| Objectives: By studying the effect of angelica polysaccharides on TGF-β1/Smads signaling pathway and renal fibrosis related factors,discussing the mechanism of action of angelica polysaccharides in the prevention and treatment of diabetic fibrosis.Methods: Clean 10-week-old SD rats were divided randomly into six groups: normal control group,model group,irbesartan group,AP high-dose treatment group,AP middle-dose group and AP low-dose group.10 rats each group.Except the control group was given normal feed,other groups were fed with high-sucrose and high-fat diet.After 4 weeks,the latter five groups were induced to be DN by intraperitoneal injection with 28mg/kg streptozoticin.The successfully established experimental rats were intragastrically administered according to the corresponding drug and dose in each treatment group.After 6 weeks,the pathological changes of renal tissue were observed using HE staining.E-caderin and α-SMA expression levels were detected by immunohistochemistry.FN,TIMP,TGF-βRI,TGF-βRII,TGF-βmRNA,collagen expression were detected by PCR.Changes of contents of Smad2 and Smad3 were observed by Western Blot.Results:1.Comparison of general conditions.The control group was better.The model group and the Western and Chinese medicine treatment groups all had symptoms of typical clinical polydipsia,polyuria,and more food and weight loss,model group in particular.In the irbesartan group,AP high-dose group and AP middle-dose group,those symptoms were eased.During the experiment,one rat of model group died and one rat of AP low-dose treatment group died.2.Histopathological observation of renal tissue: The rats of control group had clear renal tissue under the light microscope,neatly organized renal tubules and normally stained cytoplasm.In comparison,the rats of model group had disorganized and dilated renal tubules,the epithelial cells were detached and vacuolated,the cytoplasm staining shallow,and the glomerular volume decreased and pyknosis appeared.In contrast,the lumen of the tubules in each treatment group significantly decreased,no vacuolization was observed,the cytoplasm staining was relatively deep,and the glomerular volume increased.The pathological changes AP high-dose group were more obvious than those of other groups.3.Comparison of extracellular matrix composition and renal tubular epithelial–myofibroblast transition: The content of type I collagen and type III collagen in renal tissue of diabetic rats raised significantly,and the expressions of molecule E-cadherin in renal tubular epithelial cells significantly decreased while α-SMA increased,and the difference was extremely significant(P<0.01).Compared with the model group,the content of type I collagen of irbesartan group and of AP middle-dose group and high-dose group decreased,the difference was statistically remarkable(P< 0.05).Compared to the model group,type III collagen content of irbesartan group and high dose AP group decreased,the difference was statistically remarkable(P <0.05).Compared to the control group,the MMP-9 mRNA level in the model group was notably lower(P<0.05),TIMP-1 mRNA content was notably higher(P<0.05).In the irbesartan group and middle dose and high dose AP group,compared to the model group,the MMP-9 mRNA content increased and the TIMP-1 mRNA content showed a decreasing trend.Among them,the changes of irbesartan and AP high dose group were more distinct,and the differences were in statistical sense(P<0.05),while there was no notable difference between these two groups(P>0.05).Compared to the model group,in irbesartan group and AP high dose group the expression of E-cadherin protein substantially increased(P<0.05),while there was no statistically sense in the expression of E-cadherin protein of AP low and middle dose group(P> 0.05).The expression of protein α-SMA of irbesartan group and AP-treated group decreased,the difference is extremely significant(P < 0.01).There was no remarkable variance between irbesartan group and AP high-dose group in the reduction of type I collagen and type III collagen content and the expression of α-SMA protein,and in the increase of the expression of protein E-cadherin(P>0.05).4.Comparison of TGFβ1/Smads signaling pathway activation and related factors:In the model group,the expression levels of CTGF,TGF-βRI and TGF-βRII mRNA in the kidney tissue were significantly increased(P<0.05),compared with the control group.When AP dose were increased,the content of CTGF,TGF-βRI,TGF-βRII,and mRNA gradually decreased,and the decrease was more diustict in the middle dose and high dose AP group(P<0.05).In the irbesartan group,the expression of CTGF,TGF-βRI,TGF-βRII,and mRNA was obviously decreased in Tan group(P<0.05).The expression of TGF-β mRNA and protein Smad2,Smad3,Smad2 and Smad3 in the kidney tissue of diabetic rats was remarkably increased(P<0.01),compared with the control group.In the irbesartan-intervened group,TGF-βmRNA and the expression of Smad2,Smad3,Smad2+Smad3 protein was notably reduced,compared with the model group.In the AP-treated group,when AP dose raise,the content of TGF-βmRNA,Smad2,Smad3,Smad2+ Smad3 protein notably decreased,and the difference was in statistical sense(P<0.05).So the expression of TGF-βmRNA,Smad2,Smad3,Smad2 and Smad3 protein decreased in both AP high-dose group and irbesartan group,and there was no remarkable variance between these two groups(P>0.05).Conclusion:Angelica polysaccharides have an fuction of inhibiting the germination about tubular epithelial myofibriblast transdifferentiation of renal tubular epithelial cells,it can also regulate the process of the extracellular matrix components’ formation and degradation,and play a protective role in diabetic fibrosis by reducing the activity of TGF-β1/Smads signaling pathway. |
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