过氧化物还原酶4蛋白对小鼠卵巢颗粒细胞凋亡的影响及机制

doi:10.12280/gjszjk.20210596

摘要/Abstract

摘要：

目的： 探讨过氧化物还原酶4（Prdx4）蛋白对小鼠卵巢颗粒细胞凋亡的影响及相关机制。方法： 免疫荧光明确Prdx4在人卵巢颗粒细胞（KGN）中的定位;利用Prdx4小干扰RNA（siRNA）转染小鼠卵巢颗粒细胞,以空白组作为对照,蛋白质印迹法检测Prdx4表达水平以验证干扰效果,并检测2组颗粒细胞凋亡相关因子[B细胞淋巴瘤2（Bcl-2）、Bcl-2关联X蛋白（Bax）和裂解的半胱氨酸天冬氨酸蛋白酶3（cleaved caspase 3）]及内质网应激相关因子[免疫球蛋白结合蛋白（BIP）、转录激活因子4（ATF4）、ATF6和CCAAT/增强子结合蛋白同源蛋白（CHOP）]的表达。利用流式细胞仪检测2组颗粒细胞凋亡率,电镜下观察2组颗粒细胞的内质网形态。结果： 利用免疫荧光共定位明确Prdx4主要定位于颗粒细胞的内质网中。转染Prdx4-siRNA的小鼠颗粒细胞Prdx4表达水平低于空白对照组（P<0.01）;下调Prdx4蛋白表达导致小鼠颗粒细胞凋亡率增加（P<0.05）,促凋亡蛋白Bax及cleaved caspase 3蛋白表达水平增加（均P<0.01）,抗凋亡蛋白Bcl-2蛋白表达下降（P<0.05）。电镜下观察Prdx4-siRNA处理组内质网显著扩张,蛋白质印迹法显示Prdx4-siRNA处理组内质网应激相关蛋白BIP、ATF4及CHOP表达水平均高于空白对照组（均P<0.05）。结论： Prdx4蛋白表达水平下调可通过促进小鼠颗粒细胞内质网应激进而导致颗粒细胞凋亡。

关键词: 过氧化物还原酶, 内质网应激, 卵巢, 粒层细胞, 细胞凋亡

Abstract:

Objective: To explore the effects and mechanisms of peroxiredoxin 4 (Prdx4) on the apoptosis of granulosa cells in mice. Methods： The localization of Prdx4 in the human ovarian granulosa cellline (KGN) was detected by immunofluorescence staining. The expression of Prdx4 in granulosa cell was down-regulated by a Prdx4-siRNA, which was evaluated by Western blotting (WB). Apoptosis-related markers including B cell lymphoma 2 (Bcl-2), Bcl-2 associated X (Bax) and cleaved caspase 3 were also tested by WB. After that, the endoplasmic reticulum stress (ER stress)-related proteins including binding immunoglobulin protein (BIP), activating transcription factor 4 (ATF4), ATF6 and C/EBP homologous protein (CHOP) were then detected. Apoptosis was examined by flow cytometry. Electron microscopy was used to characterize the morphological changes of endoplasmic reticulum (ER). Results： In KGN cells, Prdx4 was mainly localized in ER. The expression of Prdx4 protein in the Prdx4-siRNA group was significantly reduced (P<0.01). Flow cytometry analysis revealed that the apoptosis ratio was increased significantly in the Prdx4-siRNA group (P<0.05). Compared with the control group, the Prdx4-siRNA group had the increased expressions of the proapoptotic proteins, Bax and cleaved caspase 3 (P<0.01) and the decreased expression of antiapoptotic protein, Bcl-2 (P<0.05). The electron microscopy showed that ER was swelled and dilated significantly in the Prdx4-siRNA group. Interestingly, the Prdx4-siRNA group was showed the significant increases in the expressions of BIP, ATF4 and CHOP protein (P<0.05). Conclusions： Knockdown expression of Prdx4 in granulosa cells would induce the cell apoptosis by the enhanced ER stress.

Key words: Peroxiredoxin Ⅲ, Endoplasmic reticulum stress, Ovary, Granulosa cells, Apoptosis

邹小飞, 梁秀茹, 严正杰, 崔毓桂, 刘嘉茵, 孟艳. 过氧化物还原酶4蛋白对小鼠卵巢颗粒细胞凋亡的影响及机制[J]. 国际生殖健康/计划生育, 2022, 41(2): 89-93.

ZOU Xiao-fei, LIANG Xiu-ru, YAN Zheng-jie, CUI Yu-gui, LIU Jia-yin, MENG Yan. Effects and Mechanisms of Prdx4 on Apoptosis of Granulosa Cell in Mice[J]. Journal of International Reproductive Health/Family Planning, 2022, 41(2): 89-93.

图/表 6

图1 Prdx4 在KGN细胞中的亚细胞定位（×600）注：在KGN细胞中进行免疫荧光共定位,以DAPI作为核荧光染料。蛋白质PDI阳性信号为红色荧光,特异性定位于内质网中,Prdx4阳性信号为绿色荧光,在荧光显微镜下两者位置重合。

图2 小鼠颗粒细胞下调Prdx4蛋白表达后凋亡情况注：小鼠颗粒细胞予Prdx4-siRNA处理下调Prdx4蛋白表达。A 流式细胞计数检测细胞凋亡。左侧为空白对照组,右侧为Prdx4-siRNA处理组。B Western blotting检测cleaved caspase 3、Bax和Bcl-2蛋白表达。左侧为空白对照组,右侧为Prdx4-siRNA处理组。

表1 2组小鼠颗粒细胞凋亡相关蛋白相对表达量比较（x±s）

组别	n	cleaved caspase 3	Bax	Bcl-2
空白对照组	3	0.590±0.081	0.866±0.027	1.025±0.051
Prdx4-siRNA处理组	3	1.282±0.154	1.204±0.049	0.787±0.110
t		6.909	10.450	3.409
P		0.002	0.001	0.027

图3 小鼠颗粒细胞下调Prdx4蛋白表达后内质网超微结构的改变（×3 000）注：小鼠颗粒细胞予Prdx4-siRNA处理下调Prdx4蛋白表达,左图为空白对照组,右图为Prdx4-siRNA处理组。可见与对照组内质网相比（箭头显示）,Prdx4表达下降导致内质网退行性改变（肿胀、扩张和增生,箭头显示）。

图4 小鼠颗粒细胞下调Prdx4蛋白表达后内质网应激相关蛋白表达情况注：小鼠颗粒细胞予Prdx4-siRNA处理下调Prdx4蛋白表达,以Prdx4、BIP、ATF6、ATF4和CHOP抗体检测相应蛋白。左侧为空白对照组,右侧为Prdx4-siRNA处理组。

表2 2组小鼠颗粒细胞内质网应激相关蛋白相对表达量比较（x±s）

组别	n	Prdx4	BIP	ATF6	ATF4	CHOP
空白对照组	3	1.057±0.126	0.563±0.169	0.943±0.106	0.373±0.139	0.567±0.145
Prdx4-siRNA处理组	3	0.469±0.080	0.967±0.162	0.671±0.486	0.959±0.149	1.013±0.162
t		6.859	2.982	0.948	4.975	3.549
P		0.002	0.041	0.397	0.008	0.024

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