| Object: To observe the effect of hirudin on the expression of TGF-?1,Col3,?-SMA,FN protein and p-smad2 and p-smad3 in renal stroma of rats with unilateral ureteral obstruction and to explore its mechanism.Methods: 60 healthy male SD rats weighed 180±20g.After 1 week of adaptive feeding,10 mice were randomly divided into the SOR group and 50 mice were randomly divided into the model group.There were 5 groups in the model group: 10 in the model group(UUO group),10 in the low dose hirudin group(UUO+LI group),10 in the medium dose hirudin group(UUO+MI group),10 in the high dose hirudin group(UUO+HI group),and 10 in the lotimsin group(UUO+LOT group).All rats in the model group underwent left ureteral ligation,while those in the sham group had to dissociate the left ureter without ligation.After the operation,each rat was injected with 200,000 units of penicillin,and continued anti-infection treatment for 3 days.From the first day after surgery,UUO + LI group to subcutaneous injections of hirudin 10IU·kg-1·d-1,and UUO + MI group to subcutaneous injections of hirudin 20IU·kg-1·d-1,UUO+HI group to subcutaneous injections of hirudin 40IU·kg-1·d-1,UUO+LOT group on postoperative day 1 give lotensin 1mg·kg-1·d-1 lavage,control group and model group to subcutaneous injection amount of saline solution,injection for 14 days in a row,the experiment gives ordinary feed,free water.All rats in each group were sacrificed on the 14 th day after the operation.5ml-7ml blood samples were collected from the inferior ventral aorta to detect serum Scr and BUN.The left kidney was removed for HE staining.PCR and Western Blot were used to detect the expression of TGF-?/Smad signaling pathway in renal tissues.Results: 1.Biochemical CriterionThe results of serum creatinine in each group were as follows: compared with SOR group,serum creatinine in UUO group was increased after operation(P < 0.05).The serum creatinine level of other intervention groups showed an increasing trend,but it was not significant(P > 0.05).Compared with the UUO group,the UUO+LI group had a decreasing trend,but not a significant one(P > 0.05),the serum creatinine level of the other intervention groups significantly decreased(P < 0.01).Urea nitrogen levels in the UUO group were significantly higher than those in the SOR group(P<0.05);The BUN levels of rats in UUO+LOT group were significantly increased(P<0.01),other intervention groups showed an increasing trend,but not significant.Compared with UUO group,urea nitrogen in UUO+MI group decreased significantly(P<0.05),UUO+LI group and UUO+HI group showed a downward trend in urea nitrogen,but it was not significant(P>0.05).2.Pathology Observation 1)Perusal: The kidney in SOR group was normal in size and shape and dark red in color.The obstructed kidney of rats in the UUO group was significantly larger in size and dark in color.The surface of the kidney was uneven,the capsule was significantly thickened,and the renal pelvis was dilated.The macroscopic manifestations of the obstructed kidney in the intervention group were similar to that in the UUO group,but all of them were relieved to different degrees.2)HE dyeing: There were no obvious pathological changes in the kidney of rats in SOR group,while in UUO group,there were denaturation and necrosis of epithelial cells in renal tubules,dilatation or atrophy of renal tubules,infiltration of renal interstitial inflammatory cells and hyperplasia of fibrous tissue.Compared with the UUO group,all drug intervention groups were alleviated to different degrees.3.RT-PCR Detection 1)By establishing the rat UUO model,it was found that the relative expression of TGF-?1 protein in UUO+LOT group increased significantly compared with SOR group(P<0.05),the relative expression of TGF-?1 protein in other drug intervention groups and UUO group was significantly increased(P<0.01).Compared with the UUO group,the relative expression of TGF-?1 protein in UUO+LI group and UUO+MI group was significantly decreased(P<0.05).2)By establishing the rat UUO model,it was found that the relative expression of ?-SMA in the UUO+HI group significantly increased compared with the SOR group(P<0.01),the relative expression of ?