摘要
目的探讨脯氨酸/丝氨酸丰富的卷曲螺旋蛋白1(PSRC1)与膜联蛋白A2(ANXA2)相互作用减缓动脉粥样硬化进展的可能机制。方法为检测与PSRC1结合的蛋白,将RAW264.7巨噬细胞分为Ad-GFP+ox-LDL组与Ad-PSRC1+ox-LDL组,采用非标记定量蛋白质组学方法检测与PSRC1结合的蛋白表达水平;采用免疫共沉淀(Co-IP)实验及免疫荧光实验对上述蛋白质谱结果进行验证。为检测ox-LDL刺激下过表达PSRC1对ANXA2分泌的影响,将RAW264.7巨噬细胞分为四组(对照组、ox-LDL组、Ad-GFP+ox-LDL组、Ad-PSRC1+ox-LDL组),采用酶联免疫吸附实验(ELISA)检测各组培养上清中ANXA2的水平。为检测Ad-shANXA2对AXNA2的敲低效果,将RAW264.7巨噬细胞分为Ad-GFP组与Ad-shANXA2组,采用实时荧光定量PCR(RT-qPCR)检测细胞内ANXA2 mRNA的表达情况。为检测敲低ANXA2对主动脉斑块进展及炎性因子分泌的影响,将24只ApoE-/-小鼠随机分为四组(n=6):普通饮食+Ad-GFP组、普通饮食+AdshANXA2组、高脂饮食+Ad-GFP组、高脂饮食+Ad-shANXA2组,采用油红O染色观察主动脉大体及主动脉根部动脉粥样硬化(AS)面积,并采用ELISA检测高脂饮食+Ad-GFP组与高脂饮食+Ad-shANXA2组小鼠血清中白细胞介素-1β(IL-1β)及IL-6的水平。结果蛋白质组学检测结果显示,用ox-LDL刺激后PSRC1与ANXA2的结合明显增加,且二者的结合具有特异性,免疫荧光实验也显示PSRC1与ANXA2在细胞内存在共定位现象。RT-PCR检测发现,与Ad-GFP组比较,Ad-shANXA2组的ANXA2水平明显下降(0.198±0.065 vs.1.002±0.069,P<0.05);ELISA检测发现,与对照组比较,ox-LDL组的ANXA2水平明显升高[(2027.23±93.55)pg/ml vs.(697.01±30.08)pg/ml,P<0.01],与Ad-GFP+ox-LDL组比较,Ad-PSRC1+ox-LDL组的ANXA2水平明显降低[(1061.65±68.52)pg/ml vs.(2098.67±318.41)pg/ml,P<0.01]。在动物实验中,油红O染色发现普通饮食两组间主动脉大体及主动脉根部斑块面积差异无统计学意义;而与高脂饮食+Ad-GFP组比较,高脂饮食+Ad-shANXA2组小鼠的主动脉大体斑块面积百分比明显减少(5.29%±1.14%vs.12.28%±2.48%,P<0.05),主动脉根部切片斑块面积百分比也明显减少(1.31%±0.04%vs.2.83%±0.22%,P<0.05),且血清中IL-1β水平明显降低[(122.90±9.80)pg/ml vs.(172.90±21.83)pg/ml,P<0.05],IL-6水平也明显降低[(3.65±0.12)pg/ml vs.(5.97±0.42)pg/ml,P<0.05]。结论巨噬细胞过表达PSRC1可减缓动脉粥样硬化的进展,其机制可能与PSRC1与ANXA2的结合增加,抑制ANXA2释放至胞外,从而抑制炎性因子的释放有关。
Objective To investigate the possible mechanism of proline/serine rich coil protein 1 and annexin A2(PSRC1-ANXA2)interaction to attenuate the progression of atherosclerosis.Methods To detect the PSRC1 binding proteins,RAW264.7 macrophages were divided into two groups:control group(Ad-GFP)and PSRC1 overexpression group(Ad-PSRC1),treated with ox-LDL(100μg/ml)for 24 h after adenovirus transfection,and the PSRC1-binding proteins were detected by non-labeled quantitative macrophages.The above protein spectra were verified by co-immunoprecipitation(Co-IP)and immunofluorescence assay.To detect the effect of PSRC1 overexpression on ANXA2 secretion after ox-LDL stimulation,RAW264.7 macrophages were divided into four groups:control group,ox-LDL group,Ad-GFP+ox-LDL group and Ad-PSRC1+ox-LDL group.The levels of ANXA2 in the cultured supernatant were determined by enzyme-linked immunosorbent assay(ELISA).To detect the knockdown effect of adenovirus Ad-shANXA2 on AXNA2,RAW264.7 macrophages were divided into two groups:Ad-GFP group and Ad-shANXA2 group.The mRNA levels of ANXA2 in RAW264.7 macrophages were detected by real-time fluorescence quantitative PCR(RTqPCR).To detect the effect of ANXA2 knockdown on the