Increased Phosphorylation Of P38 Research Articles

Hesperetin but not ellagic acid increases myosin heavy chain expression and cell fusion in C2C12 myoblasts in the presence of oxidative stress.

Skeletal muscle regeneration is impaired in elderly. An oxidative stress-induced decrease in differentiation capacity of muscle satellite cells is a key factor in this process. The aim of this study is to investigate whether orange polyphenol hesperetin and pomegranate polyphenol ellagic acid enhance myoblast differentiation in the presence and absence of oxidative stress, and to explore underlying mechanisms. C2C12 myoblasts were proliferated for 24 h and differentiated for 120 h while exposed to hesperetin (5, 20, 50 μM), ellagic acid (0.05, 0.1 μM) or a combination (20 μM hesperetin, 0.05 μM ellagic acid) with and without oxidative stress-inducing compound menadione (9 μM) during 24 h of proliferation and during the first 5 h of differentiation. The number of proliferating cells was assessed using fluorescent labeling of incorporated 5-ethynyl-2'-deoxyuridine. Myosin heavy chain expression was assessed by fluorescence microscopy and cell fusion index was calculated. Furthermore, protein expression of phosphorylated p38 and myomixer were assessed using Western blot. None of the compounds induced effects on cell proliferation. Without menadione, 50 μM hesperetin increased fusion index by 12.6% compared to control (p < 0.01), while ellagic acid did not affect measured parameters of differentiation. Menadione treatment did not change myosin heavy chain expression and fusion index. In combination with menadione, 20 μM hesperetin increased myosin heavy chain expression by 35% (p < 0.01) and fusion index by 7% (p = 0.04) compared to menadione. Furthermore, the combination of menadione with hesperetin and ellagic acid increased myosin heavy chain expression by 35% compared to menadione (p = 0.02). Hesperetin and ellagic acid did not change p38 phosphorylation and myomixer expression compared to control, while treatment with menadione increased p38 phosphorylation (p < 0.01) after 5 h and decreased myomixer expression (p = 0.04) after 72 h of differentiation. Hesperetin increased myosin heavy chain expression in the presence of oxidative stress induced by menadione, and increased cell fusion both in the presence and absence of menadione. Ellagic acid did not affect the measured parameters of myoblast differentiation. Therefore, hesperetin should be considered as nutritional prevention or treatment strategy to maintain muscle function in age-related diseases such as sarcopenia. Future research should focus on underlying mechanisms and translation of these results to clinical practice.

CTRP13-Mediated Effects on Endothelial Cell Function and Their Potential Role in Obesity.

Obesity, a major component of cardiometabolic syndrome, contributes to the imbalance between pro- and anti-atherosclerotic factors via dysregulation of adipocytokine secretion. Among these adipocytokines, the C1q/TNF-related proteins (CTRPs) play a role in the modulation of atherosclerosis development and progression. Here, we investigated the vascular effects of CTRP13. CTRP13 is not only expressed in adipose tissue but also in vessels/endothelial cells (ECs) of mice, rats, and humans. Obese individuals (mice, rats, and humans) showed higher vascular CTRP13 expression. Human Umbilical Vein Endothelial Cells (HUVECs), cultured in the presence of serum from obese mice, mimicked this obesity-associated effect on CTRP13 protein expression. Similarly, high glucose conditions and TNF-alpha, but not insulin, resulted in a strong increase in CTRP13 in these cells. Recombinant CTRP13 induced a reduction in EC proliferation via AMPK. In addition, CTRP13 reduced cell cycle progression and increased p53 phosphorylation and p21 protein expression, but reduced Rb phosphorylation, with the effects largely depending on alpha-2 AMPK as suggested by adenoviral overexpression of dominant-negative (DN) or wild-type (WT) alpha 1/alpha 2 AMPK. The present study demonstrates that CTRP13 expression is induced in ECs under diabetic conditions and that CTRP13 possesses significant vaso-modulatory properties which may have an impact on vascular disease progression in patients.

