目的 評價湖北金粟蘭中倍半萜二聚體化合物（+）-chlorahupetenes B[（+）-CHB]對脂多糖（LPS）誘導的小鼠巨噬細胞（RAW264.7）炎癥反應的影響及作用機制。方法 RAW264.7細胞分為對照組（給予等體積DMSO）、模型組（給予等體積DMSO）、地塞米松磷酸鈉注射液（Dex，陽性對照，1 μmol·L^-1）組和（+）-CHB低、中、高濃度（5、10、20 μmol·L^-1）組。各組分別加入相應藥物孵育細胞1 h，除對照組外，其余組加入LPS （1 μg·mL^-1）誘導24 h造成炎癥應答模型。細胞增殖檢測法用于評估細胞活力；Griess反應檢測一氧化氮（NO）濃度；酶聯(lián)免疫分析檢測炎癥細胞因子腫瘤壞死因子α（TNF-α）、白細胞介素（IL）-1β、IL-6的生成；實時熒光定量PCR （qRT-PCR）檢測IL-1β、NOD樣受體熱蛋白結(jié)構(gòu)域相關(guān)蛋白3（NLRP3）、環(huán)氧合酶-2（COX-2）、誘導型一氧化氮合酶（iNOS）、IL-6和TNF-α mRNA表達；Western blotting檢測Toll樣受體4（TLR4）、髓樣分化因子88（MyD88）、核因子κB （NF-κB） p65、p-NF-κB p65、NLRP3、嘌呤能受體（P2X7）蛋白表達水平。結(jié)果 與模型組比較，（+）-CHB減少了梭形細胞數(shù)量，使大部分細胞恢復正常形態(tài)；顯著抑制NO、TNF-α、IL-6、IL-1β生成（P<0.01），顯著降低COX-2、iNOS、IL-6、TNF-α、NLRP3、IL-1β mRNA水平（P<0.05、0.01）；顯著降低TLR4、MyD88、NF-κB p65、p-NF-κBp65、NLRP3、P2X7蛋白表達水平（P<0.05、0.01）。結(jié)論 （+）-CHB通過抑制TLR4/MyD88/NF-κB信號通路和P2X7/NLRP3/IL-1β炎癥小體軸激活緩解LPS誘導的巨噬細胞炎癥反應。

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.

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國家自然科學基金青年資助項目（82204720）；中央高校基本科研業(yè)務(wù)費資助項目（3332022085）