Extracellular Signal-regulated Kinase Research Articles

Deletion of pyruvate dehydrogenase kinases reduces susceptibility to deep vein thrombosis in mice

AbstractNeutrophils contribute to deep vein thrombosis (DVT) by releasing prothrombotic neutrophil extracellular traps (NETs). NET formation (known as NETosis) is an energy-intensive process that requires an increased rate of aerobic glycolysis. The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux from oxidative phosphorylation toward aerobic glycolysis. Herein, we identified that the combined deletion of PDK2 and PDK4 (PDK2/4–/–) renders mice less susceptible to DVT (measured by thrombus incidence, weight, and length) in the inferior vena cava–stenosis model at day 2 after surgery. Compared with wild-type (WT) mice, the venous thrombus obtained from PDK2/4–/– mice exhibited reduced citrullinated histone content, a known marker of NETs. In line with in vivo observations, phorbol 12-myristate 13-acetate (PMA)–stimulated PDK2/4–/– neutrophils displayed reduced NETosis and secretion of cathepsin G and elastase compared with PMA-stimulated WT neutrophils. The formation of platelet aggregates mediated by PMA-stimulated PDK2/4–/– neutrophils were significantly reduced compared with PMA-stimulated WT neutrophils. Finally, PDK2/4–/– neutrophils exhibited reduced levels of intracellular Ca2+ concentration, extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation, and glycolytic proton efflux rate (a measure of aerobic glycolysis), known to facilitate NETosis. Together, these findings elucidate, to our knowledge, for the first time, the fundamental role of PDK2/4 in regulating NETosis and acute DVT.

Increased PHLPP1 expression through ERK-4E-BP1 signaling axis drives nicotine induced oxidative stress related damage of cardiomyocytes

Nicotine, a key constituent of tobacco/electronic cigarettes causes cardiovascular injury and mortality. Nicotine is known to induce oxidative stress and mitochondrial dysfunction in cardiomyocytes leading to cell death. However, the underlying mechanisms remain unclear. Pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP) is a member of metal-dependent protein phosphatase (PPM) family and is known to dephosphorylate several AGC family kinases and thereby regulate a diverse set of cellular functions including cell growth, survival, and death. Our lab has previously demonstrated that PHLPP1 removal reduced cardiomyocyte death and cardiac dysfunction following injury. Here, we present a novel finding that nicotine exposure significantly increased PHLPP1 protein expression in the adolescent rodent heart. Building upon our in vivo finding, we determined the mechanism of PHLPP1 expression in cardiomyocytes. Nicotine significantly increased PHLPP1 protein expression without altering PHLPP2 in cardiomyocytes. In cardiomyocytes, nicotine significantly increased NADPH oxidase 4 (NOX4), which coincided with increased reactive oxygen species (ROS) and increased cardiomyocyte apoptosis which were dependent on PHLPP1 expression. PHLPP1 expression was both necessary and sufficient for nicotine induced mitochondrial dysfunction. Mechanistically, nicotine activated extracellular signal-regulated protein kinases (ERK1/2) and subsequent eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to increase PHLPP1 protein expression. Inhibition of protein synthesis with cycloheximide (CHX) and 4EGI-1 abolished nicotine induced PHLPP1 protein expression. Moreover, inhibition of ERK1/2 activity by U0126 significantly blocked nicotine induced PHLPP1 expression. Overall, this study reveals a novel mechanism by which nicotine regulates PHLPP1 expression through ERK-4E-BP1 signaling axis to drive cardiomyocyte injury.

Invariant Natural Killer T Cells Regulate Conventional Dendritic Cell Maturation to Re-establish Immune Tolerance to Rheumatoid Arthritis in DBA/1 Mice.

Rheumatoid arthritis (RA) is a type of autoimmune disease that results in immune disorder and excessive inflammatory response due to a reduction of self-tolerance. Invariant natural killer T (iNKT) cells can effectively alleviate clinical symptoms and hyper-inflammation in RA, but their mechanism of action is not well-defined. This study aims to investigate the mechanism of iNKT cell therapy for RA. We established a DBA/1 mouse model for RA and treated it with specific iNKT cells. A cytometric bead array was used to measure the amounts of cytokines in the serum. Flow cytometry was then employed to identify different subsets of helper T cells (Th), the frequency of conventional dendritic cells (cDC), the expression of CD80, CD86, programmed cell death ligand 1 (PD-L1), and PD-L2 on cDC surfaces, and associated pathway proteins. iNKT cell treatment reduced Th1/Th2 and Th17/ regulatory T (Treg) cell ratios while increasing interleukin-4 (IL-4) and IL-10. It enhanced the generation of immature cDCs, and it upregulated the level of PD-L2 by stimulating the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Meanwhile, it activated the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and inhibited the nuclear factor kappa B (NF-κB) pathway. According to our findings, iNKT cell treatment increased the expression of phosphates STAT3 in lymph node cDC, causing them to upregulate PD-L2 molecules. While activating the ERK1/2 pathway and inhibiting the NF-κB pathway, tolerogenic cDC was produced, restoring immune homeostasis and correcting excessive inflammation. These results deliver new insights into the treatment of RA by iNKT cells.