Aging-related alterations in mechanistic target of rapamycin signaling promote platelet hyperreactivity and thrombosis

BackgroundAging is an independent risk factor for the development of cardiovascular, thrombotic, and other chronic diseases. However, mechanisms of platelet hyperactivation in aging remain poorly understood. ObjectivesHere, we examine whether and how aging alters intracellular signaling in platelets to support platelet hyperactivity and thrombosis. MethodsQuantitative mass spectrometry with tandem mass tag labeling systematically measured protein phosphorylation in platelets from healthy aged (>65 years) and young human (<45 years) subjects. The role of platelet mechanistic target of rapamycin (mTOR) in aging-induced platelet hyperreactivity was assessed using pharmacologic mTOR inhibition and a platelet-specific mTOR-deficient mouse model (mTORplt−/−). ResultsQuantitative phosphoproteomics uncovered differential site-specific protein phosphorylation within mTOR, Rho GTPase, and MAPK pathways in platelets from aged donors. Western blot confirmed constitutive activation of the mTOR pathway in platelets from both aged humans and mice, which was associated with increased aggregation compared with that in young controls. Inhibition of mTOR with either Torin 1 in aged humans or genetic deletion in aged mice reversed platelet hyperreactivity. In a collagen–epinephrine pulmonary thrombosis model, aged wild-type (mTORplt+/+) mice succumbed significantly faster than young controls, while time to death of aged mTORplt−/− mice was similar to that of young mTORplt+/+ mice. Mechanistically, we noted increased Rac1 activation and levels of mitochondrial reactive oxygen species in resting platelets from aged mice, as well as increased p38 phosphorylation upstream of thromboxane generation following agonist stimulation. ConclusionAging-related changes in mTOR phosphorylation enhance Rac1 and p38 activation to enhance thromboxane generation, platelet hyperactivity, and thrombosis.

#1400 Acidosis-induced inflammatory response in tubule cells and fibroblasts is amplified by tubule-fibroblast crosstalk and mediated via p38 signaling

Abstract Background and Aims Tubulointerstitial kidney disease, associated with micromilieu changes such as acidification and inflammation, impacts tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Furthermore, cell-cell communication modulates the interstitial microenvironment. In the early stages of kidney failure, fibroblasts undergo a transition to an inflammatory phenotype driven by rapid and long-lasting p38 or CREB (cAMP response element-binding protein) signaling. This phenotypic shift involves an increased secretion of cytokines such as IL-6, TNF, or COX-2 metabolites, thereby changing the microenvironment. Additionally, the phenotype switch is characterized by remodelling of the cytoskeleton and alterations in cell-cell contacts. The objective of our study was to evaluate the impact of acidosis on inflammatory responses in proximal tubule cells and fibroblasts in the context of cellular crosstalk. Furthermore, we aimed to investigate the involvement of p38 and CREB signaling. Method HK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression levels of inflammation markers (IL-6, TNF, TGF-ß, and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin, and vimentin), as well as phospho- and total p38 and CREB, were determined through western blot analysis. The impact of p38 activation was assessed using pharmacological interventions. Furthermore, the influence of IL-6 incubation on the expression of phospho-p38 was investigated Results In tubule cells acidosis led, independent of culture conditions, to an increase of IL-6 after 3h and a decrease of vimentin and COX-2 after 48h. Moreover, acidosis caused an increase of TNF solely in coculture after 3h. Only in monoculture, ß-Catenin expression decreased in tubule cells during acidosis. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin and a decrease of TGF-ß expression exclusively in co-culture after 48 h. Independent of incubation or culture conditions acidosis induced a strong increase of IL-6 protein expression in fibroblasts. In co-culture, acidosis enhanced phosphorylation of p38 phosphorylation only after 3 h and that of CREB appeared after 3h and was persistent after 48h. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. Moreover, acidosis caused an increased phosphorylation of CREB independent of culture conditions and incubation time. Inhibition of p38 under co-culture conditions reduced acidosis-induced changes in fibroblasts significantly. In monoculture, incubation with IL-6 under acidic conditions caused an increase in p38 phosphorylation after 3 hours in tubule cells and after 48 hours in fibroblasts. Conclusion Our data indicate that acidosis-induced pathophysiological changes are significantly amplified by tubule-fibroblast crosstalk. The synergy between cellular crosstalk and acidosis triggers the activation of the p38, CREB, and IL-6 signaling pathways, leading to a phenotype switch in fibroblasts that promotes inflammation and fibrosis. If these findings hold true in vivo, the synergistic interplay between tubule-fibroblast crosstalk and acidosis emerges as a crucial factor in the development of kidney failure.

Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights.

Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.

Sex-Related and Brain Regional Differences of URB597 Effects on Modulation of MAPK/PI3K Signaling in Chronically Stressed Rats.

