C57BL/KsJ-db/db-Apc Mice Exhibit an Increased Incidence of Intestinal Neoplasms

The numbers of obese people and diabetic patients are ever increasing. Obesity and diabetes are high-risk conditions for chronic diseases, including certain types of cancer, such as colorectal cancer (CRC). The aim of this study was to develop a novel animal model in order to clarify the pathobiology of CRC development in obese and diabetic patients. We developed an animal model of obesity and colorectal cancer by breeding the C57BL/KsJ-db/db (db/db) mouse, an animal model of obesity and type II diabetes, and the C57BL/6J-Apc(Min/+) (Min/+) mouse, a model of familial adenomatous polyposis. At 15 weeks of age, the N9 backcross generation of C57BL/KsJ-db/db-Apc(Min/+) (db/db-Min/+) mice developed an increased incidence and multiplicity of adenomas in the intestinal tract when compared to the db/m-Min/+ and m/m-Min/+ mice. Blood biochemical profile showed significant increases in insulin (8.3-fold to 11.7-fold), cholesterol (1.2-fold to 1.7-fold), and triglyceride (1.2-fold to 1.3-fold) in the db/db-Min/+ mice, when compared to those of the db/m-Min/+ and m/m-Min/+ mice. Increases (1.4-fold to 2.6-fold) in RNA levels of insulin-like growth factor (IGF)-1, IRF-1R, and IGF-2 were also observed in the db/db- Min/+ mice. These results suggested that the IGFs, as well as hyperlipidemia and hyperinsulinemia, promoted adenoma formation in the db/db-Min/+ mice. Our results thus suggested that the db/db-Min/+ mice should be invaluable for studies on the pathogenesis of CRC in obese and diabetes patients and the therapy and prevention of CRC in these patients.     

Corticotropin-Releasing Hormone Receptor Alters the Tumor Development and Growth in Apcmin/+ Mice and in a Chemically-Induced Model of Colon Cancer

The neuroendocrine circuit of the corticotropin-releasing hormone (CRH) family peptides, via their cognate receptors CRHR1 and CRHR2, copes with psychological stress. However, peripheral effects of the CRH system in colon cancer remains elusive. Thus, we investigate the role of CRHR1 and CRHR2 in colon cancer. Human colon cancer biopsies were used to measure the mRNA levels of the CRH family by quantitative real-time PCR. Two animal models of colon cancer were used: Apcmin/+ mice and azoxymethane (AOM)/dextran sulfate sodium (DSS)-treated mice. The mRNA levels of CRHR2 and UCN III are reduced in human colon cancer tissues compared to those of normal tissues. Crhr1 deletion suppresses the tumor development and growth in Apcmin/+ mice, while Crhr2 deficiency exacerbates the tumorigenicity. Crhr1 deficiency not only inhibits the expression of tumor-promoting cyclooxygenase 2, but also upregulates tumor-suppressing phospholipase A2 in Apcmin/+ mice; however, Crhr2 deficiency does not change these expressions. In the AOM/DSS model, Crhr2 deficiency worsens the tumorigenesis. In conclusion, Crhr1 deficiency confers tumor-suppressing effects in Apcmin/+ mice, but Crhr2 deficiency worsens the tumorigenicity in both Apcmin/+ and AOM/DSS-treated mice. Therefore, pharmacological inhibitors of CRHR1 or activators of CRHR2 could be of significance as anti-colon cancer drugs.     

