Blockage of the fractalkine pathway reduces hyperalgesia and prevents morphological glial alterations-Comparison between inflammatory and neuropathic orofacial pain in male rats.

This study aimed to evaluate the effects of inhibitors of the fractalkine pathway in hyperalgesia in inflammatory and neuropathic orofacial pain in male rats and the morphological changes in microglia and satellite glial cells (SGCs). Rats were submitted to zymosan-induced arthritis of the temporomandibular joint or infraorbital nerve constriction, and treated intrathecally with a P2 X7 antagonist, a cathepsin S inhibitor or a p-38 mitogen-activated protein kinase (MAPK) inhibitor. Mechanical hyperalgesia was evaluated 4 and 6 h following arthritis induction or 7 and 14 days following nerve ligation. The expression of the receptor CX3 CR1 , phospho-p-38 MAPK, ionized calcium-binding adapter molecule-1 (Iba-1), and glutamine synthetase and the morphological changes in microglia and SGCs were evaluated by confocal microscopy. In both inflammatory and neuropathic models, untreated animals presented a higher expression of CX3 CR1 and developed hyperalgesia and up-regulation of phospho-p-38 MAPK, which was prevented by all drugs (p < .05). The number of microglial processes endpoints and the total branch length were lower in the untreated animals, but the overall immunolabeling of Iba-1 was altered only in neuropathic rats (p < .05). The mean area of SGCs per neuron was significantly altered only in the inflammatory model (p < .05). All morphological alterations were reverted by modulating the fractalkine pathway (p < .05). In conclusion, the blockage of the fractalkine pathway seemed to be a possible therapeutic strategy for inflammatory and neuropathic orofacial pain, reducing mechanical hyperalgesia by impairing the phosphorylation of p-38 MAPK and reverting morphological alterations in microglia and SGCs.

The functional role of p38 MAPK pathway in malignant brain tumors.

Gliomas are extremely debilitating malignant brain tumors with very limited response to therapies. The initiation and progression of gliomas can be attributed to several molecular abnormalities, such as mutations in important regulatory networks. In this regard, the mitogen-activated protein kinases (MAPKs) arise as key signaling pathways involved in cell proliferation, survival, and differentiation. MAPK pathway has been altered in most glial tumors. In glioma cells, the activation of p38 MAPK contributes to tumor invasion and metastasis and is positively correlated with tumor grade, being considered a potential oncogenic factor contributing to brain tumorigenesis and chemotherapy resistance. Hence, a better understanding of glioma pathogenesis is essential to the advancement of therapies that provide extended life expectancy for glioma patients. This review aims to explore the role of the p38 MAPK pathway in the genesis and progression of malignant brain tumors.

Genistein improves mitochondrial function and inflammatory in rats with diabetic nephropathy via inhibiting MAPK/NF-κB pathway

Purpose: To investigate the effect of genistein on inflammation and mitochondrial function of diabetic nephropathy. Methods: Diabetic nephropathy model was established in Sprague-Dawley rats. Automatic biochemical analyzer was employed to detect the kidney function index, serum creatinine, serum urea nitrogen, and 24 h-urine protein and blood glucose. Hematoxylin and eosin staining and periodic acid Schiff staining were used to observe renal morphology. Mitochondrial changes and podocyte integrity were monitored by transmission electron microscope. The expression levels of mfn2, NOX4, P53, MAPK, and NF-κB were detected by Western blotting. The changes of mitochondrial membrane potential were measured by JC-1. The level of mfn2 was assessed by immunofluorescence assay. Results: Genistein ameliorated the kidney function with reduced Scr and blood glucose. The expressions of NOX4, MAPK, p65 and p53 were downregulated, while the expression of mnf2 was the opposite in genistein-treated kidneys. Further investigations revealed that genistein reduced expansion of mesangial matrix and oxidative stress, protected podocyte integrity and increased mitochondrial membrane potential. Conclusions: Genistein could alleviate diabetic nephropathy through inhibiting MAPK/NF-κB pathway, improving mitochondrial function and anti-inflammatory.To investigate the effect of genistein on inflammation and mitochondrial function of diabetic nephropathy. Diabetic nephropathy model was established in Sprague-Dawley rats. Automatic biochemical analyzer was employed to detect the kidney function index, serum creatinine, serum urea nitrogen, and 24 h-urine protein and blood glucose. Hematoxylin and eosin staining and periodic acid Schiff staining were used to observe renal morphology. Mitochondrial changes and podocyte integrity were monitored by transmission electron microscope. The expression levels of mfn2, NOX4, P53, MAPK, and NF-κB were detected by Western blotting. The changes of mitochondrial membrane potential were measured by JC-1. The level of mfn2 was assessed by immunofluorescence assay. Genistein ameliorated the kidney function with reduced Scr and blood glucose. The expressions of NOX4, MAPK, p65 and p53 were downregulated, while the expression of mnf2 was the opposite in genistein-treated kidneys. Further investigations revealed that genistein reduced expansion of mesangial matrix and oxidative stress, protected podocyte integrity and increased mitochondrial membrane potential. Genistein could alleviate diabetic nephropathy through inhibiting MAPK/NF-κB pathway, improving mitochondrial function and anti-inflammatory.

