P-p38 Mitogen-activated Protein Kinase Research Articles

Propofol Inhibits Lipopolysaccharide-Induced Inflammatory Responses in Spinal Astrocytes via the Toll-Like Receptor 4/MyD88-Dependent Nuclear Factor-κB, Extracellular Signal-Regulated Protein Kinases1/2, and p38 Mitogen-Activated Protein Kinase Pathways.

In this study, we investigated the effect of propofol, a commonly used IV anesthetic, on lipopolysaccharide (LPS)-induced inflammatory responses in astrocytes and explored the molecular mechanisms by which it occurs. Astrocytes were stimulated with LPS (1.0 μg/mL) in the absence and presence of different concentrations of propofol. The expression of astrocyte marker glial fibrillary acidic protein (GFAP) in astrocytes was detected using immunofluorescence staining and Western blot analysis. The levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-α were measured using an enzyme-linked immunosorbent assay. The mRNA level of Toll-like receptor 4 (TLR4) was determined by semiquantitative reverse transcriptase-polymerase chain reaction. The protein expressions of TLR4, myeloid differentiation factor 88 (MyD88), p- extracellular signal-regulated protein kinases (ERK)1/2, p-c-Jun N-terminal kinase, p-p38 mitogen-activated protein kinase (MAPK), p-I-κBα, I-κBα, and p-nuclear factor-κB (NF-κB)p65 were detected by Western blot. Our results show that after stimulation with LPS, the levels of IL-1β, IL-6, and tumor necrosis factor-α and the expression of GFAP in astrocytes were up-regulated significantly. In addition, the expression of TLR4, MyD88, p-ERK1/2, p-c-Jun N-terminal kinase, p-p38 MAPK, and p-NF-κBp65 increased, whereas the expression of total I-κBα decreased upon stimulation with LPS. Propofol (10 μM) reduced the secretion of proinflammatory cytokines, inhibited the expressions of GFAP, TLR4, MyD88, p-ERK1/2, p-p38 MAPK, and p-NF-κBp65 in astrocytes challenged with LPS. In the present study, propofol 10 μM but not lower clinically relevant or higher supra-clinical concentrations attenuated LPS-induced astrocyte activation and subsequent inflammatory responses by inhibiting the TLR4/MyD88-dependent NF-κB, ERK1/2, and p38 MAPK pathways.

Activation of transient receptor potential vanilloid 4 induces apoptosis in hippocampus through downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways.

Transient receptor potential vanilloid 4 (TRPV4) is a calcium-permeable cation channel that is sensitive to cell swelling, arachidonic acid and its metabolites, epoxyeicosatrienoic acids, which are associated with cerebral ischemia. The activation of TRPV4 induces cytotoxicity in many types of cells, accompanied by an increase in the intracellular free calcium concentration. TRPV4 activation modulates the mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3 kinase (PI3K)/ protein kinase B (Akt) signaling pathways that regulate cell death and survival. Herein, we examined TRPV4-induced neuronal apoptosis by intracerebroventricular (ICV) injection of a TRPV4 agonist (GSK1016790A) and assessed its involvement in cerebral ischemic injury. ICV injection of GSK1016790A dose-dependently induced apoptosis in the mouse hippocampi (GSK-injected mice). The protein level of phosphorylated p38 MAPK (p-p38 MAPK) was markedly increased and that of phosphorylated c-Jun N-terminal protein kinase (p-JNK) was virtually unchanged. TRPV4 activation also decreased Bcl-2/Bax protein ratio and increased the cleaved caspase-3 protein level, and these effects were blocked by a PI3K agonist and a p38 MAPK antagonist, but were unaffected by a JNK antagonist. ICV injection of the TRPV4 antagonist HC-067047 reduced brain infarction after reperfusion for 48 h in mice with middle cerebral artery occlusion (MCAO). In addition, HC-067047 treatment attenuated the decrease in the phosphorylated Akt protein level and the increase in p-p38 MAPK protein level at 48 h after MCAO, while the increase in p-JNK protein level remained unchanged. Finally, the decreased Bcl-2/Bax protein ratio and the increased cleaved caspase-3 protein level at 48 h after MCAO were markedly attenuated by HC-067047. We conclude that activation of TRPV4 induces apoptosis by downregulating PI3K/Akt and upregulating p38 MAPK signaling pathways, which is involved in cerebral ischemic injury.

Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes

Thymic stromal lymphopoietin downregulates filaggrin expression by signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK) phosphorylation in keratinocytes

Early transplantation of mesenchymal stem cells after spinal cord injury relieves pain hypersensitivity through suppression of pain-related signaling cascades and reduced inflammatory cell recruitment.

