Phosphorylation Of ERK MAPK Research Articles

Enhanced GRP78 protein expression via the IRE1α/ASK1/p38 MAPK pathway during As2O3-induced endoplasmic reticulum stress in BEAS-2B cells

Inorganic arsenic is widely present in the environment. Exposure to moderate to high concentrations of arsenic from drinking water or air can cause various cancers and multisystem dysfunction. Glucose-regulated protein 78 (GRP78) is an endoplasmic reticulum (ER) stress sensor of unfolded protein response (UPR) under stress conditions and it enhances cell survival. The aim of this study is to investigate molecular mechanisms of arsenic-induced GRP78 expression in BEAS-2B cells model. We found that GRP78 protein expression was enhanced, while the level of GRP78 mRNA expression did not change under arsenic trioxide (As2O3)-induced ER stress condition in BEAS-2B cells. Cycloheximide, a protein synthesis inhibitor, completely inhibited As2O3-induced GRP78 protein expression. GRP78 mRNA expression was inhibited by actinomycin-D (Act-D). However, GRP78 protein expression was upregulated in the presence of Act-D under As2O3-induced ER stress condition. These data indicated that the upregulation of GRP78 protein under As2O3-induced UPR condition was possibly due to the increased biosynthesis of GRP78 protein. Moreover, both inositol-requiring enzyme 1α (IRE1α) RNase and kinase inhibitor KIRA6 and IRE1α kinase inhibitor APY29 completely inhibited As2O3-induced GRP78 protein expression and phosphorylation of JNK, ERK and p38 MAPK. Activation of apoptotic signaling kinase 1 (ASK1) is a downstream effector of IRE1α kinase. ASK1 inhibitor selonsertib and p38 MAPK inhibitor SB203580 partially inhibited As2O3-induced GRP78 protein expression, respectively. Our results suggested that As2O3 enhanced GRP78 protein expression in BEAS-2B cells via IRE1α kinase/ASK1/p38 MAPK signaling pathway. To our knowledge, this is the first report on illuminating the related mechanisms of increased GRP78 protein expression in As2O3-induced ER stress condition through a novel IRE1α pathway.

Noble 3,4-Seco-triterpenoid Glycosides from the Fruits of Acanthopanax sessiliflorus and Their Anti-Neuroinflammatory Effects.

Acanthopanax sessiliflorus (Araliaceae) have been reported to exhibit many pharmacological activities. Our preliminary study suggested that A. sessiliflorus fruits include many bioactive 3,4-seco-triterpenoids. A. sessiliflorus fruits were extracted in aqueous EtOH and fractionated into EtOAc, n-BuOH, and H2O fractions. Repeated column chromatographies for the organic fractions led to the isolation of 3,4-seco-triterpenoid glycosides, including new compounds. Ultra-high-performance liquid chromatography (UPLC) mass spectrometry (MS) systems were used for quantitation and quantification. BV2 and RAW264.7 cells were induced by LPS, and the levels of pro-inflammatory cytokines and mediators and their underlying mechanisms were measured by ELISA and Western blotting. NMR, IR, and HR-MS analyses revealed the chemical structures of the nine noble 3,4-seco-triterpenoid glycosides, acanthosessilioside G–O, and two known ones. The amounts of the compounds were 0.01–2.806 mg/g, respectively. Acanthosessilioside K, L, and M were the most effective in inhibiting NO, PGE2, TNF-α, IL-1β, and IL-6 production and reducing iNOS and COX-2 expression. In addition, it had inhibitory effects on the LPS-induced p38 and ERK MAPK phosphorylation in both BV2 and RAW264.7 cells. Nine noble 3,4-seco-triterpenoid glycosides were isolated from A. sessiliflorus fruits, and acanthosessilioside K, L, and M showed high anti-inflammatory and anti-neuroinflammatory effects.

