Density Functional Theory Study on the Interaction between Aflatoxin B1/M1 and Gold Substrate.

Aflatoxin M1 (AFM1) and its precursor, Aflatoxin B1 (AFB1), are highly pathogenic and mutagenic substances, making the detection and sensing of AFB1/M1 a long-standing focus of researchers. Among various detection techniques, surface-enhanced Raman spectroscopy (SERS) is considered an ideal method for AFB1/M1 detection due to its ability not only to enhance characteristic frequencies but also to detect shifts in these frequencies with high repeatability. Therefore, we employed density functional theory in conjunction with surface-enhanced Raman spectroscopy to investigate the interaction between AFB1/M1 and a Au substrate in the context of the SERS effect for the first time. To predict the potential binding sites of AFB1/M1 and Au within the SERS effect, we performed calculations on the molecular electrostatic potential of AFB1/M1. Considering the crucial role of the binding energy in molecular docking studies, we computed the binding energy between two molecules interacting with Au at different binding sites. The molecular frontier orbitals and related chemical parameters of AFB1/M1 and "molecular-Au" complexes were computed to elucidate the alterations in AFB1/M1 molecules under the SERS effect. Subsequently, the theoretical Raman spectra of AFB1/M1 and the complexes were compared and analyzed, enabling determination of the adsorption conformation of AFB1/M1 on the gold surface based on SERS surface selection rules. These findings not only provide a deeper understanding of the interaction mechanism between molecules and substrates in the SERS effect but also offer theoretical support for developing novel aflatoxin SERS sensors.

Discovering a New Okadaic Acid Derivative, a Potent HIV Latency Reversing Agent from Prorocentrum lima PL11: Isolation, Structural Modification, and Mechanistic Study.

Marine toxins (MTs) are a group of structurally complex natural products with unique toxicological and pharmacological activities. In the present study, two common shellfish toxins, okadaic acid (OA) (1) and OA methyl ester (2), were isolated from the cultured microalgae strain Prorocentrum lima PL11. OA can significantly activate the latent HIV but has severe toxicity. To obtain more tolerable and potent latency reversing agents (LRAs), we conducted the structural modification of OA by esterification, yielding one known compound (3) and four new derivatives (4-7). Flow cytometry-based HIV latency reversal activity screening showed that compound 7 possessed a stronger activity (EC50 = 46 ± 13.5 nM) but was less cytotoxic than OA. The preliminary structure-activity relationships (SARs) indicated that the carboxyl group in OA was essential for activity, while the esterification of carboxyl or free hydroxyls were beneficial for reducing cytotoxicity. A mechanistic study revealed that compound 7 promotes the dissociation of P-TEFb from the 7SK snRNP complex to reactivate latent HIV-1. Our study provides significant clues for OA-based HIV LRA discovery.

The outer membrane protein of Fusobacterium necrophorum, 43K OMP, stimulates inflammatory cytokine production through nuclear factor kappa B activation.

OBJECTIVE: Fusobacterium necrophorum causes bovine hepatic abscess, foot rot, mastitis, and endometritis. The 43 kDa outer membrane protein (43 K OMP) of F. necrophorum is a porin protein that plays an important role in infections by this bacterium, but the biological function and the pathogenesis of this protein are largely unknown. METHODS: In this study, we investigated the role of the 43 K OMP in bacterial infection of bovine mammary epithelial cells (MAC-T cells) by Tandem Mass Tag proteomic analysis. The RAW264.7 cells were incubated with recombinant 43 K OMP (12.5 µg/mL) for 2 h, 4 h, 6 h, and 12 h, and then the inflammatory related protein and inflammatory cytokine production were measured by Western blot analysis and ELISA, the mRNA expression levels of inflammatory cytokine were measured by Real-Time PCR. RESULTS: Proteomic analysis results demonstrated there were 224 differentially expressed proteins in the MAC-T cells stimulated with the 43 K OMP compared with control, and 118 proteins were upregulated and 106 proteins were downregulated. These differentially expressed proteins were mainly involved in NF-kappa B signaling, bacterial invasion of epithelial cells, cell adhesion, complement and coagulation cascades. The top six differentially expressed proteins were; MMP9, PLAU, STOM, PSMD13, PLAUR, and ITGAV, which were involved in a protein-protein interaction network. Furthermore, TLR/MyD88/NF-κB pathway related proteins and inflammatory cytokines (IL-6, TNF-α, and IL-1ß) were assessed by Western blot analysis and ELISA. Results showed the 43 K OMP to enhance the expression of TLR4 protein at 2 h (P < 0.01) and the MyD88 protein at 4 h (P < 0.05) post-stimulation, and to decrease IκBα expression at 4 h, 6 h and 12 h (P < 0.05) post-infection, as well as induce phosphorylation at Ser536 (P < 0.01). Levels of IL-6, IL-1ß, and TNF-α in the supernatants of mouse macrophages were increased (P < 0.05), as were mRNA expression levels of IL-6, IL-1ß, and TNF-α (P < 0.05), while IL-4 mRNA expression was decreased (P < 0.05). CONCLUSIONS: Taken together, these results suggested the important role for 43 K OMP in F. necrophorum infection, promoting the production of pro-inflammatory cytokines (IL-6 and TNF-α) by activation of the TLR/MyD88/NF-κB pathway. These findings provided a theoretical basis for a better understanding of the pathogenesis of F. necrophorum infection.

