| Objective:To observe the effect of mitochondrial ALDH2 on Notch1 signaling pathway in diabetic myocardial injury and to explore the related molecular mechanisms.Methods:Diabetic rats were induced by intraperitoneal injection of streptozotocin(55mg / kg)and divided into normal control group(Con),diabetic 4W group(DM 4W),diabetic 8W group(DM 8W)and diabetic 8W + ALDH2 agonist Ethanol group(DM + Et OH).One week after the model was established,rats in the DM+Et OH group were given daily drinking of 2.5% ethanol,and after 1 week,the concentration was changed to 5% ethanol for 8 weeks.The heart function of rats in each group was measured at different time points.After 8 weeks,the rats in each group were subjected to myocardial ischemia / reperfusion(I / R)model and divided into Con 8W I / R group,DM 8W I / R,DM + Et OH 8W I / R.The body weight and heart weight were measured to calculate the cardiac coefficient.The level of plasma creatine kinase(CK)and creatine kinase isozyme(CK-MB)were measured by automatic biochemical analyzer.Detection of cytochrome C(CytC)and TNF-a levels in rat myocardial tissue by the kit.HE staining after light microscopy myocardial tissue and cell morphology.Masson staining and PAS staining myocardial fibrosis.Western blotting was used to detect the expression of ALDH2,Notch1,jagged-1 and Hes-1 in myocardium.Immunohistochemistry was used to detect the protein of ALDH2,Notch1,Hes-1 and Jagged-1 in rat myocardium.On the basis of the animal model,primary ventricular myocytes from 1 to 3 days-old SD neonatal rats were cultured and divided into normal control group(N),normal + ALDH2 agonist(N + Alda-1)(HG + Alda-1),high glucose + Alda-1 + Daidzin group with ALDH2 inhibitor(HG + Alda-1 + Daidzin),high glucose + Daidzin group(HG + Daidzin),high glucose + Alda-1 +Notch1 inhibitor DAPT+(HG+Alda-1+DAPT).Cardiomyocytes were identified by immunofluorescence.Cardiomyocyte proliferation activity was detected by CCK-8 assay.Apoptosis of cardiomyocytes was detected by flow cytometry with Annexin V-FITC / PI apoptosis detection kit Western Blotting was used to detect the expression of ALDH2,Notch1,Hes-1 and Jagged-1 in each group.Results:(1)the general situation in rats: Compared with the Con group,all groups of diabetes have different degrees of weight loss,lack of energy,increased urine output,slow activity,body hair,lack of luster and other performance,the longer the week,the more obvious performance,DM + Et OH group The above situation has been improved to varying degrees;(2)the cardiac coefficient:Compared with Con group,the body weight and heart weight of DM group and DM + Et OH group were significantly reduced(P <0.01),and the DM 8W group had more weight(P <0.05).The cardiac coefficient continued to increase(P <0.01).Compared with DM group,the cardiac weight of DM + Et OH group decreased(P <0.05)and the cardiac coefficient decreased(P <0.01).(3)Ventricular hemodynamic parameters:Compared with Con group,LVIDd and LVIDs in DM 4W group and DM 8W group increased significantly(P <0.01),EF% and FS% decreased significantly,and heart function decreased.In different course of diabetes,Compared with DM 4W groups,DM 8W group increased further(P <0.01),EF% and FS% decreased further and heart function decreased further(P <0.01).Compared with DM 8W groups,LVIDd and LVIDs(P <0.01),EF% and FS% increased significantly,and heart function improved.(4)CK and CK-MB:Compared with Con group,the levels of CK and CK-MB in DM4 W and DM 8W group were significantly increased(P <0.01).Compared with DM 4W group,CK and CK-MB in DM 8W group increased further(P <0.01).The levels of CK and CK-MB in DM + Et OH group were significantly lower than those in DM 8W groups(P <0.01).(5)ELISA detection of myocardial tissue CytC and TNF-a levels:Compared with Con group,the expression of CytC and TNF-a in DM 4W and DM8 W groups was significantly increased(P <0.01).Compared with DM4 W,the level of CytC and TNF-a in DM 8W group increased further(P <0.01).The levels of CytC and TNF-a in DM + Et OH group were significantly lower than those in DM 8W groups(P <0.01).