Spatiotemporal regulation of the hepatocyte growth factor receptor MET activity by sorting nexins 1/2 in HCT116 colorectal cancer cells.

Cumulative research findings support the idea that endocytic trafficking is crucial in regulating receptor signaling and associated diseases. Specifically, strong evidence points to the involvement of sorting nexins (SNXs), particularly SNX1 and SNX2, in the signaling and trafficking of the receptor tyrosine kinase (RTK) MET in colorectal cancer (CRC). Activation of hepatocyte growth factor (HGF) receptor MET is a key driver of CRC progression. In the present study, we utilized human HCT116 CRC cells with SNX1 and SNX2 genes knocked out to demonstrate that their absence leads to a delay in MET entering early endosomes. This delay results in increased phosphorylation of both MET and AKT upon HGF stimulation, while ERK1/2 (extracellular signal-regulated kinases 1 and 2) phosphorylation remains unaffected. Despite these changes, HGF-induced cell proliferation, scattering, and migration remain similar between the parental and the SNX1/2 knockout cells. However, in the absence of SNX1 and SNX2, these cells exhibit increased resistance to TRAIL-induced apoptosis. This research underscores the intricate relationship between intracellular trafficking, receptor signaling, and cellular responses and demonstrates for the first time that the modulation of MET trafficking by SNX1 and SNX2 is critical for receptor signaling that may exacerbate the disease.

AI-based prediction of protein–ligand binding affinity and discovery of potential natural product inhibitors against ERK2

Determination of protein–ligand binding affinity (PLA) is a key technological tool in hit discovery and lead optimization, which is critical to the drug development process. PLA can be determined directly by experimental methods, but it is time-consuming and costly. In recent years, deep learning has been widely applied to PLA prediction, the key of which lies in the comprehensive and accurate representation of proteins and ligands. In this study, we proposed a multi-modal deep learning model based on the early fusion strategy, called DeepLIP, to improve PLA prediction by integrating multi-level information, and further used it for virtual screening of extracellular signal-regulated protein kinase 2 (ERK2), an ideal target for cancer treatment. Experimental results from model evaluation showed that DeepLIP achieved superior performance compared to state-of-the-art methods on the widely used benchmark dataset. In addition, by combining previously developed machine learning models and molecular dynamics simulation, we screened three novel hits from a drug-like natural product library. These compounds not only had favorable physicochemical properties, but also bound stably to the target protein. We believe they have the potential to serve as starting molecules for the development of ERK2 inhibitors.

EGFL6 activates the ERK signaling to improve angiogenesis and osteogenesis of BMSCs in patients with steroid-induced osteonecrosis of the femoral head.

Recently, epidermal growth factor-like domain protein 6 (EGFL6) was proposed as a candidate gene for coupling angiogenesis to osteogenesis during bone repair; however, the exact role and underlying mechanism are largely unknown. Here, using immunohistochemical and Western blotting analyses, we found that EGFL6 was downregulated in the femoral head tissue of patients with steroid-induced osteonecrosis of the femoral head (SONFH) compared to patients with traumatic femoral neck fracture (FNF), accompanied by significantly downregulation of osteogenic and angiogenic marker genes. Then, bone marrow mesenchymal stem cells (BMSCs) were isolated from the FNF and the SONFH patients, respectively, and after identification by immunofluorescence staining surface markers, the effect of EGFL6 on their abilities of osteogenic differentiation and angiogenesis was evaluated. Our results of alizarin red staining and tubular formation experiment revealed that BMSCs from the SONFH patients (SONFH-BMSCs) displayed an obviously weaker ability of osteogenesis than FNF-BMSCs, and EGFL6 overexpression improved the abilities of osteogenic differentiation and angiogenesis of SONFH-BMSCs. Moreover, EGFL6 overexpression activated extracellular signal-regulated kinases 1/2 (ERK1/2). ERK1/2 inhibitor U0126 reversed the promoting effect of EGFL6 overexpression on the expression of osteogenesis and angiogenesis-related genes in the SONFH femoral head. In conclusion, EGFL6 plays a protective role in SONFH, it promotes osteogenesis and angiogenesis of BMSCs, and its effect is likely to be related to ERK1/2 activation.

