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Article(id=1210676786367697858, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1210676785113600955, articleNumber=null, orderNo=null, doi=10.11855/j.issn.0577-7402.2021.05.03, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1599062400000, receivedDateStr=2020-09-03, revisedDate=1606838400000, revisedDateStr=2020-12-02, acceptedDate=null, acceptedDateStr=null, onlineDate=1766577439157, onlineDateStr=2025-12-24, pubDate=1622131200000, pubDateStr=2021-05-28, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1766577439157, onlineIssueDateStr=2025-12-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1766577439157, creator=13701087609, updateTime=1766577439157, updator=13701087609, issue=Issue{id=1210676785113600955, tenantId=1146029695717560320, journalId=1189873630562394117, year='2021', volume='46', issue='5', pageStart='425', pageEnd='530', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1766577438858, creator=13701087609, updateTime=1766718730270, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1211269404306838321, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1210676785113600955, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1211269404306838322, tenantId=1146029695717560320, journalId=1189873630562394117, issueId=1210676785113600955, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=440, endPage=447, ext={EN=ArticleExt(id=1210676786657104842, articleId=1210676786367697858, tenantId=1146029695717560320, journalId=1189873630562394117, language=EN, title=Mechanism of PSRC1-ANXA2 interaction in attenuating the progression of atherosclerosis, columnId=1190310110212751762, journalTitle=Medical Journal of Chinese People’s Liberation Army, columnName=Basic Research, runingTitle=null, highlight=null, articleAbstract=

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 (100 μg/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 (RT-qPCR). 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-1β level 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.

, correspAuthors=Zhi-Gang Guo, authorNote=null, correspAuthorsNote=
*E-mail:
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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+Ad-shANXA2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。

, correspAuthors=郭志刚, authorNote=null, correspAuthorsNote=
郭志刚，E-mail：
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郭中州，硕士研究生，主要从事冠状动脉粥样硬化发病机制方面的研究

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郭中州，硕士研究生，主要从事冠状动脉粥样硬化发病机制方面的研究

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郭中州，硕士研究生，主要从事冠状动脉粥样硬化发病机制方面的研究

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A. 过表达PSRC1+ox-LDL刺激组与过表达GFP+ox-LDL刺激组免疫共沉淀的质谱总离子流图；B. 采用STRING软件对变化倍数上调>1.2倍的蛋白进行的互作分析；C. 质谱鉴定到的ANXA2蛋白代表性肽段的肽指纹图；D. ANXA2蛋白全长序列(红色：图1C中肽指纹图谱所展示的肽段；绿色：质谱鉴定到的其他肽段；黑色：未鉴定到的序列)

, figureFileSmall=0ePawps2x6HkMcCaC+wFOQ==, figureFileBig=V8tUJfC9LtGwTp6UC+Nhcw==, tableContent=null), ArticleFig(id=1210676791165980831, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=EN, label=Fig.2, caption=Co-immunoprecipitation to verify the binding of PSRC1 and ANXA2, figureFileSmall=TPqN5/R74I/QHhIji8hZ1g==, figureFileBig=qlAJ82udanjdC0C96tPaoA==, tableContent=null), ArticleFig(id=1210676791237284003, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=CN, label=图2, caption=免疫共沉淀验证PSRC1与ANXA2的结合

A. 外源过表达flag-PSRC1，使用anti-flag磁珠进行免疫共沉淀；B. 使用ANXA2抗体进行免疫共沉淀，检测内源PSRC1

, figureFileSmall=TPqN5/R74I/QHhIji8hZ1g==, figureFileBig=qlAJ82udanjdC0C96tPaoA==, tableContent=null), ArticleFig(id=1210676791371501736, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=EN, label=Fig.3, caption=ANXA2 in RAW264.7 supernatant after over-expression of PSRC1 (ELISA), figureFileSmall=2Wk/D/5EY2mY2SfGjFf8VQ==, figureFileBig=gsIXauxlZ6N7tIbcwroNgA==, tableContent=null), ArticleFig(id=1210676791455387820, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=CN, label=图3, caption=RAW264.7巨噬细胞过表达PSRC1对培养上清中ANXA2水平的影响

