Expression Of P-p38 Mitogen-activated Protein Kinase Research Articles

Mechanism of Mailuo Shutong Pills on posterior limb muscle swelling caused by femoral fracture in rats based on network pharmacology and experimental verification

This study aims to investigate the mechanism of Mailuo Shutong Pills(MLST) on posterior limb muscle swelling caused by femoral fracture(SCFF) through network pharmacology and animal experiments. The plasma components of MLST were analyzed by LC-MS, and the target and signal pathway of SCFF were predicted by network pharmacology and verified by molecular docking. SCFF model rats were established through animal experiments, and different doses of MLST were administered to detect the degree of limb swelling. Hematoxylin-eosin(HE) staining was used to observe pathological changes in muscle tissue, and interleukin-6(IL-6), interleukin-1β(interleukin-1β), and tumor necrosis factor-α(TNF-α) in peripheral blood were determined by enzyme-linked immunosorbent assay(ELISA). The expression of relevant signaling pathways was measured by Western blot. Network pharmacological results showed that MLST and SCFF had a total of 153 disease targets, and the key targets were IL-6, TNF, etc., involving mitogen-activated protein kinase(MAPK) signaling pathway, phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT) signaling pathway, etc. The binding energies of the main components and key targets were lower than-7.0 kcal·mol~(-1), indicating that the network analysis results were reliable. The results of animal experiments showed that MLST could reduce the swelling degree and pathological damage of the posterior limb muscles of SCFF rats compared with the model group. ELISA results showed that MLST could reduce the levels of IL-6, IL-1β, and TNF-α in the serum of SCFF rats. Western blot results showed that MLST can reduce the expression of p-AKT, p-PI3K, p-NF-κB, p-p38 MAPK, and p-ERK in SCFF rats. MLST may reduce the content of inflammatory factors in serum by regulating the expression of PI3K/AKT and MAPK-related signaling pathway protein and improving posterior limb muscle SCFF in rats.

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

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

HET0016 inhibits neuronal pyroptosis in the immature brain post-TBI via the p38 MAPK signaling pathway

Traumatic brain injury (TBI) is a serious health threat worldwide, especially for the younger demographic. Our previous study demonstrated that HET0016 (a specific inhibitor of 20-hydroxyeicosatetraenoic acid synthesis) can decrease the lesion volume in the immature brain post-TBI; however, its mechanism of action and its association with pyroptosis post-TBI are unclear. In this study, we established a controlled cortical impact (CCI) injury rat model (postnatal day 9–10) and observed that increased expression of indicators for pyroptosis, including NLR family pyrin domain containing 3 (NLRP3), caspase-1 and gasdermin D (GSDMD) proteins and interleukin (IL)-18/IL-1β mRNA during the acute phase of TBI, especially on post-injury day (PID) 1. Additionally, we found that caspase-1 was primarily expressed in the neurons and microglia. HET0016 (1 mg/kg/d, ip, 3 consecutive days since TBI) reduced the lesion volume; neuronal death; expression of NLRP3, caspase-1, and GSDMD; and expression of IL-18/IL-1β mRNA. Bioinformatics analysis suggested involvement of mitogen-activated protein kinase (MAPK) signaling pathway in the HET0016-mediated neuroprotective role against TBI in the immature brain. Western blot analysis revealed reduced expression of p-p38 MAPK and nuclear factor-kappa B (NF-κB) p65 in the neurons and microglia upon HET0016 treatment in TBI rats. In cultured primary cortical neurons subjected to oxygen-glucose deprivation/re-oxygenation (OGD) + (lipopolysaccharide) LPS, HET0016-induced the reduction of p-p38 MAPK, NLRP3, cleaved-caspase-1, GSDMD, IL-18, and IL-1β was reversed by co-treatment with p38 MAPK activator as well as NLRP3 agonist. Therefore, we conclude that pyroptosis is involved in neuronal death in the immature brains post-TBI and that HET0016 administration can alleviate neuronal pyroptosis possibly via inhibiting the phosphorylation of p38 MAPK.

