肠道菌群介导肥胖结肠癌患者的炎症和肠道通透性改变的机制研究

目的研究肠道菌群介导肥胖结肠癌患者的炎症和肠道通透性改变的机制研究。方法收集患有(OB-CRC)和没有肥胖症(L-CRC)的肠癌患者粪便样本,肥胖健康对照组(L-HC)的粪便样本作为对照。16S rRNA测序分析各组的种群数量和种类。酶联免疫吸附测定法测定患者血清人连蛋白、IL-10和IL-1β的水平。LC-MS(ESI-MS)检测氧化三甲胺的血清浓度。结果肥胖的存在并没有引起CRC患者肠道细菌多样性和丰富度的显著变化。然而,OB-CRC患者表现出特定的肠道微生物群,其特征是产生丁酸盐的细菌减少和机会致病菌过多。促炎细胞因子IL-1β水平明显升高,有害的细菌代谢物TMAO,以及这些病人的肠道通透性明显增加。结论肥胖相关的肠道菌群可能在结肠癌的炎症反应和改变肠道通透性的发展中起到关键作用,这为临床采用新的结肠癌预防诊断工具提供了新的理论依据。     

CD34 mediates intestinal inflammation in Salmonella‐infected mice

CD34 is a highly glycosylated sialomucin expressed on a variety of cells, ranging from vascular endothelial cells to haematopoietic stem cells. Depending on its glycosylation state, CD34 has been shown to promote or inhibit cell adhesion and migration; however, a functional role for CD34 in the gut has not been determined. Using a model of Salmonella‐induced gastroenteritis, we investigated the role of CD34 in the context of infection. Upon oral infection, the number of CD34+ cells detected in the submucosa, vascular endothelium and lamina propria significantly increased in S. Typhimurium‐infected C57Bl/6 mice. The pathology of S. Typhimurium‐infected C57Bl/6 mice was characterized by recruitment of neutrophils to the site of inflammation, submucosal oedema and crypt destruction. In contrast, Cd34^?/? mice showed a delayed pathology, a defect in inflammatory cell migration into the intestinal tissue and enhanced survival. Importantly, this was not due to a lack of chemotactic signals in Cd34^?/? mice as these mice had either similar or significantly higher levels of pro‐inflammatory cytokines and chemokines post infection when compared with infected C57/Bl6 control mice. In summary, we demonstrate a novel role for CD34 in enhancing migration of inflammatory cells and thereby exacerbating host‐mediated immunopathology in the intestine of S. Typhimurium‐infected mice.     

Innate immune activation by inhaled lipopolysaccharide, independent of oxidative stress, exacerbates silica-induced pulmonary fibrosis in mice

Acute exacerbations of pulmonary fibrosis are characterized by rapid decrements in lung function. Environmental factors that may contribute to acute exacerbations remain poorly understood. We have previously demonstrated that exposure to inhaled lipopolysaccharide (LPS) induces expression of genes associated with fibrosis. To address whether exposure to LPS could exacerbate fibrosis, we exposed male C57BL/6 mice to crystalline silica, or vehicle, followed 28 days later by LPS or saline inhalation. We observed that mice receiving both silica and LPS had significantly more total inflammatory cells, more whole lung lavage MCP-1, MIP-2, KC and IL-1β, more evidence of oxidative stress and more total lung hydroxyproline than mice receiving either LPS alone, or silica alone. Blocking oxidative stress with N-acetylcysteine attenuated whole lung inflammation but had no effect on total lung hydroxyproline. These observations suggest that exposure to innate immune stimuli, such as LPS in the environment, may exacerbate stable pulmonary fibrosis via mechanisms that are independent of inflammation and oxidative stress.     

