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.

Bile acids promote lipopolysaccharide clearance via the hepato-biliary pathway in broiler chickens.

Lipopolysaccharide (LPS) acts as a trigger that disrupts metabolic functions and the immune system. While bile acids (BA) have detoxification and anti-inflammatory effects, their role in promoting LPS excretion in broiler chickens remains unclear. This study aimed to investigate the potential of exogenous BA to enhance hepatic clearance of LPS and thereby potentially alleviate LPS-induced liver injury in broiler chickens. Forty-five 21-day-old male broiler chickens were randomly assigned to three groups: the control group, which received daily intraperitoneal injections of a solvent for LPS treatment and a gavage solvent for BA treatment; the LPS group, which received daily intraperitoneal injections of 0.5 mg/kg body weight LPS and a gavage solvent for BA treatment; the LPS + BA group, which received daily intraperitoneal injections of 0.5 mg/kg body weight LPS and 60 mg/kg body weight BA by gavage. BA administered by gavage protected the broiler chickens from increases in liver and spleen indices, systemic inflammatory response, and hepatic damage induced by LPS. Hepatic clearance of LPS was enhanced, as evidenced by decreased serum LPS levels and accelerated excretion into the gallbladder. Additionally, the LPS-induced downregulation of detoxification genes, including those for the lipoprotein receptor and bile acids export pump, was reversed by BA administered by gavage. Furthermore, nuclear transcription factors such as the Farnesoid X receptor (FXR) and Liver X receptor α (LXRα) were enhanced in BA-treated broiler chickens. These findings suggest that BA administration via gavage enhances hepatic LPS clearance through the upregulation of hepatic uptake and efflux proteins, likely mediated by the activation of nuclear transcription factors FXR and LXRα.

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.

Sex-biased transgenerational transmission of betaine-induced epigenetic modifications in glucocorticoid receptor gene and its down-stream BDNF/ERK pathway in rat hippocampus.

Objectives: Glucocorticoid receptor (GR) expressed in hippocampus is critical for the homeostasis of stress responses and susceptible to epigenetic modulation caused by maternal factors. Here we show that maternal methyl nutrition causes sex-biased changes in hippocampal expression of GR exon 1 mRNA variants, associated with promoter DNA methylation, across two offspring generations in rats.Methods: Three-month-old female Sprague-Dawley rats (F0) were fed a diet supplemented with 1% betaine throughout the gestation and lactation. F0 dams and their F1 and F2 offspring of both sexes at weaning were used in the study.Results: A sex-specific transgenerational effect was observed. F2 females, but not males, followed the same pattern of their grand dams showing increased mRNA expression of total GR and its exons 1.4, 1.7, 1.10 and 1.11 variants coincided with promoter DNA hypomethylation in the hippocampus. However, F1 females, but not males, exhibited an opposite pattern, showing decreased expression of GR and its mRNA variants accompanied with promoter hypermethylation. The protein content of phospho-GR and BDNF/ERK in the hippocampus displayed the same sex and generation specificity.Discussion: These results indicate that maternal betaine exerts transgenerational effects on hippocampal GR expression and BDNF/ERK pathway in female rat offspring, with generation-dependent patterns of DNA methylation on alternative GR promoters.

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.

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.

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.

Transcriptomic responses of porcine cumulus cells to heat exposure during oocytes in vitro maturation.

The oocyte is vulnerable to various environmental stressors, including heat exposure. Cumulus-oocyte complexes (COCs) comprise functional units for oocytes in vitro maturation, and the cumulus cells provide essential supports and protect the oocyte from environmental insults. Heat exposure results in varied consequences in oocyte, presumably due to different responses of cumulus cells to heat exposure. In this study, we examined whether heat exposure of different duration affects porcine oocytes quality differently, and how such effects, if any, relate to transcriptomic profiles of cumulus cells. COCs were heat-exposed for 4 h (20-24 h, COC4) and 24 h (0-24 h, COC24), respectively, and the quality of oocytes in COC24 group showed significantly impaired with disrupted cumulus expansion and extracellular matrix (ECM) structure. The transcriptomic analysis identified 749 and 1238 differential expression genes (DEGs) in COC4 and COC24, respectively. Moreover, 852 DEGs were found when COC24 was compared with COC4, and the downregulated DEGs were mainly associated with Gene Ontology terms linked with ECM and cell proliferation. In the protein-protein interaction network, HSPE1, TNFAIP6, COL12A1, and COL18A1 were identified as hub genes playing important roles in heat-induced transcriptomic responses. These results indicate that impaired cumulus proliferation and ECM structure are responsible for heat-induced damage in oocytes quality.

