亚低温对脂多糖诱导巨噬细胞Toll样受体4mRNA转录及炎症平衡的影响

Impact of hypothermia on Toll-like receptor 4 mRNA transcription and inflammatory balance ofmacrophage induced by lipopolysaccharide

目的探讨亚低温对脂多糖（LPS）刺激下巨噬细胞Toll样受体4（TLR4）mRNA转录及下游炎症平衡的影响。方法实验用RAW264．7小鼠巨噬细胞株，将细胞分别置于37℃和32℃的温育箱中培养，干预组加入10rig／mELPS进行刺激，对照组加入等量生理盐水。分别于培养2、8、24h取细胞上清液检测白细胞介素（IL-1B、IL-10）水平；用TRIzol提取细胞总lqNA，逆转录-聚合酶链反应（RT-PCR）检测TLR4mRNA的转录水平。结果LPS刺激下，常温组和亚低温组TLR4mRNA表达水平均呈先升高、后降低的趋势（均P〈O．001）；同-时间点，亚低温组TLR4mRNA表达水平（2-AACt）均明显低于常温组（2h：9．19±0．57比10．08±1．02，f=-2．285，P=0．036；8h：13．58±1．57比15．24±1．63，f=-2．201，P=0．042；24h：6．85±1．17比8．17±1．21，f=2．353，P=O．032）。LPS刺激下，常温组和亚低温组随时间延长IL-1B、IL-10均逐步增高（均P〈0．001）；同-时间点，亚低温组IL-1B（ng／L）明显低于常温组（2h：87．08±29．35比110．53±26．58，f=1．777，P=O．047；8h：119．19±49．14比173．25±37．21，t=2．631，P=0．018：24h：208．66±53．83比346．56±64．30，t=4．933，P〈0．001）；IL-10（ng／i．）明显高于常温组（8h：170．01±24．90比140．02±15．08，f=-3．091，P=0．007；24h：423．10±52．40比165．06±31．97，t=-12．611，P〈0．001）。随着LPS刺激时间的延长，常温组IL-1β／IL-10比值逐渐升高（F=20．003，P〈0．001），亚低温组IL-1B／IL-10比值则逐渐降低（F=1．811，P=0．185）；同-时间点，亚低温组IL-1B／IL-10比值明显低于常温组（2h：0．740±0．214比0．993±0．256，t=2．275，P=0．037；8h：0．701±0．363比1．237±0．455，f=2．763，P=0．014；24h：0．493±0．292比2．099±0．428，f=9．299，P〈0．001）。结论亚低温可以降低LPS刺激下巨噬细胞TLR4mRNA的转录水平，相对降低IL-10水平，但保留了更高水平的IL-10，使炎症平衡向抗炎方向发展。

Objective To investigate the impact of hypothermia on Toll-like receptor 4 （TLR4） mRNA transcription and downstream inflammatory balance of maerophage induced by lipopolysaeeharide （LPS）. Methods RAW264.7 mouse maerophage cell line was collected and cultivated in incubator at 37 °C or 32 °C. The cells in stimulation group were incubated with 10 ng/mL LPS, while normal saline was used instead in control group. The cells culture supernatant was collected at 2, 8, 24 hours to determine the interleukin （IL-113 and IL-10） levels. Total RNA was collected with TRIzol, and TLR4 mRNA transcriptional level was determined by reverse transcription-polymerase chain reaction （RT-PCR）. Results Under the intervention of LPS, TLR4 mRNA expression in normal temperature group and hypothermia group was increased first followed by a decreasing trend （both P〈0.001 ）, and at the same time point, TLR4 mRNA expression （2-△△O ） in hypothermia group was lower than that in normal temperature group （2 hours： 9.19 ±0.57 vs. 10.08 ± 1.02, t=-2.285, P=0.036; 8 hours： 13.58 ± 1.57 vs. 15.24 ± 1.63, t=-2.201, P= 0.042 ; 24 hours ： 6.85 ± 1.17 vs. 8.17 ± 1.21, t= 2.353, P= 0.032 ）. After the administration of LPS, IL- 1β and IL- 10 levels in both normal and hypothermia group were gradually increased （all P〈0.001 ）, and at the same time point, the level of IL-113 （ng/L） in hypothermia group was lower than that in normal temperature group （2 hours： 87.08 ＋ 29.35 vs. 110.53 ± 26.58, t=-1.777, P=0.047; 8 hours： 119.19 ± 49.14 vs. 173.25 ± 37.21, t=2.631, P=0.018; 24 hours： 208.66 ± 53.83 vs. 346.56 ± 64.30, t=4.933, P〈0.001）, whereas the level of IL-10 （ng/L） of hypothermia group was higher than that in normal temperature group （8 hours： 170.01 -＋ 24.90 vs. 140.02±15.08, t=-3.091, P= 0.007; 24 hours： 423.10 β 52.40 vs. 165.06 ± 31.97, t=12.611, P〈0.001 ）. The IL-1β/L-10 ratio in normal temperature group was gradually increased （F=20.003, P〈0.001 ）, and it was decreased gradually in hvpothermia grouo（F= 1.811, P=0.185 ）. At the same time point, IL-1 [3/IL-10 ratio in hypothermia group was lower than that in normal temperature group （2 hours： 0.740 ＋0.214 vs. 0.993 -＋0.256, t=2.275, P=0.037; 8 hours： 0.701±0.363 vs. 1.237 ± 0.455, t=2.763, P=0.014; 24 hours： 0.493 ＋ 0.292 vs. 2.099 ± 0.428, t=9.299, P〈0.001 ）. Conclusion Hypothermia can reduce TLR4 mRNA transcription level of macrophage after the challenge of LPS, and it can relatively lower the level of IL-I[3, but a higher level of IL-10 is maintained, thus creatin~ a tendency of anti-inflammation.