Protective Roles Of Antioxidant N-acetylcysteine On Cardiac And Vascular Function In Streptozotocin-induced Diabetic Mice

BackgroundDiabetes mellitus(DM) is well established as a major threat to human health due to its alarming rise in incidence over the decades. This rise in incidence is largely attributed to environment and lifestyle changes. China has become one of the countries that have the most DM patients in the 21 st century. It is a common, chronic, and progressive disease characterized by insulin deficiency and/or resistance, resulting in elevated plasma glucose levels and metabolic disorders, eventually causing a variety of complications. Diabetic cardiomyopathy(DCM) is the primary cause of morbidity and mortality among the diabetic complications. In addition, structural changes of blood vessels causing diabetic vasculature diseases including coronary artery disease, stroke and peripheral vascular disease, leading to vascular stiffness and vascular compliance decreased. It has been well known that vascular stiffness is associated with increased cardiovascular mortality and morbidity.The pathogeneses underlying diabetic-induced cardiac and vascular damages are complex and multifactorial. Hyperglycaemia induces oxidative stress, as a result of excessive production of reactive oxygen species(ROS) plays a key role in the pathogeneses of DCM and diabetic vasculature diseases. ROS include free radicals, such as superoxide(O2-) and hydroxyl radical(?OH), and non-radicals which are able to generate free radicals(e.g., hydrogen peroxide H2O2). Theoretically, suppression of oxidative stress using antioxidants could reverse adverse effects in diabetic patients. N-acetylcysteine(NAC) is a Glutathione precursor of micromolecule with antioxidant properties. It contains, sulfhydryl group and is a source of cysteine to glutathione synthesis. NAC can alleviate impairment of myocardial cells, myocardial interstitium, vascular endothelial cells and smooth muscle cell metabolism, therefore NAC could protect cardiac and vascular function, but role of ROS in diabetic cardiomyopathy is still evasive, especially how ROS involved in the cardiac fibrosis. We discussed the antioxidant therapy on cardiac and vascular impairment of diabetes mellitus, it is important to look for protection strategy of diabetic cardiovascular disease.On the other hand, evaluation of the cardiac function by ultrasound is essential for the study, which could be affected by anesthesia and other procedures. Chloral hydrate as a kind of anesthetic provides sedation and immobility in experimental mouse with the advantage of safety, reliable and high cost performance, but it influences the heart rate,the cardiac structure and function and thus influences the accuracy of the results. Therefore, correct selection of the dosage and time of the anesthesia agents are very important. Objectives(1) To access the effect of anesthesia on heart rate and cardiac function of normal and DM mice, and to explore the best anesthesia condition for the current study.(2) To assess the influence of the antioxidant N-acetylcysteine on the change of myocardial cellular morphology and function, the level of oxidative stress and the changes of cardiac structure and function, and to investigate the possible mechanisms of these changes.(3) To access the influence of antioxidant N-acetylcysteine on the change of the abdominal aorta and renal artery structure, morphology and stiffness, and to analyze the correlation of these changes with the left ventricular function and myocardium oxidative stress levels. Methods(1) Forty-five normal C57BL/6 male mice, 15 mice were induced by STZ to create DM mouse model. Thirty normal and 15 DM mice were respectively divided into three groups and anaesthetized with different dosage of chloral hydrate(300mg/kg, 350mg/kg and 400mg/kg) for each group of mice. The anesthesia onset time, duration time and recovery time were recorded. The heart rate, blood glucose, cardiac structure and function were assessed by echocardiography at 10, 20 and 30 min after induction of anesthesia of normal mice. The heart rate, cardiac structure and function were assessed by echocardiography at 20 min after induction of anesthesia of DM mice.(2) Thirty male C57BL/6 mice, 25 mice were induced by five days intraperitoneal injection of streptozocin(STZ) to create DM mouse model. The mice with a blood glucose level above 13.5 mmol/L were considered DM. Another 5 mice were injected with equal volume of citrate buffer and served as controls. The 25 diabetic mice were divided into 5 groups based on the different treatment initiation time(1 week, 3 week, 5 week and 7 week)with 5 mice in each, they were DM(diabetes without NAC treatment), and NAC treatment groups, namely NAC1, NAC3, NAC5 and NAC7. Ultrasound was performed 12 weeks after STZ injection for all groups. Heart tissues were collected after ultrasound for Hematoxylin Eosin(HE) and Trichrome staining and ROS staining. Abdominal aortas and renal arteries were collected for Hematoxylin Eosin(HE) and Trichrome staining for all mice. Results(1) The heart rate, cardiac structure and function were different with different dosages and at different time point of anesthesia. It revealed that the optimal anesthesia dosage was 350mg/kg chloral hydrate and optimal echo observation time was 20 min after the initiation of the anesthesia for both normal and diabetic mice.(2) We found that both cardiac systolic function and diastolic function were impaired, coupled with excessive reactive oxygen stress and cardiac fibrosis 12 weeks after STZ induction. NAC significantly reduced ROS generation and fibrosis, together with improved cardiac systolic function and diastolic function. Strikingly, earlier and longer treatment produced significant improvement of cardiac function and less fibrosis. In the cardiac fibroblasts, NAC blocked cardiac fibroblast proliferation and collagen synthesis induced by hyperglycemia.(3) Twelve weeks after induction of DM, the endothelium and smooth muscular cell of the abdominal aorta and renal ateries were impaired, with arterial matrix fibrosis and structural and function abnormality. NAC significantly reduced abdominal and renal function impairment and remodeling. Earlier and longer treatment produced significant improvement of vascular function and remodeling Conclusions(1) The anesthesia agent of chloral hydrate has effects on mouse heart rate, cardiac structure and function. The optimal anesthesia dosage is 350mg/kg for chloral hydrate and the optimal echo observation time is 20 min after the initiation of the anesthesia for both normal and diabetic mice.(2) Our study indicates that NAC treatment in diabetes effectively protects from diabetic cardiomyopathy, possibly through inhibiting the ROS production and fibrosis, which warrants further clarification.(3) Our study indicated that early NAC treatment in diabetes effectively protects from diabetic vasculature impairment.