ATP2B3 Inhibition Alleviates Erastin-Induced Ferroptosis in HT-22 Cells through the P62-KEAP1-NRF2-HO-1 Pathway.

Ferroptosis participates in the occurrence and development of neurological disorders. Modulating ferroptosis may have therapeutic potential in nervous system diseases. Therefore, TMTbased proteomic analysis in HT-22 cells was performed to identify erastin-induced differentially expressed proteins. The calcium-transporting ATP2B3 (ATP2B3) was screened as a target protein. ATP2B3 knockdown markedly alleviated the erastin-induced decrease in cell viability and elevated ROS (p < 0.01) and reversed the up-regulation of oxidative stress-related proteins polyubiquitin-binding protein p62 (P62), nuclear factor erythroid 2-related factor2 (NRF2), heme oxygenase-1 (HO-1), and NAD(P)H quinone oxidoreductase-1 (NQO1) protein expression (p < 0.05 or p < 0.01) and the down-regulation of Kelch-like ECH-associated protein 1(KEAP1) protein expression (p < 0.01). Moreover, NRF2 knockdown, P62 inhibition, or KEAP1 overexpression rescued the erastin-induced decrease in cell viability (p < 0.05) and increase in ROS production (p < 0.01) in HT-22 cells, while simultaneous overexpression of NRF2 and P62 and knockdown of KEAP1 partially offset the relief effect of ATP2B3 inhibition. In addition, knockdown of ATP2B3, NRF2, and P62 and overexpression of KEAP1 significantly down-regulated erastin-induced high expression of the HO-1 protein, while HO-1 overexpression reversed the alleviating effects of ATP2B3 inhibition on the erastin-induced decrease in cell viability (p < 0.01) and increase in ROS production (p < 0.01) in HT-22 cells. Taken together, ATP2B3 inhibition mediates the alleviation of erastin-induced ferroptosis in HT-22 cells through the P62-KEAP1-NRF2-HO-1 pathway.

Caspase-1-dependent mechanism mediating the harmful impacts of the quorum-sensing molecule N-(3-oxo-dodecanoyl)-l-homoserine lactone on the intestinal cells.

N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), a quorum-sensing (QS) molecule produced by Gram-negative bacteria in the gastrointestinal tract, adversly impacts host cells. Our previous study demonstrated that 3-oxo-C12-HSL induced a decrease in cell viability via cell apoptosis and eventually disrupted mucin synthesis from LS174T goblet cells. However, the molecular mechanism underlying cell apoptosis and whether pyroptosis was involved in this process are still unknown. In this study, we emphasized on the caspases signal pathway and sterile inflammation to reveal the harmful effects of 3-oxo-C12-HSL on LS174T goblet cells. Our data showed that 3-oxo-C12-HSL is a major inducer of oxidative stress indicated by a high level of intracellular reactive oxygen species (ROS). However, TQ416, an inhibitor of paraoxonase 2, can effectively block oxidative stress. A higher ROS level is the trigger for activating the caspase-1 and 3 cascade signal pathways. Blockade of ROS synthesis and caspase-1 and 3 cascades can obviously rescue the viability of LS174T cells after 3-oxo-C12-HSL treatment. We also found that paralleled with a higher level of ROS and caspases activation, an abnormal expression of proinflammatory cytokines was induced by 3-oxo-C12-HSL treatment; however, the blockage of TLRs-NF-κB pathway cannot restore cell viability and secretary function. These data collectively indicate that 3-oxo-C12-HSL exposure induces damages to cell viability and secretary function of LS174T goblet cells, which is mediated by oxidative stress, cell apoptosis, and sterile inflammation. Overall, the data in this study will provide a better understanding of the harmful impacts of some QS molecules on host cells and their underlying mechanism.

Chronic Dexamethasone exposure activates the TLR4-Mediated inflammation pathway and induces epithelial apoptosis in the goat colon.

Chronic stress has a profound effect on health in both animals and humans. Dexamethasone (Dex), a synthetic glucocorticoid, is used to induce chronic stress in many studies. The impact of chronic stress on epithelial cells of hindgut of ruminants is still unknown. In this study, we investigated the effect of chronic stress induced by long term injection of low dosage of Dex on the colonic epithelium of goats. The results showed that Dex exposure increased the number of TUNEL-positive cells, upregulated caspase-3 and caspase-8 enzyme activity, but decreased protein expression of cell proliferation markers proliferating cell nuclear antigen (PCNA) and Cyclin D2(CCND2). It also activated TLR-4 and NF-κB pathway and increased the transcription levels of vital inflammatory cytokines such as interleukin-10 (IL-10), interleukin-1ß (IL-1ß), and inducible nitric oxide synthase 2 (iNOS2). Chronic stress down-regulated the methylation level of total DNA, suggesting a mechanism for the transcriptional activation of genes, such as claudin-1, claudin-4, ZO-1, and cell cycle-related genes. Taken together, long-term injection of a low dosage of Dex caused damage to the colon epithelium accompanied with the inhibition of cell proliferation and the activation of cell apoptosis and inflammation. However, a general up-regulation of genes expression induced by Dex is due to a lower level of genomic DNA methylation.

