红树莓酵素对伊犁马肠道菌群结构及其免疫调节机制的研究

Study on the effects of red raspberry enzyme on the intestinal microbiota structure and immune regulation mechanism in Yili horse

为研究红树莓酵素对伊犁马肠道形态、菌群结构及免疫系统调节作用的机制,本研究选取24匹健康状况良好、遗传背景一致,公母各半的伊犁马,随机平均分为4组,分别为空白对照组、低剂量红树莓酵素添加组(0.1 g/kg)、中剂量红树莓酵素添加组(0.2 g/kg)和高剂量红树莓酵素添加组(0.4 g/kg),实验周期28 d。饲料中给药后观察并计算各组伊犁马的腹泻率,结果显示各实验组与对照组间均无差异(P>0.05),表明红树莓酵素对伊犁马无毒副作用,可以进一步实验。各组饲喂至28 d时选取2匹中等、健康状况良好的伊犁马,麻醉后分离带有食糜的盲肠肠段和小肠肠段,测定其微生物菌群和肠道pH,采用qPCR检测肠组织中纤维素酶关键酶基因melA的转录情况,并进一步观察其组织形态,结果显示,与对照组相比,红树莓酵素添加组伊犁马空肠总菌(大肠杆菌、双歧杆菌和乳酸杆菌)含量和肠道pH值得到显著改善(P<0.05),且呈红树莓酵素剂量依赖性。红树莓酵素添加组马肠组织中纤维素酶基因melA转录水平显著提高,且呈红树莓酵剂量依赖性(P<0.05)。电镜下观察小肠黏膜形态结构结果显示,红树莓酵素高剂量添加组马小肠绒毛排列整齐且密集,长度略有增加,黏膜形态结构明显最优;其次是红树莓酵素中剂量添加组,最后是红树莓酵素低剂量添加组和空白对照组。采集各组马血液分离单核细胞,采用ELISA检测伊犁马单核细胞中细胞因子TNF-α、IFN-β、IL-1β、IL-8的含量;采用qPCR测定各组马血清中免疫相关基因的转录水平。ELISA检测结果显示,与空白对照组相比,各红树莓酵素添加组伊犁马单核细胞中细胞因子IFN-β含量均显著增加(P<0.05),而TNF-α、IL-1β、IL-8含量均显著下降(P<0.05),且呈红树莓酵剂量依赖性。qPCR结果显示,红树莓酵素添加组马血清中免疫相关基因IFN-β的转录水平均显著上调,而免疫相关基因TNF-α、IL-1β、IL-8的转录水平均显著下调,且呈红树莓酵剂量依赖性(P<0.05)。本研究首次证实红树莓酵素可通过改变马肠道p H值使其肠道菌群数量显著优化(P<0.05),改善肠道黏膜形态,且增强伊犁马的免疫力,该研究为马的无抗养殖和疫病防控奠定了基础。

This article mainly studies the regulating mechanism of red raspberry enzyme on the intestinal morphology and immune system of Yili horses. The diarrhea rate of Yili horses in each group was observed and calculated, and the results showed that there was no difference between the experimental groups(P>0.05), indicating that the red raspberry enzyme has no toxic and side effects on Yili horses. Thus, these results made further experiments possible. Two Yili horses of medium and good health were selected when each group was fed for 28 days. After anesthesia, the caecal intestine segment with chyme was taken. The microbial flora was measured, and the tissue morphology was observed. Blood was collected from each group of horses to isolate monocytes,using enzyme-linked immunoassay(ELISA) to detect the cytokine TNF-α, IFN-β, IL-1β, IL-8 content in the supernatant of Yili horse monocyte culture;using fluorescence quantitative polymerase chain reaction(real-time PCR) technology to determine the expression levels of key enzymes and immune-related genes. The results of total jejunum and intestinal p H showed that compared with the control group, the content of total jejunum(E. coli, Bifidobacterium and Lactobacillus) and intestinal p H was significantly improved(P<0.05), and is dose-dependent with red raspberry enzyme. Observation of the morphological structure of the small intestine mucosa under the electron microscope showed that the Yili horse of the red raspberry enzyme high-dose supplement group were arranged neatly and densely, and the length was slightly increased. The mucosal morphology and structure of the high-dose group were obviously the best;and the second was the red raspberry enzyme medium-dose treatment group;and the last was red raspberry enzyme low-dose treatment group and blank control group. The ELISA test results showed that the content of cytokine IFN-β in the supernatant of Yili horse monocytes in the red raspberry enzyme treatment group increased significantly(P<0.05),while the content of TNF-α, IL-1β, and IL-8 decreased significantly(P<0.05), and it is dose-dependent with red raspberry enzyme.Real-time PCR results showed that the cellulase gene melA and immune-related gene IFN-β were significantly up-regulated in the red raspberry enzyme treatment group, while the immune-related genes TNF-α, IL-1β, and IL-8 were all significantly downregulated, and it is dose-dependent with red raspberry enzyme(P<0.05). In this study, by adding red raspberry enzyme to the feed and analyzing its effects on Yili horse intestinal microbiota structure and immune regulation, we are the first to prove that red raspberry enzyme can significantly increase the number of intestinal flora by changing the pH value of the intestine(P<0.05) and optimize the morphology of the intestinal mucosa. Feeding horses with red raspberry enzyme at the same time can enhance the immune system of Yili horses and provide support for the improvement of Yili horse feed nutrition in the future.