Gender differences exist in depression incidence and antidepressant efficacy. In addition to the neurotransmission theory of depression, inflammation and disrupted signaling pathways play crucial roles in the pathophysiology of depression. Endocannabinoids offer a novel approach to treat inflammatory and emotional disorders like depression. URB597, a FAAH inhibitor, reduces endocannabinoids breakdown. In this study, URB597 effects were investigated on the pro-inflammatory cytokine interleukin-1β (IL-1β), nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3(NLRP3), and mitogen-activated protein kinase (MAPK)/ phosphatidylinositol 3-hydroxy kinase/ protein kinase B (PI3K) signaling in the hippocampus and the medial prefrontal cortex (mPFC) of male and female rats subjected to chronic unpredictable stress (CUS). The results show that CUS induces depression-like behaviors, and the URB597 exhibited antidepressant-like effects inboth sexes. URB597 reduced the CUS-induced NLRP3 and IL-1β increase in the hippocampus and mPFC of both sexes. URB597 increased the reduced pERK1/2 levels in the mPFC of both sexes and hippocampus of CUS males. URB597 also prevented the increase in p38 phosphorylation after chronic stress in the mPFC of both sexes and in the hippocampus of the females. The CUS suppressed the downstream Akt phosphorylation in the mPFC and hippocampi of both sexes. URB597 produced an up-regulation of the pAkt in the hippocampus of the CUS animals but did not affect the pAkt in the mPFC. These data demonstrated a sexual dimorphism in the neural cell signaling, and in the effects of endocannabinoids, and indicated these dimorphisms are region-specific.

High sodium promotes the secretion and synthesis of PTH through PiT-1-IKKβ pathway in parathyroid gland in vitro.

Parathyroid hormone (PTH) is secreted by the parathyroid glands (PTGs) and is an important hormone regulating mineral metabolism. Previous studies reported that high sodium diet will cause the increase in serum PTH, but the specific mechanism is unknown. Consequently, the present study aims to investigate the effects and mechanisms of high sodium on PTH synthesis and secretion from PTGs. We developed a tissue culture model using normal rat PTGs, discovered that sodium elicited and promoted concentration-dependent and time-dependent PTH secretion. Changes in sodium-associated transporters from PTGs incubated with high sodium were thoroughly examined. Increased expression of sodium-phosphate cotransporter Slc20a1 (also known as PiT-1) was observed. Further tests revealed that PiT-1 activated the NF-κB signaling pathway, resulting in increased IKKβ phosphorylation, IKBα degradation, and increased p65 phosphorylation followed by nuclear entry, which led to increased PTH transcription. Meanwhile, IKKβ phosphorylated SNAP23, promoting exocytosis and eventually led to increased PTH secretion. In conclusion, our findings indicate that PiT-1 plays an important role in the increased secretion and synthesis of PTH directly induced by high sodium under physiological conditions, and may provide a potential therapeutic target for secondary hyperparathyroidism (SHPT).

Loss of MD1 Promotes Inflammatory and Apoptotic Atrial Remodelling in Diabetic Cardiomyopathy by Activating the TLR4/NF-κB Signalling Pathway

Introduction: Myeloid differentiation protein 1 (MD1), a negative regulator of toll-like receptor 4 (TLR4), is widely expressed in the heart. Recent studies have shown that MD1 plays an important role in cardiac remodelling. However, the effects and potential mechanisms underlying MD1-mediated atrial remodelling in diabetic cardiomyopathy (DCM) remain unclear. Therefore, this study was designed to explore the role of MD1 in DCM-related atrial remodelling. Methods: MD1 knockout (MD1-KO) mice and wild-type (WT) littermates were injected with streptozotocin (STZ) to establish a diabetic mouse model. These mice were then used to evaluate MD1 expression and its effects on atrial remodelling in vivo. Results: MD1 expression was significantly decreased in STZ-induced diabetic mice. The loss of MD1 aggravated atrial fibrosis, inflammation, and apoptosis in DCM mice and promoted atrial remodelling. MD1-KO diabetic mice also showed higher susceptibility to atrial fibrillation (AF) and worse cardiac function. Mechanistically, the deletion of MD1 promoted the activation of the TLR4/NF-κB signalling pathway, resulting in atrial remodelling in DCM mice via increased p65 phosphorylation. Conclusions: The deletion of MD1 plays an important role in inflammatory and apoptotic atrial remodelling and increases susceptibility to AF in DCM mice, providing a new target for the preventive treatment of DCM-related atrial remodelling.

