A method for quantifying hepatic and intestinal ceramides on mice by UPLC-MS/MS.

BACKGROUND: Ceramide is one type of sphingolipids, is associated with the occurrence of metabolic diseases, including obesity, diabetes, cardiovascular disease, cancer, and nonalcoholic fatty liver disease. Dihydroceramide, the direct precursors of ceramide, which is converted to ceramide with the dihydroceramide desaturase, is recently regarded as involving in various biological processes and metabolic diseases. The liver and gut ceramide levels are interactional in pathophysiological condition, quantifying hepatic and intestinal ceramide levels become indispensable. The aim of this study is to establish a rapid method for the determination of ceramides including dihydroceramides in liver and small intestinal tissues for researching the mechanisms of ceramide related diseases. METHODS: The levels of Cer d18:1/2:0, Cer d18:1/6:0, Cer d18:1/12:0, Cer d18:1/14:0, Cer d18:1/16:0, Cer d18:1/17:0, Cer d18:1/18:0, Cer d18:1/20:0, Cer d18:1/22:0, Cer d18:1/24:1, Cer d18:1/24:0, dHCer d18:0/12:0, dHCer d18:0/14:0, dHCer d18:0/16:0, dHCer d18:0/18:0, dHCer d18:0/24:1 and dHCer d18:0/24:0 in mice liver and small intestine were directly quantified by ultra-high performance liquid chromatography-tandem mass spectrometry after methanol extraction. In detail, liver or small intestine tissues were thoroughly homogenized with methanol. The resultant ceramides were separated on a Waters BEH C18 column using gradient elution within 10 min. Positive electrospray ionization with multiple reaction monitoring was applied to detect. In the end, the levels of ceramides in mice liver and small intestine tissues were quantified by this developed method. RESULTS: The limits of detection and quantification of 11 ceramides and 6 dihydroceramides were 0.01-0.5 ng/mL and 0.02-1 ng/mL, respectively, and all detected ceramides had good linearities (R2 > 0.997). The extraction recoveries of ceramides at three levels were within 82.32%-115.24% in the liver and within 83.21%-118.70% in the small intestine. The relative standard deviations of intra- and inter-day precision were all within 15%. The extracting solutions of the liver and small intestine could be stably stored in the autosampler 24 h at 10 °C, the lyophilized liver and small intestine for ceramides quantification could be stably stored at least 1 week at -80 °C. The ceramides and dihydroceramides in normal mice liver and small intestinal tissues analyzed by the developed method indicated that the detected 9 ceramide and 5 dihydroceramides levels were significantly different, in which Cer d18:1/16:0, Cer d18:1/22:0, Cer d18:1/24:1, Cer d18:1/24:0 and dHCer d18:0/24:1 are the main components in the liver, whereas Cer d18:1/16:0 and dHCer d18:0/16:0 accounts for the majority of proportion in the intestinal tissues. CONCLUSION: A simple and rapid method for the quantification of 11 ceramides and 6 dihydroceramides in the animal tissues was developed and applied. The compositions of ceramides in two tissues suggested that the compositional features should to be considered when exploring the biomarkers or molecular mechanisms.

Effects of probiotics on Zebrafish model infected with Aeromonas hydrophila: spatial distribution, antimicrobial, and histopathological investigation.

Usage of "probiotics" for treatment of food-borne pathogens associated diseases, makes a significant reduction in transmission of resistant bacteria, and antimicrobial resistance genes from aquaculture environments to humans. In this research, the authors aim to evaluate the immunomodulatory, and histological effects of two probiotic strains on the Zebrafish model. Fish models were treated with Lactobacillus delbrueckii (G2), Lactobacillus acidophilus (G3) and both probiotics (G4) and compared with the control group (G1) (only infected by pathogen and receiving no probiotic). Biometric tests, height, weight, and mortality rate of the fishes were assessed. Afterward, RT-PCR was conducted for bacterial existence of probiotic strains, and quantitative assessment of alterations in targeted immune genes. Subsequently, histological sampling was done for investigation of spatial distribution, and villus length in proximal, middle, and distal sections of intestinal tissues. Based on the results, G4 showed the highest gene expression for Lactobacillus acidophilus after 28 days (P < 0.05). G4 also showed an increase in the number of goblet cells and villus length in the middle and distal sections of intestinal tissue after 56 days. Furthermore, after 56 days, the highest number of intraepithelial cells was observed in the proximal sections of intestinal tissue in G4. G2 and G3 showed significant differences in comparison with G1 (P < 0.05). After 60 days, the highest gene expression for Lactobacillus bulgaricus was found in group treated with only this probiotic bacteria. The highest expression level of IL-1ß and TNF-α were found in G1. The highest survival rate was in the case of groups only treated with Lactobacillus bulgaricus (G2). To sum up, it seems that usage of probiotics for the improvement of public health and fisheries industries can be helpful.

The change of the electrophysiological parameters using human intestinal tissues from ulcerative colitis and Crohn's disease.

