Background  Microglia polarization mediates many central nervous system inflammatory processes, and NOD-like receptor family X1 (NLRX1) protein is a negative regulator of inflammatory response. Currently, the regulatory effects of hypoxia and NLRX1 on microglia polarization are unclear.

  Objective  To explore the effect of hypoxia on microglia polarization and NLRX1 protein expression, and investigate whether NLRX1 is involved in microglia polarization.

  Methods   The microglia BV-2 cells of mice were divided into control group (21% O₂, 5% CO₂, 37℃), treatment group (1% O₂, 5% CO₂, 37℃ for 6 h, 12 h, 24 h, 48 h), and positive control group for M1 type polarization (LPS 1 mg/mL treatment for 24 h). BV-2 cells in which NLRX1 was knocked down were also divided into BV-2 sh-Control normoxic (21% O₂, 5% CO₂, 37℃), BV-2 sh-Control hypoxic group (1% O₂, 5% CO₂, 37℃ treatment for 6 h, 24 h, 48 h), BV-2 sh-NLRX1 group (1% O₂, 5% CO₂, 37℃ treatment for 6 h, 24 h, 48 h). The morphological changes of microglia were photographed with the phase contrast microscope, and the cell viability was determined by CCK-8. Microglia polarization was analyzed by flow cytometry, and the mRNA levels of tumor necrosis factor, interleukin-6, interleukin-1b, interleukin-10 and transforming growth factor-β were examined by real-time fluorescent quantitative PCR, and the level of NLRX1 protein was determined by Western blotting.

  Results  Cell ecptomas were increased and shortened, and amebocyte morphological-like cells increased when BV-2 cell was treated with hypoxia or LPS. The relative cell viability increased after treatment under hypoxia for 2 h and 6 h, but decreased under hypoxia for 12 h, 24 h, 48 h and in LPS group (F=459.1, P＜0.05). Compared with control group, the percentage of CD11b⁺+CD86⁺ cells decreased while that of CD11b⁺+CD86⁺+CD206⁺ cells increased when the cells were treated under hypoxia for 48 h, which was in contrast to the significant increase in percentage of CD11b⁺+CD86⁺ cells in LPS group; but the percentage of CD11b⁺+CD206⁺ cells did not change when cells treated with either hypoxia or LPS. The mRNA levels of pro-inflammatory factors TNF-α, IL-6 and IL-1β decreased gradually with hypoxia treatment time and were the lowest at 24 h (P＜0.05), while mRNA level of anti-inflammatory factor IL-10 increased (F=11.38, P＜0.05). NLRX1 protein level increased with the extension of hypoxia time. Compared with the sh-Control normoxic group, the level of IL-1β mRNA in the sh-Control hypoxia group decreased (P＜0.05), and the level of IL-10 mRNA increased at both 6 h and 48 h after hypoxia treatment. Compared with the sh-Control hypoxia group, the level of IL-1β mRNA in the sh-NLRX1 group decreased at 24 h and 48 h after hypoxia treatment (P＜0.05), and the level of IL-10 mRNA decreased after hypoxia treatment (P＜0.05).

  Conclusion  Hypoxia can upregulate the level of NLRX1 protein and reduce the M1-type polarization of microglia. This process may be mediated by down-regulation of anti-inflammatory factors through NLRX1.