Procyanidin B2 alleviates liver injury caused by cold stimulation through Sonic hedgehog signalling and autophagy.

Procyanidin B2 (PB2), a naturally occurring flavonoid abundant in a wide range of fruits, has been shown to exert antioxidant, anti-inflammatory and anticancer properties. However, the role of PB2 in the prevention of cold stimulation (CS)-induced liver injury. The present study was undertaken to determine the effects of PB2 on liver injury induced by cold stimulation and its potential molecular mechanisms. The present study results showed that treatment with PB2 significantly reduced CS-induced liver injury by alleviating histopathological changes and serum levels of alanine transaminase and aspartate transaminase. Moreover, treatment with PB2 inhibited secretion of inflammatory cytokines and oxidative stress in cold-stimulated mice. PB2 reduced cold stimulation-induced inflammation by inhibiting TLR4/NF-κB and Txnip/NLRP3 signalling. Treatment with PB2 reduced oxidative stress by activating Nrf-2/Keap1, AMPK/GSK3ß signalling pathways and autophagy. Furthermore, simultaneous application of Shh pathway inhibitor cyclopamine proved that PB2 targets the Hh pathway. More importantly, co-treatment with PB2 and cyclopamine showed better efficacy than monotherapy. In conclusion, our findings provide new evidence that PB2 has protective potential against CS-induced liver injury, which might be closely linked to the inhibition of Shh signalling pathway.

O-GlcNAc / Akt pathway regulates glucose metabolism and reduces apoptosis in liver of piglets with acute cold stress.

Cold stress is one of the serious factors restricting the development of animal husbandry in cold areas. Cold exposure can easily lead to cold stress, slow growth and even death of newborn animals. O-GlcNAcylation modification can act as type of "stress receptor" and"nutrition sensor" in a variety of stress responses, however, it is not clear how O-GlcNAcylation can regulate glucose metabolism in the liver of piglets under cold stress. In this study, piglets 21 days of age were exposed to 4 °C for 4 h or 8 h in a phytotron. Serum cortisol and other stress hormones were used to assess body status to establish a cold stress piglet model. The changes of glycogen in liver were detected by PAS. FDP and PA were also measured to study the glycolysis level of liver. To characterize potential mechanisms of O-GlcNAcylation on the livers of cold stress piglets, AKT, GSK3ß, GS, PFKFB2, AS160 and their corresponding phosphorylation were determined by Western blotting. Results show O-GlcNAcylation increased and apoptosis levels increased in the liver following cold exposure during excessive CORT or metabolic dysfunction. It is suggested that the acute cold exposure of piglets induced a sequential change in the level of O-GlcNAcylation, which may be one of the factors mediating liver cell apoptosis and glucose metabolism regulation by the O-GlcNAc/AKT pathway. These findings provide new insight into the mechanisms of the cold stress response, which can facilitate the development of new strategies to combat the effects of hypothermia.

Down-regulation of miR-383-5p suppresses apoptosis in oxidative stress rat hepatocytes by targeting Bcl2.

miRNAs are a class of small non-coding RNAs that are involved in various biological processes. In the preliminary work of the laboratory, found that miR-383-5p was down-regulated in the liver tissue of acute cold stress rats and has been shown to be an important regulatory factor in tumour proliferation, but there are very few studies involving the mediation of cold stress in rat liver tissues. Therefore, the purpose of this study was to determine the effect of miR-383-5p on the livers of cold stress rats by simulating the cold stress state of rat liver tissues in vitro using H2 O2 to induce rat hepatocyte oxidative stress. The results showed that MDA content, Caspase 3 and Cyto C protein levels increased significantly; GPx activity and SOD1 protein levels decreased significantly and miR-383-5p expression was significantly down-regulated in rat liver tissues after cold stress. Different concentrations of H2 O2 was added to rat hepatocytes, and the results showed that the expression of miR-383-5p, the ROS level, and the apoptosis rate in rat hepatocytes was increased significantly in a concentration-dependent fashion. Transfection of miR-383-5p inhibitor revealed that the apoptosis rate of rat hepatocytes, and the protein level of apoptosis-related protein Caspase 3 were reduced; the results of the dual-luciferase reporter gene assay showed that miR-383-5p targeted regulation of Bcl2. The results suggested that the expression of miR-383-5p was up-regulated in oxidative stress rat hepatocytes and may aggravate the apoptosis of rat hepatocytes induced by targeting inhibition of Bcl2 translation.

