具粘附特性的动物双歧杆菌对便秘模型小鼠血清中胃肠调节肽水平的影响
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摘要
目的:研究具有较好粘附上皮细胞能力和耐受胃肠液的动物双歧杆菌CCFM625对便秘模型小鼠血清中胃肠调节肽(胃动素、胃泌素、乙酰胆碱酯酶、P物质、内皮素、生长抑素以及血管活性肠肽)水平的影响。方法:将BALB/c雄性小鼠随机分为空白组、模型组、阳性对照组(酚酞和植物乳杆菌ST-Ⅲ)、2个剂量的阴性对照(不具备粘附特性的动物双歧杆菌CCFM624)和2个剂量的给药组。给药组分别经灌胃连续给予小鼠14 d低、高剂量(4×10~8、4×10~(10)CFU/mL)的双歧杆菌CCFM625菌悬液0.25 mL;空白组和模型组给予0.25 mL的无菌生理盐水;阳性药物对照组给予7 mg/mL酚酞溶液0.25 mL,阳性对照组给予低、高剂量(4×10~8,4×10~(10)CFU/mL)的植物乳杆菌ST-Ⅲ菌悬液0.25 mL,阴性对照组低、高剂量(4×10~8,4×10~(10) CFU/mL)的动物双歧杆菌CCFM624菌悬液0.25 mL。第15～17天用盐酸洛哌丁胺灌胃小鼠造便秘模型,建模后于第18天上午处死小鼠,应用酶联免疫法检测小鼠血清中便秘相关肠肽类递质的表达情况。结果:具有较强粘附能力的动物双歧杆菌CCFM625可以显著提高血清中促进性递质(胃动素、胃泌素、乙酰胆碱酯酶和P物质)的分泌,同时降低抑制性递质(内皮素、生长抑素和血管活性肠肽)的水平。表明动物双歧杆菌CCFM625通过粘附于肠上皮细胞达到调节肠道内便秘相关的胃肠调节肽的分泌来缓解便秘。
Objective:To study the effect of Bifidobacterium animalis CCFM625 which has good adhesion properties and tolerance capacity to simulated gastric and small intestine juices on the levels of gastrointestinal peptide neurotransmitter(motilin(MTL),gastrin(Gas),acetylcholinesterase(AchE),substance P(SP),endothelin(ET),somatostatin(SS) and vasoactive intestinal peptide(VIP) in serum of mice with constipation.Methods:The mice were randomly divided with eight mice in each of 9 groups:the normal group(saline solution or distilled water),the constipation control group(loperamide 10 mg/kg body weight),two groups with different doses of B.animalis CCFM 624 or 625(i.e.,1×10~(10) CFU or 1×10~8 CFU B.animalis CCFM 624 or 625;for a total of four groups),a positive drug control group(phenolphthalein 70 mg/kg body weight) and two positive control groups(L.plantarum ST-III at either a high dose of 1×10~(10) CFU or a low dose of 1×10~8 CFU).The control groups were given normal saline solution once per day via gavage for 17 days.The high-dose B.animalis CCFM 624 and CCFM 625 and L.plantarum ST-III groups and the low-dose B.animalis CCFM 624 and CCFM 625 and L.plantarum ST-III groups received 0.25 mL of 4×10~(10) CFU/mL or 4×10~8 CFU/mL bacterial suspension,respectively,in the same manner as the treatment groups for 14 days,and the positive drug control group was treated with a 70 mg/kg body weight dose of phenolphthalein once per day for 14 days.The control and treatment groups were treated with loperamide(10 mg/kg body weight;0.25 mL) via gavage from Day 15 to 17 to induce constipation.At day 18,the mice were killed with light ether anesthesia,and their blood was collected in tubes for almost 2 h and centrifuged at 3 000×g for 15 min to obtain serum.MTL,Gas,AchE,ET,SS,SP and VIP levels in the serum were determined by an enzyme-linked immunosorbent assay(ELISA) instrument according to the manufacturer's instructions.Results:B.animalis CCFM625 which had strong adhesion ability could significantly improve the secretion of excitatory neurotransmitter in serum(MTL,Gas,AchE and SP),while reducd the inhibitory neurotransmitter(ET,SS and VIP) level.It is suggested that B.animalis CCFM625 can relieve constipation by adhering to intestinal epithelial cells to regulate the secretion of gastrointestinal peptide neurotransmitter.
