HO-1在巨噬细胞中的抗炎和抗氧化作用

doi:10.16431/j.cnki.1671-7236.2023.02.038

Abstract

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

CLC Number:

S852.2

FU Jingcheng, HE Junhui, TONG Jiang, WU Wei, GUO Shuang, YANG Yanbin, HAN Yingqian, WANG Yueying, LI Heping. Anti-inflammatory and Antioxidant Roles of HO-1 in Macrophages[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(2): 807-816.

References

[1] BRIEGER K,SCHIAVONE S,MILLER JR F J,et al.Reactive oxygen species:From health to disease[J]. Swiss Medical Weekly,2012,142:w13659.
[2] ZHAO S,JIANG Y,YANG X,et al.Lipopolysaccharides promote a shift from Th2-derived airway eosinophilic inflammation to Th17-drived neutrophilic inflammation in an ovalbumin sensitized murine asthma model[J]. Journal of Asthma,2017,54(5):447-455.
[3] 蔡文涛.MTT法和CCK-8法检测中药抗病毒活性成分细胞毒性的比较[J].湖北大学学报(自然科学版),2017,39(3):305-310. CAI W T.The comparision of MTT and CCK-8 by detecting cytotoxicity of antiviral active components in traditional Chinese medicine[J]. Journal of Hubei University (Natural Science),2017,39(3):305-310.(in Chinese)
[4] 熊建文,肖化,张镇西.MTT法和CCK-8法检测细胞活性之测试条件比较[J].激光生物学报,2007,5:559-562. XIONG J W,XIAO H,ZHANG Z X.An experimental research on different detection conditions between MTT and CCK-8[J]. Acta Laser Biology Sinica,2007,5:559-562.(in Chinese)
[5] RYTER S W,ALAM J,CHOI A M.Heme oxygenase-1/carbon monoxide:From basic science to therapeutic applications[J]. Physiological Reviews,2006,86(2):583-650.
[6] CHORA A A,FONTOURA P,CUNHA A,et al.Heme oxygenase-1 and carbon monoxide suppress autoimmune neuroinflammation[J]. The Journal of Clinical Investigation,2007,117:438-447.
[7] NITTI M,PIRAS S,BRONDOLO L,et al.Heme oxygenase 1 in the nervous system:Does it favor neuronal cell survival or induce neurodegeneration[J]. International Journal of Molecular Sciences,2018,19 (8):2260.
[8] LI H B,ZHANG X Z,SUN Y,et al.HO-1/PINK1 regulated mitochondrial fusion/fission to inhibit pyroptosis and attenuate septic acute kidney injury[J]. Biomed Research International,2020,2020:2148706.
[9] CHEN X,WU S,TANG L,et al.Mesenchymal stem cells overexpressing heme oxygenase-1 ameliorate lipopolysaccharide-induced acute lung injury in rats[J]. Journal of Cell Physiology, 2019,234(5):7301-7319.
[10] 程梅,董晓筠,朱彬.利多卡因对脓毒症大鼠心肌损伤及Nrf2/HO-1通路的影响[J].中国病理生理杂志,2021,37(4):634-639. CHENG M,DONG X Y,ZHU B.Effects of lidocaine on myocardial injury and Nrf2/HO-1 signaling pathway in sepsis rats[J]. Chinese Journal of Pathophysiology,2021,37(4):634-639.(in Chinese)
[11] MENG X,HU L,LI W.Baicalin ameliorates lipopolysaccharide-induced acute lung injury in mice by suppressing oxidative stress and inflammation via the activation of the Nrf2-mediated HO-1 signaling pathway[J]. Naunyn Schmiedebergs Archives Pharmacology,2019,392(11):1421-1433.
[12] WENZEL P,ROSSMANN H,MULLER C,et al.Heme oxygenase-1 suppresses a pro-inflammatory phenotype in monocytes and determines endothelial function and arterial hypertension in mice and humans[J]. European Heart Journal,2015,36(48):3437-3446.
[13] FERENBACH D A,RAMDAS V,SPENCER N,et al.Macrophages expressing heme oxygenase-1 improve renal function in ischemia/reperfusion injury[J]. Molecular Therapy, 2010,18(9):1706-1713.
[14] 李玉婷,李香云,史佳,等.内毒素攻击大鼠肺泡巨噬细胞时HO-1对高尔基体应激的影响[J].中国病理生理杂志,2022,38(3):509-516. LI Y T,LI X Y,SHI J,et al.Role of HO-1 on Golgi stress in LPS-stimulated rat alveolar macrophages[J]. Chinese Journal of Pathophysiology, 2022,38(3):509-516.(in Chinese)
