鱼腥草素钠抗LPS诱导的奶牛子宫内膜上皮细胞炎症的分子机制

发布时间：2018-03-21 10:03

本文选题：奶牛子宫内膜炎　切入点：脂多糖　出处：《中国农业大学》2015年博士论文　论文类型：学位论文

【摘要】：奶牛产后子宫内膜炎是由致病微生物上行感染母牛生殖道而引起的炎症。大肠杆菌是主要的致病菌之一,但其发病机制并不十分清楚。鱼腥草素钠(Sodium houttuyfonate, SH)是鱼腥草有效成分鱼腥草素的亚硫酸氢钠加成化合物,稳定性强,临床上用于治疗炎症疾病。目前鱼腥草素钠对抗炎症疾病的分子机制还不完全清楚,我们假设NF-κB充当关键调解物,从体外细胞水平探明其抗炎分子机制。并在体内对小鼠子宫内膜炎模型和奶牛子宫内膜炎的疗效进行观察。从而深入研究SH抗炎活性和其药理机制。本研究运用体外原代培养细胞的方法,分离鉴定了奶牛子宫内膜上皮细胞和基质细胞。并对培养获得的原代细胞进行了不同染色方法的形态学观察和透射电镜观察。在此基础上,建立LPS诱导的奶牛子宫内膜上皮细胞(bEECs)炎症模型。为了评估SH的体内外抗炎作用,首先用MTT法和LDH分析检测梯度浓度的SH对bEECs的毒性；其次用ELISA和qRT-PCR方法分别从蛋白分泌和nRNA表达两个水平检测细胞炎性因子TNF-α、IL-1β、IL-6和IL-8,同时用western blotting的方法检测细胞内炎症信号转导通路MAPKs/NF-κB的关键蛋白ERK、JNK、p38、IκBα和NF-κBp65;最后用临床诊断的方法评估SH治疗小鼠和奶牛子宫内膜炎的疗效。研究表明,运用组织培养和复合酶消化结合差时技术可以分离出纯度很高的bEECs,为后续的实验提供了足够的细胞。不同染色方法对子宫内膜细胞观察可知其细胞结构清晰,胞浆胞膜分明；超微结构观察结果显示,体外培养的子宫内膜上皮细胞可清晰看到微胞凸,胞内各种细胞器清晰可见。细胞毒性实验显示,SH (120μg/mL)浓度范围内对bEECs无毒副作用；15,30and60μg/mL高中低浓度的SH可以抑制LPS诱导bEECs的TNF-α、IL-1β、IL-6和IL-8的蛋白分泌水平,并呈浓度依赖关系；对炎性细胞因子的mRNA有同样的调控作用。而且,在LPS激活的bEECs中,SH抑制了IκBα的降解和NF-κB p65的磷酸化；压制了ERK、JNK、p38磷酸化水平。体内疗效实验证实,SH能够显著减弱模型组小鼠子宫内膜的炎性反应,子宫指数与正常对照组差异不显著,镜检子宫组织显示显微结构基本正常,表明SH对实验性子宫内膜炎模型小鼠有很好的治疗效果。用SH治疗奶牛临床型子宫内膜炎受孕率达到86%,发情头数占总头数的91.7%。而用土霉素治疗奶牛受孕率为50%,发情头数占总头数的67%。在发情头数和受孕率上SH的治疗效果优于土霉素。
[Abstract]:Postpartum endometritis in dairy cows is caused by the upward infection of pathogenic microorganisms in the reproductive tract of cows. Escherichia coli is one of the major pathogens. But its pathogenesis is not very clear. Sodium houttuyfonate (SH) is a sodium bisulfite addition compound of houttuynin, which has strong stability. Clinical use in the treatment of inflammatory diseases. The molecular mechanism of houttuynin sodium against inflammatory diseases is not fully understood, and we assume that NF- 魏 B acts as a key mediator. The anti-inflammatory molecular mechanism of SH and its pharmacological mechanism were investigated in vitro, and in vivo, the model of endometritis in mice and the therapeutic effect of endometritis in dairy cows were observed in order to study the anti-inflammatory activity of SH and its pharmacological mechanism. In this study, we used the method of primary culture of cells in vitro, The endometrial epithelial cells and stromal cells of dairy cattle were isolated and identified. The primary cells were observed by different staining methods and transmission electron microscopy. The inflammatory model of bovine endometrial epithelial cells induced by LPS was established. In order to evaluate the anti-inflammatory effect of SH in vitro and in vivo, the toxicity of gradient concentration of SH to bEECs was detected by MTT assay and LDH analysis. Secondly, ELISA and qRT-PCR were used to detect the cytokines TNF- 伪, IL-1 尾, IL-6 and IL-8, respectively, from the two levels of protein secretion and nRNA expression. Meanwhile, western blotting was used to detect MAPKs / NF- 魏 B, the key proteins of MAPKs / NF- 魏 B and NF- 魏 Bp65. The therapeutic effect of SH on endometritis in mice and cows was evaluated. The results showed that high purity bEECscould be isolated by tissue culture and enzyme digestion, which provided enough cells for subsequent experiments. Different staining methods showed that the cell structure of endometrial cells was clear. The ultrastructural observation showed that the endometrial epithelial cells cultured in vitro could clearly see the microcellular convexity. The cytotoxicity test showed that SH in the range of 120 渭 g / mL had no toxic side effect on bEECs. SH at high or low concentrations of 1530 渭 g / mL and 60 渭 g / mL could inhibit the protein secretion of TNF- 伪, IL-1 尾, IL-6 and IL-8 induced by LPS in a concentration-dependent manner. Moreover, in bEECs activated by LPS, SH inhibited the degradation of I 魏 B 伪 and phosphorylation of NF- 魏 B p65. The therapeutic effect experiment in vivo showed that SH-H could attenuate the inflammatory response of endometrium in the model group, and the uterine index was not significantly different from that of the normal control group. The microscopic examination showed that the microstructure of the uterus was basically normal, but no significant difference was found between the model group and the normal control group. The results showed that SH had a good therapeutic effect on experimental endometritis model mice. The pregnancy rate of clinical endometritis in dairy cows treated with SH was 86%, and the number of estrous heads accounted for 91.7%. The pregnant rate of cows treated with oxytetracycline was 50%, and the pregnancy rate was 50%. The therapeutic effect of SH was better than that of oxytetracycline in estrus number and pregnancy rate.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.23

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