P38alpha Mitogen-activated Protein Kinase Research Articles

Regulation of STAT1 and STAT4 Expression by Growth Factor and Interferon Supplementation in Sjögren's Syndrome Cell Culture Models.

Our goal was to investigate the effects of epidermal growth factor (EGF) and interferons (IFNs) on signal transducer and activator of transcription STAT1 and STAT4 mRNA and active phosphorylated protein expression in Sjögren's syndrome cell culture models. iSGECs (immortalized salivary gland epithelial cells) and A253 cells were treated with EGF, IFN-alpha, -beta, -gamma, or mitogen-activated protein kinase p38 alpha (p38-MAPK) inhibitor for 0-24-48-72 h. STAT1 and STAT4 mRNA expression was quantified by qRT-PCR. Untreated and treated cells were compared using the delta-delta-CT method based on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) normalized relative fold changes. phospho-tyrosine-701-STAT1 and phospho-serine-721-STAT4 were detected by Western blot analysis. STAT4 mRNA expression decreased 48 h after EGF treatment in A253 cells, immortalized salivary gland epithelial cells iSGECs nSS2 (sicca patient origin), and iSGECs pSS1 (anti-SSA negative Sjögren's Syndrome patient origin). EGF and p38-MAPK inhibitor decreased A253 STAT4 mRNA levels. EGF combined with IFN-gamma increased phospho-STAT4 and phospho-STAT1 after 72 h in all cell lines, suggesting additive effects for phospho-STAT4 and a major effect from IFN-gamma for phospho-STAT1. pSS1 and nSS2 cells responded differently to type I and type II interferons, confirming unique functional characteristics between iSGEC cell lines. EGF/Interferon related pathways might be targeted to regulate STAT1 and STAT4 expression in salivary gland epithelial cells. Further investigation is required learn how to better target the Janus kinases/signal transducer and activator of transcription proteins (JAK/STAT) pathway-mediated inflammatory response in Sjögren's syndrome.

Early chronic suppression of microglial p38α in a model of Alzheimer's disease does not significantly alter amyloid-associated neuropathology.

The p38 alpha mitogen-activated protein kinase (p38α) is linked to both innate and adaptive immune responses and is under investigation as a target for drug development in the context of Alzheimer's disease (AD) and other conditions with neuroinflammatory dysfunction. While preclinical data has shown that p38α inhibition can protect against AD-associated neuropathology, the underlying mechanisms are not fully elucidated. Inhibitors of p38α may provide benefit via modulation of microglial-associated neuroinflammatory responses that contribute to AD pathology. The present study tests this hypothesis by knocking out microglial p38α and assessing early-stage pathological changes. Conditional knockout of microglial p38α was accomplished in 5-month-old C57BL/6J wild-type and amyloidogenic AD model (APPswe/PS1dE9) mice using a tamoxifen-inducible Cre/loxP system under control of the Cx3cr1 promoter. Beginning at 7.5 months of age, animals underwent behavioral assessment on the open field, followed by a later radial arm water maze test and collection of cortical and hippocampal tissues at 11 months. Additional endpoint measures included quantification of proinflammatory cytokines, assessment of amyloid burden and plaque deposition, and characterization of microglia-plaque dynamics. Loss of microglial p38α did not alter behavioral outcomes, proinflammatory cytokine levels, or overall amyloid plaque burden. However, this manipulation did significantly increase hippocampal levels of soluble Aβ42 and reduce colocalization of Iba1 and 6E10 in a subset of microglia in close proximity to plaques. The data presented here suggest that rather than reducing inflammation per se, the net effect of microglial p38α inhibition in the context of early AD-type amyloid pathology is a subtle alteration of microglia-plaque interactions. Encouragingly from a therapeutic standpoint, these data suggest no detrimental effect of even substantial decreases in microglial p38α in this context. Additionally, these results support future investigations of microglial p38α signaling at different stages of disease, as well as its relationship to phagocytic processes in this particular cell-type.