Bone marrow-derived mesenchymal stem cells (BMSC) modulate inflammatory/immune responses and promote motor functional recovery after spinal cord injury (SCI). However, the effects of BMSC transplantation on central neuropathic pain and neuronal hyperexcitability after SCI remain elusive. This is of importance because BMSC-based therapies have been proposed for clinical treatment. We investigated the effects of BMSC transplantation on pain hypersensitivity in green fluorescent protein (GFP)-positive bone marrow-chimeric mice subjected to a contusion SCI, and the mechanisms of such effects. BMSC transplantation at day 3 post-SCI improved motor function and relieved SCI-induced hypersensitivities to mechanical and thermal stimulation. The pain improvements were mediated by suppression of protein kinase C-γ and phosphocyclic AMP response element binding protein expression in dorsal horn neurons. BMSC transplants significantly reduced levels of p-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (p-ERK1/2) in both hematogenous macrophages and resident microglia and significantly reduced the infiltration of CD11b and GFP double-positive hematogenous macrophages without decreasing the CD11b-positive and GFP-negative activated spinal-microglia population. BMSC transplants prevented hematogenous macrophages recruitment by restoration of the blood-spinal cord barrier (BSCB), which was associated with decreased levels of (a) inflammatory cytokines (tumor necrosis factor-α, interleukin-6); (b) mediators of early secondary vascular pathogenesis (matrix metallopeptidase 9); (c) macrophage recruiting factors (CCL2, CCL5, and CXCL10), but increased levels of a microglial stimulating factor (granulocyte-macrophage colony-stimulating factor). These findings support the use of BMSC transplants for SCI treatment. Furthermore, they suggest that BMSC reduce neuropathic pain through a variety of related mechanisms that include neuronal sparing and restoration of the disturbed BSCB, mediated through modulation of the activity of spinal-resident microglia and the activity and recruitment of hematogenous macrophages.

The Red Nucleus TNF-α Participates in the Initiation and Maintenance of Neuropathic Pain Through Different Signaling Pathways.

Previous studies have demonstrated that tumor necrosis factor-alpha (TNF-α) in the red nucleus (RN) plays a facilitated role in the development of neuropathic pain. Here, we further investigated the expression changes and roles of the downstream signaling molecules of the red nucleus TNF-α, including nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), in the initiation and maintenance of neuropathic pain induced by spared nerve injury (SNI). Immunohistochemistry demonstrated that increased expressions of NF-κB, phospho-ERK (p-ERK) and p-p38 MAPK were observed in the RN contralateral (but not ipsilateral) to the nerve injury side at 3days after SNI compared with sham-operated and normal rats, the up-regulations of NF-κB and p-ERK but not p-p38 MAPK remained at high levels till 14days later. An elevated expression of p-JNK occurred at 14days (but not 3 and 7days) after SNI, which was later than those of NF-κB, p-ERK and p-p38 MAPK. The up-regulations of NF-κB, p-ERK, p-p38 MAPK and p-JNK all could be abolished by microinjection of anti-TNF-α antibody into the RN of rats with SNI. Microinjection of NF-κB inhibitor PDTC, ERK inhibitor PD98059, p38 MAPK inhibitor SB203580 but not JNK inhibitor SP600125 into the RN contralateral to the nerve injury side at 3days postinjury significantly alleviated SNI-induced mechanical allodynia. In addition, microinjection of PDTC, PD98059 and SP600125 but not SB203580 into the RN at 14days postinjury significantly alleviated SNI-induced mechanical allodynia. These results suggest that the red nucleus TNF-α produces the algesic effect through activating NF-κB, ERK and p38 MAPK in the early initiation stage but relying on the activation of NF-κB, ERK and JNK in the later maintenance stage of SNI-induced neuropathic pain.

Effect of quercetin derivatives on melanogenesis stimulation of melanoma cells

Controlling melanogenesis is important for maintaining the good health and cosmetic appearance of a human body. This study aims to search the active compounds from natural products exhibiting the melanogenesis modulating activity and elucidate the mechanism underlying the observed activity. Two novel quercetin glycosides namely 4′-O-β-d-glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranoside 1 and 4′-O-β-d–glucopyranosyl-(1 → 2)-β-d-glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1 → 4)-β-d-glucopyranoside 2 were isolated and identified from Helminthostachys zeylanica roots 50 % ethanol extract. Compound 1 exhibited intracellular melanogenesis stimulatory activity, while 2 showed no effect even the structural similarity. To understand the structure–activity relationships, twelve quercetin glycosides and seven methylquercetins were synthesized from rutin as starting material. As the result of bioassay using synthesized nineteen quercetin derivatives in B16 melanoma cells, some quercetin-3-O-β-d-glucopyranosides stimulated the intracellular melanogenesis. On the other hand, synthesized 3-O-methylquercetin 12 and 3,4′,7-O-trimethylquercetin 15 increased both intra and extracellular melanin contents with no cytotoxicity. Compoud 15 increased the phosphorylated p38 mitogen-activated protein kinase (MAPK) and microphthalmia-associated transcription factor (MITF) which regulates the expression of tyrosinase, TRP-1 and TRP-2. While 12 enhanced the expression of the melanogenic enzymes without involving the MITF, as evidenced by its lack of any stimulation of the expression of MITF and p-p38 MAPK. This result indicates that 12 may stimulate the expression of tyrosinase, TRP-1, and TRP-2 by stimulating currently unidentified transcriptional factors and/or by regulating the degradation of melanogenic enzymes.

