Milk Fat Globule Epidermal Growth Factor 8a Regulates Neurogenesis in Telencephalon and Affects Larval Behavior in Zebrafish.

Mfge8, a secreted glycoprotein, is a key molecule that mediates the phagocytosis of apoptotic cells. Previous research reported that Mfge8 is critical for the proliferation and differentiation of radial glial cells (RGCs) in the dentate gyrus of adult mice. The treatment of Mfge8 is also beneficial for the repair of central nervous system (CNS) injury after cerebral ischemia. This study aimed to investigate whether the expression of mfge8a in zebrafish embryos was associated with the development of CNS and larval behavior. We found that zebrafish mfge8a was initially expressed at 48 hpf, and its expression was gradually increased in the ventricular zone. Knocking down mfge8a with antisense morpholino oligonucleotides impaired both spontaneous and photoinduced swimming locomotion in the behavioral tests. The neurogenesis analysis in telencephalon showed that mfge8a morphants excessively promoted neural differentiation over self-renewal after RGCs division, and consequently depleted proliferative RGC population during early neurogenesis. Furthermore, downregulation of mfge8a was shown to alter the expression patterns of genes associated with Notch signaling pathway. Our results demonstrated that mfge8a is involved in the maintenance of the progenitor identity of RGCs in embryonic zebrafish brain through regulating Notch signaling pathway, thereby contributing to consistent neurogenesis and locomotor development.

Green tea catechin EGCG could prevent obesity-related precocious puberty through NKB/NK3R signaling pathway.

This study aimed to explore the potential regulatory pathways of (-)-epigallocatechin-3-gallate (EGCG) in preventing obesity-related precocious puberty. A retrospective analysis on the impact of EGCG on puberty onset in obese girls was conducted on plasma samples collected from a human randomized controlled trial. In the trial, participants consumed EGCG capsules for 12 weeks. In the animal experiment, rats were divided into four groups: normal diet control (NC) group, high-fat diet (HFD) group, NC+EGCG group, and HFD+EGCG group. Blood samples were collected on postnatal days 27, 33, and 36 to detect sexual development indicators. The hypothalamic expressions of kisspeptin/Kiss1R and neurokinin B (NKB)/NK3R signaling were measured by RT-qPCR and Western blot assay. The ovary NKB protein expression was assessed by immunohistochemical assays. Serum NKB level in the EGCG group was lower than the placebo group by 0.599 ng/mL [ß=-0.599, 95% CI: (-1.005, -0.193)], at the end of intervention and after adjusting for confounders (clinical study). In the animal experiment, EGCG intervention could significantly delay the vaginal opening (VO) time of rats fed with HFD. On day 33, EGCG intervention could significantly reduce serum NKB, luteinizing hormone (LH) levels, ovarian NKB protein expression, and endometrial thickness of HFD-fed rats, while EGCG intervention could remarkably increase mRNA and protein expression of NKB/NK3R. EGCG could prevent obesity-related precocious puberty through NKB/NK3R signaling pathway, which may provide a novel insight into the role of EGCG in preventing precocious puberty in obese girls.

Trans-10-Hydroxy-2-Decenoic Acid Alleviates Dextran Sulfate Sodium-Induced Colitis in Mice via Regulating the Inflammasome-Mediated Pyroptotic Pathway and Enhancing Colonic Barrier Function.

SCOPE: The objective of this study is to explore the effects of 10-hydroxy-2-decenoic acid (10-HDA), the major fatty acid in royal jelly, on dextran sodium sulfate (DSS)-induced mice ulcerative colitis (UC) and its potential mechanism of action. METHODS AND RESULTS: Forty male C57BL/6 mice are randomly divided into five experimental groups: control, DSS, DSS + 25 (or 100)mg kg-1  d-1 10-HDA, and DSS + 200 mg kg-1  d-1 mesalazine (ME). UC is induced in mice using 2.5% DSS in drinking water for 7 days. During the induction, these UC mice are orally administrated 10-HDA or ME per day. Meanwhile, lipopolysaccharide (LPS)/adenosine-triphosphate (ATP)-stimulated THP1 cells are used as a model to test the effects of 10-HDA. 10-HDA reduces DSS-induced pathological damage, reactive oxygen species (ROS) accumulation, neutrophil infiltration, and cytokine production in colonic tissue. Compared with the DSS group, the expressions of thioredoxin interacting protein (TXNIP), NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC), cysteinyl aspartate specific proteinase-1 (Caspase-1), gasdermin-D (GSDMD), N-terminal domain of gasdermin-D (N-GSDMD), interleukin-1ß (IL-1ß), and interleukin-18 (IL-18) in the colon are decreased after administration of 10-HDA. 10-HDA also elevates the barrier integrity and the expressions of zonula occludens-1 (ZO-1) and Occludin in colonic epithelium exposed to DSS. In THP1 cells, the inflammasome-mediated pyroptosis induced by LPS/ATP is inhibited by 10-HDA pretreatment. CONCLUSION: 10-HDA alleviates DSS-induced colitis by regulating the NLRP3 inflammasome-mediated pyroptotic pathway and enhancing colonic barrier function.

