Phosphorylation Of P38 Mitogen-activated Protein Kinase Research Articles

Rose Bengal Photodynamic Therapy (RB-PDT) Modulates the Inflammatory Response in LPS-Stimulated Human Corneal Fibroblasts By Influencing NF-κB and p38 MAPK Signaling Pathways

Purpose To investigate the effect of rose bengal photodynamic therapy on lipopolysaccharide-induced inflammation in human corneal fibroblasts. Furthermore, to analyze potential involvement of the mitogen-activated protein kinase and nuclear factor kappa B signaling pathways in this process. Methods Human corneal fibroblast cultures underwent 0–2.0 µg/mL lipopolysaccharide treatment, and 24 h later rose bengal photodynamic therapy (0.001% RB, 565 nm wavelength illumination, 0.17 J/cm2 fluence). Interleukin-6, interleukin-8, intercellular adhesion molecule-1, interferon regulatory factor-3, interferon α2, and interferon β1 gene expressions were determined by quantitative PCR. Interleukin-6, interleukin-8, and C-C motif chemokine ligand-4 concentrations in the cell culture supernatant were measured by enzyme-linked immunosorbent assays and intercellular adhesion molecule-1 protein level in human corneal fibroblasts by western blot. In addition, the nuclear factor kappa B and mitogen-activated protein kinase signaling pathways were investigated by quantitative PCR and phosphorylation of nuclear factor kappa B p65 and p38 mitogen-activated protein kinase by western blot. Results Rose bengal photodynamic therapy in 2.0 µg/mL lipopolysaccharide-stimulated human corneal fibroblasts triggered interleukin-6 and interleukin-8 mRNA (p < .0001) and interleukin-6 protein increase (p < .0001), and downregulated intercellular adhesion molecule-1 expression (p < .001). C-C motif chemokine ligand-4, interferon regulatory factor-3, interferon α2, and interferon β1 expressions remained unchanged (p ≥ .2). Rose bengal photodynamic therapy increased IκB kinase subunit beta, nuclear factor kappa B p65, extracellular signal-regulated kinases-2, c-Jun amino terminal kinase, and p38 transcription (p ≤ .01), and triggered nuclear factor kappa B p65 and p38 mitogen-activated protein kinase phosphorylation (p ≤ .04) in lipopolysaccharide treated human corneal fibroblasts. Conclusion Rose bengal photodynamic therapy of lipopolysaccharide-stimulated human corneal fibroblasts can modify the inflammatory response by inducing interleukin-6 and interleukin-8 expression, and decreasing intercellular adhesion molecule-1 production. C-C motif chemokine ligand-4, interferon regulatory factor-3, and interferon α and β expressions are not affected by rose bengal photodynamic therapy in these cells. The underlying mechanisms may be associated with nuclear factor kappa B and p38 mitogen-activated protein kinase pathway activation.

Oleoylethanolamide and Palmitoylethanolamide Enhance IFNβ-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells.

Oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are endogenous lipids that act as agonists of the peroxisome proliferator-activated receptor α (PPARα). Recently, an interest in the role of these lipids in malignant tumors has emerged. Nevertheless, the effects of OEA and PEA on human neuroblastoma cells are still not documented. Type I interferons (IFNs) are immunomodulatory cytokines endowed with antiviral and anti-proliferative actions and are used in the treatment of various pathologies such as different cancer forms (i.e., non-Hodgkin's lymphoma, melanoma, leukemia), hepatitis B, hepatitis C, multiple sclerosis, and many others. In this study, we investigated the effect of OEA and PEA on human neuroblastoma SH-SY5Y cells treated with IFNβ. We focused on evaluating cell viability, cell proliferation, and cell signaling. Co-exposure to either OEA or PEA along with IFNβ leads to increased apoptotic cell death marked by the cleavage of caspase 3 and poly-(ADP ribose) polymerase (PARP) alongside a decrease in survivin and IKBα levels. Moreover, we found that OEA and PEA did not affect IFNβ signaling through the JAK-STAT pathway and the STAT1-inducible protein kinase R (PKR). OEA and PEA also increased the phosphorylation of p38 MAP kinase and programmed death-ligand 1 (PD-L1) expression both in full cell lysate and surface membranes. Furthermore, GW6471, a PPARα inhibitor, and the genetic silencing of the receptor were shown to lower PD-L1 and cleaved PARP levels. These results reveal the presence of a novel mechanism, independent of the IFNβ-prompted pathway, by which OEA and PEA can directly impair cell survival, proliferation, and clonogenicity through modulating and potentiating the intrinsic apoptotic pathway in human SH-SY5Y cells.

Inhibitory Effects of Eicosapentaenoic Acid on Vascular Endothelial Growth Factor-Induced Monocyte Chemoattractant Protein-1, Interleukin-6, and Interleukin-8 in Human Vascular Endothelial Cells.

