肠道微生态对腹泻型肠易激综合征的影响及其作用机制探讨
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摘要
目的 观察肠道微生态对腹泻型肠易激综合征(irritable bowel syndrome,IBS)的影响情况,探讨其可能的作用机制。方法 将32只SD大鼠均分为4组,每组8只。IBS-D组、益生菌组和TLR4拮抗剂组首先分别给予乙酸灌肠+束缚应激造模,同时益生菌组每日予70 mg/ml双歧杆菌三联活菌胶囊350 mg/(kg?d)灌胃,TLR4拮抗剂组予TLR4拮抗剂TAK-2423 mg/(kg?d)腹腔注射。空白对照组正常饲养。荧光定量PCR法测定各组大鼠肠道菌群含量,qRT-PCR法检测肠道上皮TLR4基因表达情况,ELISA法检测结肠组织匀浆中TNF-α及IL-10水平。结果 大便性状评分:IBS-D组和拮抗剂组均高于空白对照组,也均高于益生菌组,拮抗剂组低于IBS-D组;内脏敏感性评分:IBS-D组大鼠球囊容量均明显低于其它三组,且随着评分增加,IBS-D组大鼠球囊容量上升不明显,球囊容量为0.8 ml时,IBS-D组大鼠腹壁收缩次数明显高于其他三组大鼠;肠道菌群:IBS-D组和拮抗剂组大肠埃希菌和肠球菌含量均高于对照组,B/E比值均<1;TLR4表达:IBS-D组、益生菌组及拮抗剂组大鼠TLR4表达均高于对照组,益生菌组及拮抗剂组大鼠TLR4表达均低于IBS-D组;炎性因子:IBS-D组大鼠结肠组织中TNF-α水平均高于其他三组,IL-10水平明显低于其他三组;以上差异均有统计学意义(均P <0.05)。另外,益生菌组大鼠结肠组织中TNF-α和IL-10水平与对照组相当,拮抗剂组大鼠结肠组织中TNF-α水平与对照组相当,益生菌组与对照组各菌群含量相当,且B/E> 1,拮抗剂组大鼠TLR4表达与益生菌组相当,差异均无统计学意义(均P> 0.05)。结论 腹泻型IBS发生发展中,肠道微生态失调致病机制之一可能是活化TLR4,诱发产生炎性因子,增高内脏敏感性,进而产生或加重IBS症状。益生菌制剂可能通过调节肠道微生态,下调TLR4表达,改善肠道敏感性,从而缓解IBS患者腹痛与大便性状改变等症状。
Objectives To observe the effect of intestinal microbiota on diarrhea-predominant irritable bowel syndrome and explore its possible mechanism. Methods 32 male SD rats were divided into 4 groups. Acetic acid enema and restraint stress were given respectively to IBS-D group, probiotics group and TLR4 antagonist group. Meanwhile, the probiotic group was given intragastric administration with 70 mg/ml Bifidobacterium triple viable capsule 350 mg/(kg·d) daily, and the TLR4 antagonist group was given TLR4 antagonist TAK-242 3 mg/(kg·d) by intraperitoneal injection. Blank control group was normal feeded. The intestinal flora was analyzed by fluorescent quantificative PCR, the mRNA expression of TLR4 was detected by RT-PCR, and the levels of TNF-α and IL-10 were detected by ELISA. Results The Bristol stool scores of IBS-D group and antagonist group were significantly higher than blank control group and probiotics group, and antagonist group was lower than IBS-D group. The balloon volume of IBS-D group was significantly lower than the other three groups,and with the increase of AWR score, the balloon volume of IBS-D group did not increase significantly, and the number of abdominal contraction of IBS-D group was significantly higher than the other three groups when the balloon volume was0.8 ml. The abundance levels of E.coli and Enterococcus in IBS-D group and antagonist group were higher than control group, B/E<1; the expression levels of TLR4 in IBS-D group, probiotic group and antagonist group were higher than control group, and the expression levels of TLR4 in probiotic group and antagonist group were lower than IBS-D group. The level of TNF-α in IBS-D group was higher than the other groups,and the level of IL-10 was significantly lower than the other three groups(P<0.05). In addition, the levels of TNF-α and IL-10 in probiotic group were similar to control group,the level of TNF-α in antagonist group was similar to control group, the probiotic group and the control group had the same content of intestinal flora, B/E>1,and the expression level of TLR4 in antagonist group was similar to probiotic group, with no significant difference(P>0.05). Conclusion During the occurrence and development of IBS-D, one of the pathogenic mechanisms of intestinal microbiota dysbiosis may be activation of TLR4, inducing the production of inflammatory factors, increasing visceral sensitivity, and then producing or aggravating IBS symptoms. Probiotics may improve intestinal sensitivity by regulating intestinal microbiota and decreasing the expression of TLR4, thus alleviating symptoms such as abdominal pain and changes in stool trait in IBS patients.
