| Part 1: IN VITROObjective: Macrophage apoptosis is an important feature of atherosclerosis. A likely cause of macrophage apoptosis is accumulation of free cholesterol (FC) in the ER, leading to activation of the unfolded protein response (UPR) induced apoptosis. But the cellular mechanism for this process is largely unknown. In this study, a cell culture model was used to explore the cellular mechanisms involved in ER-stress pathway of FC-induced macrophage apoptosis.Methods: FC loaded mouse peritoneal macrophages incubated with a cholesterol transport inhibitor U18666A, a polyclonal mouse anti-GADD153 antibody, a JNK specific inhibitor SP600125, a p38-MAPK specific inhibitor SB203580 or a ERK specific inhibitor PD98059 respectively for the indicated time, a series of biochemical assays were perform.Results: The results show that FC trafficking to the ER and activation of the C/EBP homologous protein (CHOP) branch of the UPR and subsequent apoptosis are required for IRE1αactivity. P38-MAPK and JNK signaling played a role in FC-induced apoptosis of macrophages, but JNK is independent of the UPR-CHOP pathway.Conclusions: Excess FC loading of macrophages leads to macrophage apoptosis that is partially dependent on induction of the ER-stress response. The IRE1α, p38-CHOP and JNK signal pathway is involved in this process. Part:2 IN VIVOObjective: To study the pathway of FC-induced macrophage apoptosis by using the atherosclerotic mice.Methods: Six apoE-/- mice on the C57BL6/J background were fed with a high-cholesterol diet containing 21% anhydrous milk fat and cholesterol for the 13 weeks. Another six wild type C57BL6/J mice as control fed with a standard diet. After for the indicated time plasma levels of TG, TC, LDLC, HDLC and the atheromatous lesions were analyzed. Immunoblot was used to determine the expression of IRE1α,CHOP,MAPKs.Results: The results show that after fed with a high-cholesterol diet for 13 weeks, the average (percentage mean) size of the atherosclerotic lesion area in the apoE-/- mice was (50.91±4.35)% of the total aortic arch surface. There was no apparent lesion found in wild-type mice. In situ TUNEL staining of proximal aorta sections showed that macrophage apoptosis was significantly increased in atherosclerotic lesion compared with normal aortic tissue. The expression of active IRE1, CHOP, phospho-JNK and phospho-p38 were found to be markedly elevated in proximal aortic lesions from apoE-/- mice compared with wild-type mice. No phospho-ERK has been detected in the two droups.Conclusions: It can be conclusioned that hypercholesteremia is an improment factor for atherosclerosis and excess FC loading of macrophages leads to macrophage apoptosis to promote progression of atherosclerosis. IRE1α, CHOP, JNK, p38 signal pathways is involved in this process. |