In vitro and in silico studies of 7′′,8′′-buddlenol D anti-inflammatory lignans from Carallia brachiata as p38 MAP kinase inhibitors

Excessive macrophage activation induces the release of high levels of inflammatory mediators which not only amplify chronic inflammation and degenerative diseases but also exacerbate fever and retard wound healing. To identify anti-inflammatory molecules, we examined Carallia brachiata—a medicinal terrestrial plant from Rhizophoraceae. Furofuran lignans [(−)-(7′′R,8′′S)-buddlenol D (1) and (−)-(7′′S,8′′S)-buddlenol D (2)] isolated from the stem and bark inhibited nitric oxide (half maximal inhibitory concentration (IC50): 9.25 ± 2.69 and 8.43 ± 1.20 micromolar for 1 and 2, respectively) and prostaglandin E2 (IC50: 6.15 ± 0.39 and 5.70 ± 0.97 micromolar for 1 and 2, respectively) productions in lipopolysaccharide-induced RAW264.7 cells. From western blotting, 1 and 2 suppressed LPS-induced inducible nitric oxide synthase and cyclooxygenase-2 expression in a dose-dependent manner (0.3–30 micromolar). Moreover, analysis of the mitogen-activated protein kinase (MAPK) signaling pathway showed decreased p38 phosphorylation levels in 1- and 2-treated cells, while phosphorylated ERK1/2 and JNK levels were unaffected. This discovery agreed with in silico studies which suggested 1 and 2 bound to the ATP-binding site in p38-alpha MAPK based on predicted binding affinity and intermolecular interaction docking. In summary, 7′′,8′′-buddlenol D epimers demonstrated anti-inflammatory activities via p38 MAPK inhibition and may be used as viable anti-inflammatory therapies.

Abstract P2024: Novel Inhibitor Of P38 Mitogen-activated Protein Kinase Reverses Hypoxia-induced Pulmonary Hypertension In Rats

Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular changes with subsequent right ventricular (RV) failure. This work investigated the hypothesis that a new p38-alpha mitogen-activated protein kinase (p38-alpha MAPK) inhibitor, named LASSBio-1824, could interfere on PH model in rats. Methods: HP was induced in male Wistar rats through the exposure to hypoxia (10% O2) during 3 weeks, in combination to i.p. injection of an antagonist of vascular endothelial growth factor receptor (SU5416; 20 mg/kg/weekly). Control rats were kept in normoxia. After 3 weeks, echocardiography images confirmed PH using the pulmonary artery (PA) outflow waveform. After disease onset, animals were randomly divided into three groups: control + vehicle (DMSO), PH + vehicle and PH + LASSBio-1824 (50 mg/kg/day), in which vehicle and LASSBio-1824 were orally administered (gavage) during two weeks. All experiments were in accordance with the Animal Care and Use Committee at Federal University of Rio de Janeiro (039/19). Results: Pulmonary acceleration time (PAT; ms) was reduced from 40.0 ± 2.1 (control) to 24.2 ± 1.8 in PH + vehicle group (p < 0.05) and restored to 36.8 ± 2.9 after treatment with LASSBio-1824 (p < 0.05). RV afterload was detected in PH group because of the increase of systolic pressure (mmHg) from 23.2 ± 1.5 (control) to 46.7 ± 2.8 (p < 0.05) which was reduced to 19.4 ± 2.7 with LASSBio1824 treatment. Medial wall thickness (%) of distal pulmonary arteries (PA, < 50 μm) was evaluated by using immunohistochemistry for alpha smooth muscle actin (alpha-SMA) which was increased from 64.0 ± 4.9 (control) to 80.8 ± 2.7 in PH group (p < 0.05), but it was significantly reduced to 55.0 ± 1.3 after treatment with the inhibitor (p < 0.05). Acetylcholine (ACh)-induced maximal relaxation (%) of PA from normal and PH rats, was reduced from 67.5 ± 1.6 to 51.9 ± 4.9 (p < 0.05). Therefore, the treatment with the p38 inhibitor normalized vascular reactivity to 73.0 ± 3.3 %. The immunohistochemistry for c-fos protein, a product of the p38 pathway related to cell proliferation, showed an increase of stained myocyte nuclei (%) in PH animals of 36.5 ± 1.5 when compared to normal group of 20.2 ± 4.2. The inhibitor significantly reduced ratio to 24.0 ± 2.3% (p < 0.05). PH increased the inflammatory condition because the expression of inducible nitric oxide synthase altered from 10.4 ± 1.9% (control) to 31.9 ± 2.2% and it was recovered by the inhibitor to 15.5 ± 2.2% (p < 0.05). In conclusion, the new p38-alpha MAPK inhibitor represents an important approach for the future treatment of PH, since it improves the underlying remodeling and inflammation processes.

