银杏叶复方防治脾虚性断奶仔猪腹泻的作用和机理研究

Post-weaning diarrhea has long been a handicap in pig industry. Ginkgo biloba has not been well developed though ginkgo is ample in China. Based on our previous observations that Ginkgo biloba compound could increase the activity of digestive enzymes, improve the gut absorption function and correct the immune deficiency of the model mice with spleen deficiency, this subject is first to establish the mice model and the piglets model with spleen deficiency, and to determine a compound Ginkgo biloba formula that is effective for the weaned piglets model, then in vivo and vitro studies were made to determine the acute toxicity and cytotcoxicity of Ginkgo biloba compound to make sure the safety of clinical application. and then animal assay continues, observing the influences on the intestinal mucosal immunity, the activity of digestive enzyme, the rate of gastrointestinal transit and the alteration of intestinal microflora in piglets model, in order to elucidate the underlying mechanism for the effects of Ginkgo biloba compound on invigorating the spleen to arrest diarrhea. Finally, Chinese veterinary theory and orthogonal design method is used to explore the formula mechanism of the compound, and to determine the composition of herbs and dose of each herb in the compound. Our research is valuable to develop Ginkgo leaves as a new animal feed additive and to enhance the preventive effects on post-weaning diarrhea, and also provides useful hints on the utilization of Chinese medicine for the spleen deficiency, the diseases prevention and the growth promotion in the animal-feed industry.

断奶仔猪腹泻是影响养猪业的难题，我国银杏资源丰富，银杏叶尚未充分开发利用。本项目在前期研究中发现银杏叶复方能提高脾虚小鼠消化酶活性、促进肠道吸收功能、纠正免疫功能低下的基础上，首先拟建立小鼠脾虚模型、断奶仔猪脾虚模型，筛选出防治脾虚性断奶仔猪腹泻效果较好的银杏叶复方制剂，通过体内外不同途径和方法测定该复方制剂对小鼠的急性毒性和细胞毒性，以确保临床用药安全，然后进行临床验证，研究经筛选出的银杏叶复方对脾虚断奶仔猪免疫功能、肠道黏膜免疫、消化酶活性、肠道推进率和肠道菌群数量变化等的影响，阐明银杏叶复方健脾止泻的机理，最后运用中兽医理论与正交设计法，研究可增强脾虚小鼠肠道黏膜免疫功能的银杏叶复方的组方机制，确定复方中各中药的组成和剂量。本研究不仅对开发利用银杏叶、提高断奶仔猪腹泻的防治效果具有实用价值，而且为应用中药调控脾虚、开发抗病促壮中药饲料添加剂提供思路。

根据项目研究计划，成功建立了脾虚模型小鼠作为研究脾虚性断奶仔猪的标准试验动物，选用三种银杏叶复方（YXF），通过建立小鼠脾虚模型和免疫抑制模型，观察其血液学指标、肠道、肝、脾组织病理学变化、胃肠激素、血清消化酶活性等指标，经与经典方“四君子汤”(SJZ)比较，不仅筛选出了一种防治脾虚效果较好的YXF，而且发现脾虚证模型小鼠存在神经一内分泌一免疫网络之间的调节失衡，最终引起脾虚时的机体免疫功能低下，经YXF治疗后上述免疫失调得到明显纠正；同时研究发现YXF制剂通过调控细胞因子生成，对环磷酰胺（CY）介导的免疫抑制模型小鼠的细胞免疫发挥正向调节作用，能有效拮抗CY对肝脾组织造成的损伤，效果优于SJZ。YXF急性毒性及细胞毒性试验结果表明， LD50>40 g•kg-1体重，MTD>120 g•kg-1体重，小鼠无一例出现死亡，剖检未见明显肉眼可见的病理变化，说明药物毒性很小，临床使用安全。在形态学观察法中，YXF在高浓度时并不表现细胞毒性，在MTT法中YXF对CEF的最大安全质量浓度为0.78 g•L-1。临床验证结果表明，脾虚模型组的仔猪粪便异常，YXF组仔猪粪便基本成型；脾虚模型组的仔猪肝脾均有不同程度的损伤，而YXF组的损伤较小，形态与对照组较为接近，说明YXF对脾虚性腹泻断奶仔猪的肝脾损伤具有修复作用。通过观察脾虚性断奶仔猪肠道黏膜形态和上皮内淋巴细胞和杯状细胞数量的变化以及YXF的调控作用，发现YXF有维持和保护脾虚性断奶仔猪小肠黏膜的作用。对脾虚性断奶仔猪免疫功能研究表明，其血清中Th1型细胞因子如IL-1和Th2型细胞因子IL-6的含量较对照组均显著增加，Th1/ Th2细胞比例失调，可能的作用机制是由于脾虚证导致了机体免疫功能的失调，经YXF治疗后上述免疫失调得到明显纠正。探究不同浓度的YXF在LPS或PHA刺激下对脾虚小鼠肠淋巴细胞体外增殖的影响，结果表明YXF在200μg/mL、100μg/mL时能有效刺激正常和脾虚小鼠的肠淋巴细胞分裂增殖。最后从中兽医角度分析认为YXF制剂对脾虚性断奶仔猪小肠黏膜结构有保护作用，其作用与YXF组方中有效成分有关。采用正交设计，利用MTT法研究其组分对脾虚小鼠肠道淋巴细胞增殖的影响，结果与中兽医组方“君、臣、佐、使”原则并未一致，从本试验结果来看用正交设计方法来阐明中药复方的组方机制是需要更进一步研究的。