Objective:To study the effect of Bifidobacterium animalis CCFM625 which has good adhesion properties and tolerance capacity to simulated gastric and small intestine juices on the levels of gastrointestinal peptide neurotransmitter(motilin(MTL),gastrin(Gas),acetylcholinesterase(AchE),substance P(SP),endothelin(ET),somatostatin(SS) and vasoactive intestinal peptide(VIP) in serum of mice with constipation.Methods:The mice were randomly divided with eight mice in each of 9 groups:the normal group(saline solution or distilled water),the constipation control group(loperamide 10 mg/kg body weight),two groups with different doses of B.animalis CCFM 624 or 625(i.e.,1×10~(10) CFU or 1×10~8 CFU B.animalis CCFM 624 or 625;for a total of four groups),a positive drug control group(phenolphthalein 70 mg/kg body weight) and two positive control groups(L.plantarum ST-III at either a high dose of 1×10~(10) CFU or a low dose of 1×10~8 CFU).The control groups were given normal saline solution once per day via gavage for 17 days.The high-dose B.animalis CCFM 624 and CCFM 625 and L.plantarum ST-III groups and the low-dose B.animalis CCFM 624 and CCFM 625 and L.plantarum ST-III groups received 0.25 mL of 4×10~(10) CFU/mL or 4×10~8 CFU/mL bacterial suspension,respectively,in the same manner as the treatment groups for 14 days,and the positive drug control group was treated with a 70 mg/kg body weight dose of phenolphthalein once per day for 14 days.The control and treatment groups were treated with loperamide(10 mg/kg body weight;0.25 mL) via gavage from Day 15 to 17 to induce constipation.At day 18,the mice were killed with light ether anesthesia,and their blood was collected in tubes for almost 2 h and centrifuged at 3 000×g for 15 min to obtain serum.MTL,Gas,AchE,ET,SS,SP and VIP levels in the serum were determined by an enzyme-linked immunosorbent assay(ELISA) instrument according to the manufacturer's instructions.Results:B.animalis CCFM625 which had strong adhesion ability could significantly improve the secretion of excitatory neurotransmitter in serum(MTL,Gas,AchE and SP),while reducd the inhibitory neurotransmitter(ET,SS and VIP) level.It is suggested that B.animalis CCFM625 can relieve constipation by adhering to intestinal epithelial cells to regulate the secretion of gastrointestinal peptide neurotransmitter.
引文
[1] GRAY J R. What is chronic constipation? Definition and diagnosis[J]. Can J Gastroenterol, 2016, 25Suppl B:7-10.
[2] LONGSTRETH G F, THOMPSON W G, CHEY W D, et al. Functional bowel disroders[J]. Gastroenterology, 2002, 130(5):1480-1491.
[3] RONALD B, STEWART M S, MARY T MOORE,et al. Correlates of constipation in an ambulatory elderly population[J]. The Am J Gas, 1992, 87(7):859.
[4] MURRAY F E, BLISS C M.老年人便秘问题[J].国外医学老年医学分册, 1991, 12(6):271.
[5]柯美云,罗金燕,许国铭,等.慢性便秘的诊治指南[J].中华内科杂志, 2004, 43(1):73-74.
[6]陈雪梅,邱翔.双歧杆菌的研究进展[J].营养与日粮, 2007, 2(214):15-17.
[7] BIBILONI R, PEREZ P F, DE ANTONI G L. An enzymatic-colourimetric assay for the quantification of Bifidobacterium[J]. J Food Prot, 2000, 63(3):322-326.
[8] COCONNIER M, KLAENHAMMER T R, KERNEIS S, et al. Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture[J]. Appl Environ Microbiol, 1992, 58(6):2034-2039.
[9] HOOD S K, E A ZOTTOA. Effect of low pH on the ability of lactobacillus acidophilus to survive and adhere to human intestinal cells[J]. J Food Sci,1998, 53(5):1514-1516.
[10] CLARKM P A, J H MARTIN. Survival of bifidobacteria under in vitro conditions simulating the human stomach[J]. Abstract D139. J. Dairy Sci.,1993, 76(1):136.
[11]陈军,常山,张雅萍,等.双歧杆菌粘附大鼠肠上皮细胞相关因素的研究[J].第三军医大学学报,2002, 24(2):138-141.
[12] BENRET M F, BRASSART D, NEESER J R, et al. Adhesion of human Bifidobacterial strain to cultured human intestinal epithelial cells and inhibition of Enteropathogen-cell interaction[J]. Applied and Environmental Microbiology, 1993, 59(12):4121-4128.
[13] G W TANNOCK. Colonization of tissue surfaces in the gastrointestinal tract of gnotobiotic animals by Lactobacillus strains[J]. Canadian Journal of Microbiology, 1982, 28(10):1196-1198.
[14] HUUB J M. Interaction of bifidobacterial lipoteichoic acid with human intestinal epithelial cells[J]. Infection and Immunity, 1985, 47(1):332-334.
[15] KIM J E, LEE Y J, KWAK M H, et al. Aqueous extracts of Liriope platyphylla induced significant laxative effects on loperamide-induced constipation of SD rats[J]. BMC Complementary and Alternative Medicine, 2013, 13(1):333.
[16] SCHULZ S, ROCKEN C, MAWRIN C, et al. Im munocyto chemical identification of VPAC1, VPAC2,and PAC1 receptors in normal and neoplastic human tissues with subtype-specific antibodies[J]. Clin Cancer Res, 2004, 10(24):8235-8242.
[17] PERACCHI M, BASILISCO G, TAGLIABUE R, et al. Postprandial gut peptide plasma levels in women with idiopathic slow transit constipation[J]. Scand J Gastroenterol, 1999, 34(1):25-28.
