Epigenetic regulation of H3K27me3 in laying hens with fatty liver hemorrhagic syndrome induced by high-energy and low-protein diets.

BACKGROUND: Fatty liver hemorrhagic syndrome (FLHS) in the modern poultry industry is primarily caused by nutrition. Despite encouraging progress on FLHS, the mechanism through which nutrition influences susceptibility to FLHS is still lacking in terms of epigenetics. RESULTS: In this study, we analyzed the genome-wide patterns of trimethylated lysine residue 27 of histone H3 (H3K27me3) enrichment by chromatin immunoprecipitation-sequencing (ChIP-seq), and examined its association with transcriptomes in healthy and FLHS hens. The study results indicated that H3K27me3 levels were increased in the FLHS hens on a genome-wide scale. Additionally, H3K27me3 was found to occupy the entire gene and the distant intergenic region, which may function as silencer-like regulatory elements. The analysis of transcription factor (TF) motifs in hypermethylated peaks has demonstrated that 23 TFs are involved in the regulation of liver metabolism and development. Transcriptomic analysis indicated that differentially expressed genes (DEGs) were enriched in fatty acid metabolism, amino acid, and carbohydrate metabolism. The hub gene identified from PPI network is fatty acid synthase (FASN). Combined ChIP-seq and transcriptome analysis revealed that the increased H3K27me3 and down-regulated genes have significant enrichment in the ECM-receptor interaction, tight junction, cell adhesion molecules, adherens junction, and TGF-beta signaling pathways. CONCLUSIONS: Overall, the trimethylation modification of H3K27 has been shown to have significant regulatory function in FLHS, mediating the expression of crucial genes associated with the ECM-receptor interaction pathway. This highlights the epigenetic mechanisms of H3K27me3 and provides insights into exploring core regulatory targets and nutritional regulation strategies in FLHS.

Taurine Protects against the Fatty Liver Hemorrhagic Syndrome in Laying Hens through the Regulation of Mitochondrial Homeostasis.

Metabolic-associated fatty liver disease (MAFLD) is a chronic liver disease caused by fat deposition in the liver of humans and mammals, while fatty liver hemorrhagic syndrome (FLHS) is a fatty liver disease in laying hens which can increase the mortality and cause severe economic losses to the laying industry. Increasing evidence has shown a close relationship between the occurrence of fatty liver disease and the disruption of mitochondrial homeostasis. Studies have proven that taurine can regulate hepatic fat metabolism, reduce hepatic fatty deposition, inhibit oxidative stress, and alleviate mitochondrial dysfunction. However, the mechanisms by which taurine regulates mitochondrial homeostasis in hepatocytes need to be further studied. In this study, we determined the effects and mechanisms of taurine on high-energy low-protein diet-induced FLHS in laying hens and in cultured hepatocytes in free fatty acid (FFA)-induced steatosis. The liver function, lipid metabolism, antioxidant capacity, mitochondrial function, mitochondrial dynamics, autophagy, and biosynthesis were detected. The results showed impaired liver structure and function, mitochondrial damage and dysfunction, lipid accumulation, and imbalance between mitochondrial fusion and fission, mitochondrial autophagy, and biosynthesis in both FLHS hens and steatosis hepatocytes. Taurine administration can significantly inhibit the occurrence of FLHS, protect mitochondria in hepatocytes from disease induced by lipid accumulation and FFA, up-regulate the expression levels of Mfn1, Mfn2, Opa1, LC3I, LC3II, PINK1, PGC-1α, Nrf1, Nrf2, and Tfam, and down-regulate the expression levels of Fis1, Drp1, and p62. In conclusion, taurine can protect laying hens from FLHS through the regulation of mitochondrial homeostasis, including the regulation of mitochondrial dynamics, autophagy, and biosynthesis.

Berberine Protects against High-Energy and Low-Protein Diet-Induced Hepatic Steatosis: Modulation of Gut Microbiota and Bile Acid Metabolism in Laying Hens.

Berberine (BBR) is a natural alkaloid with multiple biotical effects that has potential as a treatment for fatty liver hemorrhagic syndrome (FLHS). However, the mechanism underlying the protective effect of BBR against FLHS remains unclear. The present study aimed to investigate the effect of BBR on FLHS induced by a high-energy, low-protein (HELP) diet and explore the involvement of the gut microbiota and bile acid metabolism in the protective effects. A total of 90 healthy 140-day-old Hy-line laying hens were randomly divided into three groups, including a control group (fed a basic diet), a HELP group (fed a HELP diet), and a HELP+BBR group (high-energy, high-protein diet supplemented with BBR instead of maize). Our results show that BBR supplementation alleviated liver injury and hepatic steatosis in laying hens. Moreover, BBR supplementation could significantly regulate the gut's microbial composition, increasing the abundance of Actinobacteria and Romboutsia. In addition, the BBR supplement altered the profile of bile acid. Furthermore, the gut microbiota participates in bile acid metabolism, especially taurochenodeoxycholic acid and α-muricholic acid. BBR supplementation could regulate the expression of genes and proteins related to glucose metabolism, lipid synthesis (FAS, SREBP-1c), and bile acid synthesis (FXR, CYP27a1). Collectively, our findings demonstrate that BBR might be a potential feed additive for preventing FLHS by regulating the gut microbiota and bile acid metabolism.

