Differentiation of intestinal stem cells toward goblet cells under systemic iron overload stress are associated with inhibition of Notch signaling pathway and ferroptosis.

Iron overload can lead to oxidative stress and intestinal damage and happens frequently during blood transfusions and iron supplementation. However, how iron overload influences intestinal mucosa remains unknown. Here, the aim of current study was to investigate the effects of iron overload on the proliferation and differentiation of intestinal stem cells (ISCs). An iron overload mouse model was established by intraperitoneal injection of 120 mg/kg body weight iron dextran once a fortnight for a duration of 12 weeks, and an iron overload enteroid model was produced by treatment with 3 mM or 10 mM of ferric ammonium citrate for 24 h. We found that iron overload caused damage to intestinal morphology with a 64 % reduction in villus height/crypt depth ratio, and microvilli injury in the duodenum. Iron overload mediated epithelial function by inhibiting the expression of nutrient transporters and enhancing the expression of secretory factors in the duodenum. Meanwhile, iron overload inhibited the proliferation of ISCs and regulated their differentiation into secretory mature cells, such as goblet cells, through inhibiting Notch signaling pathway both in mice and enteroid. Furthermore, iron overload caused oxidative stress and ferroptosis in intestinal epithelial cells. In addition, ferroptosis could also inhibit Notch signaling pathway, and affected the proliferation and differentiation of ISCs. These findings reveal the regulatory role of iron overload on the proliferation and differentiation of ISCs, providing a new insight into the internal mechanism of iron overload affecting intestinal health, and offering important theoretical basis for the scientific application of iron nutrition regulation.

Specifically targeted antimicrobial peptides synergize with bacterial-entrapping peptide against systemic MRSA infections.

INTRODUCTION: The design of precision antimicrobials aims to personalize the treatment of drug-resistant bacterial infections and avoid host microbiota dysbiosis. OBJECTIVES: This study aimed to propose an efficient de novo design strategy to obtain specifically targeted antimicrobial peptides (STAMPs) against methicillin-resistant Staphylococcus aureus (MRSA). METHODS: We evaluated three strategies designed to increase the selectivity of antimicrobial peptides (AMPs) for MRSA and mainly adopted an advanced hybrid peptide strategy. First, we proposed a traversal design to generate sequences, and constructed machine learning models to predict the anti-S. aureus activity levels of unknown peptides. Subsequently, six peptides were predicted to have high activity, among which, a broad-spectrum AMP (P18) was selected. Finally, the two targeting peptides from phage display libraries or lysostaphin were used to confer specific anti-S. aureus activity to P18. STAMPs were further screened out from hybrid peptides based on their in vitro and in vivo activities. RESULTS: An advanced hybrid peptide strategy can enhance the specific and targeted properties of broad-spectrum AMPs. Among 25 assessed peptides, 10 passed in vitro tests, and two peptides containing one bacterial-entrapping peptide (BEP) and one STAMP passed an in vivo test. The lead STAMP (P18E6) disrupted MRSA cell walls and membranes, eliminated established biofilms, and exhibited desirable biocompatibility, systemic distribution and efficacy, and immunomodulatory activity in vivo. Furthermore, a bacterial-entrapping peptide (BEP, SP5) modified from P18, self-assembled into nanonetworks and rapidly entrapped MRSA. SP5 synergized with P18E6 to enhance antibacterial activity in vitro and reduced systemic MRSA infections. CONCLUSIONS: This strategy may aid in the design of STAMPs against drug-resistant strains, and BEPs can serve as powerful STAMP adjuvants.

Sex-Specific Appetite Regulation of Lipocalin-2 in High-Fat-Diet-Induced Obese Mice.

