Influence Of Estradiol On Macrophage Phagocytosis Function And Its Mechanism Study

Background and ObjectiveAtherosclerosis can be considered as both composition changed in arterial wall and a lipid metabolism disorder. The process of atherosclerosis is the accumulation of lipid in the artery wall.which lead to the arterial thickening and the deep composition necrosis,disaggregation.The ultimate result is the formation of foam cells.The estrogen concentration of female gradually decreases with age.The morbidity and mortality of artherosclerosis apparently increase in women after menopause compared with before menopause. So it seems that estrogen could protect women from atherosclerosis, which was proved by a lot of animal experiments , the mechanism maybe involved in lipid metabolic pathways (such as lowering LDL, increased HDL) and non-lipid metabolic pathways (such as making atherosclerotic plaque more stable). However, some clinical investigations indicate that postmenopausal hormone replacement therapy(HRT) could increase the risk of myocardial infarction(MI), stroke, and venous thrombosis. breast carcinoma, endometrial cancer, etc.. there were also some clinical investigation indicate that although hormone therapy had reduced the mortality of artherosclerosis in premenopausal and early postmenopausal women, but in late postmenopausal women, the hormone therapy increased the risk of artherosclerosis. At present the contradictory role of estrogen has not been explained well.The initiation of atherosclerosis is macrophage invasion of modified low-density lipoprotein,lipid accumulation of plaque in the arterial wall. Various risk factors can damage endothelial cells, monocytes recruit and migrate to the endothelium aided by the endothelial expression of the adhesion molecules(VCAM, ICAM, selectins, etc.) , monocytes differentiate into macrophages aided by macrophage colony stimulating factor in endothelial. Macrophages take up low-density lipoprotein oxidation (Ox-LDL) by the scavenger receptor (SR-A and CD36, etc.)and form foam cell gradually .Macrophages are central to the initiation and progression of atherosclerosis.Atherosclerosis occurrence and the developmental process can be considered as a clean up Ox-LDL response of macrophage. Monocyte adhesion, migration, differentiation, phagocytosis and foam cell formation are the key of the process. A large amount NO generated by monocyte iNOS, can lead to oxidative stress, which oxidate LDL to Ox-LDL or NO₂-LDL. Ox-LDL activate PPARγby MAPK phosphorylation and increase PPARγactivity,subsequently enhances expression of scavenger receptor CD36, thereby promoting macrophage to take up Ox-LDL and forming foam cell . Ox-LDL can also directly result in JNK phosphorylation, thereby contributing to another scavenger receptor SR-A phosphorylation and promoting macrophage to take up Ox-LDL, promoting foam cell formation; iNOS also induced apoptosis of macrophages, NO regulates IL-8 and TNF-αproduction of macrophages through different mechanisms . The macrophages release the metalloproteinases protein (MMP) induced by TNF-α, IL^-1 , MMP and the extracellular matrix (ECM) components specifically combine to each other and lead to the degradation of ECM, reducing neovascularization, increasing plaque instability and promoting plaque rupture and thrombosis. Monocytes /macrophages are central to the initiation and progression of atherosclerosis and would be the potential targets for therapy.Studies have shown that there is the existence of estrogen receptor response element in iNOS gene of mice and human (vitellogenin ERE, AGGTCAnnnTGACCT) , estrogen can promote macrophage pro-inflammatory factor iNOS expression and activate macrophage . In addition, estrogen can promote the expression of TNF-α, increase the production of NO. Therefore, estrogen is to promote the inflammatory response in fact . So in late postmenopausal women, estrogen replacement therapy, as a result of the pro-inflammatory effects of estrogen, may result in atherosclerotic plaque instability, induced by acute vascular events.the ultimate result is to increase the risk of atherosclerotic disease. However, a single dose-effect relationship is contradictory with following phenomenon that estrogen show protective effect on women in premenopausal period, menopausal period and early postmenopausal period, but the risk of atherosclerosis increases in late postmenopausal women.To explain this paradox phenomenon RAW264. 7 are selected to study the influence of estradiol on oxidized Ox-LDL induced macrophage phagocytosis function and its mechanism. MethodsRAW 264. 7 macrophages incubated in DMEM medium containing 10% fetal bovine serum were induced by Ox-LDL and were assigned into six groups: control group,DMSO treated group , 1 pmol·L^-1 estradiol treated group, 25 pmol·L^-1 estradiol treated group, 50 pmol·L^-1 estradiol treated group, 100 pmol·L^-1 estradiol treated group. Oil red O staining was used to show the lipid droplets in macrophages cells. Reverse-transcription polymerase chain (RT-PCR) method was used to reveal the express of scavenger receptor CD36,SR-BI, iNOS and AGR1 mRNA. Nitric oxide (NO) content was measured by the method of nitric acid reductase.ResultsThere were more lipid droplets in macrophages cells which induced by Ox-LDL than that was not induced by Ox-LDL, as well as the express of CD36,SR-BI,iNOS, AGR1 mRNA, and the NO content in cell culture fluid. Among the cells induced by Ox-LDL, there was no significant difference between the DMSO treated group, l pmol·L^-1 estradiol treated groups and the control group, while more lipid droplets were in cells which incubated with 25 pmol·L^-1 , 50 pmol·L^-1 , 100 pmol·L^-1 estradiol treated group compared with the DMSO treated group, The express of CD36, iNOS mRNA and the NO content in cell culture fluid was obviously increased(P<0.05), but the express of ARG1 mRNA was decreased in 25 pmol·L^-1, 50 pmol·L^-1,100 pmol·L^-1 estradiol group. The dose-relationship shows inverse-correlation, However there was no significant difference in the express of SR-BI.. ConclusionEstradiol (1～100pmol·L^-1 ) can promote mouse monocyte-macrophage RAW264. 7 to take up the Ox-LDL , and enhance the expression of CD36, which may be due to pro-inflammatory effects of estradiol., the greatest effect was at the concentration of 25 pmol·L^-1.