全反式维甲酸通过激活BMP2的表达抑制中性粒细胞胞外诱捕网的形成  

All-Trans Rtinoic Acid Inhibits the Formation of Neutrophil Extracellular Traps by Activating BMP2

作　　者：王金丽 刘志丹 王晓敏 仲经亚 程树群 潘巍巍[1] 詹淑玉[1] 郑永霞[1] WANG Jinli;LIU Zhidan;WANG Xiaomin;ZHONG Jingya;CHENG Shuqun;PAN Weiwei;ZHAN Shuyu;ZHENG Yongxia(School of Medicine,Jiaxing University,Jiaxing 314001,China;School of Pharmacy,Zhejiang University of Technology,Hangzhou 310014,China;School of Basic Medicine,Zhejiang University of Traditional Chinese Medicine,Hangzhou 310053,China;Six Departments of Hepatology,Oriental Hepatobiliary Surgery Hospital,Shanghai 200433,China)

机构地区：[1]嘉兴学院医学院,嘉兴314001 [2]浙江工业大学药学院,杭州310014 [3]浙江中医药大学基础医学院,杭州310053 [4]东方肝胆外科医院肝外六科,上海200433

出　　处：《中国细胞生物学学报》2022年第11期2057-2070,共14页Chinese Journal of Cell Biology

基　　金：浙江省公益应用技术研究项目(批准号:GD22H163452);中国博士后科学基金(批准号:2018M633092)资助的课题

摘　　要：该研究探讨了全反式维甲酸(all-trans retinoic acid,ATRA)对中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)释放的调节作用及其可能的机制。体外提取C57BL/6小鼠的中性粒细胞,给予ATRA处理,佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导NETs产生,免疫荧光检测NETs的髓过氧化物酶(myeloperoxidase,MPO)和脱氧核糖核酸(deoxyribonucleic acid,DNA)的共定位,Sytox Green荧光定量细胞外DNA的数量,蛋白质印迹法检测MPO蛋白表达,ELISA检测细胞上清中瓜氨酸化组蛋白(citrullinated histone 3,H3Cit)含量。该研究利用RNA-seq筛选了对ATRA易感的基因,并通过q RT-PCR验证相关差异基因(Arg1、Camkk1、BMP2等)的m RNA表达情况。为了研究ATRA的靶基因BMP2对NETs发生的影响,该研究用BMP2的抑制剂(LDN193189)处理中性粒细胞,再通过Sytox Green荧光实验验证BMP2对NETs形成的作用。结果显示:ATRA处理后的NETs的MPO和DNA共定位减少,MPO蛋白表达水平降低,细胞上清中H3Cit含量显著低于PMA组(P<0.0001),Sytox Green定量NETs荧光值显著低于PMA组(P<0.01),ATRA以剂量依赖性方式抑制NETs生成;RNA-seq结果显示456个基因的表达发生了变化,其中274个基因上调,182个基因下调,qRT-PCR结果表明,与PMA组相比,ATRA预处理组中的BMP2、Arg1、Camkk1、Abca1、GSS等基因发生上调,与RNA-seq结果一致;LDN193189处理后,Sytox Green定量NETs荧光值显著高于PMA组(P<0.0001)。综上,ATRA能够抑制NETs的释放,其机制可能是通过激活BMP2的表达从而抑制NETs形成的。This study investigated the regulatory effect of ATRA(all-trans retinoic acid)on the release of NETs(neutrophil extracellular traps)and its possible mechanism.In vitro,neutrophils were extracted from C57BL/6 mice and pretreated with ATRA.Then,NETs were induced by PMA(phorbol 12-myristate 13-acetate).Afterwards,colocalization of MPO(myeloperoxidase)and DNA(deoxyribonucleotide)levels of NETs was detected by immunofluorescence,extracellular DNA was quantified by Sytox Green fluorescence,and MPO protein expression was determined by Western blot,and the level of H3Cit(citrullinated histone 3)in the supernatant was evaluated by ELISA.For the mechanism study,RNA-seq was used to screen the genes susceptible to ATRA,and qRT-PCR was used to verify the mRNA expression of ATRA related-genes(Arg1,Camkk1,BMP2,etc.).In order to study the effect of BMP2,the target gene of ATRA,on the formation of NETs,neutrophils were treated with BMP2inhibitor(LDN193189),and Sytox Green fluorescence assay was used to verify the effect of LDN193189 on NETs.The results showed that all of detected values,including the colocalization of MPO and DNA,extracellular DNA,the expression of MPO protein,the H3Cit level in the supernatant of NETs,treated with ATRA were significantly lower than those in PMA group(P<0.0001),indicating that ATRA inhibited the production of NETs in a dose-dependent manner;RNA-seq results showed that the expression of 456 genes changed,of which 274 genes were upregulated and 182 genes were down-regulated.qRT-PCR results showed that BMP2,Arg1,Camkk1,Abca1 and GSS were up-regulated in ATRA pretreated group compared with the PMA group,which was consistent with the RNAseq results.After LDN193189 treatment,the fluorescence value of Sytox Green quantitative NETs was significantly higher in ATRA pretreated group than that in PMA group(P<0.0001).In summary,ATRA can inhibit the release of NETs,and its mechanism may be through the activation of BMP2 expression.

关 键 词：中性粒细胞胞外诱捕网 全反式维甲酸 感染性疾病 

分 类 号：R392.12[医药卫生—免疫学]