Effects Of Astragaloside IV And Puerarin On ERS-mediated Apoptosis Pathway In Rat Cardiomyocytes Induced By High Glucose

Background:Cardiovascular diseases is the top chronic disease of death rate all over the world.Diabetes has shown to be a high-risk factor for cardiovascular events.During diabetic cardiomyopathy as a concept has been proposed,the incidence has been increasing.The basic reason of occurrence of diabetic cardiomyopathy is the elevated glucose levels in diabetes mellitus patients.Research finding that it can slow down the process of myocardial remodeling and the degree of heart failure in diabetic cardiomyopathy if inhibited the early apoptosis of myocardial cell.Recently,with the related studies of regulatory mechanisms of myocardial cell apoptosis are getting further,the pathway of apoptosis regulated by endoplasmic reticulum stress has caught amount of attention.The apoptosis of myocardial cell always is the key part in the research.Therefore,the research of apoptosis by endoplasmic reticulum stress can provide the new principle of traditional Chinese medicine preventing and treating diabetic cardiomyopathy.Objective:To investigate the occurrence and changes of ERS during diabetic cardiomyopathy and to observe the expression of the mRNA and protein of ERS and the apoptosis pathway regulated by ERS in H9C2 cardiomyocytes.Explore mechanism and effect of combination of astragaloside IV and puerarin to treat and prevent diabetic cardiomyopathy.Methods:(1)To explore whether each concentration of each stimulation drug be toxic to H9C2 cells via CCK-8.(2)Detect apoptosis situation of H9C2 cells after using 30.5mmol/L mannitol,35?70?140mmol/L glucose and 2?4?8?mol/L thapsigargin stimulated cells by flow cytometer machine.Also use real-time PCR to evaluate the expression of mRNA of GRP78,IRE1 and CHOP.(3)10?20?40?mol/Lastragaloside ?,125?250?500?mol/L puerarin stimulated H9C2 cells 24,48,72 hours respectively,compare the expression of mRNA GRP78?IRE1? and CHOP with normal group(low glucose stimulated)and model group(high glucose stimulated)by real-time PCR.(4)Test the mRNA expression of GRP78 in H9C2 cells that stimulated 24hours by 3 different concentration of ERS inhibitor 4-PBA via real-time PCR.(5)The myocardial cells cultured by low glucose of H9C2 rats were divided into 6 groups:normal group,model group,mannitol control group,treatment group and inhibition group.Low glucose medium stimulation in the normal group.Mannitol control group was stimulated by 30.5mmol/L low glucose medium.Model group with high glucose medium stimulation and treatment group with Puerarin and astragaloside IV and high glucose medium stimulation.Inhibition group was stimulated by high glucose medium mixing 5mmol/L ERS inhibitor 4-PBA.After used above methods stimulated H9C2 cells respectively 24 hours then detect the expression of target gene and protein GRP78/IER1?/CHOP/PUMA/XBP1u/XBP1s through Real-time PCR and Western Blotting.Result:(1)There was no significant difference between Each group of drugs,glucose and stimulation period compared with normal group.(2)The results of flow cytometer machine is that each group of different concentrations of glucose cannot cause H9C2 cells apoptosis.However,2?mol/L TG stimulated 72 hours and 4,8?mol/L TG stimulated 48 and 72 hours can cause H9C2 cells apoptosis.And there was a significant up-regulation of the mRNA expression of GRP78,IRE1? and CHOP in H9C2 cells between those different concentrations of glucose and TG stimulation groups than normal group.(3)There was a significant decline of mRNA expression of GRP78,IRE1? and CHOP between 24 and 48 hours of each concentrations of Puerarin and Astragaloside IV groups than model group.Besides,it is no significant difference between 72 hous low concentration of Puerarin group and model group.The mRNA expression of CHOP in each group of 72 hours has no significant difference with model group.(4)There was a significant decline of mRNA expression of GRP78 between each 4-PBA concentrations group than normal group.(5)Except XBP1u,each of other mRNA expression has a significant up-regulation between model group and TG group than normal and mannitol control group and a significant decline between treatment and inhibition group than model group.The mRNA expression of XBP1u has no significant difference between TG group and normal group,and has a significant decline between model group than normal group.(6)The protein expression of GRP78,IRE1?,XBP1S,CHOP and PUMA has a significant up-regulation between model and TG group than normal and monitor group,and has a significant down-regulation between treatment and inhibition group than model group.Conclusion:(1)long duration high intensity of endoplasmic reticulum stress can induce apoptosis of H9C2 cells,simply using high glucose stimulated H9C2 cells(up to 72 hours)in the experiment and early stage of using TG stimulated cells cannot induce the apoptosis of H9C2 cells.(2)Astragaloside ? and Puerarin can inhibit the ERS and apoptosis mediated by ERS in H9C2 stimulated by high glucose.However,in later period the effect of regulation toward apoptosis pathway is not good enough.(3)Astragaloside ? combined Puerarin can inhibit the mRNA and protein expression of ERS and ERS mediated apoptosis pathway in H9C2 rat cardiomyocytes stimulated by high glucose,and the effect is better than simply using Astragaloside IV or Puerarin.