| Objective:This study was designed to evaluat the effection of Trimetazidine hydrochloride on renal pathological changes of renal interstitial fibrosis (RIF) induced by unilateral ureteral obstruction (UUO).And the expression of transforming growth factor (TGF-β1) in renal tissue.The content of vascular smooth muscle actin-a (a-SMA), malondialdehyde (MDA),and superoxide dismutase (SOD) were compared with or without Trimetazidine hydrochloride.Methods:54 clean-level Wistar rats were envolved in the study (purchased from the Experimental Animal Center, Shandong University), and were divided into three groups randomly, sham-operated group(group A), model group(group B), and Trimetazidine hydrochloride-treated group(group C). After operation to Wistar rats in roup C Trimetazidine hydrochloride was given immediately(5mg/kg/d)(lml), and normal saline (1ml) was given in group A and B.6 rats were killed On 7 days,14 days and 21 days after surgery,the rats were killed in each group.First,the rats were killed after getting blood from the heart,and the blood was used to be detected for the value of urea nitrogen and creatinine by using biochemical automatic analyzer,then rat kidneys were removed rapidly. 100mg renal tissue was got and washed in ice-saline.After that,the renal tissue was placed into the glass homogenizer,4ml ice-saline was added and ground into homogenate,then the homogenate was centrifuged on the contention of 4℃,4000rpm for 10 minutes.200μl supernatant was taken and storaged in -80℃refrigerator. When specimens collection is completed, we detected the content of TGF-β1, SOD and MDA according to ELISA kit steps. After the process of formalin-fixed, dehydrated, transparent,paraffin-embedded and sliced,the remaining kidney tissue was delt with conventional staining and Masson's trichrome to observe the morphological changes,and the immunohistochemical method was used to test the content of vascular smooth muscle actin-a (a-SMA).Results:1. BUN, Cr, Blood urea nitrogen and serum creatinine did not change significantly in sham-operated group, model group and treatment group on 7 days,14 days,21 days. The difference in groups and between groups were not statistically significance (p >0.05).2. Pathologic results:The following we can see with naked eye,The rats kidney was dull-red in sham-operated groups, and the size and morphology were normal. The ligated kidneys was pale, enlarged obviously and sexy capsule in group B and group C. In the capsule,there is brown turbid liquid,the renal pelvis expansed remarkably and the renal cortical become thinner.Light microscope, HE staining showed that renal tubular epithelial cell degeneration and infiltration of foci-like mononuclear cell can occasionally be seen in group A;in group B,we can see diffused vacuolar degeneration of renal tubular epithelial cells, and some proteins or cells within the lumen tube,and part of the tubular expansion, contraction, and occasionally see necrosis,and infiltration of inflammatory cell, and renal cortical thinning.Compared with group B pathological changes were lighter in group C. We scored the injury of Renal tubular refer to , that is observed under the microscope (×200 times, HE stain), based on each field of vision of renal tubular disease (renal tubular expansion, degeneration, necrosis, etc.), involving the number of small tubes to rate.0 points,tubular structure was normal, no any lesion.1 points,the number of small tubular disease<3.2 points, a small number of tubular disease involving 3-5.3 points, the number of small tubular disease>5 or tubular structure disappeared. Each slice randomly selected 10 non-overlapping field of vision at the cortical tubules, each select five tissue sections of rat and finally averaged.Carried out on the HE staining of renal interstitial damage index score,we found that as time going, renal interstitial damage index score gradually increased.After Trimetazidine hydrochloride treatment, renal interstitial damage index score were decreased at each time point, But on 21 day, the difference between group C and group B was not statistically significant (p> 0.05).Masson's staining:It showed that the model group can be seen a large number of blue-stained interstitial collagen fibers proliferation,but glomerular lesions is not obvious.Compared with group B,renal interstitial collagen fibers in an area of positive staining decreased significantly in group C(p<0.05), the difference between groups has statistically significance. Tubulointerstitial fibrosis index can be measued according to, Masson trichrome, using MPIAS-500 multimedia color pathological graphic analysis system, and intake the image on the condition of 200 times, and selecte 10 non-overlapping field of vision in cortex randomly, then determination of renal tubular interstitial fibrosis in tubulointerstitial area accounts for a percentage of the total area and take the average. Interstitial fibrosis index (%)=(interstitial fibrosis area/tubulointerstitial area)×100%.3.Immunohistochemical staining results:Compared with group A, expression in the other two groups was significantly higher (p<0.05).After Trimetazidine hydrochloride treatment, the expression of a-SMA decreased significantly (p<0.05). In group A, only vascular smooth muscle cells were positive,and the glomerular and tubular epithelial cells were unobviously colored. Expression in group B mainly concentrate on renal interstitial sites,surrounding renal tubules and interstitial renal fibrosis. In group C,we can see a small amount of expression scattered throughout the renal interstitial fibrosis sites.4.ELISA test results (kidney tissue):(1) TGF-β1,On 7 days,14 days,21 days, in obstructed kidneys,compared with the sham-operated group,the levels of TGF-β1 increased in model group (p <0.05).Compared with the group A, the levels of TGF-β1 increased in group C (p <0.05).Compared with group B, the levels of TGF-β1 decreased in group C(p<0.05).(2) SOD, MDA, On 7 days,14 days,21 days, in obstructed kidneys,compared with group A, the content of MDA increased and the content of SOD decreased in group B(p<0.05); Compared with group A, the content of MDA increased and the content of SOD decreased in group C(p<0.05); Compared with group B, the content of MDA decreased and the content of SOD increased in group C(p<0.05).Conclusion:1. Unilateral ureteral ligation is a simple and effective method in modeling a rat model of renal interstitial fibrosis.2. Trimetazidine hydrochloride has no effect on renal function.3. The pathologic of the obstructed kidneys of rats is lighter and the collagen fiber area decreased in Masson's staining in Trimetazidine hydrochloride treated-group, indicate that Trimetazidine hydrochloride can lighten renal fibrosis.4. Trimetazidine hydrochloride can reduce the a-SMA positive area,and it may be related to the inhibition of differentiation of renal tubular epithelial cells, thus inhibit the expression of a-SMA in renal interstitial, and delay the renal interstitial fibrosis process.5. As time of obstruction going, the expression of TGF-β1 increased; TGF-β1 is the key cytokines to promote the formation and development of renal fibrosis; Trimetazidine hydrochloride can inhibit its expression in kidney.6. Oxidative stress plays an important role in the process of renal interstitial fibrosis;Trimetazidine hydrochloride can reduce the expression of MDA and increase the expression of SOD; It can prevent the formation of oxygen free radicals after injury and increased renal clearance of oxygen free radicals; Then reduce the toxic effects of oxygen free radicals on the cell membrane to protect the cell function, and slow the process of renal interstitial fibrosis. |
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