Phase II Study of Ulixertinib in Children and Young Adults With Tumors Harboring Activating Mitogen-Activated Protein Kinase Pathway Alterations: APEC1621J of the National Cancer Institute-Children's Oncology Group Pediatric MATCH Trial.

The National Cancer Institute-Children's Oncology Group (NCI-COG) Pediatric MATCH trial assigns patients age 1-21 years with refractory malignancies to phase II treatment arms of molecularly targeted therapies on the basis of genetic alterations detected in their tumor. Patients with activating alterations in the mitogen-activated protein kinase pathway were treated with ulixertinib, an extracellular signal-regulated kinase (ERK)1/2 inhibitor. As there were no previous pediatric data, ulixertinib was initially tested in a dose escalation cohort to establish the recommended phase II dose (RP2D) before proceeding to the phase II cohort. Ulixertinib was administered at 260 mg/m2/dose orally twice a day (dose level 1 [DL1], n = 15) or 350 mg/m2/dose orally twice a day (DL2, n = 5). The primary end point was objective response rate; secondary end points included safety/tolerability and progression-free survival (PFS). Twenty patients (median 12 years; range, 5-20) were treated, all evaluable for response. CNS tumors comprised 55% (11/20) of diagnoses, with high-grade glioma and low-grade glioma most common (n = 5 each). All CNS tumors except one harbored BRAF fusions or V600E mutations. Rhabdomyosarcoma (n = 5) was the most frequent non-CNS diagnosis. DL1 was declared the RP2D in the dose escalation cohort after dose-limiting toxicities in Cycle 1 occurred in 1/6 patients at DL1 and 2/5 patients at DL2, including fatigue, anorexia, rash, nausea, vomiting, diarrhea, dehydration, hypoalbuminemia, and hypernatremia. No objective responses were observed. Six-month PFS was 37% (95% CI, 17 to 58). Three patients with BRAF-altered CNS tumors achieved stable disease >6 months. Ulixertinib, a novel targeted agent with no previous pediatric data, was successfully evaluated in a national precision medicine basket trial. The pediatric RP2D of ulixertinib is 260 mg/m2/dose orally twice a day. Limited single-agent efficacy was observed in a biomarker-selected cohort of refractory pediatric tumors.

MPT0E028, a novel pan-HDAC inhibitor, prevents pulmonary fibrosis through inhibition of TGF-β-induced CTGF expression in human lung fibroblasts: Involvement of MKP-1 activation

Histone deacetylase (HDAC) inhibitors are potential candidates for treating pulmonary fibrosis. MPT0E028, a novel pan-HDAC inhibitor, has been reported to exhibit antitumor activity in several cancer cell lines. In this study, we investigated the mechanism underlying the inhibitory effects of MPT0E028 on the expression of fibrogenic proteins in human lung fibroblasts (WI-38). Our results revealed that MPT0E028 inhibited transforming growth factor-β (TGF-β)-, thrombin-, and endothelin 1-induced connective tissue growth factor (CTGF) expression in a concentration-dependent manner. In addition, MPT0E028 suppressed TGF-β-stimulated expression of fibronectin, collagen I, and α-smooth muscle actin (α-SMA). Furthermore, MPT0E028 inhibited the TGF-β-induced phosphorylation of c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK). MPT0E028 reduced the increase in SMAD3 and c-Jun phosphorylation, and SMAD3-and activator protein-1 (AP-1)-luciferase activities under TGF-β stimulation. Transfection with mitogen-activated protein kinase phosphatase-1 (MKP-1) siRNA reversed the suppressive effects of MPT0E028 on TGF-β-induced increases in CTGF expression; JNK, p38, and ERK phosphorylation; and SMAD3 and AP-1 activation. Moreover, MPT0E028 increased MKP-1 acetylation and activity in WI-38 cells. Pretreatment with MPT0E028 reduced the fibrosis score and fibronectin, collagen, and α-SMA expression in bleomycin-induced pulmonary fibrosis mice. In conclusion, MPT0E028 induced MKP-1 acetylation and activation, which in turn inhibited TGF-β-stimulated JNK, p38, and ERK phosphorylation; SMAD3 and AP-1 activation; and subsequent CTGF expression in human lung fibroblasts. Thus, MPT0E028 may be a potential drug for treating pulmonary fibrosis.