Interactive effects of temperature and salinity on the apoptosis, antioxidant enzymes, and MAPK signaling pathway of juvenile pufferfish (Takifugu fasciatus)

Takifugu fasciatus is an anadromous fish widely distributed in East Asia and often experiences fluctuations in temperature and salinity. This study investigated the effects of the interactions between temperature and salinity on apoptosis, antioxidant enzymes, and mitogen-activated protein kinase (MAPK) signaling pathways in the liver of T. fasciatus. The temperature (25, 21, 17, and 13 ℃) and salinity (0, 10, and 20 ppt salinity) were conducted with three replicates for each treatment. Quantitative reverse transcription-PCR (qRT-PCR), western blotting, and TdT-mediated dUTP Nick-End Labeling (Tunel) assays were used to detect mRNA expression, protein levels, and apoptosis levels. The expressions of caspase 3, 7, 9, and the apoptosis index increased in T. fasciatus as the temperature decreased, while the expression of B-cell lymphoma-2 (BCL-2) decreased. The correlation results in this study showed that after the exposure to 10 ppt salinity at 13 and 17 ℃, the glutathione peroxidase (GSH-PX) activity and malondialdehyde (MDA) concentration, caspase3, 7, and 9 expressions, and apoptosis index were decreased as compared with those in the control. In addition, there were no deaths and no significant change in apoptosis in the group at 21 ℃, and there were a small number of deaths in the 20 ppt, 25 ℃ groups. Correspondingly, MDA concentration and total superoxide dismutase (T-SOD) activity, caspase7 and 9 expressions, and apoptosis index in the liver were significantly higher in the 20 ppt, 25 ℃ groups than in the 0 ppt, 25 ℃, and 10 ppt, 25 ℃ groups. In addition, salinity had no significant influence on the expression of p-p38 MAPK and its phosphorylation ratio (p-p38/p38) at low temperatures but significantly affected its downstream transcription factors (ATF2, ElK-1, MEF2, and P53). Our study provides a reference for healthy cultures of T. fasciatus.

Possible Implication of Nrf2, PPAR-γ and MAPKs Signaling in the Protective Role of Mangiferin against Renal Ischemia/Reperfusion in Rats.

Mangiferin (Mang) is a known glucosylxanthone that has proven its shielding effect against ischemia/reperfusion (Is/R). However, its full underlying mechanistic perspective against renal Is/R induced lesions is not fully revealed. Consequently, the purpose of this study is to track further non-investigated modulatory signals of Mang against the renal Is/R model involving nuclear factor erythroid 2-related factor (Nrf)2/heme oxygenase (HO)-1, peroxisome proliferator-activated receptor (PPAR)-γ/nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) signaling. To ratify our aim, Mang was administrated (20 mg/kg, i.p for seven days) before the induction of bilateral Is/R. Mechanistic maneuver revealed that Mang balanced oxidative state via increasing the expression of the antioxidant Nrf2/HO-1 cue with subsequent enhancement of GSH besides MDA lessening. Additionally, Mang enhanced PPAR-γ mRNA expression and declined p-p38 MAPK and p-JNK expression with concomitant NF-κB downsizing leading to iNOS/NOx and TNF-α rebating. Furthermore, the Mang anti-apoptotic trait was affirmed by enriching Bcl-2 expression as well as decreasing Bax and caspase-3 expression. All these potentials were in the line with the molecular docking results and the improved histopathological findings and renal function biomarkers. Consequently, Mang provided plausible protective mechanisms against renal Is/R-related events, possibly by amending oxidative status, inflammatory mediators, and apoptotic cell death through the involvement of Nrf2, PPAR-γ, MAPK, JNK, and NF-κB signaling.

Neuronal Nitric Oxide Synthase Suppression Confers the Prolonged Analgesic Effect of Sciatic Nerve Block With Perineural Dexamethasone in Postoperative Pain Model Mice