Integrated analysis of the methylome and transcriptome of chickens with fatty liver hemorrhagic syndrome.

BACKGROUND: DNA methylation, a biochemical modification of cytosine, has an important role in lipid metabolism. Fatty liver hemorrhagic syndrome (FLHS) is a serious disease and is tightly linked to lipid homeostasis. Herein, we compared the methylome and transcriptome of chickens with and without FLHS. RESULTS: We found genome-wide dysregulated DNA methylation pattern in which regions up- and down-stream of gene body were hypo-methylated in chickens with FLHS. A total of 4155 differentially methylated genes and 1389 differentially expressed genes were identified. Genes were focused when a negative relationship between mRNA expression and DNA methylation in promoter and gene body were detected. Based on pathway enrichment analysis, we found expression of genes related to lipogenesis and oxygenolysis (e.g., PPAR signaling pathway, fatty acid biosynthesis, and fatty acid elongation) to be up-regulated with associated down-regulated DNA methylation. In contrast, genes related to cellular junction and communication pathways (e.g., vascular smooth muscle contraction, phosphatidylinositol signaling system, and gap junction) were inhibited and with associated up-regulation of DNA methylation. CONCLUSIONS: In the current study, we provide a genome-wide scale landscape of DNA methylation and gene expression. The hepatic hypo-methylation feature has been identified with FLHS chickens. By integrated analysis, the results strongly suggest that increased lipid accumulation and hepatocyte rupture are central pathways that are regulated by DNA methylation in chickens with FLHS.

Osteocalcin activates lipophagy via the ADPN-AMPK/PPARα-mTOR signaling pathway in chicken embryonic hepatocyte.

Fatty liver hemorrhage syndrome (FLHS) is the leading cause of noninfectious mortality in caged layers worldwide. Osteocalcin (OCN) is a protein secreted by osteoblasts, and its undercarboxylated form (ucOCN) acts as a multifunctional hormone that protects laying hens from FLHS. Lipophagy is a form of selective autophagy that breaks down lipid droplets (LDs) through lysosomes, and defective lipophagy is associated with FLHS. The aim of this study was to investigate the effects of ucOCN on the lipophagy of chicken embryonic hepatocytes and associated the function of the adiponectin (ADPN) signaling pathway. In this study, chicken embryonic hepatocytes were divided into 5 groups: control (CONT), fat emulsion (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 4 µM AdipoRon, an adiponectin receptor agonist, was used to investigate the function of ADPN. The results showed that compared with CONT group, FE promoted the levels of phosphorylation of mammalian target of rapamycin (p-mTOR) (P < 0.05) and decreased the mRNA expression of ADNP receptors (AdipoR1 and AdipoR2). Compared with FE group, 3 and 9 ng/mL ucOCN inhibited the levels of autophagy adaptor p62 and p-mTOR (P < 0.05), increased the ratios of LC3-II/LC3-I (P < 0.05) and phosphorylated adenosine 5'-monophosphate-activated protein kinase (p-AMPK)/AMPK (P < 0.05), as well as the levels of peroxisome proliferator-activated receptor α (PPAR-α) and ADPN (P < 0.05). In addition, ucOCN at the tested concentrations increased the colocalization of LC3 and LDs in fatty hepatocytes. Administrated 4 µM AdipoRon activated AdipoR1 and AidpoR2 mRNA expression (P < 0.05), decreased the concentrations of triglyceride (P < 0.05), without effects on cell viability (P > 0.05). AdipoRon also increased the LC3-II/LC3-I ratio (P < 0.05) and the levels of p-AMPK/AMPK and PPAR-α (P < 0.05). In conclusion, the results reveal that ucOCN regulates lipid metabolism by activating lipophagy via the ADPN-AMPK/PPARα-mTOR signaling pathway in chicken embryonic hepatocytes. The results may provide new insights for controlling FLHS in laying hens.

Berberine alleviates high-energy and low-protein diet-induced fatty liver hemorrhagic syndrome in laying hens: insights from microbiome and metabolomics.