Zinc-α2-glycoprotein promotes skeletal muscle lipid metabolism in cold-stressed mice.

Skeletal muscle is the most abundant tissue in the adult body and plays an essential role in maintaining heat production for the entire body. Recently, muscle-derived non-shivering thermogenesis under cold conditions has received much attention. Zinc-α2-glycoprotein (ZAG) is an adipokine that was shown to influence energy metabolism in the adipose tissue. We used ZAG knock-out (ZAG KO) and wild-type (WT) mice to investigate the effect of ZAG on the lipid metabolism of skeletal muscle upon exposure to a low temperature (6°C) for one week. The results show that cold stress significantly increases the level of lipolysis, energy metabolism, and fat browning-related proteins in the gastrocnemius muscle of WT mice. In contrast, ZAG KO mice did not show any corresponding changes. Increased expression of ß3-adrenoceptor (ß3-AR) and protein kinase A (PKA) might be involved in the ZAG pathway in mice exposed cold stress. Furthermore, expression of lipolysis-related proteins (ATGL and p-HSL) and energy metabolism-related protein (PGC1α, UCP2, UCP3 and COX1) was significantly enhanced in ZAG KO mice after injection of ZAG-recombinant plasmids. These results indicate that ZAG promotes lipid-related metabolism in the skeletal muscle when the animals are exposed to low temperatures. This finding provides a promising target for the development of new therapeutic approaches to improve skeletal muscle energy metabolism.

Melatonin alleviates hippocampal GR inhibition and depression-like behavior induced by constant light exposure in mice.

Light pollution has become a potential health risk factor worldwide. Chronic exposure to constant light (CCL) leads to depressive-like behavior, yet the mechanism remains unclear. In this study, mice exposed to CCL for 3 weeks exhibited depression-like behaviors, with decreased melatonin in plasma and increased oxidative stress in hippocampus. Meanwhile, CCL-exposed mice showed elevated plasma corticosterone (CORT) levels and diminished glucocorticoid receptor (GR) phosphorylation in hippocampus. Concurrently, glycogen synthase kinase 3 beta (GSK3ß) was inactivated with increased phosphorylation at Ser9. The interrelationship of GSK3ß and GR was clarified in mouse hippocampal neuron (HT-22) cells. GSK3ß inhibitor CHIR-99021 induced GR inhibition with diminished phosphorylation, while GR inhibitor RU486 did not affect GSK3ß expression or phosphorylation. Furthermore, GSK3ß-mediated GR inhibition was reproduced in vitro in HT-22 cells treated with melatonin receptor antagonist luzindole and H2O2 in combination. Finally, melatonin reversed GSK3ß-mediated GR inhibition in hippocampus and improved CCL-induced depression-like behavior in mice. These results indicate that CCL induces melatonin deficiency and oxidative stress in hippocampus, which in turn leads to GSK3ß-mediated GR inhibition and depression-like behavior in mice.

Effects of heat stress on posture transitions and reproductive performance of primiparous sows during late gestation.