Chronic dexamethasone exposure retards growth without altering the digestive tract microbiota composition in goats.

BACKGROUND: Dexamethasone (Dex), an artificially synthetic cortisol substitute, is commonly used as an anti-inflammatory drug, and is also employed to mimic the stress state experimentally. It is well known that chronic stress disturbs the gut microbiota community and digestive functions. However, no relevant studies have been conducted in ruminants. RESULTS: In this study, a low dosage of Dex (0.2 mg/kg body weight, Dex group, n = 5) was consecutively injected intramuscularly for 21 days to simulate chronic stress in growing goats. Goats were injected with saline (0.2 mg/kg body weight) as the control group (Con, n = 5). Dex-treated goats showed a higher number of white blood cells and blood glucose levels (p < 0.01), but lower dry matter intake (DMI) and body weight (p < 0.01) than those of saline-injected goats. Plasma cortisol concentration decreased significantly in response to the Dex injection compared to the control (p < 0.05). The Dex treatment did not change most ruminal volatile fatty acid (VFAs) concentrations before the morning feeding after 1-21 days of treatment (p > 0.05); however, ruminal VFA concentrations decreased dramatically 2, 4, 6, and 8 h after the morning feeding on day 21 of the Dex injections. In this study, chronic Dex exposure did not alter the community structure of microbes or methanogenes in the rumen, caecum, or colonic digesta. Only Prevotella increased on days 7 and 14 of Dex treatment, but decreased on day 21, and Methanosphaera was the only genus of methanogene that decreased. CONCLUSIONS: Our results suggest that chronic Dex exposure retards growth by decreasing DMI, which may be mediated by higher levels of blood glucose and lower ruminal VFA production. Microbiota in the digestive tract was highly resistant to chronic Dex exposure.

Chronic dexamethasone exposure markedly decreased the hepatic triglyceride accumulation in growing goats.

Chronic stress seriously threatens welfare and health in animals and humans. Consecutive dexamethasone (Dex) injection was used to mimic chronic stress previously. In order to investigate the effect of chronic stress on hepatic lipids metabolism, in this study, 10 healthy male goats were randomly allocated into two groups, one received a consecutive injection of Dex via intramuscularly for 3 weeks (Dex group), the other received the same volume of saline as the control group (Con group). Hepatic health and triglyceride (TG) metabolism were analyzed and compared between two groups. The data showed that a significant decrease of TG in plasma and the liver was significantly decreased by Dex (P < .05), while the hepatic nonesterified fatty acid (NEFA) concentration was increased compared to the Con group (P < .05). Consistent with the decrease of TG level, the activity of hepatic lipoprotein lipase (LPL) and hepatic lipase (HL) enzymes activities were significantly enhanced by Dex. Real-time PCR results showed that the mRNA expression of sterol regulatory element binding transcription factor 1 (SREBP-1), acyl-CoA dehydrogenase long chain (ACADL) and acyl-CoA synthetase bubblegum family member 1 (ACSBG1) genes in liver was significantly up-regulated by chronic Dex injection (P < .05), whereas perilipin 2 (PLIN2) and adipose triglyceride lipase (ATGL) mRNA expression was significantly decreased by Dex (P < .05). In addition, no obvious damages were observed in the liver in both Con and Dex groups demonstrating by the sirius red staining, HE staining as well as several biochemical parameters related to the functional status of hepatocytes. Our data indicate that chronic Dex exposure decreases TG levels in the circulation and the liver through activating lipolysis and inhibiting lipogenesis without causing hepatic damages in the growing goats.

Downregulation of prostaglandin E2 is involved in hindgut mucosal damage in lactating goats fed a high-concentrate diet.

