| Purpose:Based on the theory of qi and blood in traditional Chinese medicine,this study combined the pathogenesis of "qi deficiency and blood stasis" of diabetic cardiomyopathy with modern molecular biology.In this study,rat cardiac fibroblasts were induced by high glucose to establish a diabetic cardiomyopathy cell model.By observing the expression level of TGF-?1/Smads signaling pathway related factors,we studied the correlation between this pathway and diabetic myocardial fibrosis,and explored the mechanism of Yiqi Huoxue therapy on diabetic cardiomyopathy,in order to provide theoretical basis for the prevention and treatment of the disease by traditional Chinese medicine.Material and method:Rat cardiac fibroblasts were randomly divided into six groups after resuscitation and subculture: control group,model group,valsartan group,low dose group of Yiqi Huoxue Recipe,medium dose group of Yiqi Huoxue Recipe and high dose group of Yiqi Huoxue Recipe.The control group was treated with 5 mmol / L glucose,and the rest of the groups were treated with 25 mmol / L glucose.MTT method was used to detect the proliferation and activity of cells in the control group and model group at 6 h,12 h,24 h,48 h and 72 h after glucose treatment,expressed by absorbance(OD value).On this basis,we choose the optimal time of action for the following experiment.Low,medium and high dose groups of Yiqi Huoxue Recipe and valsartan groups were treated with the corresponding concentrations of drugs,and then the proliferation and viability of these six groups of cells were detected by MTT method;the protein expression levels of type I collagen,type III collagen and TGF-?1 were detected by Enzyme-Linked Immuno Sorbent Assay(ELISA);the protein expression levels of phospho-Smad2,phospho-Smad3 and Smad7 were detected by Western blot.Results:1.The test results of MTT method:(1)Time concentration gradient detection: The OD value of the control group gradually increased with the extension of time,and reached the peak at 48 h,and began to decline at 72h;the OD value of the model group also gradually increased with the extension of time,and reached the peak at 24 h,and began to decline at 48 h.Compared with the control group,the OD value of the model group increased significantly at 6,12 and 24 hours(P<0.01).(2)Effect of Yiqi Huoxue Recipe: Compared with the control group,the OD value of the model group was increased significantly(P<0.01);compared with the model group,the OD value of the low,medium and high dose groups of Yiqi Huoxue Recipe and valsartan group were decreased significantly(P<0.01),and the high dose group was the most obvious;compared with the Valsartan group,the OD value of the high dose group of Yiqi Huoxue Recipe was decreased significantly(P<0.01),while the low and medium dose groups were both increased significantly(P<0.01 or P<0.05).To sum up,the therapeutic effect of each treatment group was: high dose group of Yiqi Huoxue Recipe > valsartan group > medium dose group of Yiqi Huoxue Recipe > low dose group of Yiqi Huoxue Recipe.2.The test results of ELISA:(1)Compared with the control group,the expression levels of type I collagen in the model group were increased significantly(P<0.01);compared with the model group,the expression levels of type I collagen in the treatment groups were decreased significantly(P<0.01);compared with the valsartan group,the expression levels of type I collagen in the the high dose group of Yiqi Huoxue Recipe were decreased significantly(P<0.01),while the expression levels of type I collagen in the low-dose groups were increased significantly(P<0.05),and there was no significant difference between the medium-dose group and the valsartan group(P>0.05).To sum up,the therapeutic effect of each treatment group was: high dose group of Yiqi Huoxue Recipe > valsartan group > medium dose group of Yiqi Huoxue Recipe > low dose group of Yiqi Huoxue Recipe.(2)Compared with the control group,the expression levels of type III collagen in the model group were increased significantly(P<0.01);compared with the model group,the expression levels of and type III collagen in the treatment groups were decreased significantly(P<0.01);compared with the valsartan group,the expression levels of type III collagen in the the high dose group of Yiqi Huoxue Recipe were decreased significantly(P<0.01),while the expression levels of type III collagen in the low-dose and medium-dose groups were increased significantly(P<0.05).To sum up,the therapeutic effect of each treatment group was: high dose group of Yiqi Huoxue Recipe > valsartan group > medium dose group of Yiqi Huoxue Recipe > low dose group of Yiqi Huoxue Recipe.(3)Compared with the control group,the expression level of TGF-?1 in the model group was increased significantly(P<0.01);compared with the model group,the expression level of TGF-?1 in the treatment groups was decreased significantly(P<0.01);compared with the Valsartan group,the expression level of TGF-?1 in the high-dose Yiqi Huoxue Recipe group was decreased significantly(P<0.01),while the expression levels in the low-dose and medium-dose groups were increased significantly(P<0.01).To sum up,the therapeutic effect of each treatment group was: high dose group of Yiqi Huoxue Recipe > valsartan group > medium dose group of Yiqi Huoxue Recipe > low dose group of Yiqi Huoxue Recipe.3.The test results of Western blot:(1)Compared with the control group,the protein expression levels of phospho-Smad2 and phospho-Smad3 in the model group were increased significantly(P<0.01);compared with the model group,the protein expression levels of phospho-Smad2 and phospho-Smad3 in treatment groups were decreased significantly(P<0.01);compared with the valsartan group,the protein expression levels of phospho-Smad2 and phospho-Smad3 in the high-dose Yiqi Huoxue Recipe group were reduced significantly(P<0.01),and both the low-dose and medium-dose groups were increased significantly(P<0.05 or P<0.01).To sum up,the therapeutic effect of each treatment group was: high dose group of Yiqi Huoxue Recipe > valsartan group >medium dose group of Yiqi Huoxue Recipe > low dose group of Yiqi Huoxue Recipe.(2)Compared with the control group,the protein expression level of Smad7 in the model group was reduced significantly(P<0.01);compared with the model group,the protein expression level of Smad7 in treatment groups was increased significantly(P<0.01);compared with the valsartan group,the protein expression level of Smad7 in the medium-dose and high-dose Yiqi Huoxue Recipe group was increased significantly(P<0.05 or P<0.01),while there was no significant difference between the low-dose group and the valsartan group(P>0.05).To sum up,the therapeutic effect of each treatment group was: high dose group of Yiqi Huoxue Recipe > medium dose group of Yiqi Huoxue Recipe > low dose group of Yiqi Huoxue Recipe > valsartan group.Conclusion:1."qi deficiency and blood stasis" is one of the basic pathogenesis of diabetic cardiomyopathy.The Yiqi Huoxue Recipe formulated in accordance with the Yiqi Huoxue therapy,and it can significantly inhibit the proliferation and activity of rat cardiac fibroblasts under high glucose condition,and inhibit the expression of type I and type III collagen,Further improve the excessive deposition of extracellular matrix in order to inhibit myocardial fibrosis and protect the myocardium.2.The mechanism of Yiqi Huoxue Recipe against myocardial fibrosis may be down-regulating the expression of TGF-?1,inhibiting the phosphorylation of Smad2 and Smad3,and up-regulating the expression of Smad7,and they are dose-related.In other words,Yiqi Huoxue therapy can reduce the secretion of type I and III collagen by regulating the expression of TGF-?1/Smads signaling pathway related factors,so as to against diabetic myocardial fibrosis and play an effective role in the prevention and treatment of diabetic cardiomyopathy. |
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