Knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells

Increasing evidence suggests that general control nonderepressible 2 (GCN2) is a critical regulator of oxidative stress and cell apoptosis in response to various stimuli. However, the role of GCN2 in diabetic retinopathy remains unclear. The aim of the present study was to investigate the effects of GCN2 on oxidative stress and apoptosis in ARPE-19 cells exposed to high glucose. The results showed that GCN2 was highly expressed in high glucose-induced ARPE-19 cells. Moreover, knockdown of GCN2 greatly improved ARPE-19 cell viability in response to high glucose. In addition, GCN2 knockdown significantly suppressed the production of reactive oxygen species (ROS) and malondialdehyde (MDA), as well as increased superoxide dismutase (SOD) activity in high glucose-stimulated ARPE-19 cells. Furthermore, GCN2 knockdown reduced cell apoptosis and enhanced the activation of nuclear factor E2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) pathway in high glucose-stimulated ARPE-19 cells. However, knockdown of Nrf2 reversed the effects of GCN2 on oxidative stress and cell apoptosis. Taken together, our findings suggest that knockdown of GCN2 inhibits high glucose-induced oxidative stress and apoptosis in ARPE-19 cells through activation of the Nrf2/HO-1 pathway.     

Knockdown of ALK7 inhibits high glucose-induced oxidative stress and apoptosis in retinal pigment epithelial cells

Diabetic retinopathy (DR) is one of the diabetic complications associated with hyperglycaemia-mediated oxidative stress. Activin receptor-like kinase 7 (ALK7) has been proven to be a potential therapeutic approach for diabetic cardiomyopathy, which is another diabetic complication. However, the role of ALK7 in DR remains unclear. In the current study, ALK7 was found to be up-regulated in clinical samples from DR patients and high glucose (HG)-induced human retinal pigment epithelial cells (ARPE-19). In vitro studies demonstrated that knockdown of ALK7 in ARPE-19 cells through transfection with siRNA-ALK7 (si-ALK7) improved cell viability in HG-induced ARPE-19 cells. Knockdown of ALK7 suppressed HG-induced reactive oxygen species (ROS) production, as well elevating the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in ARPE-19 cells. The number of apoptotic cells was significantly decreased after transfection with si-ALK7. ALK7 knockdown also caused a significant decrease in bax expression and an increase in bcl-2 expression in HG-induced ARPE-19 cells. In addition, ALK7 knockdown resulted in remarkable increase in the expressions of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in ARPE-19 cells in response to HG induction. Taken together, knockdown of ALK7 protected ARPE-19 cells from HG-induced oxidative injury, which might be mediated by the activation of the Nrf2/HO-1 signalling pathway.     

Redox/ROS regulation of lipopolysaccharide-induced mitogen-activated protein kinase (MAPK) activation and MAPK-mediated TNF-alpha biosynthesis

Redox and ROS regulation of MAPK-mediated TNF-alpha biosynthesis is not well characterized. It was hypothesized that the involvement of the MAPK pathway in regulating LPS-mediated TNF-alpha secretion is redox-dependent, NF-kappaB-sensitive and attenuated by N-acetyl-L-cysteine (NAC) and other antioxidants. In alveolar epithelial cells, LPS induced a time- and dose-dependent phosphorylation of MAPK(p38). This was associated with the activation of MAPK-activated protein kinase, which phosphorylated the small heat-shock protein, Hsp27. MAPK(p38) inhibition (SB-203580) abrogated LPS-induced TNF-alpha production. MAPK(ERK) blockade (PD-98059) attenuated TNF-alpha secretion, an effect synergistically amplified in the presence of SB-203580. Regulation of NF-kappaB by selective inhibitors revealed that this pathway is partially involved in regulating LPS-mediated TNF-alpha secretion. Whereas the proteasome inhibitor, MG-132, had no effect on LPS-mediated TNF-alpha production, CAPE, sulfasalazine and SN-50, a cell-permeant NF-kappaB inhibitor, attenuated but did not abrogate TNF-alpha biosynthesis. LPS up-regulated ROS, an effect abrogated by 4'-hydroxy-3'-methoxy-acetophenone and NAC, which reduced TNF-alpha secretion, induced the accumulation of GSH, reduced the concentration of GSSG, and blockaded the phosphorylation/activation of MAPK(p38) pathway. ROS induced MAPK(p38) phosphorylation and selective antioxidants, including the permeant GSH precursor, gamma-GCE, reduced ROS-dependent MAPK(p38) phosphorylation. These results indicate that the MAPK pathway and MAPK-mediated regulation of TNF-alpha production is redox-dependent, GSH-mediated and requires, at least in part, a NF-kappaB/ROS-sensitive mechanism.     