Vascular endothelial growth factor (VEGF) induces monocyte chemoattractant protein-1 (MCP-1) and plays an important role in vascular inflammation and atherosclerosis. We investigated the mechanisms of VEGF-induced MCP-1 expression and the effects of eicosapentaenoic acid (EPA) in human umbilical vein endothelial cells (HUVECs). Real-time reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that VEGF enhanced MCP-1 gene expression and protein secretion in HUVECs. Western immunoblot analysis revealed that VEGF induced the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and inhibitor of nuclear factor (NF)-κB (IκB). Treatment with pharmacological inhibitors of p38 MAPK (SB203580) or NF-κB (BAY11-7085) significantly suppressed VEGF-induced MCP-1 in HUVECs. EPA inhibited VEGF-induced MCP-1 mRNA, protein secretion, phosphorylation of p38 MAPK, and the translocation of phospho-p65 to the nucleus. Additionally, VEGF also stimulated gene expressions of interleukin (IL)-6 and IL-8, which were suppressed by SB203580, BAY11-7085, and EPA. The present study has demonstrated that VEGF-induced activation of MCP-1, IL-6, and IL-8 involves the p38 MAPK and NF-κB signaling pathways and that EPA inhibits VEGF-induced MCP-1, IL-6, and IL-8 via suppressing these signaling pathways. This study supports EPA as a beneficial anti-inflammatory and anti-atherogenic drug to reduce the VEGF-induced activation of proinflammatory cytokine and chemokines.

Co-exposure to fluoride and sulfur dioxide induces abnormal enamel mineralization in rats via the FGF9-mediated MAPK signaling pathway

Fluoride (F) and sulfur dioxide (SO2) contamination is recognized as a public health concern worldwide. Our previous research has shown that Co-exposure to F and SO2 can cause abnormal enamel mineralization. Ameloblastin (AMBN) plays a crucial role in the process of enamel mineralization. However, the process by which simultaneous exposure to F and SO2 influences enamel formation by regulating AMBN expression still needs to be understood. This study aimed to establish in vivo and in vitro models of F–SO2 Co-exposure and investigate the relationship between AMBN and abnormal enamel mineralization. By overexpressing/knocking out the Fibroblast Growth Factor 9 (FGF9) gene, we investigated the impact of FGF9-mediated Mitogen-Activated Protein Kinase (MAPK) signaling on AMBN synthesis to elucidate the mechanism underlying the induction of abnormal enamel mineralization by F–SO2 Co-exposure in rats. The results showed that F–SO2 exposure damaged the structure of rat enamel and ameloblasts. When exposed to F or SO2, gradual increases in the protein expression of FGF9 and phosphorylated p38 mitogen-activated protein kinase (p-P38) were observed. Conversely, the protein levels of AMBN, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated c-Jun N-terminal kinase (p-JNK) were decreased. AMBN expression was significantly correlated with FGF9, p-ERK, and p-JNK expression in ameloblasts. Interestingly, FGF9 overexpression reduced the levels of p-ERK and p-JNK, worsening the inhibitory effect of F–SO2 on AMBN. Conversely, FGF9 knockout increased the phosphorylation of ERK and JNK, partially reversing the F–SO2-induced downregulation of AMBN. Taken together, these findings strongly demonstrate that FGF9 plays a critical role in F–SO2-induced abnormal enamel mineralization by regulating AMBN synthesis through the JNK and ERK pathways.

Electroacupuncture attenuates middle cerebral artery occlusion-induced learning and memory impairment by regulating microglial polarization in hippocampus

Background As a traditional medical therapy, electroacupuncture (EA) has been demonstrated to have beneficial effects on ischemic stroke-induced cognitive impairment. However, the underlying mechanism is largely unclear. Methods Adult rats received occlusion of the middle cerebral artery and reperfusion (MCAO/R) to establish the ischemic stroke model. Morris water maze test was performed following EA stimulation at the GV20, PC6, and KI1 acupoints in rats to test the learning and memory ability. Western blot, immunofluorescent staining, and enzyme-linked immunosorbent assay were conducted to assess the cellular and molecular mechanisms. Results EA stimulation attenuated neurological deficits. In the Morris water maze test, EA treatment ameliorated the MCAO/R-induced learning and memory impairment. Moreover, we observed that MCAO/R induced microglial activation and polarization in the ischemic hippocampus, whereas, EA treatment dampened microglial activation and inhibited M1 microglial polarization but enhanced M2 microglial polarization. EA treatment inhibited the increased expression of proinflammatory cytokines and enhanced the increased expression of anti-inflammatory cytokines. Finally, we found that EA treatment dampened microglial p38 mitogen-activated protein kinase (MAPK) phosphorylation. Conclusion Collectively, our data suggested that EA treatment ameliorated cognitive impairment induced by MCAO/R and the underlying mechanism may be p38-mediated microglia polarization and neuroinflammation.