Berberine (BBR), a well-known quaternary ammonium alkaloid, is recognized for its ability to prevent and alleviate metabolic disorders because of its anti-oxidative and anti-inflammatory properties. However, the underlying mechanisms of BBR to mitigate fatty liver hemorrhagic syndrome (FLHS) through the modulation of gut microbiota and their metabolism remained unclear. The results revealed that BBR ameliorates lipid metabolism disorder in high-energy and low-protein (HELP) diet-induced FLHS laying hens, as evidenced by improved liver function and lipid deposition of the liver, reduced blood lipids, and the expression of liver lipid synthesis-related factors. Moreover, BBR alleviated HELP diet-induced barrier dysfunction, increased microbial population, and dysregulated lipid metabolism in the ileum. BBR reshaped the HELP-perturbed gut microbiota, particularly declining the abundance of Desulfovibrio_piger and elevating the abundance of Bacteroides_salanitronis_DSM_18170. Meanwhile, metabolomic profiling analysis revealed that BBR reshaped microbial metabolism and function, particularly by reducing the levels of hydrocinnamic acid, dehydroanonaine, and leucinic acid. Furthermore, fecal microbiota transplantation (FMT) experiments revealed that BBR-enriched gut microbiota alleviated hepatic lipid deposition and intestinal inflammation compared with those chicks that received a gut microbiota by HELP. Collectively, our study provided evidence that BBR effectively alleviated FLHS induced by HELP by reshaping the microbial and metabolic homeostasis within the liver-gut axis.

Positive effects and mechanism of mulberry leaf extract on alleviating fatty liver hemorrhagic syndrome in laying hens.

This experiment was conducted to investigate the effects of mulberry leaf extract (MLE) on alleviating fatty liver hemorrhagic syndrome (FLHS) in laying hens. The 576 Jing Fen laying hens of 56 weeks of age with good health and similar weights (1.76 ± 0.17 kg) were randomly divided into 6 groups, with 8 replicates in each group and 12 chickens in each replicate. The experiment lasted 56 d. The control group was fed a corn-soybean meal diet. The FLHS group was fed a high energy-low protein (HELP) diet, and the other four experimental groups were fed HELP diets supplemented with 0.04, 0.40, 0.80, and 1.20% MLE, respectively. The results showed that HELP treatment significantly induced liver injury, which indicated that the FLHS model was successfully established. MLE supplementation could alleviate the FLHS by reducing the liver index, abdominal fat percentage, total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL) in the serum (P < 0.05), and subsequently increase the egg production rate (P < 0.05). The laying hens fed 0.8% MLE exhibited the greatest production performance (P < 0.05) and could improve serum lipid levels. In addition, the genes associated with fatty acid synthesis (ACC, HMGR and SREBP-1C) were downregulated (P < 0.05), and genes related to fatty acid oxidation (CPT1A, AMPK, and ATGL) were found to be upregulated (P < 0.05). Supplementation with 1.2% MLE significantly reduced the relative abundance of Firmicutes and Desulfurized Bacillus (P < 0.05) and significantly increased the relative abundance of Fecal Bacillus (P < 0.05). In conclusion, MLE may regulate the mRNA expression of lipid metabolism-related genes through the AMPK signaling pathway and improve cecal microbiota balance and serum lipid levels to alleviate FLHS in laying hens and subsequently improve egg production performance.

Quantitative lipidomics reveals the changes of lipids and antioxidant capacity in egg yolk from laying hens with fatty liver hemorrhagic syndrome.

In laying hens, fatty liver hemorrhagic syndrome (FLHS) is a common metabolic disorder, which can affect egg production and nutritional value. However, the impact of FLHS on the lipid content in egg yolks was not clear. In this study, FLHS model was induced by using high-energy low-protein diet, and the egg quality was evaluated. Egg yolk lipids were quantitatively analyzed by using ultra-performance liquid chromatography-mass spectrometry combined with multivariate statistical analysis. Gene expressions of the lipoprotein were determined by qRT-PCR and antioxidant capacity of the egg yolk were determined by kits. The elevated blood lipids and extensive lipid droplets observed indicated successful establishment of the FLHS model in laying hens. Measurements of egg quality showed that egg yolk weight was increased in the FLHS group. Lipidomics revealed that 1,401 lipids, comprising 27 lipid subclasses in the egg yolk. According to score plots of principal component analysis and orthogonal partial least squares discriminant analysis, different lipid profile was observed between the control and FLHS groups. A total of 97 different lipid species were screen out. Sphingolipid and glycerophospholipid metabolism were identified as key pathways. Free polyunsaturated fatty acids (PUFA) exhibited an increase in the FLHS group (P < 0.05). Notably, the form of PUFAs was changed that the FLHS group showed an increase in triacylglycerol-docosahexenoic acid and triacylglycerol-arachidonic acid in the egg yolk, while triacylglycerol-α-linolenic acid was decreased (P < 0.05). Total superoxide dismutase was decreased in the egg yolks affected by FLHS. Gene expressions of vitellogenin 2 (VTG2), VTG3, very low-density apolipoprotein II and apolipoprotein B were increased in the liver of laying hens with FLHS (P < 0.05). In conclusion, FLHS promoted the lipid transport from the liver to the yolk by upregulating lipoprotein expression, which altered lipid profile, and reduced antioxidant capacity in the yolk. This study provided a foundation for understanding the changes in lipids, lipid transport and lipid antioxidation capacity in egg yolk from laying hens with FLHS.

Sodium Butyrate Alleviates Free Fatty Acid-Induced Steatosis in Primary Chicken Hepatocytes via Regulating the ROS/GPX4/Ferroptosis Pathway.

