Alteration Of Signaling Pathways Due To GSK-3?/NF-?B Inhibitors During The Development Of Diabetic Cardiomyopathy

ObjectiveDiabetes mellitus(DM)is a disease with high morbidity and mortality,which seriously affects people's work and life.DM sustained hyperglycemia,can induce a series of cardiovascular complications,especially diabetic cardiomyopathy(DCM).DCM has the highest incidence.This paper detected the change of Wnt/?-catenin and NF-?B these two signaling pathways in type 1 diabetic rats' myocardium.By inhibiting GSK-3? or NF-?B expression use meisoindigo(Me)or PDTC.It cleared the role of Wnt/?-catenin and NF-?B signaling pathways in the occurrence and development of DCM and provide a theoretical basis for finding new therapeutic targets for DCM.Methods1.Animal model1.1.Establishment of diabetic rat models.30 SD male rats after 1 week adaptive feeding were randomly divided into two groups:control group and diabetic model group(DM group),the model group 20.After fasting 16 h,DM group injected in STZ by intraperitoneal injection of 60 mg/kg the ice bath under the condition of the current allocation,while the control group by intraperitoneal injection the same volume of buffer.After 72 h,all rats were fasted for12 h,with a syringe needle punctured the tail vein blood glucose strips and connect to blood glucose,fasting blood glucose(FBG)detection values of all subjects.Successful animal model: compared with the control group,rats in DM groups FBG was more than 16.7 mmol/L,with polydipsia,polyphagia,polyuria,weight loss.1.2.Research onthe changes of Wnt/?-catenin and NF-?B signaling pathways related proteins in T1 DM rats.DM group rats were randomly divided into 4 weeks and 8 weeks DM model groups with10 in each group.All rats were treated with standard rodent chow and watered libitum.After the specified period,rats anesthesia laparotomy,5mL syringe aortic blood in a centrifuge tube,separation of serum.Remove the heart,separate the left heart,HE staining to observe the pathological changes of myocardial tissue,IHC to detect rats myocardial expression of?-catenin,GSK-3?,p-GSK-3?,NF-?B p65,p-NF-?B p65,I?B?,p-I?B?.Western blotting(WB)to detect rat myocardial samples ?-catenin,Wnt2,GSK-3?,p-GSK-3?,TNF-?,NF-?B p65,p-NF-?B p65,I?B?,p-I?B? proteins expression changes.2.Animal treatment experiment2.1.Establishment of diabetic rat models.After 1 week adaptive feeding,80 rats were divided into two groups randomly: control group and diabetic model group(DM group),the model group 60.After fasting 16 h,DM group injected in STZ by intraperitoneal injection of 60 mg/kg the ice bath under the condition of the current allocation,while the control group by intraperitoneal injection the same volume of buffer.After 72 h,all rats were fasted for 12 h,with a syringe needle punctured the tail vein blood glucose strips and connect to blood glucose,fasting blood glucose(FBG)detection values of all subjects.Successful animal model: compared with the control group,rats in DM groups FBG was more than 16.7 mmol/L,with polydipsia,polyphagia,polyuria,weight loss.2.2.Research processDM group rats were randomly divided into 4 and 8 weeks DM model groups,4 and 8weeks Me groups,4 and 8 weeks PDTC groups with 10 in each group.All rats were treated with standard rodent chow and watered libitum.After the specified period,rats anesthesia laparotomy,5mL syringe aortic blood in a centrifuge tube,separation of serum.Remove the heart,separate the left heart,HE staining to observe the pathological changes of myocardialtissue,IHC to detect rats myocardial expression of ?-catenin,GSK-3?,p-GSK-3?,NF-?B p65,p-NF-?B p65,I?B?,p-I?B?.Western blotting(WB)to detect rat myocardial samples?-catenin,Wnt2,DKK1,GSK-3?,p-GSK-3?,TNF-?,IL-2,NF-?B p65,p-NF-?B p65,I?B?,p-I?B? proteins expression changes.Results1.The general characteristics of DM ratsCompared with the control group,DM group rats gradually depressed symptoms,and accompanied by xingsao odor and in the breeding process and found that the rats polydipsia,polyphagia,polyuria,weight loss,these are the hallmark of diabetes symptoms.The experimental rats in DM group,BW decreased sharply,while FBG,CI and LVWI were significantly increased than the control group.Compared with DM group,the BW increased slightly in Me/PDTC treatment groups,while FBG,CI and LVWI were significantly decreased.The general characteristics in Me/PDTC groups improved compared with DM group.2.Expression of Wnt/?-catenin and NF-?B signaling in DM rats' myocardiumHE staining revealed that the myocardial cells in the control group were normal,with uniform distribution,no deformation and necrosis.DM induced myocardial focal myocyte hypertrophy,solubility,necrosis,fiber tissue hyperplasia,etc.,especially in 8 weeks DM group,local coagulation necrosis of myocardial cells,strengthen eosinophilic cytoplasm,nuclear pyknosis hyperchromatic,fibrous tissue hyperplasia,focal necrosis in lymphocyte proliferation.IHC showed that compared with control group rats,the positive expression of GSK-3? decreased slightly in DM group with partial nuclear expression.Compared with control group,4 weeks of DM rats the expressions of ?-catenin,NF-?B p65,I?B? were significantly increased(P<0.05),all of which had partial nuclear expression.WB results showed that,compared with control group,the expression of NF-?B p65,I?B?,p-GSK-3?,Wnt2 in DM group increased significantly(P<0.05).Compared with control group,the expression of TNF-? in DM group increased significantly(P<0.05).3.Expression of Wnt/?-catenin and NF-?B signaling in Me/PDTC groups DM rats' myocardiumHE staining revealed that compared with DM group,the lesion in myocardial tissue of the Me/PDTC group was partial alleviated.IHC results showed that compared with DM group,the expression of p-GSK-3?,?-catenin,NF-?B p65 in Me/PDTC group decreased significantly(P<0.05)and the expression of nucleus and cytoplasm is homogeneous.Compared with DM group,the expression of p-NF-?B p65,I?B? increased significantly(P<0.05).WB results revealed that compared with DM group,the expression of NF-?B p65,p-GSK-3?,Wnt2,?-catenin in DM group decreased significantly(P<0.05),while the expression of p-NF-?B p65 and I?B? increased significantly(P<0.05)in Me/PDTC groups.Compared with control group,the expression of TNF-? and IL-2 in DM group decreased significantly(P<0.05)in Me/PDTC groups.qRT-PCR showed that compared with control group,the mRNA expression of NF-?B p65,I?B?,?-catenin increased significantly(P<0.05).Compared with DM group,the mRNA expression of NF-?B p65,?-catenin,GSK-3?decreased significantly(P<0.05)in Me/PDTC groups.Conclusion1.The rats were induced by intraperitoneal injection of STZ,and cardiac function had a tendency significant myocardial hypertrophy.CI and LVMI increase obviously,along with myocardial inflammation and fibrosis.2.In T1 DM rats' myocardium,Wnt/?-catenin and NF-?B signaling pathways were activated.With the time increased,the symptoms of myocardial inflammation and fibrosis have deepened.These results indicated that the Wnt/?-catenin and NF-?B signaling play important roles in the development of DCM.3.Treated with Me/PDTC in T1 DM rats,could inhibit the activation of Wnt/?-catenin and NF-?B signaling pathways in T1 DM rats' myocardium,which could repair the myocardial injury in DM rats.