Metabonomics analysis of Zi goose follicular granulosa cells using ENO1 gene expression interference.

The Zi goose is native to North-east China and is noted for its high egg production. Alpha enolase (ENO1) is a glycolytic enzyme which functions as a plasminogen receptor in follicular granulosa cells (FGCs), with several studies showing that FGCs can support follicular development. By transfecting the ENO1 interfering plasmid (shRNA) into FGCs, ENO1 expression in these cells was downregulated, suggesting the successful knock-down of ENO1 in these cells. In this knock-down model, we detected 13 metabolites from FGCs using LC/MS. When compared with the non-coding shRNA (NC) group, the lower level metabolites were (R)-(+)-citronellic acid, altretamine, 3-hydroxycaproic acid, heptadecanoic acid, cholecalciferol vitamin D3, indole, benzoic acid, capric acid, caffeic acid, azelaic acid, 3,4-dihydroxyhydrocinnamic acid and cholic acid, while oleic acid was detected at high levels. To further examine the results of metabolomics, six key metabolites were verified by gas chromatography-mass spectrometry (GC-MS). We found that vitamin D3, indole, benzoic acid, capric acid and cholic acid were significantly downregulated in the shRNA group, while oleic acid was significantly upregulated. This observation was consistent with the metabolomics data. Through these studies, we found that decreased ENO1 levels altered certain metabolite levels in FGCs.

HMGB1-mediated differential response on hippocampal neurotransmitter disorder and neuroinflammation in adolescent male and female mice following cold exposure.

Stress induces many different sex-specific physiological and psychological responses during adolescence. Although the impact of certain brain stressors has been reported in the literature, the influence of cold stress on the mechanisms underlying hippocampal neurotransmitter disorder and neuroinflammation remain unstudied. Adolescent male and female C57BL/6 mice were exposed to 4 °C temperatures, 3 h per day for 1 week. Serum CORT and blood gas analysis was then used to assess body status. Using western blotting, immunofluorescence and immunohistochemistry we also assessed glial cell number and microglial activation, as well as inflammatory cytokine levels and related protein expression levels. The phenomena of excessive CORT, microglial activation, increased acetylate-HMGB1 levels, NF-κB signaling pathway activation, pro-inflammatory cytokine release, neuronal apoptosis and neurotransmitter disorder were demonstrated in mouse hippocampal tissue following cold exposure. We believe that these phenomena are mediated by the HMGB1/TLR4/NFκB pathway. Finally, the male inflammatory response in hippocampal tissue was more severe and the influence of cold exposure on neurotransmitter was greater in females.

Regulating glycolysis, the TLR4 signal pathway and expression of RBM3 in mouse liver in response to acute cold exposure.

