Deoxynivalenol induces m6A-mediated upregulation of p21 and growth arrest of mouse hippocampal neuron cells in vitro.

Hippocampal neurons maintain the ability of proliferation throughout life to support neurogenesis. Deoxynivalenol (DON) is a mycotoxin that exhibits brain toxicity, yet whether and how DON affects hippocampal neurogenesis remains unknown. Here, we use mouse hippocampal neuron cells (HT-22) as a model to illustrate the effects of DON on neuron proliferation and to explore underlying mechanisms. DON exposure significantly inhibits the proliferation of HT-22 cells, which is associated with an up-regulation of cell cycle inhibitor p21 at both mRNA and protein levels. Global and site-specific m6A methylation levels on the 3'UTR of p21 mRNA are significantly increased in response to DON treatment, whereas inhibition of m6A hypermethylation significantly alleviates DON-induced cell cycle arrest. Further mechanistic studies indicate that the m6A readers YTHDF1 and IGF2BP1 are responsible for m6A-mediated increase in p21 mRNA stability. Meanwhile, 3'UTR of E3 ubiquitin ligase TRIM21 mRNA is also m6A hypermethylated, and another m6A reader YTHDF2 binds to the m6A sites, leading to decreased TRIM21 mRNA stability. Consequently, TRIM21 suppression impairs ubiquitin-mediated p21 protein degradation. Taken together, m6A-mediated upregulation of p21, at both post-transcriptional and post-translational levels, contributes to DON-induced inhibition of hippocampal neuron proliferation. These results may provide new insights for epigenetic therapy of neurodegenerative diseases.

Parental betaine supplementation promotes gosling growth with epigenetic modulation of IGF gene family in the liver.

Betaine is widely used as a feed additive in the chicken industry to promote laying performance and growth performance, yet it is unknown whether betaine can be used in geese to improve the laying performance of goose breeders and the growth traits of offspring goslings. In this study, laying goose breeders at 39 wk of age were fed basal (Control, CON) or betaine-supplemented diets at low (2.5 g/kg, LBT) or high (5 g/kg, HBT) levels for 7 wk, and the breeder eggs laid in the last week were collected for incubation. Offspring goslings were examined at 35 and 63 d of age. The laying rate tended to be increased (P = 0.065), and the feed efficiency of the breeders was improved by betaine supplementation, while the average daily gain of the offspring goslings was significantly increased (P < 0.05). Concentrations of insulin-like growth factor 2 (IGF-2) in serum and liver were significantly increased in the HBT group (P < 0.05), with age-dependent alterations of serum T3 levels. Concurrently, hepatic mRNA expression of the IGF gene family was significantly increased in goslings derived from betaine-treated breeders (P < 0.05). A higher ratio of proliferating cell nuclear antigen (PCNA)-immunopositive nuclei was found in the liver sections of the HBT group, which was confirmed by significantly upregulated hepatic expression of PCNA mRNA and protein (P < 0.05). Moreover, hepatic expression of thyroxine deiodinase type 1 (Dio1) and thyroid hormone receptor ß (TRß) was also significantly upregulated in goslings of the HBT group (P < 0.05). These changes were associated with significantly higher levels of global DNA 5-mC methylation, together with increased expression of methyl transfer genes (P < 0.05), including betaine-homocysteine methyltransferase (BHMT), glycine N-methyltransferase (GNMT), and DNA (cytosine-5-)-methyltransferase 1 (DNMT1). The promoter regions of IGF-2 genes, as well as the predicted TRß binding site on the IGF-2 gene, were significantly hypomethylated (P < 0.05). These results indicate that gosling growth can be improved by dietary betaine supplementation in goose breeders via epigenetic modulation of the IGF gene family, especially IGF-2, in the liver.

