湖北金粟兰中倍半萜二聚体化合物（+）-chlorahupetenes B对脂多糖诱导的RAW264.7细胞的抗炎作用

目的 评价湖北金粟兰中倍半萜二聚体化合物（+）-chlorahupetenes B[（+）-CHB]对脂多糖（LPS）诱导的小鼠巨噬细胞（RAW264.7）炎症反应的影响及作用机制。方法 RAW264.7细胞分为对照组（给予等体积DMSO）、模型组（给予等体积DMSO）、地塞米松磷酸钠注射液（Dex，阳性对照，1 μmol·L^-1）组和（+）-CHB低、中、高浓度（5、10、20 μmol·L^-1）组。各组分别加入相应药物孵育细胞1 h，除对照组外，其余组加入LPS （1 μg·mL^-1）诱导24 h造成炎症应答模型。细胞增殖检测法用于评估细胞活力；Griess反应检测一氧化氮（NO）浓度；酶联免疫分析检测炎症细胞因子肿瘤坏死因子α（TNF-α）、白细胞介素（IL）-1β、IL-6的生成；实时荧光定量PCR （qRT-PCR）检测IL-1β、NOD样受体热蛋白结构域相关蛋白3（NLRP3）、环氧合酶-2（COX-2）、诱导型一氧化氮合酶（iNOS）、IL-6和TNF-α mRNA表达；Western blotting检测Toll样受体4（TLR4）、髓样分化因子88（MyD88）、核因子κB （NF-κB） p65、p-NF-κB p65、NLRP3、嘌呤能受体（P2X7）蛋白表达水平。结果 与模型组比较，（+）-CHB减少了梭形细胞数量，使大部分细胞恢复正常形态；显著抑制NO、TNF-α、IL-6、IL-1β生成（P<0.01），显著降低COX-2、iNOS、IL-6、TNF-α、NLRP3、IL-1β mRNA水平（P<0.05、0.01）；显著降低TLR4、MyD88、NF-κB p65、p-NF-κBp65、NLRP3、P2X7蛋白表达水平（P<0.05、0.01）。结论 （+）-CHB通过抑制TLR4/MyD88/NF-κB信号通路和P2X7/NLRP3/IL-1β炎症小体轴激活缓解LPS诱导的巨噬细胞炎症反应。

Anti-inflammatory effect of a sesquiterpene dimer compound (+)- chlorahupetenes B from Chloranthus henryi var. hupehensis (Pamp.) K. F. Wu on lipopolysaccharides-stimulated RAW264.7 Cells

Objective To evaluate the anti-inflammatory activity and mechanism of (+)-chlorahupetenes B, a natural eudesmane-type sesquiterpenoid dimer compound obtained from Chloranthus henryi var. hupehensis, in lipopolysaccharides (LPS)-induced RAW264.7 cell. Methods RAW264.7 cells were divided into control group (given equal volume of DMSO), model group (given equal volume of DMSO), dexamethasone sodium phosphate injection (Dex, positive control, 1 μmol·L^-1) group, and (+)-CHB low, medium, and high concentrations (5, 10, 20 μmol·L^-1) groups. Each group was incubated with corresponding drugs for 1 h. Except for the control group, the other groups were induced with LPS (1 μg·mL^-1) for 24 hours to create an inflammatory response model. Cell proliferation detection method was used to evaluate cell viability. Griess reaction was employed for determining nitric oxide (NO) concentration. Enzyme-linked immunosorbent assay was utilized for detecting inflammatory cytokines including tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6. Real time fluorescence quantitative PCR (qRT PCR) was performed to analyze IL-1β, NOD like receptor heat protein domain associated protein 3 (NLRP3), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), IL-6, and TNF-α mRNA expression. Western blotting detection was used to detect the expression levels of Toll like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor κB (NF-κB) p65, p-NF-κB p65, NLRP3, and purinergic receptor (P2X7) proteins. Results Compared with the model group, (+)-CHB reduced the number of spindle cells and restored most of the cells to their normal morphology, significantly inhibited NO and TNF-α, IL-6, IL-1β generation (P< 0.01), significantly reduced COX-2, iNOS, IL-6, TNF-α, NLRP3, and IL-1β mRNA levels (P< 0.05, 0.01), significantly reduced the expression levels of TLR4, MyD88, NF-κB p65, p-NF-κB p65, NLRP3, and P2X7 proteins (P< 0.05, 0.01). Conclusion These findings suggested that(+)-CHB was an anti-inflammatory compound which could relieve LPS-stimulated macrophage inflammatory response partly by targeting TLR4/MyD88/NF-κB signal pathway and P2X7/NLRP3/IL-1β inflammasome axis.