At low temperatures, the liver increases glucose utilization and expresses RNA-binding motif 3 (RBM3) to cope with cold exposure. In this study, the expression of heat shock protein 70 (HSP70), Toll-like receptor 4 (TLR4), bone marrow differentiation factor 88 (MYD88), and phosphorylated nuclear factor-κB (NF-κB) was consistent with fluctuations in insulin in fasted cold-exposed mice. We also found up-regulation of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in acute cold exposure with a decrease in core body temperature. RBM3 transcription and translation were activated 2 h after cold exposure. The anti-apoptotic factor Bcl-2/Bax ratio also increased, while expression of apoptosis factors: cleaved caspase-3, cleaved poly(ADP-ribose)polymerase 1 (PARP-1) and cytochrome-c (Cyt-c) was unchanged. Liver glycogen was depleted after 2 h of cold exposure, and blood glucose decreased after 4 h. Glycogen synthase kinase 3ß (GSK3ß) phosphorylation continued to increase to promote hepatic glycogen synthesis. We found a high level of protein kinase B (AKT) phosphorylation after 6 h of cold exposure. In addition, we demonstrated that after cold exposure for 2 h, in the liver, continued phosphorylation of fructose-2,6-diphosphate (PFKFB2) and decreased accumulation of glycogen intermediates fructose-1,6-diphosphate (FDP) and pyruvic acid (PA). In summary, the liver responds to cold exposure through a number of different pathways, including activation of HSP70/TLR4 signaling pathways, up-regulation of RBM3 expression, and increased glycolysis and glycogen synthesis. We propose a possible signaling pathway in which regulation of RBM3 expression by the liver affects the AKT metabolic signaling pathway. Lay summary In response to changes in ambient temperature, mice regulate global metabolism and gene expression through hormones. This study focused on the effects of environmental hypothermia on molecular pathways of glucose metabolism in the liver, which is the important metabolic organ in mice. This provides a basis for further study of mice against cold exposure damage.

Effects of Cold-inducible RNA-binding Protein (CIRP) on Liver Glycolysis during Acute Cold Exposure in C57BL/6 Mice.

Cold-inducible RNA-binding protein (CIRP) is a stress-responsive protein involved in several signal transduction pathways required for cellular function, which are associated with apoptosis and proliferation. The present study aimed to investigate the possible effects of CIRP-mediated regulation of glucose metabolism in the liver following acute cold exposure. The livers and serum of male C57BL/6 mice were collected following cold exposure at 4 °C for 0 h, 2 h, 4 h, and 6 h. Glucose metabolic markers and the expression of glucose metabolic-related proteins were detected in the liver. Acute cold exposure was found to increase the consumption of glycogen in the liver. Fructose-1,6-diphosphate (FDP) and pyruvic acid (PA) were found to show a brief increase followed by a sharp decrease during cold exposure. Anti-apoptotic protein (Bcl-2) expression was upregulated. CIRP protein expression displayed a sequential increase with prolonged acute cold exposure time. Acute cold exposure also increased the level of protein kinase B (AKT) phosphorylation, and activated the AKT-signaling pathway. Taken together, these findings indicate that acute cold exposure increased the expression of CIRP protein, which regulates mouse hepatic glucose metabolism and maintains hepatocyte energy balance through the AKT signaling pathway, thereby slowing the liver cell apoptosis caused by cold exposure.

Rno-miR-425-5p targets the DLST and SLC16A1 genes to reduce liver damage caused by excessive energy mobilization under cold stress.

MicroRNAs (miRNAs) are a class of single-stranded non-coding small RNA molecules, which participate in the regulation of many physiological processes, and play a crucial role in cancer, metabolism and other processes. Rno-miR-425-5p has been shown to play a role in the response to cold stress. To explore the mechanism by which rno-miR-425-5p regulates the response to cold stress, we analysed the candidate target genes of rno-miR-425-5p. After verification in rat hepatocyte BRL cells and in rat liver tissue, we identified several target genes that were altered in expression in response to cold stress. In rat liver tissue, the expression of rno-miR-425-5p was significantly increased and the expression levels of target genes DLST and SLC16A1 were decreased under cold stress. The miRNA and mRNA levels were analysed by quantitative real-time PCR and the protein levels were detected by Western blot analysis. Combined with the results of bioinformatic analysis, we concluded that rno-miR-425-5p reduced the expression of DLST and SLC16A1, inhibiting energy release from the tricarboxylic acid cycle and preventing the liver from being injured by excessive energy mobilization.

The Favored Mechanism for Coping with Acute Cold Stress: Upregulation of miR-210 in Rats.

