脂多糖对妊娠周期小鼠子宫内膜上皮黏膜免疫屏障影响的研究

Effects of lipopolysaccharide on expression of uterine mucin and epithelial tight junction protein in pregnant mice

为评估脂多糖(LPS)对子宫内膜免疫屏障的影响,本研究将30只健康8周龄雌性小鼠随机均分为对照组LPS(-)和LPS(+)组,适应性饲养3 d后,将两组小鼠分别与雄性小鼠(15只/组)合笼,当日计为妊娠0 d,LPS(+)组雌性小鼠从合笼前15 d至0 d以LPS灌胃后检测其对小鼠繁殖性能的影响,结果显示,LPS可显著抑制雌性小鼠的受孕率和仔鼠成活率(P<0.05),造成仔鼠生长性能下降,表明本研究采用小鼠作为该研究动物模型具有可行性。进一步分别在妊娠7 d、14 d和分娩后5 d采集LPS灌服组[LPS(+)]和健康对照组[LPS(-)]母鼠子宫,采用相应ELISA试剂盒检测子宫内膜主要黏蛋白(Mucins)MUC1、MUC2和MUC4的含量;采用western blot检测子宫内膜上皮细胞紧密连接蛋白Claudin、Occludin、ZO-1、JAM、E-Cadherin的表达量。ELISA结果显示:与LPS(-)组相比,妊娠7 d时LPS(+)组母鼠子宫内膜中MUC1,MUC2和MUC4的含量均极显著减少(P<0.01),妊娠14 d时上述指标均显著升高(P<0.05),但妊娠期间LPS(+)组母鼠子宫内膜中MUC1,MUC2和MUC4的总量均极显著低于LPS(-)组(P<0.01);而分娩后两组小鼠子宫各黏蛋白含量和总量均无显著差异(P>0.05)。Western blot检测结果显示,与LPS(-)组相比,LPS(+)组雌性小鼠子宫中Occludin的表达量在妊娠7 d时显著降低(P<0.05),在妊娠14 d时显著升高(P<0.05);ZO-1的表达量在妊娠7 d时显著升高(P<0.05);JAM的表达量在妊娠14 d时显著降低(P<0.05);E-Cadherin的表达量在妊娠7 d、妊娠14 d时显著下降(P<0.05)。上述结果首次证实当子宫内膜受到LPS的刺激之后,在妊娠7 d时小鼠子宫内膜黏蛋白的含量降低,子宫内膜上皮细胞紧密连接相关蛋白Occludin、JAM和E-Cadherin的表达量显著减少;妊娠14 d小鼠子宫内膜黏蛋白的表达量上升,起到屏障和保护胎儿的作用,此时紧密连接相关蛋白Claudin、JAM和E-Cadherin仍受到不同程度的损害,但紧密连接的调节蛋白Occludin表达量上调;分娩后5 d,LPS对黏蛋白和紧密连接相关蛋白均无显著影响。本实验为研究家畜子宫内膜炎引发的繁殖性能障碍机制奠定了基础。

In order to investigate the effect of LPS on the endometrial immune barrier,the present study used LPS to gavage pregnant mice.Uterine samples of mice were collected on the 7th and the 14th day of gestation,as well as on the 5th day after delivery.ELISA and western blot were used to detect MUC1,the main MUC1 protein in endometrium.The study found that LPS significantly inhibit the pregnancy rate of mother mice and the survival rate of offspring,as well as affecting the contents of MUC2 and MUC4 and the expression of tight junction protein in endometrial epithelial cells.The results showed that the contents of MUC1,MUC2 and MUC4 in endometrium of LPS-treated mice were significantly decreased compared with those of LPS(-)group on day 7 of gestation.The expression of Occludin protein in uterus of LPS(+)group was significantly decreased compared with LPS(-)group on day 7 of gestation,and significantly increased compared with LPS(-)group on day 14 of gestation.Additionally,the expression level of ZO-1 in utero in LPS(+)group was significantly higher than that in LPS(-)group on day 7 of gestation.The expression of JAM in utero in LPS(-)group was significantly lower than that in LPS(-)group at day 14 of gestation.Compared with LPS(-)group,the expression of E-Cadherin in utero of mice in LPS(+)group decreased significantly on gestation days 7 and 14.In conclusion,when the endometrium was stimulated by LPS,the expression level of mucin in the endometrium decreased on day 7 of pregnancy,and LPS penetrating the uterine mucus layer significantly reduced the expression levels of tight junction related proteins Occludin,JAM and E-Cadherin in the endometrial epithelial cells.On the 14th day of gestation,the expression of Occludin in endometrium of LPS-treated mice increased,acting as a barrier and protecting fetus.At this time,tight junction related proteins Claudin,JAM and E-Cadherin were still damaged to varying degrees,but the expression of tight junction regulatory protein Occludin was up-regulated.At 5 days after delivery,LPS had no significant effect on mucins and tight junctions.