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. 2016 Sep;18(9):867-873.
doi: 10.7499/j.issn.1008-8830.2016.09.016.

[Association between endoplasmic reticulum stress pathway mediated by inositol-requiring kinase 1 and AECII apoptosis in preterm rats induced by hyperoxia]

[Article in Chinese]
Hui-Min Ju  1 , Hong-Yan Lu, Yan-Yu Zhang, Qiu-Xia Wang, Qiang Zhang
Affiliations

[Association between endoplasmic reticulum stress pathway mediated by inositol-requiring kinase 1 and AECII apoptosis in preterm rats induced by hyperoxia]

[Article in Chinese]
Hui-Min Ju et al. Zhongguo Dang Dai Er Ke Za Zhi. 2016 Sep.

Abstract
in English, Chinese

Objective: To study the association between endoplasmic reticulum stress (ERS) pathway mediated by inositol-requiring kinase 1 (IRE1) and the apoptosis of type II alveolar epithelial cells (AECIIs) exposed to hyperoxia.

Methods: The primarily cultured AECIIs from preterm rats were devided into an air group and a hyperoxia group. The model of hyperoxia-induced cell injury was established. The cells were harvested at 24, 48, and 72 hours after hyperoxia exposure. An inverted phase-contrast microscope was used to observe morphological changes of the cells. Annexin V/PI double staining flow cytometry was performed to measure cell apoptosis. RT-PCR and Western blot were used to measure the mRNA and protein expression of glucose-regulated protein 78 (GRP78), IRE1, X-box binding protein-1 (XBP-1), and C/EBP homologous protein (CHOP). An immunofluorescence assay was performed to measure the expression of CHOP.

Results: Over the time of hyperoxia exposure, the hyperoxia group showed irregular spreading and vacuolization of AECIIs. Compared with the air group, the hyperoxia group showed a significantly increased apoptosis rate of AECIIs and significantly increased mRNA and protein expression of GRP78, IRE1, XBP1, and CHOP compared at all time points (P<0.05). The hyperoxia group had significantly greater fluorescence intensity of CHOP than the air group at all time points. In the hyperoxia group, the protein expression of CHOP was positively correlated with the apoptosis rate of AECIIs and the protein expression of IRE1 and XBP1 (r=0.97, 0.85, and 0.88 respectively; P<0.05).

Conclusions: Hyperoxia induces apoptosis of AECIIs possibly through activating the IRE1-XBP1-CHOP pathway.

目的: 探讨需肌醇酶1（IRE1）介导的内质网应激通路与高氧暴露肺泡Ⅱ型上皮细胞（AEC Ⅱ）凋亡的关系。

方法: 原代培养早产大鼠AEC Ⅱ，随机分为空气组和高氧组，建立高氧细胞损伤模型。在24、48及72 h收集细胞，倒置相差显微镜观察细胞形态变化；Annexin V/PI双染流式细胞术检测细胞凋亡；RT-PCR及Western blot分别检测葡萄糖调节蛋白78（GRP78）、IRE1、X盒结合蛋白1（XBP1）及C/EBP同源蛋白（CHOP）mRNA及蛋白表达；免疫荧光检测CHOP表达。

结果: 随着给氧时间延长，高氧组AEC Ⅱ伸展呈不规则形，出现空泡样改变；高氧组AEC Ⅱ凋亡率与同时间点空气组比较明显增加（P < 0.05）；随着氧暴露时间延长，高氧组GRP78、IRE1、XBP1及CHOP mRNA及蛋白表达升高，且较同时间点空气组明显上升（P < 0.05）；高氧组CHOP荧光强度高于同时间点空气组。高氧组CHOP蛋白表达与AEC Ⅱ凋亡率、IRE1及XBP1蛋白表达呈显著正相关（r=0.97、0.85、0.88，均P < 0.05）。

结论: 高氧所致AEC Ⅱ凋亡可能是通过激活IRE1-XBP1-CHOP通路来实现。

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Figures

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Annexin V/PI双染流式细胞术检测AECⅡ凋亡左上象限代表机械损伤细胞，左下象限代表正常细胞，右下象限代表早期凋亡细胞，右上象限代表晚期凋亡细胞。24、48及72 h，空气组早期凋亡率均较低，同时间点高氧组早期凋亡率均明显增加。
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Western blot检测ERS相关因子蛋白表达1：空气组24 h；2：高氧组24 h；3：空气组48 h；4：高氧组48 h；5：空气组72 h；6：高氧组72 h。[GRP78]葡萄糖调节蛋白78；[IRE1]需肌醇酶1；[XBP1]X盒结合蛋白1；[CHOP]C/EBP同源蛋白。
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免疫荧光检测CHOP表达（免疫荧光，×400）CHOP呈绿色荧光。24、48及72 h，空气组荧光强度较弱，CHOP表达较少；同时间点高氧组，荧光强度增强，CHOP表达增多。
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CHOP蛋白表达与AECII凋亡率相关分析图
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CHOP蛋白表达与IRE1蛋白表达相关分析图
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CHOP蛋白表达与XBP1蛋白表达相关分析图
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