| Objective:Diabetic cardiomyopathy(DCM)is an important part of diabetic cardiovascular complications,and also one of the main causes of death in diabetic patients.Its pathogenesis is very complex,and myocardial fibrosis like changes play an important role in its occurrence and development.Dihydroartemisinin has the effect of anti renal fibrosis and anti liver fibrosis,but its related research on myocardial fibrosis is less,and the specific mechanism is not clear.Therefore,our project takes the anti tissue fibrosis characteristics of dihydroartemisinin as the breakthrough point to study its potential therapeutic effect and possible related mechanism in myocardial fibrosis.The main purpose of this study is:(1)to study the therapeutic effect of ihydroartemisinin on myocardial fibrosis in diabetic cardiomyopathy mouse model.(2)Objective to study the effect of Dihydroartemisinin on high glucose treated cardiac fibroblasts and its mechanism.Methods and methods:1.In vivo experiment: after one week of adaptive feeding,the mice(purchased from experimental animal center of China Medical University) ere randomly divided into three groups: normal control group(control group,n=10),artemisinin group(DHA group,n=10),model treatment group(STZ group,n=40).The model treatment group was injected with streptozocin(STZ)solution to make the model.After the model was successfully established,it was divided into model group,low dose group(dha-l)and artemisinin high dose group(dha-h),the experimental period was 8 eeks.The test indexes were as follows:(1)the mental state,weight,hair color,diet,defecation and other general states of each group of mice were observed and recorded regularly every day.(2)After the model was established,the fasting blood glucose of each group of mice was monitored closely.The blood glucose concentration was measured by tail vein blood sampling every 2 weeks.(3)The changes of myocardial tissue were observed by paraffin section,he staining and light microscope.(4)The expression levels of vimentin and α-SMA in myocardial tissues of each group were detected by immunofluorescence.(5)The expression levels of TGF-β1,collagenani and collagen Ⅲ were detected by protein immunoassay.2.In vitro experiment: the rat fibroblasts were cultured by resuscitation in vitro;After success,the rat cardiac fibroblasts were identified by immunofluorescence method.Fibroblasts were divided into groups(normal control group,artemisinin group,model group,artemisinin low dose group and artemisinin high dose group),and high sugar treatment(30mm,18h),and high and low-dose dihydroartemisinin was used for intervention.The results showed that:(1)MTT assay was used to detect cell viability.(2)The expression ofα-SMA in the myofibroblasts of each group was detected by immunofluorescence.(3)The expression levels of TGF-β1,α-SMA,Collagen Ⅰ and collagen Ⅲ were detected by Western blot.(4)The expression of Smad2,Smad3 and phosphorylation of TGF-β/smads pathway in each group of myocardial cells were detected by Western blot.(5)The expression levels of p38,ERK1/2,JNK and phosphorylation of MAPK pathway related proteins in each group were detected by Western blot.Results:1.In vivo experiments:(1)general state of mice: the mice in normal control group and artemisinin group were in good condition,with normal coat color and luster,normal activities,normal diet,drinking water and urine volume.After STZ injection,the mice in other groups had increased drinking water,diet and urination,poor spirit,dry and lusterless hair color,significant weight loss,and typical "three more and less" diabetic symptoms.Compared with the control group,the weight of model group decreased significantly.At the end of the experiment,compared with the model group,the mental status of mice in each DHA treatment group was improved in varying degrees,especially in the DHA-H group.(2)Blood glucose changes in mice: after modeling,the results of blood glucose values in each group showed that: compared with the normal control group,the blood glucose values in the model group and artemisinin treated group were significantly increased;compared with the model group,the blood glucose values in the DHA-L group and DHA-H group were significantly decreased.(3)He staining results of the myocardial tissue of each group: the myocardial tissue of normal control group and DHA group was regular and the muscle fibers were distributed in order.The nucleus morphology was oval,distributed evenly,and the cytoplasm staining was pink.The cross pattern of the myocardium was clear under the microscope,no muscle fiber fracture or defect was found,and no fibrosis like change was found.The myocardial tissue arrangement in model group was obviously disordered,the size of myocardial cells was irregular,and a large number of muscle fibers were broken,suggesting that the myocardial fibrosis like changes occurred.Compared with the model group,the myocardial tissue of DHA-L group and DHA-H group was relatively orderly,and the changes of cell morphology and fibrosis degree were improved to varying degrees,among which DHA-H group improved most obviously.(4)Immunofluorescence was used to detect vimentin and α-SMA in myocardial tissue of each group.Results: obvious vimentin fluorescence staining reaction was observed in myocardial fiber of model group.Vimentin positive reaction was observed in myocardium of mice in low-dose artemisinin group(DHA-L group)and high-dose artemisinin group(DHA-H group),but the number of vimentin positive reaction was less than that in model group.There was almost no fluorescence reaction of α-SMA in control group and DHA treated group,suggesting that there was almost no expression of α-SMA in normal mice.(5)Results: the expression of TGF-β1,Collagen Ⅰ and collagen Ⅲ in model group was significantly higher than that in control group and DHA group.After DHA treatment,the expression of TGF-β1,Collagen Ⅰ and collagen Ⅲ in DHA-L group and DHA-H group were significantly lower than that in model group,among which,DHA-H group was the most obvious.2.In vitro experiments:(1)MTT colorimetric assay results: when the drug concentration was 25μM and 100 μM,respectively,and the treatment time was 18 h,the cell survival rate was 80%;in 18 h treatment,when the high glucose concentration was30 mm,the cell survival rate was more than 80%.(2)The results of immunofluorescence assay were used to detect α-SMA in the fibroblasts of each group.The fluorescence response of α-SMA in control group and DHA treated group was weak.The results showed that the expression of α-SMA in the cells was increased by high glucose treatment.The fluorescence intensity of α-SMA in DHA treated group was higher than that in the control group,but lower than that in the model group.(3)Western blotting was used to detect the expression of TGF-β1,α-SMA,collagen Ⅰ and collagen Ⅲ.results: at the end of the experiment,the expression levels of TGF-β1,α-SMA,collagen Ⅰ and collagen Ⅲ in model group were significantly higher than those in control group;compared with model group,the expression levels of TGF-β1,α-SMA,collagen Ⅰ(except DHA-H group)and collagen Ⅲ in DHA treated groups were significantly higher It’s a drop in the number of people.(4)Western blotting was used to detect the expression of Smad2,Smad3 and their phosphorylation in cardiac fibroblasts of each group.Results: compared with the control group,higher levels of Smad2,Smad3 and their phosphorylation p-Smad2,p-smad3 were detected in cardiac fibroblasts of model group;the expression levels of Smad2,Smad3 and their phosphorylation p-Smad2,p-smad3 in DHA-L group and DHA-H group were higher than those in model group Compared with the control group,it was significantly lower.(5)Results: compared with the control group,the expressions of p38,ERK1 / 2,JNK and their phosphorylation in model group were significantly increased;the expressions of p38,ERK1 / 2 and their phosphorylation in DHA-L and DHA-H groups were significantly decreased,but the effects of DHA on JNK and its phosphorylation were not significant Phosphorylation had no significant effect.Conclusions1.Dihydroartemisinin can reduce the degree of myocardial fibrosis in diabetic cardiomyopathy mice.2.Dihydroartemisinin can inhibit the activation of cardiac fibroblasts induced by high glucose,inhibit the secretion of type I and type Ⅲ collagen,thereby reducing the deposition of extracellular matrix to improve the degree of myocardial fibrosis.3.The above effects of dihydroartemisinin may be realized through its regulation of TGF-β/Smads signaling pathway and MAPK signaling pathway. |
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