The goose industry plays important roles in economics, cultures, and ecosystems, yet the low laying and growth rates of many indigenous breeds hinders the development of the goose farming. Betaine, an important methyl donor, is commonly used as a feed additive in livestock and poultry to enhance animal growth. Dietary supplementation of betaine in laying hens or gestational sows has been reported to promote the growth of their offspring. Here, we sought to investigate whether and how dietary betaine supplementation affects the growth and development of offspring goslings. In this study, goose breeders, both male and female, were fed a basal diet supplemented respectively with 0, 2.5, or 5 g/kg betaine for 7 wk. Goslings hatched from the breeder eggs of different groups were raised under the same standard condition for assessing the growth performance. Parental betaine increases the growth rate of offspring goslings with decreased DNA methylation on the IGF-2 gene promoter and increased expression of the IGF-2 gene in the liver. These results provide scientific evidence for the inter-generational effect of betaine on gosling growth.

Dietary bile acids improve breast muscle growth in chickens through FXR/IGF2 pathway.

It is a common practice to provide fast-growing broilers with high-fat diets in the context of integrated farms in Northeast China. Therefore, fat digestion, absorption, and utilization efficiency are critical for broiler meat production. Bile acids (BA) promote fat digestion and absorption, but whether and how BA affects muscle growth in broilers remains unclear. In this study, 1-day-old broilers were fed diets containing varying levels of crude fat (low, medium, and high) with or without BA supplementation for 42 d. Chickens fed a high-fat diet supplemented with BA exhibited significantly (P < 0.05) higher body weight (BW) at 21 d and average daily gain (ADG) during the first 21 d compared to the other groups. Throughout the entire experiment, feed conversion rate (FCR) was significantly (P < 0.05) lower in the high-fat group without the addition of BA, which was further decreased (P < 0.05) with BA supplementation. The improved growth performance in the BA-supplemented high-fat group was associated with significantly (P < 0.05) higher lipase activity in the small intestine chyme, a decreased trend (P = 0.06) in abdominal fat ratio, and significantly (P < 0.05) higher breast muscle mass. Histological analysis revealed significant (P < 0.05) increases in myofiber diameter, cross-sectional area, and RNA and DNA concentrations in the breast muscle of BA-supplemented broilers on the high-fat diet. Additional histological analysis further revealed significant (P < 0.05) enhancements in myofiber diameter, cross-sectional area, and RNA and DNA concentrations within the breast muscles of broilers supplemented with BA and a high-fat diet. The increased insulin-like growth factor 2 (IGF2) in the breast muscle of broilers fed a BA-supplemented high-fat diet correlated with significantly (P < 0.05) increased farnesoid X factor (FXR) protein expression and binding to the IGF2 promoter. These results suggest that dietary BA supplementation improves FCR and breast muscle growth in broilers fed a high-fat diet, potentially through the FXR-mediated IGF2 pathway.

Bile Acids Promote Hepatic Biotransformation and Excretion of Aflatoxin B1 in Broiler Chickens.

Aflatoxin B1 (AFB1) is a hazardous mycotoxin that often contaminates animal feed and may potentially induce severe liver damage if ingested. The liver is the primary organ responsible for AFB1 detoxification through enzyme-catalyzed xenobiotic metabolism and bile acid (BA)-associated excretion. In this study, we sought to investigate whether exogenous BA improves hepatic AFB1 detoxification to alleviate AFB1-induced liver injury in broiler chickens. Five-day-old broiler chicks were randomly assigned to three groups. CON and AFB1 received a basal diet; AFB1 + BA received a basal diet with 250 mg/kg BA for 20 days. After a 3-day pre-feed, AFB1 and AFB1 + BA were daily gavaged with 250 µg/kg BW AFB1, while CON received gavage solvent for AFB1 treatment. Dietary BA supplementation protected chickens from AFB1-induced hepatic inflammation and oxidative stress. The hepatic biotransformation of AFB1 to its metabolite AFBO was improved, with accelerated excretion to the gallbladder and cecum. Accordantly, AFB1-induced down-regulation of detoxification genes, including cytochrome P450 enzymes, glutathione S-transferases, and the bile salt export pump, was rescued by BA supplementation. Moreover, liver X receptor α, suppressed by AFB1, was enhanced in BA-treated broiler chickens. These results indicate that dietary BA supplementation improves hepatic AFB1 detoxification and excretion through LXRα-involved regulation of xenobiotic enzymes.

