Phosphorylation Of P38 Mitogen-activated Protein Kinase Research Articles

The deleterious effect of stress-induced depression on rat liver: Protective role of resveratrol and dimethyl fumarate via inhibiting the MAPK/ERK/JNK pathway.

This study aimed to uncover the protective potentiality of resveratrol and dimethyl fumarate (DMF) in the liver of a chronic unpredictable mild stress (CUMS)-induced depression animal model. Resveratrol and DMF significantly alleviated CUMS-induced behavioral abnormalities in stressed rats through improving sucrose preference in sucrose preference test and decreasing immobility time in a forced swimming test. They also mitigated serum corticosterone levels and elevated serum serotonin levels, which were formerly disturbed in CUMS rats. The hepatoprotective effect is evidenced by improvement in hepatic histopathological examinations, as well asnormalized serum alanine aminotransferase and aspartate aminotransferase activities. Molecular signaling of resveratrol and DMF was estimated by diminishing hepatic expression of phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase1/2 (ERK1/2), and c-Jun N-terminal kinase (JNK). Consequently, they improved the hepatic antioxidant and anti-inflammatory activities as elaborated by the normalization of total antioxidant capacity, glutathione, malondialdehyde, nuclear factor-κB, tumor necrosis factor-α, and myeloperoxidase levels. In addition, they inhibited hepatocyte apoptosis as evidenced by the increased expression of B-cell lymphoma 2, the decreased expression of Bax, as well asthe suppressed activity of caspase-3. In conclusion, resveratrol and DMF purveyed a significant anti-depressant effect, which may be mediated, at least in part, via inhibiting the MAPK/ERK/JNK pathway in the CUMS rat model.

Rare earth element lanthanum protects against atherosclerosis induced by high-fat diet via down-regulating MAPK and NF-κB pathways

Rare earth elements, which are extensively used in environmental protection, medicine, food, aerospace and other fields, have attracted widespread attention in recent years. However, the effect on atherosclerosis and its biological mechanism remains unclear. To elucidate these problems, here we performed a study that Apolipoprotein E-deficient mice were fed with high-fat diet to promote the development of atherosclerosis, meanwhile, mice were received 0.1, 0.2, 1.0, 2.0 mg/kg lanthanum nitrate (La(NO3)3) for 12 weeks. The results showed that La(NO3)3 prominently inhibited aorta morphological alternations by histopathological examination. Meanwhile, La(NO3)3 regulated serum lipids, including reducing total cholesterol and increasing high-density lipoprotein. Moreover, the oxidative stress was alleviated by La(NO3)3 intervention through enhancing superoxide dismutase and glutathione, and decreasing malondialdehyde levels. In addition, enzyme-linked immunosorbent assay analysis showed La(NO3)3 could ameliorate the dysfunction of vascular endothelium with declined endothelin-1 and increased prostacyclin. Furthermore, Western blot analysis indicated that La(NO3)3 significantly down-regulated inflammation-mediated proteins including phosphorylated p38 mitogen-activated protein kinases (p-p38 MAPK), monocyte chemo-attractant protein, intercellular adhesion molecule-1, nuclear factor-kappa B p65 (NF-κB p65), tumor necrosis factor-α, interleukin-6 and interleukin-1β, whereas up-regulated the inhibitor of NF-κB protein. In conclusion, La(NO3)3 ameliorates atherosclerosis by regulating lipid metabolism, oxidative stress, endothelial dysfunction and inflammatory response in mice. The potential mechanism associates with the inhibition of MAPK and NF-κB signaling pathways.