BACKGROUND/AIMS: The main aim of this study was to determine the mechanisms by which rno-miR-210-3p affects changes in gene expression, metabolism, apoptosis and proliferation of cells under acute cold stress (ACS) conditions. METHODS: The treatment group (n=6, weight 340±20 g) was exposed to ACS (temperature 4±0.5°C, relative humidity 45±0.5%) and the control group (n=6, weight 340±20 g) to normal temperature (NT) (temperature 24±0.5°C, relative humidity 45±0.5%). Rat liver samples were collected for qRT-PCR and western blot analyses to detect relative expression of rno-miR-210-3p, ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2. For cell experiments, 100 pmol/dish rno-miR-210-3p mimic and 150 pmol/dish rno-miR-210-3p inhibitor were used. Mitochondrial glucose flux and glycolysis were measured using the XFe24 Extracellular Flux Analyzer. Cells were collected for apoptosis analysis 24 h after transfection and proliferation was quantified using the WST-1 Cell Proliferation and Cytotoxicity Assay Kit (Beyotime, Shanghai, China), according to the manufacturer's instructions. RESULTS: In the rat experiment, expression of rno-miR-210-3p under ACS was increased sharply while ISCU, E2F3, RAD52, and PSMB6 levels declined, along with protein expression of ISCU and PSMB6. In cell experiments, ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2 genes were downregulated while ISCU and PSMB6 protein expression decreased with upregulation of rno-miR-210-3p. Conversely, in response to decreased rno-miR-210-3p expression, ISCU, E2F3, RAD52, PSMB6 and GPD2 genes were upregulated, in addition to ISCU and PSMB6 proteins. Upregulation of miR-210 inhibited cell proliferation and induced cell death whereas its downregulation promoted cell proliferation. Upregulation or downregulation of miR-210 promoted glycolysis and mitochondrial respiration of BRL cells. However, downregulation of miR-210 caused acid production in cells. CONCLUSION: Expression of rno-miR-210-3p is significantly increased under ACS. Upregulation of rno-miR-210-3p inhibits the expression of ISCU, Rap1b, ATP1b1, GPD1, E2F3, RAD52, PSMB6 and GPD2 genes, promotes glycolysis of liver and enhances the mitochondrial respiratory capacity of cells, but may also cause cell death. Our findings collectively indicate that regulation of rno-miR-210-3p is a preferential mechanism of choice used by the body to cope with ACS.

Effects of Acute Cold Stress on Liver O-GlcNAcylation and Glycometabolism in Mice.

Protein O-linked ß-N-acetylglucosamine glycosylation (O-GlcNAcylation) regulates many biological processes. Studies have shown that O-GlcNAc modification levels can increase during acute stress and suggested that this may contribute to the survival of the cell. This study investigated the possible effects of O-GlcNAcylation that regulate glucose metabolism, apoptosis, and autophagy in the liver after acute cold stress. Male C57BL/6 mice were exposed to cold conditions (4 °C) for 0, 2, 4, and 6 h, then their livers were extracted and the expression of proteins involved in glucose metabolism, apoptosis, and autophagy was determined. It was found that acute cold stress increased global O-GlcNAcylation and protein kinase B (AKT) phosphorylation levels. This was accompanied by significantly increased activation levels of the glucose metabolism regulators 160 kDa AKT substrate (AS160), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2 (PFKFB2), and glycogen synthase kinase-3ß (GSK3ß). The levels of glycolytic intermediates, fructose-1,6-diphosphate (FDP) and pyruvic acid (PA), were found to show a brief increase followed by a sharp decrease. Additionally, adenosine triphosphate (ATP), as the main cellular energy source, had a sharp increase. Furthermore, the B-cell lymphoma 2(Bcl-2)/Bcl-2-associated X (Bax) ratio was found to increase, whereas cysteine-aspartic acid protease 3 (caspase-3) and light chain 3-II (LC3-II) levels were reduced after acute cold stress. Therefore, acute cold stress was found to increase O-GlcNAc modification levels, which may have resulted in the decrease of the essential processes of apoptosis and autophagy, promoting cell survival, while altering glycose transport, glycogen synthesis, and glycolysis in the liver.