Mice Harboring a Non-Functional CILK1/ICK Allele Fail to Model the Epileptic Phenotype in Patients Carrying Variant CILK1/ICK

CILK1 (ciliogenesis associated kinase 1)/ICK (intestinal cell kinase) is a highly conserved protein kinase that regulates primary cilia structure and function. CILK1 mutations cause a wide spectrum of human diseases collectively called ciliopathies. While several CILK1 heterozygous variants have been recently linked to juvenile myoclonic epilepsy (JME), it remains unclear whether these mutations cause seizures. Herein, we investigated whether mice harboring either a heterozygous null Cilk1 (Cilk1^+/−) mutation or a heterozygous loss-of-function Cilk1 mutation (Cilk1^R272Q/+) have epilepsy. We first evaluated the spontaneous seizure phenotype of Cilk1^+/− and Cilk1^R272Q/+ mice relative to wildtype littermates. We observed no electrographic differences among the three mouse genotypes during prolonged recordings. We also evaluated electrographic and behavioral responses of mice recovering from isoflurane anesthesia, an approach recently used to measure seizure-like activity. Again, we observed no electrographic or behavioral differences in control versus Cilk1^+/− and Cilk1^R272Q/+ mice upon isoflurane recovery. These results indicate that mice bearing a non-functional copy of Cilk1 fail to produce electrographic patterns resembling those of JME patients with a variant CILK1 copy. Our findings argue against CILK1 haploinsufficiency being the mechanism that links CILK1 variants to JME.     

Selenium and the 15kDa Selenoprotein Impact Colorectal Tumorigenesis by Modulating Intestinal Barrier Integrity

Selenoproteins play important roles in many cellular functions and biochemical pathways in mammals. Our previous study showed that the deficiency of the 15 kDa selenoprotein (Selenof) significantly reduced the formation of aberrant crypt foci (ACF) in a mouse model of azoxymethane (AOM)-induced colon carcinogenesis. The objective of this study was to examine the effects of Selenof on inflammatory tumorigenesis, and whether dietary selenium modified these effects. For 20 weeks post-weaning, Selenof-knockout (KO) mice and littermate controls were fed diets that were either deficient, adequate or high in sodium selenite. Colon tumors were induced with AOM and dextran sulfate sodium. Surprisingly, KO mice had drastically fewer ACF but developed a similar number of tumors as their littermate controls. Expression of genes important in inflammatory colorectal cancer and those relevant to epithelial barrier function was assessed, in addition to structural differences via tissue histology. Our findings point to Selenof’s potential role in intestinal barrier integrity and structural changes in glandular and mucin-producing goblet cells in the mucosa and submucosa, which may determine the type of tumor developing.     

16S rRNA of Mucosal Colon Microbiome and CCL2 Circulating Levels Are Potential Biomarkers in Colorectal Cancer

Colorectal cancer (CRC) is one of the most common malignancies in the Western world and intestinal dysbiosis might contribute to its pathogenesis. The mucosal colon microbiome and C-C motif chemokine 2 (CCL2) were investigated in 20 healthy controls (HC) and 20 CRC patients using 16S rRNA sequencing and immunoluminescent assay, respectively. A total of 10 HC subjects were classified as overweight/obese (OW/OB_HC) and 10 subjects were normal weight (NW_HC); 15 CRC patients were classified as OW/OB_CRC and 5 patients were NW_CRC. Results: Fusobacterium nucleatum and Escherichia coli were more abundant in OW/OB_HC than in NW_HC microbiomes. Globally, Streptococcus intermedius, Gemella haemolysans, Fusobacterium nucleatum, Bacteroides fragilis and Escherichia coli were significantly increased in CRC patient tumor/lesioned tissue (CRC_LT) and CRC patient unlesioned tissue (CRC_ULT) microbiomes compared to HC microbiomes. CCL2 circulating levels were associated with tumor presence and with the abundance of Fusobacterium nucleatum, Bacteroides fragilis and Gemella haemolysans. Our data suggest that mucosal colon dysbiosis might contribute to CRC pathogenesis by inducing inflammation. Notably, Fusobacterium nucleatum, which was more abundant in the OW/OB_HC than in the NW_HC microbiomes, might represent a putative link between obesity and increased CRC risk.     