Objectives To observe the effect of intestinal microbiota on diarrhea-predominant irritable bowel syndrome and explore its possible mechanism. Methods 32 male SD rats were divided into 4 groups. Acetic acid enema and restraint stress were given respectively to IBS-D group, probiotics group and TLR4 antagonist group. Meanwhile, the probiotic group was given intragastric administration with 70 mg/ml Bifidobacterium triple viable capsule 350 mg/(kg·d) daily, and the TLR4 antagonist group was given TLR4 antagonist TAK-242 3 mg/(kg·d) by intraperitoneal injection. Blank control group was normal feeded. The intestinal flora was analyzed by fluorescent quantificative PCR, the mRNA expression of TLR4 was detected by RT-PCR, and the levels of TNF-α and IL-10 were detected by ELISA. Results The Bristol stool scores of IBS-D group and antagonist group were significantly higher than blank control group and probiotics group, and antagonist group was lower than IBS-D group. The balloon volume of IBS-D group was significantly lower than the other three groups,and with the increase of AWR score, the balloon volume of IBS-D group did not increase significantly, and the number of abdominal contraction of IBS-D group was significantly higher than the other three groups when the balloon volume was0.8 ml. The abundance levels of E.coli and Enterococcus in IBS-D group and antagonist group were higher than control group, B/E<1; the expression levels of TLR4 in IBS-D group, probiotic group and antagonist group were higher than control group, and the expression levels of TLR4 in probiotic group and antagonist group were lower than IBS-D group. The level of TNF-α in IBS-D group was higher than the other groups,and the level of IL-10 was significantly lower than the other three groups(P<0.05). In addition, the levels of TNF-α and IL-10 in probiotic group were similar to control group,the level of TNF-α in antagonist group was similar to control group, the probiotic group and the control group had the same content of intestinal flora, B/E>1,and the expression level of TLR4 in antagonist group was similar to probiotic group, with no significant difference(P>0.05). Conclusion During the occurrence and development of IBS-D, one of the pathogenic mechanisms of intestinal microbiota dysbiosis may be activation of TLR4, inducing the production of inflammatory factors, increasing visceral sensitivity, and then producing or aggravating IBS symptoms. Probiotics may improve intestinal sensitivity by regulating intestinal microbiota and decreasing the expression of TLR4, thus alleviating symptoms such as abdominal pain and changes in stool trait in IBS patients.
引文
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[2]Brint E K,Macsharry J,Fanning A,et al.Differential expression of toll-like receptors in patients with irritable bowel syndrome[J].Am J Gastroenterol,2011,106(2):329-336.
[3]Belmonte L,Beutheu Youmba S,Bertiaux-Vandaele N,et al.Role of toll like receptors in irritable bowel syndrome:differential mucosal immune activation according to the disease subtype[J].PLoS One,2012,7(8):e42777-e42777.
[4]Ko?ak E,Akbal E,K?klüS,et al.The colonic tissue levels of TLR2,TLR4 and nitric oxide in patients with irritable bowel syndrome[J].Intern Med,2016,55(9):1043-1048.
[5]Marchesi JR,Adams DH,Fava F,et al.The gut microbiota and host health:a new clinical frontier[J].Gut,2016,65(2):330-339.
[6]Lewis SJ,Heaton KW.Stool form scale as a useful guide to intestinal transit time[J].Scand J Gastroenterol,1997,32(9):920-924.
[7]刘雁冰,袁耀宗,陶然君,等.大鼠肠道高敏性模型的建立及其内脏敏感性评估[J].中华消化杂志,2003,23(1):30-33.
