Objective
To observe the effects of maternal high fat diet (MHFD) during pregnancy and lactation on intestinal barrier function in offspring mice.
Methods
C57BL/6 pregnant mice were divided into high fat diet (MHFD) group and normal diet group (MND) randomly and were given high fat diet and normal diet during pregnancy (3 weeks) and lactation (3 weeks) respectively. Both groups of offspring mice were naturally given and bodyweight of pups was monitored at birth and weekly. After weaning, the intestinal permeability of offspring mice was detected by fluorescein isothiocyanate conjugated-dextran method (FITC-D). Immunofluorescence was used to detect the expression of ZO-1 in intestinal tissues. HE staining was used to assess the villus length and crypt depth. The intestinal cell proliferation(expression of Ki-67)and Mucin 2(MUC2) were assessed by immunohistochemistry. PAS staining was used to evaluate the goblet cells. The expression of inflammatory cytokines including IL-1β, IL-6, and TNF-α in intestinal tissue were measured by real-time PCR.
Results
At the age of 2 and 3 weeks, the offspring in MHFD group were significantly heavier than those in MND group. HE staining showed no obvious microscopic inflammation in both groups of 3 weeks old offspring mice, however, the relative expression levels of IL-1β (1.95±0.53 vs. 1.13±0.15; t=3.65, P=0.005), IL-6(1.40±0.71 vs. 0.73±0.17; t=2.72, P=0.04), and TNF-α(1.63±0.53 vs. 1.04±0.12; t=2.64, P=0.02) mRNA were significantly higher in the MHFD group. Compared with the 3 weeks old offspring mice in MND group, MHFD significantly increased the permeability of intestine and decreased the expression of ZO-1 in membrane. The number of Ki-67 positive cells (18.00±4.74 vs. 24.60±4.17; t=3.31, P=0.004) in each villus, goblet cells (14.70±2.91 vs. 28.10±4.95; t=7.38, P<0.001) and MUC2 positive cells (20.60±3.13 vs. 30.00±3.33; t=6.50, P<0.001) in each crypt were significantly lower than those in MND group.
Conclusion
Maternal high fat diet in early life of offspring mice can induce intestinal low grade inflammation and lead to the disruption of intestinal mucosal barrier in offspring mice, which may be involved in the progeny diseases.
Key words:
Early life; High fat diet; Inflammation; Intestinal mucosal barrier
Inflammatory bowel disease (IBD), characterized by sustained inflammation, is a latent risk factor of colon tumorigenesis. Silibinin has been reported to be anti-inflammatory and antineoplastic, but its efficacy on colitis-associated cancer (CAC) has not been reported. Interlukin-6/signal transducer and activator of transcription 3 (IL-6/STAT3) is the key signaling pathway involved in CAC. We evaluated the chemopreventive effect of silibinin on a CAC mouse model and determined its impact on IL-6/STAT3 signaling. Intestinal tumor cells (IMCE and HCT-116 cell lines) were also treated by graded concentration of silibinin, and cellular viability was determined. Silibinin (750 mg/kg/day) was administered to an azoxymethane/dextran sulfate sodium (AOM/DSS) C57BL/6 mouse model for 10 weeks by gavage. Body weight, colon length, and the amount and diameter of colon tumors were documented, respectively. Specimens were subjected to H&E staining for colitis and tumor scoring, immunohistochemical staining and terminal deoxynucleotidyl transferase dUTP nick end labeling for proliferation assessment, and immunofluorescent staining for intestinal mucosa barrier assessment. Production of inflammatory cytokines was determined by real-time PCR. IL-6/STAT3 pathway activation was evaluated through immunohistochemical staining and western blot. In the current study, silibinin significantly inhibited the viability of intestinal tumor cells. The production of inflammatory cytokines and the phosphorylation of STAT3 were both inhibited in intestinal tumor cells. Meanwhile, silibinin decreased the amount and size of tumors in AOM/DSS mice. Colitis and tumor scores were decreased accompanying with inhibition of colonic tumor cell proliferation and promotion of cellular apoptosis. Additionally, silibinin could reduce the production of inflammatory cytokines and attenuate the impairment of colonic mucosal barrier. Furthermore, STAT3 phosphorylation was significantly suppressed by silibinin. In conclusion, silibinin could protect against colitis-associated tumorigenesis in mice via inhibiting IL-6/STAT3, which showed promising chemopreventive potential of CAC.
Objective
To investigate the effects of Lactobacillus rhamnosus GG (LGG) colonization in early life on intestinal barrier and intestinal development in offspring mice and its possible mechanism.