The effects and mechanisms of G protein-coupled receptor 91 on blood-retinal barrier in diabetic rats

Objective To investigate the effects and mechanisms of G protein-coupled receptor 91 (GPR91) on blood-retinal barrier (BRB) in diabetic rats. Methods A lentiviral vector of shRNA targeting rat GPR91 and scrambled shRNA were constructed. Healthy male Sprague-Dawley (SD) rats were selected in this study. The 60 rats were randomized into 4 groups and treated as follows: (1) control group (Group A, n=15), the rats received injections of an equal volume of 0.1% citrate buffer; (2) streptozocin (STZ) group (Group B, n=15), the rats received injections of STZ; (3) LV.shScrambled group (Group C, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml scrambled shRNA lentiviral particles at 2 weeks after the induction of diabetes; (4) LV.shGPR91 group (Group D, n=15), diabetic rats received an intravitreal injection of 1 μl 1×108 TU/ml pGCSIL-GFP-shGPR91 lentiviral particles. At 12 weeks after intravitreal injection, immunohistochemistry and Western blot were used to assess the expression of GPR91, p-extracellular signal-regulated kinase(ERK)1/2, t-ERK1/2, p-Jun N-terminal kinase (JNK), t-JNK, p-p38 mitogen-activated protein kinase (MAPK) and t-p38 MAPK. Haematoxylin and eosin (HE) staining and Evans blue dye were used to assess the structure and function of the retinal vessel. Immunohistochemistry enzyme-linked immunosorbent assay (ELISA) was used to test the protein level of VEGF. Results Immunohistochemistry staining showed that GPR91 was predominantly localized to the cell bodies of the ganglion cell layer. Western blot showed that GPR91 expression in Group D decreased significantly compared with Group C (F=39.31, P<0.01). HE staining showed that the retina tissue in Group B and C developed telangiectatic vessels in the inner layer of retina, while the telangiectatic vessels attenuated in Group D. It was also demonstrated in Evans blue dye that the microvascular leakage in Group D decreased by (33.8±4.11)% compared with Group C and there was significant difference (F=30.35, P<0.05). The results of ELISA showed the VEGF secretion of Group B and C increased compared with Group A and the VEGF expression in Group D was significantly down regulated after silencing GPR91 gene (F=253.15, P<0.05). The results of Western blot indicated that compared with Group A, the expressions of p-ERK1/2, p-JNK and p-p38 MAPK were significantly upregulated (q=6.38, 2.94, 3.45; P<0.05). Meanwhile, the activation of ERK1/2 was inhibited by GPR91 shRNA and the difference was statistically significant (F=22.50, P<0.05). Conclusions The intravitreal injection of GPR91 shRNA attenuated the leakage of BRB in diabetic rats. GPR91 regulated the VEGF release and the leakage of BRB possibly through the ERK1/2 signaling pathway. Key words: Diabetic retinopathy/physiopathology; RNA, small interfering; Receptors, G-protein-coupled; Vascular Endothelial Growth Factors; p38 Mitogen-Activated Protein Kinases; Animal experimentation

Phospho-p38 MAPK Expression in COPD Patients and Asthmatics and in Challenged Bronchial Epithelium

Background: The role of mitogen-activated protein kinases (MAPK) in regulating the inflammatory response in the airways of patients with chronic obstructive pulmonary disease (COPD) and asthmatic patients is unclear. Objectives: To investigate the expression of activated MAPK in lungs of COPD patients and in bronchial biopsies of asthmatic patients and to study MAPK expression in bronchial epithelial cells in response to oxidative and inflammatory stimuli. Methods: Immunohistochemical expression of phospho (p)-p38 MAPK, p-JNK1 and p-ERK1/2 was measured in bronchial mucosa in patients with mild/moderate (n = 17), severe/very severe (n = 16) stable COPD, control smokers (n = 16), control non-smokers (n = 9), in mild asthma (n = 9) and in peripheral airways from COPD patients (n = 15) and control smokers (n = 15). Interleukin (IL)-8 and MAPK mRNA was measured in stimulated 16HBE cells. Results: No significant differences in p-p38 MAPK, p-JNK or p-ERK1/2 expression were seen in bronchial biopsies and peripheral airways between COPD and control subjects. Asthmatics showed increased submucosal p-p38 MAPK expression compared to COPD patients (p < 0.003) and control non-smokers (p < 0.05). Hydrogen peroxide (H₂O₂), cytomix (tumour necrosis factor-α + IL-1β + interferon-γ) and lipopolysaccharide (LPS) upregulated IL-8 mRNA at 1 or 2 h. p38 MAPKα mRNA was significantly increased after H₂O₂ and LPS treatment. JNK1 and ERK1 mRNA were unchanged after H₂O₂, cytomix or LPS treatments. Conclusion: p-p38 MAPK expression is similar in stable COPD and control subjects but increased in the bronchi of mild asthmatics compared to stable COPD patients. p38 MAPK mRNA is increased after bronchial epithelial challenges in vitro. These data together suggest a potential role for this MAPK in Th2 inflammation and possibly during COPD exacerbations.