The involvement of L-gamma-glutamyl-L-cysteinyl-glycine (glutathione/GSH) in the mechanism of redox signaling mediating MAPK(p38)-dependent regulation of pro-inflammatory cytokine production

Redox regulation of mitogen-activated protein kinase (MAPK(p38))-mediated pro-inflammatory cytokine production is not well characterized in the alveolar epithelium. It was hypothesized that the involvement of the MAPK(p38) pathway in regulating lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha and interleukin-6 secretion is redox-sensitive and affected by NAC, an antioxidant and a precursor of glutathione, and L-buthionine-(S,R)-sulfoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, the rate-limiting enzyme in GSH biosynthesis. Exposure of fetal alveolar type II epithelial cells to Escherichia coli-derived LPS induced, in a time-dependent manner, the phosphorylation/activation of MAPK(p38) (peak at 15min). In addition, LPS up-regulated the phosphorylation of MAPK(p38) in a dose-dependent manner. The effect of LPS on the MAPK(p38) pathway was associated with the activation of MAPK-activated protein kinase, which phosphorylated the small 27kDa heat-shock protein (Hsp27). LPS induced the phosphorylation of Hsp27 in a time- and dose-dependent manner. Selective blockage of the MAPK(p38) pathway by a pyridinyl-imidazole (SB-203580) abrogated LPS-induced release of TNF-alpha and IL-6. Pre-treatment with NAC reduced LPS-mediated secretion of TNF-alpha and IL-6. Incubation of cells with NAC induced intracellular accumulation of GSH, but reduced the concentration of GSSG. On the other hand, pre-treatment with BSO augmented LPS-mediated secretion of TNF-alpha and IL-6. In addition, BSO induced intracellular accumulation of GSSG, but reduced the concentration of GSH. Whereas NAC blocked the phosphorylation/activation of MAPK(p38), BSO amplified the LPS-mediated effect on MAPK(p38). These results indicated that intracellular redox signaling plays an important role in regulating LPS-induced activation of the MAPK(p38) pathway and MAPK(p38)-mediated regulation of LPS-dependent inflammatory cytokine production in the alveolar epithelium.     

High glucose-induced inhibition of 2-deoxyglucose uptake is mediated by cAMP, protein kinase C, oxidative stress and mitogen-activated protein kinases in mouse embryonic stem cells

Abnormally high glucose levels may play an important role in early embryo development and function. In the present study, we investigated the effect of high glucose on 2-deoxyglucose (2-DG) uptake and its related signalling pathway in mouse embryonic stem (ES) cells. 2. 2-Deoxyglucose uptake was maximally inhibited by 25 mmol/L glucose after 24 h treatment. However, 25 mmol/L mannitol and dextran did not affect 2-DG uptake. Indeed, 25 mmol/L glucose decreased GLUT-1 mRNA and protein levels. The glucose (25 mmol/L)-induced inhibition of 2-DG uptake was blocked by pertussis toxin (a G(i)-protein inhibitor; 2 ng/mL), SQ 22,536 (an adenylate cyclase inhibitor; 10(-6) mol/L) and the protein kinase (PK) A inhibitor myristoylated PKI amide-(14-22) (10(-6) mol/L). Indeed, 25 mmol/L glucose increased intracellular cAMP content. 3. Furthermore, 25 mmol/L glucose-induced inhibition of 2-DG uptake was prevented by 10(-4) mol/L neomycin or 10(-6) mol/L U 73,122 (phospholipase C (PLC) inhibitors) and staurosporine or bisindolylmaleimide I (protein kinase (PK) C inhibitors). At 25 mmol/L, glucose increased translocation of PKC from the cytoplasmic fraction to the membrane fraction. The 25 mmol/L glucose-induced inhibition of 2-DG uptake and GLUT-1 protein levels was blocked by SQ 22,536, bisindolylmaleimide I or combined treatment. In addition, 25 mmol/L glucose increased cellular reactive oxygen species and the glucose-induced inhibition of 2-DG uptake were blocked by the anti-oxidants N-acetylcysteine (NAC; 10(-5) mol/L) or taurine (2 yen 10(-3) mol/L). 4. Glucose (25 mmol/L) activated p38 mitogen-activated protein kinase (MAPK) and p44/42 MAPK. Staurosporine (10(-6) mol/L), NAC (10(-5) mol/L) and PD 98059 (10(-7) mol/L) attenuated the phosphorylation of p44/42 MAPK. Both SB 203580 (a p38 MAPK inhibitor; 10(-7) mol/L) and PD 98059 (a p44/42 MAPK inhibitor; 10(-7) mol/L) blocked 25 mmol/L glucose-induced inhibition of 2-DG uptake. 5. In conclusion, high glucose inhibits 2-DG uptake through cAMP, PLC/PKC, oxidative stress or MAPK in mouse ES cells.     

