受体相互作用蛋白3对自身免疫性肝炎巨噬细胞的活化及白细胞介素6的影响  

Receptor interacting protein serine threonine kinase 3 mediates macrophage activation and interleukin-6 production in autoimmune hepatitis

作　　者：张君[1] 荆洋[1] 周璐[1] 刘梦静 周思敏 李燕妮[1] 郭丽萍[1] 王邦茂[1] 

机构地区：[1]天津医科大学总医院消化科,300052

出　　处：《中华消化杂志》2018年第1期25-31,共7页Chinese Journal of Digestion

基　　金：国家自然科学基金青年项目（81200282）;国家自然科学基金面上项目（81470834）

摘　　要：摘要目的 探索自身免疫性肝炎（AIH）肝组织活化的巨噬细胞受体相互作用蛋白（RIP）3的表达水平，及其对细胞因子的调控作用。 方法 应用免疫荧光双染法观察AIH与肝囊肿旁肝组织巨噬细胞浸润程度及RIP3的表达水平。通过蛋白质印迹法检测RAW264.7巨噬细胞经不同浓度（0、1、3、6、10 μg/mL）脂多糖、RIP3通路抑制剂Necrostatin-1和硫唑嘌呤有效分子6-硫基嘌呤不同组合处理24 h后RIP1（RIP3上游信号分子）、RIP3和混合谱系激酶结构区域样蛋白（MLKL；RIP3下游底物）的表达水平。实时荧光定量PCR检测各处理组中巨噬细胞相关细胞因子mRNA表达水平。统计分析采用t检验和秩和检验，相关性采用秩相关分析。 结果 与肝囊肿旁肝组织相比，AIH肝组织CD68阳性巨噬细胞浸润显著增多（4.75±0.96比28.86±6.23），差异有统计学意义（t＝7.80，P〈0.05），且其RIP3表达水平显著增高[15（11，22）比0（0，1）]，差异有统计学意义（Z＝－2.66，P〈0.05）。体外实验发现，与对照组相比，1、3、6、10 μg/mL脂多糖组RIP1、RIP3和MLKL的蛋白质表达水平均增高，差异均有统计学意义（t＝4.00、4.90、6.40、10.30，3.80、9.30、9.80、9.00，4.90、9.90、9.30、7.70，P均〈0.05），且呈剂量依赖性（r＝0.91、0.86、0.79，P均〈0.05）。与脂多糖组相比，Necrostatin-1＋脂多糖组RIP1、RIP3、MLKL蛋白质的表达水平均下调（分别为0.73±0.11比0.47±0.13，0.60±0.07比0.37±0.05，0.65±0.22比0.38±0.04），差异均有统计学意义（t＝2.60、4.50、2.10，P均〈0.05）；IL-1β、IL-6和IL-10 mRNA表达水平也下调（分别为810.3±200.8比463.7±118.1，1 504.4±482.7比290.4±106.9，1 358.6±559.2比677.8±297.6），差异均有统计学意义（t＝5.40、12.52、5.70，P均〈0.05），而IL-4和TGF-β mRNA表达水平均上调（分别为0.3±0.2比0.6±0.3，0.4±0.1比0.9±0.4），差异均有统计学意Objective To investigate the expression level of receptor-interacting protein serine threonine kinase （RIP） 3 in macrophages/monocytes activation in autoimmune hepatitis （AIH） and its regulation on inflammatory cytokines. Methods The degree of macrophage infiltration and the expression of RIP3 in liver tissues from patients with AIH or hepatic cysts by double-immunofluorescence. After 24 hours treated with different concentrations （0, 1, 3, 6, 10 μg/mL） of lipopolysaccharide （LPS）, Necrostatin-1, the specific inhibitor of RIP3 signaling pathway, and 6-thioguanine, the active metabolite of azathioprine, the expression levels of RIP1 （RIP3 upstream signal molecule）, RIP3 and mixed-lineage kinase domain-like protein （MLKL, RIP3 downstream substrate） in RAW264.7 macrophages were detected by Western blotting. The expression of macrophage-associated cytokine at mRNA level of each treatment group was determined by real-time quantitative polymerase chain reaction （PCR）. Student′s t test and sum rank test were performed for statistical analysis. Spearman analysis was performed for the correlation analysis. Results Compared with hepatic cysts adjacent liver tissues, the infiltration of CD68 positive macrophages in liver tissue of AIH patients was significantly increased （4.75±0.96 vs 28.86±6.23）, and the difference was statistically significant （t=7.80, P〈0.05）, and the expression level of RIP3 also was significantly increased （15, 11 to 22 vs 0, 0 to 1）, and the difference was statistically significant （Z=-2.66, P〈0.05）. In vitro, compared with those of control group, the expression levels of RIP1, RIP3 and MLKL stimulated by LPS at 0, 1, 3, 6, 10 μg/mL were significantly increased, and the differences were statistically significant （t=4.00, 4.90, 6.40, 10.30; 3.80, 9.30, 9.80, 9.00; 4.90, 9.90, 9.30 and 7.70; all P〈0.05）, and were dose-dependent （r=0.91, 0.86 and 0.79, all P〈0.05）. Furthermore, compared with those of LPS-stimulated group, the expressi

关 键 词：肝炎 自身免疫性 巨噬细胞 受体相互作用蛋白3 白细胞介素6 

分 类 号：R575.1[医药卫生—消化系统]