贝壳杉烷型二萜类化合物PV006对THP-1源性巨噬细胞M1型极化的调节作用

Effects of Kaponane Diterpenoids Compound PV006 on M1 Polarization of THP-1-derived Macrophages

目的利用体外诱导巨噬细胞向M1型极化的模式,初探贝壳杉烷型二萜类化合物PV006对M1型极化的影响。方法用佛波酯(PMA)联合脂多糖(LPS)及重组人干扰素-γ(rh IFN-γ)刺激人单核细胞株THP-1细胞,促使其向M1型巨噬细胞分化。通过显微镜观察细胞形态学变化,qRT-PCR法检测诱导后的细胞内IL-1β、TNF-α、IL-6以及IL-8 mRNA表达水平,Western blot法检测核转录因子-κB磷酸化P65蛋白(NF-κB p-P65)和磷酸化信号转导与转录激活物1(p-STAT1)蛋白的表达,确认THP-1细胞向M1型极化,同时检测PV006处理后对上述指标的影响。结果经PMA联合LPS及rh IFN-γ诱导后,THP-1细胞形态由圆形转变为特征的长梭形或纺锤形,IL-1β、TNF-α、IL-6、IL-8 mRNA表达升高,NF-κB p-P65和p-STAT1蛋白表达增强。不同浓度PV006同步处理后,作M1型极化诱导的THP-1细胞未出现特征性的长梭形或纺锤形,大部分细胞仍为圆形。IL-1β、TNF-α、IL-6、IL-8 mRNA表达水平下调(P<0.05),NF-κB p-P65和p-STAT1蛋白表达减弱。结论 PMA联合LPS及rh IFN-γ可诱导THP-1源性巨噬细胞向M1型极化,PV006有抑制巨噬细胞向M1型分化的作用。

Objective To study the effects of kaurane diterpenoids compound PV006 on M1 polarization of macrophages by inducing M1 type differentiation of macrophages in vitro.Methods The human monocyte cell line THP-1 cells were stimulated to differentiate into M1-type macrophages by phorbol 12-myristate13-acetate（PMA） combined with lipopolysaccharide（LPS） and recombinant human interferon-γ（rh IFN-γ）.M1-type polarization of THP-1-derived macrophages was confirmed by observing morphological changes of cells with microscope,detecting the mRNA expression of IL-1β,TNF-α,IL-6 and IL-8 by real-time fluorescence-based quantitative PCR（qRT-PCR）,as well as analyzing expressions of nuclear transcription factor κB phospho-P65（NF-κB p-P65） and phospho signal transducer and activator of transcription-1（p-STAT1） by western blotting.After synchronously treatment with PV006,above parameters of stimulated cells were detected.Results After THP-1 cells were induced by PMA combined with LPS and rh IFN-γ,the shape of cells changed from round to characteristic long fusiform or spindle shape,the expression of IL-1β,TNF-α,IL-6,IL-8 mRNA increased,the expression of NF-κB p-P65 and p-STAT1 protiens enhanced.After treatment with different concentrations of PV006,long fusiform or spindle shape cells did not be observed and most cells remains round in M1 polarization stimulated THP-1 cells.PV006 down-regulated the expression of IL-1β,TNF-α,IL-6,IL-8 mRNA（P 0.05）,and attenuated the expression of NF-κB p-P65,p-STAT1 proteins.Conclusion M1-type polarization of macrophages derived from THP-1 cells can be induced by stimulation of PMA combined with LPS and rh IFN-γ.PV006 compound could inhibit the differentiation of macrophages into M1 subtype.