基于TLR4-NF-kB、NOD1/2-RIP2通路研究中药对ETEC诱导的断奶腹泻仔猪肠道炎性损伤的调控机制

It is often happened to the weaned stress syndrome of piglets with the technology of early weaned piglets, which would cause the functions of intestinal mucosal immunity in disorder and fall in decline, and then the weaned piglets will be infected by Enterotoxigenic Escherichia coli (ETEC) with the severe intestinal inflammation and intestinal mucosal barrier damages. Therefore, it is the key to keep the weaned piglets in healthy by reducing or eliminating the intestine inflammation and damages caused by ETEC. The experiments will be carried out to investigate the pathological changes in the intestine gut and the intestinal epithelial cells injured with ETEC in vivo or vitro, and the effects of the TLR4-NF-kB and NOD1/2-RIP2-NF-kB/MAPK signal pathway on the intestine and intestinal epithelial cells infected with ETEC will be studied via Flow Cytometry, Gene transcriptome depth sequencing technology, RNA interference technology, western-blot, real-time PCR. And then, the results of experiments in vivo or vitro will be helpful to evaluate the regulatory mechanism of Xiangsha Liujunzi decoction and even the active ingredients, which will lead to determine the regulatory mechanism and targets of the injury of ETEC to intestinal mucosal immunity in weaned piglets.

早期断奶技术易引起仔猪断奶应激综合征，导致断奶仔猪肠道黏膜免疫机能下降，继而发生肠毒素性大肠杆菌（ETEC）感染性肠道炎症和剧烈腹泻，给养猪业带来了巨大危害；由于肠道是机体最大的黏膜免疫屏障，亦是营养物质消化吸收的主要场所。因此，减轻或消除ETEC引起的断奶仔猪肠道炎症和黏膜损伤，维持肠道黏膜免疫屏障功能正常是保证仔猪健康生长的关键。本研究拟通过构建断奶仔猪体内外（猪肠道/肠上皮细胞）ETEC感染模型，观察猪肠道黏膜及肠上皮细胞免疫病理变化，结合流式细胞术、基因转录组测序技术、RNA干扰技术、免疫印迹技术、荧光定量PCR技术以研究TLR4-NF-kB、NOD1/2-RIP2-NF-kB/MAPK信号通路在ETEC诱导的猪肠道黏膜炎性损伤中的作用，评价香砂六君子汤及有效成分对ETEC诱导的仔猪肠道炎性损伤调控作用，确定ETEC感染引起断奶仔猪肠道黏膜炎性损伤的药物作用靶点及中药分子调控机制。

肠毒素性大肠杆菌（ETEC）感染会引起断奶仔猪严重肠道炎症和剧烈腹泻，给养猪业带来了巨大危害，减轻或消除ETEC引起的断奶仔猪肠道炎症和黏膜损伤，维持肠道黏膜免疫屏障功能正常是保证仔猪健康生长的关键。然而，可有效防治ETEC引起的断奶仔猪腹泻的中药及复方的调控作用机制尚未阐明。本研究构建了断奶仔猪ETEC感染腹泻动物模型、猪肠道上皮细胞ETEC感染模型，观察猪肠道黏膜及肠上皮细胞免疫机能变化，结合流式细胞术、基因转录组测序技术、免疫印迹技术、荧光定量PCR技术以研究TLR4-NF-kB、NOD1/2-RIP2-NF-kB/MAPK信号通路在ETEC诱导的猪肠道黏膜炎性损伤中的作用及香砂六君子汤及中药成分调控作用机制。本研究不仅证实了香砂六君子汤及中药成分白芍总苷及甘草多糖可有效缓解ETEC感染引起的断奶仔猪腹泻，维护肠道绒毛及黏膜完整性，减轻肠道组织炎性病理损伤，并可通过PPAR、IL-17、NF-κB、MAPK、细胞因子-细胞因子受体相互作用及NOD样受体等信号通路调节ETEC诱导的肠道炎症，提高肠道厚壁菌门细菌含量，维护仔猪肠道益生菌群稳定；而且明确了香砂六君子汤可通过NF-kB/MAPK信号通路抑制炎性细胞因子表达，缓解ETEC感染引起的仔猪肠道炎性损伤；白芍总苷及甘草多糖均可显著增加断奶腹泻仔猪肠道菌群的丰富度和多样性，并主要通过NF-kB/MAPK信号通路发挥抑制肠道炎性反应作用。同时，本研究明确了ETEC感染可通过TLR2/4、NOD1/2-RIP2-NF-kB/MAPK信号通路诱导肠道上皮细胞炎性反应，而白芍总苷、芍药苷及甘草多糖可通过以上通路发挥抑制细胞炎性反应，提高细胞活力作用；此外，也证实了黄连水提物、小檗碱可通过NF-kB/MAPK信号通路抑制LPS诱导的猪肠道上皮细胞炎性损伤，淫羊藿苷及磷酸化产物也具有抑制LPS诱导的上皮细胞炎性及氧化应激损伤作用。综上所述，本研究不仅揭示了ETEC感染引起断奶仔猪腹泻及肠道炎性损伤作用靶点，而且阐释了中药复方及成分的抗炎分子调控机制，也为筛选和研制可有效防治断奶仔猪腹泻的新药奠定了基础。