Neonatal Supplementation of Oleamide During Suckling Promotes Learning Ability and Memory in Adolescent Mice.

BACKGROUND: Fatty acid amides (FAMs) are present in breast milk. Oleamide (ODA), a member of the FAM family, has been reported to affect learning and memory-related abilities in animal experiments. OBJECTIVES: This study aimed to characterize the temporal changes of FAMs in human milk and sought to examine the effect of ODA supplementation during suckling on postweaning cognitive performance in mice. METHODS: FAMs were measured in human milk (postpartum 1-24 wk) by ultra-performance liquid chromatography-triple quadruple mass spectrometry (UPLC-TQ-MS) analysis. We supplemented neonatal C57BL/6J mice of both sexes with vehicle (control), 5 mg/(kg · day) ODA (L-ODA), or 25 mg/(kg · day) ODA (H-ODA) throughout suckling by oral gavage. After weaning, the Morris water maze test and novel object recognition test were performed. Neurogenesis, spinal morphogenesis in the dentate gyrus (DG) region, and hippocampal expression of synaptic markers were analyzed. Data were analyzed by ANOVA and repeated-measures ANOVA. RESULTS: ODA (0.566-1.31 mg/L) was the most abundant FAM in breast milk, followed by palmitamide (0.135-0.269 mg/L) and linoleamide (0.046-0.242 mg/L). Compared with the control group, the H-ODA group demonstrated shorter escape latency, shorter travel distance, 113% more platform crossing, and 48% greater discrimination index in behavioral tests (P < 0.05). Additionally, the H-ODA group showed a higher density of 5-ethynyl-2'-deoxyuridine (EdU)+ and EdU+& doublecortin (DCX)+ cells (62% and 53%, respectively), and 52% greater spine density in the DG region than the control group (P < 0.05). The synaptic markers, postsynaptic density protein 95 (PSD95) and synaptophysin (SYP), were upregulated in the H-ODA group compared with the control group (P < 0.05). The L-ODA group also showed shorter escape latency in behavioral tests and 27% greater spine density in the DG region than the control group (P < 0.05). CONCLUSIONS: ODA is the most common FAM in human milk. ODA supplementation during suckling promotes learning and memory-related abilities in adolescent mice by augmenting hippocampal neuronal proliferation and boosting synaptic plasticity.

Neonatal Milk Fat Globule Membrane Supplementation During Breastfeeding Ameliorates the Deleterious Effects of Maternal High-Fat Diet on Metabolism and Modulates Gut Microbiota in Adult Mice Offspring in a Sex-Specific Way.

