齐墩果酸抑制脂多糖诱导RAW264.7细胞炎症反应
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摘要
通过CCK-8法检测齐墩果酸对巨噬细胞增殖的影响;采用Griess试剂检测细胞培养液中NO含量,通过Western Blot法分析iNOS蛋白表达和MAPK通路p38,ERK和JNK蛋白表达和磷酸化状态,研究齐墩果酸对脂多糖(LPS)诱导巨噬细胞RAW264.7炎症反应的影响,探讨齐墩果酸抗感染作用及机制。结果表明:齐墩果酸(100.0μmol·L~(-1))明显抑制巨噬细胞增殖(P<0.05);12.5~100.0μmol·L~(-1)的齐墩果酸能够显著抑制脂多糖诱导巨噬细胞分泌NO(P<0.01),并能显著抑制LPS诱导iNOS蛋白的表达;LPS作用后能够激活ERK通路,抑制p38和JNK通路磷酸化,齐墩果酸作用后,能显著抑制ERK磷酸化,促进JNK和p38蛋白的磷酸化。证明齐墩果酸能显著降低脂多糖诱导巨噬细胞分泌NO,通过抑制iNOS的表达和双向调控MAPK通路蛋白磷酸化达到抗感染作用。
CCK-8 assay was used to detect the influence of oleanolic acid(OA)on macrophage proliferation;Griess assay was used to determine the NO release in LPS induced macrophage with or without OA treatment;Western-blot method was used to detect the iNOS protein expression and MAPK pathway p38,ERK and JNK protein expression and phosphorylation status.To study the anti-inflammatory effect of OA on lipopolysaccharide(LPS)induced RAW264.7 macrophage and its mechanism.Results:only OA(100.0μmol·L~(-1))inhibited macrophage proliferation.(P<0.05);12.5-100.0μmol·L~(-1) of OA significantly inhibited the secretion of NO in LPS stimulated macrophages(P<0.01),and strongly inhibited the expression of iNOS protein;LPS activated the phosphorylation of ERK pathway,and inhibited the phosphorylation status of JNK and p38,but OA decreased the phosphorylation of ERK,and promoted the phosphorylation status of JNK and p38 protein.This study demonstrated that OA showed strong anti-inflammatory effect by inhibition of the expression of iNOS and regulation of protein phosphorylation of MAPK pathway in dual way.
CCK-8 assay was used to detect the influence of oleanolic acid(OA)on macrophage proliferation;Griess assay was used to determine the NO release in LPS induced macrophage with or without OA treatment;Western-blot method was used to detect the iNOS protein expression and MAPK pathway p38,ERK and JNK protein expression and phosphorylation status.To study the anti-inflammatory effect of OA on lipopolysaccharide(LPS)induced RAW264.7 macrophage and its mechanism.Results:only OA(100.0μmol·L~(-1))inhibited macrophage proliferation.(P<0.05);12.5-100.0μmol·L~(-1) of OA significantly inhibited the secretion of NO in LPS stimulated macrophages(P<0.01),and strongly inhibited the expression of iNOS protein;LPS activated the phosphorylation of ERK pathway,and inhibited the phosphorylation status of JNK and p38,but OA decreased the phosphorylation of ERK,and promoted the phosphorylation status of JNK and p38 protein.This study demonstrated that OA showed strong anti-inflammatory effect by inhibition of the expression of iNOS and regulation of protein phosphorylation of MAPK pathway in dual way.
引文
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[13]HWANG Y J,SONG J,KIM H R,et al.Oleanolic acid regulates NF-κB signaling by suppressing MafK expression in RAW264.7cells[J].BMB Rep,2014,47(9):524-529.
[14]连俊江,程彬峰,高尧鑫,等.齐墩果酸对IL-1β诱导的SW982细胞炎症反应的抑制作用[J].药学学报,2016,51(11):1711-1716.
[15]刘娟,姚天佑,孔莺莺,等.NAD+抑制脂多糖诱导海马炎症因子的表达[J].扬州大学学报(农业与生命科学版),2016,37(1):16-21.
