杨再兴，梁艳，刘东红，张治宇，仲人前

（1.台州市第一人民医院 检验科，浙江 台州 318020；2.上海长征医院 实验诊断科，上海 200003；3.上海长征医院 贵宾科，上海 200003）

丙酮酸脱氢酶复合体E2亚基通过活化Toll样受体4影响单核细胞分泌细胞因子

杨再兴1，梁艳2，刘东红1，张治宇3，仲人前2

（1.台州市第一人民医院 检验科，浙江 台州 318020；2.上海长征医院 实验诊断科，上海 200003；3.上海长征医院 贵宾科，上海 200003）

目的：探讨丙酮酸脱氢酶复合体E2亚基（PDC-E2）是否可以活化Toll样受体4（TLR4）-NF-κB通路，并通过该通路诱导单核细胞分泌肿瘤坏死因子α（TNF-α）、白介素12（IL-12）和可溶性肿瘤坏死因子相关凋亡诱导配体（sTRAIL）。方法：培养单核细胞株U937，分为空白对照组、PDC-E2组、PDC-E2+HTA125组和PDCE2+PDTC组。空白对照组不予任何刺激；PDC-E2组予2、10和50 μg/mL的PDC-E2刺激；PDC-E2+HTA125组予10 μg/mL的TLR4功能抑制抗体HTA125孵育4 h后，再加入50 μg/mL的PDC-E2；PDC-E2+PDTC组加入100 nmol/mL的NF-κB抑制剂PDTC 0.5 h后，再加入50 μg/mL的PDC-E2。通过流式细胞术检测TLR4表达，EMSA实验检测NF-κB活化情况，ELISA法检测培养上清TNF-α、IL-12和sTRAIL浓度。结果：2、10和50 μg/mL浓度PDC-E2刺激U937细胞24 h，TLR4表达均明显增加，但无浓度依赖性。浓度为50 μg/mL的PDC-E2刺激U937细胞1 h，NF-κB即显著活化；至2 h，活化逐渐减少；至4 h，已明显减少。加入HTA125后，NF-κB活性较阻断前显著降低。浓度为50 μg/mL的PDC-E2刺激U937细胞24 h，培养上清TNF-α和sTRAIL的浓度显著高于空白对照组（P＜0.05），IL-12与空白对照组比差异无统计学意义；至48 h和72 h，TNF-α、IL-12和sTRAIL的浓度均显著高于空白对照组（P＜0.05）。加入HTA125或PDTC后，各时间点3种细胞因子的浓度均较阻断前显著降低（P＜0.05）。结论：PDC-E2可以活化TLR4-NF-κB通路，并通过该通路刺激单核细胞分泌细胞因子TNF-α、IL-12和sTRAIL。

丙酮酸脱氢酶复合体；Toll样受体4；核因子；细胞因子；原发性胆汁性胆管炎

Abstract: Objective:To explore whether pyruvate dehydrogenase complex E2 subunit (PDC-E2) may activate Toll-like receptor 4 (TLR4)-NF-κB pathway and induce secretion of tumor necrosis factor-α (TNF-α),interleukin-12 (IL-12) and TNF-related apoptosis-inducing ligand (sTRAIL).Methods:PDC-E2 was used to stimulate monocyte line U937, combined with HTA125 (an inhibitory antibody of TLR4) and PDTC (an inhibitor of NF-κB). The study groups included control group, PDC-E2 group, PDC-E2+HTA125 group and PDCE2+PDTC group. TLR4 on U937 was detected by flow cytometry, activity of NF-κB was determined by EMSA,TNF-α, IL-12 and sTRAIL were measured by ELISA.Results:At 24 h after stimulation by 2, 10 and 50 μg/mL of PDC-E2, TLR4 expression on U937 cell was significantly increased. At 1 h after stimulation by 50 μg/mL of PDC-E2, NF-κB was markedly activated, while at 2 h, the activation of NF-κB was gradually decreased and at 4 h, the decrease was very significant. NF-κB activity was significantly decreased after HTA125 addition compared with that before. At 24 h after stimulation by 50 μg/mL of PDC-E2, supernant TNF-α and sTRAIL levels were significantly higher than that of control, while IL-12 level was of no significant difference. At 24 h and 48 h, the levels of TNF-α, IL-12 and sTRAIL were all significantly higher than that of control. The three cytokines were significantly lower after addition of HTA125 or PDTC than before.Conclusion:PDC-E2 can activate TLR4-NF-κB pathway, by which PDC-E2 stimulates the secretion of TNF-α, IL-12 and sTRAIL by monocyte.

Key words:pyruvate dehydrogenase complex; Toll-like receptor 4; nuclear factor; cytokine; primary biliary cholangitis

原发性胆汁性胆管炎（primary biliary cholangitis，PBC）是一种以肝内门管区中小胆管炎症性损伤为主要表现的慢性自身免疫性肝病，其病因和发病机制未明。……