[关键词]
[摘要]
目的 研究菥蓂Thlaspi arvense水提醇沉部位、正丁醇部位的抗炎活性以及正丁醇部位的作用机制。方法 将菥蓂水提物经过乙醇、正丁醇萃取后制得菥蓂水提醇沉部位、正丁醇部位。采用MTT法确定水提醇沉部位、正丁醇部位(15.625、31.25、62.5、125、250、500、1 000 μg/mL)对RAW264.7细胞的毒性。采用脂多糖(LPS)1 μg/mL诱导RAW264.7细胞产生炎性反应,建立体外炎症细胞模型。Griess法检测水提醇沉部位、正丁醇部位(7.812 5、15.625、31.25、62.5、125、250、500、1 000 μg/mL)对LPS诱导RAW264.7细胞后NO释放量的影响;ELISA法检测水提醇沉部位、正丁醇部位(125、250、500 μg/mL)对白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)释放量的影响;Western Blotting法检测正丁醇部位(125、250、500 μg/mL)对环氧合酶-2(COX-2)、一氧化氮合酶(iNOS)、白细胞介素1β(IL-1β)、核因子-κB(NF-κB)p-P65、Toll样受体4(TLR-4)、磷酸化NF-κB抑制蛋白(p-IκBα)蛋白表达的影响。结果 菥蓂水提醇沉部位、正丁醇部位质量浓度在1 000 μg/mL以下时对RAW264.7细胞几乎无毒性作用。与模型组比较,菥蓂水提醇沉部位、正丁醇部位62.5、125、250、500、1 000 μg/mL组NO释放量显著下降(P<0.01);菥蓂水提醇沉部位、正丁醇部位125、250、500 μg/mL组IL-6、TNF-α的分泌量显著降低(P<0.01),且正丁醇部位作用优于水提醇沉部位;菥蓂正丁醇部位500 μg/mL组的iNOS和250、500 μg/mL组COX-2、IL-1β、NF-κB p-P65、TLR-4、p-IκBα蛋白表达量显著降低(P<0.01),作用均呈浓度相关性。结论 菥蓂正丁醇部位显著抑制LPS诱导的RAW264.7细胞NO的释放,IL-1β、IL-6、TNF-α的分泌以及COX-2、iNOS蛋白的表达,其抗炎作用机制可能与抑制TLR-4/NF-κB信号通路相关。
[Key word]
[Abstract]
Objective To study the anti-inflammatory activities of Thlaspi arvense water extraction and alcohol precipitation site and N-butanol site, and the mechanism of N-butanol site. Methods Thlaspi arvense water extracts were extracted by ethanol and N-butanol to obtain water extraction and alcohol precipitation site and N-butanol site. MTT assay was used to determine the toxicity of water extraction and alcohol precipitation site and N-butanol site (15.625, 31.25, 62.5, 125, 250, 500, 1 000 μg/mL) to RAW264.7 cells. RAW264.7 cells were induced by lipopolysaccharide (LPS) at 1 μg/mL, and the inflammatory cell model was established in vitro. Griess method was used to detect the effects of water extraction and alcohol precipitation site and N-butanol site (7.8125, 15.625, 31.25, 62.5, 125, 250, 500, 1 000 μg/mL) on NO release of RAW264.7 cells after LPS induction. ELISA method was used to detect the effects of water extraction and alcohol precipitation site and N-butanol site (125, 250, and 500 μg/mL) on the release of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α). Western Blotting was used to detect the effects of N-butanol site (125, 250, and 500 μ g/mL) on the expression of cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS), interleukin 1β (IL-1β), NF- κ B P-P65, TLR-4 and P-IκBα. Results Thlaspi arvense water extraction and alcohol precipitation site and N-butanol site had almost no toxicity to RAW264.7 cells when the concentration was less than 1 000 μg/mL. Compared with model group, the release of NO in Thlaspi arvense water extraction and alcohol precipitation site and N-butanol site of 62.5, 125, 250, 500 and 1 000 μg/mL groups was significantly decreased (P<0.01). The secretion levels of IL-6 and TNF-α were significantly decreased (P<0.01) in Thlaspi arvense water extraction and alcohol precipitation site and N-butanol site of 125, 250 and 500 μg/mL, and the effects of N-butanol were better than those of water extraction and alcohol precipitation site. The protein expression levels of iNOS in 500 μg/mL and COX-2, IL-1β, NF-κB p-P65, TLR-4, and p-IκBα in 250 and 500 μg/mL groups of N-butanol site decreased significantly (P<0.01), and the effects were correlated with concentration. Conclusion Thlaspi arvense N-butanol site was almost non-cytotoxic, inhibited the release of RAW264.7 cell NO, inhibited the secretion of inflammatory factor IL-1β, IL-6, TNF-α and the expression of COX-2、iNOS protein in a dose-dependent manner. The anti-inflammatory effect of extract may be mediated by inhibition of TLR-4/NF-κB signaling pathway.
[中图分类号]
R285.5
[基金项目]
国家自然科学基金项目(21262005);广西科技重大专项项目(桂科AA18242040)