P38 Mitogen-activated Protein Kinase Inhibitor Research Articles

EPO Deficiency Upregulates GADD45b/p38 MAPK Axis, Mediating Schizophrenia-Related Synaptic and Cognitive Impairments.

Schizophrenia (SZ) is a chronic and severe mental illness associated with psychiatric symptoms, cognitive deficits, and social dysfunction. Current clinical interventions only limit relief of psychiatric symptoms and have minimal impact on cognitive impairments. Erythropoietin (EPO), known for its role in neurogenesis and synaptic plasticity, is significantly low in SZ patients. However, the role of EPO deficiency in SZ-associated cognitive deficits remains unclear. In this study, we used the MK801-induced SZ rat model to show that EPO levels were significantly decreased, correlating with cognitive impairments. EPO supplementation mitigated apoptosis, synaptic damage, and cognitive impairments caused by MK801. RNA-sequencing and Western blot analysis revealed increased expression of growth arrest and DNA damage 45b (GADD45b) in MK801-treated rats, reversed by EPO supplementation. Moreover, overexpression of GADD45b exacerbated neuronal loss and cognitive impairments in male Sprague-Dawley rats, while downregulation of GADD45b rescued these SZ-related pathologies. Notably, the benefits of EPO supplementation on SZ pathology were blocked by GADD45b overexpression. Inhibition of p38 MAPK, a GADD45b target, reduced MK801-induced apoptosis and synaptic damage. These findings uncover a novel etiopathogenic mechanism of SZ-related cognitive impairments, driven by EPO deficiency and the activation of the GADD45b/p38 MAPK axis.

Corrigendum: CHF6297: a novel potent and selective p38 MAPK inhibitor with robust anti-inflammatory activity and suitable for inhaled pulmonary administration as dry powder

Corrigendum: CHF6297: a novel potent and selective p38 MAPK inhibitor with robust anti-inflammatory activity and suitable for inhaled pulmonary administration as dry powder

Mevastatin-Induced HO-1 Expression in Cardiac Fibroblasts: A Strategy to Combat Cardiovascular Inflammation and Fibrosis.

Mevastatin (MVS) is known for its anti-inflammatory effects, potentially achieved by upregulating heme oxygenase-1 (HO-1), an enzyme involved in cytoprotection against oxidative injury. Nonetheless, the specific processes by which MVS stimulates HO-1 expression in human cardiac fibroblasts (HCFs) are not yet fully understood. In this study, we found that MVS treatment increased HO-1 mRNA and protein levels in HCFs. This induction was inhibited by pretreatment with specific inhibitors of p38 MAPK, JNK1/2, and FoxO1, and by siRNAs targeting NOX2, p47phox, p38, JNK1, FoxO1, Keap1, and Nrf2. MVS also triggered ROS generation and activated JNK1/2 and p38 MAPK, both attenuated by NADPH oxidase or ROS inhibitors. Additionally, MVS promoted the phosphorylation of FoxO1 and Nrf2, which was suppressed by p38 MAPK or JNK1/2 inhibitor. Furthermore, MVS inhibited TNF-α-induced NF-κB activation and vascular cell adhesion molecule-1 (VCAM-1) expression via the HO-1/CO pathway in HCFs. In summary, the induction of HO-1 expression in HCFs by MVS is mediated through two primary signaling pathways: NADPH oxidase/ROS/p38 MAPK, and JNK1/2/FoxO1 and Nrf2. This research illuminates the underlying processes through which MVS exerts its anti-inflammatory effects by modulating HO-1 in cardiac fibroblasts.

Trehalose Attenuates In Vitro Neurotoxicity of 6-Hydroxydopamine by Reducing Oxidative Stress and Activation of MAPK/AMPK Signaling Pathways.