Heat shock protein 70 positively regulates transforming growth factor-α-induced hepatocellular carcinoma cell migration via the AKT signaling pathway

Heat shock proteins (HSPs) are induced in response to extracellular stress and manage the quality of proteins as molecular chaperones. HSP70, a highly conserved HSP, has been reported to correlate with the proliferation and migration of human cancer cells, such as oral, prostate, lung and liver cancer. Regarding hepatocellular carcinoma (HCC), the HSP70 levels in the tumor tissues from patients are significantly higher than those in the normal liver tissues. HSP70 reportedly upregulates the migration and invasion of HCC. The AKT, p38 mitogen-activated protein kinase (MAPK), c-jun N-terminal kinase (JNK) and Rho-kinase signaling pathways regulate the transforming growth factor (TGF)-α-induced migration of human HCC-derived HuH7 cells. However, the exact mechanism underlying the role of HSP70 in growth factor-induced HCC migration remains unclear. Therefore, in the present study, the mechanism underlying the involvement of HSP70 in TGF-α-induced HCC cell migration was investigated. Treatment with the HSP70 inhibitors VER155008 and YM-08 and the downregulation of HSP70 protein were confirmed to significantly suppress the TGF-α-induced cell migration of HuH7 cells. Both VER155008 and YM-08 reduced the TGF-α-induced phosphorylation of AKT without affecting the phosphorylation of p38 MAPK, JNK or Rho-kinase. These results strongly suggest that HSP70 positively regulates the TGF-α-induced migration of HCC cells via the AKT signaling pathway.

Environmental enrichment rescues cognitive impairment with suppression of TLR4-p38MAPK signaling pathway in vascular dementia rats

Increasing evidence demonstrated the promising effects of environmental enrichment (EE) on brain recovery and cognitive performance in animal models of various diseases. However, the effect and molecular mechanisms of EE on vascular dementia (VD) remain to be studied. The aim of this study was to explore the effect of EE on cognitive decline and its mechanism. Sprague-Dawley rats underwent 2-vessel occlusion (2-VO) surgery or sham operation. Subsequently, rats were kept in EE for 4 weeks. In Morris water maze (MWM) test, we demonstrated that EE significantly improved cognitive function in rats with VD. HE staining exhibited morphological changes of neurons and quantitative analysis of TUNEL showed increased apoptotic neurons in hippocampal CA1 region following 2-VO. Results from RT-qPCR showed up-regulation of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) after 2-VO. Western blotting analysis revealed enhanced toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MYD88) and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK) in 2-VO rats. Whereas administration of EE reduced apoptotic neurons, down-regulated inflammatory factors. Moreover, EE suppressed protein expression of TLR4-p38MAPK pathway. Spearman correlation analysis showed that improved cognitive function was associated with decreased expression of TLR4 and p-p38MAPK proteins. Thus, our study proved that EE has a prominent effect on cognitive impairment and neuronal damage following 2-VO by attenuating inflammation and apoptosis, which may be realized via inhibiting the TLR4-P38MAPK signaling pathway.

Inhibition of gastric cancer cell apoptosis by long noncoding RNA TRPM2-AS via mitogen-activated protein kinase and activators of transduction-3.

Long noncoding RNA TRPM2-AS has emerged as a novel regulator in cancer initiation and progression of various cancers. However, the function and underlying mechanism of TRPM2-AS in the progression of gastric cancer (GC) remain poorly understood. GEO and TCGA databases were used for isolation of differential lncRNA expression. TRPM2-AS expression levels in GC tissues and cells were measured by quantitative polymerase chain reaction method. TRPM2-AS subcellular location was detected by fluorescence in situ hybridization analysis. The functional roles of TRPM2-AS in cells were analyzed by loss and gain function assays. By using bioinformatics and quantitative polymerase chain reaction methods, TRPM2-AS expression levels were proved to be upregulated in GSE70880 dataset, TCGA database, and 26 GC tissues, which was partly induced by SP1. The results of clinical assays showed that TRPM2-AS could be an indicator for early-stage GC diagnosis. Fluorescence in situ hybridization analysis showed that TRPM2-AS was located in both nucleus and cytoplasm. Functional experiments displayed that knockdown of TRPM2-AS inhibited proliferation, migration, and invasion in GC cells. Furthermore, depression of TRPM2-AS suppressed cell growth though promotion of cell apoptosis. The expression levels of cleaved PARP, caspase 9, caspase 3, and Bax were significantly increased in BGC823 with TRPM2-AS knockdown. In addition, knockdown of TRPM2-AS reduced and phosphorylate signal transducer and activator of transcription 3 and increased and phosphorylate p38 mitogen-activated protein kinase. This study demonstrated that SP1-regulated TRPM2-AS is involved in GC cell apoptosis probably via p38 mitogen-activated protein kinase and signal transducer and activator of transcription 3 pathways, indicating that TRPM2-AS might be a potential therapeutic target in GC.

