Extracellular Signal-regulated Kinase Mitogen-activated Protein Research Articles

MAP kinase ERK5 modulates cancer cell sensitivity to extrinsic apoptosis induced by death-receptor agonists

Death receptor ligand TRAIL is a promising cancer therapy due to its ability to selectively trigger extrinsic apoptosis in cancer cells. However, TRAIL–based therapies in humans have shown limitations, mainly due inherent or acquired resistance of tumor cells. To address this issue, current efforts are focussed on dissecting the intracellular signaling pathways involved in resistance to TRAIL, to identify strategies that sensitize cancer cells to TRAIL-induced cytotoxicity. In this work, we describe the oncogenic MEK5-ERK5 pathway as a critical regulator of cancer cell resistance to the apoptosis induced by death receptor ligands. Using 2D and 3D cell cultures and transcriptomic analyses, we show that ERK5 controls the proteostasis of TP53INP2, a protein necessary for full activation of caspase-8 in response to TNFα, FasL or TRAIL. Mechanistically, ERK5 phosphorylates and induces ubiquitylation and proteasomal degradation of TP53INP2, resulting in cancer cell resistance to TRAIL. Concordantly, ERK5 inhibition or genetic deletion, by stabilizing TP53INP2, sensitizes cancer cells to the apoptosis induced by recombinant TRAIL and TRAIL/FasL expressed by Natural Killer cells. The MEK5-ERK5 pathway regulates cancer cell proliferation and survival, and ERK5 inhibitors have shown anticancer activity in preclinical models of solid tumors. Using endometrial cancer patient-derived xenograft organoids, we propose ERK5 inhibition as an effective strategy to sensitize cancer cells to TRAIL-based therapies.

Effects of advanced glycation end products via oxidative stress on beta cells: insights from in vitro and in vivo studies and update on emerging therapies.

Protein, lipid, and nucleic acid glycation reactions begin and continue as a result of persistent hyperglycemia in patients with diabetes mellitus. Advanced glycated end products (AGEs) are a complex group of chemical moieties that are formed as a result of the glycation process and play an important role in the pathogenesis of diabetes mellitus. When AGEs interact with their cellular receptor (RAGE), numerous signaling pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), c-Jun N-terminal kinase (JNK), and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), are activated, increasing oxidative stress. The aim of this review was to summarize in vitro and in vivo studies underlining the involvement of AGEs on beta cell dysfunction and death via oxidative stress. A literature search of publications published between 1912 and December 2022 was conducted using MEDLINE, EMBASE, and the Cochrane Library, with restrictions on articles written in English. Recent insights have revealed that oxidative stress has a crucial role in the development of beta cell dysfunction and insulin resistance, the major hallmarks of type 2 diabetes mellitus. Studies also revealed that AGEs decrease insulin synthesis and secretion in the pancreatic beta cells and induce cell apoptosis. Experimental data have shown that both AGEs and oxidative stress contribute to beta cell dysfunction and development as well as to the progression of diabetic complications. Many anti-AGE therapies are being developed; however, it remains to be seen whether these therapies can help maintain beta cell function and prevent diabetes complications.

Clostridium perfringens phospholipase C, an archetypal bacterial virulence factor, induces the formation of extracellular traps by human neutrophils.

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis

Background: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. Methods: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and β-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. Results: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and β-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. Discussion: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Nuclear ERK1/2 signaling potentiation enhances neuroprotection and cognition via Importinα1/KPNA2

Cell signaling is central to neuronal activity and its dysregulation may lead to neurodegeneration and cognitive decline. Here, we show that selective genetic potentiation of neuronal ERK signaling prevents cell death in vitro and in vivo in the mouse brain, while attenuation of ERK signaling does the opposite. This neuroprotective effect mediated by an enhanced nuclear ERK activity can also be induced by the novel cell penetrating peptide RB5. In vitro administration of RB5 disrupts the preferential interaction of ERK1 MAP kinase with importinα1/KPNA2 over ERK2, facilitates ERK1/2 nuclear translocation, and enhances global ERK activity. Importantly, RB5 treatment in vivo promotes neuroprotection in mouse models of Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) disease, and enhances ERK signaling in a human cellular model of HD. Additionally, RB5‐mediated potentiation of ERK nuclear signaling facilitates synaptic plasticity, enhances cognition in healthy rodents, and rescues cognitive impairments in AD and HD models. The reported molecular mechanism shared across multiple neurodegenerative disorders reveals a potential new therapeutic target approach based on the modulation of KPNA2‐ERK1/2 interactions.