Synergistic Antiviral Activity of Pamapimod and Pioglitazone against SARS-CoV-2 and Its Variants of Concern.

The SARS-CoV-2 pandemic remains a major public health threat, especially due to newly emerging SARS-CoV-2 Variants of Concern (VoCs), which are more efficiently transmitted, more virulent, and more able to escape naturally acquired and vaccine-induced immunity. Recently, the protease inhibitor Paxlovid® and the polymerase inhibitor molnupiravir, both targeting mutant-prone viral components, were approved for high-risk COVID-19 patients. Nevertheless, effective therapeutics to treat COVID-19 are urgently needed, especially small molecules acting independently of VoCs and targeting genetically stable cellular pathways which are crucial for viral replication. Pamapimod is a selective inhibitor of p38 Mitogen-Activated Protein Kinase alpha (p38 MAPKα) that has been extensively clinically evaluated for the treatment of rheumatoid arthritis. Signaling via p38 has recently been described as a key pathway for the replication of SARS-CoV-2. Here, we reveal that the combination of pamapimod with pioglitazone, an anti-inflammatory and approved drug for the treatment of type 2 diabetes, possesses potent and synergistic activity to inhibit SARS-CoV-2 replication in vitro. Both drugs showed similar antiviral potency across several cultured cell types and similar antiviral activity against SARS-CoV-2 Wuhan type, and the VoCs Alpha, Beta, Gamma, Delta, and Omicron. These data support the combination of pamapimod and pioglitazone as a potential therapy to reduce duration and severity of disease in COVID-19 patients, an assumption currently evaluated in an ongoing phase II clinical study.

A novel assay for screening WIP1 phosphatase substrates in nuclear extracts.

Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple invitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.

How Aconiti Radix Cocta can Treat Gouty Arthritis Based on Systematic Pharmacology and UPLC-QTOF-MS/MS.

Background: Gouty arthritis (GA) is a common metabolic disease caused by a long-term disorder of purine metabolism and increased serum levels of uric acid. The processed product of dried root of Aconitum carmichaeli Debeaux (Aconiti Radix cocta, ARC) is used often in traditional Chinese medicine (TCM) to treat GA, but its specific active components and mechanism of action are not clear. Methods: First, we used ultra-performance liquid chromatography-quadrupole/time-of-flight tandem mass spectrometry to identify the chemical spectrum of ARC. Based on this result, we explored the active components of ARC in GA treatment and their potential targets and pathways. Simultaneously, we used computer simulations, in vitro cell experiments and animal experiments to verify the prediction results of systems pharmacology. In vitro, we used aurantiamide acetate (AA) to treat monosodium urate (MSU)-stimulated THP-1 cells and demonstrated the reliability of the prediction by western blotting and real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR). ELISAs kit were used to measure changes in levels of proinflammatory factors in rats with GA induced by MSU to demonstrate the efficacy of ARC in GA treatment. Results: Forty-three chemical constituents in ARC were identified. ARC could regulate 65 targets through 29 active components, and then treat GA, which involved 1427 Gene Ontology (GO) terms and 146 signaling pathways. Signaling pathways such as proteoglycans in cancer, C-type lectin receptor signaling pathway, and TNF signaling pathway may have an important role in GA treatment with ARC. In silico results showed that the active components songoramine and ignavine had high binding to mitogen-activated protein kinase p38 alpha (MAPK14) and matrix metallopeptidase (MMP)9, indicating that ARC treatment of GA was through multiple components and multiple targets. In vitro experiments showed that AA in ARC could effectively reduce expression of MAPK14, MMP9, and cyclooxygenase2 (PTGS2) in THP-1 cells stimulated by MSU, whereas it could significantly inhibit the mRNA expression of Caspase-1, spleen tyrosine kinase (SYK), and PTGS2. Animal experiments showed that a ARC aqueous extract could significantly reduce expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and intereleukin (IL)-18 in the serum of GA rats stimulated by MSU. Hence, ARC may inhibit inflammation by regulating the proteoglycans in cancer-associated signaling pathways. Conclusion: ARC treatment of GA may have the following mechanisms, ARC can reduce MSU crystal-induced joint swelling, reduce synovial tissue damage, and reduce the expression of inflammatory factors in serum. AA in ARC may inhibit inflammation by regulating the protein expression of MAPK14, MMP9, and PTGS2 and the mRNA expression of caspase-1, SYK, and PTGS2.