LncRNA ITGB8-AS1 functions as a ceRNA to promote colorectal cancer growth and migration through integrin-mediated focal adhesion signaling

Long non-coding RNAs (lncRNAs) play critical roles in tumorigenesis and progression of colorectal cancer (CRC). However, functions of most lncRNAs in CRC and their molecular mechanisms remain uncharacterized. Here we found that lncRNA ITGB8-AS1 was highly expressed in CRC. Knockdown of ITGB8-AS1 suppressed cell proliferation, colony formation, and tumor growth in CRC, suggesting oncogenic roles of ITGB8-AS1. Transcriptomic analysis followed by KEGG analysis revealed that focal adhesion signaling was the most significantly enriched pathway for genes positively regulated by ITGB8-AS1. Consistently, knockdown of ITGB8-AS1 attenuated the phosphorylation of SRC, ERK, and p38 MAPK. Mechanistically, ITGB8-AS1 could sponge miR-33b-5p and let-7c-5p/let-7d-5p to regulate the expression of integrin family genes ITGA3 and ITGB3, respectively, in the cytosol of cells. Targeting ITGB8-AS1 using antisense oligonucleotide (ASO) markedly reduced cell proliferation and tumor growth in CRC, indicating the therapeutic potential of ITGB8-AS1 in CRC. Furthermore, ITGB8-AS1 was easily detected in plasma of CRC patients, which was positively correlated with differentiation and TNM stage, as well as plasma levels of ITGA3 and ITGB3. In conclusion, ITGB8-AS1 functions as a competing endogenous RNA (ceRNA) to regulate cell proliferation and tumor growth of CRC via regulating focal adhesion signaling. Targeting ITGB8-AS1 is effective in suppressing CRC cell growth and tumor growth. Elevated plasma levels of ITGB8-AS1 were detected in advanced-stage CRC. Thus, ITGB8-AS1 could serve as a potential therapeutic target and circulating biomarker in CRC.

Inhibitory Effect of Quercetin on Propionibacterium acnes-induced Skin Inflammation

Quercetin is a well-known antioxidant and a plant polyphenolic of flavonoid group found in many fruits, leaves, and vegetables. Propionibacterium acnes is a key skin pathogen involved in the progression of acne inflammation. Although quercetin has been applied to treat various inflammatory diseases, the effects of quercetin on P. acnes-induced skin inflammation have not been explored. This study investigated the effects of quercetin on P. acnes-induced inflammatory skin disease in vitro and in vivo. The results showed that quercetin suppressed the production of pro-inflammatory cytokines in P. acnes-stimulated HaCaT, THP-1 and RAW 264.7 cells. Additionally, quercetin reduced the production of TLR-2 and the phosphorylation of p38, ERK and JNK MAPKs in P. acnes-stimulated HaCaT and THP-1 cells. It also suppressed MMP-9 mRNA levels in two cell lines exposed to P. acnes in vitro. In the case of in vivo, P. acnes was intradermally injected into the ears of mice and it resulted in cutaneous erythema, swelling, and a granulomatous response. Treatment with quercetin markedly reduced ear thickness and swelling. These results suggested that quercetin can be a potential therapeutic agent against P. acnes-induced skin inflammation and may have diverse pharmaceutical and cosmetics applications.

New bioactive 5-arylcarboximidamidopyrazolo[3,4-c]pyridines: Synthesis, cytotoxic activity, mechanistic investigation and structure-activity relationships.

In this study, a series of novel substituted pyrazolo[3,4-c]pyridin-5-ylamidines was synthesized and their cytotoxicity against three cancer cell lines (MDA-MB-231, HT-1080, PC-3), as well as a human normal cell line (AG01523) was evaluated. A number of derivatives could strongly reduce cancer cells proliferation and exhibit apoptotic induction capability, while reasonable structure-activity relationships could be extracted. Certain analogues were endowed with low toxicity against normal cells. Cell cycle analysis revealed that most of the active compounds induced a G0/G1 arrest of HT-1080cells. Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds were investigated in HT-1080cells, upon study of their effects on the phosphorylation of Akt, ERK and p38 MAPK. Most of the active derivatives inhibit phosphorylation of Akt and ERK and/or induce p38 MAPK phosphorylation, providing a potential indication on the mode of action of this class.