Dexamethasone supplementation to local anesthetics prolongs its action, yet the underlying mechanism is unclear. Previous studies have reported that increased p-p38 mitogen-activated protein kinase (MAPK) in the dorsal root ganglia (DRG) is associated with pain-associated behavior and that nitric oxide (NO), which is known to be a pronociceptive substance, directly inhibits sciatic nerve conduction. Here, we investigated the temporal changes in the hyperalgesic effect and p-p38 MAPK and NO synthase (NOS) expression levels in the DRG when dexamethasone was added to ropivacaine used for a sciatic nerve block (SNB) in postoperative pain model mice. Dexamethasone supplementation to ropivacaine significantly prolonged the analgesic effect of SNB via glucocorticoid receptor activation. Histological examination revealed that ropivacaine suppressed p-p38 MAPK expression in the DRG regardless of dexamethasone supplementation, suggesting that p-p38 MAPK was not involved in the prolonging effect of dexamethasone on nerve block. Contrastingly, plantar incision markedly increased the expression of neuronal NOS (nNOS) in DRG, and dexamethasone supplementation to ropivacaine significantly suppressed nNOS expression. Supplementation of L-NAME, an inhibitor of NOS, to ropivacaine markedly prolonged the effect of SNB, similar to dexamethasone. These results suggest that dexamethasone supplementation to local anesthetics prolongs the analgesic effect by inhibiting nNOS activity. PerspectiveThe current study revealed that dexamethasone supplementation to local anesthetics prolongs the analgesic effect by inhibiting the activity of neuronal NOS and that p-p38 MAPK may not be involved in this phenomenon. Our findings offer a new target for the discovery of long-acting local anesthetics.

Lack of STAT6 enhances murine acute lung injury through NLRP3/p38 MAPK signaling pathway in macrophages

BackgroundSignal transducer and activator of transcription 6 (STAT6) is an intracelluar transcriotion factor and NLRP3 (Nod-like receptor containing a pyrin domain 3) is a component of NLRP3 inflammasome in pyroptotic cells. There was increased activation of STAT6 and expression of NLRP3 in mice with murine acute lung injury (ALI). However, it is unknown their roles in the development of murine ALI. We in this study, investigated the effects of STAT6 signaling on murine ALI and pyroptosis in STAT6 knock-out (KO) mice and macrophages.ResultsSTAT6 was activated in the lung tissues of mice 2 days after intratracheal treatmemt with 5 mg/kg LPS. Lack of STAT6 expression in KO mice induced more severe lung inflammation, associated with elevated neutrophil influx and expression of TNF-alpha, IL-6 and IL-1beta in the inflamed lung tissues. In addition, the expression of NLRP3, ASC (apoptosis-associated speck-like protein containing a CARD), p-p38 MAPK (p38 mitogen-activated protein kinase) and ratio of LC3-II/I (microtubule-associated protein-1 light chain-3) was increased, accompanied with the increased polarization of Siglec-F(−) subtype macrophages in KO mice with ALI. Further studies in bone marrow-derived macrophages (BMDMs) revealed that lack of STAT6 increased the expression of NLRP3 and p-p38 MAPK, in association with elevated expression of TNF-alpha, IL-1beta and Calreticulin in LPS-treated KO BMDMs.ConclusionsLack of STAT6 exacerbated murine ALI through improving the expression of NLRP3 and activation of p38 MAPK in macrophages. STAT6 has an immune suppressive role in the development of ALI and would be a promising therapeutic target in the treatment of ALI and possibly among patients with acute respiratory distress syndrome (ARDS).

Hydroxychavicol, a polyphenol from Piper betle leaf extract, induces cell cycle arrest and apoptosis in TP53-resistant HT-29 colon cancer cells.

This study aims to elucidate the antiproliferative mechanism of hydroxychavicol (HC). Its effects on cell cycle, apoptosis, and the expression of c-Jun N-terminal kinase (JNK) and P38 mitogen-activated protein kinase (MAPK) in HT-29 colon cancer cells were investigated. HC was isolated from Piper betle leaf (PBL) and verified by high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and gas chromatography-mass spectrometry (GC-MS). The cytotoxic effects of the standard drug 5-fluorouracil (5-FU), PBL water extract, and HC on HT-29 cells were measured after 24, 48, and 72 h of treatment. Cell cycle and apoptosis modulation by 5-FU and HC treatments were investigated up to 30 h. Changes in phosphorylated JNK (pJNK) and P38 (pP38) MAPK expression were observed up to 18 h. The half maximal inhibitory concentration (IC50) values of HC (30 μg/mL) and PBL water extract (380 μg/mL) were achieved at 24 h, whereas the IC50 of 5-FU (50 μmol/L) was obtained at 72 h. Cell cycle arrest at the G0/G1 phase in HC-treated cells was observed from 12 h onwards. Higher apoptotic cell death in HC-treated cells compared to 5-FU-treated cells (P<0.05) was observed. High expression of pJNK and pP38 MAPK was observed at 12 h in HC-treated cells, but not in 5-FU-treated HT-29 cells (P<0.05). It is concluded that HC induces cell cycle arrest and apoptosis of HT-29 cells, with these actions possibly mediated by JNK and P38 MAPK.