Photoprotective Effects of Epigallocatechin Gallate on Ultraviolet-Induced Zebrafish and Human Skin Fibroblasts Cells.

The long-term exposure to ultraviolet radiation (UVR) raises oxidative stress and chronic inflammation levels, which in turn has a series of deleterious effects on skin health, such as sunburn, photoaging, and skin cancer. Hence, our study was determined to investigate the effects and mechanisms of epigallocatechin gallate (EGCG) in zebrafish and human skin fibroblasts (HSF) cells to alleviate ultraviolet-induced photoaging. The 4 days postfertilization (dpf) zebrafish larvae and HSF cells were treated with 10 J/cm2 UVA + 30 mJ/cm2 UVB, or 25, or 50 μM EGCG for 72 hr. The indicators involving in oxidative stress, inflammatory, and photoaging were measured by the kits, ELISA Kits and western blot methods. EGCGs protect against UVR-induced skin damage in zebrafish and HSF cells. EGCG markedly decreased the reactive oxygen species (ROS), malondialdehyde, 8-OHdG levels, increased superoxide dismutase (SOD) activity, and significantly inhibited inflammatory factors levels including tumor necrosis factor-α (TNF-α), interleukin-1α (IL-1α), interleukin-6 (IL-6) in zebrafish, and HSF cells irradiated with UVR. We found that EGCG could reduce UVR-induced p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation and effectively inhibited the activity of the transcriptional factor nuclear factor-κB (NF-κB), thereby reducing the protein-1 (AP-1), TNF-α, IL-1α, IL-6, and matrix metalloproteinase-1 (MMP-1) expressions, which are critical mediators of skin aging cascade causing the photoaging. These results validate that EGCG for protection of photoaging in zebrafish and HSF cells induced by UVR, which is closely related to the regulation of p38 MAPK/NF-κB, AP-1 signaling pathway which relieve oxidative stress, inflammation, and collagen degradation.

Inverse relationship between platelet Akt activity and hippocampal atrophy: A pilot case-control study in patients with diabetes mellitus.

Akt plays diverse roles in humans. It is involved in the pathogenesis of type 2 diabetes mellitus (T2DM), which is caused by insulin resistance. Akt also plays a vital role in human platelet activation. Furthermore, the hippocampus is closely associated with memory and learning, and a decrease in hippocampal volume is reportedly associated with an insulin-resistant phenotype in T2DM patients without dementia. To investigate the relationship between Akt phosphorylation in unstimulated platelets and the hippocampal volume in T2DM patients. Platelet-rich plasma (PRP) was prepared from the venous blood of patients with T2DM or age-matched controls. The pellet lysate of the centrifuged PRP was subjected to western blotting to analyse the phosphorylation of Akt, p38 mitogen-activated protein (MAP) kinase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Phosphorylation levels were quantified by densitometric analysis. Hippocampal volume was analysed using a voxel-based specific regional analysis system for Alzheimer's disease on magnetic resonance imaging, which proposes the Z-score as a parameter that reflects hippocampal volume. The levels of phosphorylated Akt corrected with phosphorylated p38 MAP kinase were inversely correlated with the Z-scores in the T2DM subjects, whereas the levels of phosphorylated Akt corrected with GAPDH were not. However, this relationship was not observed in the control patients. These results suggest that an inverse relationship may exist between platelet Akt activation and hippocampal atrophy in T2DM patients. Our findings provide insight into the molecular mechanisms underlying T2DM hippocampal atrophy.

Effects of ivabradine on myocardial autophagia and apoptosis in isoprenaline-induced heart failure in mice.

To investigate the effects and mechanisms of ivabradine (IVA) on isoprenaline-induced cardiac injury. Forty male C57BL/6 mice were randomly divided into control group, model group, high-dose IVA group, and low-dose IVA group. The control group was given saline, other groups were given subcutaneous injections of isoproterenol (ISO) 5 mg/kg/d to make the myocardial remodeling model. A corresponding dose of IVA (high dose 50 mg/kg/d, low dose 10 mg/kg/d) was given by gavage (30 days). A transthoracic echocardiogram was obtained to detect the structure and function of the heart. An electron microscope was used to explore the cardiomyocytes' apoptosis and autophagy. HE staining and Masson's trichrome staining were performed to explore myocardial hypertrophy and fibrosis. Western blot was used to detect Bax, Bcl-2, cleaved caspase-3, Becline-1, LC3, phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK), phosphorylated extracellular regulated protein kinases1/2 (p-ERK1/2), phosphorylated c-Jun N-terminal kinase (p-JNK), and α-smooth muscle actin (α-SMA) in the myocardium. Heart rate in the IVA groups was reduced, and the trend of heart rate reduction was more obvious in the high-dose group. Echocardiography showed that IVA improved the cardiac structure and function compared to the model group. IVA attenuated cardiac fibrosis, decreased cardiomyocyte apoptosis, and increased autophagy. The phosphorylated MAPK in the ISO-induced groups was increased. IVA treatment decreased the p-p38MAPK level. There were no differences in p-ERK and p-JNK levels. The beneficial effects of IVA on myocardial injury are related to blocking the p38MAPK signal pathway, decreasing cardiomyocyte apoptosis, and increasing cardiomyocyte autophagy.

