复合菌-荔枝多酚对腹泻小鼠肠道形态结构和肠道菌群的影响
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摘要
为评价复合菌-荔枝多酚对肠道形态结构和肠道菌群的影响,采用蓖麻油灌胃的方法,制造小鼠腹泻模型,造模成功后灌服不同药物治疗4 d后,取小鼠十二指肠、空肠和回肠固定到福尔马林中,进行苏木精-伊红染色,倒置显微镜下观察,测量各段小肠的绒毛长度、隐窝深度,计算绒腺比。无菌采集粪便用于肠道菌群聚合酶链式反应-变性梯度凝胶电泳(denaturing gradient gel electrophoresis,DGGE)检测,对DGGE图谱上的优势条带回收、克隆并测序,对测序结果进行BLAST比对分析。结果表明,嗜酸乳杆菌和复合菌能提高十二指肠、空肠的绒毛长度与隐窝深度的比值(V/C),复合菌-荔枝多酚能增加空肠和回肠的V/C值。整个DGGE图谱和测序结果可见,嗜酸乳杆菌、复合菌和复合菌-荔枝多酚能增加小鼠菌群的数量,增强肠道菌群的微生态稳定性,而且复合菌还能增加群落的复杂程度。各给药组条带与正常组和模型组相比,增加了优势条带,复合菌组和复合菌-荔枝多酚组还出现了新的条带,复合菌体现了最优的效果。
The effects of co-administration of a mixed probiotic culture of Lactobaccillus acidophilus and Clostridium butyricum and litchi polyphenol on the intestinal morphology and intestinal flora of mice with diarrhea were explored. A mouse model of diarrhea was established by intragastrical administration of castor oil. After four days of treatment with different medications, the duodenum, jejunum and ileum of mice were harvested and fixed in formalin solution for paraffin sectioning and HE staining, and then examined under an inverted microscope. Intestinal villus length and crypt depth were measured and the ratio of villus length to crypt depth(V/C) was also calculated. Feces were sterilely obtained for analysis of the change in intestinal microbiota by polymerase chain reaction(PCR)-denaturing gradient gel electrophoresis(DGGE). The dominant DGGE bands were recovered, cloned and sequenced, and the sequences were analyzed by BLAST. Results showed that Lactobacilli acidophilus alone and in combination with Clostridium butyricum could increase the V/C ratio of duodenum and jejunum, and the combination of the mixed probiotic culture and litchi polyphenol could improve the V/C ratio of jejunum and ileum. The DGGE fingerprint and the sequencing results showed that Lactobacilli acidophilus, the mixed probiotic culture alone and in combination with litchi polyphenol could increase the abundance and micro-ecological stability of intestinal flora in mice with diarrhea. In addition, the mixed probiotics could also increase the complexity of the intestinal bacterial community. More dominant bands were observed for all three treatment groups compared with the normal and model groups. Furthermore, new bands were also obtained with the mixed probiotics and its combination with litchi polyphenol. The best effect was observed with the mixed probiotics.
The effects of co-administration of a mixed probiotic culture of Lactobaccillus acidophilus and Clostridium butyricum and litchi polyphenol on the intestinal morphology and intestinal flora of mice with diarrhea were explored. A mouse model of diarrhea was established by intragastrical administration of castor oil. After four days of treatment with different medications, the duodenum, jejunum and ileum of mice were harvested and fixed in formalin solution for paraffin sectioning and HE staining, and then examined under an inverted microscope. Intestinal villus length and crypt depth were measured and the ratio of villus length to crypt depth(V/C) was also calculated. Feces were sterilely obtained for analysis of the change in intestinal microbiota by polymerase chain reaction(PCR)-denaturing gradient gel electrophoresis(DGGE). The dominant DGGE bands were recovered, cloned and sequenced, and the sequences were analyzed by BLAST. Results showed that Lactobacilli acidophilus alone and in combination with Clostridium butyricum could increase the V/C ratio of duodenum and jejunum, and the combination of the mixed probiotic culture and litchi polyphenol could improve the V/C ratio of jejunum and ileum. The DGGE fingerprint and the sequencing results showed that Lactobacilli acidophilus, the mixed probiotic culture alone and in combination with litchi polyphenol could increase the abundance and micro-ecological stability of intestinal flora in mice with diarrhea. In addition, the mixed probiotics could also increase the complexity of the intestinal bacterial community. More dominant bands were observed for all three treatment groups compared with the normal and model groups. Furthermore, new bands were also obtained with the mixed probiotics and its combination with litchi polyphenol. The best effect was observed with the mixed probiotics.
