肠道菌群介导半乳糖促进小鼠采食的机制研究

Mechanism of Intestinal Flora Mediated Galactose Promoting Feeding in Mice

本试验旨在研究肠道菌群介导半乳糖促进小鼠采食的机制。首先选用40只3周龄C57/BL6J公鼠,随机分为4组,每组10只,对照组给予饮用双蒸水,试验组分别给予半乳糖浓度分别为0.5%、1.0%和1.5%的饮水,记录每周体重和采食量。根据剂量筛选试验结果选定1.0%为后续试验饮水中半乳糖浓度后,利用在下丘脑神经肽Y(NPY)和阿片-促黑素细胞皮质素原(POMC)神经元特异性表达绿色荧光蛋白(GFP)的转基因小鼠,研究饮水添加半乳糖对小鼠下丘脑弓状核中这2种神经元兴奋性的影响。通过实时荧光定量PCR、超敏多因子电化学发光技术(MSD)以及Western blot检测对照组和饮水添加1.0%半乳糖的小鼠肠道中炎症信号的变化。通过对肠道菌群和代谢物的分析探讨半乳糖调控小鼠生长的途径,并以猪肠上皮细胞(IPEC-J2)为模型进行验证。最后选用32只3周龄C57/BL6J公鼠,随机分为4组,每组8只,利用抗生素构建伪无菌小鼠模型,验证肠道菌群在半乳糖促进采食中的作用。结果表明:1)相比于0.5%和1.5%的添加剂量,饮水添加1.0%半乳糖在更短的时间内发挥出促采食作用,且小鼠体重与添加剂量呈显著正相关(P<0.05),并显著提高小鼠的能量代谢水平,包括氧气消耗量、二氧化碳排出量和能量消耗量(P<0.05)。2)与对照组相比,饮水添加1.0%半乳糖能够显著增加小鼠下丘脑NPY神经元兴奋(P<0.05)。3)与对照组相比,饮水添加1.0%半乳糖显著降低小鼠肠道中半乳糖转运体1(SGLT1)的基因相对表达量(P<0.05),但显著提高了半乳糖代谢酶(GALK)以及短链脂肪酸受体(GPR)41和GPR43的基因相对表达量(P<0.05),小鼠肠道中炎症因子的基因相对表达量显著降低(P<0.01)。4)与对照组相比,饮水添加1.0%半乳糖显著提高了小鼠肠道疣微菌门和奈瑟菌科相对丰度以及肠道中短链脂肪酸和有机酸的含量(P<0.05)。5)代谢组中差异代谢物异丁酸可显著降低细胞中白细胞介素(IL)-6和IL-8的基因相对表达量(P<0.05)以及蛋白酪氨酸激酶(JAK)和蛋白激酶B(AKT)蛋白的表达水平(P<0.05)。6)使用抗生素消除小鼠肠道中的微生物后,半乳糖促进采食量的作用也随之消除。综上所述,饮水添加0.5%、1.0%和1.5%的半乳糖均可显著提高小鼠采食量,并且小鼠体重有随半乳糖添加剂量增加而升高的趋势;半乳糖可以改善小鼠肠道菌群组成,并在肠道菌群作用下发酵产生短链脂肪酸,降低肠道炎症,改善肠道健康。

The aim of this study was to investigate the mechanism of intestinal microbiota mediating galactose to promote feeding in mice.Firstly,40 three-week-old C57/BL6J male mice were randomly divided into 4groups with 10 mice in each group.The control group was given distilled water,and the experimental groups were given drinking water with 0.5%,1.0% and 1.5% galactose,respectively.According to the experimental results,1.0% was selected as the added dose for the subsequent test,using transgenic mice that specifically express green fluorescent protein(GFP)in hypothalamic neuropeptide Y(NPY)and proopiomelanocortin(POMC)neurons,the effects of galactose added to drinking water on the excitability of these two neurons in the arcuate nucleus of hypothalamus in mice were studied.The changes of inflammatory signals in the intestinal tract of mice in the control group and drinking water with 1.0% galactose were detected by real-time fluorescent quantitative PCR,hypersensitive multifactor electrochemiluminescence(MSD)and Western blot.Through the analysis of intestinal flora and metabolites,the way of galactose regulating the growth of mice was discussed,and the model was verified by porcine intestinal epithelial cells(IPEC-J2).Finally,32 three-week-old C57/BL6J male mice were randomly divided into 4 groups with 8 mice in each group,the pseudo sterile mouse model was constructed with antibiotics to verify the role of intestinal flora in galactose promoting feeding.The results are as follows:1)compared with the adding dosages of 0.5% and 1.5%,the addition galactose of1.0% to drinking water had a better effect on promoting feed intake,there was a significant positive correlation between the weight of mice and the adding dosages of galactose(P<0.05),and significantly improved the level of energy metabolism in mice,including oxygen consumption,carbon dioxide emission and energy consumption(P<0.05).2)Compared with the control group,the excitability of NPY neurons in arcuate nucleus of hypothalamus increased significantly when drinking water was added with 1.0% galactose(P<0.05).3)Compared with the control group,the addition of 1.0% galactose to drinking water significantly reduced the relative expression level of galactose transporter 1(SGLT1)gene in the intestine(P<0.05),but significantly increased the relative expression levels of galactose metabolic enzyme(GALK)and short chain fatty acid receptor(GPR)41 and GPR43 genes(P<0.05),the relative expression levels of inflammatory factors in the intestinal tract of mice decreased significantly(P<0.01).4)Compared with the control group,adding 1.0%galactose to drinking water significantly increased the relative abundance of Verrucomicrobia and Neisseriaceae in intestinal tract of mice,as well as the contents of short chain fatty acids and organic acids in the intestine(P<0.05).5)Isobutyric acid,a differential metabolite in the metabolic group,could significantly reduce the relative expression levels of interleukin(IL)-6 and IL-8 genes(P<0.01)genes and the phosphorylation levels of protein tyrosine kinase(JAK)and protein kinase B(AKT)(P<0.05).6)After the microbes in the gut of mice were eliminated by antibiotics,the effect of galactose on feed intake was also eliminated.In conclusion,the addition of 0.5%,1.0% and 1.5% galactose to drinking water can increase the feed intake of mice,and the body weight of mice has a trend to increase with the increase of galactose adding dosages;galactose can improve the composition of intestinal flora in mice,ferment to produce short chain fatty acids under the action of intestinal flora,reduce intestinal inflammation and improve intestinal health.[Chinese Journal of Animal Nutrition,2022,34(11):7466-7480]