Isobaric Stable Isotope N-Phosphorylation Labeling (iSIPL) for Ultrasensitive Proteome Quantification.

Stable isotope chemical labeling methods have been widely used for high-throughput mass spectrometry (MS)-based quantitative proteomics in biological and clinical applications. However, the existing methods are far from meeting the requirements for high sensitivity detection. In the present study, a novel isobaric stable isotope N-phosphorylation labeling (iSIPL) strategy was developed for quantitative proteome analysis. The tryptic peptides were selectively labeled with iSIPL tag to generate the novel reporter ions containing phosphoramidate P-N bond with high intensities under lower collision energies. iSIPL strategy are suitable for peptide sequencing and quantitative analysis with high sensitivity and accuracy even for samples of limited quantity. Furthermore, iSIPL coupled with affinity purification and mass spectrometry was applied to measure the dynamics of cyclin dependent kinase 9 (CDK9) interactomes during transactivation of the HIV-1 provirus. The interaction of CDK9 with PARP13 was found to significantly decrease during Tat-induced activation of HIV-1 gene transcription, suggesting the effectiveness of iSIPL strategy in dynamic analysis of protein-protein interaction in vivo. More than that, the proposed iSIPL strategy would facilitate large-scale accurate quantitative proteomics by increasing multiplexing capability.

OGT upregulates myogenic IL-6 by mediating O-GlcNAcylation of p65 in mouse skeletal muscle under cold exposure.

Cold exposure is an unavoidable and severe challenge for people and animals residing in cold regions of the world, and may lead to hypothermia, drastic changes in systemic metabolism, and inhibition of protein synthesis. O-linked-N-acetylglucoseaminylation (O-GlcNAcylation) directly regulates the activity and function of target proteins involved in multiple biological processes by acting as a stress receptor and nutrient sensor. Therefore, our study aimed to examine whether O-GlcNAcylation affected myogenic IL-6 expression, regulation of energy metabolism, and promotion of survival in mouse skeletal muscle under acute cold exposure conditions. Total protein was extracted from C2C12 cells that had been cultured at 32°C for 3, 6, 9, and 12 h. Western blot analysis showed that mild hypothermia enhanced O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) expression. Furthermore, global OGT-dependent glycosylation and interleukin-6 (IL-6) levels peaked 3 h after induction of mild hypothermia. Enhanced activation of the NF-κB pathway was also observed in response to mild hypothermia. Alloxan and Thiamet G were used to reduce and increase global OGT glycosylation levels in C2C12 cells, respectively. Increased O-GlcNAcylation was associated with significant upregulation of IL-6 expression, as well as enhanced activity and nuclear translocation of p65, while decreased O-GlcNAcylation had the opposite effect. In addition, increased O-GlcNAcylation was associated with significantly increased glucose metabolism, and OGT-mediated O-GlcNAcylation of p65. We generated skeletal muscle-specific OGT knockout mice and exposed them to cold at 4°C for 3 h per day for 1 week. OGT deficiency attenuated the O-GlcNAcylation, activity, and nuclear translocation of p65, resulting in downregulation of IL-6 in mouse skeletal muscle of mice exposed to cold conditions. Taken together, our data suggested that O-GlcNAcylation of p65 enhanced p65 activity and nuclear translocation leading to the upregulation of IL-6, which maintained energy homeostasis and promotes cell survival in mouse skeletal muscle during cold exposure.

How the Dark Triad associated with internet gaming disorder? The serial mediation of basic psychological needs satisfaction and negative coping styles.

According to the I-PACE model, this study focused on the role of need satisfaction and negative coping styles in the relationship between the Dark Triad (i.e., Machiavellianism, psychopathy, and narcissism) and internet gaming disorder (IGD). In a sample of 749 emerging adult gamers, a multiple mediation model with Dark Triad as the distal variable, psychological need satisfaction and negative coping style as mediating variables, and IGD as the outcome variable was tested. Results indicated that Machiavellianism and psychopathy were found to be significant predictors of IGD when mediated by psychological need satisfaction and negative coping styles. Narcissism predicts IGD only through the indirect effect of negative coping styles. The findings enhanced our understanding that Machiavellianism and psychopathy are characterized by compensatory use of internet games, as well as added new perspectives to the understanding of addiction mechanisms in narcissists.

