基于TLR4/NF-κB与HIF-1的相互调节研究刺五加多糖缓解免疫应激仔猪肠上皮紧密连接损伤的机制

Intestinal epithelial tight junction (TJ) breakdown is the main pathological change of diarrhea in piglets. The synergic relationship between inflammation and hypoxia has been confirmed. The crosstalk between NF-κB and HIF-1 in the development of TJ damage is not clear although they can function in it respectively as the key transcription factor in the process of inflammation and hypoxia. We inferred that the alleviative TJ damage by Acanthopanax senticosus polysaccharides (ASP) do closely with the regulation of the crosstalk between NF-κB and HIF-1 based on our previous findings in which correlation occured in abnormal expressions of NF-κB and HIF-1 when diarrhea and TJ damage of immune stress piglets were relieved by ASP. However, the exact mechanism is unknown. We have isolated and identified polysaccharide from the root of Acanthopanax senticosus, and will evaluate the relationship between TJ injury and the expressions of TLR4,NF-κB and HIF-1 with the treatment of ASP by establishing immune stress model of piglets, and then the role of the crosstalk between TLR4/NF-κB and HIF-1 in the process of TJ damage relief and their promoters region binding under ASP treatment will be determined respectively by means of gene interference and overexpression, as well as reporter gene and ChIP. The project aiming to clarify the mechanism in which ASP alleviates TJ damge will help to explore the deep relationship between inflammation and hypoxia in gastric epithelium, and provide theory evidence for the application of ASP on preventing diarrhea of piglets.

肠上皮紧密连接（TJ）损伤是仔猪腹泻的主要病理改变。目前，炎症与缺氧的协同关系已经被证实。NF-κB和HIF-1作为炎症和缺氧的关键转录因子均能参与TJ损伤，但两者间的相互作用尚不清楚。我们前期发现，刺五加多糖能缓解免疫应激仔猪腹泻和TJ损伤，并使NF-κBp65和HIF-1α呈相关异常表达，推测其作用与调控NF-κB和HIF-1有关，但确切机制不明。项目组已从刺五加根中分离鉴定出多糖组分，拟从仔猪免疫应激模型入手，证明TLR4、NF-κB和HIF-1表达与刺五加多糖缓解TJ损伤的关系；进而通过基因干扰和过表达手段明确TLR4/NF-κB与HIF-1的相互调控及其在刺五加多糖缓解TJ损伤中的作用，并进一步采用报告基因和ChIP分析相互间启动子的结合。项目旨在阐明刺五加多糖缓解TJ损伤的机制，同时有望探寻肠上皮炎症和缺氧的深层次关系，为应用刺五加多糖改善仔猪腹泻奠定理论依据。

免疫应激致肠黏膜炎性损伤是仔猪腹泻的重要原因。刺五加多糖（ASPS）是刺五加的主要活性成分，具有缓解肠黏膜损伤和仔猪腹泻的作用，本试验通过LPS体内外刺激模拟免疫应激并给予ASPS干预，探讨ASPS缓解免疫应激仔猪肠黏膜损伤的作用机制。动物试验结果表明：预饲喂ASPS能改善免疫应激仔猪采食量，降低腹泻率和腹泻指数，调节免疫应激仔猪肠绒毛高度和隐窝深度，提高肠黏膜DAO和乳糖酶含量；ASPS对仔猪外周血白细胞和中性粒细胞数量有下调作用，并使肠粘膜肥大细胞数量，前炎症细胞因子TNF-α，IL-1β显著下降，抗炎细胞因子IL-10明显升高；同时对LPS刺激导致的TNF-α、HIF-1α和iNOS基因表达以及NFκB p65、HIF-1α和COX-2等蛋白表达的改变具有调节作用。细胞培养试验表明：抑制IPEC-J2细胞TLR4后，ASPS和LPS刺激对除了TAK1 mRNA表达以外的Transwell小室屏障电阻，TJ蛋白Occludin，MYLK2 mRNA表达以及细胞因子TNF-α，IL-1β，IL-6，IL-8和IL-12影响均不明显；HIF-1α不能促进TLR4启动子区pGL3报告基因萤光素酶活性且ASPS对此作用无影响。抑制IPEC-J2细胞HIF-1α后，ASPS预处理能缓解LPS诱导的Transwell小室屏障FITC-Dextran透过率升高以及电阻下降，对LPS刺激的IKKα，NF-kBp65，MYLK2和Occludin表达以及TNF-α，IL-1β和IL-8水平有显著影响。综上，预饲喂ASPS能通过影响NF-kBp65炎症通路继而间接影响HIF-1α诱导的缺氧通路从而发挥缓解免疫应激仔猪炎性肠损伤和腹泻，且这种作用的发挥与TLR4存在有关。