Methods
Six C57BL/6 pregnant mice with the same conception time of 6 weeks were selected and randomly divided into experiment group given 108 cfu/ml LGG live bacteria and control group given LGG inactivated bacteria by gavage from the 18th day of pregnancy until natural birth. The progeny mice in the two groups were continued to be gavaged with 107 cfu/ml of LGG live bacteria or LGG inactivated bacteria on days 1-5 of birth. The body weight changes of 3 week'progeny mice were recorded. The colonization of LGG bacteria in offspring mice was detected at 2nd and 3rd weeks. The mRNA of intestinal proinflammatory cytokines and tight junction molecules were evaluated by real-time PCR method. HE, immunohistochemistry, immunofluorescence staining and enzyme-linked immunosorbent assay were used to evaluate the intestinal barrier of 3-week old off spring mice.
Results
Compared with the control group, the progeny mice of the experiment group showed no significant difference in body weight at the first week, and the body weight increased at the second week and the third week [2nd week: (3.790±0.240)g vs.(4.326±0.140)g, t=3.707, P=0.006; 3rd week: (7.295±0.326)g vs. (8.040±0.370)g, t=3.130, P=0.011]. LGG colonization can be detected only in the feces of progeny mice in the experiment group. Intestinal colonization can promote the growth of small intestine villi and colon crypt depth[jejunum: (320.000±22.514)μm vs.(265.100±15.611)μm, t=8.258, P<0.001; ileum: (150.500±13.099)μm vs.(111.000±11.308)μm, t=9.958, P<0.001; colon: (295.000±15.209)μm vs.(233.100±6.678)μm, t=9.129, P<0.001]. Compared with the control group, the number of goblet cells in the colonic crypt of the experiment group increased(11.62±0.780 vs. 35.24 ±1.370, t=15.000, P<0.001), and the relative mRNA expression levels of pro-inflammatory factors as IFN-γ (1.280±0.232 vs. 0.512±0.206, t=4.970, P=0.001), IL-6(1.364±0.271 vs. 0.941±0.215, t=2.452, P=0.040), IL-10(1.341±0.320 vs. 0.744±0.294, t=2.762, P=0.025) and TNF-α(3.702±0.150 vs. 2.581±0.500, t=2.553, P=0.034) in the experiment group decreased; the expression levels of the intimate tight junction molecules (Claudin3)(1.283±0.152 vs. 1.881±0.172, t=4.932, P=0.001)and the atresia protein molecule (Occludin)( 1.164±0.342 vs. 0.812±0.224, t=3.67, P=0.016) significantly increased.
Conclusion
Early life LGG colonization protects the intestinal barrier by inhibiting low-grade intestinal inflammation. This study will lay the experimental foundation for the supplementation of probiotics in early life so as to prevent intestinal diseases.
Key words:
Early life; Lactobacillus rhamnosus GG; Low-grade inflammation; Intestinal barrier
A growing body of evidence has elucidated that the gut microbiota has a crucial impact on the pathophysiological process of atopic diseases. Eosinophilic chronic rhinosinusitis with nasal polyps (eCRSwNP) is a local atopic disease of the systemic immune response. Alterations in the gut microbiome in eCRSwNP patients remain largely undefined.
Abstract Background The overgrowth of Desulfovibrio , an inflammation promoting flagellated bacteria, has been found in ulcerative colitis (UC) patients. However, the molecular mechanism in promoting colitis remains unestablished. Methods The relative abundance Desulfovibrio vulgaris ( D. vulgaris ) in stool samples of UC patients was detected. Mice were treated with dextran sulfate sodium to induce colitis with or without administration of D. vulgaris or D. vulgaris flagellin (DVF), and the severity of colitis and the leucine-rich repeat containing 19 (LRRC19) signaling were assessed. The interaction between DVF and LRRC19 was identified by surface plasmon resonance and intestinal organoid culture. Lrrc19 −/− and Tlr5 −/− mice were used to investigate the indispensable role of LRRC19. Finally, the blockade of DVF-LRRC19 interaction was selected through virtual screening and the efficacy in colitis was assessed. Results D. vulgaris was enriched in fecal samples of UC patients and was correlated with the disease severity. D. vulgaris or DVF treatment significantly exacerbated colitis in germ-free mice and conventional mice. Mechanistically, DVF could interact with LRRC19 (rather than TLR5) in colitis mice and organoids, and then induce the production of pro-inflammatory cytokines. Lrrc19 knockdown blunted the severity of colitis. Furthermore, typhaneoside, a blockade of binding interfaces, blocked DVF-LRRC19 interaction and dramatically ameliorated DVF-induced colitis. Conclusions D. vulgaris could promote colitis through DVF-LRRC19 interaction. Targeting DVF-LRRC19 interaction might be a new therapeutic strategy for UC therapy.