A polysaccharide TKP-2-1 from Tamarindus indica L: Purification, structural characterization and immunomodulating activity

Abstract Herein, TKP-2-1, a water-soluble polysaccharide with a weight-average molecular weight of 1693 kDa, was isolated from the seed kernels of Tamarindus indica L. UV, FT-IR, GC–MS and 1D, 2D-NMR data analysis revealed that TKP-2-1 is chiefly composed of a (1 → 4)-β-D-glucan backbone with side chains of α-D-xylopyranosyl, α-D-xylopyranosyl-(1 → 4)-β-D-galactopyranosyl and β-D-galactopyranosyl-(1 → 2)-α-D-xylopyranosyl sugar moieties linked to the C-6 positions of the glucose residues. Furthermore, TKP-2-1 exhibited a potent immunostimulatory activity through enhancing phagocytosis and NO production, as well as the expression of iNOS, IL-1β, IL-6, and TNF-α in RAW264.7 macrophages. Mechanistically, TKP-2-1 caused the degradation of IκBα and nuclear translocation of NF-κB p65, and phosphorylation of ERK, JNK and p38 MAPK in RAW264.7 cells. Specific inhibitors of NF-κB, ERK, JNK, and p38 MAPK obviously abolished TKP-2-1-induced NO production in RAW264.7 cells, respectively. Thus, TKP-2-1 from the seed kernels of T. indica might potentially be used as a natural immunomodulator in functional foods.

Soluble epoxide hydrolase inhibitors improve angiogenic function of endothelial progenitor cells via ERK/p38-mediated miR-126 upregulation in myocardial infarction mice after exercise

It is well established that exercise could protect against myocardial infarction (MI). Previously, we found that epoxyeicosatrienoic acids (EETs) could be induced by exercise and has been found to protect against MI via promoting angiogenic function of endothelial progenitor cells (EPCs). However, the underling mechanism of EETs in promoting EPC functions is unclear. C57BL/6 mice were fed with a novel soluble epoxide hydrolase inhibitor (sEHi), TPPU, to increase EET levels, for 1 week before undergoing MI surgery. Mice were then subjected to exercise training for 4 weeks. Bone marrow-derived EPCs were isolated and cultured in vitro. Exercise upregulated miR-126 expression but downregulated the protein levels of its target gene, Spred1, in EPCs from MI mice. TPPU further enhanced the effects of exercise on EPCs. Spred1 overexpression abolished the protective effects of TPPU on EPC functions. Downregulation of miR-126 by antagomiR-126 impaired the inhibitor effects of TPPU on Spred1 mRNA and protein expression. Additionally, TPPU upregulated miR-126 is partially mediated through ERK/p38 MAPK pathway. This study showed that sEHi promoted miR-126 expression, which might be related to the beneficial effect of sEHi on EPC functions in MI mice under exercise conditions, by increasing ERK and p38 MAPK phosphorylation and inhibiting Spred1.

Dimethyl fumarate prevents osteoclastogenesis by decreasing NFATc1 expression, inhibiting of erk and p38 MAPK phosphorylation, and suppressing of HMGB1 release

Osteoclasts are multinucleated bone-resorbing cells derived from monocyte/macrophage progenitor cells. Excessive formation and resorbing activities of osteoclasts are involved in the bone-destructive pathologies of rheumatoid arthritis and osteoporosis. Recently, it has been found that nuclear factor erythroid 2-related factor 2 (Nrf2), a transcription factor for anti-oxidative stress genes, functions in osteoclastogenesis. Dimethyl fumarate (DMF) is a potent activator of Nrf2 and has been shown to inhibit osteoclastogenesis. Here, we investigated the mechanisms of this inhibition by examining the activation of several signalling pathways during the differentiation of bone marrow-derived macrophages into osteoclasts. DMF inhibited the differentiation of osteoclasts in a dose-dependent manner and suppressed the bone-resorbing activity of osteoclasts. DMF treatment decreased the expression of nuclear factor of activated T-cells cytoplasmic-1, and significantly decreased phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase in osteoclasts. We also found that DMF inhibited the extracellular release of high mobility group box 1, associated with an up-regulation of heme oxygenase-1, likely mediated through Nrf2 activation. Our results indicate that DMF inhibits osteoclast differentiation through multiple pathways.