Grape Seed Proanthocyanidins Induce Apoptosis and Cell Cycle Arrest of HepG2 Cells Accompanied by Induction of the MAPK Pathway and NAG-1.

Hepatocellular carcinoma (HCC) is one of the common malignancies leading to death. Although radiotherapy and chemotherapy have certain effects, their side effects limit their therapeutic effect. Phytochemicals have recently been given more attention as promising resources for cancer chemoprevention or chemotherapy due to their safety. In this study, the effects of grape seed proanthocyanidins (GSPs) on the apoptosis, cell cycle, and mitogen-activated protein kinase (MAPK) pathway-related proteins and non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) expression of HepG2 cells were investigated. The results showed that GSPs inhibited the viability of HepG2 cells in a time- and dose-dependent manner, induced apoptosis and G2/M phase cell cycle arrest, and regulated cell cycle-related proteins, cyclin B1, cyclin-dependent kinase 1, and p21. GSPs also increased reactive oxygen species production and caspase-3 activity. In addition, GSPs also increased the expression of p-ERK, p-JNK, p-p38 MAPK and NAG-1, and GSPs-induced NAG-1 expression was related to the MAPK pathway-related proteins. These data suggest that GSPs may be promising phytochemicals for HCC chemoprevention or chemotherapy.

Efficacy of Bunao Fuyuan decoction on cerebral ischemia and reperfusion injury in vitro.

To investigate the protective efficacy of Bunao Fuyuan decoction (BNFY) on cerebral Ischemia/reperfusion (I/R) injury. The mouse PC12 cells were chosen, and the oxidative-glucose deprivation/re-oxygenation (OGD/R) injury model were established to simulate cerebral I/R injury. Atorvastatin was selected as a positive drug, and a gradient dose of BNFY was given for 6, 12 and 24 h. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay were used to detect cell viability at each time point. Cell apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP-botin nick end labeling (TUNEL) staining. enzyme linked immunosorbent assay was used to detect the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1β and platelet activating factor (PAF). Western blot assay were performed to detect the expression of key regulators [toll-like receptor 4 (TLR4), nuclear factor kappa-B (NF-κB), p-p38 mitogen-activated protein kinase (MAPK) and p-Akt (also known as protein kinase B, PKB)] of cell survival and inflammatory response. The results of MTT assay and TUNEL staining assay revealed that BNFY significantly increased cell viability and inhibited cell apoptosis of PC12 cells following OGD/R, respectively. Furthermore, the expression of TNF-α, 1L-6, 1L-1 and PAF were decreased after BNFY treatment. And the results of Western blot assay showed that BNFY downregulated TLR4, NF-κB, p-p38 MAPK expression and upregulated p-Akt expression. Our findings suggest that BNFY may play a role in protecting OGD/R injured PC12 cells through inhibiting the inflammatory response and cell apoptosis.

Characterisation of a rat model of mechanical low back pain at an advanced stage using immunohistochemical methods.

Chronic low back pain (LBP) has high prevalence in the adult population which is associated with enormous disability. Hence, our aim was to further characterise our LBP rat model by using immunohistological and immunohistochemical methods at an advanced stage (day 49) of the model. Male Sprague-Dawley rats were anaesthetised and their lumbar L4/L5 and L5/L6 intervertebral discs (IVDs) were punctured (0.5mm outer diameter, 2mm-deep) 10 times per disc. Sham-rats underwent similar surgery, but no discs were punctured. For LBP- but not sham-rats, noxious pressure hyperalgesia was fully developed in the lumbar axial deep tissues on day 21 post-surgery, which was maintained until at least day 49. In the lumbar (L4-L6) dorsal root ganglia (DRGs), somatostatin (SRIF) and the somatostatin receptor type 4 (SST4 receptor) were co-localised with substance P and IB4, markers of small diameter unmyelinated peptidergic and non-peptidergic C-fibres respectively as well as with NF200, a marker of medium to large diameter neurons. On day 49, there was increased expression of SRIF but not the somatostatin receptor type 4 (SST4 receptor) in the lumbar DRGs and the spinal dorsal horns. There were increased DRG expression levels of the putative pro-nociceptive mediators: phosphorylated p38 (pp38) mitogen-activated protein kinase (MAPK) and phosphorylated p44/p42 MAPK (pp44/pp42 MAPK) as well as pp38 MAPK expression levels in the lumbar spinal cord. Taken together, the increased expression of SRIF in the lumbar DRGs and spinal cord and its co-localisation with nociceptive fibres in DRG sections suggest a potential role of SRIF in modulating chronic mechanical LBP.