[8]吴仲文,李兰娟,马伟杭,等.肠道微生物定植抗力的新指标--B/E值[J].浙江预防医学,2000,12(7):4-5.
[9]Shanahan F.The colonic microbiota in health and disease[J].Curr Opin Gastroenterol,2013,29(1):49-54.
[10]Pickard JM,Zeng MY,Caruso R,et al.Gut microbiota:role in pathogen colonization,immune responses,and inflammatory disease[J].Immunol Rev,2017,279(1):70-89.
[11]Piche T.Tight junctions and IBS-the link between epithelial permeability,low-grade inflammation,and symptom generation[J].Neurogastroenterol Motil,2014,26(3):296-302.
[12]Mayer EA,Savidge T,Shulman RJ.Brain-gut microbiome i n t e r a c t i o n s a n d f u n c t i o n a l b o w e l d i s o r d e r s[J].Gastroenterology,2014,146(6):1500-1512.
[13]Rajili?-Stojanovi?M,Biagi E,Heilig HG,et al.Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome[J].Gastroenterology,2011,141(5):1792-1801.
[14]黄汉忠,易星航.双歧三联活菌制剂对肠易激综合征患者肠运动功能影响及其疗效[J].世界临床药物,2015,36(5):327-330.
[15]Bhattarai Y,Muniz Pedrogo DA,Kashyap PC.Irritable bowel syndrome:a gut microbiota-related disorder[J].Am J Physiol Gastrointest Liver Physiol,2017,312(1):G52-G62.
[16]Bashashati M,Moossavi S,Cremon C,et al.Colonic immune cells in irritable bowel syndrome:a systematic review and meta-analysis[J].Neurogastroenterol Motil,2018,doi:10.1111/nmo.13192.
[17]Barbara G,Zecchi L,Barbaro R,et al.Mucosal permeability and immune activation as potential therapeutic targets of probiotics in irritable bowel syndrome[J].J Clin Gastroenterol,2012,doi:10.1097/MCG.0b013e318264e918.
[18]Liebregts T,Adam B,Bredack C,et al.Immune activation in patients with irritable bowel syndrome[J].Gastroenterology,2007,132(3):913-920.
[19]Villena J,Aso H,Kitazawa H.Regulation of toll-like receptorsmediated inflammation by immunobiotics in bovine intestinal epitheliocytes:role of signaling pathways and negative regulators[J].Front Immunol,2014,2(5):421-421.
[20]Rimoldi M,Chieppa M,Salucci V,et al.Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells[J].Nat Immunol,2015,16(3):326-326.
[21]Mckernan DP,Nolan A,Brint EK,et al.Toll-like receptor mRNA expression is selectively increased in the colonic mucosa of two animal models relevant to irritable bowel syndrome[J].PLoS One,2009,4(12):e8226-e8226.
[22]Tramullas M,Finger BC,Moloney RD,et al.Toll-like receptor4 regulates chronic stress-induced visceral pain in mice[J].Biol Psychiatry,2014,76(4):340-348
[23]Nozu T,Miyagishi S,Nozu R,et al.Lipopolysaccharide induces visceral hypersensitivity:role of interleukin-1,interleukin-6,and peripheral corticotropin-releasing factor in rats[J].J Gastroenterol,2017,52(1):72-80.
[24]Tang H,Zhang G,Ji Ning,et al.Toll-like receptor 4 in paraventricular nucleus mediates visceral hypersensitivity induced by maternal separation[J].Front Pharmacol,2017,8:309-320.
[2]Brint E K,Macsharry J,Fanning A,et al.Differential expression of toll-like receptors in patients with irritable bowel syndrome[J].Am J Gastroenterol,2011,106(2):329-336.
[3]Belmonte L,Beutheu Youmba S,Bertiaux-Vandaele N,et al.Role of toll like receptors in irritable bowel syndrome:differential mucosal immune activation according to the disease subtype[J].PLoS One,2012,7(8):e42777-e42777.
[4]Ko?ak E,Akbal E,K?klüS,et al.The colonic tissue levels of TLR2,TLR4 and nitric oxide in patients with irritable bowel syndrome[J].Intern Med,2016,55(9):1043-1048.