INTRODUCTION: Lipocalin 2 (Lcn2) is a key factor in appetite suppression. However, the effect of Lcn2 on appetite in terms of sex differences has not been thoroughly studied. METHODS: Young (3-month-old) whole-body Lcn2 knockout (Lcn2-/-) mice were fed a normal diet (ND) or high-fat diet (HFD) for 8 weeks to investigate obesity, food intake, serum metabolism, hepatic lipid metabolism, and regulation of gastrointestinal hormones. RESULTS: Lcn2 deficiency significantly increased the body weight and food intake of male mice when fed ND instead of HFD and females when fed HFD but not ND. Compared to wild-type (WT) male mice, the adiponectin level and phosphorylated form of adenosine 5'-monophosphate-activated protein kinase (AMPK) in the hypothalamus were both increased in ND-fed Lcn2-/- male mice but decreased in HFD-fed Lcn2-/- male mice. However, in female mice, adiponectin and its energy metabolism pathway were not altered. Instead, estradiol was found to be substantially higher in ND-fed Lcn2-/- female mice and substantially lower in HFD-fed Lcn2-/- female mice compared with WT female mice. Estradiol alteration also caused similar changes in ERα in the hypothalamus, leading to changes in the PI3K/AKT energy metabolism pathway. It suggested that the increased appetite caused by Lcn2 deficiency in male mice may be due to increased adiponectin expression and promotion of AMPK phosphorylation, while in female mice it may be related to the decrease of circulating estradiol and the inhibition of the hypothalamic ERα/PI3K/AKT energy metabolism pathway. CONCLUSION: Lcn2 plays in a highly sex-specific manner in the regulation of appetite in young mice.

Lcn2 deficiency accelerates the infection of Escherichia coli O157:H7 by disrupting the intestinal barrier function.

Bacterial infections result in intestinal inflammation and injury, which affects gut health and nutrient absorption. Lipocalin 2 (Lcn2) is a protein that reacts to microbial invasion, inflammatory responses, and tissue damage. However, it remains unclear whether Lcn2 has a protective effect against bacterial induced intestinal inflammation. Therefore, this study endeavors to investigate the involvement of Lcn2 in the intestinal inflammation of mice infected with Enterohemorrhagic Escherichia coli O157:H7 (E. coli O157:H7). Lcn2 knockout (Lcn2-/-) mice were used to evaluate the changes of inflammatory responses. Lcn2 deficiency significantly exacerbated clinical symptoms of E. coli O157:H7 infection by reducing body weight and encouraging bacterial colonization of. Compared to infected wild type mice, infected Lcn2-/- mice had significantly elevated levels of pro-inflammatory cytokines in serum and ileum, including interleukin (IL)-6, IL-1ß, and tumor necrosis factor-α (TNF-α), as well as severe villi destruction in the jejunum. Furthermore, Lcn2 deficiency aggravated intestinal barrier degradation by significantly reducing the expression of tight junction proteins occludin and claudin 1, the content of myeloperoxidase (MPO) in the ileum, and the number of goblet cells in the colon. Our findings indicated that Lcn2 could alleviate inflammatory damage caused by E. coli O157:H7 infection in mice by enhancing intestinal barrier function.

Heme oxygenase-1 increases intracellular iron storage and suppresses inflammatory response of macrophages by inhibiting M1 polarization.

Heme oxygenase-1 (HO-1) catalyzes the first and rate-limiting enzymatic step of heme degradation, producing carbon monoxide, biliverdin, and free iron. Most iron is derived from aged erythrocytes by the decomposition of heme, which happened mainly in macrophages. However, the role of HO-1 on iron metabolism and function of macrophage is unclear. The present study investigated the effect of HO-1 on iron metabolism in macrophages, and explored the role of HO-1 on inflammatory response, polarization, and migration of macrophages. HO-1 inducer Hemin or HO-1 inhibitor zinc protoporphyrin was intravenously injected to C57BL/6 J mice every 4 d for 28 d. We found that HO-1 was mainly located in the cytoplasm of splenic macrophages of mice. Activation of HO-1 by Hemin significantly increased iron deposition in the spleen, up-regulated the gene expression of ferritin and ferroportin, and down-regulated gene expression of divalent metal transporter 1 and hepcidin. Induced HO-1 by Hemin treatment increased intracellular iron levels of macrophages, slowed down the absorption of extracellular iron, and accelerated the excretion of intracellular iron. In addition, activation of HO-1 significantly decreased the expression of pro-inflammatory cytokines including interleukin (IL)-6, IL-1ß, and inducible nitric oxide synthase, but increased the expression of anti-inflammatory cytokines such as IL-10. Furthermore, activation of HO-1 inhibited macrophages to M1-type polarization, and increased the migration rate of macrophages. This study demonstrated that HO-1 was able to regulate iron metabolism, exert anti-inflammatory effects, and inhibit macrophages polarization to M1 type.