非诺贝特及罗格列酮对高糖培养肾小球系膜细胞p38MAPK信号通路调控影响

目的：探讨非诺贝特及罗格列酮对高糖培养大鼠肾小球系膜细胞（MC）p38丝裂原活化蛋白激酶（p38MAPK）信号传导通路的影响。方法：大鼠系膜细胞分别培养在正常糖浓度（5．5mmol／L）、高糖浓度（25mmol／L）、25mmol／L葡萄糖＋非诺贝特（FB）100μmol／L及25mmol／L葡萄糖＋罗格列酮（RG）20μmol／L。ELISA法检测培养细胞上清液Ⅳ型胶原（Col-Ⅳ）、纤维连接蛋白（FN）;Phospho—ELISA法检测胞浆及胞核内p38MAPK和磷酸化p38MAPK（p-p38MAPK）蛋白的表达：以及RT-PCR法检测p38MAPK的mRNA的表达。结果：与正常对照组比较,高糖组MC合成基质蛋白Col-Ⅳ、FN增多;胞浆及胞核内P—p38MAPK的表达增加。非诺贝特及罗格列酮干预能使高糖培养系膜细胞合成Col-Ⅳ、FN显著减少,胞核内p-p38MAPK表达显著下调,但对胞浆内p-p38MAPK的表达则无显著影响。各组总p38MAPK蛋白水平及p38MAPK的mRNA表达则没有明显改变。结论：非诺贝特及罗格列酮能够显著下调高糖培养的Mc核内p38MAPK信号传导通路的活化,进而减少胞外基质合成。     

MAPK regulate p53-dependent cell death induced by benzo[a]pyrene: involvement of p53 phosphorylation and acetylation

Benzo[a]pyrene (BaP) is a potentially genotoxic and cytotoxic environmental pollutant. Previous studies showed that exposure of HepG(2) cells to BaP causes necrotic cell death [Lin, T., Yang, M.S., 2007b. Cell death induced by benzo[a]pyrene in the HepG(2) cells is dependent on PARP-1 activation and NAD depletion. Toxicology 245, 147-153]. In the present study, the signaling pathways associated with this response was studied. BaP induced accumulation and activation of p53 in HepG(2) cells, which occurred as early as 12h after exposure. Activation of p53 was evidenced by its phosphorylation at serine 15 (Ser15) and acetylation at lysine 382 (Lys382). Chemical inhibition and siRNA-mediated knockdown of p53 expression suppressed its phosphorylation as well as cell death. BaP also activated p38 MAPK and ERK, but not JNK, at 6h after exposure. SB203580 and PD98059, specific inhibitors of p38 MAPK and ERK, respectively, suppressed phosphorylation of p53 at Ser15, but the accumulation of p53 was only moderately reduced. Acetylation of p53 at Lys 382 was not affected by these inhibitors, suggesting that acetylation stabilizes p53 in response to DNA damage. SB203580 and PD98059 prevented downstream energy failure and BaP-induced cell death. Similar results were obtained with siRNA against two isoforms of p38 MAPK, p38alpha and p38beta. Wortmannin, selective inhibitor of DNA-PK and ATM/ATR, abolished p53 phosphorylation, indicating an involvement of multiple pathways of p53 phosphorylation upon exposure to BaP. In summary, the current study demonstrated that both MAPK and p53 activation are required for BaP-induced necrotic cell death. The results also provide a novel model for studying the regulation between p53 and p38 MAPK in the progression of cellular necrosis.     