A. 对照组与ox-LDL组ANXA2表达情况比较，与对照组比较，(1)P<0.05；B. Ad-GFP+ox-LDL组与Ad-PSRC1+ox-LDL组ANXA2表达情况比较，与Ad-GFP+ox-LDL组比较，(2)P<0.05。

, figureFileSmall=2Wk/D/5EY2mY2SfGjFf8VQ==, figureFileBig=gsIXauxlZ6N7tIbcwroNgA==, tableContent=null), ArticleFig(id=1210676791547662513, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=EN, label=Fig.4, caption=Intracellular co-localization of ANXA2 and PSRC1 (Immunofluorescence staining), figureFileSmall=AmQ0oMAJ3DqEwZfyxKFyPQ==, figureFileBig=WhLHP2nq83HIuZx9CsaSbw==, tableContent=null), ArticleFig(id=1210676791665103031, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=CN, label=图4, caption=免疫荧光染色检测ANXA2与PSRC1在细胞内的共定位情况, figureFileSmall=AmQ0oMAJ3DqEwZfyxKFyPQ==, figureFileBig=WhLHP2nq83HIuZx9CsaSbw==, tableContent=null), ArticleFig(id=1210676791748989115, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=EN, label=Fig.5, caption=Aortas of apoE-/- mice under standard rodent chow or high-fat diet transfected with adenovirus (Oil red O staining), figureFileSmall=3u9ATpAEBAMjj0DUF613Xw==, figureFileBig=6lMs2xdtplDmAWzBbEAV8g==, tableContent=null), ArticleFig(id=1210676791849652417, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=CN, label=图5, caption=普通饮食或高脂饮食喂养+病毒转染apoE-/-小鼠主动脉大体标本油红O染色

A. 普通饮食+Ad-GFP组与普通饮食+Ad-shANXA2组主动脉大体斑块；B. 高脂饮食+Ad-GFP组与高脂饮食+Ad-shANXA2组主动脉大体斑块

, figureFileSmall=3u9ATpAEBAMjj0DUF613Xw==, figureFileBig=6lMs2xdtplDmAWzBbEAV8g==, tableContent=null), ArticleFig(id=1210676791933538503, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=EN, label=Fig.6, caption=Aortic root tissue of apoE-/- mice under standard rodent chow or an high-fat diet transfected with adenovirus (Oil red O staining,×400), figureFileSmall=yaLHRFXxf3KRVrXueBT/dg==, figureFileBig=VWIlq9QRWfj61RXLTRblPQ==, tableContent=null), ArticleFig(id=1210676792030007501, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=CN, label=图6, caption=普通饮食或高脂饮食喂养+病毒转染apoE-/-小鼠主动脉根部切片油红O染色(×400)

A. 普通饮食+Ad-GFP组与普通饮食+Ad-shANXA2组主动脉根部斑块；B. 高脂饮食+Ad-GFP组与高脂饮食+Ad-shANXA2组主动脉根部斑块

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Basic information of upregulated proteins binding to PSRC1

