【作者】 赵延涛；

【导师】 钟秀会；

【作者基本信息】 河北农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 繁殖障碍是使家畜繁殖力降低的主要原因,引起繁殖障碍的重要原因之一就是流产。哺乳动物尤其是反刍家畜又以妊娠早期的胚胎丢失几率最高。流产不仅造成胎儿夭折和母畜产奶量损失,延长母畜产仔间隔,而且常常引起胎衣滞留,造成子宫内膜炎,及母畜的习惯性流产,甚至使母畜失去繁殖性能,直接影响家畜的繁殖、品种的改良,给国家和企业造成巨大的经济损失,已经引起人们的高度重视。内毒素(感染或水平上升)是造成流产的一个重要原因,故本研究运用LPS建立小鼠肠道和肝脏损伤、流产和胚胎着床障碍的三种模型,并结合体外细胞培养的方法,探讨内毒素对肝脏的损伤机制及肝脏对内毒素的清除,同时研究LPS诱导小鼠流产和着床障碍的分子机制以及保胎中药的作用机理,为阐明LPS与妊娠母胎界面的免疫关系,寻找能够有效预防和调控妊娠早期胚胎丢失的药物并探索其作用机制,从而减少流产在动物生产中的发生,提高母畜的繁殖力和畜牧业养殖的经济效益。通过对LPS致小鼠肝损伤模型进行研究,发现LPS导致肠道的通透性增加,肠黏膜紧密连接蛋白Occludin和ZO-1的表达降低,推测破坏肠黏膜机械屏障可能是LPS对肠黏膜屏障损伤的重要机制之一。同时腹腔注射LPS引起回肠ROS大量蓄积,提示ROS可能参与调节LPS引发的肠黏膜屏障损伤过程。LPS处理组小鼠血清中的ALT/AST显著升高,说明肝细胞受损,肝功能下降。肝脏MDA含量剧增,抗氧化酶SOD、CAT、GPx活性明显降低,意味着LPS引起肝脏ROS的大量蓄积,并引起了脂质过氧化损伤。通过DHE荧光法检测发现LPS同样能引起人肝癌细胞HepG2产生大量的ROS,这一点与体内试验结果一致。同时LPS导致小鼠血中有大量的NO,肝脏的TNF-α、KC的含量明显升高,KC mRNA表达显著升高,肝脏的iNOS蛋白和肝细胞核内的NF-κB表达上升。LPS刺激HepG2之后能使其分泌大量的IL-8,且通过免疫荧光染色发现胞浆中静态的NF-κB被激活,转位于细胞核,参与调节趋化因子IL-8(或KC)的转录。LPS处理24h后,血清中内毒素水平仍明显高于对照组,说明受损的肝脏尚未能彻底清除血液中的内毒素,另一方面肠道通透性增加有可能导致肠腔内大量的内毒素被吸收入血。对LPS诱导孕鼠流产模型研究发现,LPS可显著减低孕鼠的子宫重量和子宫局部的Th2型细胞因子(IL-4和IL-10)含量,升高子宫Th1型细胞因子(主要是IFN-γ,对IL-2作用不明显)含量,使母-胎界面的Th1/Th2细胞因子平衡向着Th1方向偏移,同时诱导子宫内膜内的CD4+T细胞和F4/80巨噬细胞大量聚集,但基本不影响CD8+T细胞的含量。预口服保胎无忧散、泰山磐石散和白术散三种中药方剂的水煎液能调节子宫微环境,提高子宫重量,抑制Th1型细胞因子的分泌,提高Th2型细胞因子的水平,还能显著降低LPS引起的CD4+T细胞和巨噬细胞的增加,同时使子宫CD8+T细胞的数量显著增加,从而调节Th1/Th2的免疫平衡,使其向着有利于正常妊娠的方向发展,显著降低了孕鼠的流产率和胚胎吸收率,并对LPS造成的子宫损伤有一定的修复作用。采用孕3d小鼠腹腔注射LPS的方法成功建立胚胎着床障碍模型。研究发现,LPS处理后,小鼠血清和子宫组织中NOS活性明显增加,子宫中IFN-γ含量显著上升、IL-10含量无显著变化,且呈剂量依赖性,使Th1/Th2平衡向Th1反应方向移动,因此改变了着床期小鼠子宫免疫微环境,导致了胚胎着床障碍。口服0.5 mg或1 mg黄芩苷或槲皮素均能明显拮抗LPS诱导的着床障碍,使平均着床胚胎数显著上升,着床率升高。黄芩苷显著抑制LPS引起的子宫组织中IFN-γ水平的上升,提高子宫IL-10的含量,降低子宫和血清的NOS活性,从而减少NO的过量合成,使其接近于正常妊娠状态。以上结果在LPS处理的体外培养着床期小鼠子宫内膜细胞的模型中,也得到证实。此外槲皮素能通过显著抑制LPS诱导的子宫IFN-γmRNA的高表达,上调子宫组织中TGF-β1 mRNA的表达来调节子宫局部的免疫平衡。黄芩苷和槲皮素均调节子宫局部的免疫平衡,使其向着有利于妊娠建立的Th2反应偏移,发挥保胎促孕功效。综上,LPS通过导致肠道紧密连接蛋白表达降低,肠黏膜屏障受损,引发内毒素血症。大量的LPS刺激Kupffer细胞和肝细胞,产生大量ROS,抗氧化酶活性降低,激活NF-κB,产生大量的炎性介质,加重了肝损伤,清除内毒素的能力下降。LPS诱导的孕鼠流产和着床障碍,主要是通过破坏母胎界面的Th1/Th2免疫平衡状态;而中药方剂和中药成分能够显著拮抗LPS的破坏作用,改善和修复子宫的免疫平衡,使其有利于正常妊娠和着床,发挥其保胎促孕功效。更多还原

