PAD2抑制剂AFM-30a拮抗矽肺小鼠肺纤维化的作用机制  

The effects and mechanisms of PAD2 inhibitor AFM-30a attenuates pulmonary fibrosis in silicotic mice

作　　者：张祎梦 靳馥宇 高学敏 徐洪 朱莹 毛娜 Zhang Yimeng;Jin Fuyu;Gao Xuemin;Xu Hong;Zhu Ying;Mao Na(School of Public Health,Hebei Key Laboratory for Organ Fibrosis,North China University of Science and Technology,Tangshan 063210,China;School of Public Health,Hebei Key Laboratory for Organ Fibrosis,North China University of Science and Technology,Tangshan 063210,China Health Science Center,North China University of Science and Technology,Tangshan 063210,China)

机构地区：[1]华北理工大学公共卫生学院,河北省器官纤维化重点实验室,唐山063210 [2]华北理工大学医学部,唐山063210

出　　处：《中华劳动卫生职业病杂志》2025年第1期1-13,共13页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金(82204006);河北省自然科学基金(H2021209049);河北省高等学校科学技术研究项目(QN2022007)。

摘　　要：目的研究肽酰基精氨酸脱亚氨酶2(peptidylarginine deiminase 2,PAD2)抑制剂AFM-30a对实验性矽肺小鼠肺纤维化的作用及机制。方法于2022年5月,将40只SPF级雄性C57BL/6J小鼠随机分为对照组、AFM-30a组、矽肺模型组和AFM-30a治疗组,每组10只。矽肺模型组和AFM-30a治疗组小鼠气管一次性灌注二氧化硅(silicon dioxide,SiO_(2))悬液(10 mg/只,50μl),其余组别灌注等量生理盐水;2周后AFM-30a组和AFM-30a治疗组小鼠每日腹腔注射AFM-30a溶液(20 mg/kg,100μl),其余组别灌注等量生理盐水,至4周。体外培养小鼠RAW264.7单核/巨噬细胞,分为空白对照组、AFM-30a组(5μmol/L)、SiO_(2)组(200μg/ml)和SiO_(2)+AFM-30a组(先给予200μg/ml SiO_(2)诱导12 h,再给予5μmol/L AFM-30a处理12 h),以及空白对照组、波形蛋白(vimentin,Vim)重组肽组(2μg/ml)、瓜氨酸化波形蛋白(citrullinated vimentin,Cit-Vim)重组肽组(2μg/ml)和Cit-Vim重组肽+TLR4-C34组(先给予10μmol/L TLR4-C34处理1 h,再予以2μg/ml Cit-Vim重组肽处理24 h)。苏木精-伊红(HE)、马松(Masson)染色观察小鼠肺组织病理形态;micro-CT肺组织断层扫描;抗酒石酸酸性磷酸酶(tartrate resistant acid phosphatase,TRAP)染色标记阳性细胞;免疫荧光染色法、免疫印迹法检测PAD2、Cit-Vim、Toll样受体4(toll-like receptors 4,TLR4)和核因子κB受体活化因子配体(receptor activator of nuclear factor-κB ligand,RANKL)信号相关蛋白定位及表达。数据均以±s表示,多组间比较用完全随机设计的单因素方差分析,两两比较方差齐者用LSD检验,方差不齐者用Tamhane's检验。结果与对照组比较,矽肺模型组小鼠肺内矽结节形成伴胶原沉积,CT呈高密度影,TRAP阳性细胞数、PAD2、Cit-Vim、TLR4和RANKL信号相关蛋白表达明显升高(P<0.05);与矽肺模型组比较,AFM-30a治疗组肺纤维化病变减轻,TRAP阳性细胞数、PAD2、Cit-Vim、TLR4和RANKL信号相关蛋白表达明显降低(P<0.05)。与空白对照组RAW264.7细�Objective To observe the effects of peptidylarginine deiminase 2(PAD2)inhibitor AFM-30a on silicotic mice and its possible mechanisms.MethodsIn May 2022,40 SPF male C57BL/6J mice were randomly divided into control group,AFM-30a group,silicosis model group and AFM-30a treatment group,with 10 mice in each group.Silicosis model group and AFM-30a treatment group were perfused with silicon dioxide(SiO_(2))suspension(10 mg/piece,50μl),and the other groups were perfused with an equal amount of sodium chloride solution.After 2 weeks,AFM-30a group and AFM-30a treatment group were intraperitoneally injected AFM-30a(20 mg/kg,100μl)daily,and mice of other groups were injected with equal amounts of sodium chloride solution for 4 weeks.Mouse RAW264.7 monocytes/macrophages were cultured in vitro and divided into blank control group,AFM-30a group(5μmol/L),SiO_(2)group(200μg/ml),and SiO_(2)+AFM-30a group(200μg/ml SiO_(2)induction for 12 h,followed by 5μmol/L AFM-30a treatment for 12 h).As well as blank control group,vimentin(Vim)group(2μg/ml),citrullinated vimentin(Cit-Vim)group(2μg/ml),and Cit-Vim+TLR4-C34 group(10μmol/L TLR4-C34 treatment for 1 h,followed by 2μg/ml Cit-Vim induction for 24 h).Hematoxylin Eosin(HE)and Masson staining were used to observe the pathological morphology of lung.The lung fieldclarity and lung texture of each group was observed by micro-CT.The number of positive cells was detected by tartrate resistant acid phosphatase(TRAP)staining.The localization and expression levels of PAD2,Cit-Vim,toll-like receptor 4(TLR4)signaling and receptor activator of nuclear factor-κB ligand(RANKL)signaling proteins were measured by Immunofluorescence staining and Western blotting in vitro and in vivo.The experimental data were all presented as Mean±SD.A completely random design of one-way analysis of variance was used among the groups.The pduo comparison was performed using LSD test for homogeneity of variance and Tamhane's test for inconsistency.ResultsCompared with the control group,the silicosis model grou

关 键 词：矽肺 巨噬细胞 肽酰基精氨酸脱亚氨酶2 AFM-30a TOLL样受体4 核因子ΚB受体活化因子配体 

分 类 号：R135.2[医药卫生—劳动卫生]