免疫细胞与炎症介质在肠炎发病中的作用

肠炎的起因是多样的,但引起粘膜的损伤而出现各种临床症状的机制却是相似的。近年来免疫生物学,分子免疫学的发展对肠道粘膜免疫功能的了解有了巨大的进步。肠炎的起因是病原或过敏原刺激活化了先天免疫和特异免疫系统的细胞,由肠道上皮细胞、巨噬细胞和淋巴细胞分泌多种细胞因子,这些细胞因子再活化或动员更多的细胞,并进一步分泌更多的因子,形成病原、细胞和因子之间的级联反应。由细胞与因子的综合作用,造成肠道局部的炎症。炎症因子和抗炎因子比例的消长决定了炎症的转归和预后。对炎症因子及其拮抗剂作用机制的了解,将有助于肠炎的诊断和治疗。     

Molecular interactions between bacteria, the epithelium, and the mucosal immune system in the intestinal tract: implications for chronic inflammation

In the last few years, advances in immunology, metabolomics and microbial ecology have shown that the contribution of the intestinal microbiota to the overall health status of the host has been so far underestimated. In this context, intestinal epithelial cells play a crucial role in the maintenance of intestinal homoeostasis. Indeed, at the interface between the luminal content and host tissues, the intestinal epithelium must integrate pro- and anti-inflammatory signals to regulate innate and adaptative immune responses, i.e. to control inflammation. However, under the influence of environmental factors, disturbance of the dialog between enteric bacteria and epithelial cells contributes to the development of chronic inflammation in genetically susceptible hosts. The present review covers the state of knowledge of the host response, especially in intestinal epithelial cells, to enteric bacteria, including colitogenic and probiotic bacteria. It also seeks to give an overview of potential regulatory mechanisms involved in the maintenance of intestinal homeostasis, and discusses the clinical implications for inflammatory bowel diseases.     

Carrageenan oligosaccharides and associated carrageenan-degrading bacteria induce intestinal inflammation in germ-free mice

Carrageenans (CGNs) are widely used in foods and pharmaceuticals although their safety remains controversial. To investigate the effects of CGNs and CGN-degrading bacteria in the human colon, we screened for CGN degradation by human fecal microbiota, and for inflammatory response to CGNs and/or CGN-degrading bacteria in germ free mice. Thin-layer chromatography indicated that high molecular weight (MW) CGNs (!100 kDa) remained undegraded in the presence of human fecal microbiota, whereas low MW CGNs, i.e., k-carrageenan oligosaccharides (KCO,～4.5 kDa) were degraded when exposed to seven of eight human fecal samples, although sulfate groups were not removed during degradation.Bacteroides xylanisolvens and Escherichia coli isolates from fecal samples apparently degraded KCO synergistically, with B. xylanisolvens serving as the primary degrader. Combined treatment of KCO with KCO-degrading bacteria led to greater pro-inflammatory effects in the colon and rectum of germ-free mice than either KCO or bacteria alone. Similarly, p-p38-, CD3-, and CD79a-positive immune cells were more abundant in combined treatment group mice than in either single treatment group. Our study shows that KCO-degrading bacteria and the low MW products of KCO can promote proinflammatory effects in mice,and represent two key markers for evaluating CGN safety in foods or medicines.     

Parameters of intestinal inflammation in mice given graded infections of the nematode Trichinella spiralis

Four parameters of the intestinal inflammatory response (numbers of mucosal mast cells (MMC) and Paneth cells, villus:crypt ratios and mitotic figures) were measured in mice exposed to varying doses of infective larvae of Trichinella spiralis.The aim of the experiments was to determine whether generation of these components of inflammation required a threshold level of infection and whether, once triggered, inflammation became pan-mucosal. Near maximal MMC and Paneth cell responses were elicited even with infections as low as 35 larvae; changes in villus:crypt ratios and in mitotic indices also occurred at this level of infection, but were progressively greater with increasing levels of infection. In all infected mice, including those infected with 35 larvae, MMC and Paneth cell responses extended over most of the small intestine. These data are interpreted as showing: (i) that the intestinal mucosa is highly responsive to T. spiralis infection; (ii) that once triggered, components of the inflammatory response are amplified by T cell-dependent mechanisms, becoming pan-mucosal; and (iii) that MMC and Paneth cell responses, which require cell division and differentiation, become maximal at a lower infection threshold than changes in the villus:crypt ratio or in mitotic indices, which directly reflect increased rates of division in crypt cells.     