Regulatory mechanisms underlying interleukin-6 expression in murine brown adipocytes.

Three types of adipocytes, white, brown, and beige, regulate the systemic energy balance through the storage and expenditure of chemical energy. In addition, adipocytes produce various bioactive molecules known as adipokines. In contrast to white adipocyte-derived molecules, less information is available on the adipokines produced by brown adipocytes (batokine). This study explored the regulatory expression of interleukin (IL)-6 in cell culture studies. Norepinephrine or a nonselective β-adrenergic receptor agonist increased the expression of IL-6 in primary brown adipocytes and HB2 brown adipocytes. Treatment with forskolin (Fsk), an activator of the cAMP-dependent protein kinase (PKA) pathway (downstream signaling of the β-adrenergic receptor), efficiently stimulated IL-6 expression in brown adipocytes and myotubes. Phosphorylated CREB and phosphorylated p38 MAP kinase levels were increased in Fsk-treated brown adipocytes within 5 min. In contrast, a long-term (∼60 min and ∼4 h) treatment with Fsk was required for increase in STAT3 phosphorylation and C/EBPβ expression, respectively. The PKA, p38 MAP kinase, STAT3, and C/EBPβ pathways are required for the maximal IL-6 expression induced by Fsk, which were verified by use of various inhibitors of these signal pathways. Vitamin C enhanced Fsk-induced IL-6 expression through the extracellular signal-regulated kinase activity. The present study provides basic information on the regulatory expression of IL-6 in activated brown adipocytes.

Phosphorylated p38 MAP kinase expression by leucocytes is increased in allergic humans and associated with IgE responses.

Mitogen-activated protein kinases (MAPK) activate cascades that regulate cell proliferation, differentiation and death. Phosphorylated (phos-)p38 MAPK is a cell-signalling pathway associated with Th2 cytokine responses, which is required for immunoglobulin (Ig)E production. It is unknown whether MAPK are associated with IgE production. We examine the evidence linking p38 MAPK to inflammatory responses. Phos-p38, extracellular signal-related kinase (ERK) and c-JUN-n terminal (JNK) MAPK expression by blood leucocyte subsets and levels of serum Igs were measured in blood from adults with asthma and/or rhinoconjunctivitis (N = 28) and non-asthma (N = 10) (flow cytometry, microfluorenzymeimmunoassay). Peripheral blood mononuclear cells (PBMC) from allergic subjects were cultured for 10 days ± anti-CD40/recombinant IL-4 ± inhibitor of phos-P38. Culture supernatants were assayed for IgE (ELISA). Phos-p38 MAPK expression by all leucocyte subsets of allergic subjects was associated with serum IgE levels (p ≤ 0.01), after adjusting for cell counts, age, sex, race and smoking status (p ≤ 0.04). Leucocyte expression of phos-ERK and JNK did not correlate with IgE (p = 0.09-0.99). Instead, phos-ERK expression was associated with serum IgG. When PBMC from atopic subjects were cultured for 10 days with anti-CD40/rhIL-4, IgE levels were 26.2 ± 18 ng/mL. Inclusion of SB202190 (5-20 μg/mL), a specific inhibitor of phos-p38 MAPK, in culture suppressed IgE production in dose-dependent manner, with peak suppression obtained with SB202190 at 20 μg/mL (82.1% ± 11.8) (p = 0.0001), with virtually no cytotoxicity (<5%). Different MAPK pathways may be associated with IgE (p38) and IgG (ERK) responses. Phos-p38 MAPK can be a potential anti-allergy drug target.