Lactobacillus salivarius alleviates inflammation via NF-\u03baB signaling in ETEC K88-induced IPEC-J2 cells

BackgroundEnterotoxigenic Escherichia coli (ETEC) K88 commonly colonize in the small intestine and keep releasing enterotoxins to impair the intestinal barrier function and trigger inflammatory reaction. Although Lactobacillus salivarius (L. salivarius) has been reported to enhance intestinal health, it remains to be seen whether there is a functional role of L. salivarius in intestinal inflammatory response in intestinal porcine epithelial cell line (IPEC-J2) when stimulated with ETEC K88. In the present study, IPEC-J2 cells were first treated with L. salivarius followed by the stimulation of ETEC K88 for distinct time period. ETEC K88 adherent status, pattern recognition receptors (PRRs) mRNA, mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) activation, the release of pro-inflammation cytokines and cell integrity were examined.ResultsAside from an inhibited adhesion of ETEC K88 to IPEC-J2 cells, L. salivarius was capable of remarkably attenuating the expression levels of interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), IL-8, Toll-like receptor (TLR) 4, nucleotide-binding oligomerization domain (NOD)-like receptor pyrin domain-containing protein (NLRP) 3 and NLRP6. This alternation was accompanied by a significantly decreased phosphorylation of p38 MAPK and p65 NF-κB during ETEC K88 infection with L. salivarius pretreatment. Western blot analysis revealed that L. salivarius increased the expression levels of zona occludens 1 (ZO-1) and occludin (P < 0.05) in ETEC K88-infected IPEC-J2 cells. Compared with ETEC K88-infected groups, the addition of L. salivarius as well as extra inhibitors for MAPKs and NF-κB to ETEC K88-infected IPEC-J2 cells had the capability to reduce pro-inflammatory cytokines.ConclusionsCollectively, our results suggest that L. salivarius might reduce inflammation-related cytokines through attenuating phosphorylation of p38 MAPK and blocking the NF-κB signaling pathways. Besides, L. salivarius displayed a potency in the enhancement of IPEC-J2 cell integrity.

Magnesium lithospermate B improves pulmonary artery banding induced right ventricular dysfunction by alleviating inflammation via p38MAPK pathway

BackgroudMagnesium lithospermate B (MLB) is a major bioactive component of Slavia miltiorrhiza, which has been widely used in heart diseases on account of its anti-inflammatory, anti-oxidative, anti-proliferative and anti-fibrotic properties. Substance P(SP) is a small molecule neuropeptide, which was secreted much more during heart failure, and has an obvious function of immune enhancement and inflammation induction. This study aimed to investigate the protective effects of MLB on pulmonary artery banding (PAB) induced right ventricular (RV) dysfunction. MethodsThe mouse model of PAB was established. The mice were intraperitoneal (IP) injection treated with MLB (10 mg kg−1·d−1) for 4 weeks and p38 mitogen-activated protein kinase (MAPK) activator was given at the same time. Echocardiography were performed on day 28. Then the hearts were harvested, and substance P (SP), inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and cardiac fibrosis were detected. The macrophages and fibroblasts were stimulated by SP separately, and then treated with MLB as well as p38MAPK activator. The inflammatory cytokines from macrophage, the proliferation and fibrosis of cardiac fibroblasts were measured. The expression of p38MAPK proteins were confirmed by immunoblotting. FindingsMLB preserved RV ejection fraction (EF), FS, RV/(LV + septum), HW/BW index and blunted RV inflammation as well as fibrosis. Phosphorylated-p38 (p-p38) MAPK was up-regulated, which was partially reversed by MLB treatment. However, p38MAPK activator abolished the effects of MLB on RV dysfunction, suggesting a key role of p38MPAK pathway in the effects of MLB reversing RV dysfunction. In external experiment, MLB reversed the increase of inflammatory cytokines from macrophage, the proliferation and fibrosis of cardiac fibroblasts which was simulated by SP. In accordance with in vivo study, p38MAPK activator abolished the effects of MLB on macrophage as well as fibroblasts. InterpretationMLB improves PAB induced right ventricular remodeling by alleviating inflammation via p38MAPK pathway. Thus, MLB may offer the therapeutic potential for the patients of RV dysfunction.

