胆红素对脂多糖引起的腹膜炎及腹腔免疫细胞功能的影响  

Effects of bilirubin on lipopolysaccharide-induced peritonitis and the function of abdominal immune cells

作　　者：姚本来 姚立鹏 李晓明 李芳飘 吴刚[2] YAO Benlai;YAO Lipeng;LI Xiaoming;LI Fangpiao;WU Gang(Department of Hepatobiliary Surgery,Sanya People’s Hospital,Sanya 572000,China;Hepatobiliary Surgery and Organ Transplant Division,First Affiliated Hospital of China Medical University,Shenyang 110122,China)

机构地区：[1]三亚市人民医院肝胆外科,572000 [2]中国医科大学附属第一医院肝胆外科暨器官移植科,沈阳110122

出　　处：《免疫学杂志》2020年第8期662-669,共8页Immunological Journal

摘　　要：目的探讨胆红素(bilirubin)对脂多糖(lipopolysaccharide,LPS)引起的腹膜炎中腹腔炎性因子分泌,以及腹膜巨噬细胞和Raw264.7细胞功能的影响。方法构建LPS诱导的小鼠腹膜炎模型,采用酶联免疫吸附法(ELISA)测定小鼠腹腔灌洗液上清液中炎性因子(IL-2、IFN-γ、IL-4、IL-6)的含量;分离培养小鼠腹膜巨噬细胞并进行分组处理,培养Raw264.7细胞进行分组处理。采用MTT法检测不同处理的腹膜巨噬细胞与Raw264.7细胞的活性;实时荧光定量PCR(RT-PCR)检测不同处理的腹膜巨噬细胞与Raw264.7细胞中Nlrp3和IL-1βmRNA的表达情况;免疫印迹法(Western blot)检测不同处理的腹膜巨噬细胞中Nlrp3、IL-1β和caspase-1蛋白表达,Raw264.7细胞中Nlrp3、IL-1β、IκBα和p-p65蛋白表达,以及Raw264.7胞质与胞核中p50、p65和p-p65蛋白表达水平;免疫荧光染色检测p-p65蛋白在不同处理的Raw264.7细胞胞质与胞核中的表达情况。结果在LPS诱导的小鼠腹膜炎模型中,注射胆红素的小鼠腹腔灌洗液中IL-2、IL-4、IL-6和IFN-γ的含量明显降低。10μmol/L胆红素处理腹膜巨噬细胞后对细胞活性无影响;10μmol/L胆红素预处理可以显著抑制脂多糖诱导的腹膜巨噬细胞中Nlrp3、IL-1βmRNA和Nlrp3、IL-1β、caspase-1蛋白的表达。5μmol/L、10μmol/L胆红素处理Raw264.7细胞后对细胞活性无影响;胆红素呈剂量依赖性地抑制脂多糖诱导的Raw264.7细胞中Nlrp3、IL-1βmRNA和蛋白表达水平,并抑制IκBα蛋白表达降低与p-p65蛋白表达升高;10μmol/L胆红素预处理抑制了脂多糖诱导的Raw264.7细胞核中p50、p65和p-p65蛋白表达升高以及p-p65从细胞质向细胞核的转移。结论胆红素可抑制脂多糖引起小鼠腹膜炎中腹腔灌洗液中炎性因子的分泌,并抑制腹膜巨噬细胞和Raw264.7细胞中Nlrp3炎性体活化,该作用可能是通过抑制NF-κB通路激活实现的。This study was designed to investigate the effects of bilirubin on peritoneal inflammatory factors,macrophage and Raw264.7 cells in peritonitis caused by lipopolysaccharide(LPS).Mouse peritonitis model was established by LPS induction.Enzyme-linked immunosorbent assay(ELISA)was used to determine the inflammatory factors(IL-2,IFN-γ,IL-4,IL-6)in the supernatant of mouse peritoneal lavage fluid.The mouse peritoneal macrophages were isolated and treated in different groups;Raw264.7 cells were cultured and treated in different groups.MTT assay was used to detect the activity of the peritoneal macrophages and Raw264.7 cells;realtime quantitative PCR(RT-PCR)was used to detect the expression of Nlrp3 and IL-1βmRNA of the peritoneal macrophages and Raw264.7 cells;Western blot was used to detect the proteins Nlrp3,IL-1βand caspase-1 in the macrophages,the proteins Nlrp3,IL-1β,IκBαand p-p65 in the Raw264.7 cells,and the proteins p50,p65 and pp65 in the cytoplasm and nucleus of Raw264.7.Immunofluorescence staining was used to detect the expression of pp65 protein in the Raw264.7 cells.Data showed that the contents of IL-2,IL-4,IL-6 and IFN-γin the peritoneal lavage fluid of mice injected with bilirubin were significantly decreased.Bilirubin treatment(10μmol/L)had no effect on cell viability of peritoneal macrophages,but significantly inhibited the expression of Nlrp3,IL-1βmRNA and Nlrp3,IL-1β,caspase-1 proteins in LPS-induced peritoneal macrophages.Bilirubin treatment(5μmol/L or10μmol/L)had no effect on cell viability of Raw264.7 cells,while bilirubin inhibited the expression of Nlrp3,IL-1βmRNA and protein in LPS-induced Raw264.7 cells in a dose-dependent manner,and inhibited the decrease of IκBαprotein expression and the expression of p-p65 protein;pretreatment with 10μmol/L bilirubin inhibited the expression of p50,p65 and p-p65 proteins in the nucleus of LPS-induced Raw264.7 cells and inhibited the transfer of p-p65 from cytoplasm to nucleus.Taken together,bilirubin can inhibit the secretion of inflammator

关 键 词：胆红素 腹膜炎 腹膜巨噬细胞 RAW264.7细胞 NLRP3 

分 类 号：R965[医药卫生—药理学]