Impact of Liparis nervosa extract on neuroinflammation mediated by LPS-induced BV-2 microglial cells and its bioactive components analysis

To investigate the impact and potential mechanisms of extracts from different parts of Liparis nervosa on neuroinflammation by lipopolysaccharide(LPS)-induced BV-2 microglial cells. The materials of L. nervosa were subjected to crushing, ethanol extraction, and concentration to obtain an alcohol extract. Subsequently, the extract was further extracted to obtain petroleum ether extract, ethyl acetate extract, N-butanol extract, and aqueous phase extract. The ethyl acetate extract was separated into distillate(1)-(6)using D101 macroporous resin column chromatography. The experiment was divided into control group, LPS model group, L. nervosa extract group, and LPS + L. nervosa group. LPS was utilized to induce a neuroinflammatory cell model in BV-2 microglial cells. The Griess test was utilized for detecting the production of nitric oxide(NO) in the cell supernatant. Cell viability was detected by MTT assay. The release of interleukin-6(IL-6) and tumor necrosis factor alpha(TNF-α) in the cell supernatant was quantified using ELISA.RT-qPCR was utilized to assess the m RNA levels of pro-inflammatory cytokines inducible nitric oxide synthase(iNOS), cyclooxygenase-2(COX-2), interleukin( IL)-6, IL-1β, and TNF-α. The protein expression of i NOS, COX-2, nuclear factor kappa-B p65(p65), p-p65, extracellular signal-regulated kinase(ERK), p-ERK, c-jun N-terminal kinase(JNK), p-JNK, p38 mitogen-activated protein kinase(p38), and p-p38 MAPK(p-p38) were also evaluated by Western blot. The chemical composition of active substances in L. nervosa was analyzed using the UHPLC-Q-Exactive Orbitrap technology and literature comparison. Our findings indicate that extracts from different parts of L. nervosa exhibit a significant reduction in the release of NO from LPS-induced BV-2 microglial cells.Specifically, the ethyl acetate extract demonstrates the most notable inhibitory effect without causing cell toxicity. Additionally, the distillate(6) extracted from the ethyl acetate exhibits a reduction in the m RNA and protein levels of i NOS, COX-2, IL-6, IL-1β, and TNF-α in a dose-dependent manner, and it inhibits the protein expression of p-p65, p-ERK, p-p38, and p-JNK in LPS-induced BV-2 microglial cells. A total of 79 compounds in the distillate(6) were identified by mass spectrometry, including 12 confirmed compounds with anti-inflammatory effects. This study confirmed the remarkable efficacy of L. nervosa extract in the treatment of neuroinflammation, which may be achieved through the inhibition of NF-κB and MAPK signaling pathways.

First-in-human study of HMPL-295, an ERK1/2 inhibitor, in patients with advanced solid tumors: Dose-escalation results of monotherapy.

e15112 Background: HMPL-295 (’295) is an extracellular signal-regulated kinase 1 and 2 (ERK1/2) inhibitor with potent activity in non-clinical studies. This is a first-in-human, dose escalation study of ’295 in patients (pts) with advanced solid tumors. Here, we report the results of the mono dose escalation stage. Methods: Pts with advanced solid tumors who have failed standard therapy were enrolled to receive ’295 once daily (QD) in 28-day cycles or ’295 QD 2 weeks on/1 week off (2w on/1w off) in 21-day cycles. The mTPI-2 design was used for dose escalation, having explored from 5 to 75 mg QD in 6 cohorts, as well as 75 mg and 100 mg QD 2w on/1w off. The study aims to determine the maximum tolerated dose (MTD)/recommended phase 2 dose (RP2D), safety, tolerability, pharmacokinetics/pharmacodynamics (PK/PD), and preliminary efficacy (best of response, BOR). Results: As of Dec 11, 2023, 47 pts with advanced solid tumors were enrolled (n=1, 1, 3, 6, 12, 5, 13, 6 in 5, 10, 20, 40, 50, 75 mg QD cohorts, 75, 100 mg QD 2w on/1w off cohorts, respectively), with a median age of 58 years, 23 (48.9%) male pts. During the dose escalation from 5 to 75 mg QD cohort, 1 pt given 40 mg QD experienced a dose-limiting toxicity (DLT) of grade 3 dermatitis acneiform and 2 pts given 75 mg QD experienced DLTs of grade 3 rash and acute kidney injury (AKI). As for intermittent-administration cohorts, 2 pts given 100 mg QD 2w on/1w off experienced DLTs of grade 3 AKI and rash maculo-papular. After multiple-dose oral administration, ’295 plasma concentrations reach peak at 1.0-2.0 hours post dose and the exposure (Cmax and AUC) displayed dose dependent increase. The accumulation ratio (AUC) was 2.1–3.4 after repeat dosing, reaching steady-state around day 15. ’295 inhibited ribosomal S6 kinase (RSK) phosphorylation which is a downstream signaling molecule regulated by ERK1/2 and stimulated by phorbol 12-myristate 13-acetate (PMA). The BOR was partial response each in 1 pt with duodenal adenocarcinoma in 40 mg QD cohort,1 pt with endometrial cancer in 50 mg QD cohort and 1 pt with non-small cell lung cancer (NSCLC) in 75 mg QD 2w on/1w off cohort. Stable disease with tumor shrinkage was also observed in 2 pts with NSCLC on 50 mg QD and 75 mg QD 2w on/1w off cohort respectively, and 1 pt with pancreatic ductal adenocarcinoma in 100 mg QD 2w on/1w off cohort. 25 (53.2%) pts reported grade ≥3 TEAEs. The most common (≥10.0%) ≥ grade 3 TEAEs were rash (10.6%) and anemia (10.6%). Conclusions: Considering safety, tolerability, PK/PD and preliminary efficacy results, RP2D was determined to be 50 mg QD. Clinical trial information: NCT04908046 .