The effects of trehalose, an autophagy-inducing disaccharide with neuroprotective properties, on the neurotoxicity of parkinsonian mimetics 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpiridinium (MPP+) are poorly understood. In our study, trehalose suppressed 6-OHDA-induced caspase-3/PARP1 cleavage (detected by immunoblotting), apoptotic DNA fragmentation/phosphatidylserine externalization, oxidative stress, mitochondrial depolarization (flow cytometry), and mitochondrial damage (electron microscopy) in SH-SY5Y neuroblastoma cells. The protection was not mediated by autophagy, autophagic receptor p62, or antioxidant enzymes superoxide dismutase and catalase. Trehalose suppressed 6-OHDA-induced activation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and AMP-activated protein kinase (AMPK), as revealed by immunoblotting. Pharmacological/genetic inhibition of JNK, p38 MAPK, or AMPK mimicked the trehalose-mediated cytoprotection. Trehalose did not affect the extracellular signal-regulated kinase (ERK) and mechanistic target of rapamycin complex 1 (mTORC1)/4EBP1 pathways, while it reduced the prosurvival mTORC2/AKT signaling. Finally, trehalose enhanced oxidative stress, mitochondrial damage, and apoptosis without decreasing JNK, p38 MAPK, AMPK, or AKT activation in SH-SY5Y cells exposed to MPP+. In conclusion, trehalose protects SH-SY5Y cells from 6-OHDA-induced oxidative stress, mitochondrial damage, and apoptosis through autophagy/p62-independent inhibition of JNK, p38 MAPK, and AMPK. The opposite effects of trehalose on the neurotoxicity of 6-OHDA and MPP+ suggest caution in its potential development as a neuroprotective agent.

Acupuncture improves spatial learning and memory impairment caused by herpes simplex virus type-1 in rats through the p38 MAPK/CREB pathway

BackgroundAcupuncture can improve herpes simplex encephalitis (HSE), but the underlying mechanism is not clear. Therefore, we evaluated the cognitive function and apoptosis in hippocampus caused by herpes simplex virus type-1 (HSV-1) in rats after acupuncture and described the molecular mechanism.MethodsSprague–Dawley rats were induced into HSE models by HSV-1 infection. After 3 days, they received acupuncture at the acupoints of Xuanzhong (GB39), Baihui (GV20), Shenmen (HT7), Shenting (GV24), and Sanyinjiao (SP6), and/or intraperitoneal injection of the p38 MAPK inhibitor SB203580. Morris water maze test was performed on rats. The hippocampus of rats was obtained, and the expression of apoptosis-related genes in the tissues was detected by qRT-PCR. In addition, apoptosis-related proteins and proteins related to the p38 MAPK/CREB pathway in the tissues was detected by western blot.ResultsAfter HSV-1 induction, the rat's escape latency was increased, the time spent on the platform in the target quadrant and the number of platform crossings significantly decreased. In addition, there was an increase in apoptosis in the hippocampus, accompanied by elevated levels of p–p38 and decreased levels of p-CREB. However, these effects could be improved by acupuncture treatment. Interestingly, SB203580 plays a similar role to acupuncture, and acupuncture could further enhance the impacts of SB203580 on cognitive function and apoptosis in hippocampus in HSE rats.ConclusionAcupuncture improves spatial learning and memory impairment caused by HSV-1 in rats. The functional mechanism of acupuncture may be through the p38 MAPK/CREB pathway.

Intrinsic temperature increase drives lipid metabolism towards ferroptosis evasion and chemotherapy resistance in pancreatic cancer

A spontaneously occurring temperature increase in solid tumors has been reported sporadically, but is largely overlooked in terms of cancer biology. Here we show that temperature is increased in tumors of patients with pancreatic ductal adenocarcinoma (PDAC) and explore how this could affect therapy response. By mimicking this observation in PDAC cell lines, we demonstrate that through adaptive changes in lipid metabolism, the temperature increase found in human PDAC confers protection to lipid peroxidation and contributes to gemcitabine resistance. Consistent with the recently uncovered role of p38 MAPK in ferroptotic cell death, we find that the reduction in lipid peroxidation potential following adaptation to tumoral temperature allows for p38 MAPK inhibition, conferring chemoresistance. As an increase in tumoral temperature is observed in several other tumor types, our findings warrant taking tumoral temperature into account in subsequent studies related to ferroptosis and therapy resistance. More broadly, our findings indicate that tumoral temperature affects cancer biology.

Withanolides-enriched leaf extract of Withania somnifera exert anti-obesity effects by inducing brown adipocyte-like phenotype via tuning MAP-kinase signaling axis

