PPARγ激动剂对RSV感染的A549细胞MCP-1、MIP-1α、IL-8表达的作用及机制

Effect and Mechanism of PPARγ Agonists on the Expression of MCP-1,MIP-1α and IL-8 in A549 Cells Infected with Respiratory Syncytial Virus

目的研究A549细胞呼吸道合胞病毒(RSV)感染不同时间后单核细胞趋化蛋白-1(MCP-1)、巨噬细胞炎性蛋白-1α(MIP-1α)、白介素-8(IL-8)在mRNA和蛋白水平的变化,进一步观察15-脱氧前列腺素J2(15d-PGJ2)和罗格列酮干预后MCP-1、MIP-1α、IL-8 mRNA表达和蛋白水平的变化,探讨PPARγ激动剂对RSV感染趋化因子表达的影响及机制。方法建立RSV感染的细胞模型,将传代培养的细胞随机分成6组:A组(15d-PGJ2+RSV组)、B组(罗格列酮+RSV组)、C组(RSV组)、D组(PDTC+RSV组)、E组(PPARγ拮抗剂GW9662+罗格列酮+RSV组)、F组(细胞对照组),各组分别在培养12、24、48h收获细胞及上清液待测。应用ELISA检测MCP-1、MIP-1α、IL-8蛋白水平,实时定量RT-PCR检测MCP-1、MIP-1α、IL-8 mRNA表达。结果 C组与F组相比,MCP-1、MIP-1α、IL-8 mRNA及蛋白的表达均在12h开始升高,其中mRNA表达在24h达高峰,48h有所下降,与12h比较差异无统计学意义(P均>0.05);而蛋白表达在48h达高峰,与12h比较差异有统计学意义(P均<0.05);D组各时间点3种趋化因子蛋白和mRNA表达均明显低于C组(P均<0.05),但仍高于F组(P均<0.05)。A组、B组与C组相比,各时间点MCP-1、MIP-1α、IL-8蛋白及mRNA表达均明显降低(P均<0.01);E组与C组3种趋化因子蛋白及mRNA表达的差异无统计学意义,但仍高于F组(P均<0.05)。结论 RSV感染可导致MCP-1、MIP-1α、IL-8 mRNA及蛋白表达升高;15d-PGJ2、罗格列酮均可抑制上述作用,其作用可被GW9662所阻断;PPARγ激动剂的作用机制与抑制NF-κB通路激活有关。

Objective To investigate the expression of monocyte chemotactic protein 1 （ MCP - 1 ） , macrophage inflammatory protein -let（ MIP- let） and interleukin -8 （IL- 8） both at mRNA and protein levels in A549 cells infected with respiratory syneytial virus （RSV） at different point. Furthermore, to evaluate the changed expression of those chemokines when pretreated with 15 -deoxy- del- tal2, 14 prostaglandin J2 （ 15d - PGJ2 ） or rosiglitazone, and to figure out the mechanism of PPARy agonists. Methods RSV inoculation after A549 cells subcultured,then A549 cells were randomly divided into 6 groups ： group A （ 15d - PGJ2 ＋ RSV group） , group B （ rosigli- tazone ＋ RSV group） , group C （ RSV group） , group D （ PDTC ＋ RSV group）, group E （ GW9662 ＋ rosiglitazone ＋ RSV group）, group F （cell control group）. Cells and supernatant were harvested after the cultivation at 12h, 24h and 48h, respectively. ELISA was used to de- tect the protein levels of MCP - 1, MIP - lct and IL - 8 and quantitative real - time PCR was used to test mRNA levels. Results Com- pared with group F, the expression of MCP - 1, MIP - lα and IL -8 both at mRNA and protein levels in group C increased at the begin- ning of 12h, mRNA expression reached the peak at 24h, decreased at 48h and had no significant difference with those at 12h. While, the protein levels reached a peak at 48h and the difference was statistically significant compared with those at 12h （P all 〈 0.05）. At each time points, MCP - 1, MIP - let, IL -8 mRNA and protein levels in group D were lower than those in group C, but were still higher than those in group F （P all 〈 O. 05）. Compared with group C, MCP - 1, MIP - 1 α, IL - 8 mRNA and protein expression were remarkably decreased in group A and group B （P all 〈0.05）. Furthermore, MCP - 1, MIP - lot, IL - 8 mRNA and protein levels, which were higher than group F, indicated no significant difference in group E and group C. Conclusion RSV infections result in the increased ex-pression of MCP- 1, MIP- lα, IL- 8 both at mRNA and protein levels. 15d -PGJ2 and rosiglitazone inhibit this upregu｜atory effect. The effects of PPAR - y agonists can be reversed by GW9662. The mechanism is associated with the inhibition of NF - KB pathway activa- tion.