Hypoxic induction of CCN2 mRNA through p38 MAP kinase activation in the human chondrosarcoma‐derived cell line, HCS‐2/8

AbstractCCN2/CTGF (cellular communication network factor 2/connective tissue growth factor) plays critical roles in cartilage development, maintenance, and regeneration. Hypoxia‐induced expression of CCN2 mRNA is regulated post‐transcriptionally in the human chondrosarcoma‐derived cell line, HCS‐2/8. In the present study, the hypoxia‐induced increase in CCN2 mRNA expression was assessed with quantitative real‐time PCR in HCS‐2/8 cells in the presence of inhibitors of mitogen‐activated protein kinases (MAPKs). Subsequently, the stability of CCN2 mRNA in hypoxia was evaluated with mRNA degradation assays in the presence or absence of the selective p38 MAPK inhibitor, SB203580. We detected phosphorylation of p38 MAPK by immunoblot analysis within 30 minutes in hypoxia, and we observed ~twofold higher CCN2 mRNA levels in hypoxia compared to normoxia. Blockade of p38 MAPK activation with 10 µmol/L SB203580 abolished this CCN2 induction. Furthermore, we found that inhibition of p38 MAPK suppressed the elongation of CCN2 mRNA half‐life in hypoxia. Taken together, these findings suggest that the molecular mechanism, by which CCN2 mRNA expression is increased in hypoxia, induces the activation of p38 MAPK leading to the post‐transcriptional regulation of the stability of CCN2 mRNA.

Erythronium japonicum Alleviates Inflammatory Pain by Inhibiting MAPK Activation and by Suppressing NF-κB Activation via ERK/Nrf2/HO-1 Signaling Pathway.

Microglial activation-mediated neuroinflammation influences the development of inflammatory pain. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of aqueous Erythronium japonicum extract (EJE) in microglia activation-mediated inflammatory pain. EJE was found to suppress lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), ionized calcium-binding adapter molecule 1 (IBA-1), and pro-inflammatory cytokines in BV2 microglial cells. In addition, LPS-induced c-Jun NH2 terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation were inhibited by EJE. Intriguingly, EJE also inhibited p65 phosphorylation by activating extracellular signal-regulated kinase-1/2 (ERK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Furthermore, the effects of EJE treatment, such as HO-1 induction and the reduction of NF-ĸB activation, were reversed by ERK1/2 inhibition. In an inflammatory pain mouse model, Complete Freund’s Adjuvant (CFA)-induced mechanical allodynia and foot swelling were alleviated by the oral administration of EJE. Consistent with in vitro results, EJE increased HO-1, while decreasing CFA-induced COX-2, IBA-1, and pro-inflammatory cytokines in the spinal cord. Among the components of EJE, butanol most heavily suppressed LPS-induced microglial activation and increased HO-1 expression. These findings indicate that EJE can alleviate inflammatory pain by inhibiting p38 and JNK and by suppressing NF-ĸB via ERK/Nrf2/HO-1 signaling.

Cannabidiol induces osteoblast differentiation via angiopoietin1 and p38 MAPK.