Pulmonary hypertension: reduction of vascular and cardiac remodeling by a new inhibitor of p38-alpha MAPK

Abstract Background Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling with subsequent right ventricular (RV) failure. This work investigated the effects of a new p38-alpha mitogen-activated protein kinase (p38-α MAPK) inhibitor, named LASSBio-1824, on PAH model in rats. Methods All experiments were in accordance with the Animal Care and Use Committee at our university (039/19). Male Wistar rats was exposed to hypoxia (10% O2) during 3 weeks plus i.p. injection of an antagonist of vascular endothelial growth factor receptor (SU5416; 20 mg/kg/week) to induce PAH. Control rats were kept in normoxia (21% O2). After 21 days of protocol, echocardiography images confirmed PAH using the pulmonary artery (PA) outflow waveform. After disease onset, animals were randomly divided into three groups: control + vehicle (DMSO), HAP + vehicle and HAP + LASSBio-1824 (50 mg/kg/day). Vehicle and LASSBio-1824 were administered by oral gavage during two weeks. Results Pulmonary acceleration time (PAT; ms) was reduced from 33.2±2.7 (control) to 22.7±1.1 in PAH + vehicle group (p<0.05) and restored to 29.6±1.9 after treatment with LASSBio-1824 (p<0.05). RV afterload was detected in HAP group because of the increase of systolic pressure (mmHg) from 22.2±1.5 (control) to 47.2±3.6 (p<0.05) and reduced to 18.0±2.9 with LASSBio-1824. Medial wall thickness (%) of distal PA (<50 μm) was measured by using immunohistochemistry for alpha smooth muscle actin (α-SMA) which was increased from 62.5±5.6 (control) to 78.0±7.9 in PAH group (p<0.05), but it was significantly reduced to 54.4±2.3 in PAH + LASSBio-1824 group (p<0.05). Vascular reactivity was evaluated comparing the acetylcholine (ACh)-induced maximal relaxation (%) in PA from normal and PAH rats, which was reduced from 67.5±2.1 to 49.63±5.6 (PAH group; p<0.05). Therefore, the treatment with the p38 inhibitor normalized the ACh-induced response to 73.7±4.1%. The immunohistochemistry for c-fos protein, a product of the p38 pathway related to cell proliferation, showed an increased ratio of stained and total myocyte nuclei (%) in PAH animals of 38.0±0.5 compared to normal group of 20.2±4.2. LASSBio-1824 significantly reduced ratio to 24.0±2.3% (p<0.05). PAH increased inflammatory condition because the expression of inducible nitric oxide synthase increased from 0.31±0.01 (control) to 0.57±0.02 and it was recovered by the treatment with LASSBio-1824 to 0.31±0.10 (p<0.05). Conclusion LASSBio-1824, an inhibitor of p38-alpha MAPK represents an important approach for the future treatment of PAH which improves the underlying remodeling and inflammation processes in the cardiopulmonary system. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES; Conselho Nacional de Desenvolvimento Científico e Tecnolόgico - CNPq

Mesoporous silica SBA-15 decreases hyperammonemia and affects the gene expression of mitogen-activated protein kinases in the prefrontal cortex of rats with bile duct ligation.