Targeting p38 Mitogen Activated Protein Kinase to Ameliorate Pain in SCD

Pain is a major comorbidity of sickle cell disease (SCD). Central sensitization is associated with chronic sickle cell pain. Earlier studies showed that p38 mitogen activated protein kinase (MAPK) is activated in the spinal cords of sickle mice with nociceptor sensitization of 2˚ neurons (Cataldo et al., Pain 2015). Novel approaches targeting p38 MAPK phosphorylation or its upstream targets may alleviate chronic pain. Cannabinoids represent a novel approach for ameliorating pain, including the endogenous cannabinoid-like lipid mediator palmitoylethanolamide (PEA). PEA has been shown to reduce neuropathic pain and depression scores, while improving sleep concomitant with reduced c-reactive protein (CRP) in a clinical evaluation of diabetic peripheral neuropathic pain and inflammation (Pickering et al., Inflammopharmacology 2022). We therefore examined if, [i] p38 MAPK is activated in dorsal root ganglion (DRG) which transmits action potentials to spinal neurons; [ii] if PEA administration reduces spinal neuroinflammation, oxidative stress and neuronal p38 MAPK phosphorylation; and [iii] inhibition of p38 MAPK phosphorylation ameliorates chronic pain in SCD. Female Berkeley (BERK) sickle (HbSS) expressing &amp;gt;99% human sickle hemoglobin and/or control (HbAA) expressing normal human HbA, mice (3.5-5.0 months) were treated for 14-days with PEA (i.p., 20 mg/kg/d) or vehicle (veh; i.p., 7.5% DMSO, 7.5% Tween 20, in sterile PBS). At endpoint, spinal cords and plasma were collected. HbSS mice have constitutively higher spinal malondialdehyde (MDA) levels, a marker of oxidative stress, compared to HbAA mice (~50%, P&amp;lt;.05), and PEA treatment significantly reduced HbSS spinal MDA levels (~53%, P&amp;lt;.05, vs veh); pro-apoptotic caspase-3 (~91%, P&amp;lt;.01) and pro-inflammatory interleukin (IL)-1β (~42%, P&amp;lt;.05), which are constitutively elevated in HbSS compared to HbAA mice (~45%, P&amp;lt;.05). Concomitant with reduced spinal inflammation, PEA reduced global marker of inflammation, serum amyloid P (~42%, P&amp;lt;.01) in the circulation, an analog for human CRP, which is significantly higher in HbSS compared to HbAA mice (~700%, P&amp;lt;.01). Thus, PEA ameliorates spinal inflammation and oxidative stress, and inhibits apoptosis. DRG neurons in vitro isolated from sickle mice were treated with tumor necrosis factor α (TNF α; 1 ng/mL) and hemin (40 mM) to create a sickle microenvironment replete with inflammation and oxidative stress, which significantly increased p38 MAPK phosphorylation (~494%, P&amp;lt;.001). PEA (30 mM) treatment of the neurons significantly inhibited TNF α and hemin-induced MAPK phosphorylation and its nuclear translocation (~65%, P&amp;lt;.001). Thus, it is likely that both peripheral and central mechanisms mediated by p38 MAPK signaling contribute to the initiation and maintenance of pain in SCD which are inhibited by PEA. Neflamapimod (Nef) is a novel inhibitor of p38 MAPK phosphorylation with significant anti-inflammatory effects, mediated by IL-1β and TNF α. Nef is orally bioavailable and has been suggested to improve synaptic transmission. We evaluated the effect of orally administered Nef (6 and 12 mg/kg 2x daily; veh 0.1% pluronic acid in sterile H 2O) in female sickle mice (9.0 months) with hyperalgesia. Treatment with Nef 12 mg/kg/d for 7-days reduced mechanical (~42% &amp; ~34%, both P&amp;lt;.01, vs veh &amp; baseline, respectively) and cold hyperalgesia (~46% &amp; ~34%, both P&amp;lt;.001, vs veh &amp; baseline, respectively). A decrease was also observed in mechanical (~29%, P&amp;lt;.05, vs veh) and cold hyperalgesia (~24%, P&amp;lt;.05, vs veh) after 7 days with 6 mg/kg Nef treatment. These data suggest p38 MAPK signaling contributes to chronic pain in SCD. Together, our observations demonstrate the therapeutic potential of PEA and Nef in targeting p38 MAPK as novel interventions to ameliorate sickle cell pain and central sensitization. PEA is a safe nutraceutical which has shown effectiveness in many pain conditions including osteoarthritis. Nef has shown efficacy in improving cognitive function in Phase II clinical trials in dementia patients. Pain and cognitive impairment co-exist in SCD. Therefore, Nef has the potential to reduce pain and improve cognitive function in SCD.

Oncostatin M enhances osteoprotegerin synthesis but reduces macrophage colony‑stimulating factor synthesis in bFGF‑stimulated osteoblast‑like cells.