Perfluorooctanoic acid disrupts the blood-testis barrier and activates the TNFα/p38 MAPK signaling pathway in vivo and in vitro.

Perfluorooctanoic acid (PFOA) is correlated with male reproductive dysfunction in animals and humans, but the underlying mechanisms for this remain unknown. To explore the potential reproductive toxicity of PFOA, we studied blood-testis barrier (BTB) damage using in vivo and in vitro models. Male mice were gavage-administered PFOA (0-20 mg/kg/d) for 28 consecutive days, and breeding capacity and permeability of the Sertoli cell-based BTB were estimated. Primary Sertoli cells (SCs) were exposed to PFOA (0-500 μM) for 48 h, and transepithelial electrical resistance (TER) was assessed. Furthermore, BTB-associated protein expression, TNFα content, and phosphorylation and protein levels of the mitogen-activated protein kinase (MAPK) pathway were detected. An apparent decrease in mated and pregnant females per male mouse as well as litter weight was observed. Marked BTB damage was evidenced by increased red biotin fluorescence in the lumen tubular of the testes and the decrease in TER in SCs in vitro. The protein levels of claudin-11, connexin-43, N-cadherin, β-catenin, and occludin were significantly decreased in the testes and also in the SCs in vitro except for N-cadherin and β-catenin. TNFα content showed a dose-dependent increase in the testes and a dose- and time-dependent increase in the SCs, with the p-p38/p38 MAPK ratio also increasing in testes and SCs after PFOA exposure. Moreover, PFOA altered expressions of claudin-11, connexin-43, TNFα, and p-p38 MAPK were recovered 48 h after PFOA removal in the SCs. The SCs appeared to be target to PFOA, and the disruption of the BTB may be crucial to PFOA-induced reproductive dysfunction in mice.

Cathepsin S activity controls ischemia-induced neovascularization in mice

BackgroundEvidence from human and animal studies has demonstrated elevated levels of the cysteine protease cathepsin S (CatS) in hypoxic atherosclerotic lesions. We hypothesized that silencing of CatS gene would suppress ischemia-induced angiogenic action. Methods and resultsLeft femoral artery ligation-induced ischemia in mice showed the increased expression and activity of CatS in the ischemic muscle. The CatS-deficiency (CatS−/−) mice showed impaired functional recovery following hindlimb ischemia and reduced levels of peroxisome proliferator-activated receptor-γ (PPAR-γ), phospho-Akt (p-Akt), p-endothelial nitric oxide synthase, p-extracellular signal-regulated kinase1/2 (Erk1/2), p-p38 mitogen-activated protein kinase, and vascular endothelial growth factor (VEGF) proteins, as well as reduced levels of matrix metalloproteinase-9 and macrophage infiltration in the ischemic muscles. In vitro, CatS silencing reduced the levels of these targeted essential molecules for angiogenesis and vasculogenesis. Together, the results indicated that the effects of CatS knockdown led to defective endothelial cell invasion, proliferation, and tube formation. This notion was reinforced by the finding that CatS inhibition led to a decreased PPAR-γ level and VEGF/Erk1/2 signaling activation in response to ischemia. CatS−/− resulted in decreased circulating EPC-like CD31+/c-Kit+ cells, accompanied by the reduction of the cellular levels of PPAR-γ, p-Akt, and VEGF induced by ischemic stress. Transplantation of bone-marrow-derived mononuclear cells from CatS+/+ mice restored neovascularization in CatS−/− mice. ConclusionsCatS activity controls ischemia-induced neovascularization partially via the modulation of PPAR-γ and VEGF/Akt signaling activation.

Activation of liver X receptor protects inner retinal damage induced by N-methyl-D-aspartate.