[18] STERN H P, STROH S E, FIEDOREK S C, et al.Increased plasma levels of pancreatic polypeptide and decreased plasms levels of motilin in encopretic children[J]. Rayford PL Pediatr, 1995, 96(1):111-117.
[19] EL-SALHY M, NORRGARD O. Colonic neuroendocrine peptide levels in patients with chronic idiopathic slow transit constipation[J]. Ups. J. Med. Sci.,1998, 103(3):223-230.
[20] COLUCCI W S. Myocardial endothelin does it play a role in myocardial failure?[J]. Circulation, 1996,93(6):1069-1072.
[21] SHI Y, CHEN Q, HUANG Y, et al. Function and clinical implications of short-chain fatty acids in patients with mixed refractory constipation[J]. Colorectal Dis., 2016, 18(8):803-810.
[22] FASTH S. Neuropeptides in idiopathic chronic constipation(slow transit constipation)[J]. Neurogastroenterol Motil, 1995, 9(3):143-150.
[2] LONGSTRETH G F, THOMPSON W G, CHEY W D, et al. Functional bowel disroders[J]. Gastroenterology, 2002, 130(5):1480-1491.
[3] RONALD B, STEWART M S, MARY T MOORE,et al. Correlates of constipation in an ambulatory elderly population[J]. The Am J Gas, 1992, 87(7):859.
[4] MURRAY F E, BLISS C M.老年人便秘问题[J].国外医学老年医学分册, 1991, 12(6):271.
[5]柯美云,罗金燕,许国铭,等.慢性便秘的诊治指南[J].中华内科杂志, 2004, 43(1):73-74.
[6]陈雪梅,邱翔.双歧杆菌的研究进展[J].营养与日粮, 2007, 2(214):15-17.
[7] BIBILONI R, PEREZ P F, DE ANTONI G L. An enzymatic-colourimetric assay for the quantification of Bifidobacterium[J]. J Food Prot, 2000, 63(3):322-326.
[8] COCONNIER M, KLAENHAMMER T R, KERNEIS S, et al. Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture[J]. Appl Environ Microbiol, 1992, 58(6):2034-2039.
[9] HOOD S K, E A ZOTTOA. Effect of low pH on the ability of lactobacillus acidophilus to survive and adhere to human intestinal cells[J]. J Food Sci,1998, 53(5):1514-1516.
[10] CLARKM P A, J H MARTIN. Survival of bifidobacteria under in vitro conditions simulating the human stomach[J]. Abstract D139. J. Dairy Sci.,1993, 76(1):136.
[11]陈军,常山,张雅萍,等.双歧杆菌粘附大鼠肠上皮细胞相关因素的研究[J].第三军医大学学报,2002, 24(2):138-141.
[12] BENRET M F, BRASSART D, NEESER J R, et al. Adhesion of human Bifidobacterial strain to cultured human intestinal epithelial cells and inhibition of Enteropathogen-cell interaction[J]. Applied and Environmental Microbiology, 1993, 59(12):4121-4128.
[13] G W TANNOCK. Colonization of tissue surfaces in the gastrointestinal tract of gnotobiotic animals by Lactobacillus strains[J]. Canadian Journal of Microbiology, 1982, 28(10):1196-1198.
[14] HUUB J M. Interaction of bifidobacterial lipoteichoic acid with human intestinal epithelial cells[J]. Infection and Immunity, 1985, 47(1):332-334.
[15] KIM J E, LEE Y J, KWAK M H, et al. Aqueous extracts of Liriope platyphylla induced significant laxative effects on loperamide-induced constipation of SD rats[J]. BMC Complementary and Alternative Medicine, 2013, 13(1):333.
[16] SCHULZ S, ROCKEN C, MAWRIN C, et al. Im munocyto chemical identification of VPAC1, VPAC2,and PAC1 receptors in normal and neoplastic human tissues with subtype-specific antibodies[J]. Clin Cancer Res, 2004, 10(24):8235-8242.
[17] PERACCHI M, BASILISCO G, TAGLIABUE R, et al. Postprandial gut peptide plasma levels in women with idiopathic slow transit constipation[J]. Scand J Gastroenterol, 1999, 34(1):25-28.
[18] STERN H P, STROH S E, FIEDOREK S C, et al.Increased plasma levels of pancreatic polypeptide and decreased plasms levels of motilin in encopretic children[J]. Rayford PL Pediatr, 1995, 96(1):111-117.
[19] EL-SALHY M, NORRGARD O. Colonic neuroendocrine peptide levels in patients with chronic idiopathic slow transit constipation[J]. Ups. J. Med. Sci.,1998, 103(3):223-230.
[20] COLUCCI W S. Myocardial endothelin does it play a role in myocardial failure?[J]. Circulation, 1996,93(6):1069-1072.
[21] SHI Y, CHEN Q, HUANG Y, et al. Function and clinical implications of short-chain fatty acids in patients with mixed refractory constipation[J]. Colorectal Dis., 2016, 18(8):803-810.
[22] FASTH S. Neuropeptides in idiopathic chronic constipation(slow transit constipation)[J]. Neurogastroenterol Motil, 1995, 9(3):143-150.
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