紫茎泽兰精油化学分离物抑制脂多糖诱导的RAW264.7细胞炎性反应
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摘要
目的探究紫茎泽兰精油化学分离物对脂多糖(LPS)诱导RAW264.7细胞抗炎活性和Toll样受体4(TLR4)蛋白表达的影响。方法将巨噬细胞分为空白对照组、 LPS组、 LPS联合紫茎泽兰精油化学分离物组。CCK-8法检测紫茎泽兰精油化学分离物的细胞毒性,实时定量PCR检测RAW264.7细胞白细胞介素6(IL-6)、 IL-10的mRNA水平, ELISA检测IL-6、 IL-10的蛋白水平, Western blot法检测RAW264.7细胞TLR4蛋白水平。结果剂量<20 mg/mL的紫茎泽兰精油化学分离物对RAW264.7细胞无细胞毒性。LPS处理可引起RAW264.7细胞TLR4蛋白水平升高、 IL-6 mRNA和蛋白水平增加、 IL-10水平降低,分离产物可逆转以上作用。结论紫茎泽兰精油化学分离物抑制RAW264.7细胞TLR4蛋白表达和IL-6的分泌,促进IL-10表达,发挥抗炎作用。
Objective To investigate the anti-inflammatory activity of Ageratina adenophora essential oil(AAEO-CP) and its effects on the expression of Toll-like receptor 4(TLR4) protein in lipopolysaccharide(LPS)-induced RAW264.7 cells. Methods RAW264.7 cells were divided into control group, LPS group, and LPS combined with AAEO-CP group. The cytotoxicity of AAEO-CP was detected by CCK-8 assay. The mRNA and protein expression of interleukin-6(IL-6) and IL-10 were detected by real-time PCR and ELISA, respectively, and the protein expression of TLR4 in RAW264.7 cells was measured by Western blotting. Results AAEO-CP below 20 mg/mL was not cytotoxic to RAW264.7 cells. LPS increased the protein expression of TLR4, also increased the protein and mRNA expression of IL-6, but decrease the protein and mRNA expression of IL-10 in RAW264.7 cells. And all of the above results were reversed by AAEO-CP. Conclusion AAEO-CP can play the anti-inflammatory effects by increasing the expression of IL-10 protein and decreasing the expression of IL-6 protein, and inhibiting TLR4 protein in LPS-induced RAW264.7 cells.
Objective To investigate the anti-inflammatory activity of Ageratina adenophora essential oil(AAEO-CP) and its effects on the expression of Toll-like receptor 4(TLR4) protein in lipopolysaccharide(LPS)-induced RAW264.7 cells. Methods RAW264.7 cells were divided into control group, LPS group, and LPS combined with AAEO-CP group. The cytotoxicity of AAEO-CP was detected by CCK-8 assay. The mRNA and protein expression of interleukin-6(IL-6) and IL-10 were detected by real-time PCR and ELISA, respectively, and the protein expression of TLR4 in RAW264.7 cells was measured by Western blotting. Results AAEO-CP below 20 mg/mL was not cytotoxic to RAW264.7 cells. LPS increased the protein expression of TLR4, also increased the protein and mRNA expression of IL-6, but decrease the protein and mRNA expression of IL-10 in RAW264.7 cells. And all of the above results were reversed by AAEO-CP. Conclusion AAEO-CP can play the anti-inflammatory effects by increasing the expression of IL-10 protein and decreasing the expression of IL-6 protein, and inhibiting TLR4 protein in LPS-induced RAW264.7 cells.
引文
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[22] Ya P,Xu H,Ma Y,et al.Liver injury induced in BALB/c mice by PM 2.5,exposure and its alleviation by compound essential oils[J].Biomed Pharmacother,2018,105:590-598.
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[25] Cheng C,Yi Z,Jian P.Oregano essential oil attenuates RAW264.7 cells from Lipopolysaccharide-induced inflammatory response through regulating NADPH oxidase activation-driven oxidative stress[J/OL].Molecules,2018,23(8):1857.DOI:10.3390/molecules23081857.
[26] Lin S S,Lu T M,Chao P C,et al.In vivo cytokine modulatory effects of cinnamaldehyde,the major constituent of leaf essential oil from Cinnamomum osmophloeum kaneh[J].Phytother Res,2011,25(10):1511-1518.
[2] He Y,Mo Q,Hu Y,et al.E.adenophorum induces cell cycle arrest and apoptosis of splenocytes through the mitochondrial pathway and caspase activation in saanen goats[J/OL].Sci Rep,2015,5:15967.DOI:10.1038/srep15967.
[3] Liu X,Ouyang C,Wang Q,et al.Effects of oil extracts of Eupatorium adenophorum on phytophthora capsici and other plant pathogenic fungi in vitro[J].Pestic Biochem Physiol,2017,140:90-96.
[4] Kurade N P,Jaitak V,Kaul V K,et al.Chemical composition and antibacterial activity of essential oils of Lantana camara,Ageratum houstonianum and Eupatorium adenophorum[J].Pharm Biol,2010,48(5):539-544.
[5] Chen H,Zhou B,Yang J,et al.Essential oil derived from Eupatorium adenophorum spreng.mediates anticancer effect by inhibiting STAT3 and AKT Activation to induce apoptosis in hepatocellular carcinoma[J/OL].Front Pharmacol,2018,9:00483.DOI:10.3389/fphar.2018.00483.
