探究益生菌组合物(鼠李糖乳杆菌LR-168、嗜酸乳杆菌LA-99和动物双歧杆菌BB-115)对慢传输型便秘的作用及机制。将50只6周龄雄性BABL/c小鼠随机分为空白组(生理盐水)、模型组(洛哌丁胺)、阳性对照组(洛哌丁胺+动物双歧杆菌BB-12:5×108 CFU)、低剂量组(洛哌丁胺+益生菌组合物:5×108 CFU)和高剂量组(洛哌丁胺+益生菌组合物:5×109 CFU),灌胃21 d后处死,对小鼠生理生化指标进行测定。与模型组相比,益生菌组合物低、高剂量组显著提高小肠推进率、黑便粒数和黑便质量,显著降低首粒黑便时间;此外,灌菌处理后,血清和结肠组织的兴奋性神经递质(P物质、胃动素和胃泌素)分泌上升,抑制性神经递质(血管活性肽、生长抑素和内皮素-1)分泌下降;各组间的血清细胞因子水平无统计学差异;结肠组织的AQP3和c-kit基因转录量在益生菌干预后显著提高。益生菌组合物具有缓解慢传输型便秘的作用,这可能是通过影响胃肠调节肽分泌,以及AQP3和c-kit基因转录的水平来实现的。
As the popularity of probiotics, research on mechanisms of probiotics is becoming more and more in-depth. It is necessary to figure out what and how these strains can do. Using loperamide-induced constipation mouse model, the effects and mechanisms of probiotics complex (Lactobacillus rhamnosus LR-168, Lactobacillus acidophilus LA-99 and Bifidobacterium animalis BB-115) were investigated. Fifty BABL/c male mice aged six-week-old were used to test alleviation effect of probiotics complex. Firstly, five groups were randomly divided: blank group (saline), model (loperamide), positive control (loperamide + Bifidobacterium animalis BB12: 5×108 CFU), low-dose group (loperamide and probiotics complex: 5×108 CFU) and high-dose group (loperamide and probiotics complex: 5×109 CFU). Extended defecation time of first black stool was determined as the criterion for success of constipation modeling. Then, the data of defecation performance such as small intestinal transit rat, number of black stools and weight of black stools were collected. Based on the enzyme-linked immunosorbent assay, gastrointestinal peptide neurotransmitters and cytokines were analyzed. In regard to the potential effect of constipation, which unbalanced levels of aquaporin 3 (AQP3) and c-kit could bring out, quantitative real-time polymerase chain reaction was used to explore the mechanism behind, preliminarily. In the light of the criterion for the success of constipation modeling, it drew a conclusion that modeling of slow transit constipation mouse got success with loperamide induced. From the analysis of physiological changes, defecation performance was improved significantly in constipation mice after gavage with probiotics complex, specifically in small intestinal transit rate, number of black stools and weight of black stools. When the hypotheses about mechanisms of probiotics complex focused on the connection between gastrointestinal peptide and constipation, the secretion of excitatory neurotransmitters (substance P, gastrin and motilin) and inhibitory neurotransmitters (vasoactive intestinal peptide, somatostatin and endothelin 1) was observed. Outstanding variations were detected among model group and other groups, with the secretion of excitatory neurotransmitters increased and inhibitory neurotransmitters decreased, compared with model group. There was no obviously difference in cytokines reported in each group, but a slight decline in model group, elucidating a latent balance ability of probiotics. The transcription levels of AQP3 and c-kit, which could affect the transport of water and colonic peristalsis, were lifted notably after intervention with probiotics. With the intragastric administration of the probiotics complex, not only the defecation performance got improved, but also the levels of neurotransmitters and cytokines related to constipation were balanced. The probiotics complex could be taken as a preparation with great prospects for alleviating slow transit constipation.
[1] TILLOU J, POYLIN V.Functional disorders:Slow-transit constipation[J].Clinics in Colon and Rectal Surgery, 2017, 30(1):76-86.
[2] BHARUCHA A E, WALD A.Chronic constipation[J].Mayo Clinic Proceedings, 2019, 94(11):2 340-2 357.