NEW FINDINGS: What is the central question of this study? A high-concentrate (HC) diet results in damage to the hindgut mucosa. The aim of the study was to investigate the status of epithelial proliferation in the hindgut mucosa of goats with subacute ruminal acidosis and, simultaneously, to evaluate prostaglandin E2 synthesis and the downstream signalling pathways. What is the main finding and its importance? The downregulation of local prostaglandin E2 synthesis and its downstream signalling pathway are involved in the process of inhibiting epithelial proliferation in the hindgut epithelium of HC-fed goats. Our results provide new insight into the relationship between abnormal fermentation in the hindgut and damage to the intestinal mucosal barrier. Our previous data demonstrated that feeding a high-concentrate (HC) diet to lactating goats for a long time causes severe damage to the hindgut mucosa and parallels the activation of cell apoptosis and local oxidative stress. In the present study, changes in production of prostaglandin E2 (PGE2 ) and its signalling pathway were evaluated in the process of epithelial barrier disruption in the hindgut. Twelve goats in mid-lactation were randomly assigned to either a HC diet group or a low-concentrate (LC) diet group for 10 weeks. Cell proliferation markers, cyclooxygenase-2 activity, PGE2 content and the relative signalling pathway, including CREB and AKT, were analysed by enzyme-linked immunosorbent assay and Western blot, respectively. The mRNA and protein expressions of MKI67 and CCND2 (two proliferation markers) were significantly decreased in the caecal mucosa of HC- compared with LC-fed goats (P < 0.05). The protein content of interleukin-10 and ß-defensin in the caecal mucosa was also downregulated in HC-fed goats (P < 0.05). The HC-fed goats showed a tendency to a decrease in cyclooxygenase-2 enzyme activity (P = 0.081) and a significant decrease of local PGE2 content and EP4 (PGE2 receptor) protein expression in caecal mucosa (P < 0.05). Moreover, the protein abundance of p-CREB (P = 0.069) and p-AKT (P < 0.05) and the mRNA expression of epidermal growth factor receptor (P < 0.05) were downregulated in caecal mucosa of HC- compared with LC-fed goats. These results indicate that a reduction in epithelial cell proliferation was partly responsible for the damage to the epithelial barrier, which might be associated with the downregulation of PGE2 synthesis and its downstream signalling pathway.

Exogenous administration of chronic corticosterone affects hepatic cholesterol metabolism in broiler chickens showing long or short tonic immobility.

Tonic immobility (TI) is an innate characteristic of animals related to fear or stress response. Animals can be classified into long TI (LTI) and short TI (STI) phenotypes based on TI test duration. In this study, effect of TI phenotype, chronic corticosterone administration (CORT), and their interaction on cholesterol metabolism in liver was evaluated in broilers. LTI broilers showed higher level of cholesterol in liver compared to STI chickens (p<0.05), and CORT significantly increased hepatic cholesterol content (p<0.01). Real-time PCR results showed that both TI and CORT potentially altered ABCA1 and CYP7A1 gene expressions (0.05

Activation of cellular apoptosis in the caecal epithelium is associated with increased oxidative reactions in lactating goats after feeding a high-concentrate diet.

NEW FINDINGS: What is the central question of this study? What are the ultrastructural changes of the caecal mucosa and the status of epithelial cellular apoptosis and oxidative reactions in lactating goats after prolonged feeding with a high-concentrate diet? What is the main finding and its importance? High-concentrate diet results in ultrastructural damage to the caprine caecal epithelium. Increased oxidative and decreased antioxidative reactions are involved in the process of activating epithelial apoptosis in the caecal epithelium of goats fed a high-concentrate diet. Our results provide new insight into the relationship between abnormal fermentation in the hindgut and damage to the intestinal mucosal barrier. The effect of feeding a high-concentrate diet (HC) to lactating ruminants on their hindgut epithelial structure remains unknown. In this study, 12 lactating goats were randomly assigned to either HC (65% of dry matter as concentrate; n = 6) or a low-concentrate diet (LC; 35% of dry matter as concentrate; n = 6). After 10 weeks, the epithelial ultrastructure and cell apoptotic status in the caecal mucosa were determined by transmission electron microscopy and TUNEL, respectively. The results showed that the level of free lipopolysaccharide (P < 0.05), total volatile fatty acid concentrations (P < 0.1) and starch content (P < 0.05) in the caecal digesta were significantly increased in HC- compared with LC-fed goats. The HC-fed goats exhibited obvious epithelial cellular damage, with widened tight junction spaces, nuclear breakdown and mitochondrial swelling. Compared with their LC-fed counterparts, HC-fed goats showed greater apoptosis in the caecal epithelium, as evidenced by more TUNEL-positive apoptotic cells. Western blot analysis showed that there was no significant difference in activated caspase-3, Bax protein expression in caecal epithelial mucosa between HC- and LC-fed goats (P > 0.05). However, the level of malondialdehyde content in the caecal epithelium from HC-fed goats was markedly higher than that in LC-fed goats (P < 0.05), whereas the level of glutathione peroxidase and the superoxide dismutase activity were significantly decreased. Gene expressions of cytokines, including interleukin-1ß, interleukin-6, interleukin-10, tumour necrosis factor-α and interferon-γ, as well as myeloperoxidase activity in the caecal mucosa did not show any significant difference between HC- and LC-fed goats. These results indicate that feeding a high-concentrate diet to lactating goats for a prolonged period results in abnormal fermentation and structural disruption in the hindgut, which is accompanied by greater cellular apoptosis and an enhanced oxidative stress response.