In this study, we report the potential of cannabidiol, one of the major cannabis constituents, for enhancing osteoblastic differentiation in U2OS and MG-63 cells. Cannabidiol increased the expression of Angiopoietin1 and the enzyme activity of alkaline phosphatase in U2OS and MG-63. Invasion and migration assay results indicated that the cell mobility was activated by cannabidiol in U2OS and MG-63. Western blotting analysis showed that the expression of tight junction related proteins such as Claudin1, Claudin4, Occuludin1, and ZO1 was increased by cannabidiol in U2OS and MG-63. Alizarin Red S staining analysis showed that calcium deposition and mineralization was enhanced by cannabidiol in U2OS and MG-63. Western blotting analysis indicated that the expression of osteoblast differentiation related proteins such as distal-less homeobox 5, bone sialoprotein, osteocalcin, type I collagen, Runt-related transcription factor 2 (RUNX2), osterix (OSX), and alkaline phosphatase was time dependently upregulated by cannabidiol in U2OS and MG-63. Mechanistically, cannabidiol-regulated osteoblastic differentiation in U2OS and MG-63 by strengthen the protein-protein interaction among RUNX2, OSX, or the phosphorylated p38 mitogen-activated protein kinase (MAPK). In conclusion, cannabidiol increased Angiopoietin1 expression and p38 MAPK activation for osteoblastic differentiation in U2OS and MG-63 suggesting that cannabidiol might provide a novel therapeutic option for the bone regeneration.

CircANRIL reduces vascular endothelial injury, oxidative stress and inflammation in rats with coronary atherosclerosis.

Effects of circular antisense non-coding RNA in the INK4 locus (circANRIL) on vascular endothelial injury, oxidative stress and inflammation in rats with coronary atherosclerosis were studied by establishing a rat model of coronary atherosclerosis in which circANRIL was differentially expressed. A total of 40 healthy Sprague Dawley (SD) rats were randomly divided into research group (n=32) and control group (n=8). In research group, a rat model of coronary atherosclerosis was established without special treatment. The blood calcium (Ca2+) and lipid levels in the two groups were compared. After cell transfection, the rats were divided into blank group (untransfected), negative group (transfected with blank vector), circANRIL group (transfected with circANRIL overexpression plasmid) and circANRIL inhibitor group (transfected with circANRIL silencer). Then the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), malondialdehyde (MDA), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in each group were compared. Western blotting was adopted to detect the expressions of phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK), p38MAPK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Finally, p-p38MAPK/GAPDH, p38MAPK/GAPDH and p-p38MAPK/p38MAPK were calculated. There were significant differences in the levels of serum Ca2+ and lipid between control group and research group (P<0.05). Besides, differences in LDH, SOD, MDA, TNF-α and IL-6 in the supernatant in each group were statistically significant (P<0.05 or P<0.01). Moreover, there were statistically significant differences in the gray values of p-p38MAPK/GAPDH and p38MAPK/GAPDH and their ratio p-p38MAPK/p38MAPK in each group (P<0.05 or P<0.01). Inhibiting the expression of circANRIL in coronary heart disease cases can reduce vascular endothelial injury, oxidative stress and inflammation.

Carbon Dot Nanoparticles Exert Inhibitory Effects on Human Platelets and Reduce Mortality in Mice with Acute Pulmonary Thromboembolism.

The inhibition of platelet activation is considered a potential therapeutic strategy for the treatment of arterial thrombotic diseases; therefore, maintaining platelets in their inactive state has garnered much attention. In recent years, nanoparticles have emerged as important players in modern medicine, but potential interactions between them and platelets remain to be extensively investigated. Herein, we synthesized a new type of carbon dot (CDOT) nanoparticle and investigated its potential as a new antiplatelet agent. This nanoparticle exerted a potent inhibitory effect in collagen-stimulated human platelet aggregation. Further, it did not induce cytotoxic effects, as evidenced in a lactate dehydrogenase assay, and inhibited collagen-activated protein kinase C (PKC) activation and Akt (protein kinase B), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK) phosphorylation. The bleeding time, a major side-effect of using antiplatelet agents, was unaffected in CDOT-treated mice. Moreover, our CDOT could reduce mortality in mice with ADP-induced acute pulmonary thromboembolism. Overall, CDOT is effective against platelet activation in vitro via reduction of the phospholipase C/PKC cascade, consequently suppressing the activation of MAPK. Accordingly, this study affords the validation that CDOT has the potential to serve as a therapeutic agent for the treatment of arterial thromboembolic disorders