Multi-omics analysis reveals mechanism of Schisandra chinensis lignans and acteoside on EMT in hepatoma cells via ERK1/2 pathway.

Hepatocellular carcinoma (HCC), a globally common cancer, often presents late and shows high resistance to chemotherapy, resulting in suboptimal treatment efficacy. Components from traditional Chinese medicines have been recognized for their anti-cancer properties. Exploring the mechanism of Schisandra chinensis lignans and acteoside in suppressing Epithelial-Mesenchymal Transition (EMT) in hepatoma cells through the Extracellular signal-Regulated Kinases (ERK)1/2 pathway and identifying biomarkers, molecular subtypes, and targets via multi-omics for precision oncology. Proliferation was assessed using cell counting kit-8 (CCK-8) assays, with scratch and transwell assays for evaluating invasion and migration. Flow cytometry quantified apoptosis rates. Expression levels of CCL20, p-ERK1/2, c-Myc, Vimentin, and E-cadherin/N-cadherin were analyzed by real-time PCR and Western blot. Tumor volume was calculated with a specific formula, and growth. The Schisandra chinensis lignans and acteoside combination decreased CCL20 expression, inhibited hepatoma proliferation and migration, and enhanced apoptosis in a dose- and time-dependent manner. Molecular analysis revealed increased E-cadherin and decreased N-cadherin, p-ERK1/2, c-Myc, and Vimentin expression, indicating ERK1/2 pathway modulation. In vivo, treated nude mice showed significantly reduced tumor growth and volume. Schisandra chinensis lignans and acteoside potentially counteract CCL20-induced EMT, invasion, and migration in hepatocellular carcinoma cells via the ERK1/2 pathway, enhancing apoptosis. Multi-omics analysis further aids in pinpointing novel biomarkers for precision cancer therapy.

Effects of diet-induced metabolic syndrome on cardiac function and angiogenesis in response to the sodium-glucose cotransporter-2 inhibitor canagliflozin

IntroductionSodium-glucose cotransporter-2 inhibitors are antidiabetic medications that have been shown to decrease cardiovascular events and heart failure-related mortality in clinical studies. We attempt to examine the complex interplay between metabolic syndrome and the sodium-glucose cotransporter-2 inhibitor canagliflozin (CAN) in a clinically relevant model of chronic myocardial ischemia. MethodsTwenty-one Yorkshire swine were fed a high-fat diet starting at 6 weeks of age to induce metabolic syndrome. At 11 weeks, all underwent placement of an ameroid constrictor around the left circumflex coronary artery to induce chronic myocardial ischemia. After 2 weeks, swine received either control (CON) (n = 11) or CAN 300 mg by mouth daily (n = 10) for 5 weeks, whereupon all underwent terminal harvest. ResultsThere was a significant increase in cardiac output and heart rate with a decrease in pulse pressure in the CAN group compared with CON (all P values < .05). The CAN group had a significant increase in capillary density (P = .02). There was no change in myocardial perfusion or arteriolar density. CAN induced a significant increase in markers of angiogenesis, including Phospho-endothelial nitric oxide synthase, Endothelial nitric oxide synthase, vascular endothelial growth factor receptor-1, heat shock protein 70, and extracellular signal-regulated kinases (all P values < .05), plausibly resulting in capillary angiogenesis. ConclusionsCAN treatment leads to a significant increase in capillary density and augmented cardiac function in a swine model of chronic myocardial ischemia in the setting of metabolic syndrome. This work further elucidates the mechanism of sodium-glucose cotransporter-2 inhibitors in patients with cardiac disease; however, more studies are needed to determine if this increase in capillary density plays a role in the improvements seen in clinical studies.

