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目的:探讨低剂量电离辐射与小鼠睾丸细胞内质网应激的发生以及PERK-CHOP通路激活的相关性。方法:健康雄性昆明小鼠随机分成时程-效应(75 mGy照射后0、3、6、12和24 h)和剂量-效应(0、50、75、100和200 mGy照射后12 h)组,每组动物10只。采用H2O2和MDA试剂盒比色法检测其含量;利用实时定量逆转录PCR(quantitative RT-PCR)检测GRP78、PERK和CHOP mRNA;Western印迹和图像分析技术检测GRP78、PERK、磷酸化PERK(phosphorylated PERK,pho-PERK)和CHOP蛋白表达。结果:小鼠经75 mGy全身照射后,睾丸组织中H2O2含量随时间延长而增加,MDA含量在3和6 h稍有降低,而后随时间延长而增加,二者在12和24 h较0 h时显著增加(P<0.05,P<0.01);除了GRP78 mRNA(3和24 h)和蛋白表达(6 h)分别在照射后有降低趋势外,GRP78(12 h)、PERK(3、6、12和24 h)和CHOP(12和24 h)的mRNA表达较0 h显著增加(P<0.05,P<0.01),GRP78(12和24 h)、pho-PERK(3、12和24 h)和CHOP(3、6、12和24 h)的蛋白表达也都较0 h显著增加(P<0.05,P<0.01),PERK蛋白表达则无明显变化规律。小鼠经50～200 mGy全身照射后12 h,睾丸组织中H2O2含量在50～100 mGy照射后随剂量增加而增加,200 mGy照射后则稍有降低,MDA含量随剂量增加而增加,而且H2O2含量(75和100 mGy)和MDA含量(75、100和200 mGy)显著高于0 mGy组(P<0.05,P<0.01);除了GRP78mRNA表达在50和200 mGy照射后有降低趋势外,GRP78(75和100 mGy)、PERK(75、100和200 mGy)和CHOP(50、75、100和200 mGy)的mRNA表达都显著高于0 mGy组(P<0.05,P<0.01),GRP78(100和200 mGy)、pho-PERK(50、100和200 mGy)和CHOP(50、75、100和200 mGy)的蛋白表达也都显著高于0 mGy组(P<0.05,P<0.01),而PERK蛋白表达则无明显变化规律。结论:低剂量电离辐射能够诱导小鼠睾丸细胞发生内质网应激,并且激活PERK-CHOP信号通路。
Objective: To investigate the relationship between low dose ionizing radiation and the occurrence of endoplasmic reticulum stress and the activation of PERK-CHOP pathway in mouse testicular cells. Methods: Healthy male Kunming mice were randomly divided into time-effect group (0, 3, 6, 12 and 24 h after 75 mGy irradiation) and dose-effect , 10 animals per group. The contents of GRP78, PERK and CHOP mRNA were detected by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). The expressions of GRP78, PERK, phosphorylated PERK , pho-PERK) and CHOP protein expression. Results: After 75 mGy whole body irradiation, the content of H2O2 in testis tissue increased with time, the content of MDA decreased slightly at 3 and 6 h and then increased with the increase of time, both at 0 and 24 h (P <0.05, P <0.01). Except GRP78 mRNA (3 h and 24 h) and protein expression (6 h) after irradiation, GRP78 (12 h), PERK (P <0.05, P <0.01), GRP78 (12 and 24 h), pho-PERK (3, 12 and 24 h) And CHOP (3, 6, 12 and 24 h) also increased significantly compared with 0 h (P <0.05, P <0.01). There was no obvious change in PERK protein expression. After mice were exposed to 50 ~ 200 mGy whole body for 12 h, the content of H2O2 in testis increased with the increase of dose after 50 ~ 100 mGy irradiation, but slightly decreased after 200 mGy irradiation. The content of MDA increased with the increase of dose, (75 and 100 mGy) and MDA (75, 100 and 200 mGy) were significantly higher than those in 0 mGy group (P <0.05, P <0.01). In addition to the decreasing trend of GRP78 mRNA expression after 50 and 200 mGy irradiation, GRP78 (75 and 100 mGy), PERK (75,100 and 200 mGy) and CHOP (50,75,100 and 200 mGy) were significantly higher than those in 0 mGy group (P <0.05, P <0.01) 100 and 200 mGy), protein expressions of pho-PERK (50, 100 and 200 mGy) and CHOP (50, 75, 100 and 200 mGy) were also significantly higher than those of 0 mGy group (P < However, PERK protein expression did not change significantly. Conclusion: Low-dose ionizing radiation can induce endoplasmic reticulum stress in mouse testicular cells and activate PERK-CHOP signaling pathway.