Abstract LB281: HIRA suppression is a mechanism of chemotherapy resistance in ovarian cancer

Abstract Ovarian cancer accounts for the fifth most common cause of cancer death among women, despite being relatively rare. This is in large because majority of cases are diagnosed at advanced stage and frequently recur after primary treatment. Despite numerous efforts to develop effective targeted therapies and immunotherapies for ovarian cancer the standard of care remains chemotherapies. We postulate that part of the problem is the paucity of preclinical research and clinical trials focused on dormant persister ovarian cancer cells that remain after chemotherapy treatments. Emerging evidence supports the idea that persister cancer cells undergo a period of dormancy following chemotherapy which enables their survival and drive late recurrence. Thus, there is dire need to understand the pathways that empower the establishment of dormancy following chemotherapies in ovarian cancer. Epigenetic reprogramming is notoriously known to enable cell fate transitions and the establishment of drug resistance. An important layer of epigenetic regulation frequently overlooked in cancer is the regulation of nucleosome composition via histone variants. Importantly, analysis of data from the human protein atlas revealed that histone H3.3 chaperone, Histone Regulator Protein A (HIRA) expression inversely correlated with poor prognosis, suggesting that HIRA suppression might play a role in the development of chemotherapy resistance. Here, we demonstrate that HIRA is a major regulator of cell fate transitions in ovarian cancer cells in response to chemotherapies. Our data indicates that HIRA levels are suppressed in response chemotherapies in cancer cells. Genetic suppression of HIRA in ovarian cancer cells triggered a non-poliferative state characterized by the induction of canonical dormancy markers (p27, an increase in p38 phosphorylation) and cell cycle arrest. Accordingly, HIRA suppression in these cancer cells renders them less sensitive to the standard of care chemotherapy agent paclitaxel. Together, our data demonstrates that HIRA levels play an important role in controlling cell fate decisions in ovarian cancer and regulate chemotherapy sensitivity. Citation Format: Maya Yasukawa, Stanislav Drapela, Didem Ilter, Mian M. Shahzad, Ana P. Gomes. HIRA suppression is a mechanism of chemotherapy resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB281.

SHFM1 deficiency suppresses esophageal squamous cell carcinomas progression via modulating NF‑κB signaling and enhancing nature killer cell‑mediated tumor surveillance.

Excessive proliferation, metastasis and immune escape are considered to be hallmarks of cancer contributing to tumor progression. Split hand and foot malformation 1 (SHFM1) is highly expressed in various cancers and has been reported to increase malignant behaviors. However, the biological functions of SHFM1 in esophageal squamous cell carcinomas (ESCC) progression remain to be elucidated. An integrated bioinformatics analysis was performed to identify candidate genes in ESCC progression based on GSE microarrays. SHFM1 was found to be profoundly upregulated in ESCC tissues compared with normal tissues and SHFM1 expression was positively associated with poor prognosis. The biological effects of SHFM1 on cell growth, metastasis and immune escape were investigated following depletion or overexpression of SHFM1 in vitro. A xenograft mouse model was established to investigate the effect of SHFM1 on ESCC progression in vivo. SHFM1 overexpression promoted ESCC cell proliferation and migration in vitro as well as tumorigenesis in vivo, while SHFM1 knockdown restored those phenotype changes. Additionally, the present study demonstrated that the effects of SHFM1 on malignant behaviors of ESCC cells were achieved by activating the NF-κB signaling accompanied by increased P65 phosphorylation and nuclear translocation. Furthermore, SHFM1 was also found to regulate the sensitivity of cancer cells to natural killer (NK) cells. Specifically, inhibition of SHFM1 enhanced cell-mediated cell apoptosis and increased NK toxicity, which might involve the downregulation of c-Myc and programmed death-ligand 1, key targets in cancer immunotherapy. In conclusion, these findings suggested that SHFM1 probably promoted ESCC progression by activating the NF-κB pathway and enhancing the resistance of ESCC cells to NK cell cytotoxicity, indicating that SHFM1 may be a promising target for ESCC treatment.

Suppression of SMOC2 alleviates myocardial fibrosis via the ILK/p38 pathway.