Bone remodeling is tightly controlled by various factors, including hormones, autacoids and cytokines. Among them, oncostatin M (OSM) is a multifunctional cytokine produced by osteal macrophages, which serves as an essential modulator of bone remodeling. Macrophage colony-stimulating factor (M-CSF) and osteoprotegerin are secreted by osteoblasts, and also have pivotal roles in the regulation of the bone remodeling process. The binding of basic fibroblast growth factor (bFGF), a key regulator of bone remodeling, to the corresponding receptor [fibroblast growth factor receptor (FGFR)] triggers the dimerization and activation of FGFRs, which causes the phosphorylation of FGFR substrates and subsequent activation of downstream effectors, including mitogen-activated protein kinases (MAPKs), via Grb2. bFGF can activate MAPKs, resulting in the synthesis of osteoprotegerin and vascular endothelial growth factor in osteoblast-like MC3T3-E1 cells. In the present study, the effects of OSM on bFGF-induced osteoblast activation were investigated in the synthesis of osteoprotegerin and M-CSF in osteoblasts. The release of osteoprotegerin and M-CSF were analyzed using ELISA. The mRNA expression levels of osteoprotegerin and M-CSF were analyzed using reverse transcription-quantitative PCR. Phosphorylation of p38 MAPK, stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p44/p42 MAPK was assessed using western blotting. OSM enhanced bFGF-induced osteoprotegerin release and bFGF-stimulated mRNA expression of osteoprotegerin. By contrast, OSM suppressed the bFGF-induced release of M-CSF and bFGF-stimulated mRNA expression of M-CSF. SB203580, a p38 MAPK inhibitor, and SP600125, a SAPK/JNK inhibitor, suppressed the bFGF-stimulated M-CSF release, whereas PD98059, an upstream kinase inhibitor of p44/p42 MAPK, failed to suppress the M-CSF release stimulated by bFGF. Furthermore, OSM enhanced the bFGF-induced phosphorylation of p38 MAPK, but attenuated the bFGF-stimulated phosphorylation of SAPK/JNK. By contrast, OSM had little effect on the bFGF-induced phosphorylation of p44/p42 MAPK. SB203580 markedly reduced the amplification of bFGF-stimulated osteoprotegerin release enhanced by OSM. These results strongly suggested that OSM may possess divergent effects on bFGF-induced osteoblast activation, upregulation of p38 MAPK and downregulation of SAPK/JNK, leading to the amplification of osteoprotegerin synthesis and the attenuation of M-CSF synthesis.

Chronic Allergen Exposure Contributes to Steroid Resistance via Increased Phosphorylation of Glucocorticoid Receptors S226 and p38 MAPK in a Mouse Model of Asthma

Chronic allergen exposure can significantly induce p38 mitogen-activated protein kinase (MAPK) activation in asthma. p38 MAPK is involved in steroid resistance through phosphorylation of glucocorticoid receptors (GR) at S226. This study aims to investigate whether chronic allergen exposure can induce steroid resistance and whether it is associated with p38 MAPK activation in asthma.&#x0D; A mouse model of asthma was prepared by sensitizing and challenging mice with chronic ovalbumin (OVA) exposure. Key features of allergic asthma, encompassing bronchial hyperresponsiveness, pathology of lung tissues, cytokine profiles of inflammation in bronchoalveolar lavage fluid (BALF), and serum immunoglobulin (Ig)E concentration were evaluated. Furthermore, suppressive effects of corticosteroid on the splenocytes under stimulation of lipopolysaccharides, glucocorticoid receptor (GR) DNA binding ability of splenocytes, expression of GRα and phosphorylation of GR s226 in splenocytes, and p38 MAPK phosphorylation in splenocytes and lung tissues were determined.&#x0D; Chronic OVA exposure substantially induced airway hypersensitivity, leading to increased inflammatory infiltration in lung tissues. Additionally, it resulted in elevated levels of interleukin (IL)-4, IL-5, and IL-6 in BALF, as well as heightened levels of IgE in serum. Furthermore, OVA exposure substantially enhanced p38 MAPK phosphorylation in lung tissues. It also weakened the suppressive impacts of corticosteroids on splenocytes, impaired the GR DNA binding ability, and led to an enhanced phosphorylated state of GR S226 and p38 MAPK in splenocytes.&#x0D; Taken together, chronic allergen exposure contributes to steroid resistance in asthma, which is linked to an increased phosphorylated state of GR S226 and p38 MAPK.&#x0D;  

Amiloride alleviates morphine tolerance by suppressing ASIC3-dependent neuroinflammation in the spinal cord

BackgroundThe use of morphine in clinical medicine is severely constrained by tolerance. Therefore, it is essential to examine pharmacological therapies that suppress the development of morphine tolerance. Amiloride suppressed the expression of inflammatory cytokines by inhibiting microglial activation. Microglia play a crucial role in the establishment of morphine tolerance. Thus, we anticipated that amiloride might suppress the development of morphine tolerance. During this investigation, we assessed the impact of amiloride on mouse morphine tolerance. MethodsMice received morphine (10 mg/kg, s.c.) twice daily with intrathecally injected amiloride (0.3 μg/5 μl, 1 μg/5 μl, and 3 μg/5 μl) for nine continuous days. To assess morphine tolerance, mice underwent the tail-flick and hot plate tests. BV-2 cells were used to investigate the mechanism of amiloride. By using Western blotting, real-time PCR, and immunofluorescence labeling methods, the levels of acid-sensing ion channels (ASICs), nuclear factor kappa B (NF-kB) p65, p38 mitogen-activated protein kinase (MAPK) proteins, and neuroinflammation-related cytokines were determined. ResultsThe levels of ASIC3 in the spinal cord were considerably increased after long-term morphine administration. Amiloride was found to delay the development of tolerance to chronic morphine assessed via tail-flick and hot plate tests. Amiloride reduced microglial activation and downregulated the cytokines IL-1β and TNF-a by inhibiting ASIC3 in response to morphine. Furthermore, amiloride reduced p38 MAPK phosphorylation and inhibited NF-κB expression. ConclusionsAmiloride effectively reduces chronic morphine tolerance by suppressing microglial activation caused by morphine by inhibiting ASIC3.