Exposure to adverse events in early life increases the risk of chronic metabolic disease in adulthood. The objective of this study was to determine the significance of milk fat globule membrane (MFGM)-mediated alterations in the gut microbiome to the metabolic health of offspring in the long-term. Female C57BL/6 mice were fed either a high-fat diet (HFD) or a control diet for 3 weeks before pregnancy and throughout pregnancy and lactation. During lactation, pups from the HFD group were breast-fed with or without 1,000 mg/kg BW/day MFGM supplementation (HFD and HFD-MS group, respectively). After weaning, the offspring in each group were divided into male and female subgroups. The weaned mice were then shifted to a control diet for 8 weeks. At the eleventh week, stool samples were collected for 16S rRNA gene sequencing. Serum biochemical parameters were analyzed, and intraperitoneal glucose and insulin tolerance tests were performed. Neonatal supplementation with MFGM ameliorated metabolic disorder and improved glucose tolerance in offspring exposed to maternal HFD in a sex-specific manner. Furthermore, maternal HFD induced gut microbiota perturbation in offspring in adulthood. Neonatal MFGM supplementation significantly enriched g-Parabacteroides, g-Bifidobacterium, g-Faecalibaculum, and g-Lactobacillus in male offspring exposed to maternal HFD, while significantly enriched g-Parabacteroides and g-Alistipes in female offspring exposed to maternal HFD. These bacteria may be associated with the favorable changes in metabolism that occur in adulthood. Sex differences in the changes of metagenomic pathways related to oxidative phosphorylation, citrate cycle, electron transfer carries, and ubiquinone biosynthesis were also observed in the offspring. Maternal HFD has an adverse effect on the metabolism of offspring in later life. Neonatal MFGM supplementation could modulate the structure of gut microbiota communities and may have long-term protective effects on lipid and glucose metabolism, but these effects are sex dimorphic.

Milk Fat Globule Membrane Supplementation During Suckling Ameliorates Maternal High Fat Diet-Induced Hepatic Steatosis in Adult Male Offspring of Mice.

BACKGROUND: Exposure to a maternal high-fat diet (HFD) predisposes offspring to nonalcoholic fatty liver disease. OBJECTIVES: The aim of this study was to explore whether milk fat globule membrane (MFGM) supplementation during suckling exerts a long-term protective effect on hepatic lipid metabolism in adult offspring exposed to maternal HFD. METHODS: We fed 5-week-old female C57BL/6J mice either a HFD (60% kcal fat) or control diet (CD; 16.7% kcal fat) for 3 weeks before mating, as well as throughout gestation and lactation. After delivery, male offspring from HFD dams were supplemented with 1 g/(kg body weight·day) MFGM (HFD + MFGM group) or the same volume of vehicle (HFD group) during suckling. Male offspring from CD dams were also supplemented with vehicle during suckling (CD group). All offspring were weaned onto CD for 8 weeks. Histopathology, metabolic parameters, lipogenic level, oxidative stress, and mitochondria function in the liver were analyzed. A 1-way ANOVA and a Kruskal-Wallis test were used for multi-group comparisons. RESULTS: As compared to the CD group, the HFD group had more lipid droplets in livers, and exhibited ∼100% higher serum triglycerides, ∼38% higher hepatic triglycerides, ∼75% higher serum aspartate aminotransferase, and ∼130% higher fasting blood glucose (P < 0.05). The changes of these metabolic parameters were normalized in the HFD + MFGM group. Phosphorylated mammalian targets of rapamycin and AKT were downregulated, but phosphorylated adenosine monophosphate-activated protein kinase was upregulated in the HFD + MFGM group as compared to the HFD group (P < 0.05). As compared to the CD group, the HFD group showed an ∼80% higher malondialdehyde level, and ∼20% lower superoxide dismutase activity (P < 0.05), which were normalized in the HFD + MFGM group. Additionally, mitochondria function was also impaired in the HFD group and normalized in the HFD + MFGM group. CONCLUSIONS: MFGM supplementation during suckling ameliorates maternal HFD-induced hepatic steatosis in mice via suppressing de novo lipogenesis, reinforcing antioxidant defenses and improving mitochondrial function.

Bile salt-dependent lipase promotes the barrier integrity of Caco-2 cells by activating Wnt/ß-catenin signaling via LRP6 receptor.