Fatty liver hemorrhagic syndrome (FLHS) in laying hens is a nutritional metabolic disease commonly observed in high-yielding laying hens. Sodium butyrate (NaB) and ferroptosis were reported to contribute to the pathogenesis of fatty liver-related diseases. However, the underlying mechanism of NaB in FLHS and whether it mediates ferroptosis remains unclear. A chicken primary hepatocyte induced by free fatty acids (FFAs, keeping the ratio of sodium oleate and sodium palmitate concentrations at 2:1) was established, which received treatments with NaB, the ferroptosis inducer RAS-selective lethal 3 (RSL3), and the inhibitor ferrostatin-1 (Fer-1). As a result, NaB increased biochemical and lipid metabolism indices, and the antioxidant level, while inhibiting intracellular ROS accumulation and the activation of the ferroptosis signaling pathway, as evidenced by a reduction in intracellular iron concentration, upregulated GPX4 and xCT expression, and inhibited NCOA4 and ACSL4 expression. Furthermore, treatment with Fer-1 reinforced the protective effects of NaB, while RSL3 reversed it by blocking the ROS/GPX4/ferroptosis pathway, leading to the accumulation of lipid droplets and oxidative stress. Collectively, our findings demonstrated that NaB protects hepatocytes by regulating the ROS/GPX4-mediated ferroptosis pathway, providing a new strategy and target for the treatment of FLHS.

Enrichment efficiency of lutein in eggs and its function in improving fatty liver hemorrhagic syndrome in aged laying hens.

In this study, we evaluated the enrichment efficiency of lutein in eggs and its function in preventing fatty liver hemorrhagic syndrome (FLHS) in aged laying hens. Five groups of laying hens (65 wk old) were fed basal diets supplemented with 0, 30, 60, 90, or 120 mg/kg of lutein. The supplementation period lasted 12 wk followed by 2 wk of lutein depletion in feed. The results revealed that lutein efficiently enriched the egg yolks and improved their color with a significant increase in relative redness (P < 0.001). Lutein accumulation increased in the egg yolk until day 10, then depletion reached a minimum level after 14 d. Overall, zeaxanthin content in all the groups was similar throughout the experimental period. However, triglycerides and total cholesterol were significantly decreased in the liver (P < 0.05) but not significantly different in the serum (P > 0.05). In the serum, the lipid metabolism enzyme acetyl-CoA synthetase was significantly reduced (P < 0.05), whereas dipeptidyl-peptidase 4 was not significantly different (P > 0.05), and there was no statistical difference of either enzyme in the liver (P > 0.05). Regarding oxidation and inflammation-related indexes, malondialdehyde, tumor necrosis factors alpha, interleukin-6, and interleukin-1 beta were decreased, whereas superoxide dismutase and total antioxidant capacity increased in the liver (P < 0.001). The function of lutein for the same indexes in serum was limited. It was concluded that lutein efficiently enriched the egg yolk of old laying hens to improve their color and reached the highest level on day 10 without being subject to a significant conversion into zeaxanthin. At the same time, lutein prevented liver steatosis in aged laying hens by exerting strong antioxidant and anti-inflammatory functions, but also through the modulation of lipid metabolism, which may contribute to reducing the incidence of FLHS in poultry.

Epigenetic regulation of key gene of PCK1 by enhancer and super-enhancer in the pathogenesis of fatty liver hemorrhagic syndrome.

OBJECTIVE: Rare study of the non-coding and regulatory regions of the genome limits our ability to decode the mechanisms of fatty liver hemorrhage syndrome (FLHS) in chickens. METHODS: Herein, we constructed the high-fat diet-induced FLHS chicken model to investigate the genome-wide active enhancers and transcriptome by H3K27ac target chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-Seq) profiles of normal and FLHS liver tissues. Concurrently, an integrative analysis combining ChIP-seq with RNA-Seq and a comparative analysis with chicken FLHS, rat non-alcoholic fatty liver disease (NAFLD) and human NAFLD at the transcriptome level revealed the enhancer and super enhancer target genes and conservative genes involved in metabolic processes. RESULTS: In total, 56 and 199 peak-genes were identified in upregulated peak-genes positively regulated by H3K27ac (Cor (peak-gene correlation) ≥0.5 and log2(FoldChange) ≥1) (PP) and downregulated peak-genes positively regulated by H3K27ac (Cor (peak-gene correlation) ≥0.5 and log2(FoldChange)≤-1) (PN), respectively; then we screened key regulatory targets mainly distributing in lipid metabolism (PCK1, APOA4, APOA1, INHBE) and apoptosis (KIT, NTRK2) together with MAPK and PPAR signaling pathway in FLHS. Intriguingly, PCK1 was also significantly covered in up-regulated super-enhancers (SEs), which further implied the vital role of PCK1 during the development of FLHS. CONCLUSION: Together, our studies have identified potential therapeutic biomarkers of PCK1 and elucidated novel insights into the pathogenesis of FLHS, especially for the epigenetic perspective.

Osteocalcin and Its Potential Functions for Preventing Fatty Liver Hemorrhagic Syndrome in Poultry.