Er-Bai-Tang decoction improved the movement disorders and neuronal injury in the Parkinson's disease model rats via decreasing p38 MAPK pathway and improving the composition of intestinal flora

Purpose: Our previous study showed that Er-Bai-Tang decoction (EBT) could effectively improve Parkinson's disease (PD) patients' quality of life, sleep, mood, and cognitive disorders, but the mechanism of EBT to treat PD was unclear. So, our study aimed to explore the mechanism of EBT to treat PD via p38 mitogen-activated protein kinases (MAPK) pathway and intestinal flora. Methods: In our study, the PD rat model was established by subcutaneously injecting 2 mg/kg/d rotenone solution, and 23.43 g/kgEBT was used to treat PD model rats. Results: Behavioral test showed that EBT could reverse the motor impairment in the PD model rats. Hematoxylin and eosin result showed that EBT could reduce the cell necrosis in the SNpc area of the PD model rats. Western blotting and real time-polymerase chain reaction showed that EBT could decrease the p38 MAPK expression in the SNpc area of the PD model rats. 16s rRNA sequencing analysis showed that EBT could improve the composition of intestinal flora in the PD model rats. Rikenellaceae at family level and Alistipes and Allobaculum at the genus level were the key species in the PD development and EBT treatment to PD. KEGG showed that EBT might change the iron uptake in PD rats. Conclusions: EBT could improve the motor symptoms and neuronal injury in the PD model rat, and its mechanism may be related to decreasing p38 MAPK pathway and improving the composition of intestinal flora.

MPK6 Kinase Regulates Plasma Membrane H+-ATPase Activity in Cold Acclimation.

Cold and freezing stresses severely affect plant growth, development, and survival rate. Some plant species have evolved a process known as cold acclimation, in which plants exposed to temperatures above 0 °C trigger biochemical and physiological changes to survive freezing. During this response, several signaling events are mediated by transducers, such as mitogen activated protein kinase (MAPK) cascades. Plasma membrane H+-ATPase is a key enzyme for the plant cell life under regular and stress conditions. Using wild type and mpk3 and mpk6 knock out mutants in Arabidopsis thaliana, we explored the transcriptional, translational, and 14-3-3 protein regulation of the plasma membrane H+-ATPase activity under the acclimation process. The kinetic analysis revealed a differential profiling of the H+-ATPase activity depending on the presence or absence of MPK3 or MPK6 under non-acclimated or acclimated conditions. Negative regulation of the plasma membrane H+-ATPase activity was found to be exerted by MPK3 in non-acclimated conditions and by MPK6 in acclimated conditions, describing a novel form of regulation of this master ATPase. The MPK6 regulation involved changes in plasma membrane fluidity. Moreover, our results indicated that MPK6 is a critical regulator in the process of cold acclimation that leads to freezing tolerance and further survival.

Effect of IL-33 on pyroptosis of macrophages in mice with sepsis via NF-κB/p38 MAPK signaling pathway

ABSTRACT Purpose To demonstrate the effect of IL-33 on the macrophage pyroptosis in mice with sepsis through the NF-kB/p38 MAPK signal pathway. Methods In total, 24 C57BL/6 mice were divided into the sham operation group (sham) and the cecal ligation and puncture group (CLP). After CLP, 24 IL-33-/- mice were divided into the IL-33-/- group and the IL-33-/- intervention group. The latter group was intraperitoneally injected with IL-33. Mouse mortality was observed after CLP. Macrophage apoptosis in peritoneal lavage fluid was detected by flow cytometry. Serum inflammatory factor level was detected by ELISA. Apoptotic protein expression and NF-κB/p38 MAKP signaling pathway protein expression were detected by qRT-PCR and Western blot. Results Knocking out IL-33 significantly reduced the mortality of CLP mice, as well as the mRNA expression of IL-33 and the levels of serum inflammatory factors, including IL-33, IL-1β, and IL-18. It also reduced the rate of macrophage apoptosis and the expression of the apoptotic protein caspase-1 p10; increased the expression of IκBα; and reduced the protein expression of NF-κB and p38 MAPK. These effects were reversed after exogenous injection of IL-33. Conclusions IL-33 can increase the level of macrophage pyroptosis in mice with sepsis (by activating the NF-kB/p38MAPK signal pathway) and the mortality of these mice.