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.

Chronic corticosterone exposure disrupts hepatic and intestinal bile acid metabolism in chicken.

Objective: Chronic stress leads to a high circulating level of glucocorticoids, which disrupts lipid metabolism and causes non-alcoholic fatty liver disease in mice and humans. Meanwhile, bile acid (BA), a class of metabolites initially synthesized in the liver and further metabolized by gut microbiota, plays a vital role in lipid metabolism. This study aimed to investigate the effects of glucocorticoids on BA metabolism and gut microbiota in chickens. Methods: In this study, 35-day-old chickens were injected with 4 mg/kg/day corticosterone (Cort) for 14 days to simulate chronic stress. Results: Cort treatment significantly increased the triglyceride contents in the plasma and the liver. HE and oil-red staining showed that Cort treatment induced fatty liver in chickens. Meanwhile, Cort exposure downregulated total bile acid (TBA) content in the liver while increasing the TBA in feces. UPLC-HRMS results showed that Cort exposure significantly decreased the hepatic levels of CDCA, T-alpha-MCA, and T-beta-MCA. Moreover, Cort exposure significantly reduced the expression of genes related to BA synthesis (CYP8B1 and CYP27A1), conjugation (BACS), and regulation (KLß and FGFR4). 16s sequencing results showed that Cort treatment significantly decreased the amount of Lachnospiraceae, Eisenbergiella, Blautia, and Eubacterium and increased the abundance of Barnesiella, Lactobacillus, and Helicobacter. Spearman correlation analysis showed a significant positive correlation between fecal TBA and the abundance of Lactobacillales, Lactobacillus, and Barnesiella. In comparison, TBA in the liver was positively correlated with Eubacterium, and negatively correlated with Helicobacter. Conclusion: In summary, chronic Cort exposure disrupts hepatic and intestinal bile acid metabolism inducing gut microbiome dysbiosis, which might associate with the development of fatty liver in chickens.

Identification of POLQ as a key gene in cervical cancer progression using integrated bioinformatics analysis and experimental validation.

As the most common gynecologic malignancy worldwide, cervical cancer (CC) is a serious hazard to health. Therefore, the present study aimed to identify the key genes in CC progression using integrated bioinformatics analysis and experimental validation. The mRNA microarray GSE63514 and microRNA (miRNA) microarray GSE86100 were obtained from the Gene Expression Omnibus database, and the differentially expressed genes (DEGs) and differentially expressed miRNAs (DEMs) in the progression of CC were identified. Thereafter, GO and KEGG functional enrichment analysis, protein­protein interaction (PPI) network and significant subnetworks construction, and miRNA­target regulatory network construction were performed. Based on the results of integrated bioinformatics analysis, the DEGs structural maintenance of chromosomes 4 (SMC4), ATPase family, AAA domain­containing 2 (ATAD2) and DNA polymerase θ (POLQ) were identified as hub genes in the PPI network and were involved in the first significant subnetwork. In addition, these DEGs were predicted to be regulated by miR­106B, miR­17­5P, miR­20A and miR­20B, which were identified as DEMs. Of note, SMC4 and ATAD2 are tumor­promotors in CC. In the present study, small interfering (si)RNAs were used to knock down POLQ expression. Cell Counting Kit­8, Transwell, cell cycle and apoptosis analyses revealed that the downregulation of POLQ restrained cell proliferation, migration and invasion, and promoted apoptosis and the arrest of the cell cycle in the G2 phase. In conclusion, POLQ, which may have a close interaction with SMC4 and ATAD2, may serve a vital role in the progression of CC.

Embryonic thermal manipulation leads growth inhibition and reduced hepatic insulin-like growth factor1 expression due to promoter DNA hypermethylation in broilers.

