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[摘要]
研究川陈皮素对脂多糖(LPS)刺激的RAW 264.7巨噬细胞损伤的保护作用。MTT法检测川陈皮素的安全用药范围;乳酸脱氢酶试剂盒检测乳酸脱氢酶(LDH)的释放水平;一氧化氮试剂盒检测细胞上清液中一氧化氮(NO)的释放水平;Real-time PCR法检测Toll样受体4(TLR4)、内毒素受体14(CD14)、诱导型一氧化氮合酶(iNOS)、白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)的mRNA表达水平;Western Blot检测核转录因子-κB(NF-κB)的核蛋白表达水平。与正常组比较,LPS刺激组LDH活力和NO的释放量分别升高了4.12倍和3.21倍(p<0.01),iNOS、IL-1β、TNF-α、TLR4和CD14 mRNA表达水平分别升高了2.73倍、4.91倍、10倍、1.84倍和3.53倍(p<0.01);同时,NF-κB的核表达水平升高了2.50倍(p<0.01);给予川陈皮素干预后,与LPS刺激组比较,川陈皮素各用药组均能明显降低LDH活力和NO的释放量,减少TLR4、CD14、IL-1β、TNF-α和iNOS的mRNA表达水平和NF-κB的核表达水平(p<0.05或p<0.01)。川陈皮素可减轻LPS刺激RAW 264.7细胞的损伤,机制可能与抑制TLR4-NF-κB信号通路有关。
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[Abstract]
To investigate the protective effects of nobiletin on LPS-induced RAW 264.7 macrophage cell injury. The MTT method was used to detect the safe dosage of nobiletin. The release level of lactate dehydrogenase (LDH) was measured by the LDH assay kit. The level of nitric oxide (NO) released in the cell supernatant was evaluated by the NO assay kit. The mRNA expression levels of TNF-α, IL-1β, iNOS, TLR4 and CD14 were evaluated by real-time PCR analysis. The expression level of NF-κB nuclear protein was determined by the western blot method. Compared to the normal control group, the LDH activity and amount of released NO in the LPS-stimulated group increased by 4.12 times and 3.21 times, respectively (p<0.01), and the mRNA expression levels of iNOS, IL-1β, TNF-α, TLR4 and CD14 increased by 2.73 times, 4.91 times, 10 times, 1.84 times and 3.53 times, respectively (p<0.01). Meanwhile, the nuclear expression level of NF-κB in the LPS-stimulated group increased by 2.50 times (p<0.01). However, the treatment with nobiletin at different doses decreased the activity of LDH and amount of the released NO, while reducing the mRNA expression levels of TNF-α, IL-1β, iNOS, TLR4 and CD14 as well as the nuclear expression level of NF-κB, compared with the LPS-stimulated group (p<0.05 or p<0.01). Nobiletin attenuates LPS-induced injury in RAW 264.7 cells through mechanisms probably associated with the inhibition of TLR4-NF-κB signaling pathway.
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