| Objective: To establish an animal model of renal fibrosis induced by unilateral ureteral obst ruction(UUO)in rats,observe the effect of Liuwei Dihuang Pill on the level of renal autophagy in rats,and explore whether the mechanism of Liuwei Dihuang Pill in treating renal fibrosis is related to regulating the level of renal autophagy.Methods:Forty male SD rats were randomly divided into sham operation group,UUO model group,UUO model + Liuwei Dihuang pill group(3g/kg.d),UUO model + valsartan positive control group(30mg/kg.d),10 rats in each group.UUO model was established by dissociating and ligating the left ureter.Sham-operated group only dissociated and not ligated the left ureter and began to administer the drug the next day after operation for 6 weeks.Sham-operated group and model group were given 2 ml of normal saline every day.Six weeks later,rats were executed.24 hours urine was collected one day before execution and 24 hours urinary protein was measured.Body weight was measured.Cardiac artery blood and kidney histological specimens were collected immediately after intraperitoneal anesthesia with 10% chloral hydrate at a dose of 0.35ml/100 g.Liver function,electrolyte of renal function and 24-hour urinary protein were measured by automatic biochemical analyzer.Renal histopathological changes were observed by HE staining and Mason staining,and semi-quantitative evaluation was made.The relative area percentage of renal interstitial fibrosis was estimated,and the levels of LC3 and Beclin1,markers of renal autophagy,were detected by Western blot Western blot.The experimental results are as follows: 1.The successful establishment of the model: One week after the operation of the obstructive side of the kidney,the volume of the kidney increased and the cortex became thinner.The enlargement of the renal space was observed by HE staining,the infiltration of focal lymphocytes and monocytes in the renal interstitium,and renal interstitial fibrosis were observed.Two weeks after operation,the volume of the kidney increased further,the cortex became thinner,the epidermis was not smooth,the color became white,and the renal interstitial fibrosis was obvious.Three weeks after operation,the volume of the kidney increased significantly,showing a clear cystic shape.There were a lot of yellowish liquid in the kidney,showing diffuse renal fibrosis,indicating that the model of obstructive renal fibrosis was successful.2.Weight change: With the prolongation of feeding time,the weight of rats in each group increased in varying degrees.There was no significant difference in weight gain between the two groups(P > 0.05).After three weeks of administration,compared with the sham-operated group,the weight growth rate of rats in the other groups was relatively lower,and the difference between the groups was statistically significant(P < 0.05).Compared with the Valsartan group,the weight of rats in the Liuwei Dihuang pill group and the Valsartan group increased faster,and the difference between the two groups was statistically significant(P < 0.05);there was no significant difference in the weight change between the Liuwei Dihuang pill group and the Valsartan group(P > 0.05).3.The results of biochemical indicators: liver function test results of rats in each group showed no significant difference between groups(P>0.05).Compared with the sham operation group,the differences in serum creatinine and urea between the model group,liuwei dihuang pill group and valsartan group were statistically significant(P<0.05),while the difference in uric acid between liuwei dihuang pill group and the sham operation group was not significant(P>0.05).Compared with the model group,the differences in serum creatinine,urea and uric acid levels between the liuwei dihuang pill group and the valsartan group were statistically significant(P<0.05),while the differences between the liuwei dihuang pill group and the valsartan group were not statistically significant(P>0.05).Rats compared with control group,each group of 24 h urine protein quantitative value to some extent,and the differences between the groups with statistical significance(P < 0.05),compared with model group,liuwei dihuang pill and valsartan group were reduced,the difference was statistically significant(P < 0.05),and liuwei dihuang pill group is lower than the valsartan group,no obvious difference in the two groups(P > 0.05).Compared with the sham operation,the potassium of rats in each group was increased to varying degrees.There was no significant difference between the liuwei dihuang pill group and the sham operation group(P>0.05),and the difference between the model group and the valsartan group was statistically significant(P<0.05).Compared with the model group,potassium of rats in each group was decreased to different degrees,and the differences between groups were statistically significant(P>0.05).There was no significant difference in the binding force of sodium,chlorine,calcium and carbon dioxide between the groups.4.Histopathological changes: HE and MASSON staining showed that there were no obvious pathological changes in glomeruli,tubules and interstitium of rats in sham-operated group;compared with sham-operated group,there were thinning of renal cortex and substance,swelling of interstitium,vacuolar degeneration,atrophy of renal tubular epithelial cells,proliferation of interstitial fibrous tissue,increase of collagen fibers,irregular structure arrangement of kidney in other groups. Semi-quantitative evaluation of interstitial fibrosis aggravated in varying degrees compared with sham-operated group(P < 0.05);compared with model group,the degree of fibrosis in valsartan group and Liuwei Dihuang pill group was improved,the improvement in Liuwei Dihuang pill group was more significant than that in valsartan group,and the semi-quantitative evaluation of renal interstitial fibrosis was significantly reduced(P < 0.05).There was no significant difference between Liuwei Dihuang pill group and valsartan group.(P>0.05).5.Western blot protein detection results: Compared with the sham operation group,the LC3 and Beclin1 levels of model group,Liuwei Dihuang pill group and valsartan group all increased,except that there was no significant difference in Beclin1 levels between Liuwei Dihuang pill group and sham operation group(P > 0.05),there was significant difference among other groups(P < 0.05);compared with model group,LC3,Beclin1 levels of Liuwei Dihuang pill group and valsartan group had significant difference(P < 0.05).The levels of Beclin1 were all decreased,especially in Liuwei Dihuang pill group compared with valsartan group.Conclusion:(1)Liuwei Dihuang Pill can improve the renal function of rats and delay the progress of renal fibrosis caused by unilateral ureteral obstruction.(2)Autophagy is involved in the occurrence and development of renal fibrosis,and excessive autophagy may accelerate the progress of renal fibrosis.(3)Liuwei Dihuang Pill may improve renal fibrosis by inhibiting the level of renal autophagy. |
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