Beneficial effect of the oligomerized polyphenol oligonol on high glucose-induced changes in eNOS phosphorylation and dephosphorylation in endothelial cells

Background and purpose:

Hyperglycaemia is known to reduce nitric oxide (NO) bioavailability by modulating endothelial NO synthase (eNOS) activity, and polyphenols are believed to have cardiovascular benefit. One possible mechanism could be through interaction with eNOS.

Experimental approach:

The effects of the oligomerized polyphenol oligonol on eNOS phosphorylation status and activity were examined in porcine aortic endothelial cells cultured in high glucose concentrations.

Key results:

Exposure to high glucose concentrations strongly inhibited eNOS phosphorylation at Ser-1177 and dephosphorylation at Thr-495 in bradykinin (BK)-stimulated cells. These inhibitory effects of high glucose were significantly prevented by treatment with oligonol. Akt and p38 mitogen-activated protein kinase (MAPK) were activated in BK-stimulated cells. High glucose inhibited Akt activation but enhanced p38 MAPK activation, both of which were reversed by oligonol treatment. The phosphatidylinositol 3-kinase inhibitor wortmannin blocked the reversal by oligonol of phosphorylation at Ser-1177, but not dephosphorylation at Thr-495, in BK-stimulated cells exposed to high glucose. The effect of oligonol on BK dephosphorylation under high glucose was mimicked by protein kinase C (PKC) ε-neutralizing peptides. These data suggest that the effects of oligonol on high glucose-induced attenuation of eNOS Ser-1177 phosphorylation and Thr-495 dephosphorylation may be regulated by Akt activation and PKCε inhibition respectively. Oligonol also prevented high glucose-induced attenuation of BK-stimulated NO production.

Conclusions and implications:

Oligonol prevented the impairment of eNOS activity induced by high glucose through reversing altered eNOS phosphorylation status. This mechanism may underlie the beneficial cardiovascular health effects of this oligomerized polyphenol.     

缬沙坦对高糖培养的肾小球系膜细胞p38MAPK传导通路的影响

目的探讨高糖状态下肾小球系膜细胞中p38丝裂原活化蛋白激酶(p38 MAPK)、其上游因子MAPK激酶3/6(MKK3/6)和下游因子cAMP反应元件结合蛋白1(CREB1)的表达以及血管紧张素受体1拮抗剂(AT1Ra)缬沙坦的影响。方法体外培养大鼠肾小球系膜细胞,分别给予高糖和缬沙坦干预,采用Western bolt检测MKK3/6、p38 MAPK和CREB1及其磷酸化蛋白的表达,逆转录-聚合酶链反应(RT-PCR)检测系膜细胞内TGF-β1和FN mRNA的表达。放免法测定细胞上清液中纤维连接蛋白(FN)和IV型胶原的含量。MTT法检测缬沙坦在不同时间、不同药物浓度对细胞增殖状态的影响。结果①与低糖对照组相比,高糖组系膜细胞p-p38 MAPK、p-MKK3/6和p-CREB1表达明显上调,TGF-β1和FN mRNA的表达增加,FN和IV型胶原含量增加。②缬沙坦组p-p38 MAPK、p-MKK3/6和p-CREB1的表达明显下调,TGF-β1和FN mRNA的表达降低,同时FN和IV型胶原的含量减少。③MTT法检测显示不同浓度的缬沙坦对细胞增殖状态都有所抑制,并随药物浓度的增加而作用增强。结论缬沙坦抑制肾小球系膜细胞TGF-β1的表达和细胞外基质的分泌可能部分是通过影响p38 MAPK传导通路的激活来实现的。     

Role of MAPK signaling pathway in the activation of dendritic type cell line, THP-1, induced by DNCB and NiSO4