(6)Myocardial tissue and cell morphology under light microscope in HE staining: The myocardium in Con group was clear,the arrangement of myocardial cells was regular,and the myocardial interstitial space was normal.Compared with Con group,the myocardial fibers in DM 4W group were disordered,broadening,some mitochondrial matrix and cristae swelling;myocardial fibrosis in DM 8W group was broken,dissolved,mitochondrial swelling degeneration,and disappearance of cristae.Compared with DM 8W group,myocardial lesions in DM + Et OH group were alleviated.Compared with Con I/R group,myocardial myofibrils were dissolved and mitochondria were swollen in DM I/R group,crest lysis and most vacuoles were formed.Compared with DM I/R group,myocardial myofibrils in DM + Et OH I/R group were arranged neatly and partially,and the mitochondrial membrane was intact and the ridge was partially blurred.(7)Masson staining and PAS staining were used to observe the myocardial fibrosis of rats in each group: Compared with Con group,collagen fiber content and PAS positive substance in DM 4W and DM 8W groups were significantly higher.With the prolongation of disease course,DM 8W group had higher collagen fiber content and PAS positive substance than DM 4W group.Compared with DM 8W groups,collagen fiber content and PAS positive substances were significantly decreased in the DM+Et OH group.Compared with Con I/R group,collagen fiber content and PAS positive substance were significantly increased in DM I/R group.Compared with DM I/R group,collagen fiber content and PAS positive substance were significantly decreased in DM+Et OH I/R group.(8)The expression of ALDH2,Notch1,Hes-1 and Jagged-1 in myocardium of rats was detected by RT-PCR,Western Blotting and immunohistochemistry:Compared with Con group,the expression of ALDH2,Notch1,Hes-1 and Jagged-1 was decreased in DM 4W group and DM 8W group.Compared with DM 4W group,the expression of ALDH2,Notch1,Hes-1 and Jagged-1 in DM 8W group was further decreased.Compared with DM 8W group,the expression of ALDH2,Notch1,Hes-1 and Jagged-1 was significantly increased.(9)Myocardial cell viability and apoptosis rate: Compared with N group,the cell viability of HG group,HG+Alda-1+Daidzin group and HG+Daidzin group was decreased and apoptosis rate of HG group,HG+Alda-1+Daidzin group and HG+Daidzin group was significantly increased(p<0.01).Compared with HG group,the cell viability of HG+Alda-1 group was increased and apoptosis rate was significantly decreased(p<0.01).Compared with HG+Alda-1 group,the cell viability of HG+Alda-1+Daidzin group,HG+Daidzin group and HG+Alda-1+DAPT was decreased and apoptosis rate was significantly increased(p<0.01).Compared with N I/R group,the cell viability of HG I/R group,HG+Alda-1+Daidzin I/R group and HG+Daidzin I/R group was decreased(p<0.01).Compared with HG I/R group,the cell viability of HG+Alda-1 I/R group was increased(p<0.01).Compared with HG+Alda-1 I/R group,the cell viability of HG+Alda-1+Daidzin I/R group,HG+Daidzin I/R group and HG+Alda-1+DAPT I/R group was decreased(p<0.01).(10)The expression of ALDH2,Notch1,Hes-1 and Jagged-1 in cardiomyocytes were detected by Western Blotting: The expression of ALDH2,Notch1,Hes-1 and Jagged-1 in HG group and HG+Alda-1+Daidzin group was lower than that in N group(P<0.01).Compared with HG group,the expression of ALDH2,Notch1,Hes-1 and Jagged-1 in HG+Alda-1 group was increased.Compared with HG+Alda-1 group,the expression of ALDH2,Notch1,Hes-1,Jagged-1 in HG+Alda-1+Daidzin group and HG+Daidzin group was significantly decreased(P<0.01)Conclusions:(1)The enhanced expression of mitochondrial ALDH2 has a significant protective effect on diabetic cardiomyopathy.After inhibition of mitochondrial ALDH2 expression,myocardial fibrosis and apoptosis are increased,and myocardial injury is aggravated.(2)Mitochondrial ALDH2 may play a protective role in the heart by regulating Notch1 signaling pathway.Activation of ALDH2 can enhance the expression of Notch1,Hes-1,and Jagged-1.The heart rate of rats is decreased and the cardiac function is increased.(3)Activation of mitochondrial ALDH2 can protect myocardial ischemia/reperfusion injury in diabetic rats,and its mechanism may be related to activation of Notch1 signaling pathway.(4)Activation of mitochondrial ALDH2 can increase cardiomyocyte viability and improve cardiomyocyte apoptosis.Mitochondrial ALDH2 expression is decreased,cardiomyocyte viability is decreased,and apoptosis is increased when Notch1 signaling pathway was inhibited. |
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