N-乙酰半胱氨酸抑制牙龈卟啉单胞菌脂多糖引起的成骨细胞氧化应激与凋亡

Inhibition of Porphyromonas gingivalis lipopolysaccharide-induced oxidative stress and apoptosis in osteoblasts by N-acetylcysteine

目的探讨牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis,P.g)脂多糖(lipopolysaccharide,LPS)对成骨细胞氧化应激与凋亡的影响及N-乙酰半胱氨酸(N-acetylcysteine,NAC)对其保护作用,为临床运用NAC预防和治疗牙周炎提供理论基础和实验依据。方法CCK-8法检测不同浓度P.g LPS和NAC对小鼠成骨细胞增殖影响,确定最佳刺激浓度和时间。将细胞分为对照组、LPS组、NAC组、LPS+NAC组。流式细胞术检测各组细胞凋亡率,RT-PCR法检测各组Foxo1、Sod2、Bax基因表达水平,荧光显微镜下观察各组细胞内活性氧(reactive oxygen species,ROS)荧光强度,检测各组超氧化物歧化酶(superoxide dismutase,SOD)、丙二醛(malondialdehyde,MDA)含量变化。结果与对照组相比,LPS组成骨细胞内ROS,MDA含量增多,SOD活性下降,细胞凋亡率明显升高,Foxo1、Sod2 mRNA基因表达量降低,Bax基因表达水平上升。与LPS组相比,LPS+NAC能够提升成骨细胞增殖率,细胞凋亡率明显下降,细胞内ROS,MDA含量下降,SOD活性上升,抗氧化应激相关基因Sod2、Foxo1mRNA表达量升高,促凋亡基因Bax表达量下降,统计学上均有显著性差异(P<0.05)。结论NAC能够拮抗P.g LPS诱导的成骨细胞氧化应激与凋亡反应。

Objective To investigate the effect of lipopolysaccharide(LPS)of Porphyromonas gingivalis(P.g)on oxidative stress and apoptosis of osteoblasts and the protective effect of N-acetylcysteine(NAC)on them,and to provide a theoretical basis and experimental basis for the clinical use of NAC in the prevention and treatment of periodontitis.Methods The CCK-8 method was used to detect the effects of different concentrations of P.g LPS and NAC on the proliferation of mouse osteoblasts and to determine the optimal stimulation concentration and time.Cells were divided into control,LPS,NAC and LPS+NAC groups.The apoptosis rate of each group was detected by flow cytometry,the expression levels of Foxol,Sod2 and Bax genes in each group were detected by RT-PCR,the intracellular reactive oxygen species(ROS)fluorescence intensity in each group was observed under fluorescence microscope,and the changes of superoxide dismutase(SOD)and malondialdehyde(MDA)contents in each group were detected.Results Compared with the control group,the LPS group showed increased intracellular ROS,MDA content,decreased SOD activity,significantly higher apoptosis rate,decreased Foxol and Sod2 mRNAgene expression,and increased Bax gene expression level.Compared with LPS group,Compared with LPS group,LPS+NAC can increase the proliferation rate of osteoblasts,significantly decrease the apoptosis rate,decrease the content of ROS and MDA in cells,increase the activity of SOD,increase the expression of anti-oxidative stress-related genes Sod2 and Foxol mRNA,and decrease the expression of pro-apoptotic gene Bax,with statistically significant differences(P<0.05).Conclusion NAC can antagonize Pg LPS-induced oxidative stress and apoptosis response in osteoblasts.