The purpose of this study was to investigate how disease state of the UC and CD patients affect tissue function determined from electrophysiology viewpoint the electrophysiological parameters on normal, ulcerative colitis (UC) and Crohn's disease (CD) patients. Potential differences (PD), short circuit current (Isc) and resistance (R) as electrophysiological parameters were determined using human large intestinal tissues. The measure of autoptical abnormality was quantified on an arbitrary scale of 0-2. A severe effect of ulcer and thickened mucosa by fibrosis was scored as Grade 2. The larger number of autopsy grade on both UC and CD tissues, the lower values of PD and R than those of normal tissues were observed, although Isc values were not statistically changed irrespective of autopsy grade. This electrophysiological observation of reduced PD indicated functional impairment of active ion transport via ion pumps. Additionally, the R values of CD tissues on each autopsy grade tended to be lower than those of UC tissues. These results suggest that the effect of inflammatory bowel disease on barrier function is different between UC and CD tissues. Therefore, the fibrosis on CD patients might affect the electrophysiological parameters than that of UC patients.

Short-term toxicity of dibutyl phthalate to mice intestinal tissue.

The objective of this study was to investigate changes in intestinal histopathology and expression of heat-shock proteins (HSPs) in the small intestinal tissue of mouse after acute exposure to dibutyl phthalate (DBP). Forty-eight 60-day-old Institute of Cancer Research (ICR) mice were administered DBP by gavage once a day for 10 days. The mice were divided into three groups of 16 mice each: the high-dose group was administered 500 mg/kg body weight (BW) DBP; the low-dose group was administered 50 mg/kg BW; and the control group was not administered DBP. Significant increases in the uterine index, ovary index, and testicular index were observed in the DBP-exposed groups compared to those in the control group. Villus height and V/ C ratio significantly increased ( p < 0.05) in the duodenum and decreased ( p < 0.05) in the jejunum after the administration of DBP. The goblet cell number decreased in both the duodenum and the jejunum of mice exposed to DBP ( p < 0.05) compared to the number in the control group mice. Damage to the structure of the small intestine was accompanied by a marked increase in HSP27 expression and a decrease in the expression of HSP70 and HSP90 in both high-dose and low-dose groups. These results indicate that elevated HSP27 levels in the duodenum and jejunum may be important markers for acute DBP exposure and that HSP27 may act as a protective protein involved in intestinal mucosa repair.

Peptidome analysis reveals critical roles for peptides in a rat model of intestinal ischemia/reperfusion injury.

Intestinal ischemia/reperfusion injury (IIRI) has the potential to be life threatening and is associated with significant morbidity and serious damage to distant sites in the body on account of disruption of the intestinal mucosal barrier. In the present study, we have explored this line of research by comparing and identifying peptides that originated from the intestinal segments of IIRI model rats by using liquid chromatography-mass spectrometry (LC-MS). We also analyzed the basic characteristics, cleavage patterns, and functional domains of differentially expressed peptides (DEPs) between the IIRI model rats and control (sham-operated) rats and identified bioactive peptides that are potentially associated with ischemia reperfusion injury. We also performed bioinformatics analyses in order to identify the biological roles of the DEPs based on their precursor proteins. Enrichment analysis demonstrated the role of several DEPs in impairment of the intestinal mucosal barrier caused by IIRI. Based on the results of comprehensive ingenuity pathway analysis, we identified the DEPs that were significantly correlated with IIRI. We identified a candidate precursor protein (Actg2) and seven of its peptides, and we found that Actg2-6 had a more significant difference in its expression, a longer half-life, and better lipophilicity, hydrophobicity, and stability than the other candidate Actg2 peptides examined. Furthermore, we observed that Actg2-6 might play critical roles in the protection of the intestinal mucosal barrier during IIRI. In summary, our study provides a better understanding of the peptidomics profile of IIRI, and the results indicate that Actg2-6 could be a useful target in the treatment of IIRI.

Thiamine modulates intestinal morphological structure and microbiota under subacute ruminal acidosis induced by a high-concentrate diet in Saanen goats.

Ruminant animals are generally fed with starch-rich grain as the main energy source, and the incidence of metabolic diseases such as subacute ruminal acidosis (SARA) is high due to the intensive farming. Thiamin has been reported to alleviate SARA caused by high-concentrate diets, but the exact mechanism is not well understood. The goal of this study was to examine the role of thiamine in intestinal inflammation and microbiota caused by high-concentrate diets. The SARA model was induced by low neutral detergent fibre/starch ration to study the effects of thiamine on intestinal tissue structure and microbiota. 18 mid-lactation (148 ± 3 d in milk; milk yield = 0.71 ± 0.0300 kg/d) Saanen goats (BW = 36.5 ± 1.99 kg; body condition score = 2.73 ± 0.16, where 1 = emaciated and 6 = obese) in parities 1 or 2 were selected. The goats were randomly divided into three groups with six replicates: (1) control diet (C; concentrate:forage 30:70), (2) high-concentrate diet (H; concentrate:forage 70:30), and (3) high-concentrate diet with 200 mg of thiamine/kg of DM intake (H + T;concentrate:forage 70:30). The experimental period was lasted for 56 d. The small and large intestine, expression of inflammatory factor genes, tight junction protein genes, total antioxidant capacity, and intestinal microbiota were measured. The results showed that SARA was observed in treatment H, whereas rumen fluid pH was improved in treatment H + T. Treatment H + T also significantly repaired the intestinal tissue structure damaged by SARA, improved the total antioxidant capacity of the small intestinal mucosa, reduced mRNA expression of inflammatory factors in the small intestine tissue, and increased the mRNA expression of tight junction genes in small intestine tissue. The high-concentrate diet reduced the diversity of intestinal microbiota. When thiamine is added to the high-concentrate diet, the relative abundance of intestinal Firmicutes and beneficial bacteria represented by Lactobacilli were upregulated, and the relative abundance of Proteus, a marker of intestinal dysbacteriosis, returned to normal. In conclusion, thiamine supplementation could alleviate the damage to the intestinal tissue structure and microbial environment caused by SARA condition in dairy goats fed a high-concentrate diet.