Atorvastatina Reduz o Acúmulo de Células Musculares Lisas Vasculares para Inibir a Hiperplasia Intimal pela Inibição de Via p38 MAPK em um Modelo de Enxerto de Veia em Ratos / Atorvastatin Reduces Accumulation of Vascular Smooth Muscle Cells to Inhibit Intimal Hyperplasia via p38 MAPK Pathway Inhibition in a Rat Model of Vein Graft

Resumo Fundamento: A taxa de falha de enxerto de veia safena um ano após a cirurgia de revascularização do miocárdio varia de 10% a 25%. O objetivo deste estudo foi de investigar se a atorvastatina pode reduzir o acúmulo de células musculares lisas vasculares para inibir a hiperplasia intimal por meio da inibição da via p38 MAPK. Métodos: Quarenta e cinco ratos Sprague-Dawley foram randomizados em três grupos. Trinta ratos foram submetidos à cirurgia de enxerto de veia e randomizados para tratamento com veículo ou atorvastatina; quinze ratos foram submetidos à cirurgia sham. Detectamos a hiperplasia intimal por meio de coloração com hematoxilina-eosina e a expressão de proteínas relacionadas por meio de análise imuno-histoquímica e Western blot. Foram realizadas as comparações por análise de variância de fator único e pelo teste da diferença mínima significativa de Fisher, com p < 0,05 considerado significativo. Resultados: A íntima analisada pela coloração com hematoxilina-eosina era dramaticamente mais espessa no grupo controle que no grupo atorvastatina e no grupo sham (p < 0,01). Os resultados da coloração imuno-histoquímica de α-SMA demonstraram que a porcentagem de células positivas para α-SMA no grupo controle era mais alta que no grupo atorvastatina (p < 0,01). Nós também avaliamos α-SMA, PCNA, p38 MAPK e fosforilação de p38 MAPK após o tratamento com estatina por meio de análise de Western blot e os resultados indicaram que a atorvastatina não levou à redução de p38 MAPK (p < 0,05); no entanto, resultou na inibição da fosforilação de p38 MAPK (p < 0,01) e reduziu significativamente os níveis de α-SMA e PCNA, em comparação com o grupo controle (p < 0,01). Conclusão: Nós demonstramos que a atorvastatina pode inibir o acúmulo de células musculares lisas vasculares por meio da inibição da via p38 MAPK e é capaz de inibir a hiperplasia intimal em modelos de enxerto de veia em ratos.

Abstract Background: The rate of saphenous vein graft failure one year after coronary artery bypass grafting ranges from 10% to 25%. The aim of this study was to explore whether atorvastatin can reduce accumulation of vascular smooth muscle cells to inhibit intimal hyperplasia via p38 MAPK pathway inhibition. Methods: Forty-five Sprague-Dawley rats were randomized to three groups. Thirty rats received a vein graft operation, and they were randomized to be treated with vehicle or atorvastatin; fifteen rats received a sham operation. We detected intimal hyperplasia by hematoxylin-eosin staining and related protein expression by immunohistochemical and Western blot analysis. Comparisons were analyzed by single-factor analysis of variance and Fisher's least significant difference test, with p < 0.05 considered significant. Results: The intima analyzed by hematoxylin-eosin staining was dramatically thicker in the control group than in the atorvastatin group and sham group (p < 0.01). The outcomes of immunohistochemical staining of α-SMA demonstrated that the percentage of α-SMA-positive cells in the control group was higher than in the atorvastatin group (p < 0.01). We also evaluated α-SMA, PCNA, p38 MAPK, and phosphorylation of p38 MAPK after statin treatment by Western blot analysis, and the results indicated that atorvastatin did not lead to p38 MAPK reduction (p < 0.05); it did, however, result in inhibition of p38 MAPK phosphorylation (p < 0.01), and it significantly reduced α-SMA and PCNA levels, in comparison with the control group (p < 0.01). Conclusion: We have demonstrated that atorvastatin can inhibit accumulation of vascular smooth muscle cells by inhibiting the p38 MAPK pathway, and it is capable of inhibiting intimal hyperplasia in a rat vein graft model.

