde and part of the kidney keep at-70 ℃for extracting RNA and protein. The expression of TNF-α,NF-κB and α-SMA were detected by the way of immunohistochemistry and Western blot.Results:1 The general situation in ratsThe general situation of Sham group’s rats were in good condition:daily diet and water were stable, the growth of weight was gradual, hair was shiny,activity was agile, and response to external stimuli was sensitive. The water intake, food intake and weight were down-regulated in UUO group. Compared with UUO group,obstructed kidney weight and kidney weight/body weight were also decreased significantly(P<0.01, P<0.05) in EPL group and SLT group rats.2 The change of Pathomorphology in renal tissueHE staining showed that tubular epithelial cells arranged regularly and it had few inflammation cells in Sham group. There were a large number of inflammation cells infiltration, and renal tubular epithelial cells were denatured, shed and turgid in UUO group. The inflammation cell infiltration was significantly reduced and renal tubular epithelial cells were turgid in EPL and Shenluotong groups.Masson staining showed that a little collagen deposition in glomerular/tubular basement membrane and renal interstitium in Sham group. The structure were disordered and tubular interstitial collagen were significantly increased in UUO group. There were some degree of collagen deposition in treatment groups, but it was significantly reduced compared with the UUO group.3 The expression of ImmunohistochemistryIn Sham group, the expression were weak of TNF-α and NF-κB mainly in renal tubular epithelial cells; In UUO group,the expression was enhanced; In ELP and SLT groups, the expression of TNF-α and NF-κB were significantly decreased in range and strength.In Sham group, the positive expression of α-SMA was only in vascular smooth muscle cells;In UUO group,the expression of α-SMA were remarkably increased in renal tubular epithelial cells and interstitial; Compared with the rats in Sham group, the expression of α-SMA were enhanced in both EPL group and SLT group, but compared with UUO group was reduced obviously.4 The results of Real-time PCRCompared of the rats in Sham group,the m RNA expression of TNF-α were enhanced obviously in UUO group;Compared with the rats in UUO group, the m RNA expression of TNF-α were down-regulated significantly in EPL group and SLT group.5 The expression of Western blotUsing Western blot method to detect the expression of TNF-α and NF-κB signaling pathways, the expression of TNF-α and NF-κB were up-regulated obviously in UUO group, the difference was statistically significant(P<0.01); Compared with UUO group, the expression of TNF-α and NF-κB were down-regulated in both EPL group and SLT group, the difference was statistically significant(P<0.01).To detected the expression of α-SMA, the result showed that compared with the rats in Sham group it was up-regulated in UUO group, the difference was significant(P<0.01), compared with Sham group, the expression of α-SMA were enhanced in both EPL group and SLT group, but reduced obviously with UUO group.Conclusions:1 Obstructive nephropathy can induce the secrecion of inflammatory medium and stimulate the transformation of the renal tubular epithelial cell phenotype.2 Eplerenone can reduce the kidney injury of obstructive nephropathy by inhibit the effect of aldosterone.3 Shenluotong decoction has the function of inhibiting renal tubular epithelial cell phenotype transformation was related to down-regulating the expression of TNF-α and NF-κB. |
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