| Objective: Renal interstitial fibrosis is the common pathway by which almost all chronic kidney diseases progress into end-stage renal disease.The main performance of renal interstitial fibrosis is the tubular basement membrane thickening,the increment of renal interstitial components and the deposition of extracellular matrix(ECM),the pathological process refers to a variety of factors such as inflammatory reaction,oxidative stress,all kinds of cell factor and signaling pathway,cell apoptosis,fibroblast proliferation and activation, and mesenchymal transdifferentiation( EMT), etc. Heme oxygenase( HO), is the initial enzyme and restriction enzyme of heme in the procession of catabolism.In the past research,we found that HO has three kinds of isozyme HO-1, HO-2, HO-3, among them,HO-1,which is very easy to be induced expression,can play a variety of biological effects.HO-1 existed in microsome in a variety of mammals tissue cells( including humans),in physiological state,HO-1 have trace expression in certain organ or tissue,maybe no expression,the strongest expression organ is the spleen. In the renal tissue,it mainly expressed the proximal and distal tubule in the cortex nephron and medullary collecting duct epithelial cells. Extensive research in the past have demonstrated that the protective effects of HO-1 and heme degradation products of carbon monoxide, ferrousion, biliverdin included anti-inflammation, anti-oxidative stress, anti-apoptosis,the regulation of cell proliferation,and etc.While renal interstitial fibrosis also relates to the effect of multiple factors such as anti-inflammatory, oxidative stress, cell apoptosis.and so on.It indicates that HO-1 may play a role in anti renal interstitial fibrosis through its biological function.Foreign research have indicated that the degree of renal tubular epithelial myofibroblast transdifferentiation(EMT) and the renal interstitial fibrosis significantly increased in animal model which was shortage of HO-1,while muscle fibroblasts which are generated by EMT can express α-smooth muscle actin(α-SMA). Domestic research showed that the induction of HO-1 expression could decreased the level of monocyte chemotactic protein 1(MCP-1)and transforming growth factorβ1(TGF-β1),then delay the progression of renal interstitial fibrosis.Therefore,it maybe a method of delaying renal interstitial fibrosis through the induction of high expression in HO-1.The effect of statins include lipid lowering, anti-hypertension, anti proliferation, plaque stability,etc,which has become the commonly used medicine in cardiovascular and cerebrovascular disease in clinical treatment.The research showed that statins were able to alleviate renal ischemia reperfusion injury through inducing high expression of HO-1. Now there has been no report that if statins can induce the expression of HO-1 and effect the level of TGF- β1 and α-SMA in UUO, then alleviate renal interstitial fibrosis. The experiment is performed in unilateral ureteral obstruction rat model which leads to renal interstitial fibrosis, through intervention of rosuvastatin drug, combines with HE staining, Masson staining, immunohistochemical staining method, observes the expression of HO-1, TGF-β1, α-SMA in each experimental group in different time, then explores the new target of statins for anti renal interstitial fibrosis, provides new theory evidence for treatment of renal interstitial fibrosis in clinical.Method: Select 54 clean level healthy male Sprague-Dawley(SD) rats, body weight 180±20 g, after 1 week adaptive feeding, were randomly divided into three groups as follows: 1 sham operation group(group A), 2 unilateral ureteral obstruction group(group B),3 rosuvastatin treatment group(group C), with 18 rats in each group. 12 hours before the operation,the rats were forbidden to provide food and water. Operation process:A, B, C groups were anesthetized and disinfected,left ureter was exposed via a left abdominal incision. After layer by layer freed tissue,exposed the left kidney,found the left ureter, Group A was only separated left ureter as control.The mid-ureter was obstructed by two point ligation with 4-0 silk sutures, and then cut the ureter between the ligated two points to avoid retrograding infection in group B and group C. Each group administrated orally one day before the surgery, The group C were given rosuvastatin 10 mg/kg/d orally, the group A and group B were gavaged with isometric saline. Once a day, reveal rat’s weight per week, and adjust the administration according to their weight. The rats were allowed to eat and drink free. Randomly select 6 rats from each group respectively, and harvest their obstructed kidneys on day 3, 7, 14 after the UUO surgery. Slice the kidneys and place the slices into fixation solution, embed them in paraffin. The pathological changes were determined in the kidney tissues underwent operation(HE) staining, Masson staining, as well as immunohistochemistry to detect the expression of HO-1, TGF-β1, α-SMA. The result of immunohistochemistry would be analysed with the image analysis management system for semi-quantitative analysis. Statistical analysis software SPSS 20.0 was applied for statistical analysis of experimental data which was analyzed with one-way ANOVA, and displayed as mean ± standard deviation( x ±S),with P<0.05 as a statistical significance.Results: HE staining under light microscopy: group A, there was no abnormal signs;group B, After the third day of UUO, a small amount of inflammatory cells infiltration and mild expansion of renal tubules were found, as well as after the seventh day, marked dilation of renal tubule, epithelial cell were more degeneration and necrosis, renal interstitial infiltration of inflammatory cells obvious increased over the previous, and renal interstitial width significantly increased.The fourteenth day, it would be found that visible tubular atrophy was more, diffuse thickening of tubular basement membrane, shrinkage.group C, the same performance was found in renal interstitial and tubular with the same period comparing with group B. With the prolongation of obstruction changes gradually aggravated, however, they were still better than those in group B, but they were worse than those in group A. Masson staining under light microscopy :group A,there were no obvious collagen deposition in every period.group B and group C, after the third day of UUO, slight tubular expansion and increased renal interstitial collagenous fibers was found. After the seventh day and fourteenth day, the increase of renal tubular dilation, collagenous fibers,and the degree of interstitial fibrosis was found compared with the third day. and these phenomenon got worse as time went by, Compared with the same time, they were still better than those in group B, but they were worse than those in group A. The immunohistochemical results in renal tissue showed that:(1)Ho-1: group A, there were almost no expression in glomerular and tubular; group B, Increased expression of renal tubular epithelial cells and the expression height was found 3 days after operation, then the expression level gradually decreased with prolonged obstruction, and there was a significant difference(P<0.05) between group A and group B on the same time point.group C, The same trend with the expression of group B, the level of group C was more than group B and more than group A, and there was a significant difference(P<0.05) on the same time point.(2)TGF-β1:group A, only trace amount of TGF-β1 expression was detected; group B, the expression level of TGF-β1 went up in a time dependent manner, there was a significant difference(P<0.05)comparing with group A on the same time point; group C, there were the same trend between group B and group C, the level of group C was less than group B and more than group A,and there was a significant difference(P<0.05).(3)α-SMA:in group A, almost no expression was detected in renal tubular epithelial cells and interstitial.in group B, α-SMA significantly increased with the obstruction, there were statistically significant difference(P<0.05)compared to group A on the same time point; in group C, there were the same trend between group B and group C, the level of group C was less than group B and more than group A,and there was a significant difference(P<0.05).Conclusion: The expression of TGF-β1 and α-SMA were gradually increased with prolonged obstruction in UUO rats model.In the early stage of UUO, the expression of HO-1 increased obviously and decreased gradually with prolonged obstruction.Rosuvastatin increased the expression of HO-1 and relieved renal interstitial fibrosis.It maybe indicate the mechanism of rosuvastatin inhibited renal interstitial fibrosis should be at least in part on inducing the expression of HO-1 and decreasing the expression of TGF-β1 and α-SMA. |
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