The potential role of sesquiterpene lactones isolated from medicinal plants in the treatment of the metabolic syndrome - A review.

Metabolic syndrome comprises a cluster of metabolic disorders related to the development of cardiovascular disease and type 2 diabetes mellitus. In latter years, plant secondary metabolites have become of special interest because of their potential role in preventing and managing metabolic syndrome. Sesquiterpene lactones constitute a large and diverse group of biologically active compounds widely distributed in several medicinal plants used for the treatment of metabolic disorders. The structural diversity and the broad spectrum of biological activities of these compounds drew significant interests in the pharmacological applications. This review describes selected sesquiterpene lactones that have been experimentally validated for their biological activities related to risk factors of metabolic syndrome, together with their mechanisms of action. The potential beneficial effects of sesquiterpene lactones discussed in this review demonstrate that these substances represent remarkable compounds with a diversity of molecular structure and high biological activity, providing new insights into the possible role in metabolic syndrome management.

Novel compounds for the modulation of mTOR and autophagy to treat neurodegenerative diseases.

Most neurodegenerative diseases show a disruption of autophagic function and display abnormal accumulation of toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons clear abnormal protein aggregates and survive. The kinase mammalian target of rapamycin (mTOR) is a major regulator of the autophagic process and is regulated by starvation, growth factors, and cellular stressors. The phosphoinositide 3-kinase (PI3K)/ protein kinase B (Akt) pathway, which promotes cellular survival, is the main modulator upstream of mTOR, and alterations in this pathway are common in neurodegenerative diseases, e.g. Alzheimer's disease (AD) and Parkinson's disease (PD). In the present work we revised mammalian target of rapamycin complex 1 (mTORC1) pathway and mTORC2 as a complementary an important element in mTORC1 signaling. In addition, we revised the extracellular signal regulated kinase (ERK) pathway, which has become relevant in the regulation of the autophagic process and cellular survival through mTORC2 signaling. Finally, we summarize novel compounds that promote autophagy and neuronal protection in the last five years.

Profiling Transcriptional Regulation and Functional Roles of Schistosoma mansoni c-Jun N-Terminal Kinase.

Mitogen-activated protein kinases (MAPKs) play a regulatory role and influence various biological activities, such as cell proliferation, differentiation, and survival. Our group has demonstrated through functional studies that Schistosoma mansoni c-Jun N-terminal kinase (SmJNK) MAPK is involved in the parasite's development, reproduction, and survival. SmJNK can, therefore, be considered a potential target for the development of new drugs. Considering the importance of SmJNK in S. mansoni maturation, we aimed at understanding of SmJNK regulated signaling pathways in the parasite, correlating expression data with S. mansoni development. To better understand the role of SmJNK in S. mansoni intravertebrate host life stages, RNA interference knockdown was performed in adult worms and in schistosomula larval stage. SmJNK knocked-down in adult worms showed a decrease in oviposition and no significant alteration in their movement. RNASeq libraries of SmJNK knockdown schistosomula were sequenced. A total of 495 differentially expressed genes were observed in the SmJNK knockdown parasites, of which 373 were down-regulated and 122 up-regulated. Among the down-regulated genes, we found transcripts related to protein folding, purine nucleotide metabolism, the structural composition of ribosomes and cytoskeleton. Genes coding for proteins that bind to nucleic acids and proteins involved in the phagosome and spliceosome pathways were enriched. Additionally, we found that SmJNK and Smp38 MAPK signaling pathways converge regulating the expression of a large set of genes. C. elegans orthologous genes were enriched for genes related to sterility and oocyte maturation, corroborating the observed phenotype alteration. This work allowed an in-depth analysis of the SmJNK signaling pathway, elucidating gene targets of regulation and functional roles of this critical kinase for parasite maturation.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Involvement in the Regulation of Detoxification Mechanisms.