Antibacterial peptides inhibit MC3T3-E1 cells apoptosis induced by TNF-α through p38 MAPK pathway.

BackgroundAntimicrobial peptides (AMP), as a small molecular polypeptide with a broad antibacterial spectrum and high efficiency, have attracted more and more attention. Few pieces of research on the effect of the antimicrobial peptide on osteoblast under inflammatory conditions have so far been reported. The main aim of this work was to investigate the antiapoptosis effect of the antimicrobial peptide on MC3T3-E1 cells induced by TNF-α and its related mechanism.MethodsRat MC3T3-E1 cells were co-cultured with different concentrations of antibacterial peptide DP7 and TNF-α.MTS assay, cell scratch test, alkaline phosphatase activity, and alizarin red staining assay were used to determine osteoblast viability in this experiment. Annexin V-FITC/PI double staining cells and flow cytometry were used to analyze apoptosis and Western blot assay detection to show mitogen-activated protein kinase (MAPK) protein expression in rat MC3T3-E1 cells. Then, Realtime polymerase chain reaction (PCR) was used to examine the caspase-3 gene expression. Also, ELISA detection was used to clarify the anti-apoptotic effect of the p38 MAPK inhibitor, SB203580, on cells’ apoptosis.ResultsAntimicrobial peptide could promote the proliferation, migration, and osteogenic ability of MC3T3-E1 cells induced by TNF-α, but inhibit cell apoptosis rate (P<0.05), and the effect was concentration-dependent. Western blot results showed after TNF-αtreatment, the expression of p-p38 MAPK in the MC3T3-E1 cells increased after TNF-α and antimicrobial peptide cotreatment, TNF-α induced p-p38 MAPK phosphorylation was inhibited, and the difference was statistically significant (P<0.05). Realtime PCR results showed that the gene expression of caspase-3 mRNA was up-regulated after TNF-α treatment, while their expression was down-regulated after cultured with TNF-α and antimicrobial peptide. Elisa's analysis showed that cell apoptosis increased after TNF-α treatment alone, and cell apoptosis was reduced to the normal levels when combined with antimicrobial peptide, and cell apoptosis induced by TNF-α was partially abolished when combined with SB203580.ConclusionsAntimicrobial peptide DP7 could inhibit MC3T3-E1 cells apoptosis induced by TNF-α, and the effect was concentration-dependent. The antiapoptosis activation of the antimicrobial peptide on MC3TE-E1 cells may be related to the inhibition of the p38 MAPK pathway.

Tougu Xiaotong capsules may inhibit p38 MAPK pathway-mediated inflammation: In vivo and in vitro verification.

Tougu Xiaotong capsules (TXC) are an herbal compound commonly used to treat osteoarthritis (OA) in China. We attempted to verify TXC's therapeutic effects and mechanisms related to the p38 mitogen-activated protein kinase (MAPK) pathway in vivo and in vitro. TXC's therapeutic effects were assessed by observing cartilage degeneration and inflammatory factors in a modified Hulth's model (in vivo) and a lipopolysaccharides (LPS)-exposed cellular model (in vitro). The expression of biomarkers related to p38 MAPK pathway-mediated inflammation was also investigated. TXC treatment reversed cartilage degeneration related biomarkers (ADAMTS 4, ADAMTS 5, Col I, Col V, MMP 3, MMP 9, and MMP 13) and inflammation factors (IL-1β, TNF-α, and IL-6) in both the animal and cellular OA models. Expression of p-p38 MAPK was downregulated following TXC administration, and changes to microRNAs in the cellular models were recovered. These results indicated that the p38 MAPK pathway-related mechanism may involve therapeutic effects of TXC. This study verified TXC's efficacy to treat OA in vivo and in vitro and suggests that p38 MAPK pathway-related mechanisms may be involved in TXC's therapeutic effects.