Bile salt-dependent lipase (BSDL) within intestinal lumen can be endocytosed by enterocytes and support the intestinal barrier function. However, the epithelial-supporting effect of this protein has not been verified in a human cell line and neither the direct signaling pathway nor the function of endocytosis in this process has been clearly identified. We sought to investigate the signaling pathway and the membrane receptor through which BSDL might exert these effects using intestinal epithelial cells. Caco-2 cells were treated with recombinant BSDL, and the barrier function, cell proliferation, and activation of the Wnt signaling pathway were assessed. The effect of Wnt signaling activation induced by BSDL and BSDL endocytosis was investigated in LRP6-silenced and non-silenced cells. Moreover, caveolae- and clathrin-dependent endocytosis inhibitors were also applied respectively to analyze their effects on Wnt signaling activation induced by BSDL. BSDL treatment increased the barrier function but not proliferation of Caco-2 cells. It also induced ß-catenin nuclear translocation and activated Wnt target gene transcription. Moreover, in the Wnt pathway, BSDL increased the levels of non-phosphorylated-ß-catenin (Ser33/37/Thr41) and phosphorylated-ß-catenin (Ser552). Notably, the silencing of LRP6 expression impaired BSDL endocytosis and decreased BSDL-induced ß-catenin nuclear translocation. The inhibition of BSDL endocytosis induced by caveolae-mediated endocytosis inhibitor was stronger than that by clathrin-mediated endocytosis inhibitor, and the Wnt signaling activation associated with its endocytosis was also most likely caveolae-dependent. Our findings suggested that LRP6, a canonical Wnt pathway co-receptor, can mediate BSDL endocytosis and then activate Wnt signaling in Caco-2 cells.

Carboxyl ester lipase is highly conserved in utilizing maternal supplied lipids during early development of zebrafish and human.

Carboxyl ester lipase (Cel), is a lipolytic enzyme secreted by the pancreas, which hydrolyzes various species of lipids in the gut. Cel is also secreted by mammary gland during lactation and exists in breast milk. It facilitates dietary fat digestion and absorption, thus contributing to normal infant development. This study aimed to examine whether the Cel in zebrafish embryos has a similar role of maternal lipid utilization as in human infants, and how Cel contributes to the utilization of yolk lipids in zebrafish. The cel1 and cel2 genes were expressed ubiquitously in the blastodisc and yolk syncytial layer before 24 hpf, and in the exocrine pancreas after 72 hpf. The cel1 and cel2 morphants exhibited developmental retardation and yolk sac retention. The total cholesterol, cholesterol ester, free cholesterol, and triglyceride were reduced in the morphants' body while accumulated in the yolk (except triglyceride). The FFA content of whole embryos was much lower in morphants than in standard controls. Moreover, the delayed development in cel (cel1/cel2) double morphants was partially rescued by FFA and cholesterol supplementation. Delayed and weakened cholesterol ester transport to the brain and eyes was observed in cel morphants. Correspondingly, shrunken midbrain tectum, microphthalmia, pigmentation-delayed eyes as well as down-regulated Shh target genes were observed in the CNS of double morphants. Interestingly, cholesterol injections reversed these CNS alterations. Our findings suggested that cel genes participate in the lipid releasing from yolk sac to developing body, thereby contributing to the normal growth rate and CNS development in zebrafish.

Milk Fat Globule Membrane Ameliorates Necrotizing Enterocolitis in Neonatal Rats and Suppresses Lipopolysaccharide-Induced Inflammatory Response in IEC-6 Enterocytes.

BACKGROUND: Evidence has provided support for the beneficial effects of milk fat globule membrane (MFGM) on inflammation in the intestinal tract. The objective of this study was to investigate the effects of MFGM on a rat model of necrotizing enterocolitis (NEC) and its potential mechanism of action. METHODS: Sixty-two newborn Sprague Dawley rats were randomly divided into 4 experimental groups: Breast-fed normal, formula fed (FF), FF + 6 g/L MFGM, and FF + 12 g/L MFGM. The FF rats and the FF rats supplemented with MFGM were exposed to asphyxia/cold stress to induce NEC. Body weight, histological score, survival time, oxidative stress injury, enterocyte proliferation/apoptosis, and inflammatory response were assessed. Meanwhile, lipopolysaccharide (LPS)-stimulated IEC-6 enterocytes were used as a model to test the anti-inflammatory effects of MFGM. RESULTS: Supplementation with 12 g/L MFGM alleviated body weight loss, reduced the incidence of NEC, increased the survival rate, and attenuated the severity of bowel damage in the NEC rat model. Furthermore, 12 g/L MFGM administration inhibited the protein expression of toll-like receptor 4 (TLR4) in the animal model. In IEC-6 enterocytes, the upregulation of TLR4, myeloid differentiation primary response gene 88 (MyD88), phosphorylated nuclear factor-κB (NF-κB) p65 subunit, and the nuclear translocation of NF-κBp65 induced by LPS was partially inhibited by MFGM pretreatment. CONCLUSION: Our findings suggested that MFGM has beneficial effects on neonatal rats with NEC by suppressing inflammation via the TLR4/MyD88/NF-κB pathway.