Following the physiological complementary/parallel Celis-Plá et al., by inhibiting extracellular signal regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and cytokinin specific binding protein (p38), we assessed the role of the mitogen-activated protein kinases (MAPK) pathway in detoxification responses mediated by chronic copper (10 µM) in U. compressa. Parameters were taken at 6, 24, and 48 h, and 6 days (d). H2O2 and lipid peroxidation under copper and inhibition of ERK, JNK, or p38 alone increased but recovered by the sixth day. By blocking two or more MAPKs under copper, H2O2 and lipid peroxidation decayed even below controls. Inhibition of more than one MAPK (at 6 d) caused a decrease in total glutathione (reduced glutathione (GSH) + oxidised glutathione (GSSG)) and ascorbate (reduced ascorbate (ASC) + dehydroascorbate (DHA)), although in the latter it did not occur when the whole MAPK was blocked. Catalase (CAT), superoxide dismutase (SOD), thioredoxin (TRX) ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione synthase (GS), were downregulated when blocking more than one MAPK pathway. When one MAPK pathway was blocked under copper, a recovery and even enhancement of detoxification mechanisms was observed, likely due to crosstalk within the MAPKs and/or other signalling processes. In contrast, when more than one MAPK pathway were blocked under copper, impairment of detoxification defences occurred, demonstrating that MAPKs were key signalling mechanisms for detoxification in macroalgae.

MAPK Pathway under Chronic Copper Excess in Green Macroalgae (Chlorophyta): Influence on Metal Exclusion/Extrusion Mechanisms and Photosynthesis.

There is currently no information regarding the role that whole mitogen activated protein kinase (MAPK) pathways play in counteracting environmental stress in photosynthetic organisms. To address this gap, we exposed Ulva compressa to chronic levels of copper (10 µM) specific inhibitors of Extracellular Signal Regulated Kinases (ERK), c-Jun N-terminal Kinases (JNK), and Cytokinin Specific Binding Protein (p38) MAPKs alone or in combination. Intracellular copper accumulation and photosynthetic activity (in vivo chlorophyll a fluorescence) were measured after 6 h, 24 h, 48 h, and 6 days of exposure. By day 6, when one (except JNK) or more of the MAPK pathways were inhibited under copper stress, there was a decrease in copper accumulation compared with algae exposed to copper alone. When at least two MAPKs were blocked, there was a decrease in photosynthetic activity expressed in lower productivity (ETRmax), efficiency (αETR), and saturation of irradiance (EkETR), accompanied by higher non-photochemical quenching (NPQmax), compared to both the control and copper-only treatments. In terms of accumulation, once the MAPK pathways were partially or completely blocked under copper, there was crosstalk between these and other signaling mechanisms to enhance metal extrusion/exclusion from cells. Crosstalk occurred among MAPK pathways to maintain photosynthesis homeostasis, demonstrating the importance of the signaling pathways for physiological performance. This study is complemented by a parallel/complementary article Rodríguez-Rojas et al. on the role of MAPKs in copper-detoxification.

DUSP3/VHR: A Druggable Dual Phosphatase for Human Diseases.

Protein tyrosine kinases (PTK), discovered in the 1970s, have been considered master regulators of biological processes with high clinical significance as targets for human diseases. Their actions are countered by protein tyrosine phosphatases (PTP), enzymes yet underrepresented as drug targets because of the high homology of their catalytic domains and high charge of their catalytic pocket. This scenario is still worse for some PTP subclasses, for example, for the atypical dual-specificity phosphatases (ADUSPs), whose biological functions are not even completely known. In this sense, the present work focuses on the dual-specificity phosphatase 3 (DUSP3), also known as VH1-related phosphatase (VHR), an uncommon regulator of mitogen-activated protein kinase (MAPK) phosphorylation. DUSP3 expression and activities are suggestive of a tumor suppressor or tumor-promoting enzyme in different types of human cancers. Furthermore, DUSP3 has other biological functions involving immune response mediation, thrombosis, hemostasis, angiogenesis, and genomic stability that occur through either MAPK-dependent or MAPK-independent mechanisms. This broad spectrum of actions is likely due to the large substrate diversity and molecular mechanisms that are still under scrutiny. The growing advances in characterizing new DUSP3 substrates will allow the development of pharmacological inhibitors relevant for possible future clinical trials. This review covers all aspects of DUSP3, since its gene cloning and crystallographic structure resolution, in addition to its classical and novel substrates and the biological processes involved, followed by an update of what is currently known about the DUSP3/VHR-inhibiting compounds that might be considered potential drugs to treat human diseases.

High-Intensity Interval Training Improves Markers of Oxidative Metabolism in Skeletal Muscle of Individuals With Obesity and Insulin Resistance.

