谷氧还蛋白在牙龈卟啉单胞菌脂多糖诱导脐静脉内皮细胞氧化应激中的作用

Effect of glutaredoxin on oxidative stress of umbilical vein endothelial cell exposed to Porphyromonas gingivalis lipopolysaccharide

目的从基因和蛋白水平研究谷氧还蛋白(Grx)在牙龈卟啉单胞菌脂多糖(LPS)诱导脐静脉内皮细胞(EA-hy926细胞)时的表达变化及其对Akt通路的调控作用。方法采用牙龈卟啉单胞菌的LPS(1 000 ng·m L-1)对EA-hy926细胞进行不同时间段(4、12、18、24 h)的刺激诱导,采用实时荧光定量逆转录聚合酶链反应检测细胞grx1基因的表达变化;然后加入Grx特异性抑制剂氯化亚硝脲(BCNU),使用Western blot法检测对照组、LPS组(1 000 ng·m L^(-1)LPS刺激12 h)和BCNU组(25μmol·m L-1BCNU预处理30 min+1 000 ng·m L^(-1)LPS刺激12 h)的Grx、Akt、磷酸化Akt蛋白的表达情况。结果 LPS诱导下,EA-hy926细胞的grx1基因表达量在各个时间段均上调,12 h时grx1表达量最高。LPS诱导12 h时,LPS组的Grx蛋白表达量较对照组明显升高(P<0.05),而BCNU可有效抑制Grx蛋白的表达(P<0.05);Akt蛋白表达量在各组间无明显差异(P>0.05),而LPS组的磷酸化Akt活性升高,显著高于对照组和BCNU组(P<0.05),与Grx蛋白的表达趋势一致。结论 LPS可以从基因和蛋白水平诱导Grx的表达;Grx是Akt的潜在调节因子,可能对LPS刺激下Akt的调控具有重要意义。

Objective This study measures the glutaredoxin（Grx） gene and protein expression in umbilical vein endothelial cells upon exposure to Porphyromonas gingivalis（P. gingivalis） lipopolysaccharide（LPS）. The involvement of the Aktsignaling pathway is also determined. Methods EA-hy926 cells were pretreated with 1 000 ng·m L-1 P. gingivalis LPS for 4, 12, 18, and 24 h, and then real-time reverse transcription polymerase chain reaction was employed to detect Grx1 expression. The effect of Grx on Akt activity was investigated using Western blot for the control, LPS（1 000 ng·m L-1 LPS）, and carmustine（BCNU） groups（1 000 ng·m L-1 LPS, and the EA-hy926 cells were pretreated with 25 μmol·m L-1 BCNU for 30 min）. Results Gene expression of Grx1 significantly increased in LPS group compared with that in the control group. The Grx1 expression reached the peak level in 12 h, and the variation between the expression in 4 and 12 h was significant（P〈0.05）. After 12 h, the protein levels of Grx and phosphorylated-Akt（p-Akt） significantly increased in the LPS group（P〈0.05）, whereas the BCNU group showed a considerable decrease in both Grx and p-Akt expression levels（P〈0.05）. Moreover, a slight difference was observed in the total Akt protein levels in the three groups（P〉0.05）. Conclusion Grx expression increased upon exposure of EA-hy926 cells to the LPS. Akt activity could be inhibited by BCNU（a Grx inhibitor）, which indicated that Akt might act as a downstream regulator of Grx.