Schistosoma mansoni excretory-secretory products induce protein kinase signalling, hyperkinesia, and stem cell proliferation in the opposite sex

Adult male and female schistosomes in copula dwell within human blood vessels and lay eggs that cause the major Neglected Tropical Disease human schistosomiasis. How males and females communicate to each other is poorly understood; however, male-female physical interaction is known to be important. Here, we investigate whether excretory-secretory products (ESPs), released into the external milieu by mature Schistosoma mansoni, might induce responses in the opposite sex. We demonstrate that ESPs adhere to the surface of opposite sex worms inducing the activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) pathways, particularly in the parasite tegument. Furthermore, we show that mature worms stimulated signalling in juvenile worms. Strikingly, we demonstrate that ESPs from the opposite sex promote stem cell proliferation, in an ERK- and p38 MAPK-dependent manner, in the tegument and within the testes of males, and the ovaries and vitellaria of females. Hyperkinesia also occurs following opposite sex ESP exposure. Our findings support the hypothesis that male and female schistosomes may communicate over distance to modulate key processes underlying worm development and disease progression, opening unique avenues for schistosomiasis control.

A literature review: mechanisms of antitumor pharmacological action of leonurine alkaloid.

Leonurine refers to the desiccated aerial portion of a plant in the Labiatae family. The primary bioactive constituent of Leonurine is an alkaloid, Leonurine alkaloid (Leo), renowned for its substantial therapeutic efficacy in the treatment of gynecological disorders, in addition to its broad-spectrum antineoplastic capabilities. Over recent years, the pharmacodynamic mechanisms of Leo have garnered escalating scholarly interest. Leo exhibits its anticancer potential by means of an array of mechanisms, encompassing the inhibition of neoplastic cell proliferation, induction of both apoptosis and autophagy, and the containment of oncogenic cell invasion and migration. The key signal transduction pathways implicated in these processes include the Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL), the Phosphoinositide3-Kinase/Serine/Threonine Protein Kinase (PI3K/AKT), the Signal Transducer and Activator of Transcription 3 (STAT3), and the Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase (MAP/ERK). This paper commences with an exploration of the principal oncogenic cellular behaviors influenced by Leo and the associated signal transduction pathways, thereby scrutinizing the mechanisms of Leo in the antineoplastic sequence of events. The intention is to offer theoretical reinforcement for the elucidation of more profound mechanisms underpinning Leo's anticancer potential and correlating pharmaceutical development.

The Neuroprotective Effects of Flavonoid Fisetin against Corticosterone-Induced Cell Death through Modulation of ERK, p38, and PI3K/Akt/FOXO3a-Dependent Pathways in PC12 Cells.

The overactive hypothalamic-pituitary-adrenal (HPA) axis is believed to trigger the overproduction of corticosterone, leading to neurotoxicity in the brain. Fisetin is a flavonoid commonly found in fruits and vegetables. It has been suggested to possess various biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. This study aims to explore the potential neuroprotective properties of fisetin against corticosterone-induced cell death and its underlying molecular mechanism in PC12 cells. Our results indicate that fisetin, at concentrations ranging from 5 to 40 μM, significantly protected PC12 cells against corticosterone-induced cell death. Fisetin effectively reduced the corticosterone-mediated generation of reactive oxygen species (ROS) in PC12 cells. Fisetin treatments also showed potential in inhibiting the corticosterone-induced apoptosis of PC12 cells. Moreover, inhibitors targeting MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K) were found to significantly block the increase in cell viability induced by fisetin in corticosterone-treated cells. Consistently, fisetin enhanced the phosphorylation levels of ERK, p38, Akt, and c-AMP response element-binding protein (CREB) in PC12 cells. Additionally, it was found that the diminished levels of p-CREB and p-ERK by corticosterone can be restored by fisetin treatment. Furthermore, the investigation of crosstalk between ERK and CREB revealed that p-CREB activation by fisetin occurred through the ERK-independent pathway. Moreover, we demonstrated that fisetin effectively counteracted the corticosterone-induced nuclear accumulation of FOXO3a, an apoptosis-triggering transcription factor, and concurrently promoted FOXO3a phosphorylation and its subsequent cytoplasmic localization through the PI3K/Akt pathway. In conclusion, our findings indicate that fisetin exerts its neuroprotective effect against corticosterone-induced cell death by modulating ERK, p38, and the PI3K/Akt/FOXO3a-dependent pathways in PC12 cells. Fisetin emerges as a promising phytochemical for neuroprotection.