When broilers cannot adapt to a high-temperature environment through self-regulation, it will cause heat stress, resulting in a large number of deaths and substantial economic losses. Studies have shown that thermal manipulation (TM) during the embryonic stage can improve broilers' ability to resist heat stress later. However, different TM strategies produce different results on broilers' growth. In this study, yellow-feathered broiler eggs were selected and randomly divided into 2 groups between E10 and E18, which the control group was incubated at 37.8°C with 56% humidity, and the TM group was subjected to 39°C with 65% humidity. After hatching, all broilers were reared normally until slaughtered at 12 d of age (D12). During D1 to D12, body weight, feed intake, and body temperature were recorded. The results showed that TM significantly decreased (P < 0.05) the final body weight, weight gain, and average daily feed intake of broilers. Meanwhile, the serum levels of Triiodothyronine (T3) and free T3 were significantly decreased in the TM group (P < 0.05). The expressions of hepatic growth regulation-associated genes, growth hormone receptor (GHR), insulin-like growth factor1, and 2 (IGF1 and IGF2) were significantly down-regulated in the TM group (P < 0.05). In addition, TM altered hepatic DNA methylation, resulting in a significant increase (P < 0.05) in the methylation of the IGF1 and GHR promoter regions. The above results indicated that TM during the embryonic stage decreased the serum thyroid hormone level and increased the methylation level of the IGF1 and GHR promoter regions to down-regulate the expression of growth-related genes, resulting in early growth inhibition of broilers.

DNA 5mC and RNA m6A modification successively facilitates the initiation and perpetuation stages of HSC activation in liver fibrosis progression.

Hepatic stellate cells (HSC) are key effector cells in liver fibrosis. Upon stimulation, the quiescent HSC undergoes complex morphological and functional changes to transdifferentiate into activated collagen-producing myofibroblasts. DNA/RNA methylations (5mC/m6A) are both implicated to participate in hepatic fibrosis, yet their respective roles and specific targets in HSC activation remain elusive. Here, we demonstrate that 5mC is indispensable for the initiation stage of HSC activation (myofibroblast transdifferentiation), whereas m6A is essential for the perpetuation stage of HSC activation (excessive ECM production). Mechanistically, DNA 5mC hypermethylation on the promoter of SOCS3 and PPARγ genes leads to STAT3-mediated metabolic reprogramming and lipid loss in the initiation stage. RNA m6A hypermethylation on the transcripts of major collagen genes enhances the mRNA stability in a YTHDF1-dependent manner, which contributes to massive ECM production. Vitamin A-coupled YTHDF1 siRNA alleviates CCl4-induced liver fibrosis in mice through HSC-specific inhibition of collagen production. HIF-1α, which is transactivated by STAT3, serves as a bridge linking the initiation and the perpetuation stages through transactivating YTHDF1. These findings indicate successive roles of DNA 5mC and RNA m6A modification in the progression of HSC activation, which provides new drug targets for epigenetic therapy of liver fibrosis.

Chronic Corticosterone Exposure Suppresses Copper Transport through GR-Mediated Intestinal CTR1 Pathway in Mice.

Numerous studies have discovered that chronic stress induces metabolic disorders by affecting iron and zinc metabolism, but the relationship between chronic stress and copper metabolism remains unclear. Here, we explore the influence of chronic corticosterone (CORT) exposure on copper metabolism and its regulatory mechanism in mice. Mice were treated with 100 µg/mL CORT in drinking water for a 4-week trial. We found that CORT treatment resulted in a significant decrease in plasma copper level, plasma ceruloplasmin activity, plasma and liver Cu/Zn-SOD activity, hepatic copper content, and liver metallothionein content in mice. CORT treatment led to the reduction in duodenal expression of copper transporter 1 (CTR1), duodenal cytochrome b (DCYTB), and ATPase copper-transporting alpha (ATP7A) at the mRNA and protein level in mice. CORT treatment activated nuclear glucocorticoid receptor (GR) and down-regulated CRT1 expression in Caco-2 cells, whereas these phenotypes were reversible by an antagonist of GR, RU486. Chromatin immunoprecipitation analysis revealed that GR bound to the Ctr1 promoter in Caco-2 cells. Transient transfection assays in Caco-2 cells demonstrated that the Ctr1 promoter was responsive to the CORT-activated glucocorticoid receptor, whereas mutation/deletion of the glucocorticoid receptor element (GRE) markedly impaired activation of the Ctr1 promoter. In addition, CORT-induced downregulation of Ctr1 promoter activity was markedly attenuated in Caco-2 cells when RU486 was added. These findings present a novel molecular target for CORT that down-regulates intestinal CTR1 expression via GR-mediated trans-repression in mice.

