The Protective Effect And Mechanism Of Geniposide On Myocardial Injury In Type 1 Diabetes

Background:Diabetic cardiomyopathy(DCM)is a myocardial dysfunction that occurs without coronary artery disease and hypertension,and DCM has been considered to be one of the main reasons of heart failure in diabetic patients.Therefore,the prevention and treatment of DCM has become an urgent problem in the research field of cardiovascular diseases.The main participant in the pathophysiology of DCM is myocardial fibrosis.As an intracellular catabolic pathway,autophagy can maintain cellular homeostasis under adverse environmental conditions.In recent years,it has been found that apoptosis and autophagy are closely related to the development of DCM.Pre-clinical studies have shown that some Chinese herbal medicines and their extracts are promising therapeutic drugs for DCM.Geniposide is one of the main bioactive components of traditional Chinese medicine Gardeniae,which plays an important role in the research field of cardiovascular function and diabetic complications.Geniposide,as a glucagon-like peptide 1 receptor(GLP-1R)agonist,exerts potential protective effect on various heart diseases via the activation of AMPK.Purposes:This study aims to explore the effect and mechanism of geniposide on myocardial injury in type 1 diabetes in vivo and in vitro experiments.Contents and methods:(1)Rat cardiomyocytes H9C2 were cultured in vitro,and different concentrations of glucose and geniposide were added to the cells for intervention.Cell viability was detected by CCK8 assay,and the most suitable concentration was selected.The H9C2 cells were seeded in six-well plate at a density of 5×10~4/m L and divided into 4 groups:normal group(NC),normal+geniposide group(GE),high glucose group(HG),high glucose+geniposide group(HG+GE).Flow cytometry and Hoechst33342 staining were used to detect the apoptosis of cells in each group.Western blot was used to detect the protein expression levels of BAX,BCL2,Caspase 3 and Cleaved-Caspase 3.Then the AMPK inhibitor(Compound C)was added to the geniposide treatment group.Western blot was performed to detect the protein expression levels of autophagy-related proteins LC3BⅡ/Ⅰ,P62,Beclin1,P-AMPK and AMPK.(2)32 male C57 mice were randomly divided into 4 groups:Control group,STZ group,STZ+GE(25 mg/kg)group,STZ+GE(50 mg/kg)group.After modeling and administration,the blood glucose and body weight levels of the mice were measured,HE and MASSON staining were used to detect myocardial fibrosis,the ultrastructural morphologies of the type 1 diabetic mice were observed by transmission electron microscopy.Immunohistochemistry was conducted to detect the protein expression levels of LC3B,P62,collagenⅠand collagenⅢ,and western blot to detect the protein expression levels of autophagy-related proteins LC3BⅡ/Ⅰ,P62,Beclin1,P-AMPK,AMPK,m TOR and P-m TOR and the protein expression levels of apoptosis-related proteins BAX,BCL2,Caspase 3 and Cleaved-Caspase 3.Results:(1)In vitro experiments,the results showed that the apoptosis rate of the HG group was significantly higher than NC group.Compared with the HG group,the apoptosis rate of the HG+GE group was significantly reduced.Western blot analysis showed that compared with the NC group,the protein expression levels of BAX/BCL2and Cleaved-caspase 3/Caspase 3 in the HG group were significantly increased(P<0.01).Compared with the HG group,the protein expression levels of BAX/BCL2 and Cleaved-caspase 3/Caspase 3 in the HG+GE group were significantly decreased(P<0.01).Compared with the HG group,geniposide pretreatment activated AMPK(increased P-AMPK/AMPK ratio)and autophagy activity(increased LC3BⅡ/I,decreased expression leavels of P62),and this effect was blocked by AMPK inhibitors.(2)Compared with the control group,the blood glucose of the STZ group was significantly increased[(17.21±1.49)vs.(4.94±0.38)mmol/L,P<0.001],and the body weight was significantly reduced[(18.09±1.25)vs.(24.01±1.00)g,P<0.001].Compared with the STZ group,STZ+GE 50 group can significantly reduce blood glucose in mice[(15.13±0.84)vs.(17.21±1.49)mmol/L,P<0.01],The body weight of mice in the STZ+GE 25 group and STZ+GE 50 group increased significantly(P<0.01).HE and MASSON staining showed that,the STZ group had abnormal myocardial structure and increased collagen deposition(P<0.01),STZ+GE 25 group and STZ+GE 50 group significantly reduced myocardial damage;electron microscopy showed that compared with STZ group,STZ+GE 25 group and STZ+GE 50 group improved the abnormality of myocardial mitochondrial structure.Immunohistochemistry and western blot showed that the protein expression levels of LC3BⅡ/Ⅰ,Beclin1 and P-AMPK/AMPK decreased,and the protein expression levels of collagenⅠ,collagenⅢ,P62,P-m TOR/m TOR,BAX/BCL2 and Cleaved-caspase 3/Caspase 3 increased in the STZ group.STZ+GE 25group and STZ+GE 50 group reversed this change.Conclusion:Geniposide can improve myocardial injury and reduce myocardial fibrosis in type 1 diabetic cardiomyopathy.The mechanism may be related to inhibited cell apoptosis and activated AMPK-mediated autophagy.