Fatmass and obesity associated (FTO) gene regulates gluconeogenesis in chicken embryo fibroblast cells.

Fat mass and obesity-associated (FTO) gene was found to be associated with energy homeostasis in mammals, yet the function of chicken FTO is less clear. In this study, chicken embryo fibroblast cells (DF-1) were transiently transfected to over-express (FTO(+)) or to knockdown (FTO−) the chicken FTO gene and were used for the functional analysis. FTO expression was significantly augmented in FTO(+) cells while depressed in FTO(−) cells (P < 0.05). FTO(+) cells had significantly lower glucose yet higher lactic acid (LD) concentrations (P < 0.05) in the culture media, which was associated with significantly up-regulated (P < 0.05) mRNA expression of the rate-limiting gluconeogenic enzymes, glucose-6-phosphatase (G6PC) and the phosphoenolpyruvate carboxykinase-mitochondrial (PEPCK-m). The protein content and enzyme activity of G6PC were also significantly higher (P < 0.05) in FTO(+) cells. Moreover, CCAAT/enhancer-binding protein-beta (C/EBP-beta) and cAMP responsive element binding protein 1 (CREB1), which were found to transcriptionally regulate the expression of G6PC, were increased at the level of both mRNA (P < 0.05) and protein (P < 0.05) in FTO(+)cells. ChIP analysis revealed significantly higher (P < 0.05) binding of C/EBP-beta and phospho-CREB1 to G6PC gene promoter in FTO(+) cells. In addition, the interaction of FTO and C/EBP-beta was significantly enhanced (P < 0.05) in FTO+ cells. Opposite changes in G6PC expression and regulation were observed in FTO(−) cells. Our results indicate that chicken FTO regulates gluconeogenesis in DF-1 cells through enhanced transcriptional regulation of G6PC gene by C/EBP-beta and phospho-CREB1.

Embryonic exposure to corticosterone modifies aggressive behavior through alterations of the hypothalamic pituitary adrenal axis and the serotonergic system in the chicken.

Exposure to excess glucocorticoids (GCs) during embryonic development influences offspring phenotypes and behaviors and induces epigenetic modifications of the genes in the hypothalamic-pituitary-adrenal (HPA) axis and in the serotonergic system in mammals. Whether prenatal corticosterone (CORT) exposure causes similar effects in avian species is less clear. In this study, we injected low (0.2µg) and high (1µg) doses of CORT into developing embryos on day 11 of incubation (E11) and tested the changes in aggressive behavior and hypothalamic gene expression on posthatch chickens of different ages. In ovo administration of high dose CORT significantly suppressed the growth rate from 3weeks of age and increased the frequency of aggressive behaviors, and the dosage was associated with elevated plasma CORT concentrations and significantly downregulated hypothalamic expression of arginine vasotocin (AVT) and corticotropin-releasing hormone (CRH). The hypothalamic content of glucocorticoid receptor (GR) protein was significantly decreased in the high dose group (p<0.05), whereas no changes were observed for GR mRNA. High dose CORT exposure significantly increased platelet serotonin (5-HT) uptake, decreased whole blood 5-HT concentration (p<0.05), downregulated hypothalamic tryptophan hydroxylase 1 (TPH1) mRNA and upregulated 5-HT receptor 1A (5-HTR1A) and monoamine oxidase A (MAO-A) mRNA, but not monoamine oxidase B (MAO-B). High dose CORT also significantly increased DNA methylation of the hypothalamic GR and CRH gene promoters (p<0.05). Our findings suggest that embryonic exposure to CORT programs aggressive behavior in the chicken through alterations of the HPA axis and the serotonergic system, which may involve modifications in DNA methylation.

A high-concentrate diet induced colonic epithelial barrier disruption is associated with the activating of cell apoptosis in lactating goats.