To investigate whether activation of liver X receptors (LXRs) protects N-methyl-D-aspartic (NMDA)-induced retinal neurotoxicity in mice and to explore the underlying mechanism. Inner retinal damage was induced by intravitreal injection of NMDA. A synthetic LXR ligand TO901317 (TO90, 50 mg/kg/d) or vehicle was intragastrically administrated from 3 days before to 1 day or 7 days after NMDA injection. The severity of retinal damage was evaluated with histological analysis and TUNEL staining, and retinal functions were evaluated by ERG. The expressions of caspase-3, bax, bcl-2, TNF-α, and BACE1, the rate-limiting enzyme in the formation of amyloid β (Aβ), in the retina were examined by real-time PCR and ELISA. The levels of LXRs, NF-κB subunit p65, p-p38 mitogen-activated protein kinase (MAPK), and an LXR target gene ABCA1 were detected with real-time PCR and Western blotting. The localization and protein expression of Aβ in the retina was assessed by immunohistochemistry and Western blotting. The NMDA enhanced the expression of LXRβ but not LXRα and ABCA1 in mouse retina. Nevertheless, administration of TO90 after NMDA injection not only enhanced the expression of LXRβ but also upregulated the level of ABCA1, suggesting retinal LXRs were activated in a ligand-dependent manner. The LXRα expression was unchanged in the vehicle and the TO90-treated groups. Activation of LXRβ with TO90 inhibited cell death in the ganglion cell layer (GCL) and inner nuclear layer (INL), preserved ERG b- and a-wave amplitudes, and the b/a ratio in the NMDA-treated mice. Meanwhile, TO90 suppressed the elevation of apoptosis factors caspase-3 and bax induced by NMDA and upregulated the level of an antiapoptotic factor bcl-2. The TO90 also inhibited the increase of p-p38 MAPK and proinflammatory cytokine TNF-α after NMDA injection. Furthermore, activation of LXR attenuated the activation of NF-κB, and reduced gene expression of BACE1 and accumulation of Aβ induced by NMDA. Activation of LXRβ with a synthetic LXR ligand TO90 protects the inner retinal damage induced by NMDA in mice. We speculate the protective effect is associated with inhibition of the NF-κB signaling pathway and reduction of Aβ formation in retina. The LXR agonists may become a new class of neuroprotective agent for retinal diseases associated with glutamate-induced excitotoxicity.

Glucagon-like peptide-1 receptor agonists inhibit hepatic stellate cell activation by blocking the p38 MAPK signaling pathway.

We investigated the effects of glucagon-like peptide-1 receptor (GLP-1R) agonists on p38 mitogen-activated protein kinase (MAPK) signaling during inhibition of hepatic stellate cell (HSC) activity. Human HSCs were cultured and morphologically identified. HSC samples were collected and randomly divided into three groups (N = 20 samples per group): a control group treated with high glucose (final concentration 25 mM); a GLP-1R agonist group treated with liraglutide (final concentration 5 mM); and a p38-blocked group treated with the p38 MAPK inhibitor SB203580 (final concentration 14 μM). All cells were cultured for 120 h followed by detection of phosphorylated p38 MAPK (p-p38 MAPK) and α-smooth muscle actin (α-SMA, a measure of HSC activation) by western blot. p-p38 MAPK and α-SMA expression levels were both significantly lower in HSCs in the GLP-1R agonist and p38-blocked groups compared with the control group (all P < 0.01). GLP-1R agonists may inhibit the activation of HSCs by blocking the p38 MAPK signaling pathway.

Rosuvastatin Attenuates Atrial Structural Remodelling in Rats with Myocardial Infarction through the Inhibition of the p38 MAPK Signalling Pathway

The purpose of this study was to verify the hypothesis that rosuvastatin attenuates atrial structural remodelling in rats with myocardial infarction (MI) through the regulation of the p38 mitogen-activated protein kinase (MAPK) signalling pathway. A total of 66 rats were used in this study to establish a model of MI. The 56 rats that survived the first 24h after surgery were randomly divided into four groups: the control group (C group), the rosuvastatin group (R group), the low-dose torasemide group (T1 group), and the high-dose torasemide group (T2 group). The four groups of rats received daily intragastric administration of normal saline, rosuvastatin, or torasemide (T1: 1mg/kg body weight; T2: 2mg/kg body weight) for a total of four weeks. The rats in the sham-operated group (n=14) also received daily intragastric administration of normal saline for four weeks. After four weeks of intervention, the left ventricular end-diastolic pressure (LVEDP) was measured in all groups of rats by haemodynamic methods. The rats were then sacrificed, and the left atrial tissues were collected. The collagen volume fractions (CVFs) in the left atrial tissues were determined using Masson's trichrome staining. The expression of phosphorylated p38 (P-p38) MAPK in the left atrial tissues was examined by immunohistochemistry and western blot analysis. The results showed that LVEDP, CVF, and P-p38 MAPK expression were drastically elevated in the four MI groups in comparison to the sham-operated group (p<0.001). Rosuvastatin elevated the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF). Both rosuvastatin and torasemide improved the haemodynamic parameters. No significant difference was detected in LVEDP between the R group and the T1 group (p=0.37). In contrast, LVEDP was significantly higher in the R group than in the T2 group (p <0.05). CVF (%) was markedly decreased in the R group compared to the C, T1, and T2 groups (decreased by 47.4%, 28%, and 20.1%, respectively). Immunohistochemical analysis showed that the indices of P-p38 MAPK positive cells were significantly decreased in the R group in comparison with the C, T1, and T2 groups (decreased by 44.6%, 36.6%, and 21.4%, respectively). Western blot analysis demonstrated that P-p38 MAPK expression was markedly reduced in the R group compared with the C and T1 groups (reduced by 67% and 40.5%, respectively). The level of P-p38 MAPK in the R group was slightly higher than in the T2 group. However, the difference was not statistically significant (p>0.05). Rosuvastatin attenuates atrial structural remodelling in rats with MI. The mechanism underlying this phenomenon may be associated with the downregulation of P-p38 MAPK by rosuvastatin.

