脂肪因子Omentin-1通过磷脂酰肌醇-3激酶/蛋白激酶B通路抑制泡沫巨噬细胞炎症因子释放及凋亡的研究

Adipokine omentin-1 inhibits inflammatory cytokines secretion and apoptosis in macrophage

目的:探讨脂肪因子Omentin-1对泡沫巨噬细胞炎症因子的释放以及凋亡的影响及机制。方法:THP-1细胞系用佛波酯(PMA)诱导为巨噬细胞后,加入氧化低密度脂蛋白(ox-LDL)构建泡沫巨噬细胞模型。采用不同浓度Omentin-1孵育泡沫巨噬细胞48 h后,利用Westernblot技术测定细胞中凋亡相关蛋白bcl-2、bax、caspase-3表达情况,并利用Annexin V试剂盒测定细胞凋亡率。在泡沫巨噬细胞中加入Omentin-1以及PI3K/AKT通路阻断剂LY294002,利用RT-qPCR技术及ELISA试剂盒测定细胞内及培养液中炎症因子TNF-α与IL-6的表达与分泌情况。在巨噬细胞中加入Omentin-1,分别于加入后不同时间点提取蛋白,之后采用LY294002抑制磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/AKT)通路,并用Westernblot技术测定细胞中蛋白激酶B以及Ser473位点磷酸化蛋白激酶B的表达量。结果:与对照组相比,加入Omentin-1后泡沫巨噬细胞凋亡率显著下降(P<0.05),细胞caspase-3、bax蛋白表达显著下降,bcl-2蛋白表达显著上升(P<0.01),抑制PI3K/AKT通路后,以上效应均显著减弱(P<0.05)。与对照组相比,加入Omentin-1可显著抑制泡沫巨噬细胞炎症因子释放(P<0.05),抑制PI3K/AKT通路后,此效应显著减弱(P<0.05)。与对照组相比,加入Omentin-1后细胞内Ser473位点磷酸化蛋白激酶B/蛋白激酶B比值显著上升(P<0.01)。油红O染色提示Omentin-1可显著减少ox-LDL诱导的泡沫细胞内的脂质含量(P<0.05),抑制PI3K/AKT通路后此效应未受显著影响(P>0.05)。结论:Omentin-1可能通过磷脂酰肌醇-3激酶/蛋白激酶B通路调控泡沫巨噬细胞炎症因子释放与凋亡。

Objective: To investigate how Omentin-1 regulate macrophage-derived foam cell pro-inflammatory cytokine secretion and apoptosis. Methods: We utilized PMA at concentration of 100 ng/mL to induce THP-1 monocytes into macrophages. After 48 hours of incubation, we added 50μg/mL ox-LDL and prolonged the incubation time to another 48 hours in order to establish macrophage-derived foam cell in vitro. THP-1-derived macrophages were treated with Omentin-1 at 800 ng/mL, and the protein was extracted at 15 min, 30 min, and 60 min, respectively. LY294002 was used to block PI3K/AKT signaling pathway, and Western Blot was used to determine the expression levels of AKT and phospho-AKT(ser473) in cells. The cells were also divided into macrophage group, foam cell group, foam cell group with addition of Omentin-1 and foam cell group with addition of Ometin-1 and LY294002. RT-qPCR and ELISA was used to measure the mRNA levels and cytokine secretion levels of TNF-α and IL-6 in each group. After incubation with Omentin-1 at different concentrations for 48 h, foam cells were subjected to Western Blot analysis in order to measure the expression levels of bcl-2, bax and caspase-3. The ratio of apoptotic cells was determined by Annexin V assay kit. Results: Compared with the control group, Omentin-1 ameliorated the expression of apoptosis-related proteins caspase-3(P<0.01) in macrophages through the PI3K/AKT pathway, and inhibited the apoptosis of macrophages(P<0.05). In macrophages induced by ox-LDL and LPS, Omentin-1 inhibited the expression and release of inflammatory cytokines via PI3K/AKT pathway(P<0.05). An increase of expression of phospho-AKT(Ser473) was observed after addition of Omentin-1(P<0.01). Oil red staining revealed that Omentin-1 significantly reduced the content of lipid droplets in macrophages treated with ox-LDL(P<0.05). Conclusions: Omentin-1 inhibits foam cell inflammatory cytokine release and apoptosis via PI3K/AKT pathway.