, figureFileSmall=null, figureFileBig=null, tableContent=
Accession No.蛋白名称缩写平均分子质量(Da)鉴别肽段序号PSRC1+ox-LDL组相对丰度对照组相对丰度错误发现率变化倍数
P07356膜联蛋白A2ANXA238 6769151.19416.9800.0188.91
Q8K310Matrin-3蛋白MATR394 63010.3970.0680.0096.58
Q8VE97精氨酸/丝氨酸丰富剪接因子4SRSF455 97910.1460.0280.0425.28
Q9JHU9肌醇-3-磷酸合成酶1INO160 93222.2280.5640.0443.95
Q60872真核转录起始因子1AIF1A16 50230.1560.0480.0263.23
Q61189Methylosome亚基pIClnICLN26 02110.1750.0660.0112.63
P17742肽酰-丙酰顺反异构酶APPIA17 971450.44824.6970.0502.04
Q7TNC4推定的RNA结合蛋白Luc7样2LC7L246 58340.0580.0290.0121.96
P06330IG重链V区AC38 205.12HVM5112 93425.8383.3340.0191.75
E9Q557桥粒斑蛋白DESP332 9121319.53311.1600.0061.75
Q3UL36富含精氨酸和谷氨酸的蛋白1ARGL132 88740.5840.4270.0221.37
P07091S100-A4蛋白S10A411 72143.8802.9690.0221.31
P09103蛋白质二硫键异构酶PDIA157 058117.1895.7140.0081.26
), ArticleFig(id=1210676792201973973, tenantId=1146029695717560320, journalId=1189873630562394117, articleId=1210676786367697858, language=CN, label=表1, caption=

与PSRC1结合上调的蛋白基本信息

, figureFileSmall=null, figureFileBig=null, tableContent=
Accession No.蛋白名称缩写平均分子质量(Da)鉴别肽段序号PSRC1+ox-LDL组相对丰度对照组相对丰度错误发现率变化倍数
P07356膜联蛋白A2ANXA238 6769151.19416.9800.0188.91
Q8K310Matrin-3蛋白MATR394 63010.3970.0680.0096.58
Q8VE97精氨酸/丝氨酸丰富剪接因子4SRSF455 97910.1460.0280.0425.28
Q9JHU9肌醇-3-磷酸合成酶1INO160 93222.2280.5640.0443.95
Q60872真核转录起始因子1AIF1A16 50230.1560.0480.0263.23
Q61189Methylosome亚基pIClnICLN26 02110.1750.0660.0112.63
P17742肽酰-丙酰顺反异构酶APPIA17 971450.44824.6970.0502.04
Q7TNC4推定的RNA结合蛋白Luc7样2LC7L246 58340.0580.0290.0121.96
P06330IG重链V区AC38 205.12HVM5112 93425.8383.3340.0191.75
E9Q557桥粒斑蛋白DESP332 9121319.53311.1600.0061.75
Q3UL36富含精氨酸和谷氨酸的蛋白1ARGL132 88740.5840.4270.0221.37
P07091S100-A4蛋白S10A411 72143.8802.9690.0221.31
P09103蛋白质二硫键异构酶PDIA157 058117.1895.7140.0081.26
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PSRC1与ANXA2相互作用减缓动脉粥样硬化进展的作用机制
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郭中州 , 张亚南 , 刘季晨 , 郭志刚 *
解放军医学杂志 | 基础研究 2021,46(5): 440-447
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解放军医学杂志 | 基础研究 2021, 46(5): 440-447
PSRC1与ANXA2相互作用减缓动脉粥样硬化进展的作用机制
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郭中州, 张亚南, 刘季晨, 郭志刚*
作者信息
  • 南方医科大学南方医院心血管内科，广州 510515

    • 郭中州，硕士研究生，主要从事冠状动脉粥样硬化发病机制方面的研究

通讯作者:

郭志刚，E-mail：
Mechanism of PSRC1-ANXA2 interaction in attenuating the progression of atherosclerosis
Zhong-Zhou Guo, Ya-Nan Zhang, Ji-Chen Liu, Zhi-Gang Guo*
Affiliations
  • Department of Cardiology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China
出版时间: 2021-05-28 doi: 10.11855/j.issn.0577-7402.2021.05.03
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摘要
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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+Ad-shANXA2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。

动脉粥样硬化  /  脯氨酸/丝氨酸丰富的卷曲螺旋蛋白1  /  膜联蛋白A2  /  炎性因子

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 (100 μg/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 (RT-qPCR). 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-1β level 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.