Peptidylarginine Deiminase 4 Promotes the Renal Infiltration of Neutrophils and Exacerbates the TLR7 Agonist-Induced Lupus Mice

Peptidylarginine deiminase 4 (PAD4), encoded by PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.

HSP90 inhibitors strengthen extracellular ATP-stimulated synthesis of interleukin-6 in osteoblasts: Amplification of p38 MAP kinase.

Heat shock protein 90 (HSP90) is expressed ubiquitously in a variety of cell types including osteoblasts. HSP90 acts as a key driver of proteostasis under pathophysiological conditions. Here, we investigated the involvement of HSP90 in extracellular ATP-stimulated interleukin (IL)-6 synthesis and HSP90 downstream signalling in osteoblast-like MC3T3-E1 cells. In osteoblasts, extracellular ATP stimulates the synthesis of IL-6, a bone-remodelling agent. Geldanamycin, 17-allylamino-17-demethoxy-geldanamycin (17-AAG) and onalespib, three different HSP90 inhibitors, amplified the ATP-stimulated IL-6 release. Geldanamycin increased IL-6 mRNA expression elicited by ATP. ATP enhanced the triiodothyronine-induced osteocalcin release, but HSP90 inhibitors suppressed the release. Extracellular ATP induced the phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), p38 MAPK, c-Jun N-terminal kinase (JNK), p70 S6 kinase, Akt, and myosin phosphatase-targeting subunit (MYPT), a Rho-kinase substrate. SB203580, an inhibitor of p38 MAPK, suppressed ATP-stimulated IL-6 release. Inhibitors of MEK1/2 (PD98059), JNK (SP600125), upstream kinase of p70 S6 kinase (rapamycin) and Akt (deguelin), all increased IL-6 release. Y27632, a Rho-kinase inhibitor, failed to affect the IL-6 release stimulated by ATP. Geldanamycin and 17-AAG both amplified ATP-induced p38 MAPK phosphorylation, although geldanamycin inhibited the phosphorylation of Akt induced by ATP. In addition, SB203580 significantly reduced the amplification by geldanamycin of the IL-6 release. Taken together, our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 MAPK activation in osteoblasts. SIGNIFICANCE OF THE STUDY: Heat shock protein 90 (HSP90) acts as a key driver of proteostasis under pathophysiological conditions in a variety of cell types. We have previously shown that HSP90 is expressed at high levels in osteoblast-like MC3T3-E1 cells, even in their quiescent state, consistent with HSP90 performing an important physiological function in osteoblasts. In the present study, we investigated whether HSP90 is implicated in extracellular ATP-induced interleukin (IL)-6 synthesis in osteoblast-like MC3T3-E1 cells. Our results strongly suggest that HSP90 inhibitors up-regulate extracellular ATP-stimulated IL-6 synthesis via amplification of p38 mitogen-activated protein kinase activation in osteoblasts.

Maternal Hyperhomocysteinemia Induces Neuroinflammation and Neuronal Death in the Rat Offspring Cortex.

