日本血吸虫成虫抗原通过ROS诱导人LX-2肝星状细胞NLRP3炎症小体活化和胶原表达  

SCHISTOSOME ADULT WORM ANTIGEN INDUCES ACTIVATION OF NLRP3 INFLAMMASOME AND COLLAGEN SYNTHESIS IN HUMAN HEPATIC STELLATE CELL LINE THROUGH ROS IN VITRO

作　　者：朱燕楠 缪婷婷 王伟 陆忠奎 刘劲峰 王勇 ZHU Yan-Nan;MIAO Ting-Ting;WANG Wei;LU Zhong-Kui;LIU Jin-Feng;WANG Yong(Department of Pathogen Biology,School of Basic Medical Sciences,Nanjing Medical University,Nanjing 211100,China)

机构地区：[1]南京医科大学基础医学院病原生物学系江苏省现代病原生物学重点实验室,江苏南京211100

出　　处：《寄生虫与医学昆虫学报》2018年第2期71-79,共9页Acta Parasitologica et Medica Entomologica Sinica

基　　金：国家自然科学基金资助项目(No.81471573)

摘　　要：为了探讨日本血吸虫成虫抗原(SWA)对LX-2细胞NLRP3炎症小体活化和纤维化相关基因表达的影响,以及活性氧(ROS)在其中的作用,本文首先采用不同浓度(0、25、50、100μg/m L)的SWA刺激LX-2细胞,通过实时荧光定量PCR (Real-time PCR)和Western blot检测细胞中NLRP3、Caspase-1、IL-1β、α-SMA和ColⅠ的基因和蛋白表达, ELISA检测细胞IL-1β的分泌水平;同时使用CCK-8 (Cell Counting Kit-8)法检测细胞活性,乳酸脱氢酶(LDH)检测试剂盒检测细胞LDH的释放;流式细胞术检测SWA刺激对LX-2细胞中ROS产生的影响;之后使用ROS清除剂N-乙酰半胱氨酸(N-acetylcysteine,NAC)处理细胞,检测NLRP3、IL-1β、Caspase-1、α-SMA、ColⅠ基因和蛋白的表达水平。结果显示,当SWA的浓度达100μg/m L时,LX-2细胞内NLRP3、Caspase-1、IL-1β、α-SMA和ColⅠ的m RNA的表达与对照组相比均明显升高,同时相关蛋白的表达和IL-1β的分泌也显著升高。流式检测结果显示,SWA刺激LX-2细胞后能显著增加细胞内ROS的生成,并在NAC处理后,可见明显抑制细胞内ColⅠ和炎症小体活化相关的基因和蛋白的表达。结果提示,SWA可以诱导LX-2细胞内炎症小体活化和胶原表达,并且ROS反应可能在介导NLRP3活化中发挥重要的作用。Abstract The effects of schistosome adult worm antigen （SWA） on NLRP3 inflammasomes activation and collagen synthesis in human hepatic LX-2 stellate cells were assayed and the role of reactive oxygen species （ROS） involved was evaluated in vitro in present paper. After induced by different concentrations of SWA （0, 25, 50 and 100 μg/mL） , the mRNA and protein levels of NLRP3, Caspase-1, IL-la, α-SMA and Col I in LX-2 cells were detected by Real-time PCR, Western blot and ELISA. Moreover, cell viability was investigated by CCK-8 assay and the cell pyroptosis was analyzed by a standard lactate dehydrogenase （LDH） release assay. The generation of ROS in LX-2 cells after SWA stimulation was eoneurently detected via FCM. The results showed that, when SWA reached 100 μg/mL, the mRNA expressions increased obviously of NLRP3, Caspase-1, IL-la, α-SMA and Col I in LX-2 cells compared to that in control group. Meantime, the expressions of correlated proteins and IL-1β secretion were also increased. FCM showed that SWA could markedly increase the generation of ROS in LX-2 ceils, and significantly inhibit the expressions of Col I and correlated mRNA and proteins of inflammasomes activation after treated by N-aeetylcysteine （NAC）. Therefore, SWA may induce the NLRP3 inflammasomes activation and collagen synthesis in LX-2 cells through ROS, which may play an important role in mediating NLRP3 activation.

关 键 词：LX-2细胞 SWA NLRP3炎症小体 ROS 肝纤维化 

分 类 号：R532.21[医药卫生—内科学]