BACKGROUND: In ruminants, lower ruminal pH causes massive disruption of ruminal epithelial structure during periods of feeding high-concentrate diets. However, the influence of excessive organic fatty acids in the lumen of hindgut on the epithelial structure is unclear. In this study, twelve mid-lactating goats were randomly assigned to either a HC diet group (65% concentrate of dry matter; n = 6) or a LC diet group (35% concentrate of dry matter; n = 6) for 10 weeks. The colonic epithelial structure was detected by HE staining and transmission electron microscopy (TEM), and the apoptotic status of epithelial cells was estimated by TUNEL method and caspase activities. RESULTS: HC goats showed higher level of free lipopolysaccharide (LPS) in rumen fluid (p < 0.01) but not in colonic digesta (p > 0.05), and higher total volatile fatty acid (VFA) concentrations in rumen fluid (p < 0.05) and in colonic digesta (p < 0.01), and higher content of starch in colonic digesta (p < 0.05) compared to LC goats. HC goats demonstrated profound alterations in the colonic epithelial structure and tight junctions (TJ), apparently due to damage of the epithelium with widened TJs space and nuclear breakdown and mitochondrial swelling. HC goats showed higher level of apoptosis in the colonic epithelium with higher proportion of TUNEL-positive apoptotic cells and increases of caspase-3 and -3/7 activities, as well as the lower ratio of bcl-2/bax mRNA expression in the colonic mucosa (p < 0.05). However, ß-defense mRNA was significantly down-regulated in the colonic mucosa of HC goats compared to LC (p < 0.05). HC goats showed higher level of TJ proteins including claudin-1 and claudin-4 in the colonic mucosa than LC (p < 0.05). Neither free LPS content in the colonic digesta nor NF-κ B protein expression in tissues showed significant difference between HC and LC goats (p > 0.05). CONCLUSIONS: Our results reveal that long-term feeding HC diet to lactating goats causes severe damages to the colonic mucosa barrier associated with activating cells apoptosis.

Effects of tonic immobility (TI) and corticosterone (CORT) on energy status and protein metabolism in pectoralis major muscle of broiler chickens.

Tonic immobility (TI), which can be divided into short (STI) or long (LTI) duration, is a character related to fear. Our previous study has demonstrated LTI phenotype and chronic corticosterone (CORT) administration retarded growth of breast muscle in broiler chickens. In order to investigate the mechanism behind the negative effects of LTI and CORT on growth, the level of mRNA transcription of several key genes linked to energy and protein metabolism was measured in muscle. LTI broilers showed lower levels of ATP, energy charge (EC) (p<0.01), and lower muscle glycogen content (p<0.05) but higher level of ADP (p = 0.08) than STI birds. CORT treatment elevated EC level (p<0.05) and reduced liver glycogen content (p<0.05). Real-time PCR results showed that STI chickens had higher mRNA expression of PPAR α (p = 0.06) and AMPK α (p = 0.09) than LTI. CORT significantly down-regulated α-enolase mRNA expression in breast muscle compared to control (p<0.05). Neither TI nor CORT altered gene expression in Akt/mTOR/p70s6k cascade pathway in muscle (p > 0.05). However, western blot results showed that LTI chickens exhibited higher protein content of total Akt (p = 0.05) and phosphorylated Akt (p = 0.06) than STI. CORT treatment decreased the total protein content of Akt (p = 0.09) and p70s6k (p = 0.08). These results suggest that the retardation of muscle growth by LTI and chronic CORT administration parallels a strong alternation in energy status but slight changes of Akt/mTOR/p70s6k cascade, indicating that a decrease in muscle growth induced by LTI and CORT might not be mediated through mTOR-dependent signaling pathways.

Cholesterol deregulation induced by chronic corticosterone (CORT) stress in pectoralis major of broiler chickens.