Identification of cold-responsive miRNAs in rats by deep sequencing.

miRNA is an endogenously noncoding sRNA, which is involved in post-transcription gene expression regulation of growth, tumor development and stress survival. As a biological marker, miRNA has been used for the early diagnosis of diseases and the evaluation of some physiological state. We constructed two small RNA libraries with the serums of rats treated or not with cold conditions (4℃ for 12h) by deep sequencing, in order to understand the miRNAs' expressions of cold-exposed rats and find new cold-responsive biological markers. 485 conserved miRNAs and 287 novel miRNAs were identified in the two libraries by comparing to the known miRNAs of rat in miRBase 21.0 Differential expression analysis showed that 56 conserved miRNAs and 3 novel miRNAs were expressed differentially in low ambient temperature. The qRT-PCR results confirmed that rno-miR-151-3p, rno-miR-210-3p, rno-miR-425-5p, rno-miR-383-5p, rno-miR-92a-3p, rno-miR-98-5p and rno-miR-328a-3p decreased significantly in rats serums treated with cold exposure. The expressions of the 7 miRNAs changed significantly in cold-exposed rats' livers too. rno-miR-383-5p decreased significantly, but all the others increased significantly. Thus, the 7 miRNAs were considered as cold-responsive miRNAs of rat. 670 target genes of the 7 cold-responsive miRNAs were predicted. KEGG analysis showed that they were enriched in 28 pathways and most of them were enriched by metabolic pathway. Overall, the results of this study suggest an important role for selected miRNA's in the response to cold stress.

[Biological function prediction of mir-210 in the liver of acute cold stress rat].

The study was aimed to observe mir-210 expression in liver tissue of acute cold stress rat and predict the function of mir-210 in cold stress. Thirty SPF Wistar male rats which were 12-week-old and weighed (340 ± 20) g were used. The rats were pre-fed in normal room temperature for one week, and then were randomly divided into acute cold stress group at (4 ± 0.1) °C and normal control group at (24 ± 0.1) °C. After the rats were treated with cold stress for 12 h, the liver tissue was extracted and the gene expression of mir-210 was assayed using qRT-PCR. The results demonstrated that the gene expression of mir-210 was significantly enhanced in acute cold stress group compared with that in normal control group (n = 3, P < 0.01). The bioinformatics analysis showed that mir-210 has over hundreds of target genes and four kinds of target genes such as E2F3, RAD52, ISCU and Ephrin-A3 are more relative with liver cold stress. ISCU regulates the cell respiratory metabolism and Ephrin-A3 is related with cell proliferation and apoptosis. On the other hand, up-regulated mir-210 affects the DNA repairing mechanism which usually leads to genetic instabilities. Our results suggest that cold stress-induced up-regulation of mir-210 in liver harmfully influences cell growth, energy metabolism and hereditary.

ß-Hydroxybutyric sodium salt inhibition of growth hormone and prolactin secretion via the cAMP/PKA/CREB and AMPK signaling pathways in dairy cow anterior pituitary cells.

ß-hydroxybutyric acid (BHBA) regulates the synthesis and secretion of growth hormone (GH) and prolactin (PRL), but its mechanism is unknown. In this study, we detected the effects of BHBA on the activities of G protein signaling pathways, AMPK-α activity, GH, and PRL gene transcription, and GH and PRL secretion in dairy cow anterior pituitary cells (DCAPCs). The results showed that BHBA decreased intracellular cAMP levels and a subsequent reduction in protein kinase A (PKA) activity. Inhibition of PKA activity reduced cAMP response element-binding protein (CREB) phosphorylation, thereby inhibiting GH and PRL transcription and secretion. The effects of BHBA were attenuated by a specific Gαi inhibitor, pertussis toxin (PTX). In addition, intracellular BHBA uptake mediated by monocarboxylate transporter 1 (MCT1) could trigger AMPK signaling and result in the decrease in GH and PRL mRNA translation in DCAPCs cultured under low-glucose and non-glucose condition when compared with the high-glucose group. This study identifies a biochemical mechanism for the regulatory action of BHBA on GH and PRL gene transcription, translation, and secretion in DCAPCs, which may be one of the factors that regulate pituitary function during the transition period in dairy cows.