atherosclerosis  /  proline/serine-rich coiled-coil protein 1  /  annexin A2  /  inflammatory cytokines
郭中州, 张亚南, 刘季晨, 郭志刚. PSRC1与ANXA2相互作用减缓动脉粥样硬化进展的作用机制. 解放军医学杂志, 2021 , 46 (5) : 440 -447 . DOI: 10.11855/j.issn.0577-7402.2021.05.03
Zhong-Zhou Guo, Ya-Nan Zhang, Ji-Chen Liu, Zhi-Gang Guo. Mechanism of PSRC1-ANXA2 interaction in attenuating the progression of atherosclerosis[J]. Medical Journal of Chinese People’s Liberation Army, 2021 , 46 (5) : 440 -447 . DOI: 10.11855/j.issn.0577-7402.2021.05.03
正文
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动脉粥样硬化(atherosclerosis，AS)是一种慢性炎症性疾病[1]，降脂联合抗炎治疗可降低其主要心血管事件的发生率[2-3]。研究证实，巨噬细胞在AS的炎症反应过程中扮演了重要角色[4]。本课题组前期研究结果显示，过表达脯氨酸/丝氨酸丰富的卷曲螺旋蛋白1(proline/serine-rich coiled-coil protein 1，PSRC1)可通过抑制巨噬细胞的炎症反应延缓AS的进展[5]。PSRC1是由PSRC1基因编码的一种微管结合蛋白，其中一端可与发挥细胞支撑作用的微管蛋白结合以维持细胞骨架的稳定性，但仅存在于胞质内，且其影响巨噬细胞炎性因子释放的确切分子机制国内外均未见报道。因此，本课题组提出PSRC1的另一端与细胞内某种其他蛋白结合而抑制炎症反应，进而发挥抑制AS作用的假设，以寻找影响巨噬细胞炎症反应的新靶点，为防治AS提供新的思路。
1 材料与方法
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1.1 主要材料
小鼠巨噬细胞RAW264.7购自中国典型培养物保藏中心。重组腺病毒载体Ad-PSRC1、Ad-GFP及Ad-shANXA2由和元生物技术(上海)有限公司构建；ox-LDL购自广州奕源生物科技有限公司，ANXA2单克隆抗体购自研如玉(广州)生物科技有限公司，DAPI染色液购自上海碧云天生物科技有限公司，RNA提取试剂Trizol及反转录试剂盒购自日本TaKaRa公司，RT-PCR引物购自生工生物工程(上海)股份有限公司，DMEM培养液、胎牛血清(FBS)、胰酶购自美国Gibco公司，PBS购自美国Thermo Scientific公司；离心管、EP管购自美国Axygen公司；细胞培养瓶、培养皿购自美国Corning公司；牛血清白蛋白(BSA)、油红O染料购自美国Sigma公司；二甲基亚砜(DMSO)购自美国Biosharp公司；ANXA2 ELISA试剂盒购自广州徕智生物科技有限公司，白细胞介素-1β(IL-1β)、IL-6 ELISA试剂盒购自广州佳研生物科技有限公司。
1.2 实验方法
1.2.1 非标记定量蛋白质组学方法鉴定与PSRC1结合上调的蛋白
将RAW264.7巨噬细胞分为Ad-GFP+ox-LDL组(Ad-GFP+100 μg/ml ox-LDL)与Ad-PSRC1+ox-LDL组(Ad-PSRC1+100 μg/ml ox-LDL)。ox-LDL刺激24 h后对细胞进行裂解，使用抗-FLAG®M2磁珠(Millipore，Cat. M8823)对两组细胞裂解液进行下拉试验，所获产物在南方医科大学中心实验室使用超高分辨三合一质谱仪(Thermo ScientificTM Orbitrap FusionTM TribridTM)进行数据非依赖(data independent acquisition，DIA)的定量质谱分析，具体步骤如下。首先对样品进行还原烷基化、胰酶酶解；然后对酶解后肽段除去非挥发性盐，分别进行数据依赖(data dependent acquisition，DDA)图谱采集及数据非依赖采集(data independent acquisition，DIA)；最后，将数据导入Skyline软件(Skyline v4.2 Released on 11/01/2018)进行定量分析。将Ad-PSRC1+ox-LDL组/Ad-GFP+ox-LDL组变化倍数上调1.2倍以上，且错误发现率(false discovery rate，FDR)<0.05的蛋白质作为与PSRC1结合上调的蛋白质。
