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[石豆兰提取物对脂多糖诱导的BV-2小胶质细胞介导的神经炎症的影响及其生物活性成分分析]

[Impact of Liparis nervosa extract on neuroinflammation mediated by LPS-induced BV-2 microglial cells and its bioactive components analysis].

作者信息

Kong Shuai-Wen, Zhang Yuan, Zeng Lian, Chen Xi-Rong, Wang Hai-Jing, Cai He-Lun, Hao Xin-Cai, Zhang Yu

机构信息

Hubei Key Laboratory of Wudang Local Chinese Medicine Research, School of Pharmacy, Hubei University of Medicine Shiyan 442000, China.

Anesthesiology Institute,Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology Wuhan 430000, China.

出版信息

Zhongguo Zhong Yao Za Zhi. 2024 Jun;49(11):3050-3060. doi: 10.19540/j.cnki.cjcmm.20240122.701.

Abstract

To investigate the impact and potential mechanisms of extracts from different parts of Liparis nervosa on neuroinflammation by lipopolysaccharide(LPS)-induced BV-2 microglial cells. The materials of L. nervosa were subjected to crushing, ethanol extraction, and concentration to obtain an alcohol extract. Subsequently, the extract was further extracted to obtain petroleum ether extract, ethyl acetate extract, N-butanol extract, and aqueous phase extract. The ethyl acetate extract was separated into distillate(1)-(6)using 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 assay. 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 m RNA levels of pro-inflammatory cytokines inducible nitric oxide synthase(iNOS), cyclooxygenase-2(COX-2), interleukin( IL)-6, IL-1β, and TNF-α. The protein expression of i NOS, 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 extracts 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 distillate(6) extracted from the ethyl acetate exhibits a reduction in the m RNA and protein levels of i NOS, 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 neuroinflammation, which may be achieved through the inhibition of NF-κB and MAPK signaling pathways.

摘要

为研究石仙桃不同部位提取物对脂多糖(LPS)诱导的BV-2小胶质细胞神经炎症的影响及潜在机制。将石仙桃材料进行粉碎、乙醇提取和浓缩,得到乙醇提取物。随后,对提取物进一步萃取,得到石油醚提取物、乙酸乙酯提取物、正丁醇提取物和水相提取物。乙酸乙酯提取物通过D101大孔树脂柱色谱分离为馏分(1)-(6)。实验分为对照组、LPS模型组、石仙桃提取物组和LPS+石仙桃组。利用LPS诱导BV-2小胶质细胞建立神经炎症细胞模型。采用Griess试验检测细胞上清液中一氧化氮(NO)的产生。通过MTT法检测细胞活力。采用ELISA法定量检测细胞上清液中白细胞介素-6(IL-6)和肿瘤坏死因子α(TNF-α)的释放。利用RT-qPCR评估促炎细胞因子诱导型一氧化氮合酶(iNOS)、环氧化酶-2(COX-2)、白细胞介素(IL)-6、IL-1β和TNF-α的mRNA水平。通过蛋白质印迹法还评估了iNOS、COX-2、核因子κB p65(p65)、p-p65、细胞外信号调节激酶(ERK)、p-ERK、c-jun氨基末端激酶(JNK)、p-JNK、p38丝裂原活化蛋白激酶(p38)和p-p38 MAPK(p-p38)的蛋白表达。采用UHPLC-Q-Exactive Orbitrap技术并结合文献比较分析石仙桃中活性物质的化学成分。我们的研究结果表明,石仙桃不同部位的提取物可显著降低LPS诱导的BV-2小胶质细胞中NO的释放。具体而言,乙酸乙酯提取物表现出最显著的抑制作用且不引起细胞毒性。此外,从乙酸乙酯中提取的馏分(6)以剂量依赖性方式降低iNOS、COX-2、IL-6、IL-1β和TNF-α的mRNA和蛋白水平,并抑制LPS诱导的BV-2小胶质细胞中p-p65、p-ERK、p-p38和p-JNK的蛋白表达。通过质谱鉴定出馏分(6)中共有79种化合物,其中包括12种已确认具有抗炎作用的化合物。本研究证实了石仙桃提取物在治疗神经炎症方面具有显著疗效,这可能是通过抑制NF-κB和MAPK信号通路实现的。

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