Tussilagone inhibits allergic responses in OVA-induced allergic rhinitis guinea pigs and IgE-stimulated RBL-2H3 cells

Farfarae Flos is the dried flower buds of Tussilago farfara L. which is widely used to treat allergic and inflammatory diseases in Chinese folk. Tussilagone (TSL), a sesquiterpene compound purified from Farfarae Flos, has been confirmed the main active component in the plant. However, its anti-allergic activity hasn't been reported yet. The purpose of this study is to investigate the anti-allergic effect of TSL in ovalbumin (OVA)-induced allergic rhinitis (AR) guinea pigs and immunoglobulin E (IgE)-stimulated RBL-2H3 cells. The AR symptoms such as nasal scratching, sneezing and runny nose were scored and the histological changes of nasal mucosa were observed by H&E staining. The levels of histamine, OVA-specific IgE, IL-6 and TNF-α in the serum were measured by ELISA. In IgE-stimulated RBL-2H3 cells, the phosphoryration of Lyn, Syk, Akt, NF-κB p65, ERK and p38 MAPK were investigated by western blot analysis. The results showed that intraperitoneal injection of TSL at doses of 25 and 50 mg/kg significantly alleviated the allergic symptoms and the histological changes of nasal mucosa in OVA-induced allergic rhinitis guinea pigs. Moreover, the levels of histamine, IgE and IL-6 in the serum decreased significantly (p < .05). In vitro, TSL suppressed the phosphorylation of Lyn, Syk, Akt, NF-κB p65, ERK and p38 MAPK in IgE-stimulated RBL-2H3 cells. These results indicate TSL has therapeutic effect on allergic rhinitis in guinea pigs. The anti-allergic mechanism may be through the inhibition of allergic and inflammatory related pathways in mast cells.

Gymnema montanum improves endothelial function via inhibition of endoplasmic reticulum stress by activating Nrf2 signaling

Objective: To investigate the effects of Gymnema montanum leaf extract against endoplasmic reticulum (ER) stress-induced toxicity in endothelial cells. Methods: The immortalized endothelial hybrid cell, EA.hy926 was treated with different concentrations of Gymnema montanum leaf extract (0-100 μg/mL) and the ER stress inducer, tunicamycin. The cytotoxicity was assessed by MTT as well as lactate dehydrogenase and malondialdehyde levels were determined. The levels of ER stress markers, GRP78 and CHOP were analysed by Western blot assay. The Gymnema montanum leaf extract-mediated activation of nuclear factor erythroid 2-related factor 2 (Nrf2) was assessed by cell-based luciferase enzyme fragment complementation assay and antioxidant responsive element driven luciferase reporter assay. The levels of phosphoproteins of the MAPK pathway were analyzed using the Bioplex system. Results: A dose-dependent cytoprotective effect of Gymnema montanum leaf extract was observed in tunicamycin-induced toxicity. Gymnema montanum leaf extract significantly reduced lactate dehydrogenase activity and malondialdehyde levels in ER stress-induced endothelial cells. It also suppressed ER stress markers dose dependently and inhibited the phosphorylation of JNK, ERK, MEK and p38 MAPK in tunicamycin-induced endothelial cells. Moreover, Gymnema montanum leaf extract increased the expression of Nrf2 and its downstream targets in endothelial cells. Conclusions: Gymnema montanum leaf extract attenuates ER stress by increasing the expression of Nrf2 and its downstream genes.

Inhibition of Allergic Reactivity through Targeting FcεRI-Bound IgE with Humanized Low-Affinity Antibodies.