Maternal Folic Acid Supplementation Improves the Intestinal Health of Offspring Porcine by Promoting the Proliferation and Differentiation of Intestinal Stem Cells.

Maternal folic acid intake has important effects on offspring growth and development. The mechanism involved in the renewal of intestinal epithelial cells remains unclear. Thus, this study aimed to investigate the potential effect of maternal folic acid supplementation during gestation and lactation on the structural and functional development of the small intestine in piglet offspring. Twenty-four Duroc sows were assigned to a control group (CON) and a folic-acid-supplemented group (CON + FA, supplemented with 15 mg/kg of folic acid). The results showed that maternal folic acid supplementation throughout gestation and lactation significantly increased the body weight, serum folate level, and intestinal folate metabolism in piglets. It also improved the villus length, villus height-to-crypt depth ratio, and transcript levels of nutrient transporters (GLUT4, SNAT2, FABP2, and SLC7A5) in piglets' duodenum and jejunum. In addition, maternal folic acid supplementation increased Ki67-positive cells and the expression of proliferation-related marker genes (C-Myc, CyclinD1, and PCNA) in piglets' intestinal stem cells. It also boosted the expression of genes associated with mature secreted cells (ChrA, Muc2, Lyz, Vil1), indicating enhanced proliferation and differentiation of intestinal stem cells. These findings demonstrate that maternal folic acid supplementation enhances growth performance and gut health in piglet offspring by promoting epithelial cell renewal equilibrium.

Iron Chelator Deferoxamine Alleviates Progression of Diabetic Nephropathy by Relieving Inflammation and Fibrosis in Rats.

Diabetic nephropathy (DN) is one of the most devastating diabetic microvascular complications. It has previously been observed that iron metabolism levels are abnormal in diabetic patients. However, the mechanism by which iron metabolism levels affect DN is poorly understood. This study was designed to evaluate the role of iron-chelator deferoxamine (DFO) in the improvement of DN. Here, we established a DN rat model induced by diets high in carbohydrates and fat and streptozotocin (STZ) injection. Our data demonstrated that DFO treatment for three weeks greatly attenuated renal dysfunction as evidenced by decreased levels of urinary albumin, blood urea nitrogen, and serum creatinine, which were elevated in DN rats. Histopathological observations showed that DFO treatment improved the renal structures of DN rats and preserved podocyte integrity by preventing the decrease of transcripts of nephrin and podocin. In addition, DFO treatment reduced the overexpression of fibronectin 1, collagen I, IL-1ß, NF-κB, and MCP-1 in DN rats, as well as inflammatory cell infiltrates and collagenous fibrosis. Taken together, our findings unveiled that iron chelation via DFO injection had a protective impact on DN by alleviating inflammation and fibrosis, and that it could be a potential therapeutic strategy for DN.

Secondary iron overload induces chronic pancreatitis and ferroptosis of acinar cells in mice.