The activation of dendritic cells (DC), including Langerhans cells (LC) that reside within the epidermis, is a critical event in the induction phase of allergic contact hypersensitivity. Although recently, p38 mitogen-activated protein kinase (MAPK) has been reported to play a role in the activation of DC induced by allergens, the signaling pathways involved in this process have yet to be determined. We previously found that THP-1 cells have a high capacity to induce TNF-alpha release and CD86, CD54, and CD40 expression following allergen treatment; reflecting in vitro allergen-induced DC activation during skin sensitization. In this study, we investigated the signaling pathways in THP-1 cells activated by two representative allergens, DNCB and NiSO(4). We found that DNCB and NiSO(4) induced phosphorylation of p38 MAPK and extracellular signal-regulated kinase (ERK). Inhibition of p38 MAPK activation selectively blocked DNCB-induced TNF-alpha release, but not NiSO(4)-induced release. In contrast, inhibition of ERK pathways selectively suppressed NiSO(4)-induced TNF-alpha release but not DNCB-induced release. In addition, we found that the inhibition of p38 MAPK and ERK pathways caused a selective inhibition of CD86, CD54, and/or CD40 expression following treatment with DNCB or NiSO(4). In particular, inhibition of p38 MAPK suppressed CD86, CD54, and CD40 expression induced by DNCB and CD86 expression induced by NiSO(4) while inhibition of ERK pathways suppressed CD86, CD54 and CD40 expression induced by DNCB and NiSO(4). These data indicate that both DNCB and NiSO(4) activate p38 MAPK and ERK, and thereby stimulate TNF-alpha release and phenotypic changes through the different signal transduction pathways.     

Metformin enhances insulin signalling in insulin-dependent and-independent pathways in insulin resistant muscle cells

1 Metformin lowers blood glucose levels in type 2 diabetic patients. To evaluate the insulin sensitizing action of metformin on skeletal muscle cells, we have used C2C12 skeletal muscle cells differentiated in chronic presence or absence of insulin. 2 Metformin was added during the last 24 h of differentiation of the C2C12 myotubes. Insulin-stimulated tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) was determined. 3 Chronic insulin treatment resulted in 60 and 40% reduction in insulin-stimulated tyrosine phosphorylation of IR and IRS-1, respectively. Treatment with metformin was able to increase the tyrosine phosphorylation of IR and IRS-1 by 100 and 90% respectively. 4 Chronic insulin treatment drastically reduced (45%) insulin-stimulated phosphatidyl inositol 3-kinase (PI 3-kinase) activity. Metformin treatment restored PI 3-kinase activity in insulin-resistant myotubes. 5 Insulin-stimulated glucose uptake was impaired in chronically insulin-treated myotubes. Metformin increased basal glucose uptake to significant levels (P<0.05), but metformin did not increase insulin-stimulated glucose transport. 6 All the three mitogen-activated protein kinases (MAPK) were activated by insulin in sensitive myotubes. The activation of p38 MAPK was impaired in resistant myotubes, while ERK and JNK were unaffected. Treatment with metformin enhanced the basal activation levels of p38 in both sensitive and resistant myotubes, but insulin did not further stimulate p38 activation in metformin treated cells. 7 Treatment of cells with p38 inhibitor, SB203580, blocked insulin- and metformin-stimulated glucose uptake as well as p38 activation. 8 Since the effect of metformin on glucose uptake corresponded to p38 MAPK activation, this suggests the potential role p38 in glucose uptake. 9 These data demonstrate the direct insulin sensitizing action of metformin on skeletal muscle cells.     

Mitogen-activated protein kinase inhibitors differently affect the growth inhibition and death of a proteasome inhibitor, MG132-treated human pulmonary fibroblast cells

Carbobenzoxy-Leu-Leu-leucinal (MG132) as a proteasome inhibitor can induce growth inhibition and death in lung cancer or normal cells. However, little is known about relationship between proteasome inhibition and mitogen-activated protein kinase (MAPK) signaling in normal lung cells. Thus, in the present study, we investigated the effects of MAPK inhibitors on MG132-treated human pulmonary fibroblast (HPF) cells in relation to cell growth inhibition, cell death, reactive oxygen species (ROS) and glutathione (GSH). Treatment with 15 μM MG132 increased ROS levels including mitochondrial O(2?)(-) and GSH depleted cell numbers in HPF cells at 24 hours. MAP kinase or ERK kinase (MEK) inhibitor did not significantly affect cell growth inhibition, cell death, the loss of mitochondrial membrane potential (MMP; ΔΨ(m)), ROS level and GSH depletion in MG132-treated HPF cells. c-Jun N-terminal kinase (JNK) inhibitor attenuated the growth inhibition and death by MG132. This inhibitor also significantly decreased O(2?)(-) level in MG132-treated HPF cells. Although p38 inhibitor slightly enhanced HPF cell growth inhibition by MG132, this inhibitor and siRNA prevented HPF cell death induced by MG132. p38 inhibitor also attenuated d O(2?)(-) level and GSH depletion. Moreover, extracellular signal regulated kinase (ERK), JNK or p38 siRNA did not strongly affect ROS levels in MG132-treated HPF cells. ERK and JNK siRNAs decreased anonymous ubiquitinated protein levels in MG132-treated HPF cells. In conclusion, MAPK inhibitors differently affected the growth inhibition and death of MG132-treated HPF cells. Especially, p38 inhibitor attenuated HPF cell death by MG132, which was in part related to changes in ROS and GSH levels.     

Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways

BackgroundLanthanide cerium oxide (CeO₂) nanoparticles have extensive applications in industrial fields, and concerns regarding their potential toxicity in humans and their environmental impact have increased. We investigated the underlying molecular mechanisms by which CeO₂ nanoparticles induce toxicity in human hepatoma SMMC-7721 cells.ResultsOur results demonstrated that CeO₂ nanoparticles reduced viability, caused dramatic morphological damage, and induced apoptosis in SMMC-7721 cells. CeO₂ nanoparticles significantly increased the production of reactive oxygen species (ROS) and malondialdehyde (MDA), and significantly reduced the activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT). The phosphorylation levels of ERK1/2, JNK and p38 MAPK were significantly elevated after treatment with CeO₂ nanoparticles. Pretreatment with the antioxidant N-acetyl-cysteine (NAC): reduced the induction of ROS and MDA by CeO₂ nanoparticles; recovered the activity of SOD, GSH-px and CAT; reduced the phosphorylation levels of ERK1/2, JNK and p38; and attenuated CeO₂ nanoparticles-induced damage and apoptosis in SMMC-7721 cells.ConclusionsOur data demonstrated that CeO₂ nanoparticles induced damage and apoptosis in human SMMC-7721 cells via oxidative stress and the activation of MAPK signaling pathways.     

The mechanism of MAP kinase activation under acidic condition in feline esophageal smooth muscle cells

Reflux esophagitis results from repeated exposure of the esophagus to acidic gastric juice or bile-containing duodenal contents. In Barrett’s adenocarcinoma, acid increases proliferation via ERK and p38 MAPK activation. This study was focused on determination of the mechanism(s) underlying MAPKs (ERK 1/2, p38 MAPK, and JNK) activation induced by acidic medium at pH 4 in normal feline primary cultured esophageal smooth muscle cells (FESMCs). We detected ERK 1/2 and p38 MAPK phosphorylation after exposure to pH 4 or neutral media in the presence or absence of several inhibitors and quantified the MAPK levels using western blotting analysis and densitometry. Acidic medium markedly increased the phosphorylation of ERK 1/2 and p38 MAPK within 10 min. Acid-induced ERK 1/2 and p38 MAPK activation was inhibited by pertussis toxin (PTX-sensitive G_i/o protein inhibitor), DEDA (phospholipase (PL) A₂ inhibitor), ρCMB (PLD inhibitor), GF109203X (protein kinase C (PKC) inhibitor) and D609 (phosphatidylcholinespecific PLC inhibitor). But, genistein (tyrosine kinase inhibitor), forskolin (adenylate cyclase activator) and U73122 (phosphatidylinositol-specific PLC inhibitor) had no effect on acidinduced ERK1/2 and p38 MAPK activation. These findings indicate that the activation of ERK 1/2 and p38 MAPK pathways by acidic conditions, at least in part, may be mediated by activation of the G_i/o protein coupled receptors, PC-PLC, PLD, PLA₂, and PKC in FESMCs.     

Roles of mitogen-activated protein kinase pathways for mediator release from human cultured mast cells

Human cultured mast cells (HCMC) secrete histamine, sulfidoleukotrienes (LTs), and prostaglandin D(2) (PGD(2)), and produce a variety of cytokines after aggregation of high-affinity receptors for IgE (FcepsilonRI). With respect to the mitogen-activated protein kinase (MAPK) family, extracellular signal-regulated kinases (ERKs), c-Jun NH(2)-terminal kinases (JNKs), and p38 mitogen-activated protein kinase (p38 MAPK) are known. To investigate the roles of these kinase pathways for mediator release from human mast cells, we examined the participation of the activation of these kinases in mediator release, using 1,4-diamino-2, 3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), an ERK pathway inhibitor, and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imid azo le (SB203580), a p38 MAPK pathway inhibitor. U0126 inhibited ERK activation, LT and PGD(2) release, and granulocyte macrophage-colony stimulating factor (GM-CSF) production after stimulation of HCMC. SB203580, on the other hand, potentiated JNK activation and GM-CSF production. The findings of the present study demonstrated that: (i) the release of arachidonic acid metabolites is mediated by the ERK pathway; (ii) GM-CSF production may be driven by both the ERK and JNK pathways; and (iii) the p38 MAPK pathway negatively regulates the JNK pathway. This suggests that MAPK pathways play important roles in mediator release from human mast cells.     