Chronic Psychological Stress Disrupts Iron Metabolism and Enhances Hepatic Mitochondrial Function in Mice.

To explore the changes in iron metabolism and mitochondrial function exposed to chronic psychological stress, seventy-five male mice aged 5 ~ 6 weeks were randomly sorted into 2 groups: control group and chronic psychological stress group. Mice were conducted by communication box to induce psychological stress for 21 consecutive days. The results showed that chronic psychological stress led to a significant reduction in average daily gain (P < 0.01) and the final weight (P < 0.05). Chronic psychological stress greatly increased plasma and duodenal iron level (P < 0.05), whereas markedly decreased hepatic iron content in mice (P < 0.05). Increasing expression of duodenal DCYTB and FPN (P < 0.05) was observed in mice exposed to chronic psychological stress. Moreover, chronic psychological stress greatly enhanced hepatic TFR1, FTL, and FPN protein expression (P < 0.05) in mice. Additionally, chronic psychological stress enhanced the levels of hepatic NADH, NAD + , ATP, mtDNA content, mtDNA-encoded genes, and the activity of mitochondrial complex I and II (P < 0.05). Taken together, chronic psychological stress impairs growth, disrupts iron metabolism, and enhances hepatic mitochondrial function in mice. These results will provide new insights for understanding the mechanisms of iron metabolism and mitochondrial function during chronic psychological stress.

m6A-Mediated PPARA Translational Suppression Contributes to Corticosterone-Induced Visceral Fat Deposition in Chickens.

Excess fat deposition in broilers leads to great economic losses and is harmful to consumers' health. Chronic stress in the life cycle of chickens could be an important trigger. However, the underlying mechanisms are still unclear. In this study, 30-day-old chickens were subcutaneously injected with 2 mg/kg corticosterone (CORT) twice a day for 14 days to simulate long-term stress. It was shown that chronic CORT exposure significantly increased plasma triglyceride concentrations and enlarged the adipocyte sizes in chickens. Meanwhile, chronic CORT administration significantly enlarged the adipocyte sizes, increased the protein contents of FASN and decreased HSL, ATGL, Beclin1 and PPARA protein levels. Moreover, global m6A methylations were significantly reduced and accompanied by downregulated METTL3 and YTHDF2 protein expression by CORT treatment. Interestingly, the significant differences of site-specific m6A demethylation were observed in exon7 of PPARA mRNA. Additionally, a mutation of the m6A site in the PPARA gene fused GFP and revealed that demethylated RRACH in PPARA CDS impaired protein translation in vitro. In conclusion, these results indicated that m6A-mediated PPARA translational suppression contributes to CORT-induced visceral fat deposition in chickens, which may provide a new target for the treatment of Cushing's syndrome.

The roles and clinical applications of interleukins in endometrial carcinoma.

As a common malignant tumor of the female reproductive system, endometrial carcinoma (EC) seriously endangers women's health with an increasing incidence. The oncogenesis and progression of cancer are closely linked with immune microenvironment, of which interleukins are the important components. In order to illustrate the roles and clinical applications of interleukins in EC, literature of interleukins and EC were reviewed. Based on the present studies, interleukins play crucial roles in the oncogenesis and development of EC via regulating the proliferation, migration, invasion, angiogenesis, apoptosis, pyroptosis and autophagy of EC as well as the immune function against EC. And some of the interleukins seems to have prospective clinical applications in EC, such as evaluating the risk of tumorigenesis, discriminating the malignancy from benign disorders or normal condition, indicating cancer aggressiveness, predicting the prognosis of patients and serving as the novel therapy. However, there is still a long way to go before the clinical applications of interleukins in EC come into reality. Nevertheless, it is certain that the exploration of interleukins will definitely be of great benefit to the screening, diagnosis and treatment of EC in the future.