Cyanidin-3-glucoside protects the photooxidative damage of retinal pigment epithelium cells by regulating sphingolipid signaling and inhibiting MAPK pathway

Cyanidin-3-glucoside (C3G) is the most common anthocyanin in dark grains and berries and is a food functional factor to improve visual health. However, the mechanisms of C3G on blue light-induced retinal pigment epithelial (RPE) cell photooxidative damage needs further exploration. We investigated the effects of C3G on blue light-irradiated A2E-containing RPE cells and explored whether sphingolipid, mitogen-activated protein kinase (MAPK), and mitochondria-mediated pathways are involved in this mechanism. Blue light irradiation led to mitochondria and lysosome damage in RPE cells, whereas C3G preserved mitochondrial morphology and function and maintained the lysosomal integrity. C3G suppressed the phosphorylation of JNK and p38 MAPK and mitochondria-mediated pathways to inhibit RPE cell apoptosis. Lipidomics data showed that C3G protected RPE cells against blue light-induced lipid peroxidation and apoptosis by maintaining sphingolipids balance. C3G significantly inhibited ceramide (Cer d18:0/15:0, Cer d18:0/16:0 and Cer d18:0/18:0) accumulation and elevated galactosylceramide (GalCer d18:1/15:0 and GalCer d18:1/16:0) levels in the irradiated A2E-containing RPE cells. Furthermore, C3G attenuated cell membrane damage by increasing phosphatidylcholine and phosphatidylserine levels. C3G inhibited apoptosis and preserved the structure of mitochondria and lysosome by regulating sphingolipid signaling and suppression of MAPK activation in RPE cells. Thus, dietary supplementation of C3G prevents retinal photooxidative damage.

Arthrospira platensis Nanoparticles Mitigate Aging-Related Oxidative Injured Brain Induced by D-galactose inRats Through Antioxidants, Anti-Inflammatory, and MAPK Pathways.

Loss of normal function is an inevitable effect of aging. Several factors contribute to the aging process, including cellular senescence and oxidative stress. We investigate how Arthrospira platensis Nanoparticles (NSP) protect against aging injury induced by d-galactose (D-gal) in the rat. So, we subcutaneously (S/C) injected D-gal at 200 mg/kg BW to see if Arthrospira platensis Nanoparticles (NSP) might protect against the oxidative changes generated by D-gal. NSP (0.5 mg/kg body weight once daily by gastric gavage) was given to all groups apart from the control and D-gal groups. The d-gal + NSP group was supplemented with 200 mg of D-gal per kg BW once a day and NSP 0.5 mg/kg BW given orally for 45 days. Biochemical, mRNA expression, and histological investigations of brain tissues were used to evaluate the oxidative alterations caused by d-gal and the protective role of NSP. Our data demonstrated that d-gal was causing significant reductions in relativebrain and body weight with increased malondialdehyde (MDA) and redox oxygen species (ROS) levels and increases in serum creatine phosphokinase (CPK) and creatine phosphokinase isoenzyme BB (CPK-BB) with marked decreases in the level of antioxidant enzyme activity in the brain and acetylcholinesterase activity augmented with a phosphorylated H2A histone family member X (γ-H2AX) level increased. The D-gal group had considerably higher phosphorylated p38 mitogen-activated protein kinases (P38MAPK) and C-Jun N-terminal (JNK) kinases. The d-gal administration stimulates the apoptotic gene expression by downregulating the brain superoxide dismutase (SOD), catalase (CAT), and nuclear factor erythroid 2-related factor 2 (Nrf2). The NSP administration saved these parameters in the direction of the control. The brain histopathologic and immunohistochemistry analysis findings support our findings on NSP's protective role. The NSP may be a promising natural protective compound that can prevent aging and preserve health.

