摘要
探究见血清不同部位提取物对脂多糖(lipopolysaccharide,LPS)诱导BV-2小胶质细胞神经炎症反应的作用及潜在机制.见血清药材粉碎,提取浓缩得醇提物,经萃取得石油醚、乙酸乙酯、正丁醇和水相萃取物,进一步利用D101 大孔树脂柱色谱对乙酸乙酯萃取物进行分离得流分①～⑥.实验分为对照组、模型组、见血清提取物单独处理组及LPS联合见血清提取物干预组.采用LPS诱导BV-2建立神经炎症细胞模型,Griess法检测细胞上清中一氧化氮(nitric oxide,NO)生成;MTT法检测细胞活力;ELISA法检测细胞上清中白细胞介素(interleukin,IL)-6和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的释放;RT-qPCR检测促炎细胞因子诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、环加氧酶-2(cyclooxygenase-2,COX-2)、IL-6、IL-1β和TNF-α的mRNA表达水平;Western blot检测iNOS、COX-2、核转录因子-κB p65(nuclear factor kappa-B p65,p65)、磷酸化核转录因子-κB p65(p-p65)、细胞外调节蛋白激酶(extracellular signal-regulated kinase,ERK)、磷酸化的细胞外调节蛋白激酶(p-ERK)、c-jun N末端激酶(c-jun N-terminal kinease,JNK)、磷酸化JNK(p-JNK)、p38 丝裂原活化蛋白激酶(p38 mitogen activated protein kinase,p38),磷酸化p38 丝裂原活化蛋白激酶(p-p38)蛋白表达;UHPLC-Q-Exactive Orbitrap技术结合文献比对解析活性部位化学成分.结果表明见血清不同部位提取物均可以显著降低LPS诱导BV-2 中一氧化氮(nitric oxide,NO)的释放,其中乙酸乙酯萃取物抑制作用最显著且无细胞毒性,该部位流分⑥还可以剂量依赖性降低iNOS、COX-2、IL-6、IL-1β和TNF-α的基因及蛋白表达水平,同时对LPS诱导BV-2中p-p65、p-ERK、p-p38 和p-JNK蛋白表达具有显著抑制作用;质谱分析流分⑥共鉴定到 79 种化合物,含 12 种已被证实具有抗炎活性的化合物.该研究通过体外实验证实了见血清提取物在治疗神经炎症方面具有显著效果,并揭示了其抗炎作用可能通过抑制NF-κB及MAPK信号通路实现.
Abstract
To investigate the impact and potential mechanisms of extracts from different parts of Liparis nervosa on neuroinflamma-tion by lipopolysaccharide(LPS)-induced BV-2 microglial cells.The materials of L.nervosa were subjected to crushing,ethanol ex-traction,and concentration to obtain an alcohol extract.Subsequently,the extract was further extracted to obtain petroleum ether ex-tract,ethyl acetate extract,N-butanol extract,and aqueous phase extract.The ethyl acetate extract was separated into distillate ①-⑥using D101 macroporous resin column chromatography.The experiment was divided into control group,LPS model group,L.nervosa extract group,and LPS+L.nervosa group.LPS was utilized to induce a neuroinflammatory cell model in BV-2 microglial cells.The Griess test was utilized for detecting the production of nitric oxide(NO)in the cell supernatant.Cell viability was detected by MTT as-say.The release of interleukin-6(IL-6)and tumor necrosis factor alpha(TNF-α)in the cell supernatant was quantified using ELISA.RT-qPCR was utilized to assess the mRNA levels of pro-inflammatory cytokines inducible nitric oxide synthase(iNOS),cyclooxyge-nase-2(COX-2),interleukin(IL)-6,IL-1β,and TNF-α.The protein expression of iNOS,COX-2,nuclear factor kappa-B p65(p65),p-p65,extracellular signal-regulated kinase(ERK),p-ERK,c-jun N-terminal kinase(JNK),p-JNK,p38 mitogen-activated protein kinase(p38),and p-p38 MAPK(p-p38)were also evaluated by Western blot.The chemical composition of active substances in L.nervosa was analyzed using the UHPLC-Q-Exactive Orbitrap technology and literature comparison.Our findings indicate that ex-tracts from different parts of L.nervosa exhibit a significant reduction in the release of NO from LPS-induced BV-2 microglial cells.Specifically,the ethyl acetate extract demonstrates the most notable inhibitory effect without causing cell toxicity.Additionally,the dis-tillate ⑥ extracted from the ethyl acetate exhibits a reduction in the mRNA and protein levels of iNOS,COX-2,IL-6,IL-1β,and TNF-α in a dose-dependent manner,and it inhibits the protein expression of p-p65,p-ERK,p-p38,and p-JNK in LPS-induced BV-2 microglial cells.A total of 79 compounds in the distillate ⑥ were identified by mass spectrometry,including 12 confirmed compounds with anti-inflammatory effects.This study confirmed the remarkable efficacy of L.nervosa extract in the treatment of neuroinflamma-tion,which may be achieved through the inhibition of NF-κB and MAPK signaling pathways.
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