Background: The excess body fat characteristic of obesity is related to various metabolic alterations, which includes insulin resistance (IR). Among the non-pharmacological measures used to improve insulin sensitivity are aerobic physical training, such as high-intensity interval training (HIIT). This study investigated the effects of 8 weeks of HIIT on blood and skeletal muscle markers related to IR and oxidative metabolism in physically inactive individuals with obesity and compared the changes between insulin resistant and non-insulin resistant phenotypes. Methods: Initially to investigate the effect of obesity and IR in the analyzed parameters, insulin-sensitive eutrophic volunteers (CON; n = 9) and obese non-insulin (OB; n = 9) and insulin-resistant (OBR; n = 8) were enrolled. Volunteers with obesity completed 8 weeks of HIIT in a cycle ergometer. Venous blood and vastus lateralis muscle samples were obtained before and after the HIIT. Body composition and peak oxygen consumption (VO2peak) were estimated before and after HIIT. Results: HIIT reduced IR assessed by the homeostatic model assessment of insulin resistance (HOMA-IR) in OBR (4.4 ± 1.4 versus 4.1 ± 2.2 µU L-2), but not in OB (HOMA-IR 1.8 ± 0.5 versus 2.3 ± 1.0 µU L-2) volunteers. HIIT increased VO2peak with no change in body fat in both groups. In skeletal muscle, HIIT increased the phosphorylation of IRS (Tyr612), Akt (Ser473), and increased protein content of ß-HAD and COX-IV in both groups. There was a reduction in ERK1/2 phosphorylation in OBR after HIIT. Conclusion: Eight weeks of HIIT increased the content of proteins related to oxidative metabolism in skeletal muscle of individuals with obesity, independent of changes total body fat.

Treatment with galectin-1 eye drops regulates mast cell degranulation and attenuates the severity of conjunctivitis.

Galectin-1 (Gal-1) is a ß-galactoside-binding protein with diverse biological activities in the pathogenesis of inflammation, however the mechanisms by which Gal-1 modulates cellular responses in allergic inflammatory processes have not been fully determined. In this study, we evaluated the therapeutic potential of Gal-1 eye drops in an experimental model of conjunctivitis. Wistar rats received a topical application of compound (C)48/80 (100 mg/ml) into right eyes and a drop of vehicle into the contralateral eye. Another group of rats received Gal-1 (0.3 or 3 µg/eye) or sodium cromoglycate (SCG; 40 mg/ml) in both eyes and, after 15 min, right eye was challenged with C48/80. Conjunctivitis-induced by C48/80 was characterized by severe eyelid oedema and tearing, but clinical signs were ameliorated by eye drop doses of both Gal-1 (0.3/3 µg) and SCG. As expected, an increased proportion of degranulated mast cells (62%, P < 0.01) and lower histamine levels were observed after 6 h of C48/80 challenge, compared to control (32%). This effect was abrogated by Gal-1 and SCG, which reduced mast cell degranulation (31-36%), eosinophil migration and eosinophil peroxidase levels in the eyes. Gal-1 (3 µg) and SCG treatments also decreased IL-4 levels, as well as activation of mitogen activated protein kinases compared to untreated C48/80 eyes. Our findings suggest that Gal-1 eye drops represent a new therapeutic strategy for ocular allergic inflammation.

The Application Methods of Mitogen-activated Protein Kinase Signal Pathway Inhibitors SP600125 and SB203580 in Long-term in Vivo Experiments / Métodos de aplicación de los inhibidores SP600125 y SB203580 de la vía de señalización de la proteína quinasa activada por mitógeno en experimentos in vivo a largo plazo

ABSTRACT Objective: To explore the application methods of mitogen-activated protein kinase signal pathway inhibitors SP600125 and SB203580 in long-term in vivo experiments. Methods: A total of 55 healthy New Zealand rabbits were randomly divided into blank control group, model control group, SP low dose group, SP high dose group, SP blank group, SB low dose group, SB high dose group, SB blank group, dimethyl sulfoxide (DMSO) control group, DMSO blank group, and positive control group. Since the first day of the experiment, each group was administered the corresponding treatment for four weeks continuously. Then, the myocardial c-Jun N-terminal kinase (JNK) and the total protein of p38, protein phosphorylation and its gene expression levels were detected. Results: After intravenous treatment with adriamycin, the myocardial phosphorylate-JNK (p-JNK) and phosphorylate-p38 (p-p38) levels in all groups were increased to varying degrees, of which the model control group increased the most significantly (p < 0.05). Compared with the model control group, the myocardial p-JNK and p-p38 increased more slowly in the SP low dose group, SP high dose group, SB low dose group, SB high dose group and positive control group (p < 0.05), of which the increase in the SP high dose group and the SB high dose group was the slowest (p < 0.05). After four weeks, the total protein and messenger ribonucleic acid of the myocardial JNK and p38 in all groups had no statistically significant difference (p > 0.05). Conclusion: The continuous intravenous injection of SP600125 and SB203580 for four weeks significantly reduced the protein phosphorylation levels of JNK and p38, which provides a practical avenue for the long-term study in vivo.