【Abstract】 Reproductive disorder is one of main reasons for the reduction of domestic animal fertility, while abortion plays a causal role in reproductive disorder. Abortion during earlier pregnancy stage is very common in mammals especially ruminant. Early abortion can cause not only the death of fetus, deduction of milk yield and prolonged calving interval, but also the injury of female animals, such as inducing retention of the afterbirth, endometritis and recurrent miscarriages accompanied by decreased reproductive rate and even female infertility. The occurrence of the above pathological phenomena resulted in hindering animal reproduction and breeding improvement, which made immeasurable financial loss of country and enterprises and provoked great attentions. As we know, endotoxin plays an important role in the occurrence of animal abortion. This stud utilized Lipopolysaccharide (LPS) -induced liver and intestinal injury model, abortion and implantation failure models. The aims are to understand the molecular mechanisms of LPS induced liver and intestine injury as well as abortion, implantation failure. We also explained the anti-abortion effects of traditional Chinese herbal medicine. This study also explained the relationship between LPS and maternal-fetal immunity, and the possible roles of the effective medicine in protecting against early fetal loss.Through studying the LPS-induced mice liver injury model, we found that LPS decreased the expressions of two main tight junction proteins (Occludin and ZO-1), which resulted in increased intestinal permeability. LPS induced reactive oxygen species (ROS) accumulation in the ileum which may be related to the disrupted gut barrier. The serum Alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities were significantly increased after LPS treatment. These responses were associated with elevated malondialdehyde (MDA) and dramatically reduced superoxide diamutase, catalase, and glutathione peroxidase. Serum nitric oxide (NO) was increased, and the levels of tumor necrosis factor alpha (TNF-α) and keratinocyte chemoattractant (KC) in the liver were elevated. ROS accumulation was also detected in HepG2 cells after LPS treatment. The KC mRNA and inducible NO synthase (iNOS) and nuclear factor kappa B (NF-κB) proteins in the liver were significantly upregulated by LPS challenge. LPS-induced interleukin-8 production in HepG2 cells, which was associated with NF-κB nuclear translocation. The elevated endotoxin levels in serum after LPS challenge was associated with the liver dysfunction and the significantly increased intestinal permeability. The abortion and embryo resorption rates were significantly decreased by pre-fed the concentrated decoction of three Chinese herbal formula (bao tai wu you san, tai shan pan shi san and bai shu san) before LPS treatmetns In LPS-induced abortion mice, the weight of uterus was significantly reduced and the Th1 cytokines (IFN-γ) and the number of CD4+ T cell (but not CD8+ T cell) and macrophage in mice endometrium were dramatically elevated while the amounts of Th2 cytokines (IL-4 and IL-10) was decreased,. LPS caused the imbalance of Th1/Th2 ratio, leading to a Th1 dominant shift in uterus. And the Th1 dominant shift is known to be deleterious to normal pregnancy. The Chinese herbal formula normalized and improved the micro immune environment in mice endometrium.Mice were injected LPS intraperitoneally on Day 3 of gestation to establish the embryo implantation disruption model. LPS challenge significantly increased the serum and uterine NO synthase (NOS) activities and uterine IFN-γlevels, but not IL-10. The above results indicated that LPS also induced embryo implantation failure through altering the immune environment towards Th1 dominant in endometrium during implantation. Oral administration of Baicalin and Quercetin (0.5mg and 1.0 mg) significantly improved the number of implanted embryo and implantation rate compared with LPS treated mice. Baicalin decreased the uterine IFN-γlevel, NOS activities and increased the IL-10 level, resulting in normalized endometrial immune environment. The in vitro study confirms the above in vivo findings. Quercetin downregulated IFN-γmRNA expression and upregulated transforming growth factor-β1 (TGF-β1) mRNA levels compared with those of the LPS treated mice. In conclusion, Baicalin and Quercetin, like the Chinese herbal formula, achieved their anti-abortion and promoting implantation functions by modulating the uterine Th1/Th2 ratio to Th2 dominant which is beneficial to pregnant maintenance and embryo implantation.In conclusion, these studies demonstrate that LPS-induced intestinal barrier dysfunction, which resulting from the down-regulated tight junction proteins, plays an important role in excess LPS absorption into the blood stream; LPS stimulates Kupffer cells and hepatocytes through inducing ROS accumulation and reducing the activities of antioxidant enzymes. NF-κB activation upon LPS challenge might be a causal factor in generating pro-inflammatory cytokines and chemokines which aggravating liver damage; LPS impairs abortion and embryo implantation in mice through increasing the Th1/Th2 ratio; Chinese herbal medicine or certain ingredients in Chinese herbal medicine (Baicalin, Quercetin) demonstrate anti-abortion and beneficial of embryo implantation properties through effectively balancing the immune environment in the uterus and alleviating LPS-induced toxicity.更多还原