【作者】 张红霞；

【导师】 周栋；

【作者基本信息】 西北农林科技大学 ， 临床兽医学， 2025， 硕士

【摘要】 奶牛子宫内膜炎是一种严重的生殖系统疾病,其引发的母牛繁殖障碍性疾病一直是全球奶业亟待解决的的重大难题。奶牛分娩过程中,由于胎儿、骨盆及胎盘特点,易形成子宫内膜机械性创伤。产后奶牛常因能量负平衡和代谢紊乱,导致机体免疫防御能力下降,使得定植于生殖道的条件致病菌如大肠杆菌和化脓隐秘杆菌(Trueperella pyogenes,T.pyogenes)等,突破受损黏膜屏障,从而引起子宫微生态失衡并引发子宫内膜炎。目前,对T.pyogenes感染的防治仍普遍采用抗菌药物,但长期使用易促使耐药性的产生。直肠真杆菌(Eubacterium rectale,E.rectale)作为肠道内主要的丁酸盐产生菌,已被多项研究证明为健康机体的重要优势菌株,并具有潜在益生作用。实验室前期研究显示,E.rectale可以缓解奶牛子宫内膜炎损伤,可能成为预防和治疗子宫内膜炎新的备选益生菌菌株,其释放的膜囊泡(Membrane vesicles,MVs)可能在预防奶牛子宫内膜炎中发挥重要作用。本研究利用T.pyogenes诱导奶牛子宫内膜上皮细胞(Bovine Endometrial Epithelial Cells,BEND)炎性损伤建立炎症模型,在此基础上分别使用E.rectale及其MVs进行预处理,探寻炎性因子和上皮屏障基因的表达以及对细胞凋亡的影响。进一步利用转录组学测序筛选MVs预处理后的差异表达基因,通过GO、KEGG富集分析筛选MVs主要调控通路。根据测序结果,确定MVs的作用通路和靶向因子,利用抑制剂和siRNA双重方法验证MVs和与p-STAT 3的靶向关系。结果如下:(1)T.pyogenes以MOI=100处理BEND 6 h成功构建炎症模型,使用E.rectale预处理3 h,RT-q PCR检测炎症因子的表达,结果显示E.rectale预处理3 h可极显著下调T.pyogenes诱导的炎性因子IL-1β、IL-6和IL-8高表达,同时上调原本因感染而降低的连接黏附分子JAM-A与MUC-1的表达。流式细胞术和倒置显微镜观察证明,E.rectale预处理显著降低T.pyogenes诱导的细胞凋亡和死亡。(2)荧光标记MVs,通过观察其与BEND共定位分析内化过程,结果显示E.rectale分离的MVs与BEND共孵育3 h可内化进入细胞。RT-q PCR结果显示,MVs预处理3 h可显著抑制T.pyogenes诱导的炎性因子IL-1β、IL-6和IL-8表达,且作用趋势与E.rectale一致。ELISA检测结果进一步证明,MVs预处理可显著降低T.pyogenes诱导的IL-1β和IL-6分泌水平的升高。Western Blot分析发现,MVs预处理可以显著降低T.pyogenes导致的p-STAT3表达升高,而JAK2蛋白及p-JAK2表达水平无显著变化。(3)转录组测序结果筛选出T.pyogenes处理组相对于空白组显著上调,而MVs预处理组相较于炎症组显著下调的共同差异基因,经GO、KEGG富集分析发现,其主要聚焦于JAK/STAT信号通路。RT-q PCR检测显示,T.pyogenes感染显著上调STAT3及其下游基因SOCS3的表达。利用STAT3通路抑制剂和siRNA两种方法进一步探究MVs作用的分子机制,结果显示,抑制STAT3磷酸化后会显著抑制T.pyogenes诱导的IL-6和IL-8高表达,沉默STAT3会显著降低T.pyogenes诱导的IL-1β、IL-6和IL-8的高表达,ELISA结果与RT-q PCR结果趋势相同。综上,两种干预方法均会减弱MVs对T.pyogenes诱导的炎性因子抑制作用。综上所述,E.rectale及其释放的MVs可通过非经典激活途径,靶向抑制JAK/STAT信号通路中STAT3的磷酸化,从而缓解T.pyogenes诱导的子宫内膜炎性损伤,为细菌-MVs-宿主细胞之间的相互作用提供了新的证据,并为奶牛子宫内膜炎的预防和治疗提供了新思路。更多还原

