中国畜牧兽医 ›› 2023, Vol. 50 ›› Issue (2): 807-816.doi: 10.16431/j.cnki.1671-7236.2023.02.038

• 基础兽医 • 上一篇    下一篇

HO-1在巨噬细胞中的抗炎和抗氧化作用

付京城1,2, 何俊辉1,2, 仝江1,2, 吴魏1,2, 郭爽1,2, 杨彦宾1,2, 韩莹倩1,2, 王月影1,2, 李和平1,2   

  1. 1. 河南农业大学, 农业农村部动物生化与营养重点实验室, 郑州 450046;
    2. 河南省动物生长发育调控重点实验室, 郑州 450046
  • 收稿日期:2022-07-13 出版日期:2023-02-05 发布日期:2023-02-06
  • 通讯作者: 李和平 E-mail:liheping1972@126.com
  • 作者简介:付京城,E-mail:690878734@qq.com
  • 基金资助:
    河南省科技攻关计划(212102110356、222102110402)

Anti-inflammatory and Antioxidant Roles of HO-1 in Macrophages

FU Jingcheng1,2, HE Junhui1,2, TONG Jiang1,2, WU Wei1,2, GUO Shuang1,2, YANG Yanbin1,2, HAN Yingqian1,2, WANG Yueying1,2, LI Heping1,2   

  1. 1. Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs, Henan Agricultural University, Zhengzhou 450046, China;
    2. Key Laboratory of Animal Growth and Development of Henan Province, Zhengzhou 450046, China
  • Received:2022-07-13 Online:2023-02-05 Published:2023-02-06

摘要: 【目的】旨在揭示血红素加氧酶1(HO-1)在巨噬细胞中的抗炎和抗氧化作用。【方法】利用不同浓度脂多糖(LPS,0、3、5、10、15、20、25 μg/mL)、HO-1(0、0.02、0.04、0.06、0.08、0.10 μg/mL)及锌原卟啉(zinc protoporphyrin,ZPP;0、5、10、15、20、30 ng/mL)分别处理巨噬细胞(RAW264.7),12 h后通过CCK8法检测RAW264.7细胞活力,计算LPS、HO-1和ZPP处理RAW264.7细胞的最佳浓度。将RAW264.7细胞随机分为对照组(CT)、LPS组(LPS)、LPS+HO-1组(LH)、HO-1组(HO-1)、LPS+HO-1+ZPP组(LHZ),每组3个重复。CT组细胞用含10%胎中血清的DMEM培养基培养;LPS组细胞用LPS处理;LH组细胞用LPS和HO-1共处理;HO-1组细胞用HO-1处理;LHZ组细胞用LPS、HO-1和ZPP共处理,各组细胞的处理时间均为12 h,收集细胞和上清。采用ELISA法检测上清液中白细胞介素-6(IL-6)、IL-8、肿瘤坏死因子α(TNF-α)、活性氧(ROS)、丙二醛(MDA)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GPx)含量,实时荧光定量PCR法检测细胞中IL-6、IL-8、TNF-α、核因子E2相关因子2(Nrf2)、HO-1 mRNA表达,Western blotting检测细胞中Nrf2、HO-1蛋白的表达。【结果】与0 μg/mL LPS处理细胞相比,20 和25 μg/mL LPS均能极显著降低 RAW264.7细胞活力(P<0.01);与0 μg/mL HO-1处理细胞相比,0.08和0.10 μg/mL HO-1均极显著提高细胞活力(P<0.01);与0 ng/mL ZPP处理细胞相比,15、20、30 ng/mL ZPP极显著降低细胞活力(P<0.01),后续选用20 μg/mL LPS、0.08 μg/mL HO-1、15 ng/mL ZPP处理RAW264.7细胞。与CT组相比,LPS组细胞中IL-6、IL-8、TNF-α mRNA表达极显著上调(P<0.01),IL-6、IL-8、TNF-α、MDA、ROS含量均极显著升高(P<0.01),GPx、SOD含量均极显著降低(P<0.01);与LPS组相比,LH组细胞中IL-6、IL-8、TNF-α mRNA表达均极显著下降(P<0.01),IL-6、IL-8、TNF-α、MDA、ROS含量均极显著降低(P<0.01),GPx、SOD含量均极显著升高(P<0.01);与LH组相比,LHZ组细胞中IL-6、IL-8、TNF-α mRNA表达均极显著上调(P<0.01),IL-6、IL-8、TNF-α、MDA、ROS含量均极显著升高(P<0.01),GPx、SOD含量均极显著降低(P<0.01)。Nrf2/HO-1信号通路分子表达结果表明,与CT组相比,LPS组细胞中Nrf2和HO-1的mRNA和蛋白表达极显著上调(P<0.01);与LPS组相比,LH组细胞中Nrf2和HO-1 mRNA表达极显著下调(P<0.01),Nrf2蛋白表达极显著下降(P<0.01),而HO-1蛋白表达极显著上升(P<0.01);与LH组相比,LHZ组细胞中Nrf2和HO-1 mRNA表达均极显著上调(P<0.01),Nrf2蛋白表达极显著上升(P<0.01),HO-1蛋白表达极显著下降(P<0.01)。与CT组相比,HO-1组各项指标均差异不显著(P>0.05)。【结论】20 μg/mL LPS诱导巨噬细胞产生炎性反应和氧化应激,0.08 μg/mL HO-1具有抗炎和抗氧化作用,HO-1调节Nrf2信号通路发挥抗炎和抗氧化作用。