Therapeutic Effects of Melatonin in the Regulation of Ferroptosis: A Review of Current Evidence.

Ferroptosis is implicated in the pathogenesis of multiple diseases, including neurodegenerative diseases, cardiovascular diseases, kidney pathologies, ischemia-reperfusion injury, and cancer. The current review article highlights the involvement of ferroptosis in traumatic brain injury, acute kidney damage, ethanol-induced liver injury, and PM2.5-induced lung injury. Melatonin, a molecule produced by the pineal gland and many other organs, is well known for its anti- aging, anti-inflammatory, and anticancer properties and is used in the treatment of different diseases. Melatonin's ability to activate anti-ferroptosis pathways including sirtuin (SIRT)6/p- nuclear factor erythroid 2-related factor 2 (Nrf2), Nrf2/ antioxidant responsive element (ARE)/ heme oxygenase (HO-1)/SLC7A11/glutathione peroxidase (GPX4)/ prostaglandin-endoperoxide synthase 2 (PTGS2), extracellular signal-regulated kinase (ERK)/Nrf2, ferroportin (FPN), Hippo/ Yes-associated protein (YAP), Phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT)/ mammalian target of rapamycin (mTOR) and SIRT6/ nuclear receptor coactivator 4 (NCOA4)/ ferritin heavy chain 1 (FTH1) signaling pathways suggests that it could serve as a valuable therapeutic agent for preventing cell death associated with ferroptosis in various diseases. Further research is needed to fully understand the precise mechanisms by which melatonin regulates ferroptosis and its potential as a therapeutic target.

Effects of β-asarone on spatial learning and memory impairment by exhaustive exercise-induced fatigue: Role of NR-CaMKII-ERK/CREB signal in hippocampus of rats

ObjectiveIt is to investigate the effects of β-asarone on learning and memory, hippocampal morphology, synaptophysin (SYP) and postsynaptic density 95(PSD95) protein expression, N-methyl-D-aspartic acid receptor 2B (NR2B)- Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) - Extracellular signal-regulated kinase (ERK) / Cyclic-AMP response element binding protein (CREB) signal in hippocampus of rats with exhaustive exercise-induced fatigue. MethodsFifty Sprague-Dawley male rats were randomly divided into five groups: normal group, exercise group, exercise and β-asarone (2.5, 10, 40 mg/kg)–treated groups. The learning and memory in rats were tested by Morris water maze experiment. We measured the hippocampal morphology by Nissl staining. The levels of SYP, PSD95, NR2B, CaMKII, ERK1/2, CREB, p-NR2B, p-CaMKII, p-ERK1/2 and p-CREB expression were measured by western blot analysis. ResultsThe results demonstrated that β-asarone (10, 40 mg/kg) treatment significantly decreased the latency to find the platform, increased the time spent in the target quadrant and the number of crossing the platform of rats with exhaustive exercise-induced fatigue. β-asarone (10, 40 mg/kg) treatment increased the cell density in the hippocampus CA1 region, significantly up-regulated NR2B-CaMKII-ERK/CREB signal and improved the protein expression levels of SYP and PSD95 in hippocampus of rats with exhaustive exercise-induced fatigue. ConclusionsIt suggests that β-asarone could improve learning and memory of rats with exhaustive exercise-induced fatigue. The mechanism might be related to β-asarone protecting the morphology of hippocampus, increasing the protein expression levels of SYP and PSD95 and up-regulating NR2B-CaMKII-ERK/CREB signal in hippocampus of rats with exhaustive exercise-induced fatigue.

Genistein alleviates doxorubicin-induced cardiomyocyte autophagy and apoptosis via ERK/STAT3/c-Myc signaling pathway in rat model.

