The Protective Effect And Possible Mechanism Of Simvastatin On Myocardium Of Diabetic Rats

Objective:Diabetic myocardial injury,which can progress into diabetic cardiomyopathy,is a diseased state with changes in myocardial structure and function caused by diabetes.The pathophysiological mechanism is relatively complex and no effective treatment has been found.The member of our research group noticed in clinical practice that the heart function improved in diabetic patients with application of simvastatin.This study aimed to explore the mechanisms of the protective action by simvastatin on myocardium of diabetic rats from the aspects of cardiac structure and function,and from inflammation and myocardial apoptosis.Methods:After one week of adaptive feeding,42 Sprague-Dawley（SD）rats weighting180²20g were divided randomly into control（N）group and modeled groups,modeled groups were injected with streptozotocin intraperitoneally to induce diabeties.And then randomly divided into diabetes mellitus group（DM group）and diabetes mellitus+simvastatin group（DM+S group）,rats in DM+S group were given simvastatin by oral gavage for four weeks,while the other two groups were given the same amount of saline at the same time.During the experiment,the weight of rats was recorded once a day,and the blood glucose was measured once every three days.At the end of these processes,the contents of total cholesterol（TC）and triglyceride（TG）in rat abdominal aorta blood were measured.Heart function from 6 randomly selecteted rats were measured by Langendorff heart perfusion system,including:Left ventricular systolic pressure（LVSP）,Left ventricular end-diastolic pressure（LVEDP）,+dp/dt_max,-dp/dt_max.The heart tissues of 8rats in each group were collected and processed for the following:H&E staining of rat heart slides was used to observe the pathological changes;Sirius Red staining was used to observe inflammatory fibrosis;The content of malondialdehyde（MDA）and the activity of superoxide dismutase（SOD）in the rat heart tissues were measured by spectrophotometry;TUNEL assay was used to determine the apoptosis index of myocardial cells in each groups;the distribution of NF-κB p65,MCP-1,p53 in the heart tissues were evaluated by immunohistochemistry;western blotting was used to detect the expression of GRP78,p-IRE1α,NF-κB p65,p53,p53-Phospho-Serine 15,BAX and BCL-2 in the heart tissues.Results:1.The result of blood glucose showed no significant changes among the three groups at the beginning,after STZ modeling,the blood glucose of the DM group and DM+S group increased and remained basically stable.2.Compared with control group,the contents of TC and triglyceride TG level in DM group increased significantly（P<0.01）.After simvastatin treatment,the contents of TC（P<0.01）and TG（P<0.05）in DM+S group decreased than that of DM group.3.H&E staining showed that the myocardial cells in the DM group were disorganized,with unclear morphological structure.The myocardial morphology in DM+S group was improved significantly compared to DM group.4.Sirius Red staining under the ordinary microscope showed that the collagen fibers of DM group increased obviously and disorganized,myocardial interstitial fibrosis was remarkeble（P<0.01）,while the stains of DM+S group were obviously lighter than that of DM group（P<0.01）.5.Compared with control group,LVSP,﹢dp/dt_max,﹣dp/dt_max in DM group decreased significantly（P<0.01）,while LVEDP increased significantly（P<0.01）.LVSP（P<0.01）,﹢dp/dt_max（P<0.05）and﹣dp/dt_max（P<0.01）decreased while LVEDP（P<0.01）increased significantly after the treatment of simvastatin.6.Compared with control group,the content of MDA was increased while the activities of SOD was decreased significantly in DM groups（P<0.01）.After simvastatin administration,the activities of SOD increased and the content of MDA decreased significantly（P<0.01）compared to DM group.7.TUNEL staining results showed that the apoptosis index of myocardial cells in DM group increased significantly compared with that of control group,and the apoptosis index decreased significantly after the treatment of simvastatin（P<0.01）.8.Immunohistochemistry showed that NF-κB p65 and MCP-1 expressed in all rat heart.Compared to control group,the expression of NF-κB p65 in the nucleus significantly elevated（P<0.01）,the expression of MCP-1 in cytoplasm increased significantly（P<0.01）,the expression of p53 in DM group was increased in both cytoplasm and nucleus,while in DM+S group the expression of NF-κB p65,MCP-1,p53were decreased significantly（P<0.01）compared with DM group.9.The results of western blot showed that the expression of GRP78,p-IRE1α,NF-κB p65,p53,p53-Phospho-Serine15 and BAX were higher than that in control group（P<0.01）,and the expression of BCL-2 was lower than that in control group（P<0.01）.After simvastatin administration,GRP78,p-IRE1α（P<0.05）,NF-κB p65（P<0.01）,p53（P<0.01）,p53-Phospho-Serine 15（P<0.01）and BAX（P<0.05）decreased significantly,and the expression of BCL-2 increased（P<0.05）.Conclusion:1.Simvastatin had no significant effect on blood glucose and body weight in diabetic rats.Simvastatin can reduce the synthesis of TC,reduce the content of TG,and significantly reduce blood lipid levels in diabetic rats.2.The myocardial structure of diabetic rats was impaired,myocardial interstitial collagen accumulation caused myocardial fibrosis,and lead to myocardial contraction and diastolic dysfunction.Simvastatin can reduce the accumulation of myocardial collagen fibers,improve the myocardial structure and function of diabetic rats,thereby protecting the myocardium.3.The level of oxidative stress and endoplasmic reticulum stress in the heart of diabetic rats increased,which can aggravate the inflammatory response.Simvastatin can reduce the level of oxidative stress,inhibit the occurrence of endoplasmic reticulum stress,relieve the inflammatory response of cardiomyocytes and protect the myocardium through regulating the expression of NF-κB and MCP-1.4.The cardiomyocytes apoptosis rate was higher in DM group than that in control and in DM+S group,which means that simvastatin can protect myocardial cell from apoptosis caused by diabeties.5.The expression of p53 and BAX increased,while the expression of BCL-2decreased significantly in cardiomyocytes of diabetic rats.The mechanism of simvastatin on inhibiting cardiomyocyte apoptosis in diabetic rats is related to inhibition of the expression of p53 and BAX and activation of the expression of anti-apoptotic protein BCL-2.