关键词: 血红素加氧酶-1; 脂多糖; RAW264.7细胞; 炎症反应; 氧化应激

Abstract: 【Objective】 This study was aim to reveal the anti-inflammatory and antioxidant effects of heme oxygenase-1 (HO-1) in macrophages.【Method】 Different concentrations of lipopolysaccharide (LPS,0,3,5,10,15,20,25 μg/mL),HO-1(0,0.02,0.04,0.06,0.08,0.10 μg/mL) and zinc protoporphyrin (ZPP;0,5,10,15,20,30 ng/mL) respectively were used to treat macrophages (RAW264.7) for 12 hours.The viability of RAW264.7 cells was detected by CCK8 method,and the optimal concentration of RAW264.7 cells treated with LPS,HO-1 and ZPP was calculated. RAW264.7 cells were randomly divided into control group (CT),LPS group (LPS),LPS+HO-1 group (LH),HO-1 group (HO-1),LPS+HO-1+ ZPP group (LHZ),with three replicates in each group.The cells in CT group were seeded in DMEM medium containing 10% fetal bovine serum.The cells in LPS group were treated with LPS.The cells in LH group were treated with LPS and HO-1.The cells in HO-1 group were treated with HO-1.The cells in LHZ group were co-cultured with LPS,HO-1 and ZPP.Cells in each group was treated for 12 hours,collecting the cells and supernatant.The contents of interleukin-6 (IL-6),IL-8,tumor necrosis factor-α (TNF-α), reactive oxygen sepecies (ROS), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in the supernatant were detected by ELISA.The mRNA expression of IL-6,IL-8,TNF-α,nuclear factor-E2-related factor 2 (Nrf2) and HO-1 was measured by quantitative Real-time PCR.The expression of Nrf2,HO-1 protein was detected by Western blotting.【Result】 Compared with cells treated by 0 μg/mL LPS,the concentration of 20 and 25 μg/mL LPS extremely significantly decreased the viability of RAW264.7 cells (P<0.01).Compared with cells treated by 0 μg/mL HO-1,the concentration of 0.08 and 0.10 μg/mL HO-1 extremely significantly increased cell viability (P<0.01).Compared with cells treated by 0 ng/mL,ZPP with 15,20 and 30 ng/mL extremely significantly reduced cell viability (P<0.01).The results showed that the concentration of LPS,HO-1 and ZPP treatment was 20 μg/mL,0.08 μg/mL,15 ng/mL in subsequent experiment,respectively.Compared with CT group,IL-6,IL-8 and TNF-α mRNA expression in LPS group were extremely significantly up-regulated (P<0.01).The contents of IL-6,IL-8,TNF-α,MDA and ROS were extremely significantly increased (P<0.01),and the contents of GPx and SOD were extremely significantly decreased (P<0.01).Compared with LPS group,IL-6,IL-8 and TNF-α mRNA expression in LH group were extremely significantly decreased (P<0.01).The contents of IL-6,IL-8,TNF-α,MDA and ROS were extremely significantly decreased (P<0.01),and the contents of GPx and SOD were extremely significantly increased (P<0.01).Compared with the LH group,IL-6,IL-8 and TNF-α mRNA expression in LHZ group were extremely significantly up-regulated (P<0.01).The content of IL-6,IL-8,TNF-α,MDA and ROS were extremely significantly increased (P<0.01),while the contents of GPx and SOD were extremely significantly decreased (P<0.01).The results of Nrf2/HO-1 signal pathway molecules expression showed that the mRNA and protein expressions of Nrf2 and HO-1 in LPS group were extremely significantly up-regulated compared with CT group (P<0.01).Compared with LPS group,Nrf2 and HO-1 mRNA expression in LH group were extremely significantly down-regulated (P<0.01),Nrf2 protein expression was extremely significantly decreased (P<0.01),and HO-1 protein expression was extremely significantly increased (P<0.01).Compared with LH group,Nrf2 and HO-1 mRNA expression in LHZ group was extremely significantly up-regulated (P<0.01),Nrf2 protein expression was extremely significantly increased (P<0.01),and HO-1 protein expression was extremely significantly decreased (P<0.01).Compared with CT group,the indexes of HO-1 group were not significantly different (P>0.05).【Conclusion】 20 μg/mL LPS induced macrophages to produce inflammatory response and oxidative stress.0.08 μg/mL HO-1 had anti-inflammatory and antioxidant effects.HO-1 regulated Nrf2 signaling pathway to exert anti-inflammatory and antioxidant effects.

Key words: heme oxygenase-1; lipopolysaccharide; RAW264.7 cells; oxidative stress; inflammatory response

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