核转录因子-κB对过氧化物酶增殖体激活受体γ调控作用的体外研究

Effect of nuclear factor-κB on peroxisome proliferator activated receptor γ expression in Ana-1 cells

目的观察脂多糖（LPS）作用下小鼠巨噬细胞（Ana-1）过氧化物酶增殖体激活受体γ（PPAR-γ）的表达，并通过调控核转录因子-κB（NF-κB）表达观察PPAR7相应的变化情况。方法将体外培养的Ana-1细胞按随机数字表法分为对照组，0．1mg／LLPS作用1、2、4、8h组，50mg／LSN50作用4h组（SNS0组），NF-κB高表达质粒转染组。用逆转录-聚合酶链反应（RT—PCR）检测PPARγ、肿瘤坏死因子-α（TNF-α）的mRNA表达，用酶联免疫吸附法（EuSA）检测TNF-α蛋白表达。构建NF-κB高表达质粒，利用脂质体转染巨噬细胞并通过G418筛选出稳定表达株，用蛋白质免疫印迹法（Western blotting）检测LPS、SN50作用后及质粒转染后细胞核内PPARY表达和NF-κBp65亚基核移位情况。结果在致炎因子LPS作用下，PPARγ的mRNA和蛋白表达均下降，这一变化伴随p65核移位增加和炎症因子TNF-α的升高（P均〈0．05）。成功构建了NF-κBp65亚基高表达质粒并将其转染到巨噬细胞中实现了p65高表达。LPS刺激和NF-κBp65亚基高表达质粒转染Ana-1细胞造成p65表达升高时，核内PPAR7表达减少（r=-0．913，P〈O．05）；而使用NF-κB抑制剂SN50作用Ana-1细胞抑制p65表达后，核内PPARY表达升高（r=-0．959，P〈0．05），二者具有良好的负相关性。结论在LPS作用下，Ana-1细胞内PPARγ表达降低，而这一变化与NF—xB活化相关，调节NF-κBp65亚基表达，PPARγ会向p65改变的相反方向变化。

Objective To determine the effects of nuclear factor--κB （NF-κB） on peroxisome proliferator activated receptor γ （PPARγ） expression in the murine macrophage cell line Ana-1, based on the investigation of the PPARγ expression stimulated by lipopolysaecharide （LPS）. Methods Ana-1 cells were divided randomly into seven groups ： control group, LPS groups （cells were activated by 0. 1 mg/L LPS for 1, 2, 4, 8 hours respectively）, SN50 group （cells were stimulated by 50 mg/L SN50 for 4 hours） and NF-κB high expression plasmid transfected group. PPAR7 and tumor necrosis factor-α （TNF-α） mRNA levels were assayed by reverse transcription-polymerase chain reaction （RT-PCR） and the TNF-α protein was measured by enzyme linked immunosorbent assay （ELISA） after being activated by LPS. The eukaryotie expression vector of murine NF-κB p65 gene was constructed and stably transfected into Ana-1 cells with DOTAP liposome. The PPAR7 expression and NF-κB p65 translocation as stimulated by LPS and SN50 were assayed by Western blotting. Results Expressions of both PPARγ mRNA and protein were downregulated when cells were stimulated by LPS, which were accompanied with the activity of NF-κB and TNF-α in Ana-1 cells （all P〈0.05）. The eukaryotic expression vectors containing murine p65 gene were successfully constructed and stably transfected into Ana-1 ceils. LPS stimulation and NF-κB p65 gene overexpression resulting in upregulation of p65 could downregulate PPARγ expression in Ana-1 cells （r=- 0. 913, P〈0.05）. But downregulation of NF-κB by SN50 could upregulate PPAR7 expression in the nucleus （r =- 0. 959, P〈0.05）. Conclusion LPS can markedly decrease the expression of PPARγin Ana-1 cells, which may be related to its activity of enhancing NF-κB. NF-κB p65 gene can control PPARγ expression in the reverse direction in Ana-1 cells.