Distinct Patterns of GR Transcriptional Regulation in Liver and Muscle of LPS-Challenged Weaning Piglets.

Glucocorticoid receptor (GR), which is ubiquitously expressed in nearly all cell types of various organs, mediates the tissue-specific metabolic and immune responses to maintain homeostasis and ensure survival under stressful conditions or pathological challenges. The neonatal period is metabolically demanding, and piglets are subjected to multiple stressors in modern intensive farms, especially around weaning. The liver is more responsive to LPS challenge compared to muscle, which is indicated by significantly increased TLR4 and p-p65, TNF-α, and IL-6 levels in association with GR down-regulation at both mRNA and protein levels. GR binding to the putative nGRE on TNF-α and IL-6 gene promoters decreased in the liver, but not muscle, upon LPS stimulation. The transcriptional regulation of GR also showed striking differences between liver and muscle. GR exon 1 mRNA variants 1-4, 1-5, and 1-6 were down-regulated in both liver and muscle, but a significant up-regulation of GR exon 1-9/10 mRNA variants abolished the change of total GR mRNA in the muscle in response to LPS stimulation. The significant down-regulation of GR in the liver corresponded with significantly decreased binding of p-GR and diminished histone acetylation in GR gene promoters. These results indicate that tissue-specific GR transcriptional regulation is involved in the differential inflammation responses between liver and muscle.

The Maternal-Neonatal Outcomes of Twin Pregnancies with Preeclampsia and Their Association with Assisted Reproductive Technology: A Retrospective Study.

Objective: This study aimed to investigate the maternal−neonatal outcomes of twin pregnancies of mothers with preeclampsia and their association with assisted reproductive technology (ART). Methods: A retrospective study on the clinical and maternal−neonatal outcome data of 698 women with twin pregnancies who delivered in our hospital from December 2013 to September 2021 was conducted. Continuous variables were analyzed using a Student's t-test or Wilcoxon rank-sum test. Categorical variables were analyzed using the Chi-square test. The risk factors of twin pregnancies with preeclampsia were analyzed by logistic regression. Results: The rate of twin pregnancy complicated by preeclampsia was 17.62% (123/698). Logistic regression analysis showed that ART increased the risk of preeclampsia in twin pregnancies (AOR: 1.868, 95% CI: 1.187−2.941). Mothers with preeclampsia carrying twins conceived with ART had a higher rate of delivery at gestational week < 34 (29.9% vs. 12.5%) and asphyxia of the neonate at 5 min after delivery (13.4% vs. 1.8%) than those with preeclampsia conceived without ART (p < 0.05). Conclusions: ART increases the risk of preeclampsia in twin pregnancies and the rate of adverse maternal−neonatal outcomes for twin pregnancies with preeclampsia. The policy of single embryo transfer is a method to reduce the adverse pregnancy outcomes of ART.

Constant light in early life induces fear-related behavior in chickens with suppressed melatonin secretion and disrupted hippocampal expression of clock- and BDNF-associated genes.

BACKGROUND: Light management plays an important role in the growth and behavior of broiler chickens. Constant light in early post hatch stage has been a common practice in broiler industry for improving growth performance, while whether and how constant light in early life affects the behavior of broiler chickens is rarely reported. RESULTS: In this study, newly hatched chicks were kept in either constant (24 L:0 D, LL) or (12 L:12 D, LD) photoperiod for 7 d and then maintained in 12 L:12 D thereafter until 21 days of age. Constant light increased the average daily feed intake but not the body weight, which led to higher feed conversion ratio. Chickens in LL group exhibited fear-related behaviors, which was associated with higher corticosterone, lower melatonin and 5-HT levels. Concurrently, constant light exposure increased the mRNA expression of clock-related genes and suppressed the expression of antioxidative genes in the hippocampus. Moreover, brain derived neurotrophic factor/extracellular signal-regulated kinase (BDNF/ERK) pathway was suppressed in the hippocampus of chickens exposed to constant light in the first week post hatching. CONCLUSIONS: These findings indicate that constant light exposure in early life suppress melatonin secretion and disrupts hippocampal expression of genes involved in circadian clock and BDNF/ERK pathway, thereby contributing to fear-related behaviors in the chicken.