Present study investigated anti-obesity potential of Withania somnifera (L.) Dunal leaf extract (WSLE). Phytochemical characterization of WSLE was performed by UPLC/MS-QToF and HPLC-based analysis. WSLE was assessed for its effect on lipid metabolism and mitochondrial biogenesis in vitro using differentiated 3 T3-L1 adipocytes. WSLE was found to contain 59 phytometabolites with a total of 10.601 μg withanolides per mg of extract. WSLE (30 μg/ml) treatment decreased basal levels of intracellular lipids and triglycerides to 13.85 % and 41.58 %, respectively. WSLE downregulated the expression of PPARγ, C/EBPα, C/EBPβ, and their target genes responsible for lipogenesis dose-dependently. An upregulation in expression of lipolytic (ATGL and HSL), thermogenic (PGC1α, UCP1, and PRDM16), and glucose transporter (GLUT4) genes was also observed. Furthermore, WSLE treatment increased glucose uptake by 1.5-fold. These beneficial effects of WSLE were abolished in presence of AMPK, p38MAPK, and ERK inhibitors. These observations were then validated in vivo using Caenorhabditis elegans as a model organism. Intriguingly, WSLE diminished fat accumulation in wild-type N2 worms as evident from reduced Oil-red-O staining and reduction in GFP expression of fat-5, 6, and 7 in transgenic strains. Overall, these results highlight anti-obesity potential of WSLE exerting its effects via alterations in AMPK/p38MAPK/ERK axis.

The antiviral effect and potential mechanism of Houttuynia cordata thunb. (HC) against coxsackievirus A4

Ethnopharmacological relevanceHand, foot, and mouth disease (HFMD) is mainly caused by various of enteroviruses such as enterovirus 71 (EVA71), coxsackievirus A16 (CVA16), CVA6, and CVA10 in infants and children under 5 years old. During the past 5 years, CVA4 has become the dominant pathogen resulting in HFMD in China. However, there are no effective vaccines and antiviral drugs available. Houttuynia cordata Thunb (HC). is a Chinese herbal medicine eaten as vegetables for treating viral infection diseases, but whether HC has anti-CVA4 effect remains unclear. Aim of the studyIn this study, we want to investigate the antiviral activity of HC against CVA4 in vitro and in vivo and elucidate the potential mechanism of HC against CVA4. Materials and methodsMTT assay were used to evaluate the cytotoxicity of HC. Virus titers assay, CPE assay, violet staining and immunofluorescence were used to investigate the antiviral effect of HC against CVA4. A 13-day-old suckling mice model was established to evaluate the therapeutic efficacy of HC against CVA4 infection. Western blot, qRT-PCR and time-of-drug addition assay were performed to elucidate the potential mechanism of HC against CVA4 infection. ResultsMTT assay indicated the cytotoxicity concentration of HC on Vero cells and RD cells were more than 1 mg/ml, suggesting that the low cytotoxicity of HC. In vitro antiviral assay revealed that HC could dose-dependently prevent the CPE, suppress the release of newborn virus, and inhibit the replication of CVA4 by decreasing viral RNA transcription and protein expression with IC50 of 88.96 μg/mL. A time-of-addition assay showed that HC mainly exerted anti-CVA4 effect by inhibiting virus replication at the post-entry stage. In vivo results further demonstrated that HC could effectively prevent the lethal infection of CVA4 by promoting survival, improving clinical symptoms, prolonging the survival time, inhibiting excessive inflammatory responses, and reducing pathological injury in vivo. Mechanistic studies revealed inhibition of p38 MAPK and JNK pathway over-activation may be the primary mechanism of HC against CVA4 infection. ConclusionIn summary, our results for the first time demonstrated that HC not only effectively inhibited CVA4 replication, but also partially protected the lethal infection of CVA4 in vivo. Furthermore, pharmacological mechanism studies revealed that the primary mechanism of HC against CVA4 infection may be associated with its effect of inhibiting over-activation of p38 MAPK and JNK signaling pathways caused by enteroviruses. Our finding indicated that HC might be a potential innovative medicine for treating HFMD.

Neuroprotective Effect of Codonopsis pilosula Polysaccharide on Aβ25-35-Induced Damage in PC12 Cells via the p38MAPK Signaling Pathways.