Cooperativity and oscillations: Regulatory mechanisms of K-Ras nanoclusters

K-Ras nanoclusters (NCs) concentrate all required molecules belonging to the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway in a small area where signaling events take place, increasing efficiency and specificity of signaling. Such nanostructures are characterized by controlled sizes and lifetimes distributions, but there is a poor understanding of the mechanisms involved in their dynamics of growth/decay. Here, a minimum computational model is presented to analyze the behavior of K-Ras NCs as cooperative dynamic structures that self-regulate their growth and decay according to their size. Indeed, the proposed model reveals that the growth and the local production of a K-Ras nanocluster depend positively on its actual size, whilst its lifetime is inversely proportional to the root of its size. The cooperative binding between the structural constituents of the NC (K-Ras proteins) induces oscillations in the size distributions of K-Ras NCs allowing them to range within controlled values, regulating the growth/decay dynamics of these NCs. Thereby, the size of a K-Ras NC is proposed as a key factor to regulate cell signaling, opening a range of possibilities to develop strategies for use in chronic diseases and cancer.

Oxidative stress and inflammatory pathways in female eating disorders and borderline personality disorders with emotional dysregulation as linking factors with impulsivity and trauma

BackgroundBorderline personality disorder (BPD) and eating disorders (ED) are both disorders with emotional dysregulation that may share some similar biological underpinnings, leading to oxidative/inflammatory alterations. Unfortunately, to date, no studies have assessed the relationship between clinical features, inflammatory alterations and childhood trauma across these disorders. Our aim was to identify the potential common and disorder-specific inflammatory pathways and examine possible associationsbetween these dysregulated pathways and the clinical features. MethodsWe studied a sample of 108 women (m = 27.17 years; sd = 7.64), divided into four groups: 23 patients with a restrictive ED (ED-R), 23 patients with a bingeeating/ purging ED (ED-P) and 26 patients with BPD; whereas the control group included 23 healthy subjects. Several inflammatory/oxidative parameters: tumor necrosis factor alpha (TNFα), Thiobarbituric Acid Reactive Substances (TBARS), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), p38 mitogenactivated protein kinases, ERK mitogen-activated protein kinases and c-Jun NH2- terminal kinase (JNK), and some antiinflammatory antioxidant elements: glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), Kelch-like ECHassociated protein (Keap1) were determined in plasma or peripheral blood mononuclear cells. Furthermore, clinical, impulsivity, trauma and eating behavior questionnaires were administered. ResultsThree main inflammatory/oxidative components were extracted using principal component analysis (59.19 % of biomarker variance explained). Disorder-specific dysfunction in the inflammatory and oxidative pathways in patients with BPD and ED were revealed by means of relationships with specific principal components (p < .01). BPD patients showed higher levels of a component featured by elevated levels of JNK and lower of GPx and SOD. ED-R and impulsivity were associated with a component featured by the activation of ERK and negative influence of Keap1. The component featured by the suppression of catalase and COX2 was associated with both ED subtypes and trauma exposure. ConclusionSeveral risk factors such as trauma, impulsivity and eating disorder symptoms were transdiagnostically associated with some inflammatory alterations regardless of diagnosis. These findings suggest that the clinical profile comprising trauma exposure and an emotional dysregulation disorder might constitute a specific endophenotype highly linked with inflammatory alterations.