Betaine Alleviates High-Fat Diet-Induced Disruptionof Hepatic Lipid and Iron Homeostasis in Mice.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive fat deposition in the liver, which is often associated with disrupted iron homeostasis. Betaine has been reported to be hepatoprotective, yet whether and how betaine ameliorates high-fat diet-induced disruption of hepatic lipid and iron homeostasis remains elusive. In this study, mice were fed either standard (CON) or high-fat diet (HFD) for 9 weeks to establish a NAFLD model. Mice raised on HF diet were then assigned randomly to HF and HFB groups, HFB group being supplemented with 1% (w/v) of betaine in the drinking water for 13 weeks. Betaine supplementation significantly alleviated excessive hepatic lipid deposition and restored hepatic iron content. Betaine partly yet significantly reversed HFD-induced dysregulation of lipogenic genes such as PRARγ and CD36, as well as the iron-metabolic genes including FPN and HAMP that encodes hepcidin. Similar mitigation effects of betaine were observed for BMP2 and BMP6, the up-stream regulators of hepcidin expression. Betaine significantly rectified disrupted expression of methyl transfer gene, including BHMT, GNMT and DNMT1. Moreover, HFD-modified CpG methylation on the promoter of PRARγ and HAMP genes was significantly reversed by betaine supplementation. These results indicate that betaine alleviates HFD-induced disruption of hepatic lipid and iron metabolism, which is associated with modification of CpG methylation on promoter of lipogenic and iron-metabolic genes.

Constant light exposure in early life induces m6A-mediated inhibition of IGF gene family in the chicken.

Insulin-like growth factor (IGF) family plays important roles in regulating the development of various organ systems through stimulating cell proliferation and differentiation. Photoperiod is an important factor affecting growth and development in the chicken, yet the effect of constant light exposure in early life on IGF1 and IGF2 expression in the chicken remains unclear. In this study, one-day-old chickens were kept in either constant light (24L:0D, LL) or natural photoperiod (12L:12D, LD) for the first week of life and then maintained in constant light from 8 to 21 d of age. Constant light exposure in early life reduced mRNA expression of IGF gene family, including mRNA expression of IGF1, IGF2, and IGF2 binding proteins, in the hippocampus, hypothalamus, and liver of chickens at both 7 and 21 d of age. Moreover, constant light exposure increased mRNA expression of genes involved in RNA methylation N6-methyladenosine (m6A) in a tissue-specific manner. Interestingly, higher m6A on 3'UTR of IGF2 mRNA coincides with lower IGF2 mRNA, indicating a possible role of m6A in the post-transcriptional regulation of IGF2 expression in the hippocampus, hypothalamus, and liver of chickens. These findings suggest a m6A-mediated gene regulation of IGF gene family in different organs of chicken and expand our knowledge on mechanism of gene regulation in response to early life experience.

Light pollution has become a potential risk factor for the health of animals and humans. Aberrant light exposure (such as light at night and super-intensity light) induces sleep disturbances and mood disorders, as well as major depressive disorder. In poultry, photoperiod is an important factor affecting the growth and behavior of broiler chickens. The hippocampus is critical for the regulation of spatial memory and depression-like behaviors in birds and mammals. Insulin-like growth factor (IGF) family plays important roles in regulating the development of various organ systems through stimulating cell proliferation and differentiation in a tissue-specific manner. At present, broiler chickens are commonly reared under constant light (24 h light) in the first week after hatching, yet the effect of constant light exposure in early life on the expression of IGF family in the chicken remains unclear. In this study, 1-d-old Yellow-footed broiler chickens were kept in either constant light (24L:0D, LL) or natural photoperiod (12L:12D, LD) for the first week of life and then maintained in natural photoperiod from 8 to 21 d of age. We analyzed the mRNA expression and the post-transcriptional regulation of IGF2 expression in the hippocampus, hypothalamus, and liver of chickens. Constant light exposure in early life reduced mRNA expression of IGF gene family, including mRNA expression of IGF1, IGF2, and IGF2 binding proteins (IGF2BPs), in the hippocampus, hypothalamus, and liver of chickens at both 7 and 21 d of age. Our findings demonstrate the expression of IGF gene family in different organs of chickens and expand our knowledge on the mechanism of gene regulation in response to early-life experience.