1.2.2 免疫共沉淀(Co-IP)实验验证蛋白质谱结果
过表达Co-IP：构建带有标签蛋白的PSRC1真核表达质粒(pcDNA3.1/flag-PSRC1)并转染至RAW264.7细胞中，以ox-LDL刺激24 h。裂解细胞，提取胞质中的蛋白。采用ANTI-FLAG M2 Magnetic beads(Sigma)进行Co-IP实验，再用标签蛋白或相应蛋白抗体进行Western blotting检测，比较刺激前后ANXA2与PSRC1相互作用的变化。内源Co-IP：使用ANXA2抗体对ox-LDL刺激24 h前后的RAW264.7细胞裂解液进行Co-IP实验，再采用PSRC1抗体或ANXA2抗体进行Western blotting检测。
1.2.3 ELISA检测RAW264.7巨噬细胞过表达PSRC1后在ox-LDL刺激下的ANXA2分泌情况
实验分为四组：对照组(正常培养RAW264.7细胞培养上清)、ox-LDL组(用100 μg/ml ox-LDL刺激24 h)、Ad-GFP+ox-LDL组(外源过表达GFP后加100 μg/ml ox-LDL刺激24 h)、Ad-PSRC1+ox-LDL组(外源过表达PSRC1后加100 μg/ml ox-LDL刺激24 h)。RAW264.7巨噬细胞接种于含10%胎牛血清的DMEM完全培养基中，并置于37℃、5% CO2、饱和湿度的培养箱中培养。12 h后，根据分组情况用同体积的Ad-GFP或Ad-PSRC1转染、培养细胞48 h。实验前，各组细胞饥饿24 h处理，用100 μg/ml的ox-LDL干预细胞48 h后使其转化为泡沫细胞，收集细胞培养上清，采用ELISA法检测各组上清中ANXA2的水平。
1.2.4 免疫荧光实验检测PSRC1、ANXA2的细胞内共定位情况
胰酶消化RAW264.7细胞使其脱落，稀释至细胞浓度为1×106个/ml，将细胞定植于共聚焦皿中。将4%多聚甲醛20 μl滴至细胞表面，静置30 min，PBS洗3次；用5% BSA 100 μl对细胞进行封闭1 h，PBS洗2或3次；用5% BSA稀释一抗，加入ANXA2抗体(兔抗)，浓度为1:100(即1 μl)，同时，加入PSRC1抗体(鼠抗)，浓度为1:100(即1 μl)，避光，4℃共同孵育抗体12 h。弃去抗体，PBS轻轻冲洗细胞3次。用PBS稀释AF488羊抗鼠荧光二抗(1:100)和AF551羊抗兔荧光二抗(1:100)，室温下以100 μl稀释好的二抗孵育细胞1 h，避光。弃去二抗，PBS轻轻冲洗细胞3次。加入5% Triton破膜15 min，染DAPI 15 min后，甘油明胶封片，采用共聚焦显微镜进行观察并拍照。
1.2.5 RT-qPCR检测细胞内ANXA2 mRNA的表达情况
将RAW264.7巨噬细胞分为Ad-GFP组与Ad-shANXA2组。收集细胞并离心后，弃去旧培养基，加入无菌PBS洗涤，1000 r/min 离心5 min分离细胞，加入Trizol试剂后在4℃条件下充分裂解5 min。将获得的裂解液转移至1.5 ml EP管中，加入氯仿200 μl充分震荡，在4℃每件下静置5 min，然后4℃、12 000 r/min离心15 min，吸出上清转移至另一个1.5 ml EP管内。取500 μl异丙醇加入EP管，充分震荡后低温静置5 min，4℃、12 000 r/min离心15 min，弃上清，保留RNA沉淀物。将1 ml 75%乙醇加入RNA沉淀中，低温静置5 min，然后4℃、7500 r/min离心10 min，弃上清，保留最后的RNA沉淀。待RNA沉淀干燥后，EP管内加入20 μl焦碳酸二乙酯(DEPC)进行溶解。将所得RNA反转录为cDNA后，按照TaKaRa SYBR Green PCR Master Mix Kit试剂盒说明书进行实时荧光定量PCR。
