虾青素对脂多糖通过TLR4/MyD88/NF-κB信号通路诱导的IPEC-J2细胞炎症的影响
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摘要
【目的】研究虾青素(AST)对脂多糖(LPS)通过TLR4/My D88/NF-κB信号通路诱导的IPEC-J2细胞炎症的影响。【方法】采用MTT法确定虾青素和LPS对IPEC-J2细胞和转染IPEC-J2细胞的最佳处理浓度和处理时间,在处理后采用实时荧光定量PCR检测IPEC-J2细胞和转染IPEC-J2细胞炎症因子NF-κB、TNF-α、IL-6和IL~(-1)β的m RNA相对表达量,采用ELISA检测上述炎症因子的分泌量。【结果】当处理3 h,虾青素浓度达到150μmol·L~(-1)时,IPEC-J2细胞和转染IPEC-J2细胞活力达到峰值;当处理6 h,LPS质量浓度达到100 ng·m L~(-1)时,IPEC-J2细胞和转染IPEC-J2细胞活力达到峰值。与对照组相比,LPS组IPEC-J2细胞NF-κB、TNF-α、IL-6、IL~(-1)β的m RNA相对表达量和分泌量显著升高(P<0.05);与LPS组相比,AST+LPS组NF-κB、TNF-α、IL-6、IL~(-1)β在IPEC-J2细胞中的m RNA相对表达量和分泌量显著降低(P<0.05),而在转染IPEC-J2细胞中的炎症因子m RNA相对表达量和分泌量均无显著变化(P>0.05)。【结论】虾青素可以抑制细胞炎症反应,且对细胞的保护作用与TLR4/My D88/NF-κB信号通路相关。
【Objective】To investigate the effect of astaxanthin(AST) on IPEC-J2 cell inflammation induced by lipopolysaccharide(LPS) via TLR4/MyD88/NF-κB signaling pathway.【Method】MTT assay was performed to determine the optimal time and concentrations of astaxanthin and LPS for treating IPEC-J2 cells and transfected IPEC-J2 cells. Fluorescent quantitative RT-PCR was performed to determine the relative m RNA expressions of inflammatory factors including NF-κB, TNF-α, IL-6 and IL-1β in IPEC-J2 cells and transfected IPEC-J2 cells stimulated by LPS. ELISA assays were carried out to determine the secretion amounts of these inflammatory factors.【Result】The vitality of IPEC-J2 cells and transfected IPEC-J2 cells reached the peakwhen treated with 150 μmol·L-1 astaxanthin for 3 h or 100 ng·m L-1 lipopolysaccharide for 6 h. Compared with control group, the relative m RNA expressions and secretions of NF-κB, TNF-α, IL-6 and IL-1β in IPEC-J2 cells significantly increased in LPS treatment group(P<0.05). Compared with LPS treatment group, the relative m RNA expressions and secretions of NF-κB, TNF-α, IL-6 and IL-1β in LPS+AST group were significantly lower in IPEC-J2 cells(P<0.05), but did not differ significantly in transfected IPEC-J2 cells(P>0.05).【Conclusion】Astaxanthin can inhibit cell inflammation, and its protective effect on cells is related to TLR4/My D88/NF-κB signaling pathway.
【Objective】To investigate the effect of astaxanthin(AST) on IPEC-J2 cell inflammation induced by lipopolysaccharide(LPS) via TLR4/MyD88/NF-κB signaling pathway.【Method】MTT assay was performed to determine the optimal time and concentrations of astaxanthin and LPS for treating IPEC-J2 cells and transfected IPEC-J2 cells. Fluorescent quantitative RT-PCR was performed to determine the relative m RNA expressions of inflammatory factors including NF-κB, TNF-α, IL-6 and IL-1β in IPEC-J2 cells and transfected IPEC-J2 cells stimulated by LPS. ELISA assays were carried out to determine the secretion amounts of these inflammatory factors.【Result】The vitality of IPEC-J2 cells and transfected IPEC-J2 cells reached the peakwhen treated with 150 μmol·L-1 astaxanthin for 3 h or 100 ng·m L-1 lipopolysaccharide for 6 h. Compared with control group, the relative m RNA expressions and secretions of NF-κB, TNF-α, IL-6 and IL-1β in IPEC-J2 cells significantly increased in LPS treatment group(P<0.05). Compared with LPS treatment group, the relative m RNA expressions and secretions of NF-κB, TNF-α, IL-6 and IL-1β in LPS+AST group were significantly lower in IPEC-J2 cells(P<0.05), but did not differ significantly in transfected IPEC-J2 cells(P>0.05).【Conclusion】Astaxanthin can inhibit cell inflammation, and its protective effect on cells is related to TLR4/My D88/NF-κB signaling pathway.
