Study On The Function And Regulatory Mechanism Of Recombinant Human High Density Lipoprotein (rhHDL) To Macrophage Oxidative Stress

Atherosclerosis is one of the most fatal diseases in the world.As the living standard of people increased,the lipid content in the diet has been significantly improved.It is the main reason that cause the incidence of AS rise in recent years.So,people have pay more attention on the research about AS.The appearance of statins is a milestone for the treatment of AS.But,statins will produce serious side effects and resistance in long term treatment.In order to discover more effective and safer treatments,people begin to explore the mechanism of AS.The new targets will be found for the treatment of AS.In recent years,the study found that the accumulation of low density lipoprotein(LDL)is the starting factor of AS.However,further development and progression of AS is dependent on the inflammation which caused by macrophage cells engulfing the oxidized low density lipoprotein(ox-LDL).Therefore,anti-inflammation is becoming the new method to treat AS.People find that high density lipoprotein(HDL)can treat AS through anti-inflammation and cholesterol transfer.Along with the deepening of research,people gradually establish the mechanism of HDL in modulating different inflammatory cells.But,the function of HDL in modulating macrophage is not clear.So,in this project,the recombinant human high density lipoprotein(rhHDL)produced by the method of sodium cholate and construct foam cell model induced by ox-LDL.Then,the foam cells with different concentrations of rhHDL co-cultured to explore the mechanism and function of rhHDL in the process of macrophage oxidative stress.Firstly,the synthesis of bile acid sodium rhHDL through morphological identification of transmission electron microscopy,found that the synthetic rhHDL is relatively uniform roundness under transmission electron microscope,particle size of circular particles in 150 nm to 200 nm.Macrophage RAW264.7 and 80 mg/L High OxLDL foam cell model is established,a total of 24 h incubation,analysis to identify obviously increase the cholesterol content in the building of foam cells and cholesterol ester(CE)in cells and the ratio of total cholesterol(TC)>50%,and compared with the control group with significant difference.Morphological observation showed that model cell contains large amount of lipid droplets,in accordance with foam cells morphological characteristics.After identifying the rhHDL and foam cells,co-culture the foam cells with different concentration of rhHDL.With the increase of rhHDL concentration,the value of CE/TC and lipid content are reduced,and the process of foam cells formation is significantly inhibited.The result of time activity curve shows that rhHDL can promote the removal of cholesterol from the foam cells,and the effect is enhanced with the duration extension.Apoptosis experiments show that the process of induction foam cells formation by high ox-LDL will be accompanied by obvious apoptosis phenomenon,and rhHDL can significantly inhibit the apoptosis of macrophage.Through further experiment,found rhHDL can inhibit the apoptosis of macrophage by promoting anti-apoptotic gene expression and inhibiting the expression of apoptotic genes.The experiment of oxidative stress shows that rhHDL can increase the content of superoxide dismutase(SOD)in macrophage,and reduce the content of reactive oxygen species in macrophage.Meantime,the content of lipid oxidation product,malondialdehyde(MDA),is reduced obviously.This indicates that rhHDL can improve the antioxidant ability of macrophage.Further exploration of mechanism discovers that rhHDL can promote the expression of Nrf2 which is the key member of Keapl-Nrf2-ARE signaling pathway.Then,the high expression of Nrf2 will improve the antioxidant gene expression of its downstream,and this will benefit for macrophage to enhance the ability of resistance to oxidative stress.To sum up,found rhHDL can inhibit the process of foam cells formation through promoting the removal of cholesterol in macrophage,inhibit the apoptosis of macrophage,and improve the antioxidant ability of macrophage to treat the AS.Also,preliminary expound that rhHDL can improve the antioxidant ability of macrophage by modulating Nrf2 which is the key role of Keapl-Nrf2-ARE signaling pathway.