Osteocalcin (OCN) is synthesized and secreted by differentiating osteoblasts. In addition to its role in bone, OCN acts as a hormone in the pancreas, liver, muscle, fat, and other organs to regulate multiple pathophysiological processes including glucose homeostasis and adipic acid metabolism. Fat metabolic disorder, such as excessive fat buildup, is related to non-alcoholic fatty liver disease (NAFLD) in humans. Similarly, fatty liver hemorrhage syndrome (FLHS) is a metabolic disease in laying hens, resulting from lipid accumulation in hepatocytes. FLHS affects hen health with significant impact on poultry egg production. Many studies have proposed that OCN has protective function in mammalian NAFLD, but its function in chicken FLHS and related mechanism have not been completely clarified. Recently, we have revealed that OCN prevents laying hens from FLHS through regulating the JNK pathway, and some pathways related to the disease progression have been identified through both in vivo and vitro investigations. In this view, we discussed the current findings for predicting the strategy for using OCN to prevent or reduce FLHS impact on poultry production.

Quantitative lipidomics reveals lipid perturbation in the liver of fatty liver hemorrhagic syndrome in laying hens.

Fatty liver hemorrhagic syndrome (FLHS) is a metabolic disease that causes decreased egg production and even death in laying hens, which brings huge economic losses to the poultry industry. However, the pathogenesis of FLHS is unclear. The purpose of the present study was to identify the changes in lipid profile and the lipid species related to FLHS. In the present study, the FLHS disease model in Chinese commercial Jing Fen laying hens was induced by a high-energy low-protein diet. A lipidomics approach based on ultra-performance liquid chromatography-mass spectrometry coupled with multivariate statistical analysis was performed for the qualitative and quantitative analyses of the liver lipids. The results showed that a total of 29 lipid subclasses, including 1,302 lipid species, were detected and identified. Among them, the proportions of phosphatidylserine (Control/FLHS, 33.1% vs. 29.1%), phosphatidylethanolamine (22.7% vs. 15.5%), phosphatidylcholine (15.7% vs. 11.7%) and phosphatidylinositol (7% vs. 6%) were reduced, while triacylglycerol (7.1% vs. 18.3%) and diglyceride (3.9% vs. 11.7%) were increased. Between the Control and FLHS groups, distinct changes in lipid profile were observed in the score plots of principal component analysis and orthogonal partial least squares discriminant analysis. Twelve differential lipid species mainly involved in glycerophospholipid metabolism and linoleic acid metabolism were identified and considered to be related to the pathogenesis of FLHS. Fatty acid chain length and unsaturation were reduced, while the mRNA levels of elongation of very long chain fatty acids-2 (ELOVL2) were increased in the liver of laying hens with FLHS. Collectively, this study characterized the liver lipid profile and explored the changes in lipid species related to FLHS, which provided insights into the pathogenesis of FLHS from the view of lipid metabolism.

Protective effects of genistein on the production performance and lipid metabolism disorders in laying hens with fatty liver hemorrhagic syndrome by activation of the GPER-AMPK signaling pathways.

The aim of this study was to evaluate the beneficial effects and potential mechanisms of genistein (GEN) on production performance impairments and lipid metabolism disorders in laying hens fed a high-energy and low-protein (HELP) diet. A total of 120 Hy-line Brown laying hens were fed with the standard diet and HELP diet supplemented with 0, 50, 100, and 200 mg/kg GEN for 80 d. The results showed that the declines in laying rate (P < 0.01), average egg weight (P < 0.01), and egg yield (P < 0.01), and the increase of the ratio of feed to egg (P < 0.01) induced by HELP diet were markedly improved by 100 and 200 mg/kg of GEN treatment in laying hens (P < 0.05). Moreover, the hepatic steatosis and increases of lipid contents (P < 0.01) in serum and liver caused by HELP diet were significantly alleviated by treatment with 100 and 200 mg/kg of GEN in laying hens (P < 0.05). The liver index and abdominal fat index of laying hens in the HELP group were higher than subjects in the control group (P < 0.01), which were evidently attenuated by dietary 50 to 200 mg/kg of GEN supplementation (P < 0.05). Dietary 100 and 200 mg/kg of GEN supplementation significantly reduced the upregulations of genes related to fatty acid transport and synthesis (P < 0.01) but enhanced the downregulations of genes associated with fatty acid oxidation (P < 0.01) caused by HELP in the liver of laying hens (P < 0.05). Importantly, 100 and 200 mg/kg of GEN supplementation markedly increased G protein-coupled estrogen receptor (GPER) mRNA and protein expression levels and activated the AMP-activated protein kinase (AMPK) signaling pathway in the liver of laying hens fed a HELP diet (P < 0.05). These data indicated that the protective effects of GEN against the decline of production performance and lipid metabolism disorders caused by HELP diet in laying hens may be related to the activation of the GPER-AMPK signaling pathways. These data not only provide compelling evidence for the protective effect of GEN against fatty liver hemorrhagic syndrome in laying hens but also provide the theoretical basis for GEN as an additive to alleviate metabolic disorders in poultry.

