色素上皮衍生因子对氧化低密度脂蛋白诱导的巨噬细胞炎性反应的影响研究

Impact of Pigment Epithelium-derived Factor on Oxidized Low Density Lipoprotein-induced Macrophage Inflammatory Response

背景近年来越来越多的研究证据表明,伴有巨噬细胞浸润和极化的局部和全身炎症反应是导致动脉粥样硬化斑块不稳定的主要原因,因此减少巨噬细胞源性泡沫细胞形成、抑制巨噬细胞炎性反应对提高斑块稳定性及防治心脑血管疾病具有重要意义。目的探讨色素上皮衍生因子(PEDF)对氧化低密度脂蛋白(Ox-LDL)诱导的巨噬细胞炎性反应的影响。方法本实验于2018年1—8月完成。实验分组前将巨噬细胞分为空白对照组及A、B、C、D组,A、B、C、D组细胞分别给予终浓度为100、200、400、800 ng/ml的PEDF处理24 h,进行细胞毒性试验。细胞毒性试验完成后将巨噬细胞随机分为实验对照组、炎性反应组、低浓度组、中浓度组、高浓度组。炎性反应组细胞加OxLDL处理24 h诱导炎性反应;低浓度组、中浓度组、高浓度组分别加终浓度为100、200、400 ng/ml的PEDF处理24 h,之后加Ox-LDL处理24 h诱导炎性反应。采用CCK-8法检测细胞活力,采用Western blot法检测细胞内白介素1(IL-1)、单核细胞趋化因子1(MCP-1)蛋白表达,采用酶联免疫吸附试验(ELISA)检测细胞外IL-1、MCP-1蛋白表达,采用Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡率。结果 (1)D组细胞活力低于空白对照组(P<0.05),A、B、C组细胞活力与空白对照组比较,差异无统计学意义(P>0.05);PEDF适宜干预浓度为100、200、400 ng/ml。(2)炎性反应组、低浓度组、中浓度组、高浓度组细胞活力低于实验对照组,中浓度组、高浓度组细胞活力低于炎性反应组(P<0.05)。(3)炎性反应组、低浓度组、中浓度组、高浓度组细胞内外IL-1蛋白相对表达量高于实验对照组,低浓度组、中浓度组、高浓度组细胞内外IL-1蛋白相对表达量低于炎性反应组(P<0.05);炎性反应组、低浓度组、中浓度组、高浓度组细胞内外MCP-1蛋白相对表达量高于实验对照组,中浓度组、高浓度组细胞内外MCP-1蛋白相对表达量低于炎性反应组(P<0.05)。(4)炎性反应组、低浓度组、中浓度组、高浓度组细胞凋亡率高于实验对照组,中浓度组、高浓度组细胞凋亡率高于炎性反应组(P<0.05)。结论终浓度为200、400 ng/ml的PEDF能有效降低巨噬细胞活力,下调IL-1和MCP-1蛋白表达,促进巨噬细胞凋亡,进而抑制Ox-LDL诱导的巨噬细胞炎性反应。

Background In recent years,more and more research evidence shows that,local and systemic inflammatory response with macrophage infiltration and polarization are the major causes of atherosclerotic plaque instability.Therefore,reducing the formation of macrophage derived foam cells and inhibiting inflammatory response of macrophages are of great importance for improving the plaque stability,prevention and treatment of cardiovascular and cerebrovascular diseases.Objective To investigate the impact of pigment epithelium-derived factor(PEDF)on oxidized low density lipoprotein(Ox-LDL)-induced macrophage inflammatory response.Methods This experiment was carried out from January to August 2018.Macrophages were divided into blank control group and groups A,B,C and D before grouping,and then cells in groups A,B,C and D received PEDF with final concentration of 100,200,400,800 ng/ml for 24 hours to complete the cytotoxicity test,respectively.After cytotoxicity test,macrophages were randomly divided into experimental control group,inflammatory response group,low concentration group,medium concentration group and high concentration group.Cells in inflammatory response group received Ox-LDL for 24 hours to induce inflammatory response,cells in low concentration group,medium concentration group and high concentration group received PEDF with final concentration of 100,200 and 400 ng/ml for 24 hours,respectively,followed by Ox-LDL for 24 hours to induce inflammatory response.CCK-8 method was used to detect the cell viability,Western blot method was used to detect the intracellular expression of IL-1 protein and MCP-1 protein,ELISA was used to detect the extracellular expression of IL-1 protein and MCP-1 protein,and Annexin V-FITC/PI double staining method was used to detect the apoptotic rate.Results(1)Cell viability in D group was statistically significantly lower than that in blank control group(P<0.05),while there was no statistically significant difference in cell viability in groups A,B,C and blank control(P>0.05),thus the appropriate intervention concentration of PEDF was 100,200 and 400 ng/ml.(2)Cell viability in inflammatory response group,low concentration group,medium concentration group and high concentration group was statistically significantly lower than that in experimental control group,respectively,meanwhile cell viability in medium concentration group and high concentration group was statistically significantly lower than that in inflammatory response group,respectively(P<0.05).(3)Relative expression quantity of intracellular and extracellular IL-1 protein in inflammatory response group,low concentration group,medium concentration group and high concentration group was statistically significantly higher than that in experimental control group,respectively,meanwhile relative expression quantity of intracellular and extracellular IL-1 protein in low concentration group,medium concentration group and high concentration group was statistically significantly lower than that in inflammatory response group,respectively(P<0.05);relative expression quantity of intracellular and extracellular MCP-1 protein in inflammatory response group,low concentration group,medium concentration group and high concentration group was statistically significantly higher than that in experimental control group,respectively,meanwhile relative expression quantity of intracellular and extracellular MCP-1 protein in medium concentration group and high concentration group was statistically significantly lower than that in inflammatory response group,respectively(P<0.05).(4)Apoptotic rate in inflammatory response group,low concentration group,medium concentration group and high concentration group was statistically significantly higher than that in experimental control group,respectively,meanwhile apoptotic rate in medium concentration group and high concentration group was statistically significantly higher than that in inflammatory response group,respectively(P<0.05).Conclusion PEDF with final concentration of 200 and 400 ng/ml can effectively reduce viability of macrophage,down-regulate the expression of IL-1 protein and MCP-1 protein,promote the apoptosis of macrophage,and then inhibit the Ox-LDL-induced macrophage inflammatory response.