This study aimed to investigate the effects of heat stress on posture transitions of perinatal primiparous sows around the parturition period from 72 h prepartum to 24 h postpartum. The reproductive performance of sows was measured, and the relationship between posture transitions and reproductive performance was also analyzed. Ten primiparous sows were randomly assigned to thermoneutral (TN) (18-22 °C; n = 5) or heat stress (HS) (28-32 °C; n = 5) treatments. Posture transitioning, including the frequency of posture change, duration of dynamic posture (DP), and lateral lying with udder to the piglet creep box (PCB) during three periods (72 h prepartum, sub-partum, and 24 h postpartum, respectively), were recorded. Posture change frequency was significantly increased, starting from 24 h prepartum to the onset of farrowing in both the TN (P < 0.05) and HS (P < 0.01) groups. Moreover, the peak value of posture change frequency in the TN group was concentrated during the 12 h prepartum period, positively correlated with the quantities of head-first birth piglets and sub-partum duration, respectively. DP duration increased during the period of 24 h prepartum and then decreased sharply (P < 0.001 and P < 0.05 for TN and HS groups, respectively). The duration of facing the udder to the PCB increased during sub-partum and postpartum TN (P < 0.001). The duration of sub-partum (P < 0.05) and delivery time of single piglets (P < 0.01) in the HS group was prolonged, and piglets from the HS group had a lower weight gain than the TN group both at d10 (P < 0.001) and weaning time (P < 0.001). In conclusion, HS had obvious adverse effects on nursery behavior and reproductive abilities in perinatal primiparous sows, which resulted in poor growth performance of lactating piglets.

Zinc-α2-Glycoprotein Knockout Influenced Genes Expression Profile in Adipose Tissue and Decreased the Lipid Mobilizing After Dexamethasone Treatment in Mice.

Zinc-α2-glycoprotein (ZAG), as an adipokine, plays an important role in lipid metabolism. However, its influence on whole gene expression profile in adipose tissue is not known. Under stress condition, how ZAG affects the lipid metabolism is also unclear. Therefore, in this study ZAG systemic knockout (KO) mice were used as a model to reveal the genes expression profile in visceral fat tissues of ZAG KO mice and wild-type mice by genome-wide microarray screening. Then dexamethasone (DEX) was used to explore the effect of ZAG deletion on body fat metabolism under stress. Our results showed that 179 genes were differentially expressed more than 1.5 times between ZAG KO mice and wild type mice, of which 26 genes were upregulated dramatically and 153 genes were significantly downregulated. Under DEX simulated stress, ZAG systemic knockout in vivo resulted in a markedly decrease of triglycerides (TG) and nonesterified fatty acid (NEFA) content in in plasma. Similarly, for lipid catabolism, ZAG KO led to a significant increase of phosphorylated HSL (p-HSL) protein and a rising tendency of adipose triglyceride lipase (ATGL) protein relative to those of the DEX group. For lipid anabolism, fatty acid synthase (FAS) and adiponectin protein expression in visceral fat rose notably in ZAG KO mice after DEX treatment. In conclusion, ZAG knockout can affect the gene expression profile of adipose tissue, reduce elevated TG and NEFA levels in plasma, and alter lipid metabolism under DEX treatment. These findings provide new insights into the mechanism of lipid metabolic disorders in response to stress.

GR-mediated FTO transactivation induces lipid accumulation in hepatocytes via demethylation of m6A on lipogenic mRNAs.

Chronic stress or excessive exposure to glucocorticoids (GC) contributes to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Glucocorticoid receptor (GR) mediates the action of GC, but its downstream signalling is not fully understood. Fat mass and obesity associated (FTO) protein and its demethylation substrate N6-methyladenosine (m6A) are both reported to participate in the regulation of lipid metabolism, yet it remains unknown whether they are involved in GC-induced hepatic lipid accumulation as new components of GR signalling. In this study, we use both in vivo and in vitro models of GC-induced hepatic lipid accumulation and demonstrate that the activation of lipogenic genes and accumulation of lipid in liver cells are mediated by GR-dependent FTO transactivation and m6A demethylation on mRNA of lipogenic genes. Targeted mutation of m6A methylation sites and FTO knockdown further validated the role of m6A on 3'UTR of sterol regulatory element-binding transcription factor 1 and stearoyl-CoA desaturase mRNAs. Finally, FTO knockdown significantly alleviated dexamethasone-induced fatty liver in mice. These results demonstrate a role of GR-mediated FTO transactivation and m6A demethylation in the pathogenesis of NAFLD and provide new insight into GR signalling in the regulation of fat metablism in the liver.

Maternal betaine protects rat offspring from glucocorticoid-induced activation of lipolytic genes in adipose tissue through modification of DNA methylation.

