脂多糖致孕鼠胎盘损伤诱发早产的机制研究

Mechanism of preterm birth induced by placental injury caused by lipopolysaccharide in pregnant mice

目的探讨脂多糖(lipopolysaccharide,LPS)致胎盘损伤诱发早产的机制。方法选用CD1孕鼠,随机分为对照组16只、实验组24只,在妊娠第17天(E17)进行宫内注射,对照组注射100μl磷酸盐缓冲液,实验组注射25μg LPS。在注射药物后6 h及24 h,对照组和实验组分别取5、6只孕鼠,剖宫取胎盘样本,且对注射后24 h(E18)剖宫取出的胎鼠进行称重。对照组和实验组分别剩余6、12只孕鼠,比较两组的早产(<E19自然分娩)以及胎鼠存活情况。胎盘标本进行苏木精-伊红染色和波形蛋白免疫荧光染色观察胎盘结构,实时荧光定量聚合酶链反应检测胎盘白细胞介素(interleukin,IL)-1β、IL-6和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的mRNA水平,蛋白质印迹法检测磷酸化和总核因子κB(nuclear factor kappa-B,NFκB)蛋白水平。统计学方法采用Student's t检验、χ^(2)检验。结果实验组注射LPS后孕鼠早产率为75.0%(9/12),对照组6只孕鼠均未出现早产;实验组胎鼠总体存活率低于对照组[27.8%(42/151)与96.2%(75/78),χ^(2)=96.127,P<0.001]。在注射后24 h剖宫取出的E18胎鼠中,实验组胎鼠的平均体质量也低于对照组[(1.35±0.12)与(1.69±0.05)g,t=5.996,P<0.001]。在注射后6 h,与对照组相比,实验组胎盘迷路层结构松散,组织间空隙、空泡增多,血细胞浸润数量显著减少,波形蛋白的分布减少;在注射后24 h,实验组胎盘迷路层结构无显著变化。注射后6 h,实验组与对照组胎盘IL-1β(0.307±0.175与0.020±0.014,t=3.611,P=0.006)、IL-6(0.223±0.189与0.004±0.002,t=2.596,P=0.032)和TNF-α(0.25±0.16与0.06±0.02,t=2.684,P=0.025)的mRNA水平比较,实验组均显著高于对照组。注射后24 h,实验组与对照组胎盘IL-1β(0.045±0.035与0.009±0.006,t=2.292,P=0.048)和IL-6(0.020±0.016与0.002±0.001,t=2.413,P=0.039)的mRNA水平比较,实验组均高于对照组;但两组TNF-α的mRNA水平(0.10±0.06与0.07±0.04,t=1.071,P=0.312)比较差异无统计学意义。注射后6 h,实验组胎盘的磷酸化NFκB蛋白相对表达量高于对照组(1.23±0.48与0.66±0.13,t=2.569,P=0.030)。结论孕鼠暴露于LPS诱导的宫内炎症后,胎盘会表现出炎症诱发的结构损伤,胎盘在炎症早期的显著损伤变化可能是引起早产和死胎的重要原因之一。

Objective To explore the mechanism of preterm birth(PTB)induced by placental injury caused by lipopolysaccharide(LPS).Method Pregnant CD1 mice were utilized and randomly divided to control group(n=16)and LPS group(n=24).At the seventeenth day of gestation(E17),pregnant mice were treated by intrauterine injection of 25μg LPS for LPS group or 100μl phosphate buffered solution(PBS)for control group.At 6 h and 24 h after injection respectively,placental samples were collected in control group(n=5)and LPS group(n=6).At 24 h after injection(E18),the weight of fetal mice was recorded.In the retained 12 mice in LPS group and 6 mice in control group,the PTB(<E19)rate and fetal survival rate were compared.Hematoxylin and eosin(HE)staining and vimentin immunofluorescence staining were used to observe the placental structure.Real-time quantitative polymerase chain reaction(PCR)was applied for detecting the mRNA levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor-α(TNF-α).Western blotting was employed to measure the protein levels of phosphorylated and total nuclear factor kappa-B(NFκB).Student's t test andχ^(2) test were used to perform statistical analysis.Result The rate of PTB in LPS group was 75.0%(9/12)and there was no PTB in control group.The total survival rate of fetal mice in LPS group was significantly lower than that in control group[27.8%(42/151)vs 96.2%(75/78),χ^(2)=96.127,P<0.001].The weight of fetal mice at 24 h after injection(E18)in LPS group was significantly lighter than control group[(1.35±0.12)vs(1.69±0.05)g,t=5.996,P<0.001].At 6 h after injection,in contrast to control group,placental labyrinth zone in LPS group had loose structure,increased organizational gaps,less infiltration of blood cells and decreased vimentin distribution.At 24 h after injection,the structure of labyrinth zone was unchanged in LPS group.Additionally,at 6 h after injection,the mRNA levels of IL-1β(0.307±0.175 vs 0.020±0.014,t=3.611,P=0.006),IL-6(0.223±0.189 vs 0.004±0.002,t=2.956,P=0.032)and TNF-α(0.25±0.16 vs 0.06±0.02,t=2.684,P=0.025)in LPS group were significantly higher than those in control group.At 24 h after injection,the mRNA levels of IL-1β(0.045±0.035 vs 0.009±0.006,t=2.292,P=0.048)and IL-6(0.020±0.016 vs 0.002±0.001,t=2.413,P=0.039)in LPS group were higher than those in control group,but there was no statistical difference of the TNF-αmRNA level(0.10±0.06 vs 0.07±0.04,t=1.071,P=0.312)in LPS group and control group.At 6 h after injection,the relative protein expression of phosphorylated NFκB in LPS group was significantly higher than that of control group(1.23±0.48 vs 0.66±0.13,t=2.569,P=0.030).Conclusion After pregnant mice are exposed to intrauterine inflammation caused by LPS,the placentas appear structure injuries induced by inflammation.The obvious injury in placenta at early inflammatory stage may be one of the important causes responsible for PTB and stillbirth.