Fibrosis of the myocardium is one of the main pathological changes of adverse cardiac remodeling, which is associated with unsatisfactory outcomes in patients with heart disease. Further investigations into the precise molecular mechanisms of cardiac fibrosis are urgently required to seek alternative therapeutic strategies for individuals suffering from heart failure. SMOC2 has been shown to be essential to exert key pathophysiological roles in various physiological processes in vivo, possibly contributing to the pathogenesis of fibrosis. A study investigating the relationship between SMOC2 and myocardial fibrosis has yet to be conducted. Mice received a continuous ISO injection subcutaneously to induce cardiac fibrosis, and down-regulation of SMOC2 was achieved by adeno-associated virus-9 (AAV9)-mediated shRNA knockdown. Neonatal fibroblasts were separated and cultured in vitro with TGFβ to trigger fibrosis and infected with either sh-SMOC2 or sh-RNA as a control. The role and mechanisms of SMOC2 in myocardial fibrosis were further examined and analyzed. SMOC2 knockdown partially reversed cardiac functional impairment and cardiac fibrosis in vivo after 21 consecutive days of ISO injection. We further demonstrated that targeting SMOC2 expression effectively slowed down the trans-differentiation and collagen deposition of cardiac fibroblasts stimulated by TGFβ. Mechanistically, targeting SMOC2 expression inhibited the induction of ILK and p38 in vivo and in vitro, and ILK overexpression increased p38 phosphorylation activity and compromised the protective effects of sh-SMOC2-mediated cardiac fibrosis. Therapeutic SMOC2 silencing alleviated cardiac fibrosis through inhibition of the ILK/p38 signaling, providing a preventative and control strategy for cardiac remodeling management in clinical practice.

Posttreatment with dexmedetomidine aggravates LPS-induced myocardial dysfunction partly via activating cardiac endothelial α2A-AR in mice

BackgroundDexmedetomidine (DEX) administered before or at 30 min after sepsis induction was reported to alleviate septic cardiomyopathy in experimental models. However, sepsis is a life-threatening organ dysfunction due to infection-induced dysregulated host response, whether DEX treatment in the presence of organ dysfunction affects septic cardiomyopathy is unknown. This study investigated the effect of DEX posttreatment on septic cardiomyopathy. MethodsMale wild-type and α2A-adrenergic receptor (AR) knockout mice were exposed to lipopolysaccharide (LPS) or cecal ligation puncture (CLP), and cultured cardiac endothelial cells were used. Mouse survival, myocardial function, inflammatory response and related signaling pathways were determined. ResultsDEX treatment at 6, 9 h after LPS challenge significantly reduced survival rate of LPS-challenged mice, especially at 9 h. DEX administered at 9 h after LPS injection or CLP significantly reduced survival in LPS or CLP-induced sepsis in wild-type mice, but not in α2A-AR knockout mice. LPS treatment for 20 h decreased the left ventricle + dp/dt, increased myocardial interleukin (IL)-1β and IL-6 concentrations as well as cardiac endothelial tumor necrosis factor (TNF)-α, vascular cell adhesion molecule-1 (VCAM-1) and ICAM-1 expression, which were enhanced by DEX treated at 9 h after LPS injection in wild-type mice, but not in α2A-AR knockout mice. Furthermore, DEX posttreatment increased p38 phosphorylation, c-Fos nuclear translocation and VCAM-1 expression in LPS-treated cardiac endothelial cells, which were eliminated by α2A-AR knockout or PKC inhibitor. ConclusionsDEX posttreatment aggravates LPS-induced cardiac inflammation and myocardial dysfunction, at least in part, via activating cardiac endothelial α2A-AR-mediated PKC signal pathway.

KLF13 overexpression protectssepsis-induced myocardial injury and LPS-induced inflammation and apoptosis.

Sepsis remains a worldwide public health problem. This study aims to explore the role and mechanism of transcriptional factors (TFs) in sepsis-induced myocardial injury. Firstly, TF KLF13 was selected to explore its role in sepsis-induced myocardial injury. The caecal ligation and puncture (CLP) -induced sepsis mouse model was established and the septic mice were examined using standard histopathological methods. KLF13 expression was detected in the septic mouse heart and was also seen in a lipoploysaccharide (LPS) -induced cellular inflammation model. To explore this further both pro-apoptotic cleaved-caspase3/caspase3 and Bax levels and anti-apoptotic Bcl2 levels were examined, also in both models, In addition inflammatory cytokine (IL-1β, TNF-α, IL-8 and MCP-1) production and IκB-α protein level and p65 phosphorylationwere examined in both septic mice and LPS-induced cells. Thus three parameters - cardiomyocyte apoptosis, inflammatory response and NF-κB pathway activation were evaluated under similar conditions. The septic mice showed significant oedema, disordered myofilament arrangement and degradation and necrosis to varying degrees in the myocardial cells. KLF13 was downregulated in both the septic mouse heart and the LPS-induced cellular inflammation model. Furthermore, both models showed abnormally increased cardiomyocyte apoptosis (increased cleaved-caspase3/caspase and Bax protein levels and decreased Bcl2 level), elevated inflammation (increased production of inflammatory cytokines) and the activated NF-κB pathway (increased p65 phosphorylation and decreased IκB-α protein level). KLF13 overexpression notably ameliorated sepsis-induced myocardial injury in vivo and in vitro. KLF13 overexpression protected against sepsis-induced myocardial injury and LPS-induced cellular inflammation and apoptosis via inhibiting the inflammatory pathways (especially NF-κB signalling) and cardiomyocyte apoptosis.