A novel strategy of integrating network pharmacology and transcriptome reveals antiapoptotic mechanisms of Buyang Huanwu Decoction in treating intracerebral hemorrhage

Ethnopharmacological relevanceBuyang Huanwu Decoction (BYHWD), as a traditional Chinese medical prescription, has been used to treat intracerebral hemorrhage (ICH) for hundreds of years, but the antiapoptotic properties have not yet been studied. Aim of the studyThis study aims to elucidate the antiapoptotic mechanism of BYHWD in ICH. Materials and methodsThe therapeutic effect of BYHWD on ICH was assessed by modified neurological severity scores (mNSS), foot fault, and histopathological staining. Then, we used a modified comprehensive strategy by integrating transcriptome and network pharmacology to reveal the underlying mechanism. TUNEL assay, qRT-PCR, and western blot were further applied to evaluate the antiapoptotic effect of BYHWD on ICH. Dual-luciferase reporter assay and plasmid transfections were implemented to validate the potential competing endogenous RNAs (ceRNA) mechanism of Sh2b3. ResultsNetwork pharmacology analysis indicated that the regulation of the apoptotic process was the highest enriched GO term, and that MAP kinase activity, ERK1, and ERK2 cascade were strongly correlated. Transcriptome analysis screened 180 differentially expressed mRNAs, which were highly enriched in the immune system process and negative regulation of programmed cell death. By checking the literature, we found that Sh2b3 was of great importance to apoptosis by modulating MAPK cascades. TUNEL assay validated the anti-apoptotic effect of BYHWD. Moreover, BYHWD was proven to regulate the Sh2b3-mediated ERK1/2 signaling pathway in ICH mice by qRT-PCR and western blot. We further explored the lncRNA-miRNA-mRNA network underlying the therapeutic effect, among which 4933404O12Rik/miR-185-5p is the upstream regulatory mechanism of Sh2b3. ConclusionsWe explored the antiapoptotic mechanism of BYHWD in treating ICH by a novel integrated strategy, which involved the 4933404O12Rik/miR-185-5p/Sh2b3 ceRNAs axis.

CK2 inhibitor CX4945 inhibits collagen degradation of HaCaT human keratinocyte cells via attenuation of MMP-1 secretion.

During skin aging, the extracellular matrix (ECM) concomitantly breaks down. Out of the various protein components that comprise ECM, collagen is the most abundant one. Matrix metalloproteinase-1 (MMP-1) is a major collagenase that can degrade collagen. Therefore, the inhibition of MMP-1 may be critical for skin aging prevention. CX4945 is an inhibitor of casein kinase 2 and shows anticancer effects on various types of cancer cells. In this report, we investigated the MMP-1-inhibiting effect of CX4945 in HaCaT human keratinocyte cells. We performed zymography assays, Western blot analysis and immunoprecipitation assay to investigate the anti-MMP-1 effects of CX4945. CX4945 was found to inhibit collagen degradation via attenuation of the MMP-1 secretion out of HaCaT cells. This activity of CX4945 may be mediated by the induction of MMP-1 ubiquitylation via c-Jun N-terminal kinase (JNK) signaling. In wound healing cell migration assay, CX4945 also showed suppressive effect on the migration of HaCaT cells. This finding was closely related to the attenuation of CREB transcription factor via the downregulation of ERK mitogen-activated protein kinase as observed in Western blot analysis. Our report suggests that the inhibitory effects of CX4945 on MMP-1 in epidermal cells may offer a basis for further studying its therapeutic potential as an anti-wrinkle agent.

The role of lactoferrin in bone remodeling: evaluation of its potential in targeted delivery and treatment of metabolic bone diseases and orthopedic conditions.