Plant polysaccharides have attracted increasing attention due to their high efficiency and low toxicity. Codonopsis pilosula polysaccharide (CPP) is an essential substance extracted from Codonopsis pilosula, known for its excellent antioxidant and neuroprotective effects. However, it is still unclear how CPP improves nerve protection and what its underlying molecular mechanisms are. This study aimed to investigate the neuroprotective effect of CPP on Aβ25-35-induced damage in PC12 cells and its underlying molecular mechanisms. The neuroprotective effect of CPP was evaluated using Aβ25-35-induced damage in pheochFfromocytoma (PC12) cells as an in vitro cell model. The cells were treated with CPP alone or in combination with SB203580 (an inhibitor of p38MAPK) in Aβ25-35 culture. The cell viability was assessed using a 3-(4,5-Dimethylthiazol-2-yl)-2,diphenyltetrazolium (MTT) assay. Furthermore, reactive oxygen species (ROS) were detected using flow cytometry. The production levels of intracellular superoxide dismutase (SOD), dismutase (SOD), glutathione (GSH), catalase (CAT), and malondialdehyFde (MDA) were determined using the colorimetric method. Annexin V-FITC and propidium iodide (PI) staining, as well as 33258 were performed using fluorescence microscopy. Moreover, the effect of adding SB203580 was studied to determine the changes in cell apoptosis induced by CPP treatment and Aβ25-35 induction. The CPP markedly inhibited Aβ25-35-induced reduction in the viability and apoptosis of PC12 cells. CPP also reduced the Aβ25-35-induced increase in the expression of the apoptosis factors and the levels of free radicals (ROS and MDA) and reversed the Aβ25-35-induced suppression of antioxidant activity. Additionally, inhibition of p38MAPK via the addition of their antagonists reversed the observed anti-apoptosis effects of CPP. CPP can efficiently provide neuroprotection against Aβ25-35-induced damage in PC12 cells brought about via oxidation and apoptosis reactions, and the underlying mechanisms involve the p38MAPK pathways. Therefore, CPP could potentially be useful as a neuroprotective agent in natural medicine, pharmacy, and the food industry.

S-Methyl-L-cysteine targeting MsrA attenuates Ang II-induced oxidative stress and atrial remodeling via the p38 MAPK signaling pathway.

Atrial fibrillation (AF) is the most prevalent sustained tachyarrhythmia in patients with cardiovascular diseases. Recently, it has been discovered that oxidative stress is an important contributor to AF. Therefore, antioxidant therapies for AF have great potential for clinical applications. Methionine, a sulfur-containing amino acid residue other than cysteine, is recognized as a functional redox switch, which could be rescued from the reversible oxidation of methionine sulfoxide by methionine sulfoxide reductase A (MsrA). S-Methyl-L-cysteine (SMLC), a natural analogue of Met, which is abundantly found in garlic and cabbage, could substitute for Met oxidations and mediate MsrA to scavenge free radicals. However, whether SMLC alleviates AF is unclear. This study aims to clarify the effects of SMLC on AF and elucidate the underlying pharmacological and molecular mechanisms. In vivo, SMLC (70, 140 and 280 mg kg-1 day-1) was orally administered to mice for 4 weeks with angiotensin II (Ang II) by subcutaneous infusion using osmotic pumps to induce AF. Ang II significantly prompted high AF susceptibility and atrial remodeling characterized by oxidative stress, conductive dysfunction and fibrosis. SMLC played a remarkable protective role in Ang II-induced atrial remodeling dose-dependently. Moreover, RNA sequencing was performed on atrial tissues to identify the differentially expressed mRNA, which was to screen out MSRA, CAMK2 and MAPK signaling pathways. Western blots confirmed that Ang II-induced downregulation of MsrA and upregulation of oxidized CaMKII (ox-CaMKII) and p38 MAPK could be reversed in a concentration-dependent manner by SMLC. To investigate the underlying mechanisms, HL-1 cells (mouse atria-derived cardiomyocytes) treated with Ang II were used for an in vitro model. SMLC alleviated Ang II-induced cytotoxicity, mitochondrial damage and oxidative stress. Additionally, knockdown MsrA could attenuate the protective effects of SMLC, which were eliminated by the p38 MAPK inhibitor SB203580. In summary, the present study demonstrates that SMLC protects against atrial remodeling in AF by inhibiting oxidative stress through the mediation of the MsrA/p38 MAPK signaling pathway.

Deletion of p38 MAPK in macrophages ameliorates peritoneal fibrosis and inflammation in peritoneal dialysis

One of the most common causes of peritoneal dialysis withdrawal is ultrafiltration failure which is characterized by peritoneal membrane thickening and fibrosis. Although previous studies have demonstrated the inhibitory effect of p38 MAPK inhibitors on peritoneal fibrosis in mice, it was unclear which specific cells contribute to peritoneal fibrosis. To investigate the role of p38 MAPK in peritoneal fibrosis more precisely, we examined the expression of p38 MAPK in human peritoneum and generated systemic inducible p38 MAPK knockout mice and macrophage-specific p38 MAPK knockout mice. Furthermore, the response to lipopolysaccharide (LPS) was assessed in p38 MAPK-knocked down RAW 264.7 cells to further explore the role of p38 MAPK in macrophages. We found that phosphorylated p38 MAPK levels were increased in the thickened peritoneum of both human and mice. Both chlorhexidine gluconate (CG)-treated systemic inducible and macrophage-specific p38 MAPK knockout mice ameliorated peritoneal thickening, mRNA expression related to inflammation and fibrosis, and the number of αSMA- and MAC-2-positive cells in the peritoneum compared to CG control mice. Reduction of p38 MAPK in RAW 264.7 cells suppressed inflammatory mRNA expression induced by LPS. These findings suggest that p38 MAPK in macrophages plays a critical role in peritoneal inflammation and thickening.