Maternal hyperhomocysteinemia is one of the common complications of pregnancy that causes offspring cognitive deficits during postnatal development. In the present work, we evaluated the effect of prenatal hyperhomocysteinemia on structural and ultrastructural organization, neuronal and glial cell number, apoptosis (caspase-3 content and activity), inflammatory markers (tumor necrosis factor-α, interleukin-6, and interleukin-1β), and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation in the offspring brain cortex in early ontogenesis. Wistar female rats received methionine (0.6g/kg body weight) by oral administration during pregnancy. Histological and biochemical analyses of 5- and 20-day-old pups' cortical tissue were performed. Lysosome accumulation and other neurodegenerative changes in neurons of animals with impaired embryonic development were investigated by electron microscopy. Neuronal staining (anti-NeuN) revealed a reduction in neuronal number, accompanied by increasing of caspase-3 active form protein level and activity. Maternal hyperhomocysteinemia also elevated the number of astroglial and microglial cells and increased expression of interleukin-1β and p38 MAPK phosphorylation, which indicates the development of neuroinflammatory processes.

The NMDA receptor subunit GluN3A regulates synaptic activity-induced and myocyte enhancer factor 2C (MEF2C)-dependent transcription

N-Methyl-d-aspartate type glutamate receptors (NMDARs) are key mediators of synaptic activity-regulated gene transcription in neurons, both during development and in the adult brain. Developmental differences in the glutamate receptor ionotropic NMDA 2 (GluN2) subunit composition of NMDARs determines whether they activate the transcription factor cAMP-responsive element-binding protein 1 (CREB). However, whether the developmentally regulated GluN3A subunit also modulates NMDAR-induced transcription is unknown. Here, using an array of techniques, including quantitative real-time PCR, immunostaining, reporter gene assays, RNA-Seq, and two-photon glutamate uncaging with calcium imaging, we show that knocking down GluN3A in rat hippocampal neurons promotes the inducible transcription of a subset of NMDAR-sensitive genes. We found that this enhancement is mediated by the accumulation of phosphorylated p38 mitogen-activated protein kinase in the nucleus, which drives the activation of the transcription factor myocyte enhancer factor 2C (MEF2C) and promotes the transcription of a subset of synaptic activity-induced genes, including brain-derived neurotrophic factor (Bdnf) and activity-regulated cytoskeleton-associated protein (Arc). Our evidence that GluN3A regulates MEF2C-dependent transcription reveals a novel mechanism by which NMDAR subunit composition confers specificity to the program of synaptic activity-regulated gene transcription in developing neurons.

Syringaresinol Inhibits UVA-Induced MMP-1 Expression by Suppression of MAPK/AP-1 Signaling in HaCaT Keratinocytes and Human Dermal Fibroblasts.

Ultraviolet (UV) irradiation induces detrimental changes in human skin which result in photoaging. UV-induced intracellular changes cause degradation of extracellular matrix (ECM). UV-stimulated cleavage of collagen in ECM occurs via matrix metalloproteinases (MMPs). (±)-syringaresinol (SYR), a phytochemical which belongs to the lignan group of polyphenols, was investigated for its ability to reverse the UVA-induced changes in human HaCaT keratinocytes and dermal fibroblasts (HDFs) in vitro. Effect of SYR on UVA-induced changes was investigated by production and activation of MMPs and its transcriptional upstream effectors; mitogen-activated protein kinases (MAPKs) and pro-inflammatory mediators. Levels of expression were determined using ELISA, RT-PCR and immunoblotting. UVA irradiation stimulated the production of MMP-1 and inhibited collagen production. SYR treatment suppressed MMP-1 and enhanced collagen production in UVA-irradiated HaCaT keratinocytes and HDFs. SYR repressed the UV-induced phosphorylation of p38, ERK and JNK MAPKs in HaCaT keratinocytes while only suppressing JNK phosphorylation in HDFs. In addition, SYR was able to inhibit UVA-induced production of inflammatory cytokines; TNF-α, COX-2, IL-1β and IL-6. Moreover, SYR suppressed the activator protein-1 (AP-1), a heterodimer of phosphorylated transcription factors c-Jun and c-Fos. SYR-treatment decreased nuclear levels of activated c-Fos and c-Jun as a mechanism to inhibit UVA-induced transcriptional activities leading to MMP-1 production. In conclusion, current results demonstrated that SYR could inhibit UVA-induced upregulation of MMP-1 by suppressing MAPK/AP-1 signaling in HaCaT keratinocytes and HDFs. Therefore, SYR was suggested as a potential compound with antiphotoaging properties against UVA-induced skin aging.