Lactoferrin (Lf) is a multifunctional protein that is synthesized endogenously and has various biological roles including immunological regulation, antibacterial, antiviral, and anticancer properties. Recently, research has uncovered Lf's critical functions in bone remodeling, where it regulates the function of osteoblasts, chondrocytes, osteoclasts, and mesenchymal stem cells. The signaling pathways involved in Lf's signaling in osteoblasts include (low density lipoprotein receptor-related protein - 1 (LRP-1), transforming growth factor β (TGF-β), and insulin-like growth factor - 1 (IGF-1), which activate downstream pathways such as ERK, PI3K/Akt, and NF-κB. These pathways collectively stimulate osteoblast proliferation, differentiation, and mineralization while inhibiting osteoclast differentiation and activity. Additionally, Lf's inhibitory effect on nuclear factor kappa B (NF-κB) suppresses the formation and activity of osteoclasts directly. Lf also promotes chondroprogenitor proliferation and differentiation to chondrocytes by activating the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and phosphoinositide 3-kinase/protein kinase B(PI3K/Akt)signaling pathways while inhibiting the expression of matrix-degrading enzymes through the suppression of the NF-κB pathway. Lf's ability to stimulate osteoblast and chondrocyte activity and inhibit osteoclast function accelerates fracture repair, as demonstrated by its effectiveness in animal models of critical-sized long bone defects. Moreover, studies have indicated that Lf can rescue dysregulated bone remodeling in osteoporotic conditions by stimulating bone formation and suppressing bone resorption. These beneficial effects of Lf on bone health have led to its exploration in nutraceutical and pharmaceutical applications. However, due to the large size of Lf, small bioactive peptides are preferred for pharmaceutical applications. These peptides have been shown to promote bone fracture repair and reverse osteoporosis in animal studies, indicating their potential as therapeutic agents for bone-related diseases. Nonetheless, the active concentration of Lf in serum may not be sufficient at the site requiring bone regeneration, necessitating the development of various delivery strategies to enhance Lf's bioavailability and target its active concentration to the site requiring bone regeneration. This review provides a critical discussion of the issues mentioned above, providing insight into the roles of Lf in bone remodeling and the potential use of Lf as a therapeutic target for bone disorders.

Crebanine ameliorates ischemia-reperfusion brain damage by inhibiting oxidative stress and neuroinflammation mediated by NADPH oxidase 2 in microglia

BackgroundThe urgent challenge for ischemic stroke treatment is the lack of effective neuroprotectants that target multiple pathological processes. Crebanine, an isoquinoline-like alkaloid with superior pharmacological activities, presents itself as a promising candidate for neuroprotection. However, its effects and mechanisms on ischemic stroke remain unknown. MethodsThe effects of crebanine on brain damage following ischemic stroke were evaluated using the middle cerebral artery occlusion and reperfusion (MCAO/R) model.Mechanism of action was investigated using both MCAO/R rats and lipopolysaccharide (LPS)-activated BV-2 cells. ResultsWe initially demonstrated that crebanine effectively ameliorated the neurological deficits in MCAO/R rats, while also reducing brain edema and infarction. Treatment with crebanine resulted in the up-regulation of NeuN+ fluorescence density and down-regulation of FJB+ cell count, and mitigated synaptic damage. Crebanine attenuated the hyperactivation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) by downregulating NADP+ and NADPH levels, suppressing gp91phox and p47phox expressions, and reducing p47phox membrane translocation in Iba-1+ cells. Additionally, crebanine reduced the quantity of Iba-1+ cells and protein expression. Correlation analysis has demonstrated that the inhibition of NOX2 activation in microglia is beneficial for mitigating I/R brain injuries. Moreover, crebanine exhibited significant antioxidant properties by down-regulating the expression of superoxide anion and intracellular reactive oxygen species in vivo and in vitro, and reducing lipid and DNA peroxidation. Crebanine exerted anti-inflammatory effect, as evidenced by the reduction in the expressions of nitric oxide, interleukin 1β, tumor necrosis factor α, interleukin 6, and inducible nitric oxide synthase. The effect of crebanine was achieved through the suppression of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPK) signaling pathway. This is supported by evidence showing reduced NF-κB p65 promoter activity and nucleus translocation, as well as suppressed IκBα phosphorylation and degradation. Additionally, it inhibited the phosphorylation of ERK, JNK, and p38 MAPKs. Importantly, the anti-oxidative stress and neuroinflammation effects of crebanine were further enhanced after silencing gp91phox and p47phox. ConclusionCrebanine alleviated the brain damages of MCAO/R rats by inhibiting oxidative stress and neuroinflammation mediated by NOX2 in microglia, implying crebanine might be a potential natural drug for the treatment of cerebral ischemia.

SPRY4-dependent ERK negative feedback demarcates functional adult stem cells in the male mouse germline†.