Objective(s):We aim to examine possible ammonia lowering effects of mesoporous silica SBA-15 in rats after the common bile duct ligation (BDL). We also evaluate the effect of SBA-15 treatments during 28 days of BDL on locomotion and rearing behavior, as well as on the gene expression of Jnk3 and p38alpha (p38α) mitogen-activated protein kinases in the prefrontal cortex (PFC).Materials and Methods:SBA-15 was prepared with the hydrothermal method from the surfactant P123 and tetraethyl orthosilicate (TEOS), and calcined at 550 ºC. Then, the product was characterized by FT-IR, XRD, SEM, and BJH-BET techniques. Male Wistar rats in sham control and a group with BDL received saline but another group with BDL received SBA-15 during 28 days of BDL. We examined all groups of rats weekly for locomotion and rearing behavior; then on day 28, we sacrificed all rats, collected the blood sample, and finally dissected the PFC from the whole brain.Results:The SBA-15 treatments had no effect on locomotion but improved rearing behavior on days 7 and 14 of BDL. Biochemical analysis indicated that the SBA-15 treatments in rats with BDL significantly decreased hyperammonemia. The results also revealed that the SBA-15 treatments in rats with BDL significantly restored the decreased Jnk3 gene expression, and increased the p38α gene expression in the PFC.Conclusion:We conclude that SBA-15 can be used as an ammonia lowering agent in hepatic encephalopathy; however, its improving effects on locomotion and neuroinflammation, as well as signaling molecules in the brain need more investigations.

Opposing Oncogenic Functions of p38 Mitogen-activated Protein Kinase Alpha and Beta in Human Pancreatic Cancer Cells.

The p38 family of mitogen-activated protein kinases (MAPK) includes four isoforms: p38α, -β, -γ and -δ. The aim of this study was to elucidate possible functions of p38α and p38β in human pancreatic cancer. Isoform expression was determined in seven human pancreatic cancer cell lines. After shRNA based selective knockdown of p38α and p38β, in vitro growth and migration as well as in vivo tumorigenicity were assessed. All pancreatic cancer cells expressed p38 isoforms. Knockdown of p38α and p38β inhibited in vitro growth. Migration was markedly reduced in p38α shRNA expressing clones, but not altered by p38β knockdown. While in vivo inhibition of p38β decreased tumor formation and growth, the knockdown of p38α significantly enhanced tumorigenicity. p38 MAPKs may exert isoform specific functions in pancreatic cancer. Selective targeting may contribute to individualized treatment of pancreatic cancer in the future.

Chrysophanol, Physcion, Hesperidin and Curcumin Modulate the Gene Expression of Pro-Inflammatory Mediators Induced by LPS in HepG2: In Silico and Molecular Studies.

Hepatitis is an inflammatory condition that can develop hepatocellular carcinoma. Traditional medicine has always been the pillar of medical practice. However, it became less compatible with the current understanding of the diseases and the possible treatment. Therefore, in silico tools could be utilized for building the bridge between the legacy of the past and the current medical approaches allowing access to new therapeutic discoveries. In this work, a Chinese traditional medicine database was screened using structure-based virtual screening to identify molecules that could inhibit p38 alpha mitogen-activated protein kinase (MAPK). Out of the identified compounds, four selected compounds: chrysophanol, physcion, curcumin and hesperidin were isolated from their respective sources and their structures were confirmed by spectroscopic methods. These compounds decreased the gene expression of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1β) in lipopolysaccharide (LPS) induced inflammation in a hepatocellular carcinoma cell line (HepG2) in a dose-dependent manner. The molecular docking study revealed the specificity of these compounds towards p38 MAPK rather than other MAPKs. In conclusion, the molecular and in silico studies suggest that the isolated compounds could be a potential treatment for hepatitis by resolving inflammation controlled by MAPKs, thus limiting the development of further complications and lower side effects.

Functions of p38 mitogen-activated protein kinase alpha and beta in human pancreatic cancer cells

Objective To elucidate possible functions of p38α and p38β in human pancreatic cancer. Methods Expression of p38α, p38β, p38γ and p38δ were determined by immunoblot in 7 cultured human pancreatic cancer cell lines. To selectively inhibit p38α or p38β, cell clones were established in a stable manner expression either p38α short hairpin RNA (shRNA) or p38β shRNA. Proliferation, single cell movement, in vitro migration, colony forming ability as well as in vivo tumorigenicity were determined by methyl thiazol tetrazolium (MTT) assay, video-time laps microscopy, boyden-chamber, soft-agar assay, tumor formation and growth in nude mice respectively. Results All pancreatic cancer cells showed p38 isoforms protein expression at various levels. Selective inhibition of p38α or p38β in Mia Paca-2 and Panc-1 cells revealed that both kinases enhance cell proliferation and anchorage-independent growth. Single cell movement and cell invasion were markedly reduced in p38α shRNA expressing clones, but not altered in p38β shRNA expressing clones. Inhibition of p38α enhanced in vivo tumorigenicity profoundly in Mia Paca-2 and Panc-1 cells. In Mia Paca-2 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 18.3% and 50.0% respectively, which decreased significantly comparing to the wild type cell and null vector transfection cell. The tumor volume decreased significantly either (P<0.05). In Panc-1 cell, the in vivo tumor formation rate of two p38β shRNA expressing clones is 25.0% and 12.5% respectively. Conclusion p38 mitogen activated protein kinases (p38MAPKs) are expressed in pancreatic cancer cells and the isoforms may exert different functions. Key words: p38 mitogen-activated protein kinase; Pancreatic cancer; p38α; p38β