The role of p38 MAPK in acute paraquat-induced lung injury in rats

Context: Paraquat (PQ; 1,1'-dimethyl-4,4'-bipyridinium dichloride) is highly toxic and accounts for a large proportion of the herbicide poisonings seen in clinic. The major cause of mortality is respiratory failure. The p38 mitogen-activated protein kinase (MAPK) signal transduction pathway coordinates various cellular stress responses that have been shown to participate in the pathogenesis of PQ-induced lung injury.Objective: To evaluate the effect of the specific p38 MAPK inhibitor SB203580 on PQ-induced lung injury and cytokine secretion.Methods: In groups of 24, rats were treated with PQ, PQ and SB203580 (SB + PQ), SB203580 alone (SB) or normal saline (control group). Six rats from each group were euthanized at 1, 3, 5 or 7 d. Pathology of lung specimens was scored through hematoxylin and eosin staining. Edema in the lung was quantified from wet-to-dry weight ratios. p38 and p-p38MAPK proteins were measured via electrochemiluminescent Western blots. tumor necrosis factor (TNF)-alpha and interleukin-1 beta (IL-1β) concentrations in lung specimens and bronchoalveolar lavage fluid (BALF) were quantified via enzyme-linked immunosorbent assay.Results: The mortality rate of the SB + PQ group (16.7%) was significantly lower than that of the PQ group (33.3%; p < 0.05). The PQ group had significantly higher pulmonary histology scores, wet-to-dry weight ratios and phosphorylated p-p38 MAPK levels, as well as higher IL-1β and TNF-alpha levels in BALF and lung tissues, that did the SB + PQ and control groups (p < 0.05, all).Conclusion: The data suggest that the p38 MAPK signaling pathway has an important role in regulating the production of IL-1β and TNF-alpha in PQ-induced lung injury in rats.

Quercetin enhances adriamycin cytotoxicity through induction of apoptosis and regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling in multidrug-resistant leukemia K562 cells.

Multidrug resistance (MDR) has become a significant challenge in chemotherapeutic treatment of cancer. Quercetin, a naturally occurring flavonoid, has been found to possess anti-proliferative, anti-inflammatory and immunoregulatory bioactivities. The present study was performed to examine the effect of quercetin on human leukemic MDR K562/adriamycin (ADR) cells. Treatment of K562/ADR cells with a combination of quercetin and ADR resulted in potentiation of cytotoxicity, which was measured using a cell counting kit-8 assay. Flow cytometric analysis revealed that quercetin dose-dependently promoted cell apoptosis and treatment with a combination of quercetin and ADR caused synergistic enhancement of the apoptotic effect. In addition, treatment of K562/ADR cells with quercetin alone or in combination with ADR resulted in loss of mitochondrial membrane potential, activation of caspase-8, -9 and -3, reduced expression of the anti-apoptotic proteins B-cell lymphoma (Bcl)-2 and Bcl-extra large and enhanced expression of the pro-apoptotic proteins Bcl-2-interacting mediator of cell death, Bcl-2-associated death promoter and Bcl-2-associated X protein in the cells. Furthermore, the combined treatment of quercetin and ADR synergistically increased the expression of phosphorylated (p-)c-Jun N-terminal kinase and p-p38 mitogen-activated protein kinase and decreased the expression of p-extracellular signal-regulated kinase in the K562/ADR cells. In addition, the expression of P-glycoprotein was significantly decreased following treatment with quercetin alone or in combination with ADR. These findings demonstrated that quercetin is important in MDR and may be developed into a new reversal agent for cancer chemotherapy.

