| ObjectiveThis study aims to identify the following points:1. Whether ceramide content in arteries of diabetic rats increases? 2 the relationship between ceramide levels in arteries in diabetic rats and endothelium-dependent vasodilatation in diabetic rats 3. whether inhibition of PI3K/PKB/eNOS signaling pathways by ceramide accumulation involves in the changes of endothelial function and structure in diabetic rats? 4. whether ceramide synthesis inhibitor can improve endothelial cell structure and endothelial-dependent vasodilatation disfunction, and the formation of atherosclerosis in aorta of diabetic rats by reducing ceramide synthesisMethodsMale Sprague-Dawley (SD) rats were randomly divided into normal control group (NC group) (n=15) and diabetes mellitus (DM) model group (n=45). Diabetes mellitus (DM) model group rats were fed with high sugar and high fat diet (20% sucrose,10% refining lard,2.5% cholesterol,1% bile salt,66.5% ordinary feed) for 4 weeks, and then were intraperitoneally injected with low dose streptozotocin (STZ)-30mg/Kg to make type 2 diabetes model. The diabetic rats were randomly divided into diabetes mellitus (DM) control group (DMC group), and myriocin treated diabetic group (MTD group). Myriocin treated diabetic group rats were intraperitoneally injected with myriocin 0.3mg/kg Qod, DM control group and the normal control group rats were injected intraperitoneally with the same amount of solvent. Diabetic rats were fed a high sugar and high fat diet, and the normal control group rats were fed with normal diet.All rats were killed after 14 weeks of intervention. The fasting whole blood of each rat was collected to determine glycated hemoglobin (HbA1c), serum ceramide concentration, insulin, liver function, and blood fat biochemical indicators. Immediately after the execution of rats, thoracic aortas were isolated and sectioned for the use of endothelial-dependent vasodilation testing, ceramide determination, endothelial structure observation under the electron microscope, the observation of atherosclerosis under HE staining, signal molecules PI3K/PKB/eNOS detection with RT-PCR and Western-blot, and immunohistochemical determination of eNOS.Results1. The SD rats were successfully induced into type 2 diabetic model by fed with high sucrose-fat diet and injected with a low dose of STZ (30mg/Kg). The rats with FBS?7.0mmol/L and/or PBS?11.1mmol/L were considered as diabetic model rats. Compared with the NC group, insulin levels dramatically increased in DMC group (P<0.05), but that in MTD group obviously decreased compared to DMC group (P<0.05) Compared with the NC group, insulin resistance indexes in DMC group and MTD group increased (p<0.05), but they increased more in DMC group than in MTD group (P<0.05)?2. Compared with NC group, the FBS and PBS levels in DMC group and MTD group increased significantly which was of statistically importance (P<0.05), but the increase in MTD group was not as much as that in DMC group (P<0.05).3. Compared with NC group, the HbA1C (glycosylated hemoglobin) levels in DMC group and MTD group had a significant statistical increase (P<0.05), but the increase in MTD group was less than that in DMC group (P<0.05). Compared with NC group, liver function in DMC group and MTD group was seriously damaged (P<0.05), but the damage in MTD group was lighter than in DMC group (P<0.05). Compared with NC group, blood lipid levels in DMC group and MTD group also had a significant statistical increase (P<0.05), but the increase in MTD group was less than that in DMC group (P<0.05). Compared with NC group, liver wet weight in DMC group and MTD group increased significantly (P<0.05), but the increase in MTD group was less than that in DMC group (P<0.05).4. Compared with NC group, serum ceramide in DMC group and MTD group increased significantly (P<0.05), but the increase in MTD group was less than that in DMC group (P<0.05). Compared with NC group, vascular ceramide in DMC group increased significantly (P 0.05), but the increase in MTD group was less than that in DMC group (P <0.05).5. Compared with NC group, serum and vascular NO in DMC group and MTD group had a significant statistical decrease (P<0.05), but the decrease in MTD group was less than that in DMC group (P<0.05).6. Compared with NC group, EDVD in DMC group and MTD group decreased significantly (P<0.05), but the decrease in MTD group was less than that in DMC group (P<0.05).7. Compared with NC group, in DMC group and MTD group there was obvious lipid deposition under endothelium and in DMC group there was obviously atherosclerotic plaque formation, but in MTD group there was no obvious atherosclerotic plaque formation and the subendothelial lipid sediments decreased significantly than in DMC group (P<0.05).8. Compared with NC group, in DMC group the endodermis structure was not complete and endothelial cell cytoplasm was edema, part of endothelial cells were necrosis and fell off, but in MTD group endothelial cell lesions reduced obviously, and endodermis structure was complete.9. Compared with NC group, in DMC group expression of vascular eNOS mRNA, eNOS protein, phosphorylated eNOS-Ser1177 protein, phosphorylated PKB-Ser473 protein and phosphorylated PI3K-P85 protein declined obviously (P<0.05), in MTD group the indexes expressed significantly higher (P<0.05). The expression of AKT mRNA, PI3K - P85 mRNA and AKT protein in three groups had no significant difference (P>0.05).Conclusions:1. In type 2 diabetic rats model there is obvious accumulation of ceramide in arteries and serum. PI3K/PKB/eNOS signal pathway activity declines in vascular endothelial cells, so NO generation decreases, endothelium-dependent vasodilation function drops, endothelial cell structure disorders, and atherosclerotic plaque forms.2. Myriocin which is an inhibitor of ceramide basal synthetic pathway reduces ceramide accumulation in vascular tissues and serum of type 2 diabetic rats, and it helps to improve insulin resistance and liver function, and decrease blood sugar and blood lipid levels.3. Myriocin which is an inhibitor of ceramide basal synthetic pathway reduces ceramide accumulation in vascular tissues and serum of type 2 diabetic rats, and it helps to improve PI3K/PKB/eNOS pathways activation, endothelial-dependent vasodilation disfunction and endothelial cell structure disorder, and increase NO production, and meanwhile decrease the fat deposition under endothelium and without obvious atherosclerotic plaque formation.4. Vascular endothelial cell structural and functional disorder and atherosclerotic plaque formation are related with ceramide accumulation in vascular tissue of diabetic rats. Ceramide accumulation directly or indirectly influences the insulin signaling pathways PI3K/PKB/eNOS activity in endothelial cells, causes endothelial dysfunction, and promotes atherosclerotic plaque formation. Inhibition of ceramide synthesis in vascular tissue of diabetic rats may become a new target for treatment of diabetes vascular lesions. |
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