Abstract 16678: A Novel p38-Alpha MAPK Inhibitor Reverses Sugen/Hypoxia-Induced Pulmonary Arterial Hypertension in Rats

Introduction: Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular remodeling with subsequent right ventricular (RV) failure. Hypothesis: The p38-alpha mitogen-activated protein kinase (p38-α MAPK) plays a pathogenic role in PAH. This work investigated the effects of a selective p38-alpha MAPK inhibitor, named LASSBio-1824, on hypoxia + SU5416 -induced PAH. Methods: Male Wistar rats (180-220 g) were exposed to 3 weeks of hypoxia and i.p. injection of VEGFR antagonist (SU5416; 20 mg/kg/week). Experimental groups were: Control + vehicle (DMSO), SuHx + vehicle (PAH group) and SuHx + LASSBio-1824 (20 mg/kg/day for 2 weeks). Results: Pulmonary acceleration time (ms) was reduced from 44.7 ± 1.1 (control) to 20.6 ± 1.7 in SuHx + vehicle group (P &lt; 0.05) and restored to 38.9 ± 2.9 in SuHx + LASSBio-1824 group (P&lt; 0.05). RV systolic pressures (mmHg) were increased from 30.1 ± 3.2 (control) to 109.5 ± 7.5 in SuHx group and reduced to 47.5 ± 5.3 after treatment with LASSBio-1824 (P &lt; 0.05). The medial wall thickness of distal pulmonary arterioles (&lt; 50 μm) was measured by immunohistochemistry for alpha-SMA expression which was increased from 40.7 ± 3.5 % (control) to 64.1 ± 3.1 % in PAH animals (P &lt; 0.05) and was reduced in the treated group (52.2 ± 2.9). Acetylcholine-induced maximal relaxation (% ) in pulmonary arteries was reduced from 77.9 ± 5.2 to 17.2 ± 3.5 in PAH group. LASSBio-1824 increased that response to 67.8 ± 5.6 % (P &lt; 0.05), indicating the ame- lioration of endothelial function. RV hypertrophy was detected because its wall thickness (mm) increased from 0.56 ± 0.02 (control) to 1.3 ± 0.03 in SuHx + vehicle group (P &lt; 0.05) and the ratio between RV and left ventricle + septum increased from 1.50 ± 0.03 to 2.0 ± 0.2. Treatment with LASSBio-1824 significantly reduced the RV hypertrophy (ratio of 1.7 ± 0.07). Conclusion: The inhibition of p38-alpha MAPK isoform by LASSBio-1824 represents an impor- tant approach for the treatment of PAH in the future, which improves the underlying remodeling and inflammation processes.