Disruption of iron homeostasis is associated with multiple diseases. It has been found that patients with genetic iron overload develop massive iron deposition in the pancreas. However, few studies have focused on the effect of secondary iron overload on the pancreas. The objective of the present study was to investigate the pathogenic consequences of secondary iron overload in mice. An iron overload mouse model was constructed by intraperitoneal injection of 120 mg/kg body weight of iron dextran every other week for 12 weeks. Iron deposition, immunocyte infiltration, fibrosis, oxidative stress and ferroptosis were assessed using Prussian blue staining, immunohistochemical analysis, Masson staining, Sirius red staining, RT­qPCR analysis and western blot analysis. It was found that iron­overloaded mice showed pancreatic iron overload, together with elevated gene expression of the iron storage factor ferritin H, and decreased expression of the iron transportation mediator divalent metal transporter 1, ferroportin 1 and transferrin receptor. Iron­overloaded mice developed mild pancreatitis with increased serum amylase and lipase activities, as well as elevated gene expression levels of pro­inflammatory cytokines, including interleukin (IL)­1ß, IL­6 and inducible nitric oxide synthase. Acinar atrophy, massive immunocyte infiltration and pancreatic fibrosis were noted in the iron­overloaded mice. As an underlying mechanism, iron­overloaded mice showed increased pancreatic oxidative stress, with an elevated malondialdehyde level, and decreased SOD and glutathione peroxidase activity. Furthermore, iron overload led to ferroptosis with promoted expression of cytochrome c oxidase subunit II, and decreased transcripts of glutathione peroxidase 4 and solute carrier family 7 member 11. These results provided evidence that multiple intraperitoneal injections of iron dextran in mice lead to iron overload­induced chronic pancreatitis, which suggested that secondary iron overload is a risk factor for pancreatitis and highlights the importance of iron in maintaining the normal functions of the pancreas.

Regulation of a High-Iron Diet on Lipid Metabolism and Gut Microbiota in Mice.

Iron homeostasis disorder is associated with the imbalance of lipid metabolism, while the specific interaction remains unclear. In the present study, we investigated the effect of a high-iron diet on lipid metabolism in mice. The C57BL/6 mice were fed with a normal diet (WT) or a high-iron diet (WT + Fe) for 12 weeks. We found that mice in the WT + Fe group showed a significant decrease in body weight gain, body fat and lipid accumulation of liver when compared with mice in the WT group. Accordingly, serum total cholesterol and triglyceride levels were both reduced in mice with a high-iron diet. Moreover, mice in the WT + Fe group exhibited a significant decrease in expression of genes regulating adipogenesis and adipocyte differentiation, and a significant increase in expression of fat hydrolysis enzyme genes in both liver and adipose tissues, which was consistent with their dramatic reduction in adipocyte cell size. In addition, a high-iron diet decreased the relative abundance of beneficial bacteria (Akkermansia, Bifidobacterium and Lactobacillus) and increased the relative abundance of pathogenic bacteria (Romboutsia and Erysipelatoclostridium). Thus, our research revealed that a high-iron diet reduced lipid deposition by inhibiting adipogenesis and promoting lipolysis. Altered gut microbial composition induced by a high-iron diet may not play a critical role in regulating lipid metabolism, but might cause unwanted side effects such as intestinal inflammation and damaged villi morphology at the intestinal host-microbe interface. These findings provide new insights into the relationship among iron, lipid metabolism and gut microbiota.

Dietary iron modulates hepatic glucose homeostasis via regulating gluconeogenesis.

Iron exerts significant influences on glucose metabolism. However, the regulatory mechanisms underlying disordered glucose response remains largely unclear. The aim of this study was to examine the impact of dietary iron on hepatic gluconeogenesis in mice and in rat liver-derived cells. High iron models of C57BL/6J mice were fed with 1.25 g Fe/kg diets for 9 weeks, and high-iron BRL-3A cell models were treated with 250 µmol/L FeSO4 for 12 h and 24 h. Our data showed that higher iron intake resulted in higher hepatic iron without iron toxicity, and reduced body weight gain with no difference of food intakes. High dietary iron significantly increased 61% of hepatic glycogen deposition, but exhibited impairment in glucose responses in mice. Moreover, high dietary iron suppressed hepatic gluconeogenesis by repressing the expression of key gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Meanwhile, mice fed with higher iron diets exhibited both decreased AMP-activated protein kinase (AMPK) activity and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) protein levels. Furthermore, in BRL-3A cells, iron treatment increased cellular glucose uptake, and altered gluconeogenesis rhythmically by regulating the activation of AMPK and expression of PGC-1α successively. This study demonstrated that dietary high iron was able to increase hepatic glycogen deposition by enhancement of glucose uptake, and suppress hepatic gluconeogenesis by regulation of AMPK and PGC-1α.

Intraperitoneal supplementation of iron alleviates dextran sodium sulfate-induced colitis by enhancing intestinal barrier function.