The perturbations of gut microbiota could interact with excessively activated immune responses and play key roles in the etiopathogenesis of ulcerative colitis (UC). Desulfovibrio, the most predominant sulfate reducing bacteria (SRB) resided in the human gut, was observed to overgrow in patients with UC. The interactions between specific gut microbiota and drugs and their impacts on UC treatment have not been demonstrated well.This study aimed to elucidate whether Desulfovibrio vulgaris (D. vulgaris, DSV) and its flagellin could activate nucleotide-binding oligomerization domain-like receptors (NLR) family of apoptosis inhibitory proteins (NAIP) / NLR family caspase activation and recruitment domain-containing protein 4 (NLRC4) inflammasome and promote colitis, and further evaluate the efficacy of eugeniin targeting the interaction interface of D. vulgaris flagellin (DVF) and NAIP to attenuate UC.The abundance of DSV and the occurrence of macrophage pyroptosis in human UC tissues were investigated. Colitis in mice was established by dextran sulfate sodium (DSS) and gavaged with DSV or its purified flagellin. NAIP/NLRC4 inflammasome activation and macrophage pyroptosis were evaluated in vivo and in vitro. The effects of eugeniin on blocking the interaction of DVF and NAIP/NLRC4 and relieving colitis were also assessed.The abundance of DSV increased in the feces of patients with UC and was found to be associated with disease activity. DSV and its flagellin facilitated DSS-induced colitis in mice. Mechanistically, RNA sequencing showed that gene expression associated with inflammasome complex and pyroptosis was upregulated after DVF treatment in macrophages. DVF was further demonstrated to induce significant macrophage pyroptosis in vitro, depending on NAIP/NLRC4 inflammasome activation. Furthermore, eugeniin was screened as an inhibitor of the interface between DVF and NAIP and successfully alleviated the proinflammatory effect of DVF in colitis.Targeting DVF-induced NAIP/NLRC4 inflammasome activation and macrophage pyroptosis ameliorates UC. This finding is of great significance for exploring the gut microbiota-host interactions in UC development and providing new insights for precise treatment.
// Duochen Jin 1, * , Tianyu Liu 1, * , Wenxiao Dong 1 , Yujie Zhang 2 , Sinan Wang 1 , Runxiang Xie 1 , Bangmao Wang 1 and Hailong Cao 1 1 Department of Gastroenterology and Hepatology, General Hospital, Tianjin Medical University, Tianjin, China 2 Department of Pathology, General Hospital, Tianjin Medical University, Tianjin, China * These authors contributed equally to this work Correspondence to: Hailong Cao, email: cao_hailong@163.com Keywords: cranberry; intestinal tumor; gut barrier function; epidermal growth factor receptor; Apc min/+ mouse Received: April 11, 2017 Accepted: October 11, 2017 Published: October 26, 2017 ABSTRACT It is increasingly perceived that dietary components have been linked with the prevention of intestinal cancer. Cranberry is a rich source of phenolic constituents and non-digestible fermentable dietary fiber, which shows anti-proliferation effect in colorectal cancer cells. Herein, we investigated the efficacy of long-term cranberry diet on intestinal adenoma formation in Apc min/+ mice. Apc min/+ mice were fed a basal diet or a diet containing 20% (w/w) freeze-dried whole cranberry powder for 12 weeks, and the number and size of tumors were recorded after sacrifice. Our results showed that cranberry strongly prevented the growth of intestinal tumors by 33.1%. Decreased cell proliferation and increased apoptosis were observed in tumors of cranberry-fed mice. Cranberry diet reduced the expression profile of colonic inflammatory cytokines (IFN-γ, IL-1β and TNF-α) accompanied with increased levels of anti-inflammatory cytokines (IL-4 and IL-10). Moreover, the number of colonic goblet cells and MUC2 production were increased, and the intestinal barrier function was also improved. In addition, cranberry diet increased caecal short chain fatty acids concentrations, and down-regulated epidermal growth factor receptor signaling pathway. These data firstly show the efficacy and associated mechanisms of cranberry diet on intestinal tumor growth in Apc min/+ mice, suggesting its chemopreventive potential against intestinal cancer.
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