A Plasmonic Mass Spectrometry Approach for Detection of Small Nutrients and Toxins.

Nutriology relies on advanced analytical tools to study the molecular compositions of food and provide key information on sample quality/safety. Small nutrients detection is challenging due to the high diversity and broad dynamic range of molecules in food samples, and a further issue is to track low abundance toxins. Herein, we developed a novel plasmonic matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) approach to detect small nutrients and toxins in complex biological emulsion samples. Silver nanoshells (SiO2@Ag) with optimized structures were used as matrices and achieved direct analysis of ~ 6 nL of human breast milk without any enrichment or separation. We performed identification and quantitation of small nutrients and toxins with limit-of-detection down to 0.4 pmol (for melamine) and reaction time shortened to minutes, which is superior to the conventional biochemical method currently in use. The developed approach contributes to the near-future application of MALDI MS in a broad field and personalized design of plasmonic materials for real-case bio-analysis.

Bile salt dependent lipase promotes intestinal adaptation in rats with massive small bowel resection.

Intestinal adaptation is important for the short bowel syndrome (SBS) patients. Growing evidence has suggested that bile salt dependent lipase (BSDL) not only has the lipolytic activity, but also the immune-modulating and pro-proliferative activities. The purpose of the present study was to investigate the effects of BSDL on intestinal adaptive growth and gut barrier function in a rat model of SBS. Twenty-four male Sprague-Dawley rats were randomly divided into three experimental groups: sham group (rats underwent bowel transection and re-anastomosis), SBS group (rats underwent 80% bowel resection), SBS-BSDL group (SBS rats orally administered BSDL). The animals were weighed daily. The intestinal morpho-histochemical changes and intestinal barrier function were determined 14 days after the operations. Meanwhile, the expressions of Wnt signaling molecules in enterocytes were also analyzed by immunohistochemistry and Western blot. The postoperative weight gain was faster in the SBS rats treated with BSDL than in the SBS/untreated group. The SBS rats treated with BSDL had significantly greater villus height, crypt depth, and enterocyte proliferation in their residual intestines, as compared with the SBS/untreated group. The recovery of intestinal barrier function was promoted and the expressions of tight-junction proteins were increased in the SBS rats treated with BSDL. Additionally, the data indicated that the proadaptive activities of BSDL might be mediated by Wnt signaling activation in the enterocytes. These observations suggested that enteral BSDL administration promoted intestinal adaptive growth and barrier repairing by activating Wnt signaling pathway in SBS rats.

Metabolomic Approaches to Explore Chemical Diversity of Human Breast-Milk, Formula Milk and Bovine Milk.

Although many studies have been conducted on the components present in human breast milk (HM), research on the differences of chemical metabolites between HM, bovine milk (BM) and formula milk (FM) is limited. This study was to explore the chemical diversity of HM, BM and FM by metabolomic approaches. GC-TOFMS and UPLC-QTOFMS were applied to investigate the metabolic compositions in 30 HM samples, 20 FM samples and 20 BM samples. Metabolite profiling identified that most of the non-esterified fatty acids, which reflected the hydrolysis of triglycerides, were much more abundant in HM than those in FM and BM, except for palmitic acid and stearic acid. The levels of tricarboxylic acid (TCA) intermediates were much higher in FM and BM than those in HM. Each type of milk also showed its unique composition of free amino acids and free carbohydrates. In conclusion, higher levels of non-esterified saturated fatty acids with aliphatic tails <16 carbons, monounsaturated fatty acids and polyunsaturated fatty acids and lower levels of TCA intermediates are characteristic of HM, as compared with FM and BM. The content of non-esterified fatty acids may reflect the hydrolysis of triglycerides in different milk types.

CUGBP1 and HuR regulate E-cadherin translation by altering recruitment of E-cadherin mRNA to processing bodies and modulate epithelial barrier function.