Impacts of functional oligosaccharide on intestinal immune modulation in immunosuppressive mice

In order to research the role of soybean oligosaccharides (SBOSs) on improvements in the microenvironment of intestinal flora and immune function of cyclophosphamide (CTX) immunosuppressive mice. Via giving intragastric administration of Soybean oligosaccharide (SBOS) at the low dose (50/(kg·BW)/d), the middle dose (200 mg/(kg·BW)/d) and the high dose (500 mg/(kg·BW)/d) partly once a day, which is also 28 days in a row. At the same time, (SBOS) mice in the drug group and (CG) mice in the positive control group were given intraabdominal injection of CTX (200 mg/kg/d).The immunosuppressive mouse model (CY) was established after 72 h in the model group and the positive control group (CG) was given intragastric administration of levamisole hydrochloric acid (LMS) for 3 days, with the data of 80ug/kg/d after injection of CTX (for actually 72 h). On the 8th, 15th and 22nd day, the number of Bifidobacterium, Lactobacillus, Enterococcus and Clostridium perfringens m in the feces of mice in each dose of drug group were determined. After the test resulted, the cellular immune function, humoral immune function, monocyte/macrophage function, NK cell activity and cytokine secretion (tumor necrosis factor-α, interferon-gamma and IL-4) were measured in immunosuppressive mice each group. The results showed that 200 mg/(kg BW) soybean oligosaccharide could significantly promote the proliferation and inhibit the increase of Enterococcus in immunosuppressive mice. The soybean oligosaccharide of 500 mg/(kg BW) could dramatically promote the proliferation of both Bifidobacillus and Lactobacillus, and also inhibit the increase of both Enterobacteriaceae and Enterococcus in immunosuppressive mice. The regulatory function of SBOS on intestinal flora was positive. Soybean oligosaccharide (500 mg/(kg BW) could significantly promote the proliferation of Bifidobacillus and Lactobacillus in immunosuppressive mice and inhibit the increase of Enterococcus and Enterococcus. The proliferation of spleen lymphocytes induced by ConA, LPS in immunosuppressive mice was dose-dependent. But it was still lower than that of the normal group (CG0) (p > 0.05). The serum hemolysin level of immunosuppressive mice was significantly increased in each dose group (p < 0.05), and the level of antibody forming cells in spleen cells of each dose group was significantly increased (P < 0.05), and the level of antibody forming cells in spleen cells of each dose group was significantly higher than that of low dose group (p < 0.005), and the level of serum hemolysin in immunosuppressive mice was significantly increased in each dose group (p < 0.05). In the detection of immune effector cell activity in immunosuppressive mice, the phagocytic function of macrophages in high dose group and the natural killing activity of spleen NK cells in high dose drug group were significantly increased, which were not significantly different from those in positive control group (P < 0.05), but the expression of TNF-α, INF-γ and IL-4 cytokines in serum was increased in a dose dependent manner (p < 0.05). In conclusion, soybean oligosaccharide can significantly increase the diversity of intestinal microecology, increase the number of intestinal beneficial bacteria, has a correlation with the proliferation of Bifidobacterium and Lactobacillus in the intestinal tract, and inhibit the proliferation of harmful bacteria. The results showed that SBOS had a direct effect on the proliferation of intestinal flora under immunosuppression. Based on the improvement of intestinal microenvironment in immunosuppressive mice by soybean oligosaccharide for 25 days, the results showed that compared with the positive control group, the nonspecific and specific immunity of immunosuppressive mice in the drug group had a regulatory effect, which improved the phagocytic function of monocytes/macrophages, developed the level of antibody forming cells, enhanced the standard of the killing activity of NK cells, and promoted the expression of cytokines as well. Compared with the model group, the transformation and proliferation of spleen lymphocytes in the high and middle dose groups were remarkably increased, but all of the indexes did not reach the level of the normal blank group. By studying the improvement of intestinal microenvironment in immunosuppressive mice, to some extent, it is concluded that the proliferation of intestinal flora can improve the immunomodulatory function of the body, but it still lowers the normal immune degree, which reflects the immunomodulatory effect of the body on the stimulation of continuous external intake. The results demonstrate that the immunomodulatory ability of immunosuppressive body was insensitive to SBOS and provided a theoretical basis for the study of health care function of intestinal microenvironment improvement when SBOS acted on abnormal immune function. The results also improved the practical application value of SBOS.     