Insights into the Identification of p38-alpha Mitogen activated Protein Kinase against Pyridazinopyridinone Derivatives in the Treatment of Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a long-term disease that leads to inflammation of the joints and surrounding tissues. It also affects other organs and considered as an autoimmune disease. The body's immune system fights off foreign substances, like viruses by producing cytokines. But in an autoimmune disease, the immune system confuses healthy tissue for foreign substances and the body attacks its own tissues. The p38 Mitogen Activated Protein Kinase (MAPK) plays an important role in the production of proinflammatory cytokines, making it an attractive target for the treatment of Rheumatoid Arthritis and various inflammatory diseases. Currently Methotrexate, Leflunomide, and anti-inflammatory drugs like Aspirin, Ibuprofen are given to patients affected with Rheumatoid Arthritis, but these drugs have serious side effects. Recently, pyridazinopyridinone and its derivatives were found to be effective in inhibiting the p38 alpha MAP kinase and thereby help in treating inflammatory diseases. Docking analysis was performed using Schrodinger’s GLIDE and potent ligands were selected from the literature. The structures of the ligands were determined and energy was minimized using OPLS force field. These energy minimized ligand structures were then subjected to High throughput virtual screening against the target protein p38-a. The ligands which showed best energy and score were selected for Induced Fit Docking (IFD). The interactions between the ligands and the protein were observed in different poses and the final docking results were observed. The ligand31 has a good docking score of -11.20 and the Glide energy -72.00 with hydrogen bonding between ASP 168 and GLU 71 in the active site region. Hence, pyridazinopyridinone derivatives were found to be potent inhibitors of the target p38 Mitogen Activated Protein.

Decreased microRNA-125a-3p contributes to upregulation of p38 MAPK in rat trigeminal ganglions with orofacial inflammatory pain.

Orofacial inflammatory pain is a difficult clinical problem, and the specific molecular mechanisms for this pain remain largely unexplained. The present study aimed to determine the differential expression of microRNAs (miRNAs) and disclose the underlying role of miR-125a-3p in orofacial inflammatory pain induced by complete Freund's adjuvant (CFA). Thirty-two differentially expressed miRNAs were first screened using a microarray chip in ipsilateral trigeminal ganglions (TGs) following CFA injection into the orofacial skin innervated by trigeminal nerve, and a portion of them, including miR-23a*, -24-2*, -26a, -92a, -125a-3p, -183 and -299 were subsequently selected and validated by qPCR. The target genes were predicted based on the miRWalk website and were further analyzed by gene ontology (GO). Further studies revealed miR-125a-3p expression was down-regulated, whereas both the expression of p38 MAPK (mitogen-activated protein kinase) alpha and CGRP (calcitonin gene-related peptide) were up-regulated in ipsilateral TGs at different time points after CFA injection compared with control. Furthermore, mechanistic study revealed that miR-125a-3p negatively regulates p38 alpha gene expression and is positively correlated with the head withdrawal threshold reflecting pain. Luciferase assay showed that binding of miR-125a-3p to the 3′UTR of p38 alpha gene suppressed the transcriptional activity, and overexpression of miR-125a-3p significantly inhibited the p38 alpha mRNA level in ND8/34 cells. Taken together, our results show that miR-125a-3p is negatively correlated with the development and maintenance of orofacial inflammatory pain via regulating p38 MAPK.

The p38alpha mitogen-activated protein kinase limits the CNS proinflammatory cytokine response to systemic lipopolysaccharide, potentially through an IL-10 dependent mechanism.

BackgroundThe p38α mitogen-activated protein kinase (MAPK) is a well-characterized intracellular kinase involved in the overproduction of proinflammatory cytokines from glia. As such, p38α appears to be a promising therapeutic target for neurodegenerative diseases associated with neuroinflammation. However, the in vivo role of p38α in cytokine production in the CNS is poorly defined, and prior work suggests that p38α may be affecting a yet to be identified negative feedback mechanism that limits the acute, injury-induced proinflammatory cytokine surge in the CNS.MethodsTo attempt to define this negative feedback mechanism, we used two in vitro and two in vivo models of neuroinflammation in a mouse where p38α is deficient in cells of the myeloid lineage.ResultsWe found that p38α in myeloid cells has an important role in limiting amplitude of the acute proinflammatory cytokine response to a systemic inflammatory challenge. Moreover, we identified IL-10 as a potential negative feedback mechanism regulated by p38α.ConclusionsOur data suggest that p38α regulates a proper balance between the pro- and anti-inflammatory cytokine responses to systemic inflammation, and that if circulating IL-10 levels are not elevated to counter-balance the increased systemic proinflammatory responses, the spread of the inflammatory response from the periphery to the CNS is exaggerated.

TNFα inhibits IGFBP-3 through activation of p38α and casein kinase 2 in human retinal endothelial cells.

We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment.