Effect of Long-Term Dietary Arginyl-Fructose (AF) on Hyperglycemia and HbA1c in Diabetic db/db Mice

We have previously reported that Amadori compounds exert anti-diabetic effects by lowering sucrose-induced hyperglycemia in normal Sprague-Dawley rats. In the present study we extended our recent findings to evaluate whether α-glucosidase inhibitor arginyl-fructose (AF) lowers blood glucose level in diabetic db/db mice, a genetic model for type 2 diabetes. The db/db mice were randomly assigned to high-carbohydrate diets (66.1% corn starch) with and without AF (4% in the diet) for 6 weeks. Changes in body weight, blood glucose level, and food intake were measured daily for 42 days. Dietary supplementation of AF resulted in a significant decrease of blood glucose level (p < 0.001) and body weight (p < 0.001). The level of HbA1c, a better indicator of plasma glucose concentration over prolonged periods of time, was also significantly decreased for 6-week period (p < 0.001). Dietary treatment of acarbose^® (0.04% in diet), a positive control, also significantly alleviated the level of blood glucose, HbA1c, and body weight. These results indicate that AF Maillard reaction product improves postprandial hyperglycemia by suppressing glucose absorption as well as decreasing HbA1c level.     

Vitamin B Mitigates Thoracic Aortic Dilation in Marfan Syndrome Mice by Restoring the Canonical TGF-β Pathway

Thoracic aortic aneurysm (TAA) formation is a multifactorial process that results in diverse clinical manifestations and drug responses. Identifying the critical factors and their functions in Marfan syndrome (MFS) pathogenesis is important for exploring personalized medicine for MFS. Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), and methionine synthase reductase (MTRR) polymorphisms have been correlated with TAA severity in MFS patients. However, the detailed relationship between the folate-methionine cycle and MFS pathogenesis remains unclear. Fbn1^C1039G/+ mice were reported to be a disease model of MFS. To study the role of the folate-methionine cycle in MFS, Fbn1^C1039G/+ mice were treated orally with methionine or vitamin B mixture (VITB), including vitamins B6, B9, and B12, for 20 weeks. VITB reduced the heart rate and circumference of the ascending aorta in Fbn1^C1039G/+ mice. Our data showed that the Mtr and Smad4 genes were suppressed in Fbn1^C1039G/+ mice, while VITB treatment restored the expression of these genes to normal levels. Additionally, VITB restored canonical transforming-growth factor β (TGF-β) signaling and promoted Loxl1-mediated collagen maturation in aortic media. This study provides a potential method to attenuate the pathogenesis of MFS that may have a synergistic effect with drug treatments for MFS patients.     

The CD200 Regulates Inflammation in Mice Independently of TNF-α Production

Inflammatory bowel disease is characterized by the infiltration of immune cells and chronic inflammation. The immune inhibitory receptor, CD200R, is involved in the downregulation of the activation of immune cells to prevent excessive inflammation. We aimed to define the role of CD200R ligand-CD200 in the experimental model of intestinal inflammation in conventionally-reared mice. Mice were given a dextran sodium sulfate solution in drinking water. Bodyweight loss was monitored daily and the disease activity index was calculated, and a histological evaluation of the colon was performed. TNF-α production was measured in the culture of small fragments of the distal colon or bone marrow-derived macrophages (BMDMs) cocultured with CD200⁺ cells. We found that Cd200^−/− mice displayed diminished severity of colitis when compared to WT mice. Inflammation significantly diminished CD200 expression in WT mice, particularly on vascular endothelial cells and immune cells. The co-culture of BMDMs with CD200⁺ cells inhibited TNF-α secretion. In vivo, acute colitis induced by DSS significantly increased TNF-α secretion in colon tissue in comparison to untreated controls. However, Cd200^−/− mice secreted a similar level of TNF-α to WT mice in vivo. CD200 regulates the severity of DSS-induced colitis in conventionally-reared mice. The presence of CD200⁺ cells decreases TNF-α production by macrophages in vitro. However, during DDS-induced intestinal inflammation secretion of TNF-α is independent of CD200 expression.     