Deubiquitinase USP14 is upregulated in Crohn's disease and inhibits the NOD2 pathway mediated inflammatory response in vitro.

The nucleotide binding oligomerization domain containing 2 (NOD2) protein and its ligand N-acetyl muramyl dipeptide (MDP) are crucially involved in Crohn's disease (CD). However, the mechanism by which NOD2 signaling is regulated in CD patients remains unclear. Ubiquitin specific protease (USP14) is a deubiquitylase that plays an important role in immunity. This study aimed to investigate the mechanism by which UPS14 regulates NOD2 induced inflammatory response in CD and inflammatory bowel diseases (IBD). Our results showed that USP14 protein and mRNA levels in intestinal tissues of CD patients were significantly higher than those in healthy controls. In addition, USP14 was upregulated in IBD mouse model. While treatment with MDP, TNF-α or the Toll-like receptor 1/2 agonist Pam3CSK4 all led to significantly higher mRNA levels of TNF-α, IL-8 and IL-1β in THP-1 cells, pretreatment with USP14 inhibitor IU1 could stimulate further upregulation of TNF-α, IL-8 and IL-1β. In particular, MDP promoted the activation of JNK, ERK1/2 and p38 as well as NF-kB in THP-1 cells, and IU1 significantly enhanced the MDP-induced activation of these proteins without effects on USP14 protein level. Furthermore, the JNK inhibitor sp600125, ERK1/2 inhibitor U0126 or P38 MAPK inhibitor PD169316 significantly decreased the mRNA levels of TNF-α, IL-8 and IL-1β in THP-1 cells stimulated by both IU1 and MDP. In conclusion, our findings suggest that USP14 could inhibit MDP-induced activation of MAPK signaling and the inflammation response involved in IBD, and that USP14 is a potential therapeutic target for IBD.

The neo-potential therapeutic strategy in preeclampsia: downregulated miR-26a-2-3p motivates endothelial cell injury by targeting 15-LOX-1

Preeclampsia (PE) poses a life-threatening risk for both mothers and babies, and its onset and progression are linked to endothelial injury. The enzyme 15-lipoxygenase-1 (15-LOX-1), critical in arachidonic acid metabolism, is implicated in various diseases, yet its specific role and precise mechanisms in PE remain largely unknown. In this study, we found that 15-LOX-1 and its main metabolite, 15-HETE, were significantly increased in both the placenta and serum of PE patients. This increase was accompanied by elevated levels of endothelial injury markers, including intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). A positive correlation between 15-LOX-1 and those markers in the placenta. In Alox15-/- mice, Alox15 deficiency reduced endothelial cell injury in PE-like mice induced by L-NAME. In vitro studies showed that hypoxia-induced upregulation of 15-LOX-1 reduced the cell viability, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs), while increasing apoptosis and inflammatory cell adhesion. Mechanistically, the p38 MAPK pathway was identified as a downstream target of 15-LOX-1. Knocking down 15-LOX-1 or inhibiting p38 MAPK activation improved endothelial cell injury in hypoxia-treated HUVECs. Furthermore, downregulation of miR-26a-2-3p was found to correlate negatively and colocalize with 15-LOX-1 upregulation in the placenta of PE patients. Luciferase reporter assays further confirmed that miR-26a-2-3p directly bind to the 3’UTR of 15-LOX-1, targeting its expression. Moreover, miR-26a-2-3p agomir ameliorated the PE-like phenotype in mice through the 15-LOX-1/p38 MAPK axis, improving endothelial dysfunction. Therefore, our study provides novel insights into the pathogenesis of PE and highlight modulating the miR-26a-2-3p/15-LOX-1/p38 MAPK axis as a potential therapeutic target for PE.