[Cold inducible RNA-binding protein inhibits hippocampal neuronal apoptosis under hypothermia by regulating redox system].

In this study, we intend to confirm our hypothesis that cold inducible RNA-binding protein (CIRP) can inhibit neuronal apoptosis through suppressing the formation of oxygen free radicals under hypothermia. Primary rat hippocampal neurons were isolated and cultured in vitro, and were divided into five groups: (1) normal control group (37 °C), (2) cells infected by empty viral vector group, (3) CIRP over-expressed group, (4) CIRP knock-down group, and (5) hypothermia control group. Cells in groups 2-5 were cultured under 32 °C, 5% CO2. Apoptosis of hippocampal neurons were detected by Annexin V-FITC/PI staining and flow cytometry; Expression of CIRP was determined by Western blot; Redox-related parameters (T-AOC, GSH-Px, SOD, MDA) were detected by ELISA kits. Results showed that CIRP expression levels were significantly increased (P < 0.01) and the apoptotic rates were significantly decreased (P < 0.01) in hypothermia control group and CIRP over-expressed group when compared with normal control group. On the other hand, the apoptotic rate was significantly increased (P < 0.05) in CIRP knock-down group compared with that in hypothermia control group. The levels of redox parameters in hypothermia control group and CIRP over-expressed group were significantly changed in comparison with those in normal control group, CIRP knock-down group and empty viral vector infected group, respectively (P < 0.05 or P < 0.01). These results suggest that up-regulation of CIRP by hypothermia treatment can protect the neuron from apoptosis through suppressing the formation of oxygen free radicals.

Ubiquitinome Analysis Uncovers Alterations in Synaptic Proteins and Glucose Metabolism Enzymes in the Hippocampi of Adolescent Mice Following Cold Exposure.

Cold exposure exerts negative effects on hippocampal nerve development in adolescent mice, but the underlying mechanisms are not fully understood. Given that ubiquitination is essential for neurodevelopmental processes, we attempted to investigate the effects of cold exposure on the hippocampus from the perspective of ubiquitination. By conducting a ubiquitinome analysis, we found that cold exposure caused changes in the ubiquitination levels of a variety of synaptic-associated proteins. We validated changes in postsynaptic density-95 (PSD-95) ubiquitination levels by immunoprecipitation, revealing reductions in both the K48 and K63 polyubiquitination levels of PSD-95. Golgi staining further demonstrated that cold exposure decreased the dendritic-spine density in the CA1 and CA3 regions of the hippocampus. Additionally, bioinformatics analysis revealed that differentially ubiquitinated proteins were enriched in the glycolytic, hypoxia-inducible factor-1 (HIF-1), and 5'-monophosphate (AMP)-activated protein kinase (AMPK) pathways. Protein expression analysis confirmed that cold exposure activated the mammalian target of rapamycin (mTOR)/HIF-1α pathway. We also observed suppression of pyruvate kinase M2 (PKM2) protein levels and the pyruvate kinase (PK) activity induced by cold exposure. Regarding oxidative phosphorylation, a dramatic decrease in mitochondrial respiratory-complex I activity was observed, along with reduced gene expression of the key subunits NADH: ubiquinone oxidoreductase core subunit V1 (Ndufv1) and Ndufv2. In summary, cold exposure negatively affects hippocampal neurodevelopment and causes abnormalities in energy homeostasis within the hippocampus.

Statistical models for jointly analyzing multiple allometries.

As the reciprocal of simple allometry equation, power allometry equation can also be used to define allometry scaling but the scaling exponent has an opposite meaning to that of simple allometry equation. Based on this observation, a joint static allometry scaling model of entire body size on multiple partial body size is established, which can not only simultaneously evaluate allometry scaling of multiple partial body sizes, but also take into account the correlations among multiple partial body sizes, facilitating subsequent statistical inference and practice. Since ontogenetic allometry may be time-dependent, ontogenetic allometry is estimated by jointly analyzing changes of entire and multiple partial body sizes as growth time using multivariate stepwise analysis. Joint analysis of allometry scaling is suitable for multiple biological traits and functions with same property or comparability, which is illustrated by two examples.

Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (Anser anser domestica) Gene Expression.

UNLABELLED: Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don't know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. METHODOLOGY/PRINCIPAL FINDINGS: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), ß-actin (ACTB), ß-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. CONCLUSIONS: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese.

Integrated metabolism and epigenetic modifications in the macrophages of mice in responses to cold stress.

The negative effects of low temperature can readily induce a variety of diseases. We sought to understand the reasons why cold stress induces disease by studying the mechanisms of fine-tuning in macrophages following cold exposure. We found that cold stress triggers increased macrophage activation accompanied by metabolic reprogramming of aerobic glycolysis. The discovery, by genome-wide RNA sequencing, of defective mitochondria in mice macrophages following cold exposure indicated that mitochondrial defects may contribute to this process. In addition, changes in metabolism drive the differentiation of macrophages by affecting histone modifications. Finally, we showed that histone acetylation and lactylation are modulators of macrophage differentiation following cold exposure. Collectively, metabolism-related epigenetic modifications are essential for the differentiation of macrophages in cold-stressed mice, and the regulation of metabolism may be crucial for alleviating the harm induced by cold stress.

ActivinA activates Notch1-Shh signaling to regulate proliferation in C2C12 skeletal muscle cells.

The myoblast is a precursor cell that rebuilds muscle tissue after trauma in human and animal skeletal muscle tissue. Proliferation of myoblasts is important for skeletal muscle damage repair and is controlled by numerous transcription factors and signals. The regulation of these signaling pathways and their complex interactions are not fully understood. This study aims to determine the physiological functions of Activin A, Notch and Sonic Hedgehog (Shh) signaling in the proliferation of mouse C2C12 myoblasts and to explore their interactions. Activin A facilitated proliferation of C2C12 cells and promoted the conversion of G1 into S phase in cell cycle, whereas addition of the receptor inhibitor SB431542 attenuated the proliferation activity of rActA on C2C12 cells. Activin A also activated Notch and Shh signaling, while blockage of these pathways attenuated the function of Activin A in cell cycle. Inhibition of the Notch signaling by Notch response inhibitor DAPT significantly down-regulated the expression of Shh signaling molecules, whereas exogenous rShh reversed the inhibition of C2C12 cells proliferative activity induced by DAPT, indicating Notch signaling act upstream of the Shh pathway. Furthermore, inhibition of Notch signaling weakened the activation of Activin A-mediated Shh signaling. Taken together, our results provide a novel role of Activin A in regulating the proliferation of C2C12 skeletal muscle cells, which impacts ActivinA-Notch1-Shh signaling pathways.

Effects of prenatal cold stress on maternal serum metabolomics in rats.

Our previous studies have shown that prenatal cold stress leads to placental inflammatory response and induces anxiety-like behavior reduced in offspring rats. However, the role and mechanisms by which prenatal cold stress affects offspring remain unclear. The aim of this study was to determine the metabolic profiles from the maternal serum and helpful in understanding the role and mechanisms by which prenatal cold stress affects the offspring. In this study, liquid chromatography-mass spectrometry (LC/MS) was used to analyze serum metabolites, and PCA, PLS-DA, and OPLS-DA were performed to analyze changes in metabolites in the maternal serum after cold stress of 3 or 7 days. The results showed that 19 metabolites in the CS (cold stress 7 days)-NS (control) group and 23 metabolites in the CT (cold stress 3 days)-NT (control) group were significantly altered. These metabolites were mainly associated with unsaturated fatty acid synthesis, and arachidonic acid, linoleic acid, and glutamine and glutamate metabolism. The data indicated that prenatal cold stress not only affected the maternal neuroendocrine system, but also affected the immune system, and lipid and amino acid metabolism. These results further supported the findings of our previous studies on the effects of prenatal cold stress on the mother and offspring. A more comprehensive understanding of these data may lead to maternal intervention that can reverse the damage of prenatal stressors.