3,5-dicaffeoyl‑epi-quinic acid from Atriplex gmelinii enhances the osteoblast differentiation of bone marrow-derived human mesenchymal stromal cells via WnT/BMP signaling and suppresses adipocyte differentiation via AMPK activation

BackgroundImpaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment. PurposeIn this context, 3,5-dicaffeoyl‑epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro. MethodsAnti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways. ResultsAt 10 μM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of β-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes. ConclusionDCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.

CDDO-Me Inhibits Microglial Activation and Monocyte Infiltration by Abrogating NFκB- and p38 MAPK-Mediated Signaling Pathways Following Status Epilepticus.

Following status epilepticus (SE, a prolonged seizure activity), microglial activation, and monocyte infiltration result in the inflammatory responses in the brain that is involved in the epileptogenesis. Therefore, the regulation of microglia/monocyte-mediated neuroinflammation is one of the therapeutic strategies for avoidance of secondary brain injury induced by SE. 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid methyl ester (CDDO-Me; RTA 402) is an activator of nuclear factor-erythroid 2-related factor 2 (Nrf2), which regulates intracellular redox homeostasis. In addition, CDDO-Me has anti-inflammatory properties that suppress microglial proliferation and its activation, although the underlying mechanisms have not been clarified. In the present study, CDDO-Me ameliorated monocyte infiltration without vasogenic edema formation in the frontoparietal cortex (FPC) following SE, accompanied by abrogating monocyte chemotactic protein-1 (MCP-1)/tumor necrosis factor-α (TNF-α) expressions and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Furthermore, CDDO-Me inhibited nuclear factor-κB (NFκB)-S276 phosphorylation and microglial transformation, independent of Nrf2 expression. Similar to CDDO-Me, SN50 (an NFκB inhibitor) mitigated monocyte infiltration by reducing MCP-1 and p38 MAPK phosphorylation in the FPC following SE. Therefore, these findings suggest, for the first time, that CDDO-Me may attenuate microglia/monocyte-mediated neuroinflammation via modulating NFκB- and p38 MAPK-MCP-1 signaling pathways following SE.

Monomeric C-reactive protein affects cell injury and apoptosis through activation of p38 mitogen-activated protein kinase in human coronary artery endothelial cells.

C-reactive protein (CRP) is an important predictor of cardiovascular events and plays a role in vascular inflammation and vessel damage. The aim of this study was to investigate the effect of pentameric CRP (pCRP) and monomeric CRP (mCRP) on the production of atherosclerosis-related factors in cultured human coronary artery endothelial cells (HCAECs). HCAECs were treated with pCRP, mCRP, p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580, or transfected with p38 MAPK siRNA. Western blotting was performed to detect the expression of vascular endothelial growth factor (VEGF), cyclooxygenase-2 (COX-2), intercellular adhesion molecule-2 (ICAM-2) and vascular cell adhesion molecule-1 (VCAM-1). Proliferation, damage, and apoptosis of HCAECs were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, lactate dehydrogenase (LDH), and flow cytometry, respectively. mCRP suppressed VEGF and COX-2 expression and enhanced ICAM-2 and VCAM-1 expression in HCAECs, in both dose-dependent and time-dependent manner. Except at 100 µg/ml concentration and 20-hour or 24-hour incubation, pCRP had no apparent effects. mCRP but not pCRP induced HCAEC injury and phosphorylation of p38 MAPK, and the inhibitor SB203580 reversed the effects of mCRP. mCRP promotes injury and apoptosis of HCAECs through a p38 MAPK-dependent mechanism, which provides a new therapy for the injury of HCAECs in atherosclerosis.