粉防己碱对脂多糖诱导下RAW264.7细胞炎症模型细胞因子的作用

Effect of tetrandrine on cytokines in LPS-stimulated RAW264.7 cell

目的观察粉防己碱(tetrandrine,Tet)对脂多糖(LPS)诱导RAW264.7细胞炎症模型促炎细胞因子和抗炎细胞因子的影响。方法 LPS(1μg/mL)刺激生长良好的RAW264.7细胞,建立细胞炎症模型。四甲基偶氮唑盐(MTT)法检测不同浓度Tet对RAW264.7细胞增殖的影响,激光共聚焦显微镜观察Tet对细胞核因子-κB(NF-κB)核转运的作用,酶联免疫吸附试验(ELISA)检测细胞上清中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、白细胞介素-10(IL-10)的变化。结果当Tet浓度<1μmol/L时,单独或与1μg/mL LPS共培养,均对RAW264.7细胞生长无影响;而当Tet浓度>10μmol/L时,单独或与LPS共培养均表现出明显的细胞生长抑制效应;Tet可使IL-6、TNF-α的释放受到抑制,相反可促进IL-10的表达。激光共聚焦显微镜观察Tet大剂量组细胞核红染的阳性细胞显著减少,以阴性细胞(红染p65亚基主要集中在胞浆部位,使胞浆染色较深,胞核染色较浅,整个细胞成空泡状)为主。结论 Tet可以抑制LPS所致的RAW264.7细胞炎症反应,其抗炎作用与抑制NF-κB活化,进一步减少炎性细胞因子IL-6、TNF-α的产生,并促进抗炎细胞因子IL-10的表达等有关。

Objective To investigate the effects of tetrandrine（Tet） on expressions of proinflammatory and anti-inflammatory cytokine in lipopolysaccharide（LPS）-stimulated RAW264.7 cells.Methods RAW 264.7 cells were cultured in media containing 1 μg/mL LPS and different doses of Tet.The proliferation of cells was examined by MTT assay,and nuclear translocation of NF-κB was detected and analyzed by the laser scanning confocal microscope（LSCM）,and the levels of IL-6,TNF-α,and IL-10 were tested by ELISA.Results Compared with the control group,Tet had no influence on the proliferation of RAW264.7 cells no matter with LPS or not when its concentration lower than 1 μmol/L;But when its concentration higher than 10 μmol/L,Tet significantly inhibited the growth of the cells whether LPS existed or not.Meanwhile,Tet significantly decreased the levels of IL-6,and TNF-α induced by LPS,and upregulated IL-10 in supernatant.At the mean time,results from LSCM showed that in Tet 1 μmol/L group,positive cells（dyeing-red p65 of NF-κB was translocated into nucleuses and made the nuclueses red） decreased siglificantly,while negative cells（dyeing-red p65 of NF-κB was collectted in endochylema and nucleuses became vacant） were predominantly founded.Conclusion Tet has anti-inflammatory effect,which might be mediated by downregulating inflammatory factors IL-6,TNF-α through NF-κB signal pathway as well as increasing levels of anti-inflammatory factor IL-10