Doxorubicin (Dox) is a highly effective anti-neoplastic agent. Still, its utility in the clinic has been hindered by toxicities, including vomiting, hematopoietic suppression and nausea, with cardiotoxicity being the most serious side effect. Genistein (Gen) is a natural product with extensive biological effects, including anti-oxidation, anti-tumor, and cardiovascular protection. This study evaluated whether Gen protected the heart from Dox-induced cardiotoxicity and explored the underlying mechanisms. Male Sprague-Dawley (SD) rats were categorized into control (Ctrl), genistein (Gen), doxorubicin (Dox), genistein 20 mg/kg/day + doxorubicin (Gen20 + Dox) and genistein 40 mg/kg/day + doxorubicin (Gen40 + Dox) groups. Six weeks after injection, immunohistochemistry (IHC), transmission electron microscopy (TEM), and clinical cardiac function analyses were performed to evaluate the effects of Dox on cardiac function and structural alterations. Furthermore, each heart histopathological lesions were given a score of 0-3 in compliance with the articles for statistical analysis. In addition, molecular and cellular response of H9c2 cells toward Dox were evaluated through western blotting, Cell Counting Kit-8 (CCK8), AO staining and calcein AM/PI assay. Dox (5 μM in vitro and 18 mg/kg in vivo) was used in this study. In vivo, low-dose Gen pretreatment protected the rat against Dox-induced cardiac dysfunction and pathological remodeling. Gen inhibited extracellular signal-regulated kinase1/2 (ERK1/2)'s phosphorylation, increased the protein levels of STAT3 and c-Myc, and decreased the autophagy and apoptosis of cardiomyocytes. U0126, a MEK1/2 inhibitor, can mimic the effect of Gen in protecting against Dox-induced cytotoxicity both in vivo and in vitro. Molecular docking analysis showed that Gen forms a stable complex with ERK1/2. Gen protected the heart against Dox-induced cardiomyocyte autophagy and apoptosis through the ERK/STAT3/c-Myc signaling pathway.

Peripheral Rho-associated protein kinase activation mediates acupuncture analgesia

BackgroundAcupuncture has been proven effective for various types of pain, and peripheral molecular signals around acupuncture-treated areas have been suggested to contribute to the analgesic effects of acupuncture. However, the underlying mechanism from these peripheral molecular signals to central ones remains unclear. The purpose of this study was to investigate whether peripheral Rho-associated protein kinase (ROCK) activation induced by acupuncture treatment mediates acupuncture analgesia, and also to investigate the relationship between ROCK activation and extracellular signal-regulated kinase (ERK), which has previously been proven to mediate acupuncture analgesia and other related molecular changes during acupuncture. MethodsAcupuncture was treated at the bilateral GB34 acupoints of C57BL/6 mice, after which changes in ROCK activation and the location of its expression in the skin were analyzed. To verify the role of ROCK in acupuncture analgesia, we administrated ROCK inhibitor Y-27632 (0.3 μg/ul) into the skin before acupuncture treatment with formalin and complete Freund adjuvant (CFA) induced pain models, then the nociceptive responses were analyzed. ResultsAcupuncture treatment produced ROCK2 activation in the skin after 30 and 60 min, and the histological analyses revealed that ROCK2 was activated in the fibroblast of the dermis. The acupuncture-induced ROCK2 expression was significantly attenuated by the ERK inhibitor, whereas phospho-ERK expression was not inhibited by ROCK inhibitor. In both the formalin- and CFA-induced mouse pain models, acupuncture analgesia was blocked by ROCK inhibitor administration. ConclusionAcupuncture treatment-induced ROCK2 expression is a downstream effector of phospho-ERK in the skin and plays a crucial role in acupuncture analgesia.

Functional Role of DUSP6 in the EGFR-mediated Progression of Head and Neck Squamous Cell Carcinoma.

Epidermal growth factor receptor (EGFR) over-expression is commonly observed in advanced head and neck squamous cell carcinoma (HNSCC) and is correlated with poor patient outcomes. However, the role of dual-specificity phosphatase 6 (DUSP6) in EGFR-associated HNSCC progression remains poorly understood. This study aimed to investigate the correlation between DUSP6 expression and EGFR signaling in malignant HNSCC tissues. Data mining and in vitro assays were employed to assess DUSP6 expression levels in HNSCC tissues compared to normal tissues. Additionally, the correlation between DUSP6 and EGFR expression was examined. Functional assays were conducted to investigate the modulation of DUSP6 expression by EGFR signaling and its involvement in EGF-induced cell migration and anoikis resistance. Our analysis revealed a significant elevation in DUSP6 expression in HNSCC tissues compared to normal tissues and a strong correlation between DUSP6 and EGFR expression. EGFR signaling modulated DUSP6 expression in a dose- and time-dependent manner, primarily through the extracellular signal-regulated kinase (ERK) pathway. Knockdown experiments demonstrated the functional role of DUSP6 in EGF-induced cell migration and anoikis resistance. The findings of this study elucidate the intricate signaling networks governing DUSP6 expression and its interplay with EGFR signaling in HNSCC. Moreover, the results provide insights into the potential role of DUSP6 as a therapeutic target and highlight the importance of personalized treatment strategies in HNSCC management.