Downregulation of nuclear receptor-binding SET domain protein 1 induces proinflammatory cytokine expression via mitogen-activated protein kinase pathways in U87MG cells

Haploinsufficiency of the nuclear receptor binding SET domain-containing protein 1 gene (NSD1) leads to a neurodevelopmental disorder known as Sotos syndrome (SOTOS). This study investigated the effects of NSD1 knockdown in glial cells. U87MG glioma cells were transfected with siRNA targeting NSD1, which resulted in morphological changes characteristic of activated astrocytes. These activated phenotypes were accompanied by specific activation of mitogen-activated protein kinase (MAPK) signaling pathways, particularly those mediated by p38 MAPK and c-Jun N-terminal kinase (JNK). Transcriptome analysis showed increased expression of proinflammatory cytokine genes, particularly interleukin (IL)-1α, IL-1β, and IL-6, following NSD1 knockdown. Treatment with MAPK inhibitors significantly reduced the cytokine induction caused by NSD1 knockdown, with the p38 MAPK inhibitor being more effective than the JNK inhibitor. These findings provide new insights into the role of NSD1 loss in neurological dysfunctions associated with SOTOS.

β-glucan improves intestinal health of pearl gentian grouper via activation of the p38 mitogen-activated protein kinase signaling pathway

Our previous study has demonstrated that supplementation of yeast β-glucan improves intestinal health in pearl gentian grouper (Epinephelus lanceolatus♂ × Epinephelus fuscoguttatus♀), accompanied by the activation of the mitogen-activated protein kinase (MAPK) signaling pathway. In this study, we investigated the effects of perturbing p38 MAPK activity using an inhibitor on the intestinal health of β-glucan-injected pearl gentian grouper to elucidate the potential molecular mechanism underlying the protective effects of β-glucan on the fish gut. The pearl gentian grouper was categorized into four groups: PBS injected (CD group), β-glucan injected at a dose of 80 mg/kg (βG group), p38 MAPK inhibitor SB203580 injected at a dose of 1 mg/kg (SB203580 group), and a combination of β-glucan (80 mg/kg) and SB203580 (1 mg/kg) injected together (βG + SB203580 group). The results revealed that the introduction of SB203580 significantly suppressed the β-glucan-induced increase in p38α and p38β mRNA expression, as well as the phosphorylation of p38 MAPK. Both the βG group and SB203580 group exhibited reduced plica height and muscularis thickness. The βG + SB203580 group displayed a significant reduction in mucin cell level; interleukin 1β (il1β) mRNA expression; induced nitric oxide synthase, tumor necrosis factor α, and IL1β concentration; catalase and total antioxidant capacity activities. Additionally, there was a significant increase in the levels of intestinal malondialdehyde in the βG + SB203580 group compared to the βG group. The inhibition of the p38 MAPK signaling halted the trend of apoptosis-related caspase molecular expression induced by β-glucan. In conclusion, β-glucan injection resulted in elevated levels of mucous cells, nonspecific immunity, antioxidant capacity, and anti-apoptosis in grouper by modulating the p38 MAPK pathway. This study offers insights into the potential molecular mechanism underlying the protective effects of β-glucan on intestinal health in pearl gentian grouper.

Porous Gelatin Methacrylate Gel Engineered by Freeze-Ultraviolet Promotes Osteogenesis and Angiogenesis.

Alveolar bone defect reconstruction is a common challenge in stomatology. To address this, a thermosensitive/photosensitive gelatin methacrylate (GelMA) gel was developed based on various air solubilities and light-curing technologies. The gel was synthesized by using a freeze-ultraviolet (FUV) method to form a porous and quickly (within 15 min) solidifying modified network structure. Unlike other gel scaffolds limited by complex preparation procedures and residual products, this FUV-GelMA gel shows favorable manufacturing ability, promising biocompatibility, and adjustable macroporous structures. The results from a rat model suggested that this gel scaffold creates a conducive microenvironment for mandible reconstruction and vascularization. In vitro experiments further confirmed that the FUV-GelMA gel promotes osteogenic differentiation of human bone marrow mesenchymal stem cells and angiogenesis of human umbilical vein endothelial cells. Investigation of the underlying mechanism focused on the p38 mitogen-activated protein kinase (MAPK) pathway. We found that SB203580, a specific inhibitor of p38 MAPK, abolished the therapeutic effects of the FUV-GelMA gel on osteogenesis and angiogenesis, both in vitro and in vivo. These findings introduced a novel approach for scaffold-based tissue regeneration in future clinical applications.

