蘘荷根茎乙醇提取物对LPS诱导巨噬细胞RAW264.7炎症反应的影响

Effects of Zingiber mioga Rhizome Ethanol Extract on Lipopolysaccharide-Induced Inflammatory Response of RAW264.7 Macrophages

目的:探究蘘荷根茎乙醇提取物的抗炎活性及作用机制。方法:以70%乙醇为溶剂对蘘荷根茎回流提取制得提取物,测定其总酚酸和总黄酮含量,采用超高效液相色谱-四极杆静电场轨道阱串联质谱(UPLC-Q-Orbitrap MS)鉴定其化学成分;MTT法测定蘘荷根茎乙醇提取物对RAW264.7细胞和L929细胞存活率的影响;用脂多糖(LPS)对RAW264.7细胞进行诱导建立体外炎症模型,通过NO检测试剂盒、ROS检测试剂盒、ELISA、免疫印迹法检测其对丝裂原活化蛋白激酶(MAPK)和核转录因子κB(NF-κB)信号通路中一氧化氮(NO)、白细胞介素6(IL-6)和肿瘤坏死因子-α(TNF-α)的释放量、活性氧(ROS)水平及诱导型一氧化氮合酶(iNOS)表达的影响,探究蘘荷根茎乙醇提取物的抗炎作用及可能的作用机理。结果:蘘荷根茎乙醇提取物富含酚类成分[(15.07±0.13)mg GAE/g],鉴定出酚类化合物为L-酪氨酸、原儿茶酸、原儿茶醛、4-甲基水杨酸、对羟基安息香醛、咖啡酸、香兰素、对羟基肉桂酸、白果新酸;蘘荷根茎乙醇提取物在31.3、62.5、125、250μg/mL时对RAW264.7和L929细胞的存活率无显著影响;与空白对照组比较,模型对照组NO、IL-6、TNF-α和ROS含量显著升高(P<0.01),iNOS表达、P38、JNK、ERK、IκBα的磷酸化表达以及细胞核NF-κB p65表达明显上调(P<0.05或P<0.01),IκBα和细胞质NF-κB p65的表达显著下调(P<0.01);与模型对照组比较,蘘荷根茎乙醇提取物31.3、62.5、125、250μg/mL组NO、IL-6的释放量明显降低(P<0.05或P<0.01);蘘荷根茎乙醇提取物62.5、125、250μg/mL组ROS水平显著降低,iNOS表达、JNK、ERK、IκBα的磷酸化表达以及细胞核NF-κB p65表达显著下调,细胞质NF-κB p65表达显著上调(P<0.01);蘘荷根茎乙醇提取物125、250μg/mL组IκBα表达明显上调(P<0.05或P<0.01);蘘荷根茎乙醇提取物250μg/mL组TNF-α的释放量明显降低和P38的磷酸化表达明显下调(P<0.05)。结论:蘘荷根茎乙醇提取物具有良好的抗炎作用,其抗炎机制可能与抑制LPS诱导的MAPK和NF-κB通路的活化有关。

Objective:To explore the anti-inflammatory effects and mechanism of Zingiber mioga rhizome ethanol extract.Methods:The ethanol extract was prepared by reflux extraction of Z.mioga rhizome using 70%ethanol.The total phenol content and the total flavonoid content were measured.The chemical composition of the extract was identified by ultra-high-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap-MS).The cytotoxicity of the extract on RAW264.7 cells and L929 cells was determined by MTT assay.An inflammation model was induced by lipopolysaccharide(LPS)in RAW264.7 cells.The release levels of nitric oxide(NO),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α),the level of reactive oxygen species(ROS),and the expression of inducible nitric oxide synthase(iNOS)related to the mitogen-activated protein kinase(MAPK)and nuclear factor Kappa B(NF-κB)signaling pathways were detected using NO detection kit,ROS detection kit,ELISA,and Western blot to explore the anti-inflammatory effect and the mechanism of the Z.mioga rhizome ethanol extract.Results:Z.mioga rhizome ethanol extract was rich in phenols(15.07±0.13 mg GAE/g),including L-tyrosine,protocatechuic acid,protocatechualdehyde,4-methoxysalicylic acid,p-hydroxybenzaldehyde,caffeic acid,vanillin,p-coumaric acid,and ginkgolic acid.The extract at 31.25,62.5,125,and 250μg/mL showed no significant effect on the survival of RAW264.7 and L929 cells.Compared with the normal control group,the model group showed increased content of NO,IL-6,TNF-α,and ROS(P<0.01),up-regulated iNOS expression,phosphorylation of p38,JNK,ERK,and IκBα,and expression of NF-κB p65 in the cell nucleus(P<0.05 or P<0.01),and down-regulated expression of IκBαand NF-κB p65 in the cytoplasm(P<0.01).Compared with the model group,the Z.mioga rhizome ethanol extract groups at 31.25,62.5,125,and 250μg/mL showed decreased release of NO and IL-6(P<0.05 or P<0.01).The Z.mioga rhizome ethanol extract groups at 62.5,125,and 250μg/mL showed reduced the ROS level(P<0.01),down-regulated iNOS expression and phosphorylation of JNK,ERK,and IκBα,and NF-κB p65 expression in the cell nucleus(P<0.01),and up-regulated NF-κB p65 expression in the cytoplasm(P<0.01).The Z.mioga rhizome ethanol extract groups at 125 and 250μg/mL showed increased expression of IκBα(P<0.05 or P<0.01).The Z.mioga rhizome ethanol extract groups at 250μg/mL showed reduced release of TNF-αand the phosphorylation level of p38(P<0.05).Conclusion:Z.mioga rhizome ethanol extract shows remarkable anti-inflammatory effects,and its anti-inflammatory mechanism may be related to the inhibition of LPS-induced activation of MAPK and NF-κB pathways.