【Abstract】 Bovine endometritis is a serious uterine disease,which can lead to infertility of dairy cows and cause serious economic losses to dairy industry in China and even the world.The mechanical injury of dairy cows after parturition needs to be repaired through a series of inflammatory purification and tissue regeneration.In this process,the imbalance of intrauterine environment homeostasis is often caused by the decline of body resistance and the invasion of pathogenic microorganisms,and then endometritis occurs.The most common pathogens were Escherichia coli and Trueperella pyogenes(T.pyogenes).At present,antimicrobial agents are still widely used in the prevention and treatment of T.pyogenes infection,which can lead to drug resistance.Eubacterium rectale(E.rectale)is the main butyrate producing bacteria in the intestinal tract.Many studies have proved that E.rectale is a dominant strain in healthy individuals and has potential probiotic effects.Previous studies in our laboratory have found that E.rectale can alleviate endometritis injury in dairy cows,which may become a new alternative strain for probiotics to prevent and treat endometritis.The Membrane vesicles(MVs)released by E.rectale may play an important role in preventing endometritis in dairy cows.In this study,T.pyogenes was used to induce inflammatory damage in Bovine Endometrial Epithelial Cells(BEND)to construct an inflammatory model.On this basis,E.rectale and its MVs were pretreated for different times to detect the expression of inflammatory factors and epithelial barrier genes and their effects on apoptosis at different times.Furthermore,transcriptome sequencing was used to screen the differentially expressed genes after MVs pretreatment,and GO and KEGG enrichment analysis were used to screen the action pathways of MVs.According to the sequencing results,the pathways and targeting factors of MVs were determined,and the targeting relationship between MVs and p-STAT3was verified by inhibitor and siRNA dual methods.Results were as follows:(1)T.pyogenes was treated with BEND at MOI=100 for 6 h to construct an inflammatory model,and E.rectale was used to pre-treat bend for 3 h,and RT-q PCR was used to detect the expression of inflammatory factors.The results showed that E.rectale pretreatment for 3 h significantly down-regulated the high expression of inflammatory factors IL-1β,IL-6 and IL-8 induced by T.pyogenes,and significantly up-regulated the low expression of junctional adhesion molecules JAM-A and MUC-1 induced by T.pyogenes.E.rectale pretreatment significantly reduced T.pyogenes-induced apoptosis and death as observed by flow cytometry and inverted microscopy.(2)The degree of internalization of fluorescently labeled MVs was analyzed by observation of their co-localization with BEND.The results showed that MVs of E.rectale could be internalized into cells after co-incubation with BEND for 3 h.The results of RT-q PCR showed that pretreatment with MVs for 3 h significantly down-regulated the expression of inflammatory factors IL-1β,IL-6 and IL-8 induced by T.pyogenes,which was consistent with the effect of E.rectale.The results of ELISA showed that MVs pretreatment significantly reduced the increase of IL-1βand IL-6 secretion induced by T.pyogenes.Western Blot analysis of inflammation-related pathways showed that MVs pretreatment significantly reduced the increase of p-STAT3 expression induced by T.pyogenes,while there was no significant change in the expression of JAK2 protein and p-JAK2.(3)Transcriptome sequencing analysis was used to screen the common differential genes that were significantly up-regulated in the inflammation group of T.pyogenes compared with the blank group,and significantly down-regulated in the MVs pretreatment group compared with the inflammation group.GO and KEGG enrichment analysis showed that these genes were mainly involved in the JAK/STAT signaling pathway.RT-q PCR showed that T.pyogenes infection significantly up-regulated the expression of STAT3 and its downstream gene SOCS3.STAT3 pathway inhibitor and siRNA were used to further explore the molecular mechanism of MVs action.RT-q PCR results showed that inhibition of STAT3 phosphorylation inhibited the high expression of IL-6 and IL-8 induced by T.pyogenes.Silencing STAT3 significantly reduced the high expression of IL-1β,IL-6 and IL-8 induced by T.pyogenes.ELISA results showed the same trend as RT-q PCR results,showing that,Both results and the two intervention methods attenuated the inhibitory effect of MVs on inflammatory factors induced by T.pyogenes.In conclusion,E.rectale and its MVs can target and inhibit the phosphorylation of STAT3through the non-classical activation pathway of JAK/STAT signaling pathway,alleviate the inflammatory injury of endometrium induced by T.pyogenes,and establish a bacterial-MVs-host cell link.To provide new ideas for the prevention of endometritis in dairy cows.更多还原