Losmapimod ameliorates doxorubicin-induced cardiotoxicity through attenuating senescence and inflammatory pathways

Irreversible cardiotoxicity limits the clinical application of doxorubicin (DOX). DOX-induced cardiotoxicity has been associated with induction of senescence and activation of the p38 MAPK pathway. Losmapimod (LOSM), an orally active p38 MAPK inhibitor, is an anti-inflammatory agent with cardioprotective effects. Nevertheless, the effect of LOSM against DOX-induced cardiotoxicity has not been reported. In this study, we determined the effects of LOSM on DOX-induced chronic cardiotoxicity in C57BL/6 N mice. Five-week-old C57BL/6 N mice were fed diet containing LOSM (estimated daily intake 12 mg/kg/day) or a control diet for four days. Thereafter, mice were randomized to receive six weekly intraperitoneal injections of either DOX (4 mg/kg) or saline. Three days after the last injection, cardiac function was assessed by trans-thoracic echocardiography. Activation of p38, JNK, and ERK1/2 MAPKs were assessed by immunoblotting in the heart and liver. Gene expressions of senescence, inflammatory, oxidative stress, and mitochondrial function markers were quantified using real-time PCR and serum inflammatory markers were assessed by Luminex. Our results demonstrated that LOSM attenuated p38 MAPK activation, ameliorated DOX-induced cardiac dysfunction, and abrogated DOX-induced expression of the senescence marker p21Cip1. Additionally, LOSM demonstrated anti-inflammatory effects, with reduced cardiac Il-1α and Il-6 gene expression in DOX-treated mice. Systemic inflammation, assessed by serum cytokine levels, showed decreased IL-6 and CXCL1 in both DOX-treated mice and mice on LOSM diet. LOSM significantly increased mitofusin2 gene expression, which may enhance mitochondrial fusion. These findings underscore the potential therapeutic efficacy of p38 MAPK inhibition, exemplified by LOSM, in ameliorating DOX-induced cardiotoxicity, senescence, and inflammation.

Abstract Mo001: Inhibition of TAOK1-mediated Cardiomyocyte Death Attenuates Doxorubicin-Induced Cardiotoxicity

Background and Objective: Doxorubicin (Dox)-induced cardiotoxicity (DIC) is one of the most serious side effects of cancer treatments. However, no effective treatment is currently available. In this study, we aimed to identify novel therapeutic targets using CRISPR/Cas9-based screening of human cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Methods and Results: We performed a kinase-focused CRISPR gene knockout screen in hPSC-CMs and identified thousand and one amino acid protein kinase 1 (TAOK1 ) as a potential regulator of DOX-induced cardiomyocyte death. We generated TAOK1-knockout (KO) hPSC-CMs using CRISPR/Cas9 and found that TAOK1-KO hPSC-CMs showed higher cell viability than control KO cells after 1μM DOX treatment (49.3% vs. 30.5%, P = 0.03). TAOK1-KO decreased DOX-induced ROS production (-58.8%, P = 0.01) and DNA damage (-28.8%, P < 0.01) in hPSC-CMs. Transcriptome analysis using RNA-seq of TAOK1-KO hPSC-CMs identified 483 differentially expressed genes, and Hallmark pathway analysis revealed upregulation of the oxidative phosphorylation pathway (FDR < 0.01) in TAOK1-KO hPSC-CMs compared to the control. Extracellular flux analysis demonstrated higher basal and maximal respiratory capacity in TAOK1-KO hPSC-CMs compared to control after DOX treatment (1.2 vs. 0.6, and 3.8 vs. 2.0 pmol/min/μg, respectively, P < 0.01 for all). Next, we overexpressed TAOK1 in hPSC-CMs using adenovirus vector and found that TAOK1 overexpression (OE) augmented DOX-induced cell viability reduction in hPSC-CMs compared to control after 0.5μM DOX treatment (-63.4 % vs. -19.2%, P < 0.01). Western blot analysis showed that DOX-induced p38 phosphorylation was suppressed by TAOK1-KO (P < 0.01) and enhanced (P = 0.01) by TAOK1-OE in hPSC-CMs. Augmented DOX-induced cardiomyocyte death in TAOK1-OE hPSC-CMs was partially rescued by inhibitors of p38 MAPK or apoptosis. Lastly, we demonstrated that TAOK1 suppression using AAV-mediated gene silencing mitigated the decline in the left ventricular ejection fraction in a mouse model of DIC (-30.0% vs. -17.7%, P < 0.01). Histological analysis of heart tissue revealed that TAOK1 suppression reduced myocardial fibrosis and apoptosis of cardiac cells in a mouse model of DIC (6.6% vs. 4.3%, P = 0.02, and 0.31% vs. 0.18%, P< 0.01, respectively). Conclusion: Our results suggest that TAOK1 regulates DOX-induced cardiomyocyte death; thus, its inhibition could be a potential therapeutic approach for DIC.

