高氧对脂多糖诱导N9小胶质细胞促炎症作用的影响

The effect on the pro-inflammatory role of N9 microglia exposured to hyperoxia after preconditioning with lipopolysaccharide in vitro

目的:观察常压高浓度氧对脂多糖诱导N9小胶质细胞Toll受体4、TNF-α表达时序变化,初步探讨高氧对小胶质细胞促炎反应的作用及调控机制。方法:体外培养N9小胶质细胞,随机分为6组(n=3):空气组、sLPS组、hLPS组、高氧组、高氧+sLPS组、高氧+hLPS组。LPS浓度为100 ng/mL(sLPS)、1 mg/L(hLPS)。高氧(900 mL/L)暴露时间分别为2 h、6 h、12 h、16 h、24 h。RT-PCR检测TLR4的表达时序变化;Western blot检测处理12 h后各组TLR4蛋白表达;ELISA检测各组处理6 h、12 h、16 h、24 h培养上清中TNF-α的含量。结果:RT-PCR显示hLPS组在6 h后、sLPS组在16 h后TLR4 mRNA均表达上调,并随LPS浓度增加、暴露时间延长逐渐升高(P<0.05),24 h时hLPS组表达最高。6 h后在各个时间点高氧+hLPS组与hLPS比较TLR4 mRNA下调(P<0.05),在16 h、24 h最为明显(P<0.05)。Westernblot检测12 h高氧+sLPS组、高氧+hLPS组TLR4蛋白水平均低于相应浓度的LPS组(P<0.05)。ELISA结果示在各个时间点高氧+sLPS组、高氧+hLPS组与相应浓度的LPS组比较TNF-α均明显上调(P<0.05)。结论:高浓度氧暴露促进LPS诱导N9小胶质细胞的促炎症反应,TLR4可能参与此过程的负向调控。

AIM： To observe the time-dapendcrt expression of TLR4 and TNF-α of N9 microglia exposured to normobaric hyperoxia after preconditioning with lipopolysaccharide in vitro and to explore the role of hyperoxia on the pro-flammation response of microglia and mechanism.METHODS： N9 microglia cell line cultured in vitro was randomly divided into six groups（n=3）： normoxia group,sLPS group（100 ng/mL）,hLPS group（1 mg/L）,hyperoxia group,hyperoxia＋sLPS group（100 ng/mL）,hyperoxia＋hLPS group（1 mg/L）.Each of the last two groups,30 min after pretreatment with different level of LPS,was subjected to 900 mL/L hyperoxia for various times（2 h,6 h,12 h,24 h and 48 h）.The remanent groups was cultured in ambient O2 in which sLPS group and hLPS group respectively was treated with 100 ng/mL and 1 mg/mL LPS in the cell supernatant.After treatment,at each time point,the cells of each group was harvested and TLR4 gene expression were observed by RT-PCR.TLR4 protein expression at 12 h was observed by Western blotting.TNF-α concentrations in the supernatant of cultured microglia N9 cells at different time points were tested with ELISA.RESULTS： After 6 h in hLPS group and 16 h in sLPS group,the expression of TLR4 mRNA was gradually increased（P〈0.05）,following with increasing time and concentration of LPS,which reached to the maximum at 24 h in hLPS group.Compared with hLPS group,hyperoxia＋hLPS group showed downregulation of TLR4 mRNA at each time point after 6 h（P〈0.05）,especially at 16 h and 24 h.At 12 h,the level of TLR4 protein of hyperoxia＋sLPS group and hyperoxia＋hLPS group respectively was lower than the corresponding concentration of LPS group.The result of ELISA show that at each time point,compared with the corresponding concentration of LPS group respectively,the expression of TNF-α of hyperoxia＋sLPS group and hyperoxia＋hLPS significantly increased（P〈0.05）.CONCLUSION： Hyperoxia enhance the pro-flammation response of N9 microglia triggered by LPS and TLR4 may be the important negative-control target molecule.