Iron supplementation is necessary for the treatment of anemia, one of the most frequent complications in inflammatory bowel disease (IBD). However, oral iron supplementation leads to an exacerbation of intestinal inflammation. Gut barrier plays a key role in the pathogenesis of IBD. The aim of this study was to characterize the interrelationship between systemic iron, intestinal barrier and the development of intestinal inflammation in a dextran sulfate sodium (DSS) induced experimental colitis mice model. We found that DSS-treated mice developed severe inflammation of colon, but became much healthy when intraperitoneal injection with iron. Iron supplementation alleviated colonic and systemic inflammation by lower histological scores, restorative morphology of colonic villi, and reduced expression of pro-inflammatory cytokines. Moreover, intraperitoneal supplementation of iron enhanced intestinal barrier function by upregulating the colonic expressions of tight junction proteins, restoring intestinal immune homeostasis by regulating immune cell infiltration and T lymphocyte subsets, and increasing mucous secretion of goblet cells in the colon. High-throughput sequencing of fecal 16 S rRNA showed that iron injection significantly increased the relative abundance of Bacteroidetes, which was suppressed in the gut microbiota of DSS-induced colitis mice. These results provided evidences supporting the protective effects of systemic iron repletion by intraperitoneal injection of iron on intestinal barrier functions. The finding highlights a novel approach for the treatment of IBD with iron injection therapy.

Lipocalin-2 Alleviates LPS-Induced Inflammation Through Alteration of Macrophage Properties.

PURPOSE: Lipocalin-2 (Lcn2) is an acute-phase protein and elevated in several inflammatory diseases. This study aimed to determine whether Lcn2 alleviates inflammation and explore the underlying cellular mechanisms. METHODS: C57BL/6 Lcn2-deficient (Lcn2-/-) male mice were intraperitoneally injected with lipopolysaccharide (LPS) to build systemic inflammation model. The inflammatory processes were investigated. The morphology of villi was measured by scanning electron microscopy (SEM). The levels of inflammatory factors were detected by ELISA and qPCR analysis. The production of Lcn2 was determined with immunofluorescence staining by confocal microscope. The molecular mechanism of Lcn2 in bone marrow-derived macrophages (BMDMs) was analyzed by mass spectrometry (MS)-based quantitative proteomic analysis. RESULTS: Compared to wild-type (WT) mice injected with LPS, Lcn2-/- mice injected with LPS showed increased inflammatory damage in jejunum and ileum, and significantly elevated the levels of multiple pro-inflammatory cytokines. After determining that Lcn2 was mainly located in the cytoplasm of macrophages, we isolated BMDMs from Lcn2-/- mice to evaluate their function. During LPS challenge, transcripts of pro-inflammatory cytokines were all significantly increased in BMDMs from Lcn2-/- mice, while those of anti-inflammatory cytokines were significantly decreased when compared with the cytokines in BMDMs from WT mice. A label-free relative quantitation proteomics analysis showed that LPS-treated BMDMs from Lcn2-/- mice had elevated levels of pro-inflammatory pathways, but reduced phagocytosis and autophagy when compared with LPS-treated BMDMs from WT mice. CONCLUSION: These findings demonstrated that Lcn2 was a potent protective factor in response to systemic inflammation and might be an indispensable factor for macrophage functions.

Erastin­induced ferroptosis causes physiological and pathological changes in healthy tissues of mice.

Ferroptosis is a non­apoptotic form of cell death that relies on iron and lipid peroxidation, which is associated with multiple pathological processes in several diseases. Erastin is a small molecule capable of initiating ferroptotic cell death in cancer cells, which has shown great potential for cancer therapy. However, the physiological and pathological role of erastin­induced ferroptosis on healthy tissues has not been well characterized. The present study intraperitoneally injected erastin into healthy mice to detect the metabolic changes of several tissues of mice. Erastin injection induced typical characteristics of ferroptosis with higher level of serum iron and malondialdehyde and lower level of glutathione and glutathione peroxidase 4 protein. Erastin injection enhanced iron deposition in the brain, duodenum, kidney and spleen of mice. Erastin­induced ferroptosis altered the blood index values, causing mild cerebral infarction of brain and enlarged glomerular volume of kidney. It also promoted the growth of duodenal epithelium with thicker, longer and denser villi in erastin­treated mice. The findings provided evidence that erastin induced ferroptosis and caused pathological changes in healthy tissues of mice. This suggested that the anti­tumor drug erastin was somewhat toxic to healthy tissues.

