腹膜间皮细胞过氧化物酶体增殖物活化受体γ的表达及其配体对CD40和细胞间黏附分子1的影响

Peroxisome proliferator-activated receptor γ(PPAR-γ) expresses in rat peritoneal mesothelial cells and PPAR-γ ligands down-regulate LPS-induced CD40 and ICAM-1 production

目的 观察过氧化物酶体增殖物活化受体γ(PPAR-γ)在大鼠腹膜间皮细胞 (RPMCs)中的表达以及脂多糖(LPS)的调节作用,并探讨PPAR-γ天然配体15d-PGJ2及人工合 成配体cightazone对RPMCs表达CD40和ICAM-1的影响。方法 分离及培养RPMCs,常规传代 及鉴定,取第2代细胞用于实验研究。LPS不同浓度(0．1、1．0、10、50及100μg／ml)、LPS(1 μg／ml)处理后不同时间点及15d-PGJ2(3 μmol／L)、ciglitazone(10μmol／L)作用细胞36h后收 集细胞。RT-PCR检测PPAR-γ、CD40以及ICAM-1 mRNA表达。免疫细胞化学检测PPAR-γ在 RPMCs中的分布。Western印迹检测PPAR-γ及ICAM-1蛋白表达。结果 (1)常规培养的RPMCs 表达一定量PPAR-γ,表达部位主要分布于RPMCs细胞核内,在细胞浆微弱表达。(2)随着LPS 浓度逐渐增大,PPAR-γ蛋白表达水平呈逐渐增高的趋势,LPS浓度为10μg／ml时其表达为最 高峰。LPS(1μg／ml)作用12 h时PPAR-γ蛋白表达最强,PPAR-γ1表达高于PPAR-γ2;之后显 著降低,持续至72 h。(3)LPS刺激后RPMCs CD40mRNA表达显著增强;15d-PGJ2、ciglitazone显著 降低CD40 mRNA表达(P均<0．01)。(4)LPS刺激后RPMCs ICAM-1蛋白表达显著增加;15d-PGJ2 增加LPS介导的ICAM-1 mRNA表达(P<0．01),但显著抑制ICAM-1蛋白表达(P<0．05)。 ciglitazone对LPS介导的ICAM-1 mRNA和蛋白表达均有显著抑制作用(P均<0．05)。结论 RPMCs结构性表达PPAR-γ。LPS调节PPAR-γ表达呈现先增强后抑制趋势。PPAR-γ配体可显著 抑制LPS介导的CD40 mRNA和ICAM-1蛋白的表达。提示RPMCs功能性表达PPAR-γ,可能通 过负性调节炎症介质分泌而参与腹腔局部防御。

Objective To detect the expression of PPAR-γ in rat peritoneal mesothelial cells （RPMCs） and to investigate the effect of PPAR-γ hgands, ciglitazone and 15-deoxy-△^12,14-prostaglandin J2 （15d-PGJ2） on the production of CD40 and ICAM-1 induced by lipopolysaccharide （LPS）. Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro conditions. The cells were exposed respectively to different concentrations of LPS （0.1, 1.0,10,50 and 100 μg/ml） for 36 h or treated with LPS （1 μg/ml） from 0 h to 72 h. PPAR-γ mRNA expression was detected by reverse transcription-polymerase chain reaction （RT-PCR）. The expression of PPAR-γ at the protein level and their intraeellular distribution was detected by Western blot and immunocytoehemistry respectively. Expression of ICAM-1 and CD40 on RPMCs under normal culture or stimulation respectively with LPS（ 1 μg/ml）, LPS＋15d-PGJ2 （ 3 μmol/L）, LPS＋ciglitazone （10 μmol/L） and LPS＋vehiele （DMSO 2 μl） was examined by RT-PCR and Western blot respectively. Results A single band with a predicted size （339 bp） was detected in RT-PCR products from RPMCs, which demonstrates PPAR-γ mRNA expression. Cultured RPMCs were further immunostained to confirm that specific signals of PPAR-γ were mainly localized in the nucleus with a weak staining in the cytoplasm. Stimulation of RPMCs with LPS resulted in a concentration-dependent increase in the protein level of PPAR-γ, with the peak at 10μg/ml. Treatment with 1 μg/ml LPS resulted in a time-dependent increase in the protein level of PPAR-γ, with the peak at 12 h. However, after that time point, the protein level of PPAR-γ was strongly attenuated by LPS. The expression of ICAM-1 mRNA and protein was upregulated significantly following stimulation with LPS （1 μg/ml）. Both 15d-PGJ2 and eiglitazone significantly decreased the expression of ICAM-1 protein, but not mRNA. LPS increased the expression of CD40 mRNA significantly. Both 15d-PGJ2 and eiglitazone decreased the expression of CD40 mRNA markedly. Conclusions There is constitutive expression of PPAR-γ in cultured RPMCs and LPS can be a factor that regulates the expression of PPAR-γ. PPAR-γ ligands can strongly inhibit LPS-indueed CD40 and ICAM-1 production in RPMCs. PPAR-γ may take part in the local defense of peritoneal cavity by down-regulating inflammatory mediators.