Large-scale control over collective cell migration using light-controlled epidermal growth factor receptors.

Receptor tyrosine kinases (RTKs) are thought to play key roles in coordinating cell movement at single-cell and tissue scales. The recent development of optogenetic tools for controlling RTKs and their downstream signaling pathways suggested these responses may be amenable to engineering-based control for sculpting tissue shape and function. Here, we report that a light-controlled EGF receptor (OptoEGFR) can be deployed in epithelial cell lines for precise, programmable control of long-range tissue movements. We show that in OptoEGFR-expressing tissues, light can drive millimeter-scale cell rearrangements to densify interior regions or produce rapid outgrowth at tissue edges. Light-controlled tissue movements are driven primarily by PI 3-kinase signaling, rather than diffusible signals, tissue contractility, or ERK kinase signaling as seen in other RTK-driven migration contexts. Our study suggests that synthetic, light-controlled RTKs could serve as a powerful platform for controlling cell positions and densities for diverse applications including wound healing and tissue morphogenesis.

Equine Endothelial Cells Show Pro-Angiogenic Behaviours in Response to Fibroblast Growth Factor 2 but Not Vascular Endothelial Growth Factor A.

Understanding the factors which control endothelial cell (EC) function and angiogenesis is crucial for developing the horse as a disease model, but equine ECs remain poorly studied. In this study, we have optimised methods for the isolation and culture of equine aortic endothelial cells (EAoECs) and characterised their angiogenic functions in vitro. Mechanical dissociation, followed by magnetic purification using an anti-VE-cadherin antibody, resulted in EC-enriched cultures suitable for further study. Fibroblast growth factor 2 (FGF2) increased the EAoEC proliferation rate and stimulated scratch wound closure and tube formation by EAoECs on the extracellular matrix. Pharmacological inhibitors of FGF receptor 1 (FGFR1) (SU5402) or mitogen-activated protein kinase (MEK) (PD184352) blocked FGF2-induced extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and functional responses, suggesting that these are dependent on FGFR1/MEK-ERK signalling. In marked contrast, vascular endothelial growth factor-A (VEGF-A) had no effect on EAoEC proliferation, migration, or tubulogenesis and did not promote ERK1/2 phosphorylation, indicating a lack of sensitivity to this classical pro-angiogenic growth factor. Gene expression analysis showed that unlike human ECs, FGFR1 is expressed by EAoECs at a much higher level than both VEGF receptor (VEGFR)1 and VEGFR2. These results suggest a predominant role for FGF2 versus VEGF-A in controlling the angiogenic functions of equine ECs. Collectively, our novel data provide a sound basis for studying angiogenic processes in horses and lay the foundations for comparative studies of EC biology in horses versus humans.

Beneficial effect of heat-killed Lactiplantibacillus plantarum L-137 on intestinal barrier function of rat small intestinal epithelial cells

Heat-killed Lactiplantibacillus plantarum L-137 (HK L-137) has been suggested to enhance the intestinal barrier in obese mice, leading to improvement of metabolic abnormalities and adipose tissue inflammation, and in healthy humans with overweight, leading to improvement of systemic inflammation. However, its detailed mechanism of action has not been clarified. Therefore, this study investigated the effects of HK L-137 on the permeability of rat small intestinal epithelial IEC-6 cells, tight junction-related gene and protein expression and localization, and intracellular signaling pathways involved in barrier function. Treatment of IEC-6 cells with HK L-137 for 26 h significantly reduced the permeability to fluorescein isothiocyanate-dextran (FD-4). HK L-137 also increased gene and protein expression of zonula occludens-1 (ZO-1), an important tight junction protein, without affecting the localization. Furthermore, inhibition of the extracellular signal-regulated kinase (ERK)1/2 pathway in IEC-6 cells canceled the HK L-137-related reduction in permeability to FD-4. Phosphorylation of ERK in IEC-6 cells was induced 15 min after the addition of HK L-137. These results suggest that HK L-137 reduces intestinal permeability partly through activating the ERK pathway and increasing expression of the ZO-1 gene and protein. Enhancement of intestinal barrier function with HK L-137 might be effective in preventing and treating leaky gut, for which no specific therapeutic tool has been established.