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.

Chemical Enhancement Effect of Icotinib-Au Complex Studied by Combined Density Functional Theory and Surface-Enhanced Raman Scattering.

Icotinib is an epidermal growth factor receptor tyrosine kinase inhibitor used in the treatment of non-small cell lung cancer. The charge transfer effect between gold nanoparticles (AuNPs) and icotinib molecules can be used as a model to study the adsorption mechanism between molecules and metal. The adsorption of icotinib on the AuNP surface was confirmed by UV-vis and transmission electron microscopy (TEM) experiments. To explain the nature of chemisorption between icotinib and AuNPs from a theoretical perspective, the molecular correlation properties of the complex model of icotinib-Au6 were studied by the density functional theory method. By studying the molecular electrostatic potential of an icotinib molecule, four potential binding sites of the icotinib molecule were predicted. The calculation results of binding energy showed that the complex formed by chemisorption of icotinib through acetylene group and Au6 was the most stable one. The molecular frontier orbitals of icotinib and icotinib-Au6 confirmed that the charge transfer effect occurred on the acetylene group, benzene ring, and quinazoline ring of the icotinib molecule. The Herzberg-Teller surface selection rule was used to explain selective enhancement in the theoretically calculated Raman spectra. By comparing the spectra of theory and experiment, the cause of spectral peak shift and broadening that appeared in the surface-enhanced Raman scattering spectrum compared with the normal Raman spectrum was explained as well. This work would contribute to the development and application of the icotinib-Au drug carrier system.

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.

Integrated transcriptomics and metabolomics analysis of catechins, caffeine and theanine biosynthesis in tea plant (Camellia sinensis) over the course of seasons.

BACKGROUND: Catechins, caffeine, and theanine as three important metabolites in the tea leaves play essential roles in the formation of specific taste and shows potential health benefits to humans. However, the knowledge on the dynamic changes of these metabolites content over seasons, as well as the candidate regulatory factors, remains largely undetermined. RESULTS: An integrated transcriptomic and metabolomic approach was used to analyze the dynamic changes of three mainly metabolites including catechins, caffeine, and theanine, and to explore the potential influencing factors associated with these dynamic changes over the course of seasons. We found that the catechins abundance was higher in Summer than that in Spring and Autumn, and the theanine abundance was significantly higher in Spring than that in Summer and Autumn, whereas caffeine exhibited no significant changes over three seasons. Transcriptomics analysis suggested that genes in photosynthesis pathway were significantly down-regulated which might in linkage to the formation of different phenotypes and metabolites content in the tea leaves of varied seasons. Fifty-six copies of nine genes in catechins biosynthesis, 30 copies of 10 genes in caffeine biosynthesis, and 12 copies of six genes in theanine biosynthesis were detected. The correlative analysis further presented that eight genes can be regulated by transcription factors, and highly correlated with the changes of metabolites abundance in tea-leaves. CONCLUSION: Sunshine intensity as a key factor can affect photosynthesis of tea plants, further affect the expression of major Transcription factors (TFs) and structural genes in, and finally resulted in the various amounts of catechins, caffeine and theaine in tea-leaves over three seasons. These findings provide new insights into abundance and influencing factors of metabolites of tea in different seasons, and further our understanding in the formation of flavor, nutrition and medicinal function.

Liver X receptor α participates in LPS-induced reduction of triglyceride synthesis in bovine mammary epithelial cells.

Lipopolysaccharides (LPS) could induce milk fat depression via regulating the body and blood fat metabolism. However, it is not completely clear how LPS might regulate triglyceride synthesis in dairy cow mammary epithelial cells (DCMECs). DCMECs were isolated and purified from dairy cow mammary tissue and treated with LPS. The level of triglyceride synthesis, the expression and activity of the liver X receptor α (LXRα), enzymes related to de novo fatty acid synthesis, and the expression of the fatty acid transporters were investigated. We found that LPS decreased the level of triglyceride synthesis via a down-regulation of the transcription, translation, and nuclear translocation level of the LXRα. The results also indicated that the transcription level of the LXRα target genes, sterol regulatory element binding protein 1 (SREBP1), fatty acid synthetase (FAS), acetyl-CoA carboxylase-1 (ACC1), were significantly down-regulated in DCMECs after LPS treatment. Our data may provide new insight into the mechanisms of milk fat depression caused by LPS.

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.

[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.