Options for effective prevention and treatment of epidemic allergic diseases remain limited, and particularly so for IgE-mediated food allergies. We previously found that mouse low-affinity anti-human IgE mAbs with KD in the 10-6-10-8 M range were capable of blocking allergic reactivity without triggering immediate allergic mediator release. In this study, we humanized three parent low affinity allergic response inhibitor (LARI) mouse anti-human IgE mAbs and characterized their biological and immunological features, refined the lead candidate for further clinical development, examined their safety profiles, determined their therapeutic efficiency, and explored the mechanism of action potentially responsible for their therapeutic effects. LARI profoundly blocked cat- and peanut-allergic IgE-mediated basophil activation, inhibited acute release of both prestored and newly synthesized mediator from human mast cells, suppressed peanut-specific IgE-mediated passive cutaneous anaphylaxis, and attenuated dansyl IgE-mediated systemic anaphylaxis in human FcεRIα transgenic mice. Safety testing demonstrated that concentrations of LARI well above therapeutic levels failed to trigger immediate release of prestored and newly synthesized allergic mediators, failed to promote robust cytokine/chemokine production from allergic effector cells, and did not elicit allergic reactivity in an animal model of cutaneous and systemic anaphylaxis. Mechanistic studies revealed that LARI downregulated surface FcεRI receptors and IgE via internalization of the IgE/FcεRI, promoted a partial mediator depletion pathway leading to slow release of small amount of mediators, and functioned as a partial antagonist to inhibit FcεRI signaling phosphorylation of Syk, Akt, Erk, and p38 MAPK. These studies demonstrate that targeting surface-bound IgE with LARI profoundly suppresses human allergic reactivity while displaying an excellent safety profile.

Hydrogen sulfide inhibits cigarette smoke-induced inflammation and injury in alveolar epithelial cells by suppressing PHD2/HIF-1α/MAPK signaling pathway

Chronic obstructive pulmonary fibrosis (COPD) is a chronic and fatal lung disease with few treatment options. Sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H2S), was found to alleviate cigarette smoke (CS)-induced emphysema in mice, however, the underlying mechanisms have not yet been clarified. In this study, we investigated its effects on COPD in a CS-induced mouse model in vivo and in cigarette smoke extract (CSE)-stimulated alveolar epithelial A549 cells in vitro. The results showed that NaHS not only relieved emphysema, but also improved pulmonary function in CS-exposed mice. NaHS significantly increased the expressions of tight junction proteins (i.e., ZO-1, Occludin and claudin-1), and reduced apoptosis and secretion of pro-inflammatory cytokines (i.e., TNF-α, IL-6 and IL-1β) in CS-exposed mouse lungs and CSE-incubated A549 cells, indicating H2S inhibits CS-induced inflammation, injury and apoptosis in alveolar epithelial cells. NaHS also upregulated prolyl hydroxylase (PHD)2, and suppressed hypoxia-inducible factor (HIF)-1α expression in vivo and in vitro, suggesting H2S inhibits CS-induced activation of PHD2/HIF-1α axis. Moreover, NaHS inhibited CS-induced phosphorylation of ERK, JNK and p38 MAPK in vivo and in vitro, and treatment with their inhibitors reversed CSE-induced ZO-1 expression and inflammation in A549 cells. These results suggest that NaHS may prevent emphysema via the suppression of PHD2/HIF-1α/MAPK signaling pathway, and subsequently inhibition of inflammation, epithelial cell injury and apoptosis, and may be a novel strategy for the treatment of COPD.

GluN2A-NMDA receptor–mediated sustained Ca2+ influx leads to homocysteine-induced neuronal cell death

Homocysteine, a metabolite of the methionine cycle, is a known agonist of N-methyl-d-aspartate receptor (NMDAR), a glutamate receptor subtype and is involved in NMDAR-mediated neurotoxicity. Our previous findings have shown that homocysteine-induced, NMDAR-mediated neurotoxicity is facilitated by a sustained increase in phosphorylation and activation of extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK MAPK). In the current study, we investigated the role GluN1/GluN2A-containing functional NMDAR (GluN2A-NMDAR) and GluN1/GluN2B-containing functional NMDAR (GluN2B-NMDAR) in homocysteine-induced neurotoxicity. Our findings revealed that exposing primary cortical neuronal cultures to homocysteine leads to a sustained low-level increase in intracellular Ca2+ We also showed that pharmacological inhibition of GluN2A-NMDAR or genetic deletion of the GluN2A subunit attenuates homocysteine-induced increase in intracellular Ca2+ Our results further established the role of GluN2A-NMDAR in homocysteine-mediated sustained ERK MAPK phosphorylation and neuronal cell death. Of note, the preferential role of GluN2A-NMDAR in homocysteine-induced neurotoxicity was distinctly different from glutamate-NMDAR-induced excitotoxic cell death that involves overactivation of GluN2B-NMDAR and is independent of ERK MAPK activation. These findings indicate a critical role of GluN2A-NMDAR-mediated signaling in homocysteine-induced neurotoxicity.

