乳酸杆菌脂磷壁酸通过下调脂筏中Lck激酶水平抑制大鼠肝脏Kupffer细胞TLR4通路  

Lipoteichoic Acid of Lactobacillus bulgaricus Inhibits TLR4 Pathway in Rat Kupffer Cells via the Downregulation of Lck Kinase in Lipid Rafts

作　　者：周超[1] 王璞[1] 黄燚[2] 王晗[1] 吴越[3] 高采平[1] 李良平[1] 

机构地区：[1]四川省人民医院消化内科,四川成都610072 [2]四川省人民医院检验科,四川成都610072 [3]四川省人民医院药剂科,四川成都610072

出　　处：《现代生物医学进展》2016年第9期1639-1643,共5页Progress in Modern Biomedicine

基　　金：四川省卫生厅资助项目(100496)

摘　　要：目的:探讨保加利亚乳酸杆菌脂磷壁酸(Lipoteichoic Acid of Lactobacillus bulgaricus,LBG-LTA)是否能下调细胞膜脂筏中的淋巴细胞特异性蛋白酪氨酸激酶(Lymphocyte-specific protein tyrosine kinase,Lck),进而抑制大鼠肝脏Kupffer细胞Toll样受体4(Toll-like receptor 4,TLR4)通路。方法:分离培养10只雄性Wistar大鼠的Kupffer细胞;培养LBG并制备LBG-LTA;有或无LBG-LTA、脂筏裂解剂甲基-β-环糊精(methyl-β-cyclodextrine,MβCD)、Lck抑制剂PP2分别预处理情况下,以脂多糖(lipopolysaccharide,LPS)刺激Kupffer细胞,提取各组细胞的膜-浆蛋白及核蛋白,蔗糖密度梯度离心法提取膜-浆蛋白中的脂筏及非脂筏组分,Western blot检测脂筏及非脂筏组分中TLR4、TANK结合激酶1(TANK binding kinase-1,TBK1)、Lck及核蛋白中的核因子B(nuclear factor-κB,NF-κB),酶联免疫吸附法检测培养上清中的肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)和白介素1β(interleukin-1β,IL-1β)。结果:LPS上调的TLR4、Lck主要在脂筏内(与对照孔脂筏相应数值之比为0.95 0.23 vs 0.0120.0023,1.05 0.26 vs 0.022 0.0052,P均<0.05),TBK1主要在非脂筏组分中(与对照孔非脂筏组分数值之比1.02 0.21 vs 0.0480.011,P<0.05),核蛋白中的NF-B及培养上清中的TNF-α和IL-1β亦明显升高(与对照孔相应数值之比为0.78 0.16 vs 0.0760.014,189.2 27.1 vs 5.62 0.82,131.6 18.8 vs 7.24 1.14,P均<0.05)。与MαCD或PP2一样,LBG-LTA也明显抑制LPS的作用(LTA+LPS孔脂筏中TLR4、Lck与LPS孔相应数值之比为0.15 0.036 vs 0.95 0.23,0.17 0.052 vs 1.05 0.26,非脂筏组分中TBK1与LPS孔的比较为0.25 0.062 vs 1.02 0.21,NF-B、TNF-α及IL-1β与LPS孔相应数值之比为0.17 0.035 vs 0.78 0.16,32.2 4.37 vs189.2 27.1,23.4 3.29 vs 131.6 18.8,P均<0.05)。结论:LBG-LTA下调大鼠Kupffer细胞膜脂筏中的Lck,进而抑制其TLR4通路。Objective： To study whether Lipoteichoic Acid of Lactobacillus bulgaricus（LBG-LTA） inhibits Toll-like receptor 4（TLR4） pathway in rat Kupffer cells by decreasing lymphocyte-specific protein tyrosine kinase（Lck） in lipid rafts. Methods： Ten male Wistar rats, 2 months old, 250~300 g, were killed for the isolation of Kupffer cells. Kupffer cells were treated by lipopolysaccharide（LPS） with or without a pretreatment of LBG-LTA, methyl-β-cyclodextrine（MβCD） or PP2（Lck inhibitor）. Membrane-cytosol protein and nuclear protein were extracted from Kupffer cells. Lipid rafts and non-raft fractions were extracted from membrane-cytosol protein via sucrose density gradient centrifugation. TLR4, TANK binding kinase-1（TBK1） and Lck in rafts and non-raft fractions as well as nuclear factor-κB（NF-κB） in nuclear protein were measured by Western blot. Tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β）were quantified by ELISA. Results： The majority of TLR4 and Lck up-regulated by LPS was detected in rafts（the comparison of level of TLR4 or Lck in rafts between LPS and control group： 0.95 0.23 vs 0.012 0.0023, 1.05 0.26 vs 0.022 0.0052, P〈0.05）. However, TBK1 increased by LPS was mainly found in non-raft fractions（the comparison of TBK1 level in non-raft fractions between LPS and control group： 1.02 0.21 vs 0.048 0.011, P〈0.05）. NF-κB in nuclear protein, TNF-α and IL-1β were obviously augmented by LPS（the comparison of level of NF- B, TNF-α or IL-1β between LPS and control group： 0.78 0.16 vs 0.076 0.014, 189.2 27.1 vs 5.62 0.82, 131.6 18.8 vs7.24 1.14, P〈0.05）. Similar to MβCD or PP2, LBG-LTA also obviously inhibited these effects of LPS（the comparison of level of TLR4 or Lck in rafts, TBK1 in non-raft fractions, NF- B, TNF-α or IL-1β between LTA＋LPS and LPS group： 0.15 0.036 vs 0.95 0.23, 0.170.052 vs 1.05 0.26, 0.25 0.062 vs 1.02 0.21, 0.17 0.035 vs 0.78 0.16, 32.2 4.37 vs 189.2 27.1, 23.4 3.29 vs 131.6 18.8, P〈0.05）. Conclusions�

关 键 词：KUPFFER细胞 TOLL样受体4 淋巴细胞特异性蛋白酪氨酸激酶 脂筏 乳酸杆菌 脂磷壁酸 

分 类 号：R575.1[医药卫生—消化系统] R333.4[医药卫生—内科学]