Iron Overload Protects from Obesity by Ferroptosis.

Dysregulation in iron metabolism is associated with obesity, type 2 diabetes, and other metabolic diseases, whereas the underlying mechanisms of imbalanced glycolipid metabolism are still obscure. Here, we demonstrated that iron overload protected mice from obesity both with normal diets (ND) or high-fat diets (HFD). In iron-overload mice, the body fat was significantly decreased, especially when fed with HFD, excessive iron mice gained 15% less weight than those without iron supplements. Moreover, glucose tolerance and insulin sensitivity were all significantly reduced, and hepatic steatosis was prevented. Furthermore, these mice show a considerable decrease in lipogenesis and lipidoses of the liver. Compared with control groups, iron treated groups showed a 79% decrease in the protein level of Perilipin-2 (PLIN2), a protein marker for lipid droplets. These results were consistent with their substantial decrease in adiposity. RNA-seq and signaling pathway analyses showed that iron overload caused ferroptosis in the liver of mice with a decrease in GPX4 expression and an increase in Ptgs2 expression, resulting in a high level of lipid peroxidation. Overall, this study reveals the protective function of iron overload in obesity by triggering the imbalance of glucolipid metabolism in the liver and highlights the crucial role of ferroptosis in regulating lipid accumulation.

The role of iron homeostasis in adipocyte metabolism.

Iron plays a vital role in the metabolism of adipose tissue. On the one hand, iron is essential for differentiation, endocrine, energy supply and other physiological functions of adipocytes. Iron homeostasis affects the progression of many chronic metabolic diseases such as obesity, type 2 diabetes mellitus, and non-alcoholic fatty liver disease. In adipose tissue, iron deficiency is associated with obesity, mainly due to inflammation. Nevertheless, excessive iron in adipose tissue leads to decreased insulin sensitivity owing to mitochondrial dysfunction and adipokine changes. On the other hand, iron has an effect on the thermogenesis of adipocytes. Iron deficiency affects the production of beige fat and the direction of the differentiation of brown fat. In this review, we summarize the current understanding of the crosstalk between iron homeostasis and metabolism in adipose tissue.

[Polarized activation affects iron metabolism in macrophages].

The aim of this study was to investigate the effects of polarization program on the ability of macrophages to regulate iron metabolism. M1 and M2 macrophages were propagated in vitro from porcine alveolar macrophages 3D4/2 and polarized by cytokines. The 3D4/2 macrophages were treated with 20 ng/mL interferon gamma (IFN-γ) and 10 ng/mL interleukin-4 (IL-4) combined with 10 ng/mL macrophage colony-stimulating factor (M-CSF) to induce polarization to M1 and M2, respectively. After incubation for 24 h, the expression levels of inflammatory factors and iron-metabolism genes were determined using real-time qPCR, Western bot and immunofluorescence. The M1/M2 macrophages culture media supernatant was collected and used to treat porcine intestinal epithelial cells IPEC-J2. The proliferation ability of IPEC-J2 was detected using CCK-8 assay kit. Following exogenous addition of ammonium ferric citrate (FAC) to M1/M2 macrophages, the phagocytic function of macrophages was detected using fluorescein isothiocyanate-dextran (FITC-dextran) and flow cytometry. The results showed that, compared with control, M1 macrophages had higher mRNA levels of iron storage proteins (ferritin heavy and light polypeptide, i.e. FtH and FtL), hepcidin and lipocalin-2, as well as iron content. Moreover, iron enhanced the ability of M1 macrophages to phagocytize FITC-dextran. There was no significant change in these mRNA expression levels in M2 macrophages, but the mRNA expression levels of ferroportin and transferrin receptor were up-regulated. In addition, the conditioned media supernatant from M2 macrophages promoted cell proliferation of IPEC-J2. These findings indicate that M1 macrophages tend to lock iron in the cell and reduce extracellular iron content, thereby inhibiting the proliferation of extracellular bacteria. While M2 macrophages tend to excrete iron, which contributes to the proliferation of surrounding cells and thus promotes tissue repair.

