The Effect Of Autophagy On The Inflammation Induced By Ox-LDL

Cardiovascular disease is one of the main causes of human death at present. Atherosclerosis is the pathological basis of cardiovascular and cerebrovascular diseases. Atherosclerosis can be caused by many factors and its pathogenesis is not completely clear. Many studies showed that inflammation and abnormal lipid metabolism were involved in the formation and development of atherosclerosis. Low density lipoprotein(LDL) in the blood plays an important role in the occurrence of atherosclerosis. LDL can cause endothelial cell dysfunction and endothelial cell injury. It can also increase the expression of ICAM1and VCAM1on endothelial cell surface.The expression of ICAM1and VCAM1can promote the adhesion of monocyte with endothelial cells and the migration of monocyte into the subendothelial. Monocytes engulf LDL, and oxidize it into oxidized low density(ox-LDL). Meanwhile, monocytes transform into macrophages. Macrophages can secrete a variety of cytokines. These cytokines promote local vessel inflammation, promote the proliferation of vascular smooth muscle cells, aggravate the vessel injury, and eventually lead to the development of atherosclerosis.With many studies revealed that autophagy plays an important role in many disease, the role of autophagy in the atherosclerosis become the focus of study. Recent study showed that autophagy was induced in the atherosclerotic plaque cells and autophagy can also influence the process of atherosclerosis. Autophagy is used to degrade and recycle their cytoplasmic contents in the eukaryotic cells. Basal levels of autophagy plays a very important role in the maintenance of vascular function and morphology. In this study, we observed whether ox-LDL could induce autophagy in HUVEC cells and THP1cells, and investigate the role of autophagy in the inflammation induced by ox-LDL. Part1The effect of autophagy on the ox-LDL induced inflammation in the HUVEC cellsObjective:To examine whether ox-LDL can induce autophagy in the HUVEC cells and investigate the effect of autophagy on the expression of ICAM1、VCAM1and MCP-1induced by ox-LDL.Methods:1. The expression of LC3in the HUVEC cells treated with20,40,80μg/ml ox-LDL was detected by western-blot. The distribution of LC3protein was detected by immunofluorescence.2. The acidic vesicles in the HUVEC cells treated with20,40,80μg/ml ox-LDL was analyzed by MDC staining. The fluorescence intensity of MDC was analyzed by flow cytometry. The acidic vesicles in the cells was observed under fluorescence microscopy.3.In order to determine the effects of autophagy on the inflammation induced by ox-LDL, the cells were divided into4groups including (1) control group;(2) ox-LDL treatment group (80μg/ml);(3)3-MA treatment group (5mmol/L);(4)3-MA (5mmol/L)+ox-LDL (80μg/ml) treatment group. The expression of LC3, ICAM1and VCAM1was detected by western-blot. The changes of MCP-1secretion was determined by ELISA analysis.Results:1. Ox-LDL treatment led to the upregulation of LC3and makes more LC3I transform into LC3II. Ox-LDL treatment increase the punctate fluorescent signals of LC3in HUVEC cells. These results indicated that ox-LDL could induce autophagy in the HUVEC cells2. The result of flow cytometry analysis showed that ox-LDL treatment could increase fluorescence intensity of MDC. Ox-LDL treatment could increase the formation of acidic vesicular organelles detected by fluorescence microscopy. These results confirmed that ox-LDL could induce autophagy in the HUVEC cells.3. The result of western-blot showed that ox-LDL treatment could increase the expression of ICAM1and VCAM1. Inhibition of autophagy by3-MA further increased the expression of ICAMland VCAM1induced by ox-LDL. The result of ELISA showed that ox-LDL treatment could increase the secretion of MCP-1and inhibition of autophagy by3-MA further increased the secretion of MCP-1induced by ox-LDL.Conclusion:Ox-LDL treatment could induce autophagy in the HUVEC cells and inhibition of autophagy could enhance the ox-LDL induced inflammation. Autophagy played a protective role in the ox-LDL induced inflammation.Part2The effect of autophagy on the ox-LDL induced inflammation in the THP1cellsObjective:To examine whether ox-LDL can induce autophagy in the THP1cells and investigate the effect of autophagy on the secretion of TNF-α IL-1and IL-6induced by ox-LDL.Methods:1. The expression of LC3in the THP1cells treated with20,40,80μg/ml ox-LDL was detected by western-blot. The distribution of LC3protein was detected by immunofluorescence.2. The acidic vesicles in the THP1cells treated with20,40,80μg/ml ox-LDL was analyzed by MDC staining. The fluorescence intensity of MDC was analyzed by flow cytometry. The acidic vesicles in the cells was observed under fluorescence microscopy.3.In order to determine the effects of autophagy on the inflammation induced by ox-LDL, the cells were divided into4groups including (1) control group;(2) ox-LDL treatment group (80μg/ml);(3)3-MA treatment group(5mmol/L);(4)3-MA (5mmol/L)+ox-LDL (80μg/ml) treatment group. The expression of LC3was detected by western-blot. The changes of TNF-α、IL-1and IL-6secretion was determined by ELISA analysis. Results:1. Ox-LDL treatment led to the upregulation of LC3and makes more LC3I transform into LC3Ⅱ. Ox-LDL treatment increase the punctate fluorescent signals of LC3in THP1cells. These results indicated that ox-LDL could induce autophagy in the THP1cells2. The result of flow cytometry analysis showed that ox-LDL treatment could increase fluorescence intensity of MDC. Ox-LDL treatment could increase the formation of acidic vesicular organelles detected by fluorescence microscopy. These results confirmed that ox-LDL could induce autophagy in the THP1cells.3. The result of ELISA showed that ox-LDL treatment could increase the secretion of TNF-α、IL-1and IL-6. Inhibition of autophagy by3-MA further increased the secretion of TNF-α、IL-1and IL-6induced by ox-LDL.Conclusion:Ox-LDL treatment could induce autophagy in the THP1cells and inhibition of autophagy could enhance the inflammation induced by ox-LDL. Autophagy played a protective role in the ox-LDL induced inflammation.