[5]Marchesi JR,Adams DH,Fava F,et al.The gut microbiota and host health:a new clinical frontier[J].Gut,2016,65(2):330-339.
[6]Lewis SJ,Heaton KW.Stool form scale as a useful guide to intestinal transit time[J].Scand J Gastroenterol,1997,32(9):920-924.
[7]刘雁冰,袁耀宗,陶然君,等.大鼠肠道高敏性模型的建立及其内脏敏感性评估[J].中华消化杂志,2003,23(1):30-33.
[8]吴仲文,李兰娟,马伟杭,等.肠道微生物定植抗力的新指标--B/E值[J].浙江预防医学,2000,12(7):4-5.
[9]Shanahan F.The colonic microbiota in health and disease[J].Curr Opin Gastroenterol,2013,29(1):49-54.
[10]Pickard JM,Zeng MY,Caruso R,et al.Gut microbiota:role in pathogen colonization,immune responses,and inflammatory disease[J].Immunol Rev,2017,279(1):70-89.
[11]Piche T.Tight junctions and IBS-the link between epithelial permeability,low-grade inflammation,and symptom generation[J].Neurogastroenterol Motil,2014,26(3):296-302.
[12]Mayer EA,Savidge T,Shulman RJ.Brain-gut microbiome i n t e r a c t i o n s a n d f u n c t i o n a l b o w e l d i s o r d e r s[J].Gastroenterology,2014,146(6):1500-1512.
[13]Rajili?-Stojanovi?M,Biagi E,Heilig HG,et al.Global and deep molecular analysis of microbiota signatures in fecal samples from patients with irritable bowel syndrome[J].Gastroenterology,2011,141(5):1792-1801.
[14]黄汉忠,易星航.双歧三联活菌制剂对肠易激综合征患者肠运动功能影响及其疗效[J].世界临床药物,2015,36(5):327-330.
[15]Bhattarai Y,Muniz Pedrogo DA,Kashyap PC.Irritable bowel syndrome:a gut microbiota-related disorder[J].Am J Physiol Gastrointest Liver Physiol,2017,312(1):G52-G62.
[16]Bashashati M,Moossavi S,Cremon C,et al.Colonic immune cells in irritable bowel syndrome:a systematic review and meta-analysis[J].Neurogastroenterol Motil,2018,doi:10.1111/nmo.13192.
[17]Barbara G,Zecchi L,Barbaro R,et al.Mucosal permeability and immune activation as potential therapeutic targets of probiotics in irritable bowel syndrome[J].J Clin Gastroenterol,2012,doi:10.1097/MCG.0b013e318264e918.
[18]Liebregts T,Adam B,Bredack C,et al.Immune activation in patients with irritable bowel syndrome[J].Gastroenterology,2007,132(3):913-920.
[19]Villena J,Aso H,Kitazawa H.Regulation of toll-like receptorsmediated inflammation by immunobiotics in bovine intestinal epitheliocytes:role of signaling pathways and negative regulators[J].Front Immunol,2014,2(5):421-421.
[20]Rimoldi M,Chieppa M,Salucci V,et al.Intestinal immune homeostasis is regulated by the crosstalk between epithelial cells and dendritic cells[J].Nat Immunol,2015,16(3):326-326.
[21]Mckernan DP,Nolan A,Brint EK,et al.Toll-like receptor mRNA expression is selectively increased in the colonic mucosa of two animal models relevant to irritable bowel syndrome[J].PLoS One,2009,4(12):e8226-e8226.
[22]Tramullas M,Finger BC,Moloney RD,et al.Toll-like receptor4 regulates chronic stress-induced visceral pain in mice[J].Biol Psychiatry,2014,76(4):340-348
[23]Nozu T,Miyagishi S,Nozu R,et al.Lipopolysaccharide induces visceral hypersensitivity:role of interleukin-1,interleukin-6,and peripheral corticotropin-releasing factor in rats[J].J Gastroenterol,2017,52(1):72-80.
[24]Tang H,Zhang G,Ji Ning,et al.Toll-like receptor 4 in paraventricular nucleus mediates visceral hypersensitivity induced by maternal separation[J].Front Pharmacol,2017,8:309-320.