Abstract Tu055: Prominent effects of p38 MAPK inhibition on the phosphoproteome of a guinea pig model of heart failure and sudden cardiac death

Introduction: The p38 mitogen-activated protein kinase (p38 MAPK), activated by stress, plays a role in cardiac hypertrophy and heart failure (HF). The precise mechanism of action of p38 MAPK remains unclear, which could explain the mixed outcomes in P38 inhibitor trials. Here, we examine the impact of p38 MAPK inhibition on cardiac function and the proteome/phosphoproteome of the failing heart. Objective: To ascertain p38 MAPK’s impact on the progression of HF by inhibiting it with SB203580 (SB) in a guinea pig model of HF and sudden cardiac death. Methods: We assessed the proteomic changes in a previously described guinea pig HF model of ascending aortic constriction and once-daily isoproterenol administration (ACi). Three groups were included: sham-operated controls (Control), ACi, and ACi plus SB treatment (ACiSB). Cardiac function was evaluated by M-Mode echocardiography and proteome/phosphoproteome analysis was performed by multiplexed TMT-labeling and LC-MS/MS. Results: Inhibition of p38 MAPK with SB attenuated but did not fully block HF progression. At 5 weeks, fractional shortening was significantly improved in the ACiSB group compared to ACi (p=0.0037). Furthermore, it significantly decreased cardiac hypertrophy and lung edema (p-values <0.0001 and 0.0003 respectively). Compared to our previous study on the impact of a mitochondrially-targeted antioxidant (MitoTEMPO), SB protected a smaller subset of protein expression changes in HF; However, its impact on the phosphoproteome was substantial, extending beyond known p38 substrates. SB suppressed HF-induced inflammatory pathway proteins, modestly protected against downregulation of some mitochondrial respiratory chain proteins, and prevented maladaptive phosphorylation of several proteins involved in excitation-contraction coupling, including the ryanodine receptor, phospholamban, and cytoskeletal protein phosphorylation. Conclusion: p38 MAPK inhibition offered significant protection against HF, primarily affecting the phosphoproteome with modest effects on protein expression. The data suggests that narrowly targeted p38 MAPK-driven phosphoproteome modifications contribute to cardiac decompensation, despite broad protein expression changes in HF. Differences between MitoTEMPO and SB effects on the HF proteome highlight the plethora of remodeling pathways triggered by mitochondrial ROS including p38 MAPK activation. Our findings emphasize the importance of preventing p38 MAPK to preserve cardiac function.

Abstract Mo002: Pharmacological Inhibition of p38 MAPK Attenuates Doxorubicin-induced Cardiotoxicity, Senescence, and Inflammation in C57BL/6 mice

Introduction: Doxorubicin (DOX)-induced cardiotoxicity has been associated with induction of senescence, inflammation, and activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Losmapimod (LOSM), an orally active p38 MAPK inhibitor, is an anti-inflammatory agent with cardioprotective effects. Nevertheless, the effect of LOSM against DOX-induced cardiotoxicity has not been reported. The objective of the current work is to determine the effects of LOSM on DOX-induced chronic cardiotoxicity in C57BL/6 mice with particular focus on senescence and inflammatory pathways. Methods: Five-week-old C57BL/6 mice were fed diet containing LOSM (estimated daily intake 12 mg/kg/day) or a control diet for four days. Thereafter, mice were randomized to receive six weekly intraperitoneal injections of either DOX (4 mg/kg/week for 6 weeks) or saline. Three days after the last injection, cardiac function was assessed by trans-thoracic echocardiography. Gene expressions of senescence and inflammatory markers were quantified using real-time PCR and serum inflammatory markers were assessed by Luminex. Results: LOSM ameliorated DOX-induced cardiac dysfunction as evidenced by mitigated DOX-induced reduction in left ventricular fraction and fractional shortening. Notably, LOSM attenuating DOX-induced upregulation of p21 Cip1 , a marker of senescence, in the heart and the liver. Additionally, LOSM demonstrated anti-inflammatory effects, with reduced cardiac interleukin-1α and interleuken-6 gene expression in DOX-treated mice suggesting a senomorphic effect of LOSM. Systemic inflammation, assessed by serum cytokine levels, showed decreased interleukin-6 concentration in both DOX-treated mice and LOSM-treated mice. Conclusion: This is the first study to determine the effect of pharmacological inhibition of p38 MAPK against DOX-induced cardiotoxicity in vivo. Our study underscores the potential therapeutic efficacy of p38 MAPK inhibition in ameliorating DOX-induced cardiotoxicity, offering insights into novel strategies for mitigating chemotherapy-related cardiac complications, and suggesting a possible senomorphic effects of losmapimod.