引文
[1]ADEREM A,ULEVITCH R J.Toll-like receptors in the induction of the innate immune response[J].Nature,2000,406(6797):782-787.
[2]KOLEK M J,CARLQUIST J F,MUHLESTEIN J B,et al.Toll-like receptor 4 gene Asp299Gly polymorphism is associated with reductions in vascular inflammation,angiographic coronary artery disease,and clinical diabetes[J].Am Heart J,2004,148(6):1034-1040.
[3]MANDREKAR P,PRUETT S,ARTEEL G,et al.RSA2004:Combined basic research satellite symposium-session two:Toll-like receptors and organ damage[J].Alcohol Clin Exp Res,2005,29(9):1744-1748.
[4]ZULUAGA M,GUEGUEN V,LETOURNEUR D,et al.Astaxanthin-antioxidant impact on excessive reactive oxygen species generation induced by ischemia and reperfusion injury[J].Chem Biol Interact,2018(279):145-158.
[5]吴旻.β-胡萝卜素对脂多糖诱导的IPEC-J2细胞炎症的保护作用及其机制[D].长春:吉林农业大学,2016.
[6]张晓音,张珊珊,吴旻,等.β-胡萝卜素对脂多糖刺激巨噬细胞RAW264.7炎症因子的影响及其机制[J].中国免疫学杂志,2017,33(6):838-843.
[7]张晓音,吴旻,张珊珊,等.β-胡萝卜素的不同添加方式对脂多糖刺激的RAW264.7细胞炎症的影响[J].中国农业大学学报,2017,22(11):114-120.
[8]BAUD V,DERUDDER E.Control of NF-κB activity by proteolysis[J].Curr Top Microbiol Immunol,2011,349:97-114.
[9]BOSMANN M,GRAILER J J,RUEMMLER R,et al.Extracellular histones are essential effectors of C5a R-and C5L2-mediated tissue damage and inflammation in acute lung injury[J].FASEB J,2013,27(12):5010-5021.
[10]GOKCINAR D,ERGIN V,CUMAOGLU A,et al.Effects of ketamine,propofol,and ketofol on proinflammatory cytokines and markers of oxidative stress in a rat model of endotoxemia-induced acute lung injury[J].Acta Biochim Pol,2013,60(3):451-456.
[11]AOKI Y,NAKAHARA T,ASANO D,et al.Preventive effects of rapamycin on inflammation and capillary degeneration in a rat model of NMDA-induced retinal injury[J].Biol Pharm Bull,2015,38(2):321-324.
[12]INOUE M,TANABE H,MATSUMOTO A,et al.Astaxanthin functions differently as a selective peroxisome proliferator-activated receptorγmodulator in adipocytes and macrophages[J].Biochem Pharmacol,2012,84(5):692-700.
[13]CHAN K C,MONG M C,YIN M C.Antioxidative and anti-inflammatory neuroprotective effects of astaxanthin and canthaxanthin in nerve growth factor differentiated PC12 cells[J].J Food Sci,2009,74(7):H225-H231.
[14]TRIPATHI D N,JENA G B.Astaxanthin intervention ameliorates cyclophosphamide-induced oxidative stress,DNA damage and early hepatocarcinogenesis in rat:Role of Nrf2,p53,p38 and phase-II enzymes[J].Mutat Res,2010,696(1):69-80.
[15]XIAO M,ZHU T,ZHANG W,et al.Emodin ameliorates LPS-induced acute lung injury,involving the inactivation of NF-κB in mice[J].Int J Mol Sci,2014,15(11):19355-19368.
[16]ZHANG X,XIONG H,LI H,et al.Protective effect of taraxasterol against LPS-induced endotoxic shock by modulating inflammatory responses in mice[J].Immunopharm Immunotoxicol,2014,36(1):11-16.
[17]HUANG Z Y,STABLER T,PEI F X,et al.Both systemic and local lipopolysaccharide(LPS)burden are associated with knee OA severity and inflammation[J].Osteoarthr Cartilage,2016,24(10):1769-1775.
[18]KIM Y W,ZHAO R J,PARK S J,et al.Anti-inflammatory effects of liquiritigenin as a consequence of the inhibition of NF-κB dependent i NOS and proinflammatory cytokines production[J].Brit J Pharmacol,2008,154(1):165-173.
[19]FENG Y,XU Z X,BUYS E S,et al.Innate immune adaptor My D88 mediates neutrophil recruitment and myocardial injury after ischemia-reperfusion in mice[J].Am J Physiol Heart Circ Physiol,2008,295(3):H1311-H1318.
[20]LI Y H,WANG J B,LI M H,et al.Quantification of brain edema and hemorrhage by MRI after experimental traumatic brain injury in rabbits predicts subsequent functional outcome[J].Neurol Sci,2012,33(4):731-740.