1.2.6 油红O染色检测主动脉大体及主动脉根部斑块情况
构建ANXA2敲低的ApoE-/-小鼠模型：将24只8周龄雄性ApoE-/-小鼠随机分为四组(n=6)：普通饮食+Ad-GFP组、普通饮食+Ad-shANXA2组、高脂饮食+Ad-GFP组、高脂饮食+Ad-shANXA2组，分别给予普通饲料或高脂饲料喂养。8周后分别经尾静脉注射Ad-GFP或Ad-shANXA2，注射剂量为5×108 pfu。继续普通饮食或高脂饮食喂养2周，再次分别经尾静脉注射Ad-GFP或Ad-shANXA2，继续普通饮食或高脂饮食喂养2周。分离各组小鼠的主动脉大体标本，并取主动脉根部行冷冻切片，对大体及切片行油红O染色。心尖取血，3000 r/min离心10 min，取上清，分装后于–80℃保存备用。
1.2.7 ELISA法检测血清中IL-1β及IL-6蛋白表达水平
由于普通饮食两组小鼠斑块差异并无统计学意义，而高脂饮食两组小鼠的斑块面积差异有统计学意义，因此采用ELISA法进一步对1.2.6中收集的高脂饮食+Ad-GFP组与高脂饮食+Ad-shANXA2组小鼠血清中的IL-1β、IL-6表达水平进行检测，严格按照试剂盒说明书操作。
1.3 统计学处理
采用GraphPad Prism Version 8.3.0软件进行统计分析。所有计量资料进行方差齐性检测，均为正态分布，以$\bar{x}±s$表示，两组间比较采用Student-t检验；多组间比较采用单因素方差分析，进一步两两比较采用SNK-q检验。P<0.05为差异有统计学意义。
2 结果
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2.1 ox-LDL刺激对PSRC1与ANXA2结合的影响
通过非标记定量蛋白质组学鉴定与PSRC1结合上调的蛋白(表1)，结果发现RAW264.7巨噬细胞转染腺病毒过表达PSRC1后，在ox-LDL刺激下，与GFP对照组比较，Ad-PSRC1组免疫共沉淀的质谱总离子流明显改变，STRING软件分析发现PSRC1与ANXA2结合明显升高，增加了8.91倍(图1)。对上述质谱结果进行Co-IP验证，发现与蛋白质谱结果一致，在ox-LDL刺激下，PSRC1与ANXA2的结合明显增加，且其结合具有特异性(图2)。
2.2 RAW264.7巨噬细胞过表达PSRC1对ANXA2细胞外分泌的影响
ELISA检测结果显示，与对照组比较，ox-LDL组ANXA2的水平明显升高[(2027.23±93.55) pg/ml vs. (697.01±30.08) pg/ml，P<0.01，图3A]；与Ad-GFP+ox-LDL组比较，Ad-PSRC1+ox-LDL组ANXA2的水平明显降低[(1061.65±68.52) pg/ml vs. (2098.67±318.41) pg/ml，P<0.01，图3B)。
2.3 PSRC1与ANXA2的细胞内共定位情况
免疫荧光染色后对细胞切片进行共聚焦观察发现，ANXA2与PSRC1在细胞内存在明显的共定位现象(图4)。
2.4 腺病毒Ad-shANXA2对ANXA2的敲低效果
RT-qPCR检测结果显示，与Ad-GFP组比较，Ad-shANXA2组ANXA2 mRNA的表达水平明显降低(0.198±0.065 vs. 1.002±0.069，P<0.05)。
2.5 ANXA2敲低后对主动脉大体及主动脉根部斑块面积的影响
油红O染色结果显示，普通饮食与高脂饮食喂养小鼠主动脉产生部分AS斑块，普通饮食+Ad-shANXA2组与普通饮食+Ad-GFP组的斑块面积百分比差异无统计学意义(2.08%±0.16% vs.2.01%±0.10%，P>0.05，图5A)，而与高脂饮食+Ad-GFP组比较，高脂饮食+Ad-shANXA2组斑块面积百分比明显减少(5.29%±1.14% vs. 12.28%±2.48%，P<0.05，图5B)。显微镜下观察发现，普通饮食+Ad-shANXA2组主动脉根部斑块面积百分比与普通饮食+Ad-GFP组比较差异无统计学意义(0.80%±0.02%vs. 0.79%±0.02%，P>0.05，图6A)，而高脂饮食+Ad-shANXA2组主动脉根部斑块面积百分比与高脂饮食+Ad-GFP组比较明显降低(1.31%±0.04% vs.2.83%±0.22%，P<0.05，图6B)。