Niche-derived growth factors support self-renewal of mouse spermatogonial stem and progenitor cells through ERK MAPK signaling and other pathways. At the same time, dysregulated growth factor-dependent signaling has been associated with loss of stem cell activity and aberrant differentiation. We hypothesized that growth factor signaling through the ERK MAPK pathway in spermatogonial stem cells is tightly regulated within a narrow range through distinct intracellular negative feedback regulators. Evaluation of candidate extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK)-responsive genes known to dampen downstream signaling revealed robust induction of specific negative feedback regulators, including Spry4, in cultured mouse spermatogonial stem cells in response to glial cell line-derived neurotrophic factor or fibroblast growth factor 2. Undifferentiated spermatogonia in vivo exhibited high levels of Spry4 mRNA. Quantitative single-cell analysis of ERK MAPK signaling in spermatogonial stem cell cultures revealed both dynamic signaling patterns in response to growth factors and disruption of such effects when Spry4 was ablated, due to dysregulation of ERK MAPK downstream of RAS. Whereas negative feedback regulator expression decreased during differentiation, loss of Spry4 shifted cell fate toward early differentiation with concomitant loss of stem cell activity. Finally, a mouse Spry4 reporter line revealed that the adult spermatogonial stem cell population in vivo is demarcated by strong Spry4 promoter activity. Collectively, our data suggest that negative feedback-dependent regulation of ERK MAPK is critical for preservation of spermatogonial stem cell fate within the mammalian testis.

The release of zearalenone-induced heterophil extracellular traps in chickens is associated with autophagy, glycolysis, PAD enzyme, and P2X1 receptor

Zearalenone (ZEA) is produced mainly by fungi belonging to genus Fusarium in foods and feeds. Heterophil extracellular traps (HETs) are a novel defense mechanism of chicken innate immunity involving activated heterophils. However, the conditions and requirements for ZEA-triggered HET release remain unknown. In this study, immunostaining analysis demonstrated that ZEA-triggered extracellular fibers were composed of histone and elastase assembled on DNA skeleton, showing that ZEA can induce the formation of HETs. Further experiments indicated that ZEA-induced HET release was concentration-dependent (ranging from 20 to 80 μM ZEA) and time-dependent (ranging from 30 to 180 min). Moreover, in 80 μM ZEA-exposed chicken heterophils, reactive oxygen species (ROS) level, catalase (CAT), superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione (GSH) content were increased. Simultaneously, ZEA at 80 μM activated ERK and p38 MAPK signaling pathways by increasing the phosphorylation level of ERK and p38 proteins. Pharmacological inhibition assays revealed that blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ERK, and p38 mitogen-activated protein kinase (MAPK) reduced ZEA-induced ROS levels but had no impact on HET formation. Furthermore, immunostaining analysis indicated that the heterophil underwent the formation of autophagosome based on being stained with LC3B. The pharmacological inhibition assays demonstrated that rapamycin-, wortmannin-, and 3-methyladenine (3-MA)-treatments modulated ZEA-triggered HET formation, indicating that heterophil autophagy played a key role in ZEA-induced HET formation. Further studies on energy metabolism showed that inhibition of lactate/glucose transport, hexokinase-2 (HK-2), fructose-2,6-biphosphatase 3 (PFKFB3) in glycolysis abated ZEA-induced HETs, implying that glycolysis was one of the factors influencing the ZEA-induced HET formation. Besides, inhibition of the peptidylarginine deiminase (PAD) enzyme and P2X1 significantly reduced the ZEA-induced HET formation. In conclusion, we demonstrated that ZEA-triggered HET formation, which was associated with glycolysis, autophagy, PAD enzyme, and P2X1 receptor activation, providing valuable insight into the negative effect of ZEA on chicken innate immunity.

Anti-oxidant and Anti-inflammatory Effects of Ethanol Extract from Polygala sibirica L. var megalopha Fr. on Lipopolysaccharide-Stimulated RAW264.7 Cells.

To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE2) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O2-) radical and nitrite scavenging activity were also measured. The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE2 production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O2- radical and nitrite scavenging activity. EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.

