| Objective:(1)To observe the expressions and locations of transient receptor potential cation channel,subfamily C,member 6(TRPC6)and calcineurin(CaN)in the kidney tissues of Adriamycin model rats;(2)To explore the clinical effects of traditional Chinese medicine(Modified Zhuling Decoction)in the treatment of Adriamycin nephropathy model rats;(3)To preliminarily discuss the relationships between TCM(Modified Zhuling Decoction)and TRPC6,CaN,and to analyze the possible molecular mechanism of TCM in the treatment of nephrotic syndrome.Methods:(1)Establishing Adriamycin nephropathy model:90 healthy male SD rats were randomly divided into a healthy group,a control group and a treatment group(n=30).The control group and the treatment group were treated with two intravenous injection of doxorubicin(the first time:4.0mg/Kg,1 week after the injection:3.5mg/Kg).Intervention was given 3 weeks after the first injection:gastric lavage was performed with 5 ml normal saline every morning and evening in the health group;gastric lavage was performed with 5 ml normal saline every morning and evening in the control group;in the treatment group,gastric lavage was performed with 5 ml TCM decoction every morning and evening.(2)Rats’ urine was collected respectively before the modeling,one week,two weeks and three weeks after the modeling for the twenty-four hours urinary protein quantitative test.(3)At the end of the third week when the experiment was over,blood samples were collected from hearts to detect serum cholesterol,albumin,creatinine and urea nitrogen.(4)After the kidneys were removed,morphological changes were observed under light microscopes and the changes of glomerular podocyte structure were observed under electron microscopes.(5)TRPC6 mRNA and CaN mRNA were detected using the NGS technique.The expressions of TRPC6 and CaN mRNA were detected using the fluorescence quantitative method.(6)The expressions of TRPC6 and CaN protein in rats’ kidney tissue were detected with immunohistochemical assay.Results:(1)General condition comparisons:the rats in the healthy control group had no obvious changes in mental state,body weight,color and so on.In the model control group,the rats became depressed after the injection of doxorubicin,with decreased activities,active aggression,different degrees of hair loss and other symptoms.As the experiment went on,some rats developed symptoms of scrotum edema.The rats in the treatment group also had the same symptoms as the rats in the model control group after the modeling.However,after the intervention treatment with TCM,the rats’ mental state was significantly better than before,with obviously increased water intake,food intake and urine volume.Some rats’ symptom of scrotal edema disappeared or was relived.(2)The twenty-four hours urinary protein content and the blood biochemistry comparisons:before the modeling,there was no significant difference in the twenty-four hours urinary protein content among the three groups(p>0.05).However,one week after the modeling,the urinary protein content in the control group and the treatment group were significantly higher than that in the health group(P<0.01),but there was no significant difference between the control group and the treatment group(p>0.05).After two weeks of intervention,the urinary protein content in the treatment group was significantly lower than that in the control group(p<0.01),and this trend lasted until the third week(p<0.01).Serum cholesterol,albumin,urea nitrogen and creatinine in the three groups were compared.The results showed that serum cholesterol,urea nitrogen and creatinine in the healthy group,in the treatment group and in the control group increased in turn,while the albumin level in the healthy group,in the treatment group and in the control group decreased in turn.The comparisons among groups had statistical differences(P<0.01).(3)Comparisons of renal histopathology:under light microscopes:the size of glomeruli of rats in the healthy group was normal with complete structure.No obvious congestion can be seen in glomerular capillaries;kidney tubules structure was complete and normal,without obvious tubular structure.Under electron microscopes,the rats in the control group were observed with segmental glomerulosclerosis.The balloon structure had obvious adhesions and part of the basement membrane was broken.The kidney tubules structure was damaged with a large amount of protein casts.Massive inflammatory cell infiltration and some proliferation of fibrotic tissue were also observed.The glomeruli of rats in the treatment group were also damaged,but the degree was significantly lighter than that of rats in the control group.The glomerulosclerosis was mild;the mesangial area was slightly widened;the mesangial matrix proliferations were observed;the interstitial changes were not obvious.Under electron microscopes:the structure of the glomerular basement membrane of rats in the healthy group was complete.The arrangement of podocytes was complete and the distribution among the pores of endothelial cells was uniform.In the control group,the glomerular basement membrane of rats was damaged obviously and some glomerular basement membrane lost,most of which had uneven thickness.Podocytes showed diffuse fusion,and even partially disappeared.In the treatment group,the basement membrane of rats did not loss significantly,only having uneven thickness.Podocytes also showed diffuse fusion,but no disappearance was observed,and the fusion degree was significantly slighter than that in the control group.(4)The fluorescence quantitative expressions of TRPC6,CaN mRNA in renal cortex:the expression of TRPC6 mRNA in the health group,in the treatment group and in the control group increased in turn and the differences among the groups had statistical significance(P<0.01).The expression of CaN mRNA in the treatment group and in the control group increased significantly(P<0.01),but there was no statistical difference between the treatment group and the control group(p>0.05).(5)Immunohistochemical comparisons of TRPC6 and CaN in renal tissue:the expression of TRPC6 was mainly in podocytes,glomerular capillary loop,mesangial and renal tubular epithelial cells.The expression of TRPC6 can also be observed in some medulla but it was weak.The expression of CaN was mainly in glomeruli,and partly in vascular connective tissue and tubulointerstitium.There was no positive expression in the blank control group.In the healthy group,TRPC6 and CaN were basically expressed in glomeruli and tubulointerstitium.The expression of TRPC6 and CaN in the control group and the treatment group were significantly higher than that in the healthy group(p<0.01).The expression of TRPC6 and CaN in glomeruli in the control group was significantly higher than that in the treatment group(p<0.01).However,the expression of TRPC6 in the renal tubules in the control group was significantly higher than that in the treatment group(p<0.01).There was no significant difference in CaN expression between the control group and the treatment group(p>0.05).(6)The correlation analysis between the twenty-four hours urinary protein content and the expression of TRPC6、CaN protein showed that the twenty-four hours urinary protein content had positive correlations with both the expression of TRPC6 and the expression of CaN Protein and the correlation coefficients were respectively(r=0.653,0.569,p=0.005,0.009).Conclusion:(1)The TCM(Modified Zhuling Decoction)can significantly improve the clinical symptoms of rats with Adriamycin nephropathy.(2)The TCM(Modified Zhuling Decoction)can significantly reduce the twenty-four hours urinary protein content and the serum cholesterol in rats with Adriamycin nephropathy..At the same time,it can improve kidney functions of rats and the serum albumin levels;(3)The expression of TRPC6 was mainly found in podocytes,glomerular capillary loop,mesangial and renal tubular epithelial cells.It was also observed in some medulla,but the expression was weak.The expression of CaN was mainly in glomeruli,and also partial expression in vascular connective tissue and the tubulointerstitium.(4)The expression of the twenty-four hours urinary protein content had a good correlation with the expression of TRPC6 and the expression of CaN protein.(5)The TCM(Modified Zhuling Decoction)may be effective by reducing the expression of TRPC6 and the expression of CaN protein. |
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