乌司他丁在脂多糖/尼日利亚菌素诱导的巨噬细胞焦亡中的作用  

Ulinastatin represses lipopolysaccharides/nigericin-induced pyroptosis of macrophages

作　　者：周俊杰[1] 吴实正 段鹏凯 袁媛[1] 何璇 童华生[2] ZHOU Jun-jie;WU Shi-zheng;DUAN Peng-kai;YUAN Yuan;HE Xuan;TONG Hua-sheng(Department of Intensive Care Unit,Heyuan People＇s Hospital Affiliated to Jinan University,Heyuan,Guangdong 517000,China;Department of Intensive Care Unit,Guangzhou General Hospital of Guangzhou Command,Guangzhou 510010,China)

机构地区：[1]暨南大学附属河源医院重症医学科,广东河源517000 [2]广州军区总医院重症医学科,广州510010

出　　处：《解放军医学杂志》2018年第7期584-588,共5页Medical Journal of Chinese People's Liberation Army

基　　金：国家自然科学基金(81471839;81671896);广东省科技计划项目(2017A020215055)~~

摘　　要：目的探讨乌司他丁(UTI)对脂多糖(LPS)/尼日利亚菌素(nigericin)诱导的巨噬细胞焦亡的作用。方法分离小鼠骨髓细胞,应用小鼠巨噬细胞集落刺激因子(MCSF)将其诱导分化为巨噬细胞,并分为对照组、UTI组、模型组和模型+UTI组4组。UTI组和模型+UTI组在建模前给予1000U/ml UTI预处理,其余两组给予等量PBS;模型组和模型+UTI组先用LPS 100ng/ml刺激培养4h,再加入10μmol/L尼日利亚菌素继续刺激2h;对照组及UTI组在此期间仅给予等体积PBS。采用噻唑蓝(MTT)法评价各组细胞活力;应用分光光度法检测细胞乳酸脱氢酶(LDH)释放率;Western blotting检测半胱天冬酶-1(caspase-1)及Gasdermin D蛋白(GSDMD)的表达变化;应用RT-q PCR检测白细胞介素(IL)-1β、IL-18 mRNA水平;酶联免疫吸附(ELISA)法检测细胞上清IL-1β、IL-18、肿瘤坏死因子(TNF)-α、IL-6水平。结果与模型组比较,UTI预处理明显提高了LPS/尼日利亚菌素刺激后的巨噬细胞存活率(73.40%vs.86.30%,P<0.05),降低了LDH释放率(22.45%vs.14.80%,P<0.05),caspase-1和GSDMD片段化水平,IL-1β、IL-18 mRNA水平及蛋白分泌量,以及细胞上清TNF-α(311.30±33.02pg/ml vs.206.10±28.84pg/ml,P<0.05)和IL-6水平(212.60±32.72pg/ml vs.143.77±6.36pg/ml,P<0.05)。结论乌司他丁可抑制巨噬细胞焦亡,降低炎症反应。Objective To explore the effect of ulinastatin on the pyroptosis of macrophages induced by lipopdysaccharides（LPS）/nigericin. Methods Bone marrows（BM） were isolated from the mice, and then induced into macrophages using macrophage colony-stimulating factor（MCSF）. BM macrophages were pretreatment with 1000 U/ml ulinastatin. Then, macrophages were primed with 100 ng/ml LPS for 4 hours, followed by 10μmol/L nigericin treatment for 2 hours before the analysis. Cells without ulinastatin treatment or without LPS/nigericin prime were used as control. The cell viability was measured by MTT and LDH release rate was also measured using spectrophotometry. The alterations of caspase-1 and Gasdermin D protein（GSDMD） expression and the mRNA of interleukin（IL）-1β, IL-18 were detected using Western blotting and RT-q PCR, respectively. The concentrations of IL-1β, IL-18, TNF-α, and IL-6 were measured by ELISA. Results LPS combined with nigericin were used to establish the cell pyroptosis model in vitro. Our results showed that, ulinastatin pretreatment increased the sur vival rate of macrophage after LPS/nigericin stimuli（LPS/nigericin＋PBS group vs. LPS/nigericin＋UTI group, 73.40% vs. 86.30%, P〈0.05）, decreased the release rate of LDH（LPS/nigericin＋PBS group vs. LPS/nigericin＋U TI group, 22.45% vs. 14.80%, P〈0.05）, downregulated the protein expression of cleaved caspase-1 and cleaved GSDMD, reduced the mRNA and protein levels of IL-1βand IL-18. Furthermore, compared with LPS/nigericin＋PBS group, the concentrations of TNF-α [LPS/nigericin＋PBS group vs.LPS/nigericin＋UTI group, 311.30±33.02 pg/ml vs. 206.10±28.84 pg/ml, P〈0.05] and IL-6 [LPS/nigericin＋PBS group vs. LPS/nigericin＋UTI group, 212.60±32.72 pg/ml vs. 143.77±6.36 pg/ml, P〈0.05] in LPS/nigericin＋UTI group were lower. Conclusion Ulinastatin prevents the macrophages pyroptosis and inflammation.

关 键 词：乌司他丁 细胞焦亡 巨噬细胞 

分 类 号：R378[医药卫生—病原生物学]