MicroRNA-125b-5p regulates IL-1\u03b2 induced inflammatory genes via targeting TRAF6-mediated MAPKs and NF-\u03baB signaling in human osteoarthritic chondrocytes

Abnormal post-transcriptional modulations in inflammatory genes by microRNAs (miRNAs) play a crucial role in human disorders including arthritis. In this study, we determined the effect of hsa-miR-125b-5p on interleukin (IL)-1β induced inflammatory genes in human osteoarthritic (OA) chondrocytes. Bioinformatics algorithms showed 3′untranslated region (3′UTR) of TRAF6 mRNA (NM_004620.3) has perfectly matched ‘seed-sequence’ for hsa-miR-125b-5p. Treatment of cells with IL-1β up-regulates TRAF6 mRNA and down-regulates hsa-miR-125b-5p expression. This negative correlation between TRAF6 and hsa-miR-125b-5p was verified by transfection with miR-125b mimic (pre-miR-125b). Moreover, transfection with miR-125b mimic caused marked inhibition of IL-1β-induced phosphorylation of p38-MAPK, JNK-MAPKs and ERK-MAPKs and also suppressed the nuclear levels of NF-κBp50, NF-κBp65 and inhibited the activation of IκBα. Furthermore, transfected chondrocytes with miR-125b mimic in the presence of IL-1β also showed marked inhibition in the secretion of several proinflammatory cytokines, chemokines and growth factors including IL-6, IL-8, INF-γ, TGF-β1, IGFBP-1 and PGDF-BB. Importantly, this transfection also significantly inhibited IL-1β- induced MMP-13 expression/production. In short, this study concludes that hsa-miR-125b-5p acts as a negative co-regulator of inflammatory genes including MMP-13 via targeting TRAF6/MAPKs/NF-κB pathway in human OA chondrocytes.

Broussonin E suppresses LPS-induced inflammatory response in macrophages via inhibiting MAPK pathway and enhancing JAK2-STAT3 pathway.

Macrophages play an important role in inflammation, and excessive and chronic activation of macrophages leads to systemic inflammatory diseases, such as atherosclerosis and rheumatoid arthritis. In this paper, we explored the anti-inflammatory effect of broussonin E, a novel phenolic compound isolated from the barks ofBroussonetia kanzinoki, and its underlying molecular mechanisms. We discovered that Broussonin E could suppress the LPS-induced pro-inflammatory production in RAW264.7 cells, involving TNF-α, IL-1β, IL-6, COX-2 and iNOS. And broussonin E enhanced the expressions of anti-inflammatory mediators such as IL-10, CD206 and arginase-1 (Arg-1) in LPS-stimulated RAW264.7 cells. Further, we demonstrated that broussonin E inhibited the LPS-stimulated phosphorylation of ERK and p38 MAPK. Moreover, we found that broussonin E could activate janus kinase (JAK) 2, signal transducer and activator of transcription (STAT) 3. Downregulated pro-inflammatory cytokines and upregulated anti-inflammatory factors by broussonin E were abolished by using the inhibitor of JAK2-STAT3 pathway, WP1066. Taken together, our results showed that broussonin E could suppress inflammation by modulating macrophages activation statevia inhibiting the ERK and p38 MAPK and enhancing JAK2-STAT3 signaling pathway, and can be further developed as a promising drug for the treatment of inflammation-related diseases such as atherosclerosis.

Hyperhomocysteinemia leads to exacerbation of ischemic brain damage: Role of GluN2A NMDA receptors