Anti-inflammatory of disenecionyl cis-khellactone in LPS-stimulated RAW264.7 cells and the its inhibitory activity on soluble epoxide hydrolase

The objective of the present study was to investigate anti-inflammatory effects of disenecionyl cis-khellactone (DK) isolated from Peucedanum japonicum Thunberg, a traditional edible plant, in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Anti-inflammatory effects of DK were analyzed using various techniques, including NO assay, Western blot analysis, enzyme-linked immunosorbent assay (ELISA), real-time PCR, and immunofluorescence staining. It was revealed that DK reduced the production of pro-inflammatory cytokines including Monocyte chemoattractant protein-1 (MCP-1), Tumor necrosis factor-α (TNF-α), Interleukin 1β (IL-1β), and Interleukin 6 (IL-6) in RAW264.7 cells stimulated with LPS. It was revealed that DK effectively downregulated expression levels of iNOS and COX-2 due to inhibition of NF-κB activation and suppressing the phosphorylation of p38 and jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) phosphorylation. Also, soluble epoxide hydrolase activity and expression were decreased by the proinflammatory inhibitor, DK. Finally, findings of this study suggest that DK isolated from P. japonicum might have potential as a therapeutic candidate for inflammatory diseases.

C-reactive protein impairs immune response of CD8+ T cells via FcγRIIb-p38MAPK-ROS axis in multiple myeloma

BackgroundC-reactive protein (CRP) is a prototypical acute phase protein in humans with the function of regulating immune cells. Serum CRP levels are elevated in multiple myeloma (MM), associated with MM...

Activation of p38 MAPK hinders the reactivation of visual cortical plasticity in adult amblyopic mice

ObjectiveTo investigate the impact of p38 mitogen-activated protein kinase (MAPK) signaling on reactivating visual cortical plasticity in adult amblyopic mice. Materials and methodsReverse suture (RS), environment enrichment (EE), and combined with left intracerebroventricular injection of p38 MAPK inhibitor (SB203580, SB) or p38 MAPK agonist (dehydrocorydaline hydrochloride, DHC) were utilized to treat adult amblyopic mice with monocular deprivation (MD). The visual water task, visual cliff test, and Flash visual-evoked potential were used to measure the visual function. Then, Golgi staining and transmission electron microscopy were used to assess the reactivation of structural plasticity in adult amblyopic mice. Western blot and immunohistochemistry detected the expression of ATF2, PSD-95, p38 MAPK, and phospho-p38 MAPK in the left visual cortex. ResultsNo statistically significant difference was observed in the visual function in each pre-intervention group. Compared to pre-intervention, the visual acuity of deprived eyes was improved significantly, the impairment of visual depth perception was alleviated, and the P wave amplitude and C/I ratio were increased in the EE + RS, the EE + RS + SB, and the EE + RS + DMSO groups, but no significant difference was detected in the EE + RS + DHC group. Compared to EE + RS + DHC group, the density of dendritic spines was significantly higher, the synaptic density of the left visual cortex increased significantly, the length of the active synaptic zone increased, and the thickness of post-synaptic density (PSD) thickened in the left visual cortex of EE + RS, EE + RS + SB, and EE + RS + DMSO groups. And that, the protein expression of p-p38 MAPK increased while that of PSD-95 and ATF2 decreased significantly in the left visual cortex of the EE + RS + DHC group mice. ConclusionRS and EE intervention improved the visual function and synaptic plasticity of the visual cortex in adult amblyopic mice. However, activating p38 MAPK hinders the recovery of visual function by upregulating the phosphorylation of p38 MAPK and decreasing the ATF2 protein expression.

Toll-like receptor 4 promotes the inflammatory response in septic acute kidney injury by promoting p38 mitogen-activated protein kinase phosphorylation

Septic acute kidney injury (AKI) contributes to the mortality and morbidity of sepsis patients. Toll-like Receptor 4 (TLR4) has prominent roles in septic AKI. This study investigated the functions of TLR4 in septic AKI. A septic AKI mouse model was established by cecal ligation and puncture surgery. Mouse kidney function and kidney tissue lesion were examined using corresponding kits and H&E staining. The in vitro cell model of septic AKI was established by lipopolysaccharide induction. Cell viability, inflammatory factor (TNF-α, IL-6, IL-4, IL-1β, IL-18) levels, pyroptotic cell number changes, lactate dehydrogenase (LDH) activity, myeloperoxidase (MOP) concentration, and levels of pyroptosis-associated protein and MyD88, TRIF and p38 MAPK phosphorylation were determined by MTT, ELISA, FAM-FLICA Caspase-1 Detection kit, other corresponding kits, and Western blot. TLR4 was highly expressed in septic AKI mouse kidney tissues and human septic AKI cells. TLR4 knockdown alleviated kidney injury, increased cell viability, and reduced LDH activity and MPO concentration. TLR4 knockdown reduced cell pyroptosis by repressing p38 MAPK phosphorylation through MyD88/TRIF, suppressed pro-inflammatory factor (TNF-α, IL-6, IL-4, IL-1β, IL-18) levels, promoted anti-inflammatory factor (IL-4) level, and reduced inflammatory response, thus playing a protective role in septic AKI. Briefly, TLR4 promoted the inflammatory response in septic AKI by promoting p38 MAPK phosphorylation through MyD88/TRIF.