Wang Zhi-Cheng, Wang Jian-Feng, Li Yan-Bo, Guo Cai-Xia, Liu Yang, Fang Fang, Gong Shou-Liang
Key Laboratory of Radiobiology of Ministry of Health, School of Public Health, Jilin University, Changchun, 130021, China.
School of Public Health and Family Medicine, Capital Medical University, Beijing, 100069, China.
J Huazhong Univ Sci Technolog Med Sci. 2013 Aug;33(4):551-558. doi: 10.1007/s11596-013-1157-0. Epub 2013 Aug 1.
The study examined the role of endoplasmic reticulum stress (ERS) and signaling pathways of inositol-requiring enzyme-1 (IRE1), RNA-activated protein kinase-like ER kinase (PERK) and activating transcription factor-6 (ATF6) in apoptosis of mouse testicular cells treated with low-dose radiation (LDR). In the dose-dependent experiment, the mice were treated with whole-body X-ray irradiation at different doses (25, 50, 75, 100 or 200 mGy) and sacrificed 12 h later. In the time-dependent experiment, the mice were exposed to 75 mGy X-ray irradiation and killed at different time points (3, 6, 12, 18 or 24 h). Testicular cells were harvested for experiments. H(2)O(2) and NO concentrations, and Ca(2+)-ATPase activity were detected by biochemical assays, the calcium ion concentration ([Ca(2+)]i) by flow cytometry using fluo-3 probe, and GRP78 mRNA and protein expressions by quantitative real-time RT-PCR (qRT-PCR) and Western blotting, respectively. The mRNA expressions of S-XBP1, JNK, caspase-12 and CHOP were measured by qRT-PCR, and the protein expressions of IRE1α, S-XBP1, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP by Western blotting. The results showed that the concentrations of H2O2 and NO, the mRNA expressions of GRP78, S-XBP1, JNK, caspase-12 and CHOP, and the protein expressions of GRP78, S-XBP1, IRE1α, p-PERK, p-eIF2α, ATF6 p50, p-JNK, pro-caspase-12, cleaved caspase-12 and CHOP were significantly increased in a time- and dose-dependent manner after LDR. But the [Ca(2+)]i and Ca(2+)-ATPase activities were significantly decreased in a time- and dose-dependent manner. It was concluded that the ERS, regulated by IRE1, PERK and ATF6 pathways, is involved in the apoptosis of testicular cells in LDR mice, which is associated with ERS-apoptotic signaling molecules of JNK, caspase-12 and CHOP.
本研究探讨了内质网应激（ERS）以及肌醇需求酶1（IRE1）、RNA激活蛋白激酶样内质网激酶（PERK）和激活转录因子6（ATF6）信号通路在低剂量辐射（LDR）处理的小鼠睾丸细胞凋亡中的作用。在剂量依赖性实验中，对小鼠进行不同剂量（25、50、75、100或200 mGy）的全身X射线照射，并在12小时后处死。在时间依赖性实验中，将小鼠暴露于75 mGy的X射线照射下，并在不同时间点（3、6、12、18或24小时）处死。收集睾丸细胞进行实验。通过生化分析检测H(2)O(2)和NO浓度以及Ca(2+)-ATP酶活性，使用fluo-3探针通过流式细胞术检测钙离子浓度（[Ca(2+)]i），分别通过定量实时RT-PCR（qRT-PCR）和蛋白质免疫印迹法检测GRP78 mRNA和蛋白质表达。通过qRT-PCR测量S-XBP1、JNK、caspase-12和CHOP的mRNA表达，通过蛋白质免疫印迹法检测IRE1α、S-XBP1、p-PERK、p-eIF2α、ATF6 p50、p-JNK、前体caspase-12、裂解的caspase-12和CHOP的蛋白质表达。结果表明，LDR后，H2O2和NO浓度、GRP78、S-XBP1、JNK、caspase-12和CHOP的mRNA表达以及GRP78、S-XBP1、IRE1α、p-PERK、p-eIF2α、ATF6 p50、p-JNK、前体caspase-12、裂解的caspase-12和CHOP的蛋白质表达均呈时间和剂量依赖性显著增加。但[Ca(2+)]i和Ca(2+)-ATP酶活性呈时间和剂量依赖性显著降低。得出结论，由IRE1、PERK和ATF6通路调节的ERS参与LDR小鼠睾丸细胞的凋亡，这与ERS凋亡信号分子JNK、caspase-12和CHOP有关。