Chronic endogenous glucocorticoid (GC) excess in mammals is associated with metabolic dysfunction and dyslipidemia that are characterized by increased plasma triglyceride and total cholesterol (Tch) levels. However, the effects of chronic GC administration on cholesterol metabolism, particularly in muscle tissues of broiler chickens, are unknown. In this study, broiler chickens were treated chronically with vehicle (CON) or corticosterone (CORT) for 2 weeks. Chronic CORT treatment significantly increased Tch levels in pectoralis major muscle (PMC) (p<0.001) as well as in leg muscle (p<0.01), and CORT enhanced triglyceride levels in the PMC (p<0.001). Real-time PCR results showed that HMGCR (p<0.05) mRNA expression was up-regulated by CORT in PMC, and 11ß-HSD1 gene transcription (p=0.08) was not significantly downregulated, whereas glucocorticoid receptor (GR) mRNA expression, 11ß-HSD2, CYP7A1, CYP27A1, ApoB and LDLR were unchanged by CORT (p>0.05). Western blot results showed that the levels of total GR (p=0.08) tended to be increased and nuclear GR protein (p<0.05) was increased in PMC by CORT administration. Parallel to an increase in gene expression, HMGCR protein expression in PMC was significantly increased (p<0.05) by CORT. Moreover, LDLR (p<0.05), ApoA1 (p=0.06) and 11ß-HSD2 (p=0.07) protein expression in PMC tended to be increased by CORT compared to control. These results indicate that chronic CORT administration causes cholesterol accumulation in PMC tissues of broiler chickens by increasing cholesterol synthesis and uptake.

Comparative proteomic analysis of the breast muscle response to chronic corticosterone administration in broiler chickens showing long or short tonic immobility.

Broilers of the same genetic origin were classified as short or long tonic immobility duration (STI and LTI, respectively) phenotypes and treated chronically with vehicle (control) or corticosterone (CORT) dissolved in drinking water between 27 and 42 d of age. Differential expression of proteins and mRNA was examined using 2-dimensional gel electrophoresis and real-time PCR to elucidate the mechanism behind the severe retardation of broiler breast muscle growth caused by LTI and CORT. The majority of the 13 proteins found to be differentially expressed in breast muscle of STI and LTI broilers are involved in either glycolysis (5 proteins) or myofilament formation (5 proteins). Of the 16 proteins differentially expressed in breast muscle following CORT treatment, 6 are structural proteins, 5 are categorized as cellular defense and stress proteins, and 3 (pyruvate kinase, l-lactate dehydrogenase, and creatine kinase) are involved in responses to stress and muscle damage. Real-time PCR results indicated that expression of these proteins is transcriptionally and posttranscriptionally regulated. Protein synthesis capacity, estimated by the RNA-to-protein ratio, was significantly lower in the breast muscle of CORT-treated broilers than in untreated control broilers. The level of Leu, Gly, and Ser in serum was significantly higher in CORT-treated broilers than in the control birds. Therefore, we conclude that CORT treatment retards the growth of skeletal muscle by suppressing protein synthesis and augmenting protein catabolism, paralleling the response to severe stress and muscle damage, and the negative effect of LTI on muscle growth is likely mediated through glucose metabolism. No interaction was observed between CORT and tonic immobility affecting growth performance or any parameter examined in the current study.

Dietary bile acids supplementation improves the growth performance and alleviates fatty liver in broilers fed a high-fat diet via improving the gut microbiota.

This experiment aims to evaluate the effect of bile acids (BAs) in alleviating fatty liver disease induced by a high-fat diet (HFD) in broilers, and the modulation of the gut microbiota involved in this process. A total of 192 one-day-old Arbor Acres (AA) commercial male broilers were randomly divided into 4 groups and treated with the following diet: a basal-fat diet (BFD), a basal-fat diet plus bile acids (BFD + BAs), an HFD, and a high-fat diet plus bile acids (HFD + BAs). Bile acids were supplemented at the early growth stage (3-7 d), middle stage (17-21 d), and late stage (31-35 d). Results showed that BAs treatment had a significant effect on body weight on 14 d and 35 d, and increased the breast muscle weight and its index, but decreased the liver weight and abdominal fat weight on 35 d (P < 0.05). The supplementation of BAs significantly improved the serum lipid profile and decreased the level of triglycerides (TG), total cholesterol (TCHO), and nonesterified fatty acids (NEFA) on 35 d (P < 0.05). Dietary BAs supplementation significantly alleviated the hepatic TG deposition induced by HFD (P < 0.05), which was accompanied by upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL) gene expression (P < 0.05). Moreover, the expression levels of hepatic gene adipose triglyceride lipase (ATGL), peroxisome proliferator-activated receptor α (PPARα), and apolipoprotein B (APOB) were greatly increased by BAs treatment. The analysis of 16S rRNA sequencing showed that the microbial diversity of the cecal digesta was increased by BAs in broilers with elevated abundances of Firmicutes, Lactobacillus, Anaerostipes, Sellimonas, and CHKCI002 and decreased abundances of Barnesiella and Akkermansia genus (P < 0.05). Hepatic TG content was positively correlated with the abundance of Oscillospiraceae, but it was negatively correlated with the abundance of Lactobacillus in cecal digesta (P < 0.05). These results indicate that dietary BAs can improve growth performance and alleviate fatty liver disease induced by an HFD via modulating gut microbiota in broilers.

