Study On The Mechanism Of PI3K/Akt Signaling Mediated Nε-Carboxymethyl Lysine In Atherosclerosis

Background and objectiveIn recent years, the incidence of diabetes in C hina has continued to grow rapidly, The number of diabetic patients in C hina is the first in the world. As everyone knows, diabetes can cause many complications, especially atherosclerosis(AS) because of its high mortality rate and high disability rate has been widely concerned by the global medical community. The most critical step in the development of atherosclerosis is macrophage swallow oxidized low density lipoprotein. The final fate of foam cells is currently believed to occur apoptosis or necrosis of the core in the formation of lipid necrosis under the fibrous cap. With the development of modern medical research on atherosclerosis, people gradually know of mono nuclear macrophages to endothelium migration and foam cell relocation presented a dynamic equilibrium under normal physiological conditions. But in pathological conditions the process will be broken down to an increase in the number of cells in the endothe lium, and the relocation of the cells is less and less, forming gathered a large number of cells, lead to the formation of atherosclerotic plaque, accelerated malignant remodeling of the vascular wal.This study mainly included the establishment of diabetic atherosclerosis model in Apo E knockout mice and RAW264.7 cells to establish foam cell model.we observe the high fat diet, carboxymethyl lysine amino acid and PI3 K specific agonist 740Y-P on mouse atherosclerotic plaque and related protein expression. To observe the effect of CML on cell migration, cell activity and related protein expression of RAW264.7 derived foam cells and the role of PI3K/Akt signaling pathway in cell migration and cell activity. MethodThis experiment mainly consists of two parts, in vivo experiment and in vitro experiment. In vivo experiment we used Apo E knockout mice as the research object, diabetes model was prepared by intraperitoneal injection of STZ(40mg/kg/day). The qualified animal models were divided into four groups as follows: Control group(normal diet), model group(high fat diet), CML group(model group + CML: 10mg/kg/day), 740Y-P group(group CML + injection 740Y-P: 5μmol/kg/day); To observe the formation of atherosclerotic plaque of aorta in each experimental group. Hematoxylin and eosin staining(HE staining) to detect Apo E gene knockout mice tissue morphological changes of aortic plaque, Oil red O staining was used to detect the distribution of foam cells in the aortic plaque of mice, immunofluorescence, immunohistochemistry, real-time fluorescence quantitative PC R detection of plaque CD68(macrophage derived important markers), CML expression, At the same time, the expression of p-Akt in aortic plaque of mice was observed by immunofluorescence,C hanges of serum lipids in mice under different intervention conditions were detected by collecting the serum of mice.RAW264.7 cells were used as the research object in the cell experiment, the foam cell model was formed by CO2 incubation with ox LDL and RAW264.7 cells. O il red O staining was used to detect the foam cell model, and the enzyme method was used to determine the cellular cholesterol content. The foam cells were first treated with different concentrations of CML(0μmol、1 μmol/L 、10μmol/L、100μmol/L), effect of MTT and flow cytometry to detect different concentrations of CML in RAW264.7 macrophage derived foam cell activity; Transwell to detect the effects of different concentrations of CML on the migration of RAW264.7 derived foam cells; the aim is to select the appropriate intervention conditions for the next experiment. The following experiments are grouped as follows: Control group, ox LDL group, CML group, 740Y-P group; Transwell assay to detect RAW264.7 derived foam cell migration, western blot and cell immune fluorescence detection in each experimental group cells associated protein change, the change in phalloidin labeled F-actin was detected in the different experimental groups RAW264.7 source of foam cells fiber shaped actin. Result(1) In the model group, the aortic HE staining showed typical atherosclerotic plaque formation, and obvious fiber cap. In group CML, the atherosclerotic plaque was more obvious, and obviously the fibrous cap was formed, the core of lipid necrosis appeared, and even the rupture of the fibrous plaque. In group 740Y-P, the area of aortic plaque in mice was significantly less than that in CML group, but it was slightly larger than that of model group, and the typical plaque was formed. In the control group, early plaque formation and relatively complete vascular wall, vascular endothelial occasionally with slight damage, no obvious fiber cap and necrotic core in the mice aorta.(2) Compared with the control group, the plaque area of the model group was increased, the fluorescence intensity of CD68 and CML was increased, and the area of atherosclerotic plaque was increased significantly;Compared with the 740Y-P group and the model group, the CML group and the model group were larger, and the fluorescence intensity was stronger(especially the patch area); Compared with CML group 740Y-P group of aortic plaque area is significantly reduced, fluorescence decrease; but compared with the model group, the plaque area slightly increased, enhanced fluorescence intensity compared with the model group. At the same time, CML can be down regulated the expression of p-Akt in plaque fluorescence expression, 740Y-P can partially reverse this process. The overall p-Akt fluorescence change trend and CML, CD68 fluorescence is the trend. With the control group, immunohistochemistry and RT-PCR confirmed the expression of CML, CD68 in CML group and 740Y-P group plaque increased compared with the model group, the results and the fluorescence intensity were similar(P<0.05).(3) Ox LDL can induce RAW264.7 cells to form RAW264.7 derived foam cells. CML intervention in different concentrations can affect the activity and proliferation of RAW264.7 derived foam cells. And with the increase of CML concentration, enhance the inhibitory effect on cell activity.(4) CML can inhibit RAW264.7 derived foam cell migration, this process can be partially reversed by 740Y-P.(5) CML could significantly increase the expression of cleavedcaspase-3 protein in RAW264.7 derived foam cells, reduce the expression of p-Akt protein, and the results can be partly reversed by 740Y-P.(6) CML can decrease the fluorescence intensity of RAW264.7 in F-actin cells, which can affect the cell migration, and 740Y-P can enhance the fluorescence intensity of F-actin. Conclusion:(1) CML can promote Apo E gene knockout mice aortic atherosclerotic plaque formation, increased macrophage derived foam cel s in plaque accumulation.(2) CML can inhibit RAW264.7 derived foam cell migration, this process can be partially reversed by 740Y-P.(3) CML promote the atherogenic effect may be related to the inhibition of CML macrophage derived foam cell migration, and its mechanism may be through inhibition of PI3K/Akt signaling inhibits foam cell migration.