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 O(2); 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.