Effects of soothing liver and invigorating spleen recipes on LPS-induced hepatocytes injury of rats and TLR4/p38MAPK signal pathway

To explore the effects of soothing liver and invigorating spleen recipes on lipopolysaccharide(LPS) induced hepatocyte inflammation of rats and TLR4/p38MAPK signal pathway. The hepatocytes of SD rats were cultured and identified in vitro. The medicated serum of soothing liver and invigorating spleen recipes was prepared. The hepatocytes were treated with soothing liver and invigorating spleen recipes. Then Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression in cultural supernatants were assayed by ELISA. The expressions of Toll-Like 4 (TLR4), p38 mitogen activated protein kinases (p38MAPK) and p-p38 mitogen-activated protein kinase (p-p38MAPK) were detected by Western blot. The rat medicated serum of soothing liver and invigorating spleen recipes was extracted for 2-3 mL. The purified rat hepatocytes were 1.5 x 10(8)-2.0 x 10(8). The cell viability was above 95% detected by Typan blue staining. The hepatocytes were identified by immumofluorescence assay. The detection of hepatocyte cultural supernatants: compared with that of the control group, IL-6 and TNF-α expression were increased in the LPS group (P < 0.01). While compared with that of the LPS group, the expressions of IL-6 and TNF-α were decreased after soothing liver and invigorating spleen recipes intervention (P < 0.01). The detection of hepatocyte proteins: compared with that of the control group, the protein expressions of p38MAPK, p-p38MAPK and TLR4 were all increased significantly in the LPS group (P < 0.01). Compared with that of the LPS group, the protein expressions of p38MAPK was decreased significantly in SB239063 group and it was also decreased in the soothing liver and invigorating spleen recipes group, but with no significant difference. Compared with that of the LPS group, p38MAPK expression was reduced significantly in the soothing liver and invigorating spleen recipes group and the SB239063 (p38MAPK pathway inhibitor) group (P < 0.01). TLR4 protein expression was decreased markedly in the soothing liver and invigorating spleen recipes group (P < 0.01) but had no difference between the SB239063 group and the LPS group. The soothing liver and invigorating spleen recipes may regulate hepatocyte inflammatory injury of rats through TLR4/p38MAPK signaling pathway.

Neuroprotective effects of bilobalide on cerebral ischemia and reperfusion injury are associated with inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation.

BackgroundMitogen-activated protein kinase (MAPK) signaling pathways are implicated in inflammatory and apoptotic processes of cerebral ischemia and reperfusion (I/R) injury. Hence, MAPK pathways represent a promising therapeutic target. Exploring the full potential of inhibitors of MAPK pathways is a useful therapeutic strategy for ischemic stroke. Bilobalide, a predominant sesquiterpene trilactone constituent of Ginkgo biloba leaves, has been shown to exert powerful neuroprotective properties, which are closely related to both anti-inflammatory and anti-apoptotic pathways. We investigated the neuroprotective roles of bilobalide in the models of middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen-glucose deprivation and reoxygenation (OGD/R) of cerebral I/R injury. Moreover, we attempted to confirm the hypothesis that its protection effect is via modulation of pro-inflammatory mediators and MAPK pathways.MethodsMale Sprague-Dawley rats were subjected to MCAO for 2 h followed by reperfusion for 24 h. Bilobalide was administered intraperitoneally 60 min before induction of middle cerebral artery occlusion (MCAO). After reperfusion, neurological deficit scores, infarct volume, infarct weight, and brain edema were assessed. Ischemic penumbrae of the cerebral cortex were harvested to determine superoxide dismutase (SOD), malondialdehyde (MDA), nitric oxide, TNF-α, interleukin 1β (IL-1β), p-ERK1/2, p-JNK1/2, and p-p38 MAPK concentration. Similarly, the influence of bilobalide on the expression of nitric oxide, TNF-α, IL-1β, p-ERK1/2, p-JNK1/2, and p-p38 MAPK was also observed in an OGD/R in vitro model of I/R injury.ResultsPretreatment with bilobalide (5, 10 mg/kg) significantly decreased neurological deficit scores, infarct volume, infarct weight, brain edema, and concentrations of MDA, nitric oxide, TNF-α, IL-1β, and increased SOD activity. Furthermore, bilobalide (5, 10 mg/kg) pretreatment significantly down-regulated both p-JNK1/2 and p-p38 MAPK expression, whereas they had no effect on p-ERK1/2 expression in the ischemic penumbra. Supporting these observations in vivo, pretreatment with bilobalide (50, 100 μM) significantly down-regulated nitric oxide, TNF-α, IL-1β, p-JNK1/2, and p-p38 MAPK expression, but did not change p-ERK1/2 expression in rat cortical neurons after OGD/R injury.ConclusionsThese data indicate that the neuroprotective effects of bilobalide on cerebral I/R injury are associated with its inhibition of pro-inflammatory mediator production and down-regulation of JNK1/2 and p38 MAPK activation.