Fatty liver hemorrhagic syndrome (FLHS) is a nutritional and metabolic disease that seriously threatens the health and performance of laying hens, which is characterized by hepatic steatosis and lipid metabolism disorders. As an isoflavone phytoestrogen, genistein (GEN) exerts many beneficial functions, including alleviating lipid metabolism disorders and anti-inflammatory properties. However, further research is needed on the protective effect and potential mechanism of GEN on the FLHS in laying hens. Here, we found that GEN treatment improved liver injury and decline of production performance in laying hens with FLHS. Moreover, GEN treatment alleviated hepatic steatosis and lipid metabolism disorders through reducing the expression levels of mRNA related to fatty acid transport and synthesis and enhancing the mRNA expression levels of factors associated with fatty acid oxidation in FLHS layers, which may be achieved by activation of the G protein-coupled estrogen receptor­adenosine 5'-monophosphate (AMP)-activated protein kinase signaling pathways. These data not only provide compelling evidence for the protective effects and mechanisms of GEN against FLHS in laying hens but also provide the theoretical basis for GEN to alleviate other metabolic disorders in poultry.

Dietary supplementation of salidroside alleviates liver lipid metabolism disorder and inflammatory response to promote hepatocyte regeneration via PI3K/AKT/Gsk3-ß pathway.

Fatty liver hemorrhagic syndrome (FLHS) is a chronic hepatic disease which occurs when there is a disorder in lipid metabolism. FLHS is often observed in caged laying hens and characterized by a decrease in egg production and dramatic increase of mortality. Salidroside (SDS) is an herbal drug which has shown numerous pharmacological activities, such as protecting mitochondrial function, attenuating cell apoptosis and inflammation, and promoting antioxidant defense system. We aimed to determine the therapeutic effects of SDS on FLHS in laying hens and investigate the underlying mechanisms through which SDS operates these functions. We constructed oleic acid (OA)-induced fatty liver model in vitro and high-fat diet-induced FLHS of laying hens in vivo. The results indicated that SDS inhibited OA-induced lipid accumulation in chicken primary hepatocytes, increased hepatocyte activity, elevated the mRNA expression of proliferation related genes PCNA, CDK2, and cyclinD1 and increased the protein levels of PCNA and CDK2 (P < 0.05), as well as decreased the cleavage levels of Caspase-9, Caspase-8, and Caspase-3 and apoptosis in hepatocytes (P < 0.05). Moreover, SDS promoted the phosphorylation levels of PDK1, AKT, and Gsk3-ß, while inhibited the PI3K inhibitor (P < 0.05). Additionally, we found that high-fat diet-induced FLHS hens had heavier body weight, liver weight, and abdominal fat weight, and severe steatosis in histology, compared with the control group (Con). However, hens fed with SDS maintained lighter body weight, liver weight, and abdominal fat weight, as well as normal liver without hepatic steatosis. In addition, high-fat diet-induced FLHS hens had high levels of serum total cholesterol (TC), triglyceride (TG), alanine transaminase (ALT), and aspartate aminotransferase (AST) compared to the Con group, however, in the Model+SDS group, the levels of TC, TG, ALT, and AST decreased significantly, whereas the level of superoxide dismutase (SOD) increased significantly (P < 0.05). We also found that SDS significantly decreased the mRNA expression abundance of PPARγ, SCD, and FAS in the liver, as well as increased levels of PPARα and MTTP, and decreased the mRNA expression of TNF-α, IL-1ß, IL-6, and IL-8 in the Model+SDS group (P < 0.05). In summary, this study showed that 0.3 mg/mL SDS attenuated ROS generation, inhibited lipid accumulation and hepatocyte apoptosis, and promoted hepatocyte proliferation by targeting the PI3K/AKT/Gsk3-ß pathway in OA-induced fatty liver model in vitro, and 20 mg/kg SDS alleviated high-fat-diet-induced hepatic steatosis, oxidative stress, and inflammatory response in laying hens in vivo.

Dehydroepiandrosterone activates the GPER-mediated AMPK signaling pathway to alleviate the oxidative stress and inflammatory response in laying hens fed with high-energy and low-protein diets.