Silibinin Inhibits LPS-Induced Macrophage Activation by Blocking p38 MAPK in RAW 264.7 Cells

We demonstrate herein that silibinin, a polyphenolic flavonoid compound isolated from milk thistle (Silybum marianum), inhibits LPS-induced activation of macrophages and production of nitric oxide (NO) in RAW 264.7 cells. Western blot analysis showed silibinin inhibits iNOS gene expression. RT-PCR showed that silibinin inhibits iNOS, TNF-α, and IL1β. We also showed that silibinin strongly inhibits p38 MAPK phosphorylation, whereas the ERK1/2 and JNK pathways are not inhibited. The p38 MAPK inhibitor abrogated the LPS-induced nitrite production, whereas the MEK-1 inhibitor did not affect the nitrite production. A molecular modeling study proposed a binding pose for silibinin targeting the ATP binding site of p38 MAPK (1OUK). Collectively, this series of experiments indicates that silibinin inhibits macrophage activation by blocking p38 MAPK signaling.     

Additive effects of heat and p38 mapk inhibitor treatment on melanin synthesis

It has been reported that the activation of extracellular signal-regulated kinase (ERK) reduces melanin synthesis. Recently, we also found that heat treatment induces ERK activation and inhibits melanogenesis in Mel-Ab cells (a mouse melanocyte cell line). In addition, it was reported that p38 MAPK (mitogen-activated protein kinase) inhibition blocks melanogenesis. Thus, we investigated the effects of heat and of the p38 MAPK inhibitor, SB203580, on melanogenesis. In this study, we found that heat treatment activates ERK and reduces melanin production in human melanocytes, and that this is accompanied by a reduction in tyrosinase activity. To regulate the ERK and p38 MAPK pathways simultaneously, we combined heat treatment and SB203580 and measured melanin synthesis. The results obtained showed that heat treatment and SB203580 reduced melanin synthesis more effectively than heat or SB203580 alone. We conclude that ERK activation and p38 MAPK inhibition can work in an additive manner to decrease melanogenesis.     

Naringin Inhibits ROS‐activated MAPK Pathway in High Glucose‐induced Injuries in H9c2 Cardiac Cells

Naringin, an active flavonoid isolated from citrus fruit extracts, exhibits biological and pharmacological properties, such as antioxidant activity and antidiabetic effect. Mitogen‐activated protein kinase (MAPK) signalling pathway has been shown to participate in hyperglycaemia‐induced injury. The present study tested the hypothesis that naringin protects against high glucose (HG)‐induced injuries by inhibiting MAPK pathway in H9c2 cardiac cells. To examine this, the cells were treated with 35 mM glucose (HG) for 24 hr to establish a HG‐induced cardiomyocyte injury model. The cells were pre‐treated with 80 μM naringin for 2 hr before exposure to HG. The findings of this study showed that exposure of H9c2 cells to HG for 24 hr markedly induced injuries, as evidenced by a decrease in cell viability, increases in apoptotic cells and reactive oxygen species (ROS) production, as well as dissipation of mitochondrial membrance potential (MMP). These injuries were significantly attenuated by the pre‐treatment of cells with either naringin or SB203580 (a selective inhibitor of p38 MAPK) or U0126 (a selective inhibitor of extracellular signal regulated kinase 1/2, ERK1/2) or SP600125 (a selective inhibitor of c‐jun N‐termanal kinase, JNK) before exposure to HG, respectively. Furthermore, exposure of cells to HG increased the phosphorylation of p38 MAPK, ERK1/2 and JNK. The increased activation of MAPK pathway was ameliorated by pre‐treatment with either naringin or N‐acetyl‐L‐cysteine (NAC), a ROS scavenger, which also reduced HG‐induced cytotoxicity and apoptosis, leading to increase in cell viability and decrease in apoptotic cells. In conclusion, our findings provide new evidence for the first time that naringin protects against HG‐induced injuries by inhibiting the activation of MAPK (p38 MAPK, ERK1/2 and JNK) and oxidative stress in H9c2 cells.