The effectiveness and stability of epithelial barrier depend on apical junctional complexes, which consist of tight junctions (TJs) and adherens junctions (AJs). E-cadherin is the primary component of AJs, and it is essential for maintenance of cell-to-cell interactions and regulates the epithelial barrier. However, the exact mechanism underlying E-cadherin expression, particularly at the posttranscriptional level, remains largely unknown. RNA-binding proteins CUG-binding protein 1 (CUGBP1) and HU antigen R (HuR) are highly expressed in the intestinal epithelial tissues and modulate the stability and translation of target mRNAs. Here, we present evidence that CUGBP1 and HuR interact directly with the 3'-untranslated region of E-cadherin mRNA and regulate E-cadherin translation. CUGBP1 overexpression in Caco-2 cells inhibited E-cadherin translation by increasing the recruitment of E-cadherin mRNA to processing bodies (PBs), thus resulting in an increase in paracellular permeability. Overexpression of HuR exhibited an opposite effect on E-cadherin expression by preventing the translocation of E-cadherin mRNA to PBs and therefore prevented CUGBP1-induced repression of E-cadherin expression. Elevation of HuR also abolished the CUGBP1-induced epithelial barrier dysfunction. These findings indicate that CUGBP1 and HuR negate each other's effects in regulating E-cadherin translation by altering the recruitment of E-cadherin mRNA to PBs and play an important role in the regulation of intestinal barrier integrity under various pathophysiological conditions.

Human ß-defensin-3 promotes intestinal epithelial cell migration and reduces the development of necrotizing enterocolitis in a neonatal rat model.

BACKGROUND: The aim of this study was to investigate the effects of human ß-defensin-3 (hBD3) on intestinal wound healing and in a neonatal rat model of necrotizing enterocolitis (NEC). METHODS: Enterocyte migration and proliferation were detected in vitro and in vivo. The role of chemokine receptor CCR6 and its downstream signaling pathway was assessed. Newborn Sprague-Dawley rats were randomly divided into four groups: Control+NS, Control+hBD3, NEC+NS, and NEC+hBD3. Body weight, histological score, survival time, cytokines expression, and mucosal integrity were evaluated. RESULTS: hBD3 could stimulate enterocyte migration, but not proliferation, both in cultured enterocytes and in the NEC model. Neutralizing antibody and small interfering RNA confirmed this stimulatory effect was mediated by CCR6. Furthermore, hBD3 induced Rho activation, myosin light chain 2 phosphorylation, and F-actin accumulation. The bactericidal activity of hBD3 was maintained throughout a broad pH range. Strikingly, hBD3 administration decreased the incidence of NEC, increased the survival rate, and reduced the severity of NEC. Moreover, hBD3 reduced the proinflammatory cytokines expression in ileum and serum and preserved the intestinal barrier integrity. CONCLUSION: This study provided evidence that the antimicrobial peptide hBD3 might participate in intestinal wound healing by promoting enterocyte migration and show beneficial effects on newborn rats with NEC.

Alkylglycerols modulate the proliferation and differentiation of non-specific agonist and specific antigen-stimulated splenic lymphocytes.

Alkylglycerols (AKGs) are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed that oral AKGs administration significantly increased the immune response in mice. The aim of the present study was to investigate the in vitro immunomodulatory effect of AKGs on stimulating splenic lymphocyte responses. C57BL/6 mice were immunized with hepatitis B surface antigen (HBsAg). Splenic B cells were purified and stimulated with anti-BCR and anti-CD38. Meanwhile, splenic CD4+ T cells were purified and stimulated with anti-CD3 and anti-CD28. For antigen specific stimulation, the purified CD4+ T cells were cocultured with HBsAg -pulsed dendritic cells. The stimulated lymphocytes were treated with different concentrations of AKGs. The cell proliferation was assessed by [3H]-thymidine incorporation assay. The maturation of B cells was assessed by examining the germline (GL) transcription of IgG (γ1) mRNA expression, and the surface expressions of CD80/CD86 markers were examined by flow cytometry analysis. Th1/Th2 polarity was assessed by T-BET (Th1)/GATA-3 (Th2) flow cytometry assay and by characteristic cytokines ELISA assay (TNF-α and IFN-γ for Th1; IL-4 and IL-10 for Th2). It was found that AKGs significantly increased the BCR/CD38 -stimulated B cell proliferation. The T cell proliferation in response to CD3/CD28 or specific antigen stimulation was also increased by AKGs. The transcriptional level of IgG (γ1) and the expressions of CD80/CD86 molecules were markedly increased by AKGs in BCR/CD38 -stimulated B cells. Meanwhile, the results showed that AKGs increased the expression of T-BET transcriptional factor and the production of Th1 cytokines (TNF-α and IFN-γ) upon CD3/CD28 stimulation; whereas, levels of Th2 cytokines (IL-4 and IL-10) were decreased by AKGs. Our study demonstrated that AKGs can modulate immune responses by boosting the proliferation and maturation of murine lymphocytes in vitro.