Puerarin induces the upregulation of glutathione levels and nuclear translocation of Nrf2 through PI3K/Akt/GSK-3β signaling events in PC12 cells exposed to lead

Oxidative stress is thought to be involved in lead-induced toxicity, especially affecting the brain. We reported previously that puerarin possesses antioxidative properties in the nervous system. Therefore, the aim of the present study was to test the hypothesis that puerarin inhibits lead acetate-induced oxidative stress in PC12 cells by interrupting phosphatidylinositol-3 kinase (PI3K)/Akt signaling through increasing glutathione (GSH) synthesis. Our results showed that puerarin attenuates oxidative stress in a concentration-dependent manner in PC12 cells exposed to lead acetate demonstrated by scavenging reactive oxygen species (ROS) and reducing lipid peroxidation (LPO). Treatment with puerarin significantly up-regulates glutamate cysteine ligase catalytic subunit (GCLc) expression both at its mRNA and protein levels, but not glutamate cysteine ligase modifier (GCLm) subunit, accompanying the elevation of cellular glutathione level. The increased nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) was not because of increased transcription of Nrf2 as Nrf2 transcript levels did not change after puerarin treatment. The effects of puerarin could be partially blocked by pharmacologic inhibition of PI3K and the glycogen synthase kinase 3β (GSK-3β) pathways with LY294002 and LiCl, respectively. On the other hand, puerarin treatment promoted Akt and GSK-3β phosphorylation in PC12 cells exposed to lead acetate. Moreover, puerarin failed to modulate the phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), p-c-Jun N-terminal kinases (JNK), and p-p38 mitogen-activated protein kinase (MAPK) demonstrating some specificity for its action on the PI3K/GSK-3β pathway. These findings suggest that puerarin as a phytoestrogen might be an attractive agent for prevention and treatment of chronic diseases related to lead neurotoxicity.

Attenuation of neuropathic pain by saikosaponin a in a rat model of chronic constriction injury.

Despite immense advances in the treatment strategies, the effective treatment of patients suffering from neuropathic pain remains challenging. Saikosaponin a possesses anti-inflammatory activity. However, the role of saikosaponin a in neuropathic pain is still unclear. Therefore, the objective of this study was to investigate the effects of saikosaponin a on neuropathic pain. Neuropathic pain was induced by chronic constriction injury (CCI) of the sciatic nerve in rats. After CCI, rats were administered saikosaponin a (6.25, 12.50 and 25.00 mg/kg intraperitoneal, once daily) for 14 days. Mechanical withdrawal threshold and thermal withdrawal latency were assessed before surgery and on days 1, 3, 7, and 14 after CCI. Our results showed that CCI significantly decreased mechanical withdrawal threshold and thermal withdrawal latency on days 1, 3, 7 and 14, as compared with sham groups, however, saikosaponin a reversed this effects. In addition, saikosaponin a inhibited CCI-induced the levels of TNF-α, IL-1β, IL-2 in spinal cord. Western blot analysis demonstrated that saikosaponin a reduced the elevated expression of p-p38 mitogen-activated protein kinase (MAPK) and NF-κB in the spinal cord induced by CCI. These results suggest that saikosaponin a could effectively attenuate neuropathic pain in CCI rats by inhibiting the activation of p38 MAPK and NF-κB signaling pathways in spinal cord.

Inhibition of p38 mitogen-activated protein kinase activation in the rostral anterior cingulate cortex attenuates pain-related negative emotion in rats.

The emotional components of pain are far less studied than the sensory components. Previous studies have indicated that the rostral anterior cingulate cortex (rACC) is implicated in the affective response to noxious stimuli. Activation of p38 mitogen-activated protein kinase (MAPK) in the spinal cord has been documented to play an important role in diverse kinds of pathological pain states. We used formalin-induced conditioned place aversion (F-CPA) in rats, an animal model believed to reflect the emotional response to pain, to investigate the involvement of p38 MAPK in the rACC after the induction of affective pain. Intraplantar formalin injection produced a significant activation of p38 MAPK, as well as mitogen-activated kinase kinase (MKK) 3 and MKK6, its upstream activators, in the bilateral rACC. p38 MAPK was elevated in both NeuN-positive neurons and Iba1-positive microglia in the rACC, but not GFAP-positive cells. Blocking p38 MAPK activation in the bilateral rACC using its specific inhibitor SB203580 or SB239063 dose-dependently suppressed the formation of F-CPA. Inhibiting p38 MAPK activation did not affect formalin-induced two-phase spontaneous nociceptive response and low intensity electric foot-shock induced CPA. The present study demonstrated that p38 MAPK signaling pathway in the rACC contributes to pain-related negative emotion. Thus, a new pharmacological strategy targeted at the p38 MAPK cascade may be useful in treating pain-related emotional disorders.