Sulfiredoxin-1 accelerates erastin-induced ferroptosis in HT-22 hippocampal neurons by driving heme Oxygenase-1 activation.

Ferroptosis, a recently identified non-apoptotic form of cell death, is strongly associated with neurological diseases and has emerged as a potential therapeutic target. Nevertheless, the fundamental mechanisms are still predominantly unidentified. In the current investigation, sulfiredoxin-1 (SRXN1) has been identified as a crucial regulator that enhances the susceptibility to ferroptosis in HT-22 mouse hippocampal cells treated with erastin. Utilizing TMT-based proteomics, a significant increase in SRXN1 expression was observed in erastin-exposed HT-22 cells. Efficient amelioration of erastin-induced ferroptosis was achieved via the knockdown of SRXN1, which resulted in the reduction of intracellular Fe2+ levels and reactive oxygen species (ROS) in HT-22 cells. Notably, the activation of Heme Oxygenase-1 (HO-1) was found to be crucial for inducing SRXN1 expression in HT-22 cells upon treatment with erastin. SRXN1 increased intracellular ROS and Fe2+ levels by activating HO-1 expression, which promoted erastin-induced ferroptosis in HT-22 cells. Inhibiting SRXN1 or HO-1 alleviated erastin-induced autophagy in HT-22 cells. Additionally, upregulation of SRXN1 or HO-1 increased the susceptibility of HT-22 cells to ferroptosis, a process that was counteracted by the autophagy inhibitor 3-Methyladenine (3-MA). These results indicate that SRXN1 is a key regulator of ferroptosis, activating the HO-1 protein through cellular redox regulation, ferrous iron accumulation, and autophagy in HT-22 cells. These findings elucidate a novel molecular mechanism of erastin-induced ferroptosis sensitivity and suggest that SRXN1-HO-1-autophagy-dependent ferroptosis serves as a promising treatment approach for neurodegenerative diseases.

LPS-induced inflammation in the chicken is associated with CCAAT/enhancer binding protein beta-mediated fat mass and obesity associated gene down-regulation in the liver but not hypothalamus.

BACKGROUND: The fat mass and obesity associated gene (FTO) is widely investigated in humans regarding its important roles in obesity and type 2 diabetes. Studies in mammals demonstrate that FTO is also associated with inflammation markers. However, the association of FTO with inflammation in chickens remains unclear. In this study, male chickens on day 28 posthatching were injected intraperitoneally with lipopolysaccharide (LPS) or saline to investigate whether the FTO gene is involved in LPS-induced inflammation. RESULTS: We detected significant down-regulation of FTO mRNA in the liver (P < 0.01), but not in the hypothalamus, 2 and 24 h after LPS challenge. Toll-like receptor (TLR) 2 (P < 0.01) and TLR4 (P < 0.01) followed the same pattern as FTO, being suppressed significantly in liver but not in hypothalamus. IL-1ß was dramatically up-regulated (P < 0.01) in both liver and hypothalamus 2 h after LPS challenge, while activation of IL-6 was observed in the liver (P < 0.01), but not in hypothalamus. The 5'-flanking sequence of the chicken FTO gene contains nine predicted binding sites for CCAAT/enhancer binding protein beta (C/EBP beta) and one for signal transducer and activator of transcription 3 (STAT3). Significant elevation of C/EBP beta was detected in the liver (P < 0.01), but not in the hypothalamus, 2 h after LPS challenge. Lipopolysaccharide challenge increased the C/EBP beta binding to FTO promoter in the liver (P < 0.01 for fragment 1, P < 0.05 for fragment 2), although the protein content of C/EBP beta was not altered. Moreover, injection of LPS resulted in enhanced phosphorylation of liver STAT3, a downstream transcription factor in IL-6 signaling. Although phosphorylated STAT3 was not detected to directly bind to FTO promoter, it was found to interact with C/EBP beta. CONCLUSION: Our results reveal that FTO expression in liver, but not in hypothalamus, is affected by the i.p. injection of LPS, which may be mediated through tissue-specific FTO transcriptional regulation by C/EBP beta and STAT3 interaction.

Effect of corticosterone on growth and welfare of broiler chickens showing long or short tonic immobility.