Distribution of Lactoferrin Is Related with Dynamics of Neutrophils in Bacterial Infected Mice Intestine.

Lactoferrin (Lf) is a conserved iron-binding glycoprotein with antimicrobial activity, which is present in secretions that recover mucosal sites regarded as portals of invaded pathogens. Although numerous studies have focused on exogenous Lf, little is known about its expression of endogenous Lf upon bacterial infection. In this study, we investigated the distribution of Lf in mice intestine during Escherichia coli (E. coli) K88 infection. PCR and immunohistology staining showed that mRNA levels of Lf significantly increased in duodenum, ileum and colon, but extremely decreased in jejunum at 8 h and 24 h after infection. Meanwhile, endogenous Lf was mostly located in the lamina propria of intestine villi, while Lf receptor (LfR) was in the crypts. It suggested that endogenous Lf-LfR interaction might not be implicated in the antibacterial process. In addition, it was interesting to find that the infiltration of neutrophils into intestine tissues was changed similarly to Lf expression. It indicated that the variations of Lf expression were rather due to an equilibrium between the recruitment of neutrophils and degranulation of activated neutrophils. Thus, this new knowledge will pave the way to a more effective understanding of the role of Lf in intestinal mucosal immunity.

Dynamics of the Physicochemical Characteristics, Microbiota, and Metabolic Functions of Soybean Meal and Corn Mixed Substrates during Two-Stage Solid-State Fermentation.

Substantial annual economic loss in livestock production is caused by antinutritional factors in soybean meal and corn mixed substrates, which can be degraded by microbial fermentation. Although considerable efforts have been made to explain the effects of fermentation on soybean meal and corn-based feed, the dynamics of the physicochemical characteristics, microbiota, and metabolic functions of soybean meal and corn mixed substrates during solid-state fermentation remain unclear. Here, multiple physicochemical analyses combined with high-throughput sequencing were performed to reveal the dynamic changes that occur during a novel two-stage solid-state fermentation process. Generally, inoculated bacteria rapidly proliferated in the initial 12-h aerobic fermentation (P = 0.002). Notably, most nutritional changes occurred during 12 to 24 h compared to 0 to 12 h. Second-stage anaerobic fermentation increased the bacterial abundance and lactic acid content (P < 0.00). Bacillus spp., Enterococcus spp., and Pseudomonas spp. were predominantly involved in the maturation of the fermented mixed substrates (P < 0.05). Additionally, the available phosphorus exhibited the greatest interaction with the microbial community structure. Cellular processes and environmental information processing might be the main metabolic processes of the microbiota during this fermentation. An in vivo model further evaluated the growth-promoting effects of the fermented products. These results characterized the dynamic changes that occur during two-stage solid-state fermentation and provided potential references for additional interventions to further improve the effectiveness and efficiency of solid-state fermentation of feed.IMPORTANCE Solid-state fermentation (SSF) plays pivotal roles not only in human food but also farm animal diets. Soybean meal (SBM) and corn account for approximately 70% of the global feed consumption. However, the nutritional value of conventional SBM and corn mixed substrates (MS) is limited by antinutritional factors, causing substantial economic loss in livestock production. Although emerging studies have reported that SSF can improve the nutritional value of SBM-based substrates, the dynamic changes in the physicochemical features, microbiota, and metabolic functions of MS during SSF remain poorly understood, limiting further investigation. To provide insights into the dynamics of the physicochemical characteristics and the complex microbiome during the two-stage SSF of MS, multiple physicochemical analyses combined with high-throughput sequencing were applied here. These novel insights shed light on the complex changes that occur in the nutrition and microbiome during two-stage SSF of MS and are of great value for industrial feed-based practices and metabolomic research on SSF ecosystems.

