Effects And Mechanism Of DZJTC On Myocardial Injury Based On TLR4/MyD88/NF-κB Signaling Pathway

Objective This topic aims to observe the protective effect of DZJTC on myocardial injury in type 2 diabetic rats,study the pathological mechanism of TLR4-MyD88-NF-κB signaling pathway in regulating diabetic myocardial injury and reveal the molecular mechanism of DZJTC on the prevention and treatment of DCM.Methods in vivo The model of type 2 diabetic rats was established by high fat diet combined with streptococci(STZ).DZJTCs were respectively treated with 270,540,and 1080mg/kg for 8 consecutive weeks.The effects of DZJTC on the general signs like body weight,water and food intake and the level of blood glucose and blood lipid,hemodynamic indexes and myocardial histopathology by HE and Masson staining were observed to study the effect of DZJTC on the prevention and treatment of type 2 diabetes complicated by myocardial injury.Western blot,RT-q PCR,immunohistochemistry and other modern molecular biology experimental methods and techniques were used to study the changes of related protein and mRNA expressions of TLR4-MyD88-NF-κB signaling pathway and its downstream cytokines in myocardium and observe the changes of apoptosis in myocardium and the effect of DZJTC.in vitro H9c2 cells injury model was induced by high glucose in vitro.TAK-242 and DZJTC containing serum were added.MTT and flow cytometry were used to observe the effect of the viability and apoptosis of H9c2 cells induced by high glucose and detect the changes of related proteins of MyD88-NF-κB signaling pathway and apoptosis regulatory proteins.Thereby,the pathological mechanism of TLR4-MyD88-NF-κB signaling pathway regulating the necrosis and apoptosis of H9c2 cells induced by high glucose could be elucidated,the relationship between the effect of DZJTC on the apoptosis of H9c2 cells induced by high glucose and its regulation of TLR4-MyD88-NF-κB signaling pathway could be revealed.Results The first part,the mechanism of TLR4-MyD88-NF-κB signaling pathway in regulating diabetic myocardial injury and the protective effect of DZJTD Compared with normal group,the fasting blood glucose of 0w,4w and 8w,8w glycerogelatin hemoglobin,total cholesterol and triglyceride in model group significantly increased(P<0.01).The body weight of rats decreased with time,while the intake of food and water increased,and the observed results were significantly different at certain time(P<0.01,P<0.05).The heart weight index and the left ventricular mass index increased significantly(P<0.01).LVSP,+dp/dtmax and-dp/dtmax lowered significantly(P<0.01),while LVEDP increased significantly(P<0.01).The results of HE and Masson staining showed that cardinal tissue in model rats appeared significant necrosis of myocardial cell,interstitial fibrosis,inflammatory cell infiltration and other pathological changes.Compared with model group,the fasting blood glucose,glycerogelatin hemoglobin,total cholesterol and triglyceride of DZJTC groups decreased significantly(P<0.01,P<0.05).Meanwhile,it could improve the weight loss of model rats,increase the intake of food and water,and lower the heart weight index and left ventricle mass index significantly(P<0.01,P<0.05).LVSP,+dp/dtmax and-dp/dtmax increased significantly(P<0.01,P<0.05),while LVEDP decreased significantly(P<0.01,P<0.05).The results of HE and Masson staining also illustrated that the injury of myocardial tissue has been improved significantly.Compared with normal group,the protein and mRNA expressions of TLR4,MyD88 and NF-κB in of model group increased significantly,the contents of TNF-α,IL-1βand IL-6 in serum increased significantly,and the rate of apoptosis in myocardium by TUNEL staining increased(P<0.01).Compared with model group,the protein and mRNA expressions of TLR4,MyD88 and NF-κB in myocardium of DZJTC groups decreased significantly,the contents of TNF-α,IL-1βand IL-6 in serum decreased significantly(P<0.01,P<0.05),and the rate of apoptosis in myocardium by TUNEL staining decreased(P<0.01,P<0.05).The second part,the mechanism of DZJTC on the apoptosis of cardiomyocyte induced by high glucose through TLR4-MyD88-NF-κB signaling pathway.Compared with normal group(5.5mmol/L),the results of the survival and apoptosis rates of H9c2 cells intervened by different concentrations of glucose(5.5,15,30,45,60mmol/L)and different intervention times(12,24,48h)demonstrated that high glucose group(30mmol/L)which intervened for 48 h could significantly reduce the survival rate of H9c2 cells and increased its apoptosis rate(P<0.01).Compared with normal group,the protein expressions of TLR4 and MyD88 and the mRNA expressions of MyD88 and NF-κB p65 increased significantly(P<0.01),the phosphorylation level of NF-κBp65(phosphorylation p65/p65 ratio)increased significantly(P<0.01).The apoptosis rate of H9c2 cells detected by flow cytometry increased significantly(P<0.01),the protein expression of caspase-3 and the ratio of Bax/Bcl-2(protein relative expression ratio)increased significantly(P<0.01).Compared with model group,the protein expressions of MyD88,the mRNA expressions of MyD88 and NF-κB p65 and the level of P-NF-κB p65 decreased significantly in TAK-242 group(P<0.01).The Apoptosis rate of H9c2 cells,the protein expressions of caspase-3 and the ratio of Bax/Bcl-2 lessened significantly(P<0.01).Compared with model group,the protein expressions of TLR4 and MyD88,the mRNA expressions of MyD88 and NF-κBp65 and the level of P-NF-κBp65 in H9c2 cells incubated by DZJTC containing serum decreased significantly(P<0.01).The apoptosis rate of H9c2 cells,the protein expression of caspase-3 and the ratio of Bax/Bcl-2 lessened significantly(P<0.01).Compared with normal group,there was no significant difference in the protein expressions of TLR4,MyD88 and NF-κB in H9c2 cells incubated by DZJTC containing serum(P > 0.05).Compared with TAK-242 group,there was no significant difference in the related protein and mRNA expressions of TLR4-MyD88-NF-κB signaling pathway,the apoptosis rate of H9c2 cells,the protein expressions of caspase-3 and the Bax/Bcl-2 ratio(P > 0.05).Conclusion: The over-activation of TLR4-MyD88-NF-κB signaling pathway is an important molecular mechanism of myocardial injury induced by high glucose.DZJTC could inhibit the over-activation of TLR4-MyD88-NF-κB signaling pathway,suppress inflammatory response and cell apoptosis,protect cardiomyocyte,improve the changes of cardiac morphology,structure and function.Therefore,DZJTC possesses the effect of preventing and treating DCM significantly.