丙酮酸脫氫酶復合體E2亞基通過活化Toll樣受體4影響單核細胞分泌細胞因子

楊再興，梁艷，劉東紅，張治宇，仲人前

（1.臺州市第一人民醫院 檢驗科，浙江 臺州 318020；2.上海長征醫院 實驗診斷科，上海 200003；3.上海長征醫院 貴賓科，上海 200003）

丙酮酸脫氫酶復合體E2亞基通過活化Toll樣受體4影響單核細胞分泌細胞因子

楊再興1，梁艷2，劉東紅1，張治宇3，仲人前2

（1.臺州市第一人民醫院 檢驗科，浙江 臺州 318020；2.上海長征醫院 實驗診斷科，上海 200003；3.上海長征醫院 貴賓科，上海 200003）

目的：探討丙酮酸脫氫酶復合體E2亞基（PDC-E2）是否可以活化Toll樣受體4（TLR4）-NF-κB通路，并通過該通路誘導單核細胞分泌腫瘤壞死因子α（TNF-α）、白介素12（IL-12）和可溶性腫瘤壞死因子相關凋亡誘導配體（sTRAIL）。方法：培養單核細胞株U937，分為空白對照組、PDC-E2組、PDC-E2+HTA125組和PDCE2+PDTC組?？瞻讓φ战M不予任何刺激；PDC-E2組予2、10和50 μg/mL的PDC-E2刺激；PDC-E2+HTA125組予10 μg/mL的TLR4功能抑制抗體HTA125孵育4 h后，再加入50 μg/mL的PDC-E2；PDC-E2+PDTC組加入100 nmol/mL的NF-κB抑制劑PDTC 0.5 h后，再加入50 μg/mL的PDC-E2。通過流式細胞術檢測TLR4表達，EMSA實驗檢測NF-κB活化情況，ELISA法檢測培養上清TNF-α、IL-12和sTRAIL濃度。結果：2、10和50 μg/mL濃度PDC-E2刺激U937細胞24 h，TLR4表達均明顯增加，但無濃度依賴性。濃度為50 μg/mL的PDC-E2刺激U937細胞1 h，NF-κB即顯著活化；至2 h，活化逐漸減少；至4 h，已明顯減少。加入HTA125后，NF-κB活性較阻斷前顯著降低。濃度為50 μg/mL的PDC-E2刺激U937細胞24 h，培養上清TNF-α和sTRAIL的濃度顯著高于空白對照組（P＜0.05），IL-12與空白對照組比差異無統計學意義；至48 h和72 h，TNF-α、IL-12和sTRAIL的濃度均顯著高于空白對照組（P＜0.05）。加入HTA125或PDTC后，各時間點3種細胞因子的濃度均較阻斷前顯著降低（P＜0.05）。結論：PDC-E2可以活化TLR4-NF-κB通路，并通過該通路刺激單核細胞分泌細胞因子TNF-α、IL-12和sTRAIL。

丙酮酸脫氫酶復合體；Toll樣受體4；核因子；細胞因子；原發性膽汁性膽管炎

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

原發性膽汁性膽管炎（primary biliary cholangitis，PBC）是一種以肝內門管區中小膽管炎癥性損傷為主要表現的慢性自身免疫性肝病，其病因和發病機制未明?！?br>