[3] DIMIDI E, CHRISTODOULIDES S, SCOTT S M, et al.Mechanisms of action of probiotics and the gastrointestinal microbiota on gut motility and constipation[J].Advances in Nutrition, 2017, 8(3):484-494.
[4] GE X L, ZHAO W, DING C, et al.Potential role of fecal microbiota from patients with slow transit constipation in the regulation of gastrointestinal motility[J].Scientific Reports, 2017, 7:441.
[5] DIMIDI E, SCOTT S M, WHELAN K.Probiotics and constipation:Mechanisms of action, evidence for effectiveness and utilisation by patients and healthcare professionals[J].The Proceedings of the Nutrition Society, 2020, 79(1):147-157.
[6] ZHANG X Y, YANG H B, ZHENG J P, et al.Chitosan oligosaccharides attenuate loperamide-induced constipation through regulation of gut microbiota in mice[J].Carbohydrate Polymers, 2021, 253:117218.
[7] EOR J Y, TAN P L, LIM S M, et al.Laxative effect of probiotic chocolate on loperamide-induced constipation in rats[J].Food Research International, 2019, 116:1 173-1 182.
[8] MORI T, SHIBASAKI Y, MATSUMOTO K, et al.Mechanisms that underlie μ-opioid receptor agonist-induced constipation:Differential involvement of μ-opioid receptor sites and responsible regions[J].The Journal of Pharmacology and Experimental Therapeutics, 2013, 347(1):91-99.
[9] VAN DER WERF M J, VENEMA K.Bifidobacteria:Genetic modification and the study of their role in the colon[J].Journal of Agricultural and Food Chemistry, 2001, 49(1):378-383.
[10] WANG L L, HU L J, XU Q, et al.Bifidobacteria exert species-specific effects on constipation in BALB/c mice[J].Food and Function, 2017, 8(10):3 587-3 600.
[11] LIANG Y X, WEN P, WANG Y, et al.The constipation-relieving property of d-tagatose by modulating the composition of gut microbiota[J].International Journal of Molecular Sciences, 2019, 20(22):5721.
[12] ZHAI X C, LIN D H, ZHAO Y, et al.Bacterial cellulose relieves diphenoxylate-induced constipation in rats[J].Journal of Agricultural and Food Chemistry, 2018, 66(16):4 106-4 117.
[13] LI T, YAN Q J, WEN Y P, et al.Synbiotic yogurt containing konjac mannan oligosaccharides and Bifidobacterium animalis ssp. lactis BB12 alleviates constipation in mice by modulating the stem cell factor (SCF)/c-Kit pathway and gut microbiota[J].Journal of Dairy Science, 2021, 104(5):5 239-5 255.
[14] WANG L L, HU L J, XU Q, et al.Bifidobacterium adolescentis exerts strain-specific effects on constipation induced by loperamide in BALB/c mice[J].International Journal of Molecular Sciences, 2017, 18(2):318.
[15] OPAL S M, DEPALO V A.Anti-inflammatory cytokines[J].Chest, 2000, 117(4):1 162-1 172.
[16] MAHAPATRO M, ERKERT L, BECKER C.Cytokine-mediated crosstalk between immune cells and epithelial cells in the gut[J].Cells, 2021, 10(1):111.
[17] ZHANG X, ZHENG F J, ZHANG Z.Therapeutic effect of Cistanche deserticola on defecation in senile constipation rat model through stem cell factor/C-kit signaling pathway[J].World Journal of Gastroenterology, 2021, 27(32):5 392-5 403.
[18] IKARASHI N, KON R, SUGIYAMA K.Aquaporins in the colon as a new therapeutic target in diarrhea and constipation[J].International Journal of Molecular Sciences, 2016, 17(7):1172.
[19] KON R, TSUBOTA Y, MINAMI M, et al.CPT-11-induced delayed diarrhea develops via reduced aquaporin-3 expression in the colon[J].International Journal of Molecular Sciences, 2018, 19(1):170.
[20] YIN J Q, LIANG Y C, WANG D L, et al.Naringenin induces laxative effects by upregulating the expression levels of c-Kit and SCF, as well as those of aquaporin 3 in mice with loperamide-induced constipation[J].International Journal of Molecular Medicine, 2018, 41(2):649-658.