Deoxynivalenol hijacks the pathway of Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT-3) to drive caspase-3-mediated apoptosis in intestinal porcine epithelial cells

Deoxynivalenol (DON) can easily injure the intestinal tract, which represents the first barrier against food contaminants. The intestinal toxicity induced by DON was mainly focused on mitogen-activated protein kinase (MAPK) activation, however, the underlying mechanisms by which DON triggers apoptosis by other pathways remain poorly understood. In this study, the Janus kinase 2/signal transducers and activators of transcription 3 (JAK2/STAT-3) pathway was proposed to regulate the intrinsic apoptosis induced by DON and thoroughly investigated in intestinal porcine epithelial cells (IPEC-J2). First, DON was found to be able to efficiently inhibit cell viability and increase the release of lactate dehydrogenase. It could also enhance the activity of the cleaved caspase-3 in a time-dependent manner, accompanied by a loss of mitochondrial membrane potential and an up-regulation of the apoptosis rate. Then, the expression of genes associated with inflammation and apoptosis were investigated. DON increased the expression of IL-6, IL-1β, TNF-α, SOCS3 and Bax, but decreased the expression of Bcl-2 and Bcl-xL. Moreover, we discovered that DON robustly inhibited STAT-3 activity together with the down-regulation of JAK2, Bcl-2 and Bcl-xL, paralleling the increase in p38 phosphorylation. Furthermore, a pharmacological activation of JAK2/STAT-3 alleviated DON induced-apoptosis. Concurrent with the apoptotic pathway, during the initial exposure to DON (first 4 h), a survival pathway involving phosphorylated Erk1/2, Akt, and FoxO1 was also observed. Thus, apoptosis induced by DON was Janus faced: although the survival pathway was activated, the DON-induced apoptotic JAK2/STAT-3/caspase-3 pathway dominated, leading to an imbalance in cell homeostasis. This study provides a novel avenue to comprehensively reveal the pathological mechanisms of DON-induced intestinal disorders, which is promising for future applications to other contaminants in food and feed.

Role of ATP5G3 in sodium nitroprusside-induced cell death in cervical carcinoma cells.

We identified a gene, subunit C3 (ATP5G3) of mitochondrial ATP synthase, that displayed changes in gene expression under oxidative stress. We examined the role of ATP5G3 and its molecular mechanisms in sodium nitroprusside (SNP)-induced cell death using ATP5G3 small interfering RNA (siATP5G3)-transfected HeLa cells. A significant increase in cytotoxicity was observed in the transfected cells treated with SNP, which suggests a protective role of ATP5G3 in SNP-induced cytotoxicity in the cells. The transfected cells treated with photodegraded SNP showed equal cytotoxicity to SNP, and pretreatment with deferoxamine (DFO) completely inhibited this cytotoxicity. Further, cytotoxicity was significantly inhibited by pretreatment with a p38 inhibitor and was accentuated by the p38 activator in cells. Pretreatment with the Bcl-xL inhibitor also significantly accentuated cytotoxicity. The increase in p38 phosphorylation was significantly higher in siATP5G3-transfected cells treated with SNP in immunoblotting, which was inhibited by pretreatment with DFO. The increase in cytotoxicity with siATP5G3 transfection was completely blocked by cotransfection with sip38, and the blocking effect disappeared by cotransfection with additional siBcl-xL, which suggests that the protective role of ATP5G3 is mediated by Bcl-xL via the inhibition of p38 activity. Cytotoxicity was completely blocked by the cotransfection of siATP5G3 with siBax. No change in apoptotic parameters was observed during cytotoxicity. However, pretreatment with lysosomal inhibitors significantly inhibited cytotoxicity and increased p62 protein levels. These findings suggest that ATP5G3 plays a protective role in autophagic cell death/lysosome-associated cell death induced by SNP via the sequential signaling of ROS/p38/Bcl-xL/Bax in HeLa cells.