progression of aortic plaque and the secretion of inflammatory factors,24 ApoE-/-mice were randomly divided into four groups(6 each):chow diet+Ad-GFP group,chow diet+Ad-shANXA2 group,high fat diet+Ad-GFP group and high fat diet+Ad-shANXA2 group.The atherosclerosis areas(AS)of aorta and aortic root were detected by oil red O staining.The serum levels were determined by ELISA assay of interleukin-1β(IL-1β)and IL-6 in high fat diet+Ad-GFP group and high fat diet+Ad-shANXA2 group.Results The results of proteomics analysis showed that,after stimulation with ox-LDL,the binding of PSRC1 and ANXA2 increased significantly,and the combination was of specificity.Immunofluorescence also showed that the co-localization of PSRC1 and ANXA2 existed in the cells.RT-PCR revealed that,compared with Ad-GFP group,the ANXA2 level decreased significantly in Ad-shANXA2 group(0.198±0.065 vs.1.002±0.069,P<0.05).ELISA results showed that,compared with the control group,the ANXA2 level increased significantly in the ox-LDL group[(2027.23±93.55)pg/ml vs.(697.01±30.08)pg/ml,P<0.01],and compared with Ad-GFP+ox-LDL group,the ANXA2 level reduced significantly in Ad-PSRC1+ox-LDL group[(1061.65±68.52)pg/ml vs.(2098.67±318.41)pg/ml,P<0.01].In animal experiments,oil red O staining revealed that no statistical difference existed in the area of aortic gross and aortic root plaques between the two groups of chow diet.Compared with high-fat diet+Ad-GFP group,the percentage of aortic plaque area decreased significantly,and of aortic root section also decreased significantly in high-fat diet+Ad-shANXA2 group(5.29%±1.14%vs.12.28%±2.48%,P<0.05;1.31%±0.04%vs.2.83%±0.22%,P<0.05).ELISA test found that,compared with high-fat diet+Ad-GFP group,the IL-1βlevel decreased significantly[(122.90±9.80)pg/ml vs.(172.90±21.83)pg/ml,P<0.05],and the IL-6 level decreased also[(3.65±0.12)pg/ml vs.(5.97±0.42)pg/ml,P<0.05]in high-fat diet+Ad-shANXA2 group.Conclusion Over-expression of PSRC1 in macrophages can attenuate the progression of atherosclerosis,where the increased binding of PSRC1 to ANXA2,and the inhibition of ANXA2 release,thereby inhibiting the release of inflammatory factor may be the possible related mechanism.
作者
郭中州
张亚南
刘季晨
郭志刚
Guo Zhong-Zhou;Zhang Ya-Nan;Liu Ji-Chen;Guo Zhi-Gang(Department of Cardiology,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China)
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2021年第5期440-447,共8页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金青年基金(81900398,81700388)
广东省自然科学基金面上项目(2019A1515010666)
广东省教育厅高水平大学建设经费南方医科大学临床研究启动项目(LC2016PY002)
南方医院临床研究专项(2018CR051)。