RESUMEN Objetivo: Explorar los métodos de aplicación de los inhibidores SP600125 y SB203580 de la vía de señalización de la proteína quinasa activada por mitógeno en experimentos in vivo a largo plazo. Métodos: Un total de 55 conejos sanos de Nueva Zelandia fueron divididos aleatoriamente en los grupos siguientes: grupo de control en blanco, grupo de control modelo, grupo de dosis baja SP, grupo de dosis alta SP, grupo en blanco SP, grupo de dosis baja SB, grupo de dosis alta SB, grupo en blanco SB, grupo de control dimetilsulfóxido (DMSO), grupo en blanco DMSO, y grupo de control positivo. Desde el primer día del experimento, a cada grupo se le administró el tratamiento correspondiente por cuatro semanas continuas. Entonces, se detectaron la quinasa c-Jun N-terminal (JNK) miocárdica y la proteína p38 total, así como la fosforilación proteica y sus niveles de expresión génica. Resultados: Después del tratamiento intravenoso con adriamicina, los niveles de fosfo-JNK (p-JNK) y fosfo-p38 (p-p38) del miocardio aumentaron en todos los grupos en diversos grados, siendo el aumento del grupo de control modelo el más significativo (p < 0.05). En comparación con el grupo de control modelo, p-JNK y p-p38 miocárdicos aumentaron más lentamente en el grupo de dosis baja SP, el grupo de dosis alta SP, el grupo de dosis baja SB, el grupo de dosis alta SB, y el grupo de control positivo (p < 0.05). De estos, el aumento en el grupo de dosis alta SP y el grupo de dosis alta SB fue el más lento (p < 0.05). Después de cuatro semanas, la proteína total y el ácido ribonucleico mensajero de JNK y p38 miocárdicos en todos los grupos, no tuvieron diferencias significativas (p > 0.05). Conclusión: La inyección intravenosa continua de SP600125 y SB203580 durante cuatro semanas redujo significativamente los niveles de fosforilación proteica de JNK y p38, lo que proporciona una vía práctica para el estudio a largo plazo in vivo.

Mitogen activated protein kinases (MAPK) and protein phosphatases are involved in Aspergillus fumigatus adhesion and biofilm formation.

The main characteristic of biofilm formation is extracellular matrix (ECM) production. The cells within the biofilm are surrounded by ECM which provides structural integrity and protection. During an infection, this protection is mainly against cells of the immune system and antifungal drugs. A. fumigatus forms biofilms during static growth on a solid substratum and in chronic aspergillosis infections. It is important to understand how, and which, A. fumigatus signal transduction pathways are important for the adhesion and biofilm formation in a host during infection. Here we investigated the role of MAP kinases and protein phosphatases in biofilm formation. The loss of the MAP kinases MpkA, MpkC and SakA had an impact on the cell surface and the ECM during biofilm formation and reduced the adherence of A. fumigatus to polystyrene and fibronectin-coated plates. The phosphatase null mutants ΔsitA and ΔptcB, involved in regulation of MpkA and SakA phosphorylation, influenced cell wall carbohydrate exposure. Moreover, we characterized the A. fumigatus protein phosphatase PphA. The ΔpphA strain was more sensitive to cell wall-damaging agents, had increased ß-(1,3)-glucan and reduced chitin, decreased conidia phagocytosis by Dictyostelium discoideum and reduced adhesion and biofilm formation. Finally, ΔpphA strain was avirulent in a murine model of invasive pulmonary aspergillosis and increased the released of tumor necrosis factor alpha (TNF-α) from bone marrow derived macrophages (BMDMs). These results show that MAP kinases and phosphatases play an important role in signaling pathways that regulate the composition of the cell wall, extracellular matrix production as well as adhesion and biofilm formation in A. fumigatus.

Dysregulated mitogen-activated protein kinase and matrix metalloproteinase in ethanol-induced cavernosal dysfunction.

