High Glucose And High Lipid Stimulate Monocytes To Produce Microvesicles And Its Preliminary Mechanism

Objective:Hypercoagulable state of diabetes caused by thrombosis tendency play an important role in diabetic vascular complications. Microvesicles (MV) is a factor which lead to hypercoagulable state in diabetes, and play a vital role in arterial thrombosis. Oxidative stress is the important common mechanism in mcrovesicles producing and diabetes. In this study, we use THP-1cell cultured in vitro as the cell model, to research whether high glucose and high lipid state can stimulate monocytes to produce microvesicles and its preliminary mechanism, in order to explore new theoretical foundation for the occurrence and development of diabetic vascular thrombosis formation lead to vascular diseaseMethods:We use THP-1cell cultured in vitro as the cell model,30mM glucose as high glucose group stimulate factor, and25μg/mL ox-LDL as the high lipid group stimulate factor.1. ELISA was to detect the MV content after the high glucose and high lipid stimulating in THP-1cells for20hours, and to detect the changes in the MV content after the ERK inhibitor U0126and antioxidants NAC pretreatment1hours of high glucose and high lipid stimulation of THP-1cells.2. Western Blot was supplied to detect the impact of high sugar and high lipid stimulating in THP-1cells for2hours,4hours,6hours on the protein expression changes of p-ERK, ERK, p-p38, p38in MAPK pathway.3. Using flow cytometry instrument to detect the cell apoptosis rate after high glucose and high lipid processing20hours, and the change of intracellular ROS levels of high sugar and high lipid processing different time.Results:1. ELISA results showed that:compared with the control group, after30mM glucose and25μg/mL ox-LDL stimulation for20hours, the MV generated by THP-1cells significantly increased (P<0.01); After the antioxidant NAC pretreatment for1hour, the MV content in the high glucose+NAC group significantly lower than the high glucose group (P<0.01), high lipid+NAC group significantly was also lower than the high lipid group (P<0.01); After ERK inhibitor U0126pretreatment1hour, the MV content in the high glucose+U0126group and the high glucose group had no significant change (P>0.05), and there was no significant difference between the high lipid+U0126group and the high lipid group, either(P>0.05).2.Western Blot results show that:Compared with the control group, with high glucose stimulation time extended, the phosphorylation levels of ERK and p38protein were significantly increased(P<0.01). The phosphorylation of p38protein was the strongest at4hours. Compared with the control group, the phosphorylation level of ERK protein increased significantly after high lipid stimulation for4hours(P<0.01); the phosphorylation level of p38protein were increased after stimulate for2hours and4hours(P<0.01). Among them, the phosphorylation level of ERK protein was strongest at4hours(P<0.01); for the phosphorylation level of p38protein, the strongest role was in2hours, and then gradually decreased with the time extension at4hours(P<0.01) and6hours (P>0.05).3. Flow cytometry apoptosis rate:Compared with the control group, after30mM glucose25μg/mL ox-LDL treatmented THP-1cells for20hours, the cell apoptotic rate was significantly higher(P<0.01).4. Flow cytometry instrument detection of intracellular ROS results:After30mM glucose stimulating THP-1cells for2hour, the generation of ROS reached a peak of about3times in the control group, and then demonstrated a downward trend with time24hours, returned to baseline levels in the control group at48hours (.P<0.01). After25ug/ml ox-LDL stimulating THP-1cells for2hours, the generation of ROS reached its peak, about7times in the control group, and along with the time extension gradually decreasing, to24hours were higher than normal control group (P<0.01) in48hours back to baseline levels.Conelutions:1. High glucose and high lipid can significantly increase the generation of MV in monocytes THP-1.2. High glucose and high lipid get through the oxidative stress pathway to stimulate THP-1cells produce MV, along with the occurrence of cell apoptosis.3. High glucose and high lipid activated ERK/MAPK and p38/MAPK-path in THP-1cells.