Disruption of TAB1/p38α Interaction Using a Cell-permeable Peptide Limits Myocardial Ischemia/Reperfusion Injury

Targeting the adaptor protein (transforming growth factor-β (TGF-β)-activated protein kinase 1 (TAK1)-binding protein 1) (TAB1)-mediated non-canonical activation of p38α to limit ischemia/reperfusion (I/R) injury after an acute myocardial infarction seems to be attractive since TAB1/p38α interaction occurs specifically in very limited circumstances and possesses unique structural basis. However, so far no TAB1/p38α interaction inhibitor has been reported due to the limited knowledge about the interfaces. In this study, we sought to identify key amino acids essential for the unique mode of interaction with computer-guided molecular simulations and molecular docking. After validation of the predicted three-dimensional (3-D) structure of TAB1/p38α complex, we designed several peptides and evaluated whether they could block TAB1/p38α interaction with selectivity. We found that a cell-permeable peptide worked as a selective TAB1/p38α interaction inhibitor and decreased myocardial I/R injury. To our knowledge, this is the first TAB1/p38α interaction inhibitor.

Structure- and ligand-based drug design of novel p38-alpha MAPK inhibitors in the fight against the Alzheimer’s disease

Alzheimer’s disease (AD) is characterized microscopically by the presence of amyloid plaques, which are accumulations of beta-amyloid protein inter-neurons, and neurofibrillary tangles formed predominantly by highly phosphorylated forms of the microtubule-associated protein, tau, which form tangled masses that consume neuronal cell body, possibly leading to neuronal dysfunction and ultimately death. p38α mitogen-activated protein kinase (MAPK) has been implicated in both events associated with AD, tau phosphorylation and inflammation. p38α MAPK pathway is activated by a dual phosphorylation at Thr180 and Tyr182 residues. Drug design of p38α MAPK inhibitors is mainly focused on small molecules that compete for Adenosine triphosphate in the catalytic site. Here, we used different approaches of structure- and ligand-based drug design and medicinal chemistry strategies based on a selected p38α MAPK structure deposited in the Protein Data Bank in complex with inhibitor, as well as others reported in literature. As a result of the virtual screening experiments performed here, as well as molecular dynamics, molecular interaction fields studies, shape and electrostatic similarities, activity and toxicity predictions, and pharmacokinetic and physicochemical properties, we have selected 13 compounds that meet the criteria of low or no toxicity potential, good pharmacotherapeutic profile, predicted activities, and calculated values ​​comparable with those obtained for the reference compounds, while maintaining the main interactions observed for the most potent inhibitors.

Deletion of p38-alpha mitogen-activated protein kinase within the intestinal epithelium promotes colon tumorigenesis

p38-Alpha mitogen-activated protein kinase (p38-MAPK) is a tumor suppressor often mutated in human cancers, but its specific role in colorectal cancer is not completely understood. Previous studies have found that p38-MAPK activity inhibits epithelial proliferation and promotes apoptosis in the intestine. Therefore, we sought to test the hypothesis that intestinal disruption of p38-MAPK would lead to increased tumorigenesis in the colon. p38-MAPK was deleted in mice within the intestinal epithelium using a tamoxifen-inducible Cre system under control of the villin promoter [villin-Cre ERT2(+), MAPK14(f/f)]. An azoxymethane and dextran sodium sulfate protocol was used to drive intestinal tumor development. Tumor measurements were made using computer software from photographs of excised colon specimens. The number of mice that developed tumors was not statistically different when comparing wild-type mice (7/14) to inducible, intestine epithelial-deleted p38-MAPK (9/11) mice after azoxymethane/dextran sodium sulfate treatment (P = .21). However, the epithelial-deleted p38-MAPK mice developed significantly more tumors (3.7 vs 1.1; P = .008) and nearly 4 times the total tumor burden as wild-type mice (17.4 vs 4.8mm(2); P = .03). Wild-type and epithelial-deleted p38-MAPK groups demonstrated a similar degree of colon inflammation. Deletion of p38-MAPK within the colonic mucosa leads to a hyperplastic state promoting greater tumor development. Because the severity of colitis was not augmented in mice with p38-MAPK deficiency, tumor development is likely mediated by impaired cell cycle regulation within the colonic epithelium. Manipulation of p38-MAPK activity may provide a novel treatment and/or prevention strategy in the management of colorectal cancer, particularly in the setting of inflammatory bowel disease.