Cellular immune responses in mice infected with the intestinal nematode Trichuris muris.

CBA and B10.BR mice show variation in immune response to the intestinal nematode Trichuris muris. CBA mice develop strong resistance, eliminating worms from the intestine; B10BR mice are permissive and develop chronic infections. It is already known that resistance and permissiveness reflect differential T helper responses. The data reported here show that resistant CBA mice express good antigen-specific lymphocyte proliferative responses to infection, whereas cells from B10.BR mice are relatively anergic, although still responsive to Concanavalin A (ConA). The possibility that the altered proliferative responsiveness seen in infected B10.BR mice reflected quantitative or qualitative changes in T helper cells was examined by comparing cytokine production and expression of cell surface markers (CD4, CD8, and CD28) in mesenteric lymph node cells and spleen cells from both strains and comparing these with the characteristics of cells from resistant CBA mice and from CBA mice that had been rendered permissive to infection by a combination of irradiation and corticosteroid treatment. As expected, cells from B10.BR mice produced high levels of interferon-gamma (IFN-gamma), whereas those from CBA mice released high levels of IL-5, whether stimulated with adult worm somatic antigens, excretory/secretory antigens, or ConA. Immunosuppressed CBA mice produced high levels of both IFN-gamma and IL-5 throughout the experiment. FACS analysis revealed a decrease of CD4+ and an initial increase in CD8+ cells in all infected mice. No major changes occurred in the relative proportion of CD28(+) cells. Further evaluation of the CD28 costimulatory molecule, measured as mean fluorescence intensity, displayed down-regulation in permissive and immunosuppressed mice. The data obtained suggest that lymphocyte unresponsiveness and permissiveness to T. muris infection may be associated with a down-regulation or an initially reduced expression of costimulatory CD28 molecules.     

Effect of dietary cyclic nigerosylnigerose on intestinal immune functions in mice

We examined the dietary effects of cyclic nigerosylnigerose (CNN), a dietary indigestible oligosaccharide with four D-glucopyranosyl residues linked by alternating alpha-(1-->3)- and alpha-(1-->6) glucosidic linkages, on the intestinal immune function of mice, and the effects were compared with those of alpha-(1-->3)-linked oligosaccharide (nigerooligosaccharides, NOS) or alpha-(1-->6)-linked oligosaccharide (isomaltooligosaccharides, IMO). BALB/c mice were fed with 1-5% CNN, 5% IMO, or 12.5% NOS for 4 weeks, and the intestinal mucosal immune responses were determined. In the 1-5% CNN fed groups, the amounts of IgA in feces increased significantly. In addition, IgA, transforming growth factor-beta1 (TGF-beta1), and interleukin-6 (IL-6) secretion by Peyer's patch (PP) cells were enhanced in CNN fed mice. In the 5% CNN group, pH in the cecum decreased, and the amounts of lactic acid and butyric acid increased. These findings were not observed in the NOS- or IMO-fed group of mice. They suggest that CNN supplementation changes the intestinal environment of microflora and indirectly enhances the immune function in the gut.     