In ovo injection of betaine promotes adrenal steroidogenesis in pre-hatched chicken fetuses.

Corticosterone is critical for the maturation and survival of chicken fetus around hatching. Betaine is used as a feed additive in poultry industry to promote growth and mitigate stress. However, it remains unknown whether betaine could affect adrenal corticosterone synthesis in pre-hatching chicken fetuses. In this study, betaine (2.5 mg/egg) was injected into developing chicken fetuses at d 11 of incubation (E11) and its impact on adrenal steroidogenesis was investigated at day 19 (E19). Plasma corticosterone concentration was significantly (P < 0.05) elevated in betaine-treated fetuses, together with increased adrenal expression of melanocortin 2 receptor and steroidogenic acute regulatory protein. Accordingly, the corticosterone biosynthetic enzymes, such as cytochrome P450 family 11 subfamily A member 1, 3ß-hydroxysteroid dehydrogenase and cytochrome P450 family 21 subfamily A member 2, as well as cholesterol biosynthesis or regulation-related genes, such as sterol regulatory element-binding protein 1, 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase and low-density lipoprotein receptor, were all significantly (P < 0.05) upregulated in betaine group. Meanwhile, steroidogenic factor-1 and glucocorticoid receptor were significantly (P < 0.05) enhanced, whereas expression of dosage-sensitive sex reversal-adrenal hypoplasia congenita critical region on the X chromosome gene, a nuclear receptor known as a repressor of adrenal steroidogenesis, was significantly (P < 0.05) downregulated. Betaine significantly (P < 0.05) increased adrenal expression of genes involved in one-carbon metabolism and DNA methylation, such as S-adenosyl homocysteine hydrolase, betaine-homocysteine-methyltransferase, methionine adenosyl transferase and DNA methyltransferases, yet the promoter regions of most steroidogenic genes were significantly (P < 0.05) hypomethylated. These results indicate that in ovo injection of betaine promotes adrenal glucocorticoid synthesis in chicken fetuses before hatching, which involves alterations in DNA methylation.

Chronic corticosterone disrupts the circadian rhythm of CRH expression and m6A RNA methylation in the chicken hypothalamus.

BACKGROUND: Corticotropin-releasing hormone (CRH), the major secretagogue of the hypothalamic-pituitary-adrenal (HPA) axis, is intricately intertwined with the clock genes to regulate the circadian rhythm of various body functions. N6-methyladenosine (m6A) RNA methylation is involved in the regulation of circadian rhythm, yet it remains unknown whether CRH expression and m6A modification oscillate with the clock genes in chicken hypothalamus and how the circadian rhythms change under chronic stress. RESULTS: Chronic exposure to corticosterone (CORT) eliminated the diurnal patterns of plasma CORT and melatonin levels in the chicken. The circadian rhythms of clock genes in hippocampus, hypothalamus and pituitary are all disturbed to different extent in CORT-treated chickens. The most striking changes occur in hypothalamus in which the diurnal fluctuation of CRH mRNA is flattened, together with mRNA of other feeding-related neuropeptides. Interestingly, hypothalamic m6A level oscillates in an opposite pattern to CRH mRNA, with lowest m6A level after midnight (ZT18) corresponding to the peak of CRH mRNA before dawn (ZT22). CORT diminished the circadian rhythm of m6A methylation with significantly increased level at night. Further site-specific m6A analysis on 3'UTR of CRH mRNA indicates that higher m6A on 3'UTR of CRH mRNA coincides with lower CRH mRNA at night (ZT18 and ZT22). CONCLUSIONS: Our results indicate that chronic stress disrupts the circadian rhythms of CRH expression in hypothalamus, leading to dysfunction of HPA axis in the chicken. RNA m6A modification is involved in the regulation of circadian rhythms in chicken hypothalamus under both basal and chronic stress conditions.