Protective effects of hydrogen-rich saline on necrotizing enterocolitis in neonatal rats.

PURPOSE: The aim of this study was to test the hypothesis that hydrogen-rich saline (HRS) might have protective effects on the development of necrotizing enterocolitis (NEC) in a neonatal rat model. METHODS: NEC was induced in male newborn Sprague-Dawley rats by formula feeding, exposure to asphyxia and cold stress. Sixty-four rat pups were divided randomly into four groups: C+NS (n=11), C+H2 (n=11), NEC+NS (n=20), and NEC+H2 (n=22). Rats in the former two groups were mother-fed. Pups received intra-peritoneal injection of HRS (10 ml/kg, 10 min before asphyxia stress twice a day) or the same dose of normal saline. Rats were monitored until 96 h after birth. Body weight, histological NEC score, survival time, malondialdehyde, antioxidant capacity, inflammatory mediators, and mucosal integrity were assessed. RESULTS: HRS treatment maintained the body weight, reduced the incidence of NEC from 85% (17/20) to 54.5% (12/22), increased the survival rate from 25% (5/20) to 68.2% (15/22), and attenuated the severity of NEC. In addition, HRS inhibited the mRNA expression of pro-inflammatory mediators (inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin-6), down-regulated lipid peroxidation, enhanced total antioxidant capacity, and prevented the increase of diamine oxidase in serum. However, no significant influence of HRS on the interleukin-10 mRNA expression was observed. CONCLUSIONS: HRS showed beneficial effects on neonatal rats with NEC via decreasing oxidative stress, increasing antioxidant capacity, suppressing inflammation, and preserving mucosal integrity.

Oral administration of alkylglycerols differentially modulates high-fat diet-induced obesity and insulin resistance in mice.

Alkylglycerols (AKGs) from shark liver oil (SLO) were demonstrated to have strong potency to stimulate immune response. However, no study has been conducted on the effects of AKGs on diet-induced obesity and metabolic inflammatory disorder. The purpose of the present study was to investigate the effect of two AKGs isoforms on obesity and insulin resistance in mice fed high-fat (HF) diet. Forty-eight C57BL/6 mice were divided into normal, HF, HF + 20 mg/kg selachyl alcohol (SA), HF + 200 mg/kg SA, HF + 20 mg/kg batyl alcohol (BA), and HF + 200 mg/kg BA groups. Body weight, fasting glucose, lipids, insulin and leptin levels, serum IL-1ß, and TNF- α levels were compared among different groups. Our results showed that high-dose SA decreased body weight, serum triglyceride, cholesterol, fasting glucose level, insulin level, and serum leptin level of the HF fed mice, while high-dose BA increased fasting insulin level of the HF fed mice. Pretreatment of primary adipocytes with 10 µ M SA or BA differentially modulates LPS-mediated MAPK and NF- κ B signaling. Our study demonstrated that oral administration of AKGs has differential effects on HF-induced obesity and metabolic inflammatory disorder in mice.

Polymorphisms of SLC30A2 and selected perinatal factors associated with low milk zinc in Chinese breastfeeding women.

The variability of breast-milk zinc concentration is high among breastfeeding women, and it is known to be independent of dietary zinc intake. As a result, transient neonatal zinc deficiency is not rare in the breastfed infants due to low milk zinc concentration in their breastfeeding mothers. Up to now, SLC30A2 has been documented the only candidate gene showing correlation with human milk zinc trait. In this study, 750 breastfeeding women were recruited and 10ml foremilk was collected on 42nd postpartum day. The milk zinc concentration was measured, and genomic DNA was isolated from breast-milk. Direct sequencing and Taqman assay were used to identify the SLC30A2 polymorphisms associated with low-milk-zinc. Subsequently, the factors associated with breast-milk zinc were investigated using regression model. The correlation study showed that SLC30A2/-697G>T and SLC30A2/1031A>G polymorphisms were associated with low-milk-zinc in our subjects. These two polymorphisms explained 3.23% of total variance in milk zinc level. For non-genetic variables, the obese breastfeeding women (BMI>25) secreted less zinc into their breast-milk. The variation of milk zinc was independent of pregnant age, birth weight, infant gender, cesarean delivery, preterm delivery and vitamin D supplementation. In conclusion, our results indicated that -697G>T and 1031A>G polymorphisms in the SLC30A2 gene may be associated with low-milk-zinc in Chinese breastfeeding women. Maternal BMI is significantly correlated with milk zinc level in negative manner. Our study demonstrated that both genetic and non-genetic factors could modulate milk zinc level.