A food-based approach that targets interleukin-6, a key regulator of chronic intestinal inflammation and colon carcinogenesis

Studies have shown a causal link between high-calorie diet (HCD) and colon cancer. However, molecular mechanisms are not fully elucidated. To understand etiology of HCD-induced colon carcinogenesis, we screened 10 pathways linked to elevated colonic cell proliferation and chronic inflammation in an HCD-consuming human-relevant pig model. We observed elevated colonic mucosal interleukin-6 (IL-6) expression in HCD-consuming pigs compared to standard diet controls (SD, P=.04), and IL-6 strongly correlated with Ki-67 proliferative index and zone, early biomarkers of colon cancer risk (r=0.604 and 0.743 and P=.017 and .002, respectively). Liquid chromatography–tandem mass spectrometry-based proteomic analysis and Ingenuity Pathway Analysis showed that HCD consumption altered IL-6 signaling pathway proteins (PI3KR4, IL-1α, Mapk10, Akt3, PIK3CG, PIK3R5, Map2k2). Furthermore, these proteins also correlated with Ki-67 proliferative index/zone. Anti-IL-6 therapeutics are available for treating colon cancer; however, they are expensive and induce negative side effects. Thus, whole foods could be a better way to combat low-grade chronic colonic inflammation and colon cancer. Whole plant foods have been shown to decrease chronic diseases due to the potential of anti-inflammatory dietary compounds acting synergistically. We observed that supplementation of HCD with anthocyanin-containing purple-fleshed potatoes (10% w/w), even after baking, suppressed HCD-induced IL-6 expression (P=.03) and the IL-6-related proteins IL-1α and Map2k1 (P≤.1). Our results highlight the importance of IL-6 signaling in diet-linked induction/prevention of colonic inflammation/cancer and demonstrate the potential of a food-based approach to target IL-6 signaling.     

Acanthopanax senticosus total flavonoids alleviate lipopolysaccharide-induced intestinal inflammation and modulate the gut microbiota in mice

Here, we study the therapeutic effect of Acanthopanax senticosus total flavonoids (ASTFs) using a mouse intestinal inflammation model. The inflammation model used in the present study was developed through lipopolysaccharide (LPS) treatment of mice. The experimental mice were divided into a control group, model group (10 mg/kg LPS), dexamethasone group (1 mg/kg DEX) and ASTF low-, medium- and high-dosage groups (200, 400 and 800 mg/kg, respectively). The morphological and structural changes in the ileum, jejunum and duodenum were observed using HE staining. The number of intestinal goblet cells (GCs) was calculated based on PAS staining. The contents of interleukin (IL)-1β, IL-6, prostaglandin E2 (PGE2) and tumor necrosis factor α (TNF-α) were determined using enzyme-linked immunosorbent assay (ELISA) and the related mRNA expression level were measured by RT-PCR. The protein expression levels of Toll-like receptor 4 (TLR4), MyD88, p65 and p-p65 were measured using Western blotting. In addition, the 16S rRNA sequences of bacterial taxa were amplified and analyzed to assess changes in the intestinal microbes of LPS-induced mice and also in response to regulation by ASTF. Following intervention with ASTF, different therapeutic effects were shown according to the various dosages tested, all of which resulted in improved intestinal morphology and an increased number of intestinal GCs, while the contents of IL-1β, IL-6, PGE2 and TNF-α and the related mRNA expression level were significantly reduced. The TLR4, MyD88 and p-p65/p-65 protein expression levels were also significantly reduced. In addition, 16S rRNA sequencing results show that LPS disrupts the structure of mouse gut microbes, though we observed that normal microbial status can be restored through ASTF intervention.     

基于肠道菌群代谢和免疫炎症分析中药缓解抑郁症的研究现状

抑郁症是以连续而长期的心情低落为主要临床特征的一类最常见的精神疾病。肠道菌群的变化可导致肠道炎症,进而影响外周与中枢神经系统炎症。现代研究表明抑郁症的发生发展与肠道菌群的改变密切相关,肠道菌群已成为缓解抑郁症的关键靶点。在一定程度上中药能够调节肠道菌群及其代谢产物,修复肠道屏障,降低全身炎症水平,从而缓解抑郁症。本综述以肠道菌群为切入点,通过分析肠道菌群的代谢、免疫炎症与抑郁症发生发展的关系以及中药在调节肠道菌群缓解抑郁症方面的研究,总结中药通过影响肠道菌群的代谢,降低炎症水平,缓解抑郁症的作用和机制,以期为中药缓解抑郁症及相关药物的研发提供新思路。

     

Moderate zinc restriction affects intestinal health and immune function in lipopolysaccharide-challenged mice