Docosahexaenoic Acid Attenuates Radiation-Induced Myocardial Fibrosis by Inhibiting the p38/ET-1 Pathway in Cardiomyocytes

Radiation-induced myocardial fibrosis (RIMF) is a severe delayed complication of thoracic irradiation (IR). Endothelin-1 (ET-1) is critical in cardiac fibroblast activation, and docosahexaenoic acid (DHA) is protective against various cardiac diseases. This study aimed to explore the roles of ET-1 in RIMF and the potential of DHA in preventing RIMF. Hematoxylin and eosin, sirius red, and Masson trichrome staining were carried out to evaluate the histopathologic conditions in mouse models. Enzyme-linked immunosorbent assays were used to detect the concentration of ET-1 in serum and cell supernatants. Western blotting, immunofluorescence, and immunohistochemistry were used to assess the protein levels. The phenotypic alterations of cardiac fibroblasts were evaluated by cell proliferation/migration assays and α-smooth muscle actin (α-SMA) detection. Radiation increased ET-1 expression and secretion by increasing p38 phosphorylation in cardiomyocytes, and ET-1 markedly promoted the activation of cardiac fibroblasts, which were characterized by enhanced fibroblast proliferation, migration, and α-SMA expression. Cardiomyocyte-derived ET-1 mediated radiation-induced fibroblast activation by targeting the PI3K-AKT and MEK-ERK pathways in fibroblasts. DHA suppressed ET-1 levels by blocking p38 signaling in cardiomyocytes and significantly attenuated the activation of cardiac fibroblasts induced by the IR/ET-1 axis. Importantly, DHA decreased collagen deposition and α-SMA expression, alleviating cardiac fibrosis caused by radiation in mouse models. Our findings demonstrate that radiation facilitates cardiac fibroblast activation by enhancing p38/ET-1 signaling in cardiomyocytes, revealing the IR/p38/ET-1 regulatory axis in RIMF for the first time. DHA effectively inhibits fibroblast activation by targeting p38/ET-1 and can be recognized as a promising protective agent against RIMF.

Grass carp (Ctenopharyngodon idella) DYRK2 modulates cell apoptosis through phosphorylating p53

In mammals, DYRK2 increases p53 phosphorylation level by interacting with it and then promotes cell apoptosis. However, the function of fish DYRK2 has not yet been elucidated. In this paper, we cloned and identified the coding sequence (CDS) of a grass carp DYRK2 (CiDYRK2) which is 1773 bp in length and encodes 590 amino acids. SMART predictive analysis showed that CiDYRK2 possesses a serine/threonine kinase domain. Subsequently, we used the dsRNA analog polyinosinic-polycytidylic acid (poly (I:C) and Grass carp reovirus (GCRV) to stimulate grass carp and CIK cells for different times and found that CiDYRK2 mRNA was significantly up-regulated both in fish tissues and cells. To explore the function of CiDYRK2, we carried out overexpression and knockdown experiments of CiDYRK2 in CIK cells. Real-time quantitative PCR (Q-PCR), TdT-mediated dUTP nick end labeling (TUNEL) assay and flow cytometry were used to detect the ratio of BAX/BCL-2 mRNA, the number of TUNEL positive cells, the proportion of Annexin V-positive cells respectively. The results showed that CiDYRK2 significantly up-regulated BAX/Bcl-2 mRNA ratio and increased the number of TUNEL-positive cells, as well as the proportion of Annexin V-positive cells. On the contrary, knock-down of CiDYRK2 significantly down-regulated BAX/Bcl-2 mRNA ratio in the cells. Therefore, CiDYRK2 promoted cell apoptosis. To study the molecular mechanism by which CiDYRK2 promoting cell apoptosis, subcellular localization and immunoprecipitation experiments were used to study the relationship between grass carp DYRK2 and the pro-apoptotic protein p53. The results showed that CiDYRK2 and Cip53 were located and co-localized in the nucleus. Co-immunoprecipitation experiment also showed that CiDYRK2 and Cip53 can bind with each other. We further found that DYRK2 can increase the phosphorylation level of p53. In a word, our results showed that grass carp DYRK2 induces cell apoptosis by increasing the phosphorylation level of p53.