We evaluated the effects of ethanol consumption on the mitogen-activated protein kinases (MAPK) and metalloproteinases (MMP) pathways in the rat cavernosal smooth muscle (CSM). Male Wistar rats were treated with ethanol (20% v/v) for 6 weeks. Quantitative real-time polymerase chain reaction experiments showed that ethanol consumption did not alter mRNA levels of p38MAPK, SAPK/JNK, ERK1/2, MMP-2, or MMP-9 in the rat CSM. Western immunoblotting experiments revealed decreased protein expression of p38MAPK and phosphorylation of SAPK/JNK in the CSM from ethanol-treated rats. Additionally, ethanol consumption decreased the expression of MMP-2. Functional assays showed that SP600125, an inhibitor of SAPK/JNK, prevented the increase in endothelin (ET)-1-induced contraction in the CSM from ethanol-treated rats. Treatment with ethanol decreased MMP-2 activity, but did not change net MMP activity in the rat CSM. Ethanol consumption increased the circulating levels of MMP-2, MMP-9, and TIMP-2 as well as the MMP-9/TIMP-1 ratio. The major finding of our study is that ethanol consumption down-regulates both MAPK and MMP pathways in the rat CSM, whereas it increases the circulating levels of MMP-9. Additionally, we found that SAPK/JNK plays a role in ethanol-induced increase on ET-1 contraction in the isolated rat CSM.

The p38 MAPK and NF-κB Pathways are Involved in Cyclic Compressive Force-induced IL-6 Secretion in MLO-Y4 Cells

ABSTRACT We previously revealed the involvement of extracellular regulated protein kinases 1/2 (ERK1/2) in interleukin-6 (IL-6) secretion induced by cyclic compressive force (CCF) in MLO-Y4 cells. In this study, we investigated the contributions of the p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) pathways to IL-6 secretion by stimulating MLO-Y4 cells with CCF. At 80% confluence, different magnitudes (1000μstrain, 2000 μstrain and 4000 μstrain), frequencies (0.5 Hz, 1.0 Hz and 2.0 Hz) and durations (10 min, 30 min, 1 h, 3 h and 6 h) of CCF were loaded onto cells using a four-point bending system. Flow Cytometry (FCM) analysis was used to analyze cell mortality rates after CCF loading. p38 and p65 phosphorylation as well as IκBα degradation in MLO-Y4 cells were detected by Western blotting (WB). Changes in IL-6 secretion after inhibitor treatment were assessed by enzyme-linked immunosorbent assays (ELISAs). Cellular viability was over 90 percent after CCF. p38 and p65 phosphorylation increased under all conditions, whereas IκBα protein levels decreased. However, phosphorylation and degradation were not completely dependent on the loading magnitude, frequency or duration. Furthermore, p38 inhibition using the specific inhibitor SB203580 reduced both p38 phosphorylation and IL-6 secretion. Similarly, NF-κB inhibition using BAY 11-7082 decreased p65 phosphorylation and IL-6 secretion but increased the concentration of IκBα. These findings reveal significant roles for the p38 and NF-κB signaling pathways in IL-6 secretion induced by CCF in MLO-Y4 cells.

Mesenchymal stem cells decrease lung inflammation during sepsis, acting through inhibition of the MAPK pathway.

BACKGROUND: Sepsis is a severe medical condition that ranks among the top 10 causes of death worldwide and which has permanently high incidence rates. Mesenchymal stem cells (MSCs) have been found to be potent modulators of immune responses. More importantly, there is evidence that MSCs have a beneficial effect on preclinical models of polymicrobial sepsis. However, the changes caused by the MSCs in the effector cells of the host immune system remain unclear. METHODS: A mouse model of sepsis (male C57BL/6 mice) with three experimental groups was used for experiments in vivo: a control group, an untreated septic group, and a septic group treated with MSCs. In vitro experiments were performed using a cell line of pulmonary macrophages (RAW 264.7) co-cultured with MSCs and stimulated with lipopolysaccharide (LPS). RESULTS: In vivo we demonstrated that treatment with MSCs was able to reduce the expression of cyclooxygenase-2 (COX-2) and nuclear factor kappa B (NF-κB), and thereby decrease the production of inflammatory cytokines. In vitro experiments using a co-culture of macrophages with MSCs showed a decrease in COX-2 and NF-κB, and showed that this reduction was directly related to the ability of MSCs to inhibit phosphorylation of ERK, RSK, and p38, enzymes that belong to the family of mitogen-activated protein kinases (MAPKs). CONCLUSIONS: This study demonstrated that MSCs are able to inhibit the MAPK pathway activation, modulating the inflammatory response during sepsis. This understanding that MSCs can remodel the response of host cells and improve the course of sepsis is essential for developing new treatments for this pathology.