壳聚糖微球对断奶仔猪肠道上皮的免疫调节机制研究

Segregated early weaning (SEW), introduced with the objectives of achieving higher production, is becoming increasingly popular in swine producers. However, the occurrence of post weaning diarrhea (PWD) in early-weaned piglets is inevitable. PWD is the main cause of the death of piglets, which strongly limits the development of the swine breeding. Maintaining the piglet intestinal homeostasis through immunomodulation is an effective way of reducing the occurrences of PWD. The chitosan microsphere(CM) has been constructed successfully by the applicant and the preliminary studies have revealed that CM have more immunocompetence than chitosan. However, the immunomodulation function of CM in the intestinal tract of the early-weaned piglets has not been deliberated on. In this project, the effects of CM on growth performance and intestinal structure and function of early-weaned piglets will be evaluated with the comparation of chitosan molecules through a feeding experiment. The immunomodulation mechanisms of CM on intestinal epithelium will be dissected through assaying the secretion and expression level of the inflammatory cytokines and activation level of the key molecues in TLR4/NF-κB pathyway in the intestinal epithelial . These results will provide theoretical basis for application of CM in piglet daily to reduce the occurrences of PWD and therefore improve the piglets’ performance.

现代养猪业为了进一步提高生猪的生产潜力而采用仔猪隔离早期断奶技术（SEW）。而SEW不可避免地会引起仔猪断奶后腹泻综合征（PWD）。PWD是引起仔猪死亡的重要原因，严重制约着养猪业的发展。通过免疫调节，维持仔猪肠道稳态是减少PWD发生的有效途径。申请者已成功构建了壳聚糖微球（CM），前期研究发现CM具有比壳聚糖更强的免疫调节活性。但是，CM针对仔猪肠道的免疫调节作用尚不明确。因此，本项目拟通过饲养试验检测CM对早期断奶仔猪生长性能和肠道结构功能的影响，并与非微球化的壳聚糖分子进行比较，评价CM对仔猪肠道的免疫调节效应；通过检测肠上皮中炎症相关因子的分泌和炎症通路TLR4/NF-κB关键分子活化程度，解析CM对仔猪肠道黏膜免疫调节机制。为CM应用于仔猪饲料中减少PWD的发生，提高仔猪生产性能提供理论依据。

本研究成功制备出可添加于仔猪日粮中的高浓度壳聚糖微球（CM）粉体。为了评估CM作为一种饲料添加剂对断奶仔猪的应用效果，将144只仔猪分成基础饲粮添加0、100、200、400 mg/kg CNP4个饲粮处理，每个处理分6栏，每栏6头仔猪。饲喂28 d后测生长性能指标。结果显示，平均日增重（ADG）随CM添加浓度的升高而身高, 料肉比（F/G）和腹泻率则随CM添加浓度的升高而降低。这表明CM能改善仔猪的生长性能，并具有一定的量效关系。CM的最适添加范围为200-400mg/kg。血浆中免疫球蛋白和补体浓度检测结果显示CM能提高血浆中IgA、IgG、C3、C4浓度，而对IgM浓度没有影响。在免疫应激实验中，随机选取对照组和400mg/kg CM添加组的24只仔猪。每处理组一半的猪 (n = 6)腹腔注射大肠杆菌脂多糖(LPS)。另一半的猪在注射无菌生理盐水。检测血浆中应激轴相关激素和免疫因子浓度，结果发现CM能缓解血浆中LPS应激引起的皮质醇、PEG2,IL-6,和IL-1β的上升，但对TNF-α的分泌有轻微刺激作用。结肠内容物16SrRNA测序分析结果显示。日粮中添加CM可改善肠道菌群结构，增加一些可能有益的肠道细菌的数量，同时抑制潜在细菌病原体的生长。这些结果表明，饲粮中添加CNP可改善断奶仔猪的生长性能和免疫状态，减轻免疫应激，调节肠道生态。.肠黏膜免疫因子分析结果显示CM可通过刺激肠道免疫效应因子sIgA的分泌和抑制肠道前炎性因子的分泌而抑制由LPS引起的肠道损伤和炎症的发生和发展。CM的抗炎机制与其抑制NF-κB活化转位有关。细胞试验结果显示CM可减轻LPS诱导的Caco-2单层跨上皮电阻(transepielectrical resistance, TEER)的下降，并以剂量依赖性的方式显著抑制LPS诱导的TNF-a、MIF、IL-8和MCP-1等前炎性因子的生成。West-blot结果分析显示CM可以抑制LPS刺激的Caco-2细胞中IκB-α的降解和p65的转位。 结合动物试验结果，CM可能通过NF-κB信号通路部分减轻LPS诱导的炎症反应。CNP通过抑制IκB-α的降解和p65的易位，导致NF-κB活化的减少，进而导致LPS诱导的促炎细胞因子的生成减少，减轻对肠上皮的炎症损伤。