胎盘滋养细胞内质网应激促炎作用对妊娠期糖尿病发病的影响

Pro-inflammatory effect induced by endoplasmic reticulum stress in placental trophoblast cells participates in genesis of gestational diabetes mellitus

目的探讨内质网应激在胎盘组织中是否通过促炎作用对妊娠期糖尿病(gestational diabetes mellitus, GDM)的发病产生影响。方法选取2016年1月至12月于华中科技大学同济医学院附属同济医院产科定期产前检查并分娩的40例孕妇,其中20例确诊为GDM(GDM组),其余20例为与GDM组孕妇年龄、孕周相匹配的正常孕妇对照组。收集2组孕妇胎盘组织,透射电镜下观察胎盘滋养细胞内质网超微结构,并采用蛋白质印迹法检测胎盘组织中内质网应激标志蛋白—葡萄糖调节蛋白-78(glucose-regulated protein-78, GRP-78)及C/EBP同源蛋白(C/EBP homologous protein, CHOP)的表达情况。收集5例正常孕妇胎盘组织进行外植体培养,其中给予白细胞介素(interleukin, IL)-1β(5 ng/ml)培养胎盘组织20 h,称为IL-1β模型组;胎盘组织给予IL-1β(5 ng/ml)培养18 h后再加入内质网应激激动剂—毒胡萝卜素(thapsigargin, TG)(30 μmol/L)培养2 h,称为IL-1β+TG干预组;未给予任何刺激的胎盘组织为空白对照组。采用蛋白质印迹法检测各组胎盘组织外植体GRP-78、CHOP、葡萄糖转运蛋白4(glucose transporter 4, GLUT4)的表达,采用实时荧光定量聚合酶链反应技术检测促炎细胞因子—IL-6、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)的mRNA表达情况。采用单因素方差分析、t检验和LSD检验等进行统计学分析。结果(1)透射电镜下可见GDM组滋养细胞内质网池数量增多、体积增大,内质网扩张明显并出现大小不等的片段和大小泡;对照组胎盘组织滋养细胞内质网无明显肿胀。(2)GDM组胎盘组织中GRP-78、CHOP蛋白相对表达量分别为0.90±0.17和0.85±0.13,均高于对照组(0.48±0.08和0.46±0.12),t值分别为2.24和2.10,P值均<0.05。(3)IL-1β模型组外植体培养胎盘组织中GRP-78、CHOP蛋白相对表达量均明显高于空白对照组(0.87±0.18与0.36±0.07,1.14±0.09与0.78±0.06),GLUT4蛋白相对表达量明显低于空白对照组(1.00±0.14与2.21±0.49),IL-6、TNF-α mRNA相对表达量均明显高于空白对照组(0.89±0.23与0.30±0.06,0.62±0.16与0.17±0.09),t值分别为2.67、3.20、2.40、2.31和2.29,P值均<0.05;与IL-1β模型组比较,IL-1β+TG干预组外植体培养胎盘组织中GRP-78、CHOP蛋白相对表达量明显增加(2.02±0.32与0.87±0.18、2.18±0.31与1.14±0.09),GLUT4蛋白相对表达量明显降低(0.39±0.19与1.00±0.14),IL-6、TNF-α mRNA相对表达量均明显增加(1.67±0.25与0.89±0.23,1.42±0.27与0.62±0.16),t值分别为3.11、3.16、2.66、2.26和2.51,P值均<0.05。结论内质网应激可能参与部分GDM患者胎盘组织促炎细胞因子释放,进而参与GDM的发生及发展。

Objective To explore whether the pro-inflammatory effect of endoplasmic reticulum stress in placental tissues involves in the genesis of gestational diabetes mellitus (GDM). Methods Forty gravidas who underwent regular prenatal examinations and delivered at Tongji Hospital were recruited from January to December, 2016. Among them, 20 were GDM women (GDM group), and the remaining twenty were served as the control, which were selected from those without GDM and matched for age and gestational weeks to the GDM group. Placental tissues were collected from the two groups. The ultrastructure of endoplasmic reticulum in trophoblast cells was observed under transmission electron microscope. The expression of glucose-regulated protein-78 (GRP-78), a marker protein for endoplasmic reticulum stress, and C/EBP homologous protein (CHOP) were detected using Western blotting. Five placental tissue samples were collected from normal gravidas for explant culture. Three subgroups were set up according to different culturing methods including culturing with IL-1β(5 ng/ml) for 20 h (IL-1β model group), 30 μmol/L thapsigargin (TG, an endoplasmic reticulum stress agonist) for 2 h after treating with IL-1β(5 ng/ml) for 18 h (IL-1β+TG intervention group) or with no stimulation (blank control group). Western blotting was used to detect the expressions of GRP-78, CHOP and glucose transporter 4 (GLUT4) in placenta explants. The mRNA expressions of interleukin-6 (IL-6) and tumor necrosis factor-α(TNF-α) were determined by real-time fluorescence quantitative polymerase chain reaction (RT-PCR). Statistical analysis was performed using one-way analysis of variance, LSD and t test. Results(1) In the GDM group, increased number and size of endoplasmic reticulum cisternae were observed in trophoblast cells. Moreover, obviously dilated endoplasmic reticulum and different size of fragments and vesicles were also seen under electron microscope. While the endoplasmic reticulum in the placental tissues of the control group showed no obvious swelling.(2) The expression of GRP-78 and CHOP protein in the GDM group were higher than those in the control group (0.90±0.17 vs 0.48±0.08, t=2.24;0.85±0.13 vs 0.46±0.12, t=2.10;both P<0.05).(3) Compared with the blank control group, the expression of GRP-78 and CHOP protein in the IL-1β model group increased significantly (0.87±0.18 vs 0.36±0.07, t=2.67;1.14±0.09 vs 0.78±0.06, t=3.20;both P<0.05);but the expression of GLUT4 protein significantly decreased (1.00±0.14 vs 2.21±0.49, t=2.40, P<0.05);the expressions of IL-6 and TNF-α mRNA significantly increased (0.89±0.23 vs 0.30±0.06, t=2.31;0.62±0.16 vs 0.17±0.09, t=2.29;both P<0.05). Compared with the IL-1β model group, the expression of GRP-78 and CHOP protein significantly increased in IL-1β+TG group (2.02±0.32 vs 0.87±0.18, t=3.11;2.18±0.31 vs 1.14±0.09, t=3.16;both P<0.05);the expression of GLUT4 protein significantly decreased (0.39±0.19 vs 1.00±0.14, t=2.66, P<0.05);the expression of IL-6 and TNF-α mRNA increased significantly (1.67±0.25 vs 0.89±0.23, t=2.26;1.42±0.27 vs 0.62±0.16, t=2.51;both P<0.05). Conclusions Endoplasmic reticulum stress may be associated with increased release of pro-inflammatory cytokines in placental tissues of some GDM women and involved in the onset and development of GDM.