Hyperhomocysteinemia has been implicated in several neurodegenerative disorders including ischemic stroke. However, the pathological consequences of ischemic insult in individuals predisposed to hyperhomocysteinemia and the associated etiology are unknown. In this study, we evaluated the outcome of transient ischemic stroke in a rodent model of hyperhomocysteinemia, developed by subcutaneous implantation of osmotic pumps containing L-homocysteine into male Wistar rats. Our findings show a 42.3% mortality rate in hyperhomocysteinemic rats as compared to 7.7% in control rats. Magnetic resonance imaging of the brain in the surviving rats shows that mild hyperhomocysteinemia leads to exacerbation of ischemic injury within 24 h, which remains elevated over time. Behavioral studies further demonstrate significant deficit in sensorimotor functions in hyperhomocysteinemic rats compared to control rats. Using pharmacological inhibitors targeting the NMDAR subtypes, the study further demonstrates that inhibition of GluN2A-containing NMDARs significantly reduces ischemic brain damage in hyperhomocysteinemic rats but not in control rats, indicating that hyperhomocysteinemia-mediated exacerbation of ischemic brain injury involves GluN2A-NMDAR signaling. Complementary studies in GluN2A-knockout mice show that in the absence of GluN2A-NMDARs, hyperhomocysteinemia-associated exacerbation of ischemic brain injury is blocked, confirming that GluN2A-NMDAR activation is a critical determinant of the severity of ischemic damage under hyperhomocysteinemic conditions. Furthermore, at the molecular level we observe GluN2A-NMDAR dependent sustained increase in ERK MAPK phosphorylation under hyperhomocysteinemic condition that has been shown to be involved in homocysteine-induced neurotoxicity. Taken together, the findings show that hyperhomocysteinemia triggers a unique signaling pathway that in conjunction with ischemia-induced pathways enhance the pathology of stroke under hyperhomocysteinemic conditions.

Capsaicin Induces Apoptosis in KSHV-Positive Primary Effusion Lymphoma by Suppressing ERK and p38 MAPK Signaling and IL-6 Expression.

Primary effusion lymphoma (PEL) is defined as a rare subtype of non-Hodgkin's B-cell lymphoma which is caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. PEL is an aggressive lymphoma and is frequently resistant to conventional chemotherapies. Therefore, it is critical to investigate novel therapeutic options for PEL. Capsaicin is a pungent component of chili pepper and possesses unique pharmacological effects, such as pain relief, anti-microbial and anti-cancer properties. Here, we demonstrate that capsaicin markedly inhibited the growth of KSHV latently infected PEL cells by inhibiting ERK, p38 MAPK and expression hIL-6, which are known to contribute to PEL growth and survival. The underlying mechanism of action by capsaicin was through the inhibition of ERK and p38 MAPK phosphorylation and signaling that affected hIL-6 expression. As a result, capsaicin induced apoptosis in PEL cells in a caspase-9 dependent manner. In line with these results, ERK (U0126) and p38 MAPK (SB203580) specific signaling inhibitors suppressed hIL-6 expression and attenuated cell growth in PEL cells. Furthermore, the addition of hIL-6 neutralizing antibody to culture medium suppressed the growth of PEL cells. We also demonstrate that capsaicin suppressed PEL cell growth in the absence of nascent viral replication. Finally, we confirmed ex vivo treatment of capsaicin attenuated PEL development in SCID mice. Taken together, capsaicin could represent a lead compound for PEL therapy without the risk of de novo KSHV infection.

ZAP70 deficiency promotes reverse cholesterol transport through MAPK/ERK pathway in Jurkat cell

BackgroundLots of studies have demonstrated that immune cells could regulate reverse cholesterol transport (RCT). However, neither T cell receptor (TCR) signalling nor Zeta-chain associated protein 70 (ZAP70) have been demonstrated to be associated with RCT. To investigate this association, we used a ZAP70-deficient Jurkat-derived mutant, P116 cell line, to detect the effect of ZAP70 on RCT and inflammatory response. ZAP70 deficiency improved cholesterol efflux capacity by 14%. Meanwhile, mRNA and proteins expression of RCT regulatory proteins such as ABCA1, ABCG1 and SR-BI were increased in P116 cells. ZAP70-deficiency had no influence on LXR-α and PPAR-γ. Regarding the inflammatory response, the mRNA expression and secretion of pro-atherosclerotic cytokines, TNF-α, IFN-γ, IL-2 and IL-6, were significantly decreased in the ZAP70-deficient cell line. Activation of MAP kinases cascades, as determined by of ERK, JNK and p38 MAPK phosphorylation, were found to be inhibited in the absence of ZAP70. Specific inhibition of ERK, JNK and p38 MAPK activity was also found to decreased TNF-α, IFN-γ, and IL-6 secretion. However, only the ERK inhibition was observed to reduce IL-2 secretion, improve cholesterol efflux capacity and increase expression of ABCA1, ABCG1 and SR-BI without increasing LXR-α and PPAR-γ. Using ChIP assay to detect the binding of LXR-α to LXRE, which promotes the expression of ABCG1, we found that inhibiting ERK improved binding without increasing LXR-α levels. Thus, we speculate that ZAP70-deficiency may improve RCT and decrease the inflammatory response of T cells. Furthermore, these effects are probably achieved via ERK signalling pathway.

