| Objective: Through establishing type 2 diabetes mellitus rat models and curing them by Di Dang decoction in different disease stages,we investigate the role of Di Dang decoction in macrovascular fibrosis through the interfering of Di Dang decoction to TGF-?/Smad pathway and the regulation to CTGF and Ang II.Combined function of broken blood stasis of Di Dang decoction and traditional Chinese medicine "treatment of potential disease" theory,we reveal protection mechanism of Di Dang decoction to large blood vessels in type 2 diabetes mellitus.Methods: We used 150 healthy SD male rats.A week after adaptive feeding,20 randomly selected healthy SD male rats were used as normal control and were fed with normal diet.The other 130 were model group and fed with high fat diet.After 8weeks,the levels of fasting blood glucose(FBG)and serum insulin(FINS)were measured in canthal venous blood of fasting mice.20 rats with insulin resistance were randomly selected from the model group as a Didang decoction early group and start with gavage and begin modeling after 4 weeks.The rat tail vein of the model group was injected with streptozotocin(STZ)and the normal control group was injected with the same dose of citrate buffer.After a week,the level of blood glucose of rat tail vein blood higher than 16.7mmol / L are modeling success.The rats in the model success(117 rats),not including Didang decoction early group(19 rats,4 weeks before modelling to be administered),were randomly divided into five groups:diabetic group(20 rats),Didang decoction middle group(20 rats,modelled when administerd),Didang decoction late group(20 rats,4 weeks after modeling to be administered).Aminoguanidine group(19 rats,modelled when administered).Pioglitazone group(19 rats,modelled when administerd).Diabetes model group and normal control group were given the same amount of aseptic drinking water every day.The levels of TGF-?,Ang? and CTGF in the serum of rats were detected by enzyme-linked immunosorbent assay(ELISA).Real-time quantitative PCR(real time q PCR)was used to detect the gene expression level of TGF-?,CTCF,Ang II,Smad2 and Smad3.The expression level of TGF-?1 protein in rat aorta was detected by Western Blot.The morphological changes of thoracic aorta were observed under light microscope.During the course of the experiment,the general condition and blood glucose and blood lipid were monitored before and after the intervention.Results: Elisa showed that compared with the normal control group,the levels of TGF-?,CTGF and Ang? in the serum of diabetic rats were significantly higher(P<0.01).Compared with the diabetic model group,the levels of TGF-?,CTGF and Ang ? were significantly lower in the early and middle-stage intervention group,aminoguanidine group and pioglitazone group(P <0.05),and the levels of TGF-?,CTGF and Ang?are lower than those in the middle and late-stage intervention group(P<0.01).Real time PCR showed that compared with the normal control group,the gene expression level of TGF-?,CTGF,Ang,Smad2 and Smad3 were significantly higher in the diabetic model group(P <0.01).Compared with the diabetic model group,the gene expression level of TGF-?,CTGF,Ang? and Smad3 in the early,middle,late intervention group and aminoguanidine group was significantly lower(P<0.05),and the gene expression level of TGF-?,CTGF,Ang?and Smad3 in the early intervention group was significantly lower than those in the middle and late intervention group(P <0.05).Western blotting showed that compared with the control group,the gene expression levels of TGF-? in the diabetic model group were significantly higher(P <0.01).Compared with the diabetic model group,the gene expression level of TGF-? was significantly lower in the early,middle intervention group and the aminoguanidine group(P <0.05).And the gene expression levels of TGF-? was significantly lower in early intervention group than those in late stage intervention group(P <0.05).We next observed the morphological change of the thoracic aorta under the light microscope.Compared with the normal control group,the incomplete arteries intima,mostly absent endothelial cells,lipid deposition,disorder in the medial membrane degeneration and necrosis of smooth muscle cells and significant inflammatory cell infiltration can be observed in diabetic models.In the early intervention group,relatively complete artery intima,small number of absent endothelial cells,small amount of lipid deposition,and rare neointed neoplasm,smooth muscle cell degeneration and necrosis were obeserved.But complete intima,the loss of different degrees of endothelial cell,disorder in the medial membrane,a small number of cell degeneration or necrosis in the middle and late stage intervention group.Conclusion: Didang decoction early stage intervention can reduce the levels of TGF-?,Ang? and CTGF in the serum in T2 DM rats and also down-regulate the gene expression levels of TGF-?,Smad2,Smad3,Ang?,CTGF and the levels of TGF-?1 protein in the aorta.We conclude that Didang decoction early stage invention can regulate the balance of synthesis and decomposition of extracellular and reduce the occurrence of macrovascular fibrosis through inhibiting the Ang ?,TGF-? /Smads signaling pathway and CTGF factor. |
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