2.6 ANXA2敲低对APOE-/-小鼠血清炎性因子IL-1β、IL-6释放的影响
由于普通饮食两组小鼠斑块差异并无统计学意义，而高脂饮食两组小鼠的斑块面积差异有统计学意义，因此进一步在高脂饮食两组小鼠中进行炎性因子检测，结果发现与高脂饮食+Ad-GFP组比较，高脂饮食+Ad-shANXA2组小鼠血清IL-1β[(122.90±9.80) pg/ml vs. (172.90±21.83) pg/ml]及IL-6水平[(3.65±0.12) pg/ml vs. (5.97±0.42) pg/ml]均明显降低，差异有统计学意义(P<0.05)。
3 讨论
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AS与血脂代谢紊乱及免疫炎症息息相关[6-7]。自从21世纪人类基因图谱公布以后，Samani等[8]发表了全基因组关联研究(Genome-Wild Association Studies，GWAS)，证实PSRC1基因表达水平与冠心病的发生有关。有研究发现，PSRC1基因表达水平升高可引起HDL-C水平升高、LDL-C水平降低，从而使患冠心病的风险降低[9]。除影响血脂水平外，PSRC1基因还可调节人体对他汀类药物治疗的反应性[10]。以上研究结果表明，PSRC1与脂质代谢紊乱有关，并可通过调节血脂来影响AS的发生发展过程。PSRC1不仅可影响血脂，还可能与炎症反应有关[9]。本课题组前期研究结果提示，PSRC1的表达可能与AS的发生发展有关[5]，但具体作用及机制并不完全明了。因此，本课题组前期通过腺病毒转染RAW264.7巨噬细胞观察了PSRC1过表达对泡沫细胞形成的影响，并通过尾静脉注射腺病毒，使ApoE-/-小鼠全身过表达PSRC1基因，观察其对AS发生发展的影响，结果发现过表达PSRC1可通过抑制APOE-/-小鼠的炎症反应及影响血脂代谢而抑制AS的进展，但其确切的分子机制尚不明确。为了寻找能与PSRC1结合的蛋白，本研究进行了非标记定量蛋白质组学分析，发现在RAW264.7巨噬细胞过表达PSRC1后，再用ox-LDL刺激，PSRC1与一种名为ANXA2的蛋白结合最多，且与蛋白质谱Co-IP及免疫荧光检测的结果一致。
ANXA2是膜联蛋白家族(Annexins)的成员之一，其主要特点为能够与钙离子结合，从而参与一系列依赖于钙离子的生物膜转运过程。ANXA2广泛存在于身体各个部位，但不同组织与细胞中的ANXA2含量相差甚远。在组织水平上，ANAX2在肺、胰腺、肾上腺等组织中高表达，在肝脏及肾脏组织中低表达；在细胞水平上，ANAX2在单核细胞及巨噬细胞中高表达[11]。此外，ANXA2广泛存在于细胞核、细胞质及细胞外，既能在细胞内发挥胞膜相关的生理功能，又能释放至胞外发挥作用。研究表明，细胞膜上的ANXA2可作为受体与纤溶酶原结合，并使其与组织型纤溶酶原激活剂(tissue plasminogen activator，tPA)结合，促进纤溶酶的形成[12-13]。此外，ANXA2还可促进肿瘤细胞的迁移及新生血管的形成[12]。而在AS的发生发展中，ANXA2所扮演的角色尚未明确。有研究发现，在肝脏细胞胞膜上，ANXA2可与前蛋白转化酶枯草杆菌蛋白酶/kexin 9型(proprotein convertase subtilisin/kexin type 9，PCSK9)结合，抑制PCSK9对低密度脂蛋白受体(low density lipoprotein receptor，LDLR)的降解[14-15]。然而，也有研究发现，ANXA2可介导基质金属蛋白酶9(metalloproteinase-9，MMP-9)的释放，他汀类药物则能够通过抑制ANXA2的表达减少MMP-9的释放，稳定粥样硬化斑块[16]。此外，Wang等[17]研究发现，ANXA2可介导AS斑块处巨噬细胞的迁移，并增加炎性因子的释放，促进炎症反应。Swisher等[18-19]发现，胞外的ANXA2可与巨噬细胞表面的TLR4受体结合，促进巨噬细胞释放IL-1β、IL-6及TNF-α。因此，我们推测PSRC1过表达可抑制巨噬细胞炎症反应，可能是因为ANXA2与PSRC1的结合增加，从而改变了下游炎症信号通路所致。