TIGIT immune checkpoint blockade enhances immunity of human peripheral blood NK cells against castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) patients have a 14-month median survival, emphasizing the need for alternative treatments. Previously, we demonstrated that expanded high-dose natural killer (NK) cells derived from human peripheral blood exhibit therapeutic efficacy against CRPC. However, which immune checkpoint blockade promotes NK cell antitumor immunity against CRPC remains unknown. Here, we explored immune checkpoint molecule expression in NK and CRPC cells during their interactions, and identified that the T cell immunoreceptor with immunoglobulin and immunoreceptor tyrosine-based inhibition motif domain (TIGIT) monoclonal antibody (mAb), vibostolimab, significantly enhanced NK cell cytotoxicity against CRPC cells and cytokine production in vitro, demonstrated by upregulation of degranulation marker CD107a and Fas-ligand (Fas-L) and increased interferon-gamma (IFN-γ) and tumor necrosis factor-alpha secretion. TIGIT blockade increased Fas-L expression and IFN-γ production via the NF-κB signaling pathway and restored degranulation via the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase) kinase/ERK pathway in activated NK cells. Vibostolimab significantly enhanced NK cell antitumor effects against CRPC in two xenograft mouse models. Vibostolimab also increased T cell chemotaxis induced by activated NK cells in vitro and in vivo. Overall, blocking TIGIT/CD155 signaling enhances the antitumor effect of expanded NK cells against CRPC; this finding supports the translational application of TIGIT mAb and NK cell combination strategies from bench to bedside for CRPC treatment.

Sphingosine 1-phosphate elicits a ROS-mediated proinflammatory response in human endometrial stromal cells via ERK5 activation.

Endometriosis is a chronic gynecological disease affecting ~10% women in the reproductive age characterized by the growth of endometrial glands and stroma outside the uterine cavity. The inflammatory process has a key role in the initiation and progression of the disorder. Currently, there are no available early diagnostic tests and therapy relies exclusively on symptomatic drugs, so that elucidation of the complex molecular mechanisms involved in the pathogenesis of endometriosis is an unmet need. The signaling of the bioactive sphingolipid sphingosine 1-phosphate (S1P) is deeply dysregulated in endometriosis. S1P modulates a variety of fundamental cellular processes, including inflammation, neo-angiogenesis, and immune responses acting mainly as ligand of a family of G-protein-coupled receptors named S1P receptors (S1PR), S1P1-5 . Here, we demonstrated that the mitogen-activated protein kinase ERK5, that is expressed in endometriotic lesions as determined by quantitative PCR, is activated by S1P in human endometrial stromal cells. S1P-induced ERK5 activation was shown to be triggered by S1P1/3 receptors via a SFK/MEK5-dependent axis. S1P-induced ERK5 activation was, in turn, responsible for the increase of reactive oxygen species and proinflammatory cytokine expression in human endometrial stromal cells. The present findings indicate that the S1P signaling, via ERK5 activation, supports a proinflammatory response in the endometrium and establish the rationale for the exploitation of innovative therapeutic targets for endometriosis.

Activation of neurons and satellite glial cells in the DRG produces morphine-induced hyperalgesia.

Activation of neurons and glial cells in the dorsal root ganglion is one of the key mechanisms for the development of hyperalgesia. The aim of the present study was to examine the role of neuroglial activity in the development of opioid-induced hyperalgesia. Male rats were treated with morphine daily for 3days. The resultant phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 in the dorsal root ganglion was analyzed by immunohistochemistry and Western blotting. Pain hypersensitivity was analyzed using behavioral studies. The amount of cytokine expression in the dorsal root ganglion was also analyzed. Repeated morphine treatment induced hyperalgesia and marked induction of phosphorylated ERK1/2 in the neurons and satellite glial cells on day 3. An opioid receptor antagonist, toll like receptor-4 inhibitor, MAP/ERK kinase (MEK) inhibitor and gap junction inhibitor inhibited morphine-induced hyperalgesia and ERK1/2 phosphorylation. Morphine treatment induced alteration of cytokine expression, which was inhibited by the opioid receptor antagonist, toll like receptor-4 inhibitor, MEK inhibitor and gap junction inhibitor. Dexamethasone inhibited morphine-induced hyperalgesia and ERK1/2 phosphorylation after morphine treatment. The peripherally restricted opioid receptor antagonist, methylnaltrexone, inhibited hyperalgesia and ERK1/2 phosphorylation. Morphine activates ERK1/2 in neurons and satellite glial cells in the dorsal root ganglion via the opioid receptor and toll like receptor-4. ERK1/2 phosphorylation is gap junction-dependent and is associated with the alteration of cytokine expression. Inhibition of neuroinflammation by activation of neurons and glia might be a promising target to prevent opioid-induced hyperalgesia.