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中国应用生理学杂志 ›› 2021, Vol. 37 ›› Issue (6): 632-637.doi: 10.12047/j.cjap.6177.2021.096

• 研究论文 • 上一篇    下一篇

低氧联合LPS刺激对星形胶质细胞中炎性因子和BNIP3表达的影响*

丁利平1, 韩莹2, 成祥2, 赵彤2, 朱玲玲2,3△, 廖红1△   

  1. 1.中国药科大学药物科学研究院, 南京 210009;
    2.军事医学研究院军事认知与脑科学研究所, 北京 100850;
    3.江苏省神经再生协同创新中心, 南京 210009
  • 收稿日期:2020-10-15 修回日期:2021-03-05 出版日期:2021-11-28 发布日期:2021-11-25
  • 通讯作者: Tel: 13681099909; E-mail: linglingzhuamms@126.com, hliao@cpu.edu.cn
  • 基金资助:
    *国家自然科学基金重点项目(81430044);北京市科学与技术委员会项目(Z161100000216134)

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

DING Li-ping1, HAN Ying2, CHENG Xiang2 , ZHAO Tong2, ZHU Ling-ling 2,3△, LIAO Hong1△   

  1. 1. Institute of Pharmaceutical Sciences, China Pharmaceutical University, Nanjing 210009;
    2. Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Beijing 100850;
    3. Collaborative Innovation Center for Neural Regeneration, Nanjing 210009, China
  • Received:2020-10-15 Revised:2021-03-05 Online:2021-11-28 Published:2021-11-25

摘要: 目的:探讨在低氧联合脂多糖(LPS)作用下,星形胶质细胞中B淋巴细胞瘤-2/腺病毒E1B 19-kD相互作用蛋白3(BNIP3)的表达和炎症反应变化。方法:将体外培养的原代星形胶质细胞和神经元进行下列分组:常氧组、LPS组、低氧组和LPS+低氧组(每组设置3个复孔)。LPS处理后,低氧组和LPS+低氧组放入低氧细胞孵箱,LPS组和常氧组放入正常的细胞孵箱。LPS浓度:100 ng/ml,氧气浓度为0.3%。处理时间为24 h。原代的星形胶质细胞进行上述的分组,时间点设为6 h、12 h和24 h。Western blot检测BNIP3的表达变化,RT-PCR和ELISA分别检测星形胶质细胞的肿瘤坏死因子-ɑ(TNF-ɑ)、白细胞介素-1β(IL-1β)和白细胞介素6(IL-6)mRNA水平变化和分泌情况。结果:与常氧组比较,低氧组炎症因子的表达没有变化,LPS组和LPS+低氧组的炎症因子TNF-ɑ、IL-1β和IL-6 mRNA水平升高(P<0.01);与LPS组比较,LPS+低氧组炎症因子IL-1β和IL-6 mRNA水平进一步升高(P<0.05,P<0.01)。与常氧组比较,低氧组炎症因子的分泌水平没有变化,LPS组和LPS+低氧组的炎症因子TNF-ɑ和IL-6 分泌水平升高(P<0.01),IL-1β的水平没有变化;与LPS组比较,LPS+低氧组炎症因子TNF-ɑ和IL-6分泌水平没有进一步升高。BNIP3在体外培养的神经元和星型胶质细胞中都有表达;在星形胶质细胞中,与常氧组比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达明显增加(P<0.01);在神经元中,与常氧组比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达增加(P<0.05,P<0.01);与神经元的低氧组比较,星形胶质细胞的低氧组BNIP3的表达增加更明显(P<0.01)。在星形胶质细胞中LPS联合低氧刺激6、12、24 h后BNIP3蛋白的表达,与常氧组相同时间点比较,LPS组BNIP3的表达没有变化,低氧组和LPS+低氧组BNIP3的表达增加(P<0.05,P<0.01);与低氧组相同时间点比较,6 h和12 h的LPS+低氧组BNIP3的表达增加的更高(P<0.01)。结论:低氧联合LPS刺激可以增强星形胶质细胞的炎症反应,LPS能增加低氧下星形胶质细胞中BNIP3的表达,提示BNIP3在星形胶质细胞的炎性反应中可能具有一定的调节作用。

关键词: 星形胶质细胞, BNIP3, 炎症, 低氧, 脂多糖

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.

Key words: astrocyte, BNIP3, inflammation, hypoxia, LPS

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