Zinc (Zn) is an essential nutrient that affects immune function, especially within the digestive system, although the underlying mechanisms are not well understood. This study examined the effects of short-term moderate Zn restriction on intestinal health and immune function in lipopolysaccharide (LPS)-challenged mice through plasma cytokine profiling and histological evaluation of intestinal tissue sections. Adult male mice were fed with a Zn-adequate (40 ppm) or a Zn-marginal (4 ppm) diet for 4 weeks, and then a bacterial challenge was simulated by intraperitoneal injection of LPS (10 microg/g body weight [BW]) or saline (control). BW was recorded weekly, and feed intake was recorded daily over the last week. Voluntary locomotor activity was assessed 6 and 24 h after the challenge. Plasma and tissues were collected 0, 6 or 24 h after the challenge for analysis. Histological analysis of intestinal samples included evaluation of villi length and width, lamina propria (LP) width, crypt depth and intraepithelial as well as LP leukocyte numbers. Plasma was analyzed for IL-1beta, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, interferon gamma and tumor necrosis factor alpha. Diet did not affect BW and feed intake. The LPS challenge led to decreased voluntary locomotor activity (P<.05). Moderate Zn restriction led to greater leukocyte infiltration in the LP after the LPS challenge (P<.05) and higher plasma IL-6 and IL-10 levels 24 h after the LPS challenge (P<.01). Results indicate that Zn status impacts intestinal responses to LPS through modulation of the cytokine response and leukocyte recruitment, and this impact is evident even with short-term (4 weeks) moderate Zn restriction.     

A peptide derived from the parasite receptor,complement C2 receptor inhibitor trispanning,suppresses immune complex-mediated inflammation in mice

Complement C2 receptor inhibitor trispanning (CRIT) is a Schistosoma protein that binds the human complement protein, C2. We recently showed that peptides based on the ligand binding region of CRIT inhibit the classical pathway (CP) of complement activation in human serum, using hemolytic assays and so speculated that on the parasite surface CRIT has the function of evading human complement. We now show that in vitro the C2-binding 11-aa C terminus of the first extracellular domain of CRIT, a 1.3-kDa peptide termed CRIT-H17, inhibits CP activation in a species-specific manner, inhibiting mouse and rat complement but not that from guinea pig. Hitherto, the ability of CRIT to regulate complement in vivo has not been assessed. In this study we show that by inhibiting the CP, CRIT-H17 is able to reduce immune complex-mediated inflammation (dermal reversed passive Arthus reaction) in BALB/c mice. Upon intradermal injection of CRIT-H17, and similarly with recombinant soluble complement receptor type 1, there was a 41% reduction in edema and hemorrhage, a 72% reduction in neutrophil influx, and a reduced C3 deposition. Furthermore, when H17 was administered i.v. at a 1 mg/kg dose, inflammation was reduced by 31%. We propose that CRIT-H17 is a potential therapeutic agent against CP complement-mediated inflammatory tissue destruction.     

Sublethal infection with Salmonella Enteritidis by the natural route induces intestinal and joint inflammation in mice

Reactive arthritis (ReA) is a sterile inflammation triggered by a distal mucosal infection, which suggests a contribution from bacterial products. Investigation on the pathogenesis of ReA is difficult because of the limited studies that can be performed in humans; therefore the availability of animal models is crucial. We hereby describe a murine model for studying the early stages of Salmonella-induced ReA. BALB/c mice infected by the natural route with a sublethal dose of S. Enteritidis showed long lasting gut inflammation, synovitis in the knee joint and a significant increase of CD4+ lymphocytes in the draining popliteal lymph nodes. S. Enteritidis infection induced histological changes in intact knees and exacerbated inflammation in previously damaged joints. Experiments performed with S. Enteritidis ΔinvG mutant suggest that the proinflammatory signalling mediated by Salmonella TTSS-1 in the gut is required for the induction of joint sequelae. Since this model is highly reproducible and easy to perform, it provides great potential for investigating both host and bacterial contributions to the early stages of ReA.