PURPOSE: Excessive exposure of glucocorticoids activates adipose lipolysis, increases circulating free fatty acids, and contributes to ectopic lipid deposition in liver and skeletal muscle. Our previous study demonstrated that maternal betaine supplementation attenuates glucocorticoid-induced hepatic lipid accumulation in rat offspring. However, it is unclear whether maternal betaine supplementation is effective in preventing glucocorticoid-induced lipolysis in the adipose tissue of offspring. METHODS: In this study, 20 pregnant rats were fed with basal or betaine-supplemented (10 g/kg) diets throughout gestation and lactation, and the offspring rats were raised on the basal diet from weaning till 3 months of age followed by daily intraperitoneal injection of saline or 0.1 mg/kg dexamethasone (DEX) for 3 weeks. RESULTS: Chronic DEX treatment significantly (P < 0.05) decreased serum corticosterone level and increased proinflammatory cytokines, such as TNFα, IL-1ß, and IL-6. Meanwhile, GR protein content in adipose tissue was increased in response to DEX treatment, which was associated with a significant (P < 0.05) up-regulation of ATGL and HSL expression at both mRNA and protein levels. All these DEX-induced changes were significantly (P < 0.05) attenuated in progeny rats derived from betaine-supplemented dams. Furthermore, DEX-induced hypomethylation of ATGL and HSL gene promoters was reversed by maternal betaine supplementation. CONCLUSIONS: Taken together, these results suggest that maternal betaine supplementation is effective in alleviating glucocorticoid-induced lipolysis in adipose tissue with modification of DNA methylation on the promoter of lipolytic genes.

Grandmaternal betaine supplementation enhances hepatic IGF2 expression in F2 rat offspring through modification of promoter DNA methylation.

BACKGROUND: We reported previously that maternal betaine promotes hepatic insulin-like growth factor (IGF2) expression in F1 offspring rats through hypermethylation of the IGF2/H19 imprinting control region (ICR). It remains unknown whether this acquired trait can be transmitted to the F2 generation. This study aimed to determine whether dietary betaine supplementation to grand dams affects the hepatic IGF2 expression in F2 rat offspring and how it is related to alterations in DNA methylation. F2 rat offspring derived from grand dams fed basal or betaine-supplemented diet (10 g kg-1 ) were examined at weaning. Serum IGF2 concentration was measured with enzyme-linked immunosorbent assay (ELISA). Hepatic expression of IGF2, together with other proliferation and apoptosis markers, was determined by using quantitative polymerase chain reaction (qPCR), western blot, and immunohistochemistry. The methylation status of the IGF2/H19 ICR and the promoters of IGF2 gene were detected by methylated DNA immunoprecipitation quantitative polymerase chain reaction (MeDIP-qPCR). RESULTS: The maternal betaine-induced up-regulation of hepatic IGF2 expression in F1 rat offspring was transmitted to the F2 generation. The F2 rats from the betaine group demonstrated enhanced hepatic IGF2 expression at both mRNA and protein levels, in association with higher serum IGF2 concentration. No alterations were observed in the ICR methylation of the IGF2/H19 locus, and hypomethylation was detected in promoters of IGF2 gene in betaine group. CONCLUSION: These results indicate that maternal betaine enhances hepatic IGF2 expression in F2 rat offspring through modification of DNA methylation on IGF2 promoters. © 2019 Society of Chemical Industry.

Heat stress induces distinct responses in porcine cumulus cells and oocytes associated with disrupted gap junction and trans-zonal projection colocalization.

