Poly Ⅰ:C刺激对裸鼹鼠和小鼠巨噬细胞PKR/eIF2α信号通路影响的比较研究

Comparative Study of Effect of Poly Ⅰ:C on PKR/eIF2α Signal Path in Naked Mole-rats and Mouse Macrophages

目的比较研究聚肌胞苷酸(Poly Ⅰ:C)对裸鼹鼠和小鼠巨噬细胞双链RNA依赖性蛋白激酶R(PKR)/真核细胞翻译起始因子2α(EIF2α)信号通路的影响。方法分别提取裸鼹鼠和C57BL/6J小鼠巨噬细胞,随机分成对照组、Poly Ⅰ:C给药组、2-氨基嘌呤(2-AP)给药组、Poly Ⅰ:C+2-AP给药组。给药后,蛋白印迹检测细胞中磷酸化PKR(Pho-PKR)、PKR、磷酸化EIF2α(Pho-EIF2α)、EIF2α、Caspase-8蛋白的表达情况。结果 Poly Ⅰ:C给药后,小鼠巨噬细胞的PKR和e IF2α磷酸化水平显著降低(P<0.01),而裸鼹鼠相反则显著升高(P<0.01),表明Poly Ⅰ:C能抑制小鼠的PKR活性,而裸鼹鼠的PKR活性被显著激活;Poly Ⅰ:C干预的基础上通过2-AP抑制PKR活性,结果显示,2-AP给药后下调了小鼠和裸鼹鼠PKR和磷酸化PKR的表达(P<0.01),尤其是裸鼹鼠的PKR,2-AP给药后显著地抑制裸鼹鼠PKR的表达;单独2-AP给药后小鼠巨噬细胞的活性PKR和活性eIF2α比例显著下降(P<0.01),裸鼹鼠巨噬细胞的活性PKR比例也显著下降(P<0.01);通过2-AP抑制PKR活性后,裸鼹鼠细胞的Caspase-8蛋白表达水平显著下降,但小鼠细胞的表达却无显著差异。结论与小鼠比较,裸鼹鼠细胞对Poly Ⅰ:C刺激具有较高的抗性;细胞的PKR对2-AP的敏感性更强;PKR活性对Caspase-8表达有促进作用。

Objective To compare the affect of polyinosinic-polycytidylic acid(Poly Ⅰ:C) on double-stranded RNA-dependent protein kinase R(PKR)/eukaryotic initiation factor-2α(EIF2α)signaling pathway in macrophages of naked mole-rats and mouse. Methods The naked mole-rat and C57 BL/6 J mice which physiological age-matched were randomly divided into control group, Poly Ⅰ:C administration group, 2,6-Diaminopurine(2-AP) administration group and Poly Ⅰ:C+2-AP administration group. After administration, the expression of phosphorylated PKR(Pho-PKR), PKR, phosphorylated EIF2α(Pho-EIF2α), EIF2α and Caspase-8 protein in the cells were detected by Western blotting. Results After Poly Ⅰ:C administration, the phosphorylation levels of PKR and eIF2α in mice macrophages were significantly decreased(P<0.01), while those in naked mole-rat were significantly increased(P<0.01).This phenomelon indicated that Poly Ⅰ:C could inhibit PKR activity in mice,but activite PKR in naked mole-rat;Poly Ⅰ:C and 2-AP intervention group showed that PKR and phosphorylated PKR were down-regulated both in mice and naked mole-rats(P<0.01), especially PKR in naked mole-rats. Active PKR and active eIF2α was significantly decreased(P<0.01) in mouse macrophages, after 2-AP administration. Active PKR in naked mole-rat was significantly decreased(P<0.01) too. After inhibition of PKR activity by 2-AP, Caspase-8 expression levels in naked mole-rat were significantly reduced(P<0.01), but there were no significant differences in mouse cells.Conclusion Compared to mouse, naked mole-rat’s cells were highly resistant to Poly Ⅰ:C stimulation,PKR in naked mole-rats was more sensitive to 2-AP, PKR activity in naked-mole rat cells promoted the expression of Caspase-8.