-SMA in other drug intervention groups and UUO group was significantly increased(P<0.05).Compared with the UUO group,the relative expression of transsma was significantly decreased in all the drug intervention groups(P<0.05).3)By establishing the rat UUO model,it was found that the relative expression of FN in the UUO+LOT group increased significantly compared with the SOR group(P<0.05),the relative expression of FN in other drug intervention groups and UUO group was significantly increased(P<0.01).Compared with the UUO group,the relative expression of FN in UUO+LI group and UUO+MI group decreased significantly(P<0.05).4)By establishing the rat UUO model,it was found that the relative expression of Col3 in UUO group and UUO+LI group increased significantly compared with SOR group(P<0.01),UUO+MI significantly increased(P<0.05).Compared with the UUO group,UUO+LI group significantly decreased the expression level(P<0.05),UUO+MI group,UUO+HI group and UUO+LOT group significantly reduced the relative expression of Col3(P<0.01).4.Western Blot Detection 1)Through the establishment of the rat UUO model,it was found that the relative expression of TGF-?1 protein in the renal tissues of the rats increased after the model construction.Compared with SOR group,TGF-?1 protein expression was significantly increased in UUO group(P<0.01),UUO+MI group significantly increased(P<0.05),TGF-?1 protein in other groups showed an increasing trend,but no significant difference(P>0.05).Compared with the UUO group,TGF-?1 protein expression was significantly decreased in the UUO+LI group(P<0.05),UUO+HI group and UUO+LOT group significantly decreased the expression(P<0.01).2)It was found that the relative expression of UUO,UUO+LI and UUO+MI significantly increased(P<0.01),the relative expression of ?-SMA protein in UUO+LOT group was significantly increased(P<0.05).Compared with the UUO group,the relative expression of UUO+LI group was significantly decreased(P<0.05),the expression of ?-SMA protein decreased significantly in other treatment groups,showing a statistically significant difference(P<0.01).3)The expression of Col3 protein in UUO group and UUO+HI group increased significantly compared with SOR group(P<0.01),the relative expression of histones in UUO+LI group and UUO+LOT group increased significantly(P<0.05).Compared with the UUO group,UUO+LI histone expression was significantly decreased(P<0.05),the relative expression levels of histones in UUO+MI group and UUO+LOT group were significantly decreased(P<0.01).4)By establishing the rat UUO model,it was found that the expression of p-smad2 protein in the UUO group was significantly increased compared with that in the SOR group(P<0.01),histone expression levels of UUO+LI,UUO+MI and UUO+LOT increased significantly(P<0.01);UUO+HI histone expression increased significantly(P<0.05).Compared with the UUO group,p-smad2 protein expression was significantly decreased in all the intervention groups(P<0.01)..5)Through the establishment of rat UUO model,it was found that compared with SOR group,the expression of p-smad3 protein increased significantly in UUO group and each drug intervention group(P<0.01).Compared with UUO group,the relative expression of histones in UUO+LI group and UUO+LOT group decreased significantly(P<0.05),the relative expression of p-smad3 in UUO+MI group and UUO+HI group decreased significantly(P<0.01).6)By establishing the rat UUO model,it was found that the relative expression of FN protein in UUO group and UUO+LI group was significantly increased compared with SOR group(P<0.01),histone expression levels of UUO+HI group and UUO+LOT group were significantly increased(P<0.05).Compared with the UUO group,the relative expression of FN protein in all the drug intervention groups was significantly decreased(P<0.01).Conclusion 1.Hirudin can reduce blood Scr and BUN levels in UUO rats,improve renal interstitial injury,and delay renal interstitial fibrosis.2.Hirudin UUO+HI,UUO+MI and UUO+LI could down-regulate the expression of related gene and protein in renal stroma of UUO model rats.UUO+MI group had more significant effect 3.Differences in efficacy for the same protein and gene are related to the optimal drug dose. |
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