[6] Zou L,Zhang Y,Li W,et al.Comparison of chemical profiles,anti-Inflammatory activity,and UPLC-Q-TOF/MS-Based metabolomics in endotoxic fever rats between synthetic borneol and natural borneol[J/OL].Molecules,2017,22(9):1446.DOI:10.3390/molecules22091446.
[7] Zhang X,Xu F,Liu L,et al.(+)-Borneol improves the efficacy of edaravone against DSS-induced colitis by promoting M2 macrophages polarization via JAK2-STAT3 signaling pathway[J].Int Immunopharmacol,2017,53:1-10.
[8] Kim D S,Lee H J,Jeon Y D,et al.Alpha-pinene exhibits anti-Inflammatory activity through the suppression of MAPKs and the NF-κB pathway in mouse peritoneal macrophages[J].Am J Chin Med,2015,43(4):731-742.
[9] Varga Z V,Matyas C,Erdelyi K,et al.β-Caryophyllene protects against alcoholic steatohepatitis by attenuating inflammation and metabolic dysregulation in mice[J].Br J Pharmacol,2017,175(2):320-334.
[10] Fairweather D,Cihakova D.Alternatively activated macrophages in infection and autoimmunity[J].J Autoimmun,2009,33(3):222-230.
[11] Hoogendijk A J,Pinhanos S S,van der Poll T,et al.Intrapulmonary administration of a p38 mitogen activated protein kinase inhibitor partially prevents pulmonary inflammation[J].Immunobiology,2013,218(4):435-442.
[12] Beutler B,Rietschel E T.Innate immune sensing and its roots:the story of endotoxin[J].Nat Rev Immunol,2003,3(2):169-176.
[13] Ko Y J,Ahn G,Ham Y M,et al.Anti-inflammatory effect and mechanism of action of Lindera erythrocarpa essential oil in lipopolysaccharide-stimulated RAW264.7 cells[J].EXCLI J,2017,16:1103-1113.
[14] Medzhitov R.Origin and physiological roles of inflammation[J].Nature,2008,454(7203):428-435.
[15] Chiurchiù V,Leuti A,Maccarrone M.Bioactive lipids and chronic inflammation:managing the fire within[J/OL].Front Immunol,2018,9:38.DOI:10.3389/fimmu.2018.00038.eCollection 2018.
[16] Abdulkhaleq L A,Assi M A,Abdullah R,et al.The crucial roles of inflammatory mediators in inflammation:a review[J].Vel World,2018,11(5):627-635.
[17] Park J,Ha S H,Abekura F,et al.4-O-Carboxymethylascochlorin inhibits expression levels of on inflammation-Related cytokines and matrix metalloproteinase-9 through NF-κB/MAPK/TLR4 signaling pathway in LPS-activated RAW264.7 cells[J/OL].Front Pharmacol,2019,10:304.DOI:10.3389/fphar.2019.00304.eCollection 2019.
[18] Ko W,Sohn J H,Jang J H,et al.Inhibitory effects of alternaramide on inflammatory mediator expression through TLR4-MyD88-mediated inhibition of NF-κB and MAPK pathway signaling in lipopolysaccharide-stimulated RAW264.7 and BV2 cells[J].Chem Biol Interact,2016,244:16-26.
[19] Yamamoto M,Takeda K,Akira S.TIR domain-containing adaptors define the specificity of TLR signaling[J].Mol Immunol,2004,40(12):861-868.
[20] Jiang M P,Xu C,Guo Y W,et al.β-arrestin 2 attenuates lipopolysaccharide-induced liver injuryviainhibition of TLR4/NF-κB signaling pathway-mediated inflammation in mice[J].World J Gastroenterol,2018,24(2):216-225.
[21] 刘静,刘瑛,李宾,等.槐定碱抑制脂多糖诱导的RAW264.7细胞炎症因子分泌及机制[J].细胞与分子免疫学杂志,2015,31(5):585-589.Liu J,Liu Y,Li B,et al.Sophoridine suppresses inflammatory cytokine secretion by lipopolysaccharide-induced RAW264.7 cells and its mechanism[J].Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi,2015,31(5):585-589.
[22] Ya P,Xu H,Ma Y,et al.Liver injury induced in BALB/c mice by PM 2.5,exposure and its alleviation by compound essential oils[J].Biomed Pharmacother,2018,105:590-598.
[23] Ghasemi H.Roles of IL-6 in ocular inflammation:a review[J].Ocul Immunol Inflamm,2018,26(1):37-50.
[24] Mosser D M,Zhang X.Interleukin-10:new perspectives on an old cytokine[J].Immunol Rev,2010,226(1):205-218.
[25] Cheng C,Yi Z,Jian P.Oregano essential oil attenuates RAW264.7 cells from Lipopolysaccharide-induced inflammatory response through regulating NADPH oxidase activation-driven oxidative stress[J/OL].Molecules,2018,23(8):1857.DOI:10.3390/molecules23081857.
[26] Lin S S,Lu T M,Chao P C,et al.In vivo cytokine modulatory effects of cinnamaldehyde,the major constituent of leaf essential oil from Cinnamomum osmophloeum kaneh[J].Phytother Res,2011,25(10):1511-1518.