[2]KOLEK M J,CARLQUIST J F,MUHLESTEIN J B,et al.Toll-like receptor 4 gene Asp299Gly polymorphism is associated with reductions in vascular inflammation,angiographic coronary artery disease,and clinical diabetes[J].Am Heart J,2004,148(6):1034-1040.
[3]MANDREKAR P,PRUETT S,ARTEEL G,et al.RSA2004:Combined basic research satellite symposium-session two:Toll-like receptors and organ damage[J].Alcohol Clin Exp Res,2005,29(9):1744-1748.
[4]ZULUAGA M,GUEGUEN V,LETOURNEUR D,et al.Astaxanthin-antioxidant impact on excessive reactive oxygen species generation induced by ischemia and reperfusion injury[J].Chem Biol Interact,2018(279):145-158.
[5]吴旻.β-胡萝卜素对脂多糖诱导的IPEC-J2细胞炎症的保护作用及其机制[D].长春:吉林农业大学,2016.
[6]张晓音,张珊珊,吴旻,等.β-胡萝卜素对脂多糖刺激巨噬细胞RAW264.7炎症因子的影响及其机制[J].中国免疫学杂志,2017,33(6):838-843.
[7]张晓音,吴旻,张珊珊,等.β-胡萝卜素的不同添加方式对脂多糖刺激的RAW264.7细胞炎症的影响[J].中国农业大学学报,2017,22(11):114-120.
[8]BAUD V,DERUDDER E.Control of NF-κB activity by proteolysis[J].Curr Top Microbiol Immunol,2011,349:97-114.
[9]BOSMANN M,GRAILER J J,RUEMMLER R,et al.Extracellular histones are essential effectors of C5a R-and C5L2-mediated tissue damage and inflammation in acute lung injury[J].FASEB J,2013,27(12):5010-5021.
[10]GOKCINAR D,ERGIN V,CUMAOGLU A,et al.Effects of ketamine,propofol,and ketofol on proinflammatory cytokines and markers of oxidative stress in a rat model of endotoxemia-induced acute lung injury[J].Acta Biochim Pol,2013,60(3):451-456.
[11]AOKI Y,NAKAHARA T,ASANO D,et al.Preventive effects of rapamycin on inflammation and capillary degeneration in a rat model of NMDA-induced retinal injury[J].Biol Pharm Bull,2015,38(2):321-324.
[12]INOUE M,TANABE H,MATSUMOTO A,et al.Astaxanthin functions differently as a selective peroxisome proliferator-activated receptorγmodulator in adipocytes and macrophages[J].Biochem Pharmacol,2012,84(5):692-700.
[13]CHAN K C,MONG M C,YIN M C.Antioxidative and anti-inflammatory neuroprotective effects of astaxanthin and canthaxanthin in nerve growth factor differentiated PC12 cells[J].J Food Sci,2009,74(7):H225-H231.
[14]TRIPATHI D N,JENA G B.Astaxanthin intervention ameliorates cyclophosphamide-induced oxidative stress,DNA damage and early hepatocarcinogenesis in rat:Role of Nrf2,p53,p38 and phase-II enzymes[J].Mutat Res,2010,696(1):69-80.
[15]XIAO M,ZHU T,ZHANG W,et al.Emodin ameliorates LPS-induced acute lung injury,involving the inactivation of NF-κB in mice[J].Int J Mol Sci,2014,15(11):19355-19368.
[16]ZHANG X,XIONG H,LI H,et al.Protective effect of taraxasterol against LPS-induced endotoxic shock by modulating inflammatory responses in mice[J].Immunopharm Immunotoxicol,2014,36(1):11-16.
[17]HUANG Z Y,STABLER T,PEI F X,et al.Both systemic and local lipopolysaccharide(LPS)burden are associated with knee OA severity and inflammation[J].Osteoarthr Cartilage,2016,24(10):1769-1775.
[18]KIM Y W,ZHAO R J,PARK S J,et al.Anti-inflammatory effects of liquiritigenin as a consequence of the inhibition of NF-κB dependent i NOS and proinflammatory cytokines production[J].Brit J Pharmacol,2008,154(1):165-173.
[19]FENG Y,XU Z X,BUYS E S,et al.Innate immune adaptor My D88 mediates neutrophil recruitment and myocardial injury after ischemia-reperfusion in mice[J].Am J Physiol Heart Circ Physiol,2008,295(3):H1311-H1318.
[20]LI Y H,WANG J B,LI M H,et al.Quantification of brain edema and hemorrhage by MRI after experimental traumatic brain injury in rabbits predicts subsequent functional outcome[J].Neurol Sci,2012,33(4):731-740.