Tonic immobility (TI) test is commonly used to assess fear. Animals showing different TI durations demonstrate distinct behavior and biochemical responses to stress. However, less is known about how TI phenotype affects growth and welfare of domestic fowl. In this study, broiler chickens (Gallus gallus) were classified into short and long TI duration (STI and LTI) phenotypes and treated chronically with vehicle (CON) or corticosterone (CORT). STI broilers demonstrated significantly higher growth rate with higher breast muscle yield (P<0.05) and liver weight relative to BW tended to be lower (P=0.053), which was accompanied by higher serum concentration of CORT (P<0.05) and uric acid (P<0.01), but lower serum level of T4 (P=0.01). CORT severely reduced body weight, as well as the relative weight of muscle, bursa of Fabricius and spleen (P<0.001), but relative liver weight was increased (P<0.001). CORT-treated chickens had reduced serum CORT, elevated heterophile/lymphocyte ratio, and increased serum levels of total and free T3. STI broilers displayed more preening behavior (P<0.05), yet CORT elicited more walking behavior (P<0.05). No difference was observed in the welfare assessment scores between STI and LTI phenotypes under basal situation, while LTI chickens showed significantly increased incidence of pad dermatitis compared to STI under CORT exposure. The results suggest that STI broilers demonstrate better growth performance and higher adaptability to stress compared to LTI chickens.

Differences in egg deposition of corticosterone and embryonic expression of corticosterone metabolic enzymes between slow and fast growing broiler chickens.

Glucocorticoids (GCs) are vital for embryonic development and their bioactivity is regulated by the intracellular metabolism involving 11ß-hydroxysteroid dehydrogenases (11ß-HSDs) and 20-hydroxysteroid dehydrogenase (20-HSD). Here we sought to reveal the differences in egg deposition of corticosterone and embryonic expression of corticosterone metabolic enzymes between slow and fast growing broiler chickens (Gallus gallus). Eggs of fast-growing breed contained significantly higher (P<0.05) corticosterone in the yolk and albumen, compared with that of a slow-growing breed. 11ß-HSD1 and 11ß-HSD2 were expressed in relatively higher abundance in the liver, kidney and intestine, following similar tissue-specific ontogenic patterns. In the liver, expression of both 11ß-HSD1 and 11ß-HSD2 was upregulated (P<0.05) towards hatching, yet 20-HSD displayed distinct pattern showing a significant decrease (P<0.05) on posthatch day 1 (D1). Hepatic mRNA expression of 11ß-HSD1 and 11ß-HSD2 was significantly higher in fast-growing chicken embryos at all the embryonic stages investigated and so was the hepatic protein content on embryonic day of 14 (E14) for 11ß-HSD1 and on E14 and D1 for 11ß-HSD2. 20-HSD mRNA was higher in fast-growing chicken embryos only on E14. Our data provide the first evidence that egg deposition of corticosterone, as well as the hepatic expression of glucocorticoid metabolic enzymes, differs between fast-growing and slow-growing chickens, which may account, to some extent, for the breed disparities in embryonic development.

Breed-dependent transcriptional regulation of phosphoenolpyruvate carboxylase, cytosolic form, expression in the liver of broiler chickens.

Hepatic gluconeogenesis is the main source of glucose during chicken embryonic development, and it plays a major role in glucose homeostasis for developing embryos. Phosphoenolpyruvate carboxylase (PEPCK) catalyzes the rate-limiting step of gluconeogenesis, yet how hepatic PEPCK expression is differentially regulated between chicken breeds remains elusive. In this study, fertile eggs from a slow-growing Chinese Yellow Feathered Chicken and a fast-growing White Recessive Rock Chicken were incubated under the same standard conditions, and serum and liver samples were collected on embryonic d 18 (18E). The fast-growing breed had a significantly higher fetal weight (P < 0.01) and serum glucose concentration (P < 0.05) compared with the slow-growing breed. The fast-growing breed also had significantly higher hepatic mRNA expression levels of the cystolic form of PEPCK (PEPCK-c; P < 0.05) and significantly higher hepatic mRNA and protein expression levels of cAMP response element binding protein 1 (CREB-1; P < 0.05). Moreover, the binding of phosphorylated CREB-1 to the PEPCK-c promoter tended to be higher in the fast-growing breed (P = 0.08). Breed-specific epigenetic modifications of the PEPCK-c promoter were also observed; the fast-growing breed demonstrated lower CpG methylation (P < 0.05) and histone H3 (P < 0.05) levels but more histone H3 acetylation (H3ac) and histone H3 lysine 27 trimethylation (H3K27me3; P < 0.05) compared with the slow-growing breed. Our results suggest that hepatic PEPCK-c expression is transcriptionally regulated in a breed-specific manner and that fast- and slow-growing broiler chicken fetuses exhibit different epigenetic modifications on their PEPCK-c promoter regions.