[16]GAESTEL M.MAPK-activated protein kinases(MKs):novel insights and challenges[J].Front Cell Dev Biol,2016,3:88.
[17]PHULL A R,EO S H,KIM S J.Oleanolic acid(OA)regulates inflammation and cellular dedifferentiation of chondrocytes via MAPK signaling pathways[J].Cellular and molecular biology,2017,63(3):12-17.
[2]MENG Y Q,FENG C Q,ZHANG L F,et al.Synthesis and antitumor activity of oleanolic acid derivatives[J].Acta Pharm Sin,2015,50:469-474.
[3]SHANMUGAM M K,NGUYEN A H,KUMAR A P,et al.Targeted inhibition of tumor proliferation,survival,and metastasis by pentacyclic triterpenoids:potential role in prevention and therapy of cancer[J].Cancer Lett,2012,320(2):158-170.
[4]KANG G D,LIM S,KIM D H.Oleanolic acid ameliorates dextran sodium sulfate-induced colitis in mice by restoring the balance of Th17/Treg cells and inhibiting NF-κB signaling pathway[J].Int Immunopharmacol,2015,29(2):393-400.
[5]MA X H,ZHAO Y C,YIN L,et al.Studies on the effect of oleanolic acid on experimental liver injury(author′s transl)[J].Yao Xue Xue Bao,1982,17(2):93-97.
[6]HWANG Y J,SONG J,KIM H R,et al.Oleanolic acid regulates NF-kappaB signaling by suppressing MafK expression in RAW 264.7cells[J].BMB Reports,2014,47(9):524-529.
[7]崔树娜,姜俊杰,艾浩,等.金雀异黄素对巨噬细胞RAW 264.7增殖、形态和细胞周期的影响[J].北京中医药,2010,29(3):218-221.
[8]崔树娜,姜俊杰,李彧,等.金雀异黄素对脂多糖诱导RAW264.7细胞分泌功能的影响[J].中医药学报,2010,38(2):13-16.
[9]崔树娜,Ursula Bilitewski.金雀异黄素对脂多糖诱导的巨噬细胞MAPK和TLR信号转导通路的影响[J].细胞与分子免疫学杂志,2014,30(3):233-236.
[10]劳雪芬,邹慧,徐筱萌,等.富硒女贞子水提物、粗多糖对小鼠免疫的影响[J].扬州大学学报(农业与生命科学版),2015,36(1):16-20.
[11]LIN C,WEN X,SUN H.Oleanolic acid derivatives for pharmaceutical use:apatent review[J].Expert Opinion on Therapeutic Patents,2016,26(6):643-655.
[12]SHI Y,SONG Q,HU D,et al.Oleanolic acid induced autophagic cell death in hepatocellular carcinoma cells via PI3K/Akt/mTOR and ROS-dependent pathway[J].Korean J Physiol Pharmacol,2016,20(3):237-243.
[13]HWANG Y J,SONG J,KIM H R,et al.Oleanolic acid regulates NF-κB signaling by suppressing MafK expression in RAW264.7cells[J].BMB Rep,2014,47(9):524-529.
[14]连俊江,程彬峰,高尧鑫,等.齐墩果酸对IL-1β诱导的SW982细胞炎症反应的抑制作用[J].药学学报,2016,51(11):1711-1716.
[15]刘娟,姚天佑,孔莺莺,等.NAD+抑制脂多糖诱导海马炎症因子的表达[J].扬州大学学报(农业与生命科学版),2016,37(1):16-21.
[16]GAESTEL M.MAPK-activated protein kinases(MKs):novel insights and challenges[J].Front Cell Dev Biol,2016,3:88.
[17]PHULL A R,EO S H,KIM S J.Oleanolic acid(OA)regulates inflammation and cellular dedifferentiation of chondrocytes via MAPK signaling pathways[J].Cellular and molecular biology,2017,63(3):12-17.