Effects of hypoxia combined with LPS on the expression of pro- inflammatory cytokines and BNIP3 in primary cultured astrocyte

Abstract

Objective: To investigate the expression of Bcl-2/E1B-19K-interacting protein 3 (BNIP3) and inflammation in astrocytes under lipopolysaccharide ( LPS ) combined with hypoxia. Methods: Primary cultured astrocytes and neurons in vitro were divided into four groups: normoxia group; hypoxia group; LPS group; LPS plus hypoxia group (each group is provided with 3 duplicate holes). After treated with LPS(100 ng/ml), hypoxia group and LPS plus hypoxia group were placed in hypoxia cell incubator with 0.3% O2, and normoxia group and LPS group were placed in normal cell incubator for 24 h. Primary astrocytes were divided four groups as above for 6 h,12 h and 24 h. The expression of BNIP3 in astrocytes was detected by Western blot. The expressions of tumor necrosis factor-α(TNF-ɑ), interleukin-1β (IL-1β) and interleukin-6 (IL-6) mRNA in astrocytes were detected by RT-PCR. The levels of TNF-ɑ, IL-1β and IL-6 in cultured medium were detected by ELISA. Results: Compared with the normoxia group, the expressions of inflammatory cytokines TNF-ɑ, IL-1β and IL-6 mRNA had no change in hypoxia group and were increased in LPS group and LPS plus hypoxia group (P＜0.01). Compared with the LPS group, the expressions of inflammatory cytokines IL-1β and IL-6 mRNA were increased in LPS plus hypoxia group (P＜0.05, P＜0.01). Compared with the normoxia group, the levels of inflammatory cytokines had no change in hypoxia group and the levels of TNF-ɑ and IL-6 were increased in LPS group and LPS plus hypoxia group (P＜0.01), the level of IL-1β had no change in LPS group and LPS plus hypoxia group. Compared with the LPS group, the levels of TNF-ɑ and IL-6 had no more change in LPS plus hypoxia group. BNIP3 was expressed in primary neurons and astrocytes in vitro. Compared with astrocytes in the normoxia group, the expression of BNIP3 in LPS group had no change and was increased markedly in hypoxia group and LPS plus hypoxia group (P＜0.01). Compared with neurons in the normoxia group, the expression of BNIP3 in LPS group had no change and was increased in hypoxia group and LPS plus hypoxia group (P＜0.05, P＜0.01). Compared with neurons in the hypoxia group, the expression of BNIP3 in astrocytes of hypoxia group was increased (P＜0.01). Compared with the normoxia group at the same time point, the expression of BNIP3 in LPS group had no change and was increased in hypoxia group and LPS plus hypoxia group (P＜0.05, P＜0.01). Compared with the hypoxia group at the same time point, the expression of BNIP3 was increased markedly in LPS plus hypoxia group at 6 h and 12 h (P＜0.01). Conclusion: The combination of hypoxia with LPS augmented inflammation in astrocyte and LPS enhanced the expression of BNIP3 in astrocyte under hypoxia, suggesting BNIP3 might be involved in regulating astrocyte inflammation.