Fatty liver hemorrhagic syndrome (FLHS) seriously threatens the layer industry due to it can cause a sudden decline in egg production and acute death, and dietary supplement with bioactive substance is considered an effective way to prevent the FLHS occurrence. Dehydroepiandrosterone (DHEA) is a popular dietary supplement and it possesses anti-oxidative and anti-inflammatory functions; however, the effect and underlying mechanism about DHEA in protecting against the occurrence and development of FLHS remain elucidated. The current results showed that DHEA relieved HELP-induced decrease of egg productivity and liver injury in laying hens. Meanwhile, DHEA markedly enhanced the antioxidant capacity and then alleviated oxidative stress via activation of nuclear factor (erythroid-derived 2)-like 2 (NRF-2) signal in laying hens fed with HELP diets. In addition, DHEA significantly alleviated HELP-stimulated systemic inflammatory response by suppressing the overproduction of hepatic pro-inflammatory factors in laying hens, and further found this beneficial effect was achieved by blocking the activation of NF-κB pathway. Furthermore, we found that DHEA promoted the AMP-activated protein kinase α (AMPKα) activation and increased the G-protein-coupled estrogen receptor (GPER) expression level in laying hens fed with HELP diets. In summary, our data demonstrated that DHEA attenuates oxidative stress and inflammation through the activation of GPER-AMPK signal axis in laying hens fed with HELP diets. These results might facilitate an understanding of the benefits and mechanism of DHEA on the development of FLHS, and provide sufficient data to support it as a dietary supplement to control the FLHS-related metabolic diseases in chickens.

Osteocalcin reduces fat accumulation and inflammatory reaction by inhibiting ROS-JNK signal pathway in chicken embryonic hepatocytes.

Osteocalcin (OCN) has a function in preventing fatty liver hemorrhagic syndrome (FLHS) in poultry. The aim of this study was to investigate the effects of OCN on fat emulsion stimulated chicken embryonic hepatocytes and related signaling pathways. The primary chicken embryonic hepatocytes were isolated from the incubated 15-day (E15) pathogen free eggs and cultured with dulbecco's modified eagle medium (DMEM). After the hepatocyte density reached 80%, the cells were divided into 5 groups: control group (CONT), fat emulsion group (FE, 10% FE, v/v), FE with ucOCN at 1 ng/mL (FE-LOCN), 3 ng/mL (FE-MOCN), and 9 ng/mL (FE-HOCN). In addition, 2 mM N-Acetyl-L-cysteine (NAC) a reactive oxygen species (ROS) scavenger, and 5 µM SP600125, a Jun N-terminal kinase (JNK) inhibitor, were added separately in to the DMEM with 10% FE to test effects of FE on the function of ROS-JNK signal pathway. The number of hepatocytes, cell ultra-microstructure, viability, and apoptosis were detected after 48 h treatment, and the protein expressions and enzyme concentrations were detected after 72 h treatment. The results showed that, compared to the control group, FE increased the triglyceride (TG) concentration and lipid droplets (LDs) in chicken embryonic hepatocytes (P < 0.05), and induced hepatocytic edema with obviously mitochondrial swelling, membrane damage, and cristae rupture. FE also decreased ATP concentration, increased ROS concentrations and mitochondrial DNA (mtDNA) copy number, promoted inflammatory interleukin-1 (IL-1), IL-6, tumor necrosis factor-alpha (TNF-α) concentrations and hepatocytic apoptosis rate, and raised phospho-c-Jun N-terminal kinase (p-JNK) protein expressions. Compared to the FE group, ucOCN significantly increased hepatocyte viability, reduced hepatocytic TG concentrations and LDs numbers, and alleviated hepatocytic edema and mitochondrial swelling. Furthermore, ucOCN significantly decreased ROS concentrations, increased ATP concentrations, reduced IL-1, IL-6, TNF-α concentrations and hepatocytic apoptosis rate, and inhibited p-JNK protein expressions (P < 0.05). NAC had the similar functions of ucOCN reduced the ROS concentration and inhibited the TNF-α protein expression and p-JNK/JNK ration. Similarly, SP600125 reduced p-JNK/JNK protein expression, IL-1, IL-6, TNF-α, and TG concentrations without effects on ROS concentration and hepatocytic apoptosis. These results suggest that ucOCN alleviates FE-induced mitochondrial damage, cellular edema, and apoptosis of hepatocytes. These results reveal that the functions of ucOCN in reducing fat accumulation and inflammatory reaction in chicken embryonic hepatocytes are mostly via inhibiting the ROS-JNK signal pathway.

Activated AMP-activated protein kinase prevents hepatic steatosis, oxidative stress and inflammation in primary chicken hepatocytes.

Fatty liver hemorrhagic syndrome (FLHS) in laying hens, a nutritional metabolic disorder disease, can lead to the decline of laying rate, shortening of laying peak period and increase of mortality, which seriously constrain the sustainable development of layer industry. Until now, there is no effective strategies can prevent and control the occurrence of fatty liver hemorrhagic syndrome in laying hens. The AMP-activated protein kinase (AMPK), a major sensor of cellular energy status, acts a crucial role in regulating lipid metabolism, oxidative stress and inflammatory responses in body. However, the potential molecular mechanisms about AMP-activated protein kinase signal in controlling the occurrence of fatty liver hemorrhagic syndrome are remain unclear. In present study, we found that the phosphorylated AMP-activated protein kinase (Thr172) protein level was markedly reduced in palmitic acid plus oleic acid (PO)-induced primary chicken hepatocytes. Moreover, blocked AMP-activated protein kinase signal by AMP-activated protein kinase inhibitor compound C obviously exacerbated lipid metabolism disorders, oxidative stress and inflammatory response triggered by palmitic acid plus oleic acid in primary chicken hepatocytes. Nevertheless, the lipid metabolism disorders, oxidative stress and inflammatory response challenged by palmitic acid plus oleic acid were obviously alleviated through activation of AMP-activated protein kinase signal with AMP-activated protein kinase activator AICAR in hepatocytes. In addition, we found that the beneficial effects of AMP-activated protein kinase signal in relieving lipid metabolism disorders, oxidative stress and inflammatory response are achieved by activating the nuclear factor erythroid 2-related factor 2 (NRF-2)/kelch-like ECH-associated protein 1 (KEAP1) pathway and inhibiting the NF-κB pathway in PO-stimulated primary chicken hepatocytes. Collectively, our data demonstrated that AMP-activated protein kinase acts as a potential target for the prevention of fatty liver hemorrhagic syndrome occurrence in laying hens.