引文
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[2]GEVER D,KUGATHASAN S,DENSON L A,et al.The treat-mentnaive microbiome in new-onset Crohn’s disease[J].Cell Host&Microbe,2014,15(3):382-392.DOI:10.1016/j.chom.2014.02.005.
[3]AHN J,SINHA R,PEI Zhiheng,et al.Human gut microbe-ome and risk for colorectal cancer[J].National Cancer Institute,2013,105(24):1907-1911.DOI:10.1093/jnci/djt300.
[4]SAULNIER D M,RIEHLE K,MISTRETTA T A,et al.Gastrointestinal microbiome signatures of pediatric patients with irritable bowel syndrome[J].Gastroenterology,2011,141(5):1782-1791.DOI:10.1053/j.gastro.2011.06.072.
[5]MAZMANIAN S K,ROUND J L,KASPER D L.A microbial symbiosis factor prevents intestinal inflammatory disease[J].Nature,2008,453:620-625.DOI:10.1038/nature07008.
[6]孙芝兰.卷曲乳杆菌益生特性及黏附机理研究[D].济南:山东大学,2012:4.
[7]DAVID L A,MAURICE C F,CARMODY R N,et al.Diet rapidly and reproducibly alters the human gut microbiome[J].Nature,2014,505:559-563.DOI:10.1038/nature12820.
[8]GIBSON G R,PROBERT H M,LOO J V,et al.Dietary modulation of the human colonic microbiota:updating the concept of prebiotics[J].Nutrition Research Reviews,2004,17(2):259-275.DOI:10.1079/NRR200479.
[9]NICHOLSON J K,HOLMES E,KINROSS J,et al.Host-gut microbiota metabolic interactions[J].Science,2012,336:1262-1267.DOI:10.1126/science.1223813.
[10]DASTAR B,KHOSRAVI A,BOLDAJIE F,et al.Effect of calcium with and without probiotic,lactose,or both on organ and body weights,immune response and caecal microbiota in moulted laying hens[J].Animal Physiology and Animal Nutrition,2016,100(2):243-250.DOI:10.1111/jpn.12358.
[11]姜东京,张丽,曹雨诞,等.肠道菌群在中药研究中的应用[J].中国中药,2016,41(17):3218-3225.
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[15]王丽凤.益生菌L.plantarum P-8对肉鸡肠道菌群、肠道免疫和生长性能影响的研究[D].呼和浩特:内蒙古农业大学,2014:2-10.
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[19]CASPARY W F.Physiology and pathophysiology of intestinal absorption[J].American Journal of Clinical Nutrition,1992,55(1):299-303.
[20]王春荣,刘学飞,程永刚.肠功能恢复汤对感染性多器官功能障碍综合征大鼠肠黏膜损伤的保护作用[J].郑州大学学报,2008,43(6):13-15.DOI:10.13705/j.issn.1671-6825.2008.06.015.
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[23]JOHNSON K C,MITCHELL D J,AVITZUR Y,et al.Probiotics reduce bacterial colonization and gastric inflammation in H.pyloriinfected mice[J].Digestive Diseases and Sciences,2004,49(7):1095-1102.DOI:10.1023/B:DDAS.0000037794.02040.c2.
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[27]张扬,袁杰利.肠道益生菌对机体免疫功能的影响[J].中国微生态学杂志,2003,15(4):246-248.DOI:10.13381/j.cnki.cjm.2003.04.036.
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