Tissue Responses to Shiga Toxin in Human Intestinal Organoids.

BACKGROUND & AIMS: Shiga toxin (Stx)-producing Escherichia coli (eg, O157:H7) infection produces bloody diarrhea, while Stx inhibits protein synthesis and causes the life-threatening systemic complication of hemolytic uremic syndrome. The murine intestinal tract is resistant to O157:H7 and Stx, and human cells in culture fail to model the complex tissue responses to intestinal injury. We used genetically identical, human stem cell-derived intestinal tissues of varying complexity to study Stx toxicity in vitro and in vivo. METHODS: In vitro susceptibility to apical or basolateral exposure to Stx was assessed using human intestinal organoids (HIOs) derived from embryonic stem cells, or enteroids derived from multipotent intestinal stem cells. HIOs contain a lumen, with a single layer of differentiated epithelium surrounded by mesenchymal cells. Enteroids only contain epithelium. In vivo susceptibility was assessed using HIOs, with or without an enteric nervous system, transplanted into mice. RESULTS: Stx induced necrosis and apoptotic death in both epithelial and mesenchymal cells. Responses that require protein synthesis (cellular proliferation and wound repair) also were observed. Epithelial barrier function was maintained even after epithelial cell death was seen, and apical to basolateral translocation of Stx was seen. Tissue cross-talk, in which mesenchymal cell damage caused epithelial cell damage, was observed. Stx induced mesenchymal expression of the epithelial marker E-cadherin, the initial step in mesenchymal-epithelial transition. In vivo responses of HIO transplants injected with Stx mirrored those seen in vitro. CONCLUSIONS: Intestinal tissue responses to protein synthesis inhibition by Stx are complex. Organoid models allow for an unprecedented examination of human tissue responses to a deadly toxin.

Chemokines and receptors in intestinal B lymphocytes.

Recent studies indicate that chemoattractant cytokines (chemokines) and their receptors modulate intestinal B lymphocytes in different ways, including regulating their maturity and differentiation in the bone marrow and homing to intestinal target tissues. Here, we review several important chemokine/chemokine receptor axes that guide intestinal B cells, focusing on the homing and migration of IgA antibody-secreting cells (IgA-ASCs) to intestinal-associated lymphoid tissues. We describe the selective regulation of these chemokine axes in coordinating the IgA-ASC trafficking in intestinal diseases. Finally, we discuss the role of B cells as chemokine producers serving dual roles in regulating the mucosal immune microenvironment.

Lactobacillus rhamnosus GG supernatant upregulates serotonin transporter expression in intestinal epithelial cells and mice intestinal tissues.

BACKGROUND: The role that probiotics play in relieving irritable bowel syndrome (IBS) has been demonstrated; however, the mechanism by which IBS is affected remains unclear. In this study, serotonin transporter (SERT) mRNA and serotonin transporter protein (SERT-P) levels in HT-29, Caco-2 cells, and mice intestinal tissues were examined after treatment with Lactobacillus rhamnosus GG supernatant (LGG-s). METHODS: HT-29 and Caco-2 cells were treated with different concentrations of LGG-s for 12 and 24 h and C57BL/6 mice received supplements of different concentrations for 4 weeks. SERT mRNA and SERT-P levels were detected by real-time PCR and Western blotting. KEY RESULTS: SERT mRNA and SERT-P levels in HT-29 and Caco-2 cells were higher than those in the control 24 h after treatment. Undiluted LGG-s upregulated SERT mRNA levels by 9.4-fold in the first week, which dropped in the second week. The double-diluted LGG-s upregulated SERT mRNA by 2.07-fold in the first week; levels dropped to 1.75-fold within the second week and under base expression levels by the third week, while they again climbed to 1.56-fold in the fourth week. The triple-diluted LGG-s could not upregulate SERT mRNA expression until the end of the fourth week. The SERT-P levels in the double-diluted LGG-s group were higher than that in the control but fluctuated with time. SERT-P levels in the triple-diluted LGG-s were higher than that in the control in the last 2 weeks and increased with time. CONCLUSIONS & INFERENCES: LGG-s can upregulate SERT mRNA and SERT-P levels in intestinal epithelial cells and mice intestinal tissues.