目的：探讨棕榈酸诱导MC3T3-E1成骨细胞的凋亡是否与核因子（ NF）-κB的激活有关。方法观察不同浓度（0～1000μmol／L）棕榈酸孵育MC3T3-E1成骨细胞24 h后对细胞活力和细胞凋亡的影响。细胞活力用四甲基偶氮唑盐法（ MTT法）检测，细胞凋亡用Hochest 33258染核和Western印迹法检测活化型（ cleaved ） caspase-3蛋白验证。用500μmol／L棕榈酸分别孵育细胞0～240 min，Western印迹法检测IκBα、p-NF-κB p65蛋白表达。结果棕榈酸呈时间和浓度依赖的方式抑制MC3T3-E1细胞活性和促进细胞凋亡。500μmol／L棕榈酸在24 h时细胞活力仅为对照组的54％，cleaved caspase-3蛋白表达则较对照组高3.1倍。棕榈酸诱导p-NF-κB p65在60 min开始升高，120 min时达到高峰增加了2.96倍（ P＜0.05）；IκBα表达水平逐渐下降，以120 min最为明显，下降57％（P＜0.05）。10和20μmol／L浓度的NF-κB抑制剂吡咯烷二硫代氨基甲酸盐（PDTC）均能明显抑制棕榈酸诱导的p-NF-κB p65蛋白（1.39±0.12、1.25±0.10比1.76±0.14，均P＜0.05）；PDTC将cleaved caspase-3和caspase-3蛋白逐渐恢复至接近对照组水平（2.24±0.28比1.29±0.27，0.63±0.01比1.13±0.10，均P＜0.05）。结论棕榈酸诱导的MC3T3-E1成骨细胞凋亡可能通过激活NF-κB转导途径起作用。%Objective To explore whether palmitate-induced apoptosis of osteoblastic MC 3T3-E1 cell is mediated by an activation of nuclear factor-kappa B ( NF-κB).Methods Cell viability was assessed with methyl thiazolyl tetrazolium ( MTT) assay and cell apoptosis by Hochest 33258 staining.Palmitate was added at different timepoints and dosages.Western blot was used to evaluate the expression levels of IκBα, p-NF-κB p65 and NF-κB p65 protein.Results Palmitate led to a dose-and time-dependent decreases in cell viability and increase in cell apoptosis.Cell viability dropped to 54% and cleaved caspase-3 increased 3.1-fold in cells treated with 500 μmol/L palmitate compared to control.The level of p-NF-κB p65 protein markedly increased at 60 min post-stimulation and reached a 2.96-fold increase of baseline level at 120 min ( P<0.05).The IκBαlevel markedly declined at 60 min post-stimulation and decreased by 57%at 120 min ( P <0.05 ).Compared to the group with palmitate treatment alone , pyrrolidinedithiocarbamic acid (10/20 μmol/L) significantly inhibited the palmitate-induced increase of p-NF-κB p65 ( 1.39 ±0.12, 1.25 ±0.10 vs 1.76 ±0.14, both P<0.05), restored the palmitate-induced decrease of caspase-3(2.24 ± 0.28 vs 1.29 ±0.27, P<0.05)and inhibited the palmitate-induced increase of cleaved caspase-3(0.63 ± 0.01 vs 1.13 ±0.10, P <0.05).Conclusion Palmitate induces apoptosis of MC3T3-E1 cell by an activation of NF-κB.
展开▼