[15] GANDINI N A,ALONSO E N,FERMENTO M E,et al.Heme oxygenase-1 has an antitumor role in breast cancer[J]. Antioxidants Redox Signaling,2019,30:2030-2049.
[16] 罗影,左中夫,程雪,等.葛花总黄酮通过Nrf2/HO-1信号通路对Ⅰ型糖尿病大鼠肾脏的保护作用[J].中成药,2021,1-6. LUO Y,ZUO Z F,CHENG X,et al.Protective effect of total flavonoids of pueraria flower on kidney of typeⅠdiabetic rats through Nrf2/HO-1 signaling pathway[J]. Chinese Traditional Patent Medicine, 2021,1-6.(in Chinese)
[17] QIU Z,HE Y,MING H,et al.Lipopolysaccharide aggravates high glucose-and hypoxia/reoxygenation-induced injury through activating ROS-dependent NLRP3 inflammasome-mediated pyroptosis in H9C2 cardiomyocytes[J]. Journal of Diabetes Research,2019,2019:8151836.
[18] 钟树林,卢智健,张婷,等.脐血IL-1β、IL-6、IL-8、IL-10、TNF-α与母体、胎儿炎症反应及早产预后的相关性[J].海南医学,2016,24:4036-4039. ZHONG S L,LU Z J,ZHANG T,et al.Correlation analysis of IL-1 β,IL-6,IL-8,IL-10,TNF-α with maternal,fetal inflammatory response and prognosis of preterm birth[J]. Hainan Medical Journal,2016,24:4036-4039.(in Chinese)
[19] ZHANG D D.Mechanistic studies of the Nrf2-keap1 signaling pathway[J]. Drug Metabolism Reviews,2006,38(4):769-789.
[20] FREIGANG S,AMPENBERGER F,SPOHN G,et al.Nrf2 is essential for cholesterol crystal-induced inflammasome activation and exacerbation of atherosclerosis[J]. European Journal of Immunology,2011,41(7):2040-2051.
[21] WU Q Q,WANG Y,SENITKO M,et al.Bardoxolone methyl (BARD) ameliorates ischemic AKI and increases expression of protective genes Nrf2,PPAR,and HO-1[J]. American Journal of Physiology-Renal Physiology,2011,300(5):F1180-F1192.
[22] PARFENOVA H,BASUROY S,BHATTACHARYA S,et al.Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells:Contributions of HO-1 and HO-2 to cytoprotection[J]. American Journal of Physiology-Cell Physiology,2006,290(5):C1399-C1410.
[23] MILBURY P E,GRAF B,CURRAN-CELENTANO J M,et al.Bilberry (vaccinium myrtillus) anthocyanins modulate heme oxygenase-1 and glutathione S-transferase-pi expression in ARPE-19 cells[J]. Investigative Ophthalmology Visual Science,2007,48(5):2343-2349.
[24] XUE H,GUO H,LI Y C,et al.Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats[J]. World Journal of Gastroenterology,2007,13(40):5384-5390.
[25] SHEFTEL A D,KIM S F,PONKA P.Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism[J]. Journal of Biological Chemistry,2007,282(14):10480-10486.
[26] 张雁林,赵金垣.血红素加氧酶-1的细胞保护作用研究进展[J].环境与职业医学,2008,25(2):197-202. ZHANG Y L,ZHAO J H.Advances in studies on the cytoprotection of heme oxygenase-1[J]. Journal of Environmental and Occupational Medicine,2008,25(2):197-202.(in Chinese)
[27] ITOH K,WAKABAYASHI N,KATOH Y,et al.Keap1 regulates both cytoplasmic-nuclear shuttling and degradation of Nrf-2 in response to electrophiles[J]. Genes Cells,2003,8(4):379-391.
[28] MCMAHON M,ITOH K,YAMAMOTO M,et al.Keap1-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression[J]. Journal of Biological Chemistry,2003,278(24):21592-21600.
[29] KOBAYASGI M,ITOH K,SUZUKI T,et al.Identification of the interactive interface and phylogenic conservation of the Nrf2-Keap1 system[J]. Genes Cells,2002,7(8):807-820.
[30] LIN X,BAI D,WEI Z,et al.Curcumin attenuates oxidative stress in RAW264.7 cells by increasing the activity of antioxidant enzymes and activating the Nrf2-keap1 pathway[J]. PLoS One,2019,14(5):e0216711.
[31] YANG S,SUN J,GU D,et al.Antioxidant activities of sulfated codonopsis polysaccharides in acute oxidative stress[J]. Journal of Food Biochemistry,2021,45(12):e13974.

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

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