Viability of L. acidophilus microcapsules and their application to buffalo milk yoghurt

Lactobacillus acidophilus was microencapsulated by complex coacervation followed by lyophilisation to be applied in buffalo milk yoghurt. The viability of the probiotics was evaluated for the microencapsulation process, during storage and under simulated gastrointestinal conditions. Four buffalo milk yoghurt-test trials were prepared varying the form of the microorganisms (free and microencapsulated) and the final fermentation pH (4.5 and 5). The acidity, pH and microorganisms’ viability in the yoghurts were evaluated. Results showed that the microencapsulation technique used in this work preserved the counts of L. acidophilus higher than 10⁷ UFC/g at refrigerated conditions, but it did not provide protection to the microorganisms at a pH similar to the human stomach. For the yoghurts, the final fermentation pH did not show significant effects on the variables evaluated. Yoghurts prepared with microencapsulated cultures presented lower values for post-acidification and greater stability compared to the product prepared with the addition of the free culture. The obtained product could represent an alternative for buffalo milk intake provided by the benefits of consumption of L. acidophilus.     

Heat Killed Lactobacillus reuteri GMNL-263 Reduces Fibrosis Effects on the Liver and Heart in High Fat Diet-Hamsters via TGF-β Suppression

Obesity is one of the major risk factors for nonalcoholic fatty liver disease (NAFLD), and NAFLD is highly associated with an increased risk of cardiovascular disease (CVD). Scholars have suggested that certain probiotics may significantly impact cardiovascular health, particularly certain Lactobacillus species, such as Lactobacillus reuteri GMNL-263 (Lr263) probiotics, which have been shown to reduce obesity and arteriosclerosis in vivo. In the present study, we examined the potential of heat-killed bacteria to attenuate high fat diet (HFD)-induced hepatic and cardiac damages and the possible underlying mechanism of the positive effects of heat-killed Lr263 oral supplements. Heat-killed Lr263 treatments (625 and 3125 mg/kg-hamster/day) were provided as a daily supplement by oral gavage to HFD-fed hamsters for eight weeks. The results show that heat-killed Lr263 treatments reduce fatty liver syndrome. Moreover, heat-killed Lactobacillus reuteri GMNL-263 supplementation in HFD hamsters also reduced fibrosis in the liver and heart by reducing transforming growth factor β (TGF-β) expression levels. In conclusion, heat-killed Lr263 can reduce lipid metabolic stress in HFD hamsters and decrease the risk of fatty liver and cardiovascular disease.     

Dietary PlsEtn Ameliorates Colon Mucosa Inflammatory Stress and ACF in DMH-Induced Colon Carcinogenesis Mice: Protective Role of Vinyl Ether Linkage

Ethanolamine plasmalogen (PlsEtn), a sub-class of ethanolamine glycerophospholipids (EtnGpl), is a universal phospholipid in mammalian membranes. Several researchers are interested in the relationship between colon carcinogenesis and colon PlsEtn levels. Here, we evaluated the functional role of dietary purified EtnGpl from the ascidian muscle (87.3 mol% PlsEtn in EtnGpl) and porcine liver (7.2 mol% PlsEtn in EtnGpl) in 1,2-dimethylhydrazine (DMH)-induced aberrant crypt foci (ACF) in vivo, and elucidated the possible underlying mechanisms behind it. Dietary EtnGpl-suppressed DMH-induced aberrant crypt with one foci (AC1) and total ACF formation (P < 0.05). ACF suppression by dietary ascidian muscle EtnGpl was higher compared with dietary porcine liver EtnGpl. Additionally, dietary EtnGpl decreased DMH-induced oxidative damage, overproduction of TNF-α, and expression of apoptosis-related proteins in the colon mucosa. The effect of dietary ascidian muscle EtnGpl showed superiority compared with dietary porcine liver EtnGpl. Our results demonstrate the mechanisms by which dietary PlsEtn suppress ACF formation and apoptosis. Dietary PlsEtn attained this suppression by reducing colon inflammation and oxidative stress hence a reduction in DMH-induced intestinal impairment. These findings provide new insights about the functional role of dietary PlsEtn during colon carcinogenesis.     