Ganomycin I from Ganoderma lucidum attenuates RANKL-mediated osteoclastogenesis by inhibiting MAPKs and NFATc1

Many bone-related diseases such as osteoporosis and rheumatoid arthritis are commonly associated with excessive activity of the osteoclast. Ganomycin I (GMI), a meroterpenoid isolated from Vietnamese mushroom Ganoderma lucidum, possesses a variety of beneficial effects on human health. However, its impact and underlying mechanism on osteoclastogenesis remain unclear. In the present study, we investigated the effect of GMI on RANKL-induced osteoclast formation in mouse BMMs and RAW264.7 cells. BMMs or RAW264.7 cells were treated with GMI followed by an evaluation of cell viability, RANKL-induced osteoclast differentiation, actin-ring formation, and resorption pits activity. Effects of GMI on RANKL-induced phosphorylation of MAPKs as well as the expression levels of NFATc1 and c-Fos were evaluated by Western blot analysis. Expression levels of osteoclast marker genes were evaluated by Western blot analysis and reverse transcription-qPCR. GMI significantly inhibited RANKL-induced osteoclast differentiation by decreasing the number of osteoclasts, osteoclast actin-ring formation, and bone resorption in a dose-dependent manner without affecting cell viability. At molecular level, GMI inhibited the RANKL-induced phosphorylation of ERK, JNK, and p38 MAPKs, as well as the expression levels of c-Fos and NFATc1, which are known to be crucial transcription factors for osteoclast formation. In addition, GMI decreased expression levels of osteoclastogenesis specific marker genes including c-Src, CtsK, TRAP, MMP-9, OSCAR, and DC-STAMP in RANKL-stimulated BMMs. Our findings suggest that GMI can attenuate osteoclast formation by suppressing RANKL-mediated MAPKs and NFATc1 signaling pathways and the anti-osteoclastogenic activity of GMI may extend our understanding of molecular mechanisms underlying biological activities and pharmacological use of G. lucidum as a traditional anti-osteoporotic medicine.

Total flavones of fermentation broth by co-culture of Coprinus comatus and Morchella esculenta induces an anti-inflammatory effect on LPS-stimulated RAW264.7 macrophages cells via the MAPK signaling pathway

The inflammatory cellular model of RAW264.7 cells induced by lipopolysaccharide (LPS) has always been used to investigate the effect of anti-inflammatory agents in vitro. In the present study, the anti-inflammatory activity of total flavones extracted from the fermentation broth of the co-culture of Coprinus comatus and Morchella esculenta (MCF-F), and its potential molecular mechanism in LPS-challenged RAW264.7 macrophage cells were investigated. The data revealed that MCF-F exhibited anti-inflammatory activity in LPS-stimulated RAW264.7 cells. At the same time, MCF-F was less cytotoxic under a concentration of 16 μg/ml in RAW264.7 cells. The anti-inflammatory activity of MCF-F was detected using the Griess method and ELISA assay, and the results well-corroborated with the observed decrease in expression in pro-inflammatory mediators, including nitric oxide, tumor necrosis factor-α and inteleukin-1β (IL-1β). In addition, the expression of inducible NO synthase (iNOS) and cyclooxygenase2 (COX-2) were confirmed by RT-PCR and western blot, and it was found that both mRNA and protein levels were downregulated after MCF-F treatment. The data also revealed that MCF-F downregulated the phosphorylation of JNK, ERK and P38 MAPK. Collectively, these results lead to the conclusion that MCF-F exerts an anti-inflammatory effect against LPS-challenged RAW264.7 cells via the MAPK pathway.