| Objective: Diabetic cardiomyopathy （DC）is frequent cardiovascularcomplications in patients with type2diabetes and is one of major causes ofdiabetes related mortality. The molecluar mechanisms of DC are not fullyunderstood. The purpose of this study is to investigate the relationship ofROCK activation and myocardial oxidative stress and the expression ofendothelial nitric oxide synthase (eNOS) in rates with type2diabetes and todetermine whether the RhoA/ROCK pathway is involved in the pathogenesisof DC,with the objective of providing a novel strategy for treatment of DC.Methods: Female Sprague–Dawley (SD) rats weighed180–200g wererandomised into two groups. control group rats received untreated chow anddrinking water. The experimental type rats were injected with low dose (30mg/kg) streptozotocin(STZ) in enterocoelia and sequentially given a high-fatdiet. At week10,insulin resistance in the diabetic rats was confirmed byhyperinsulinemic–euglycemic clamp.At the same time, we needed to measurebiochemical index(the fasting blood glucose、 fasting insulin、 triglyceridesand cholesterol).Then the rats were randomly divided into three groups:control group, diabetic group and fasudil group. At week24,biochemicalindex were measured. The rats were killed, The heart were quickly excisedand subjected to the measurements described below. Body weight ratio(HW BW) was calculated for each rat. The cardiac histological changes wereobserved by hematoxylin-eosin staining, transmission electron microscopyand masson staining. The volume of collagen was evaluated byhydroxyproline(HYP). The activities of the anti-oxidant enzymes (SOD) andmalondialdehyde (MDA) in cardiac tissues were estimated by standardmethods using commercially available kits.The eNOS activity in cardiactissues was assessed by immunohistochemistry staining. The level of p-MYPT1protein expression were examined by western blot.Results:1Changes of body weightAt week10, the body weight of the hypercholesterolemic rats wassignificantly higher compared with the NC group（P＜0.05）.At week24,thebody weight of the rats in DM group and the DF group were significantlylower than the control group rats（P＜0.05）.No significant differences wereobserved between DM group and DF group（P＞0.05）.2Changes of biochemical indexAt week10,the levels of FBG,FINS, TGand TC of the diabetic rats werehigher than those in NC group（both P＜0.01）.At week24,the levels ofFBG,FINS,TG, TC and HbA1c of the rats in DM group and DF group werehigher than those in NC group（P＜0.01）.There were no statistical differencebetween the DM group and the DF group（P＞0.05）.No significant differenceswere observed in SABP among the three groups （P＞0.05）.3Sensitivity to insulinAt week10,the levels of glucose infusion rate（GIR）of the diabetic ratswere significantly lower than those in NC group（P＜0.01）.At week24,Thelevels of HOMA-IR of the rats in DM group and DF group were higher thanthose in NC group（P＜0.05）. There were no statistical difference betweenDM group and DF group（P＞0.05）.4Heart weight, Heart weight/body weight ratioAt week24,the heart weight and HW/BW ratio of DM group were higherthan the DF group（both P＜0.01）.The DF group were higher than the NCgroup（both P＜0.01）. There were no difference difference between DMgroup and DF group（P＞0.05）.5HE stainingThe myocardial structure of rats in control group was clearly evident. Themyocardial structure of control group was normal and myofibrils are the maincomponents of cytoplasm and they appeared orderly. The myocardial structurein diabetic rats model group was characterized by obvious increased in myofibril content, disordered and breaking myofilaments. Diabetic ratsshowed dense packages of collagen fibrils between cardiomyocytes.Thepathological changes of diabetic rats treated with fasudil groups were slighterthan that of diabetic rats model group.6Transmission electron microscopy(TEM) studyThe myocardial structure of rats in control group was clearly evident.Myofibrils are the main components of cytoplasm and they appeared orderly,with bright and dark areas clearly evident. The myocardial structure indiabetic rats model group was characterized by obvious increaed in myofibrilcontent,disordered and breaking myofilaments. Diabetic rats showed densepackages of collagen fibrils between cardiomyocytes.The pathologicalchanges of diabetic rats treated with fasudil groups were slighter than that ofdiabetic rats model group.7Masson stainingThere was a small quantity of collagen in myocardial interstitial andaround vascular in myocardial tissue of rats of NC group. Compared with NCgroup, the collagen fiber in myocardial interstitial and around vascular inmyocardial tissue of rats significantly increased in DM group（P＜0.01）.Thecollagen fiber in myocardial tissues of rats was markedly reduced in DF groupcompared with DM group（P＜0.01）. There were no significant differences ofthe collagen fiber between NC group and DF group (P>0.05).8The results of myocardial collagen concentration(MCC)At week24,the concentrations of MCC in myocardial tissue of the DMgroup were higher than those of the DF group and the NC group（P＜0.01）,There was no significant differences between the NC group and the DFgroup（P＞0.05）.9Activities of anti-oxidant enzymes and lipid peroxidationThe mean activities of SOD in sera of the DM group rats were significantlylower than those of the NC group rat（sP＜0.01）. Rats of DFgroup showedhigher SOD than those of the DM group rats (P＜0.01).The DF group werehigher than the DM group(P＜0.01). The mean content of MDA in DM group rats were significantly higher than those of the NC rats（P＜0.01）. Thecontent of MDA in DM group rats were higher than those of the DF rats（P＜0.05）. No significant differences were observed between DF group and NCgroup（P＞0.05）.10Immunohistochemistry stainingA brown color denotes positive staining. Compared with diabetic group,the protein expression of eNOS in myocardial tissue was increased in controlgroup(P<0.01).Treatment of diabetic rats with fasudil was significantlyincreased than diabetic group(P<0.01).There was no significant differencesbetween fasudil group and control group(\P>0.05).11Protein expression in myocardial tissueThe level of p-MYPT1was significantly elevated in the hearts of thediabetic group compared with that of the control group (P <0.05),while it wasdramatically suppressed by treatment with fasudil (P <0.05). There was nosignificant differences between NC group and F group（P＞0.05）.Conclusion：1The rat model of type2diabetes was established by high fat dietcombined with one-time intraperitoneal injection of low-dose streptozotocin.2Hyperglycemia potently elevated ROCK activity and augmentedoxidative stress and down-regulated the expression of eNOS,all of thesemediated myocardial fibrosis in diabetic rats.3ROCK inhibitor,fasudi, ameliorates myocardial fbrosis in diabetic ratsat least in part by inhibiting oxidative stress and up-regulating eNOSexpression, which is independent of blood pressure and glycemic control. Thisstudy suggests ROCK may be a new therapeutic target for DC. |
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