| 周超 王璞 黄燚 王晗 吴越 高采平 李良平.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[J].,2016,16(9):1639-1643 |
| 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 Inhibits TLR4 Pathway in RatKupffer Cells via the Downregulation of Lck Kinase in Lipid Rafts* |
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| DOI： |
| 中文关键词: 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 |
| 英文关键词: Kupffer cells Toll-likereceptor 4 Lymphocyte-specific proteintyrosine kinase Lipidrafts LipoteichoicAcid |
| 基金项目:四川省卫生厅资助项目（100496） |
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| 中文摘要: |
| 目的：探讨保加利亚乳酸杆菌脂磷壁酸（Lipoteichoic Acid of L ，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，M-betaCD）、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-资B，NF-资B），酶联免疫吸附法检测培养上清中的肿瘤坏死因子alpha（tumor necrosis factor-alpha，TNF-alpha）和白
介素1beta（interleukin-1beta，IL-1beta）。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 0.012
0.0023，1.05 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 0.048
0.011，P<0.05），核蛋白中的NF- B及培养上清中的TNF-alpha和IL-1beta亦明显升高（与对照孔相应数值之比为0.78 0.16 vs 0.076
0.014，189.2 27.1 vs 5.62 0.82，131.6 18.8 vs 7.24 1.14，P 均<0.05）。与M-alpha-CD 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-alpha及IL-1beta与LPS 孔相应数值之比为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。 |
| 英文摘要: |
| Objective:To study whether Lipoteichoic Acid of (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-kB (NF-kB) in nuclear protein were measured by Western blot. Tumor necrosis factor-alpha(TNF-alpha) and interleukin-1beta(IL-1beta)
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-kB in nuclear protein, TNF-alpha and IL-1beta were obviously augmented by LPS (the comparison
of level of NF- B, TNF-alpha or IL-1beta 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 vs
7.24 1.14, P<0.05). Similar to Mbeta CD 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-alpha or IL-1beta 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).Conclusion:LBG-LTA inhibits LPS-activated TLR4 pathway in rat Kupffer cells by decreasing Lck in rafts. |
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