Dusquetide modulates innate immune response through binding to p62

SQSTM1/p62 is an autophagic receptor that plays a major role in mediating stress and innate immune responses. Preclinical studies identified p62 as a target of the prototype innate defense regulator (IDR); however, the molecular mechanism of this process remains unclear. Here, we describe the structural basis and biological consequences of the interaction of p62 with the next generation of IDRs, dusquetide. Both electrostatic and hydrophobic contacts drive the formation of the complex between dusquetide and the ZZ domain of p62. We show that dusquetide penetrates the cell membrane and associates with p62 invivo. Dusquetide binding modulates the p62-RIP1 complex, increases p38 phosphorylation, and enhances CEBP/B expression without activating autophagy. Our findings provide molecular details underlying the IDR action that may help in the development of new strategies to pharmacologically target p62.

Inhalative as well as Intravenous Administration of H2S Provides Neuroprotection after Ischemia and Reperfusion Injury in the Rats' Retina.

Background: Neuronal ischemia-reperfusion injury (IRI), such as it can occur in glaucoma or strokes, is associated with neuronal cell death and irreversible loss of function of the affected tissue. Hydrogen sulfide (H2S) is considered a potentially neuroprotective substance, but the most effective route of application and the underlying mechanism remain to be determined. Methods: Ischemia-reperfusion injury was induced in rats by a temporary increase in intraocular pressure (1 h). H2S was then applied by inhalation (80 ppm at 0, 1.5, and 3 h after reperfusion) or by intravenous administration of the slow-releasing H2S donor GYY 4137. After 24 h, the retinas were harvested for Western blotting, qPCR, and immunohistochemical staining. Retinal ganglion cell survival was evaluated 7 days after ischemia. Results: Both inhalative and intravenously delivered H2S reduced retinal ganglion cell death with a better result from inhalative application. H2S inhalation for 1.5 h, as well as GYY 4137 treatment, increased p38 phosphorylation. Both forms of application enhanced the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and inhalation showed a significant increase at all three time points. H2S treatment also reduced apoptotic and inflammatory markers, such as caspase-3, intracellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor (VEGF), and inducible nitric oxide synthase (iNOS). The protective effect of H2S was partly abolished by the ERK1/2 inhibitor PD98059. Inhalative H2S also reduced the heat shock response including heme oxygenase (HO-1) and heat shock protein 70 (HSP-70) and the expression of radical scavengers such as superoxide dismutases (SOD1, SOD2) and catalase. Conclusion: Hydrogen sulfide acts, at least in part, via the mitogen-activated protein kinase (MAPK) ERK1/2 to reduce apoptosis and inflammation. Both inhalative H2S and intravenous GYY 4137 administrations can improve neuronal cell survival.

TCF3 regulates human endometrial stromal cell proliferation and migration in RPL.

Recurrent pregnancy loss (RPL) is a multifactorial condition with no explanation of miscarriage in approximately half of the RPL patients, consequently leaving deep physical and emotional sequels. Transcription factor 3 (TCF3 or E2A), is a unique member of the LEF/TCF family and plays an important role in embryogenesis. However, its function in RPL is poorly understood. Using real-time polymerase chain reaction (qRT-PCR), western blotting, and immunohistochemistry, we demonstrated that TCF3 was downregulated in decidual tissues from RPL patients compared with healthy control (HC). Further, TCF3 knockdown inhibited proliferation, induced G0/G1 phase arrest, and promoted migration in human endometrial stromal cells (HESCs), while overexpression of TCF3 exhibited the opposite effects. RNA-sequencing analysis combined with gene-set enrichment analysis results showed that the mitogen-activated protein kinase pathway is potentially downstream of TCF3. Knockdown of TCF3 confirmed increased p38 phosphorylation, while overexpression of TCF3 inhibited p38 phosphorylation. Furthermore, we found that TCF3 protein level was decreased in HESCs under hypoxic incubation, while hypoxia-inducible factor-1α (HIF1A) knockdown increased the expression of TCF3. TCF3 overexpression recovered the proliferation ability of HESCs inhibited by hypoxia and reversed hypoxia-induced migration. Consistently, we found that RPL patients had a significantly higher level of HIF1A in the decidual tissue than HC. Overall, this study clarifies that increased HIF1A in the decidua contributes to the occurrence of RPL through the TCF3/p38 signaling pathway.