Liver-specific cytochrome P450 CYP2C22 is a direct target of retinoic acid and a retinoic acid-metabolizing enzyme in rat liver.

Several cytochrome P450 (CYP) enzymes catalyze the C4-hydroxylation of retinoic acid (RA), a potent inducer of cell differentiation and an agent in the treatment of several diseases. Here, we have characterized CYP2C22, a member of the rat CYP2C family with homology to human CYP2C8 and CYP2C9. CYP2C22 was expressed nearly exclusively in hepatocytes, where it was one of the more abundant mRNAs transcripts. In H-4-II-E rat hepatoma cells, CYP2C22 mRNA was upregulated by all-trans (at)-RA, and Am580, a nonmetabolizable analog of at-RA. In comparison, in primary human hepatocytes, at-RA increased CYP2C9 but not CYP2C8 mRNA. Analysis of the CYP2C22 promoter region revealed a RA response element (5'-GGTTCA-(n)5-AGGTCA-3') in the distal flanking region, which bound the nuclear hormone receptors RAR and RXR and which was required for transcriptional activation response of this promoter to RA in CYP2C22-luciferase-transfected RA-treated HepG2 cells. The cDNA-expressed CYP2C22 protein metabolized [3H]at-RA to more polar metabolites. While long-chain polyunsaturated fatty acids competed, 9-cis-RA was a stronger competitor. Our studies demonstrate that CYP2C22 is a high-abundance, retinoid-inducible, hepatic P450 with the potential to metabolize at-RA, providing additional insight into the role of the CYP2C gene family in retinoid homeostasis.

Prolactin regulates ZNT2 expression through the JAK2/STAT5 signaling pathway in mammary cells.

The zinc transporter ZnT2 (SLC30A2) plays an important role in zinc secretion into milk during lactation. The physiological process of mammary gland secretion is regulated through complex integration of multiple lactogenic hormones. Prolactin plays a primary role in this regulation through the activation of various signaling cascades including Jak2/STAT5, mitogen-activated protein kinase (MAPK), p38, and phosphatidylinositol 3-kinase (PI3K). The precise mechanisms that regulate the transfer of specific nutrients such as zinc into milk are not well understood. Herein we report that prolactin increased ZnT2 abundance transcriptionally in cultured mammary epithelial (HC11) cells. To delineate the responsible mechanisms, we first determined that prolactin-mediated ZnT2 induction was inhibited by pretreatment with the Jak2 inhibitor AG490 but not by the MAPK inhibitor PD-98059. Using a luciferase reporter assay, we demonstrated that ZnT2 promoter activity was increased by prolactin treatment, which was subsequently abolished by expression of a dominant-negative STAT5 construct, implicating the Jak2/STAT5 signaling pathway in the transcriptional regulation of ZnT2. Two putative consensus STAT5 binding sequences in the ZnT2 promoter were identified (GAS1:-674 to -665 and GAS2:-377 to -368). Mutagenesis of the proximal GAS2 element resulted in complete abrogation of PRL-induced ZnT2 promoter activity. The promoter incorporating the distal GAS1 mutation was only able to respond to very high PRL concentrations. Results from both the mutagenesis and gel shift assays indicated that a cooperative relationship exists between GAS1 and GAS2 for PRL-induced activation; however, the proximal GAS2 plays a more critical role in STAT5-mediated signal transduction compared with the GAS1 element. Finally, chromosome immunoprecipition assay further confirmed that prolactin activates STAT5 binding to the ZnT2 promoter in vivo. Taken together, these results illustrate that prolactin regulates the transcription of ZnT2 through activation of the Jak2/STAT5 signaling pathway to assist in providing optimal zinc for secretion into milk during lactation.