Effects of chitosan nanoparticle supplementation on growth performance, humoral immunity, gut microbiota and immune responses after lipopolysaccharide challenge in weaned pigs.

In this study, we aimed to determine the effects of dietary supplementation with chitosan nanoparticles (CNP) on growth performance, immune status, gut microbiota and immune responses after lipopolysaccharide challenge in weaned pigs. A total of 144 piglets were assigned to four groups receiving different dietary treatments, including basal diets supplemented with 0, 100, 200 and 400 mg/kg CNP fed for 28 days. Each treatment group included six pens (six piglets per pen). The increase in supplemental CNP concentration improved the average daily gain (ADG) and decreased the feed and gain (F/G) and diarrhoea rate (p < .05). However, significant differences in the average daily feed intake (ADFI) among different CNP concentrations were not observed. CNP also increased plasma immunoglobulin (Ig)A and IgG, and C3 and C4 concentrations in piglets in a dose-dependent manner on day 28, whereas IgM concentration was not affected by CNP. A total of 24 piglets in the control diet and control diet with 400 mg/kg CNP supplementation groups were randomly selected for the experiment of immunological stress. Half of the pigs in each group (n = 6) were injected i.p. with Escherichia coli lipopolysaccharide (LPS) at a concentration of 100 µg/kg. The other pigs in each group were injected with sterile saline solution at the same volume. Plasma concentrations of cortisol, prostaglandin E2 (PEG2), interleukin (IL)-6, tumour necrosis factor (TNF)-α and IL-1ß dramatically increased after LPS challenge. However, CNP inhibited the increase in cortisol, PEG2, IL-6 and IL-1ß levels in plasma, whereas TNF-α level slightly increased. Moreover, the effects of CNP on the gut microbiota were also evaluated. Our results showed that dietary supplementation with CNP modified the composition of colonic microbiota, where it increased the amounts of some presumably beneficial intestinal bacteria and suppressed the growth of potential bacterial pathogens. These findings suggested CNP supplementation improved the growth performance and immune status, alleviated immunological stress and regulated intestinal ecology in weaned piglets. Based on these beneficial effects, CNP could be applied as a functional feed additives supplemented in piglets diet.

LPS Inhibits Fatty Acid Absorption in Enterocytes through TNF-α Secreted by Macrophages.

Diarrhea, such as steatorrhea, could result from fat absorption disorders, which could be caused by many factors, including Escherichia coli infection. However, it is not clear how E. coli affects fatty acid absorption in animals. Lipopolysaccharide (LPS), as one of the main pathogenic components of E. coli, is the main cause of the virulence of E. coli. Therefore, we used LPS to explore the underlying mechanism of E. coli that causes the inhibition of fatty acid absorption in the intestine. In this study, we found that LPS caused apoptosis of intestinal epithelial cells in mice. Further, caspase-3 activation caused the inhibition of fatty acid absorption in the intestinal porcine enterocyte cell line (IPEC-J2). However, direct treatment of LPS did not induce any significant change in fatty acid absorption in IPEC-J2. We then prepared conditioned medium of LPS-treated porcine macrophage cell line (3D4/2) for incubating IPEC-J2, as LPS initiates inflammation by activating immune cells. The conditioned medium decreased fatty acid absorption and caspase-3 activation in IPEC-J2. While inhibiting the activation of caspase-3 in IPEC-J2, conditioned medium no longer caused serious deficiency of fatty acid absorption. As IL-1ß, IL-6, and TNF-α in conditioned medium increase significantly, IPEC-J2 was treated with IL-1ß, IL-6, and TNF-α, respectively. Only TNF-α induced caspase-3 activation in IPEC-J2. Reducing the secretion of TNF-α in 3D4/2, there was no obvious activation of caspase-3 in IPEC-J2, and fatty acid absorption recovered effectively. Based on the above results, we hold the opinion that LPS does not suppress fatty acid absorption directly in the intestine, but may work on macrophages that secrete cytokines, such as TNF-α, inducing caspase-3 activation and finally leading to the inhibition of fatty acid absorption in intestine.