Study On Mechanism Of P53/GLUT1-mediated Endothelial Cell Injury Due To Disorders Of Glycolipid Metabolism

OBJECTIVE:In metabolic diseases,glycotoxicity and lipotoxicity caused by excessive caloric intake and increased nutrient availability are among the causes of endothelial dysfunction.Endothelial dysfunction is a key step in the initiation of atherosclerosis.The tumor suppressor p53 has been shown to be involved in many important cellular processes,including the regulation of energy metabolism and oxidative stress.The aim of this study was to investigate the role and mechanism of p53 in high glucose/high fat-induced metabolic disorders in endothelial cells.METHODS:In the first part,patients with ischemic cardiomyopathy combined with diabetes mellitus were selected for a 3-year follow-up and major adverse cardiovascular events were recorded.Insulin resistance metabolic score was calculated by clinically relevant metabolic indices such as fasting glucose,triglycerides,high-density lipoprotein cholesterol and body mass index.Survival analysis was used to assess the correlation between different metabolic indices and patient prognosis by comparing patients’baseline data.In the second part,atherosclerotic plaque tissues from patients with carotid artery stenosis were collected and immunohistochemical staining was performed to detect the expression level of p53 in the atherosclerotic plaque tissues of patients with carotid artery stenosis.The morphology of carotid atherosclerotic plaques in patients with carotid artery stenosis was clarified by carotid Doppler ultrasound.The correlation between the expression of p53 and plaque vulnerability in atherosclerotic plaque tissues of patients with carotid artery stenosis was analyzed.In the third part,a high glucose and high fat model was established using glucose and palmitic acid,and the expression levels of p53 and GLUT1 in endothelial cells were detected by Western blot.The transcript levels of key enzymes for glucose uptake and glycolysis were detected.The levels of reactive oxygen species in endothelial cells were detected using the fluorescent probe DCFH-DA,and the levels of antioxidant enzymes in anti-endothelial cells were detected using ELISA.Mitochondrial kinetic-related proteins were detected by Western blot,and mitochondria were labeled with a mitochondrial far-infrared fluorescent probe to simultaneously detect the level of mitochondrial membrane potential and mitochondrial membrane permeability transition pore opening.Apoptosis levels were detected using Western blot and flow cytometry.To clarify the mechanism of action of endothelial cell injury by p53/GLUT1 under high sugar and high fat conditions.In the fourth part,an atherosclerosis model was established using Apo E^-/-mice,which were given a high-calorie diet,and the body weight,blood glucose,and adipose tissue weight were recorded.Total cholesterol and triglyceride levels in plasma of mice were also detected.The atherosclerosis of mice was clarified by oil red O staining.Oxidative stress level of aorta was assessed using superoxide anion fluorescent probe,and mitochondrial morphology of vascular endothelium was observed by transmission electron microscopy.RESULTS:In the first part,fasting glucose,triglycerides,high-density lipoprotein cholesterol,body mass index,and metabolic score for insulin resistance were associated with poor prognosis in patients with ischemic cardiomyopathy,and had a predictive value for the occurrence of clinical endpoint events.In the second part,p53 expression was increased in carotid artery vulnerable plaques.p53 expression level had a predictive value for carotid atherosclerotic plaque vulnerability.In the third part,p53 expression was increased under high glucose and high fat conditions,resulting in endothelial cell dysfunction.knockdown of p53 regulates endothelial cell glycolysis,oxidative stress,and mitochondrial homeostasis by negatively regulating the expression of GLUT1.In the fourth part,p53 expression was increased in the aorta of atherosclerotic mice fed a high-calorie diet.p53 inhibitors improved metabolic disorders,reduced oxidative stress,and delayed atherosclerosis progression in mice.CONCLUSION:In the first part,fasting glucose,triglycerides,high-density lipoprotein cholesterol,body mass index,and metabolic score for insulin resistance are risk factors for poor prognosis in patients with diabetes mellitus combined with ischemic cardiomyopathy.In the second part,p53expression levels in atherosclerotic plaques were significantly associated with plaque vulnerability.In the third part,Knockdown of endothelial cell p53 under high-glucose and high-fat conditions increases glucose metabolism,maintains redox homeostasis in endothelial cells,inhibits mitochondrial dysfunction,and reduces apoptosis by regulating GLUT1.In the fourth part,inhibition of p53 improved high-calorie-induced metabolic disorders and attenuated atherosclerosis.