Effect of Quercetin on Paraoxonase 2 Levels in RAW264.7 Macrophages and in Human Monocytes�C�CRole of Quercetin Metabolism

There is increasing evidence that the intracellular antioxidant enzyme paraoxonase 2 (PON2) may have a protective function in the prevention of atherogenesis. An enhancement of PON2 activity by dietary factors including flavonoids is therefore of interest. In the present study we determined the effect of quercetin on paraoxonase 2 levels in cultured murine macrophages in vitro and in overweight subjects with a high cardiovascular risk phenotype supplemented with 150 mg quercetin/day for 42 days in vivo. Supplementation of murine RAW264.7 macrophages in culture with increasing concentrations of quercetin (1, 10, 20 μmol/L) resulted in a significant increase in PON2 mRNA and protein levels, as compared to untreated controls. Unlike quercetin, its glucuronidated metabolite quercetin-3-glucuronide did not affect PON2 gene expression in cultured macrophages. However the methylated quercetin derivative isorhamnetin enhanced PON2 gene expression in RAW264.7 cells to similar extent like quercetin. Although supplementing human volunteers with quercetin was accompanied by a significant increase in plasma quercetin concentration, dietary quercetin supplementation did not change PON2 mRNA levels in human monocytes in vivo. Current data indicate that quercetin supplementation increases PON2 levels in cultured monocytes in vitro but not in human volunteers in vivo.     

Absence of Adiponutrin (PNPLA3) and Monoacylglycerol Lipase Synergistically Increases Weight Gain and Aggravates Steatohepatitis in Mice

Altered lipid metabolic pathways including hydrolysis of triglycerides are key players in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Whether adiponutrin (patatin-like phospholipase domain containing protein-3—PNPLA3) and monoacylglycerol lipase (MGL) synergistically contribute to disease progression remains unclear. We generated double knockout (DKO) mice lacking both Mgl and Pnpla3; DKO mice were compared to Mgl^−/− after a challenge by high-fat diet (HFD) for 12 weeks to induce steatosis. Serum biochemistry, liver transaminases as well as histology were analyzed. Fatty acid (FA) profiling was assessed in liver and adipose tissue by gas chromatography. Markers of inflammation and lipid metabolism were analyzed. Bone marrow derived macrophages (BMDMs) were isolated and treated with oleic acid. Combined deficiency of Mgl and Pnpla3 resulted in weight gain on a chow diet; when challenged by HFD, DKO mice showed increased hepatic FA synthesis and diminished beta-oxidation compared to Mgl^−/−. DKO mice exhibited more pronounced hepatic steatosis with inflammation and recruitment of immune cells to the liver associated with accumulation of saturated FAs. Primary BMDMs isolated from the DKO mice showed increased inflammatory activities, which could be reversed by oleic acid supplementation. Pnpla3 deficiency aggravates the effects of Mgl deletion on steatosis and inflammation in the liver under HFD challenge.     

Sasa quelpaertensis Leaf Extract Inhibits Colon Cancer by Regulating Cancer Cell Stemness in Vitro and in Vivo

A rare subpopulation of cancer cells, termed cancer stem cells (CSCs), may be responsible for tumor relapse and resistance to conventional chemotherapy. The development of a non-toxic, natural treatment for the elimination of CSCs is considered a strategy for cancer treatment with minimal side effects. In the present study, the potential for Sasa quelpaertensis leaf extract (SQE) and its two bioactive compounds, tricin and p-coumaric acid, to exert anti-CSC effects by suppressing cancer stemness characteristics were evaluated in colon cancer cells. CD133⁺CD44⁺ cells were isolated from HT29 and HCT116 cell lines using flow-activated cell sorting (FACs). SQE treatment was found to significantly suppress the self-renewal capacity of both cell lines. SQE treatment was also associated with the down-regulation of β-catenin and phosphorylated GSK3β, while significantly enhancing cell differentiation by up-regulating CK20 expression and blocking the expression of several stem cell markers, including DLK1, Notch1, and Sox-2. In vivo, SQE supplementation suppressed tumor growth in a xenograft model by down-regulating stem cell markers and β-catenin as well as HIF-1α signaling. Compared with two bioactive compounds of SQE, SQE exhibited the most effective anti-CSC properties. Taken together, these results provide evidence that SQE inhibits colon cancer by regulating the characteristics of CSCs.     