为明确PSRC1对ANXA2的影响，本研究在细胞水平进行了探讨，以ox-LDL刺激RAW264.7巨噬细胞后，采用ELISA试剂盒检测培养上清中ANXA2含量，发现巨噬细胞释放的ANXA2增加，而过表达PSRC1后再用ox-LDL刺激，与Ad-GFP组比较，巨噬细胞培养上清中的ANXA2明显减少，提示PSRC1对ox-LDL诱导的ANXA2释放起负调控作用。随后本研究在动物水平进行了分析，构建了小鼠AS模型并用腺病毒尾静脉注射以敲低小鼠体内的ANXA2水平，结果发现抑制ANXA2的表达可明显减少斑块面积，且可降低血清中炎性因子IL-1β、IL-6的水平。
综上所述，本研究结果证实PSRC1可通过与ANXA2结合而抑制ANXA2的细胞外释放，进而抑制炎症反应，并最终延缓AS的进展，为AS的发病机制提供了新的理论依据，也为AS的抗炎治疗提供了新靶点。后续可通过构建PSRC1-/-ANXA2-/-双敲除小鼠模型来进行PSRC1敲除情况下的验证。
基金
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  • 国家自然科学基金青年基金(81900398)
  • 国家自然科学基金青年基金(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)
文献
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参考文献 引证文献
排序方式：
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2021年第46卷第5期
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文章信息
doi: 10.11855/j.issn.0577-7402.2021.05.03
  • 接收时间：2020-09-03
  • 首发时间：2025-12-24
  • 出版时间：2021-05-28
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  • 收稿日期：2020-09-03
  • 修回日期：2020-12-02
基金
National Natural Science Foundation of China(81900398)
国家自然科学基金青年基金(81900398)
National Natural Science Foundation of China(81700388)
国家自然科学基金青年基金(81700388)
Natural Science Foundation of Guangdong Province(2019A1515010666)
广东省自然科学基金面上项目(2019A1515010666)
Guangdong Provincial Department of Education High-level University Construction Fund Southern Medical University Clinical Research Initiation Project(LC2016PY002)
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)
Nanfang Hospital Clinical Research Project(2018CR051)
南方医院临床研究专项(2018CR051)
作者信息
    南方医科大学南方医院心血管内科，广州 510515

通讯作者:

郭志刚，E-mail：
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2种不同金属材料的力学参数
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Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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