黄芪多糖协同硫酸化淫羊藿多糖增强仔猪肠道黏膜免疫的作用机理研究

The combination of Astragalus polysaccharide and sulfated Epimedium polysaccharide (APS-sEPS) could enhance the immune effect of classical swine fever vaccine and porcine epidemic diarrhea vaccine, and promote the secretion of IFN-γ and sIgA in intestinal tissue. But the regulatory mechanism of APS-sEPS on piglets’ intestinal mucosa immunity has not been clearly elucidated. Based on the preliminary studies, newborn piglets will be selected as the experimental model and drenched of APS-sEPS, with using APS, sEPS and the glucose as controls. The intestinal tissue structure and epithelial cell layer integrity will be observed by histology and immunohistochemisty methods. Changes of intestinal mucosal immunocytes and related cytokines will be assayed by flow cytometry and ELISA methods. The expression of TLR-4, IRF-3 and IRF-7 gene in piglet intestinal mucosal will be determined by Real-time PCR method. These results will reveal the action site and pattern of the synergetic local mucosal immune functions, and explore their relations with TLR-4 pathway. This study will be expected to clarify the immune enhancement mechanism of APS-sEPS on intestinal mucosa by TLR-4 signaling pathway in piglets. It can provide new ideas for research of Chinese materia medica preparation to improve the mucosal immune effect. At the same time, it can also supply scientific basis for the clinical application of traditional Chinese medicine to prevent and treat the diarrhea diseases of piglets, and to establish a green healthy model of breeding pigs. In summary, our results would have a very good practical significance for development of green ecological animal husbandry in China.

黄芪多糖和硫酸化淫羊藿多糖合用（APS-sEPS）能提高猪瘟疫苗和猪流行性腹泻疫苗的免疫效果，促进肠组织IFN-γ和分泌型IgA的分泌。但二者合用增强猪肠道黏膜局部免疫的作用机制还不明确。本项目拟以前期试验结果为基础，以新生仔猪为研究对象，采用组织学和免疫组化法观察仔猪肠道组织结构和上皮细胞层完整性的变化，流式细胞法和ELISA法检测仔猪肠道黏膜免疫细胞及相关细胞因子水平的变化，实时定量 PCR法测定仔猪小肠黏膜TLR-4、IRF-3和IRF-7基因mRNA表达量，揭示APS-sEPS调节仔猪肠黏膜局部免疫的作用部位、方式及与TLR-4通路的关系，阐释APS-sEPS介导TLR-4信号通路增强仔猪肠黏膜免疫的作用机理。试验结果为研制提高黏膜免疫效果的中药制剂提供了研究思路和方法，为临床应用中药防治仔猪腹泻性疾病、建立绿色健康养猪新模式提供科学依据，对发展我国绿色生态畜牧业有很好的现实意义。

前期研究结果表明，黄芪多糖和硫酸化淫羊藿多糖合用能显著提高鸡新城疫疫苗、禽流感疫苗的免疫效果，对鸡免疫抑制有较好的拮抗作用；能提高猪瘟疫苗和猪流行性腹泻疫苗的免疫效果，促进肠组织IFN-γ和分泌型IgA的分泌。但二者合用增强猪肠道黏膜局部免疫的作用机制还不明确。本项目拟在前期工作的基础上，研究黄芪多糖（APS）和硫酸化淫羊藿多糖（sEPS）配伍增强仔猪黏膜免疫效果的作用机理，通过动物试验测定APS和sEPS对仔猪小肠发育和上皮组织完整性的影响，并通过测定肠道黏膜免疫功能及其相关因子基因表达的变化，验证APS和sEPS协同调节仔猪肠黏膜局部免疫的作用，并阐明其作用机理。为临床应用中药成分复方防控仔猪腹泻性疾病提供科学依据。.试验结果得出：试验组仔猪各肠段绒毛完整、纤细、排列紧密，对照组绒毛有轻微损伤；APS-sEPS组空肠和回肠的绒毛长度明显变长，十二指肠、空肠和回肠的隐窝深度明显变短，并且APS-sEPS组绒毛长度和隐窝深度的比值（V/C值）在空肠段和回肠段显著高于对照组；流式细胞术结果发现，试验组肠系膜淋巴结CD4+ T和CD8+ T细胞百分率与对照组差异不显著，且CD4/CD8比值下降，但是，试验组中不同肠段肠黏膜中IL-4、IL-10及分泌型IgA的分泌量明显升高，尤其在回肠段与对照组有显著差异。应用荧光定量PCR法测定小肠黏膜TLR-4、IRF-3和NF-κB基因mRNA表达量的变化，结果发现，APS-sEPS组TLR4、NF-κB、IRF-3 mRNA的表达量在不同的小肠段均显著高于对照组。.通过以上结果得出APS-sEPS是一种肠道黏膜免疫增强剂，能促进仔猪小肠的发育，增强肠道黏膜B细胞的免疫功能。APS-sEPS的免疫增强作用可能通过激活TLR4信号转导途径来实现。根据IRF3分泌量升高的结果，推测APS-sEPS增强免疫可能还和巨噬细胞和胞内信号分子的传递有关。