Cumulus cells (CCs), the granulosa cells surrounding the oocytes, play critical roles in oocytes maturation through intercellular communication by extending trans-zonal projections (TZPs) to contact oocytes via gap junctions (GJs). The adverse effect of heat stress (HS) on oocyte maturation has been well documented, whereas the HS responses of CCs and the oocytes in association with GJ/TZP colocalization remain unclear. In this study, porcine cumulus-oocyte complexes (COCs) were subjected to HS at 41.5°C for 24 hr during in vitro maturation. Cumulus expansion was impaired and oocyte quality was reduced with lower survival rate, polar body extrusion rate, and early embryo developmental potentials. CCs and oocytes isolated from COCs demonstrated distinct responses to HS. The messenger RNA abundance of heat shock protein-related genes and mitochondrial DNA-encoded genes, together with ATP content, were significantly increased in CCs, yet decreased in oocytes, despite activation of caspase 3 detected in both CCs and oocytes. Similar changes were observed when denuded oocytes and isolated CCs subjected to HS separately, except mitochondria reactive oxygen species (mROS). In heat-stressed COCs, mROS was significantly increased only in oocytes. However, when isolated CCs and denuded oocytes were heat-stressed separately, mROS was significantly increased only in CCs. Moreover, F-actin, a TZP marker, and its colocalization with a GJ protein connexin-45, were significantly reduced in heat-exposed COCs. These results indicate that HS induces distinct responses in porcine CCs and oocytes in association with disrupted GJ and TZP colocalization.

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

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

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

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

Homocysteine impairs porcine oocyte quality via deregulation of one-carbon metabolism and hypermethylation of mitochondrial DNA†.

Homocysteine (Hcy) is an intermediate in the one-carbon metabolism that donates methyl groups for methylation processes involved in epigenetic gene regulation. Although poor oocyte quality in polycystic ovarian syndrome (PCOS) patients is associated with elevated Hcy concentration in serum and follicular fluid, whether Hcy directly affects oocyte quality and its mechanisms are poorly understood. Here we show that Hcy treatment impaired oocyte quality and developmental competence, indicated by significantly reduced survival rate, polar body extrusion rate, and cleavage rate. Hcy treatment resulted in mitochondrial dysfunction, with increased production of mitochondrial ROS, reduced mtDNA copy number, and the expression of 7 out of 13 mtDNA-encoded genes and 2 ribosome RNA genes, 12S rRNA and 16S rRNA. Upon Hcy treatment, the expression of one-carbon metabolic enzymes and DNMT1 was enhanced. Interestingly, DNA methyltransferase inhibitor 5'AZA rescued Hcy-induced mitochondrial dysfunction, impaired oocyte quality and developmental competence. Concurrently, expression of one-carbon metabolic enzymes and methylation status of mtDNA coding sequences were also normalized, at least partially, by 5'AZA treatment. Our findings not only extend the understanding about how Hcy induces poor oocyte quality, but also contribute to a novel angle of identifying targets for enhancing the quality of oocyte from PCOS patients.

Adipokine zinc-α2-glycoprotein alleviates lipopolysaccharide-induced inflammatory responses through the ß3-AR/PKA/CREB pathway.

Humans and animals frequently experience dysmetabolism induced by inflammation. Zinc-α2-glycoprotein (ZAG), a newly identified adipokine, is potentially involved in lipid metabolism. Our previous study revealed that the ZAG content increased after lipopolysaccharide (LPS) treatment. To clarify ZAG's possible effects on inflammatory responses and lipid metabolism, we used gene overexpression and knockout mice as models to investigate the function of ZAG during inflammation. The results showed that LPS increased plasma triglyceride, non-esterified fatty acid and hepatic triglyceride, while ZAG overexpression decreased these effects. Furthermore, ZAG overexpression weakened inflammatory responses, suppressed lipogenesis, and improved mitochondrial function during inflammation. ZAG overexpression also increased ß3-adrenoreceptor, protein kinase A, and phosphorylated cyclic adenosine monophosphate-response element binding protein (CREB), promoted the combination of CREB and CREB-binding protein (CBP), and competitively inhibited the combination of nuclear factor-κB and CBP. After ZAG knockout, LPS-induced the hyperlipidemia worsened. ZAG knockout aggravated inflammatory responses, promoted lipogenesis, and weakened mitochondrial function during inflammation. ZAG knockout also decreased ß3-adrenoreceptor and protein kinase A. The present study demonstrated that ZAG alleviated lipid metabolism disorders by weakening inflammatory responses. The ß3-adrenoreceptor/protein kinase A/CREB pathway mediated the effects of ZAG on inflammation. These results will provide new insight for research on anti-inflammation.