中性粒细胞来源MMP-8对腐皮镰刀菌性角膜炎的组织损伤作用及其机制  

Investigation of the tissue-damaging effects and mechanisms of neutrophil-derived MMP-8 in Fusarium keratitis

作　　者：董军璐 金鑫 刘华 简守珺 岳娟 张红敏 王丽娅 Dong Junlu;Jin Xin;Liu Hua;Jian Shoujun;Yue Juan;Zhang Hongmin;Wang Liya(Department of Ophthalmology,Henan Provincial People's Hospital,Henan Eye Hospital,Henan Eye Institute,Henan University People's Hospital,Zhengzhou University People's Hospital,Zhengzhou 450003,China)

机构地区：[1]河南省人民医院眼科,河南省立眼科医院,河南省眼科研究所,河南大学人民医院,郑州大学人民医院,郑州450003

出　　处：《中华实验眼科杂志》2023年第10期961-969,共9页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金河南联合基金重点支持项目(U1704283);国家自然科学基金面上项目(81770902);河南省立眼科医院基础研究重点项目(21JCZD003)。

摘　　要：目的研究小鼠腐皮镰刀菌性角膜炎中中性粒细胞来源的基质金属蛋白酶8(MMP-8)对角膜组织的损伤作用及其机制。方法取108只6~8周龄雄性SPF级C57BL/6J小鼠,采用腐皮镰刀菌感染法制备右眼真菌性角膜炎(FK)模型,裂隙灯显微镜下观察小鼠角膜炎症情况并评分,依据FK模型眼角膜炎症评分将模型眼分为造模后0、12、24、48和72 h组。于相应时间点处死小鼠并取材角膜组织,采用Western blot法检测MMP-8、腺苷酸激活蛋白激酶α(AMPKα)及其丝氨酸172位点磷酸化形式(p-AMPKα)蛋白在角膜中的相对表达量;采用苏木精-伊红染色法检测各时间点组小鼠角膜中中性粒细胞数量;采用免疫荧光染色法观察角膜中中性粒细胞与MMP-8蛋白的共定位表达。体外角膜胶原降解实验中将角膜组织分为MMP-8组、缓冲液组和生理盐水组,分别采用100μl活化的重组MMP-8、检测缓冲液和生理盐水处理角膜,采用羟脯氨酸检测试剂盒测定并比较各组角膜组织中羟脯氨酸质量分数。分别提取人外周血中性粒细胞和采集培养的腐皮镰刀菌孢子,将人中性粒细胞分为4个组,其中阴性对照组为培养的中性粒细胞,共培养组为中性粒细胞加孢子共培养,AICAR处理组和化合物C处理组分别向中性粒细胞和孢子共培养体系内加入p-AMPK蛋白酶激动剂AICAR和抑制剂化合物C,采用免疫荧光染色法检测各组人中性粒细胞中MMP-8蛋白的表达水平。结果造模后24 h组小鼠模型眼出现角膜混浊,造模后72 h组出现角膜穿孔。造模后24、48和72 h组角膜炎症评分均高于12 h组,差异均有统计学意义(均P<0.001)。造模后12、24和48 h组角膜中MMP-8蛋白相对表达量高于0 h组,差异均有统计学意义(均P<0.001);模型眼角膜中MMP-8蛋白相对表达量与炎症评分呈中等强度正相关(r s=0.50,P<0.05)。造模后24、48和72 h组角膜内p-AMPKα(Thr 172)/AMPKα值高于0 h组,差异均有统计学意义Objective To investigate the mechanism of tissue damage caused by neutrophil matrix metalloproteinase-8(MMP-8)in Fusarium keratitis.Methods A total of 108 male C57BL/6J SPF grade mice,6-8 weeks old,were selected to establish a model of Fusarium keratitis(FK)in the right eyes.Corneal inflammation in mice was observed and scored under a slit lamp microscope.Based on the corneal inflammation scores,the modeling eyes were divided into 0,12,24,48,and 72-hour groups post-modeling.At the corresponding time points,mice were euthanized,and corneal tissues were collected.The expressions of MMP-8,adenylate-activated protein kinase(AMPKα)and its serine 172-site phosphorylated form(p-AMPKα)proteins in corneal tissues were detected by Western blot.The neutrophil count in mice corneal tissues at each time point was determined using hematoxylin and eosin staining.The co-localization of neutrophils and MMP-8 protein in the cornea was observed by immunofluorescence staining.In the in vitro corneal collagen degradation experiment,corneal tissues were divided into MMP-8 group,buffer group,and normal saline group,which were treated with 100μl of activated recombinant MMP-8,detection buffer,and normal saline,respectively.Hydroxyproline content in corneal tissues was determined using a hydroxyproline assay kit,and the mass fractions of hydroxyproline were compared among the groups.Peripheral blood neutrophils were isolated from human blood samples,and Fusarium spores were collected for experiments.Human neutrophils were divided into four groups,negative control group(cultured neutrophils),co-culture group(neutrophils co-cultured with spores),AICAR-treated group(neutrophils co-cultured with spores and treated with p-AMPK protein kinase activator AICAR),and compound C-treated group(neutrophils co-cultured with spores and treated with the inhibitor compound C).The MMP-8 protein expression levels in each group of human neutrophils were assessed via immunofluorescence staining.The use and care of animals complied with the ARVO statement

关 键 词：角膜炎 真菌 感染 基质金属蛋白酶8 中性粒细胞 近交系小鼠 人 

分 类 号：R772.21[医药卫生—眼科]