Microcirculatory Function during Endotoxemia—A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 μg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3^−/− compared to Nos2^−/− mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2^−/− mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3^−/− or Nos2^−/−/Nos3^−/− mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2^−/− mice), as this beneficial effect was absent in Nos3^−/− or Nos2^−/−/Nos3^−/− mice.     

Treatment with the Probiotic Product Aviguard® Alleviates Inflammatory Responses during Campylobacter jejuni-Induced Acute Enterocolitis in Mice

Prevalences of Campylobacter (C.) jejuni infections are progressively rising globally. Given that probiotic feed additives, such as the commercial product Aviguard^®, have been shown to be effective in reducing enteropathogens, such as Salmonella, in vertebrates, including livestock, we assessed potential anti-pathogenic and immune-modulatory properties of Aviguard^® during acute C. jejuni-induced murine enterocolitis. Therefore, microbiota-depleted IL-10^−/− mice were infected with C. jejuni strain 81-176 by gavage and orally treated with Aviguard^® or placebo from day 2 to 4 post-infection. The applied probiotic bacteria could be rescued from the intestinal tract of treated mice, but with lower obligate anaerobic bacterial counts in C. jejuni-infected as compared to non-infected mice. Whereas comparable gastrointestinal pathogen loads could be detected in both groups until day 6 post-infection, Aviguard^® treatment resulted in improved clinical outcome and attenuated apoptotic cell responses in infected large intestines during acute campylobacteriosis. Furthermore, less distinct pro-inflammatory immune responses could be observed not only in the intestinal tract, but also in extra-intestinal compartments on day 6 post-infection. In conclusion, we show here for the first time that Aviguard^® exerts potent disease-alleviating effects in acute C. jejuni-induced murine enterocolitis and might be a promising probiotic treatment option for severe campylobacteriosis in humans.     

Screening of Human Gut Bacterial Culture Collection Identifies Species That Biotransform Quercetin into Metabolites with Anticancer Properties

We previously demonstrated that flavonoid metabolites inhibit cancer cell proliferation through both CDK-dependent and -independent mechanisms. The existing evidence suggests that gut microbiota is capable of flavonoid biotransformation to generate bioactive metabolites including 2,4,6-trihydroxybenzoic acid (2,4,6-THBA), 3,4-dihydroxybenzoic acid (3,4-DHBA), 3,4,5-trihyroxybenzoic acid (3,4,5-THBA) and 3,4-dihydroxyphenylacetic acid (DOPAC). In this study, we screened 94 human gut bacterial species for their ability to biotransform flavonoid quercetin into different metabolites. We demonstrated that five of these species were able to degrade quercetin including Bacillus glycinifermentans, Flavonifractor plautii, Bacteroides eggerthii, Olsenella scatoligenes and Eubacterium eligens. Additional studies showed that B. glycinifermentans could generate 2,4,6-THBA and 3,4-DHBA from quercetin while F. plautii generates DOPAC. In addition to the differences in the metabolites produced, we also observed that the kinetics of quercetin degradation was different between B. glycinifermentans and F. plautii, suggesting that the pathways of degradation are likely different between these strains. Similar to the antiproliferative effects of 2,4,6-THBA and 3,4-DHBA demonstrated previously, DOPAC also inhibited colony formation ex vivo in the HCT-116 colon cancer cell line. Consistent with this, the bacterial culture supernatant of F. plautii also inhibited colony formation in this cell line. Thus, as F. plautii and B. glycinifermentans generate metabolites possessing antiproliferative activity, we suggest that these strains have the potential to be developed into probiotics to improve human gut health.