Osteocalcin prevents insulin resistance, hepatic inflammation, and activates autophagy associated with high-fat diet-induced fatty liver hemorrhagic syndrome in aged laying hens.

The aim of this study was to investigate the effects of osteocalcin (OCN) on fatty liver hemorrhagic syndrome (FLHS) in aged laying hens. Thirty 68-week-old White Plymouth laying hens were randomly assigned into conventional single-bird cages, and the cages were randomly allocated into one of 3 treatments (n = 10): normal diet (ND + vehicle, ND + V), high-fat diet (HFD + vehicle, HFD + V), and HFD + OCN (3 µg/bird, 1 time/2 d, i.m.) for 40 d. At day 30, oral glucose tolerance tests (OGTT) and insulin tolerance tests (ITT) were performed. At the end of experiment, the hens were euthanized followed by blood collection. The plasma aspartate transaminase (AST), alkaline phosphatase (ALP), total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were measured using an automatic biochemistry analyzer. Pathological changes in the liver were examined under both light and transmission electron microscopes. The plasma inflammatory factors including interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-α) were analyzed by ELISA, and the gene expressions of these inflammatory factors in the liver were analyzed by real-time PCR. The level of oxidative stress was evaluated using malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) assay kits, respectively. The results showed that HFD + V hens had more severe liver hemorrhage and fibrosis than ND + V hens (P < 0.05). The ultramicrostructural examination showed that hepatocytes of HFD + V hens exhibited necrotic pyknosis showing great intracellular electron, mitochondrial swelling, shrunk nucleus, and absence of autolysosomes. Osteocalcin mitigated HFD + V-induced pathological changes in aged laying hens. High-fat diet + OCN hens had higher insulin sensitivity; lower liver concentrations of MDA (P = 0.12) but higher GSH-Px (P < 0.05); and lower blood TNF-α concentrations (P < 0.05) and mRNA expressions (P < 0.05) than HFD + V hens. These results suggest OCN functions in preventing the FLHS process in old laying hens through inhibiting excessive energy diet-induced metabolic disorder, oxidative stress, and related pathological damage.

Untargeted and targeted metabolomics profiling reveals the underlying pathogenesis and abnormal arachidonic acid metabolism in laying hens with fatty liver hemorrhagic syndrome.

As a metabolic disease, fatty liver hemorrhagic syndrome (FLHS) has become the major factor responsible for the noninfectious cause of mortality in laying hens, which lead to huge economic losses to poultry industry. However, the pathogenesis of FLHS remains unclear. The aim of present study was to identify novel liver metabolites associated with FLHS. Twenty healthy Chinese commercial Jing Fen laying hens aged 90 d were used in present study. After acclimatization for 2 wk, the hens were divided into 2 treatments (n = 10): control group (normal diet) and FLHS group (high-energy low-protein diet). The experiment lasted for 48 d, and the laying hens were killed for blood and liver sampling at the end of the experiment. Blood biochemical indicators and liver pathological changes were examined. Meanwhile, the changes in liver metabolic profile were investigated with the application of metabolomics approach. Significant increased levels of alanine aminotransferase, aspartate aminotransferase, low density lipoprotein, total cholesterol and triglycerides, decreased high density lipoprotein (P < 0.01), and hepatic steatosis were observed in hens of FLHS group, which suggested FLHS was successfully established in this study. Distinct changes in metabolite patterns in liver between control and FLHS group were observed by partial least-squares discriminant analysis. In total, 42 liver metabolites including tyrosine, glutathione, carnitine, linoleic acid, uric acid, arachidonic acid (ARA), lactate and lysophosphatidylcholine (14: 0) were identified and considered to be related with pathogenesis of FLHS. Pathway analysis revealed that these metabolites were mainly involved in amino acid metabolism, fatty acid metabolism, ARA metabolism, glucose metabolism and glycerophospholipid metabolism. Furthermore, targeted metabolomics found that ARA metabolites such as prostaglandins and hydroxyeicosatetraenoic acids were significantly increased in FLHS group (P < 0.05). In conclusion, our data showed that liver metabolites and ARA metabolism were linked to the pathophysiology of FLHS, which provided a basis for understanding the pathogenesis of FLHS in laying hens.