| Objective:In this paper,the renal protective effects of lumbrokinase extract on kidney fibrosis in rat models were observed through biochemical assays,pathomorphological detection,immunohistochemistry,and computer image semi-quantitative analysis.The mechanism provides a reliable base for the treatment of renal fibrosis by Chinese medicine for better clinical guidance.Methods:In the first part,50 healthy SD rats were randomly divided into five groups: normal group,UUO model group,low-dose lumbrokinase group,medium-dose lumbrokinase group,high-dose lumbrokinase group.There are 10 rats in each group,general health dietary fitness was maintained for one week.Unilateral ureteral ligation(UUO)models were used and 14 days after the model was cast to make a model of renal interstitial fibrosis.In the second part,the drug intervention was performed on the treatment group by the gavage method from the second day after the model was established.The dose was calculated as the ratio of body surface area per kilogram of body weight between humans and rats.The low dose group of lumbrokinase was given 90,000 lumbrokinase.U·kg-1,mid-dose lumbrokinase dose group was given 180,000 U·kg-1,lumbrakinase high dose group was given 360,000 U·kg-1,normal group and UUO model group daily physiological saline 1ml·100g-1,Administration was started on the second day after the model was established,and the stomach was administered once a day for 14 consecutive days.In the third part,on the 14 th day after the operation,the abdominal cavity was anesthetized.Abdominal aorta blood was rapidly taken and the ligature side of the kidney was extracted into formalin to prepare a wax block.Serum urea nitrogen(BUN)and creatinine(Scr)were measured with a kit.HE staining and Masson staining were used to observe the pathological changes of renal tissue.TGF-β1 and α-SMA were detected by immunohistochemistry.Semi-quantitative analysis of PAI-1,Col-1,and Nox4 protein expression were used in this part.In the forth part,The experimental data of each group of rats were analyzed and processed by SPSS 17.0 statistical software.The measurement data is firstly analyzed by one-way ANOVA.The differences between the groups were statistically significant and the samples were in normal distribution.An independent sample t-test was performed.The results were expressed as mean±standard deviation(`x±s),and p<0.05 was considered statistically significant.According to the analysis results,the protective effect and possible mechanism of lumbrokinase on renal interstitial fibrosis were explored.Results:1.Compared with the normal group,serum creatinine(Scr)and urea nitrogen(BUN)increased slightly in the UUO model group,but there was no statistical difference(p>0.05).Compared with the UUO model group,there was no significant difference in the Scr and BUN parameters between the groups treated with lumbrokinase(p>0.05).2.Microscopy results showed no visible changes in the kidneys of the sham group.In the UUO model group and the lumbrokinase group,the size of the kidneys in the obstructed side increased to varying degrees,and hydronephrosis,thinning of the renal parenchyma,or cystic symphysis were evident.The ureters were thicker above the ligation site.There was a turbid urine with a tan color,and the renal capsule was thickened significantly,suggesting that the unilateral ureteral obstruction model was successfully established.HE staining showed normal renal tissue structure,no expansion and atrophy of renal tubules and collecting ducts,interstitial fibrous tissue hyperplasia,and inflammatory cell infiltration;In the UUO model group,the renal pelvis dilated,the renal parenchyma became thinner,a few normal kidney tissues remained,some glomeruli shrank,most renal tubules and collecting ducts shrunk or even disappeared,fibrosis occurred in the interstitial fibrous tissue,and there were more lymphoid and pulpy tissues,plasma cells,neutrophils,visible abscess formation,partial renal pelvic urothelial hyperplasia;Compared with the model group,the tubular atrophy and inflammatory cell infiltration in the lumbrokinase group were lower than those in the model group.The renal tubules and the collecting tube were slightly atrophied,and the fibrosis degree of the interstitial fibrous tissue was dose-dependently reduced.Masson staining showed that the normal group stained mainly in the glomerular basement membrane,Bowman’s capsule,mesangial area,and tubular basement membrane.No blue collagen fibers were found in the interstitium surrounding the tubules.Masson staining of the UUO model group showed more glomeruli,renal tubules,and collecting ducts.The spacing was widened.The interstitial collagen fibers were more proliferated,the blue collagen fibers were stained,and the collagen fibers were thicker;In the lumbrokinase-administered group,the blue staining area was reduced in a dose-dependent manner compared with the model group.3.Immunohistochemical results showed that there were no obvious pathological changes in the normal group.A large number of positive reaction points were seen in the UUO model group.The positive expression of TGF-β1,α-SMA,Col-Ⅰ,PAI-1 and Nox4 were significantly increased.(p<0.05).Compared with the UUO model group,the positive expression of each dose of lumbrokinase was dose-dependently reduced(p<0.05).Conclusions:The experimental results show that lumbrokinase can protect renal interstitial fibrosis in rats with unilateral ureteral obstruction to a certain extent.The mechanism may be to delay the progression of renal fibrosis by inhibiting the expression of pro-fibrogenic factors TGF-β1,α-SMA,PAI-1 and Nox4,and the synthesis of Col-Ⅰ collagen. |
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