IgG plasma cells initiate changes in the protein Csystem inmouse ulcerative colitis through CD14+CD64+ macrophage activation

Inhibition of the protein C system (PCS) might be one of the mechanisms of ulcerative colitis (UC). The aim of the study was to explore the role of IgG plasma cells in changes in the PCS in UC. Dextran sulfate sodium (DSS) was chosen to induce mouse UC. Inflammation was assessed using hematoxylin & eosin (H&E) staining and immunofluorescence. The profiling of colonic plasma cells and macrophages from colitis mice was analyzed with flow cytometry. After stimulation of macrophages with IgG type immune complex (IgG-IC), western blot was used to determine tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6) protein levels. After co-incubation of colonic mucosa microvascular endothelial cells (MVECs) with TNF-α or IL-6, mitogen-activated protein kinase (MAPK) expression was detected. The DSS-colitis mice showed higher inflammatory indexes (p < 0.05 or p < 0.01), accompanied by greater infiltration of CD38+IgG+ plasma cells (p < 0.01), CD14+CD64+ macrophages (p < 0.01) and IgG-IC than healthy mice. Enhancement of TNF-α and IL-6 protein expression was demonstrated in this subset of macrophages when stimulated by IgG-IC (p < 0.01). After MVECs were incubated with TNF-α or IL-6, the expression of β-arrestin1, pP38 MAPK and pJNK MAPK exhibited an increase (p < 0.05 or p < 0.01), but downregulation of endothelial protein C receptor (EPCR) expression was observed (p < 0.05 or p < 0.01); this inhibition of EPCR expression was reversed by SB203580, SP600125 or U0126 (p < 0.05 or p < 0.01). In addition, changes in activated protein C (APC) presented results similar to those for EPCR expression (p < 0.05 or p < 0.01). These results reveal that the PCS is inhibited during UC processing. There is a possibility that the interaction between IgG plasma cells and CD14+CD64+ macrophages, as well as further secretion of cytokines from CD14+CD64+ macrophages by the formation and stimulation of IgG-IC, subsequently influence MVECs through the β-arrestin-MAPK pathway. Enhancement of PCS activity may represent a novel approach for treating UC.

Role of M3 receptor in penehyclidine hydrochloride-induced reduction of increased permeability of human pulmonary microvascular endothelial cells caused by endotoxin: the relationship with MAPK signaling pathway

Objective To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells(PMVECs)caused by endotoxin and the relationship with mitogen-activated protein kinase(MAPK)signaling pathway. Methods Human PMVECs were seeded in 6-well plates(2 ml/hole)or in culture flasks(4 ml/flask)at the density of 1×105 cells/ml and randomly divided into 6 groups(n=5 each): control group(group C), M3 receptor shRNA transfection group(group shRNA), lipopolysaccharide(LPS)group, penehyclidine plus LPS group(group P+ LPS), LPS plus M3 receptor shRNA transfection group(group LPS+ shRNA)and PHC plus LPS plus M3 shRNA transfection group(group P+ LPS+ shRNA). The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA, LPS+ shRNA and P+ LPS+ shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+ shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation, cells were incubated for 1 h, then LPS at the final concentration of 0.1 μg/ml was added, and cells were incubated for another 1 h in P+ LPS and P+ LPS+ shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK(p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2(p-ERK1/2)was detected by Western blot, the expression of heat shock protein 27(HSP27)using immunofluorescent staining, and the expression of M3receptor mRNA by real-time polymerase chain reaction. Results Compared with group C, M3 receptor mRNA expression was significantly down-regulated in group shRNA, and the permeability of cells was significantly increased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was up-regulated in group LPS(P<0.05). The permeability of cells was significantly decreased, and the expression of p-p38 MAPK, p-ERK1/2, HSP27 and M3 receptor mRNA was down-regulated in P+ LPS, LPS+ shRNA and P+ LPS+ shRNA groups as compared with group LPS, and in group P+ LPS+ shRNA as compared with group LPS+ shRNA(P<0.05). Conclusion The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor. Key words: Receptor, muscarinic M3; Cholinergic agent; Endothelial cells; Blood capillary; Lung; Endoxemia; p38 MAPKs; Extracellular Signal-Regulated MAP Kinases

TMPYP4 exerted antitumor effects in human cervical cancer cells through activation of p38 mitogen-activated protein kinase

BackgroundThe aim of the present study was to investigate the potential effects of the 5,10,15,20-tetrakis (1-methylpyridinium-4-yl) porphyrin (TMPyP4) on the proliferation and apoptosis of human cervical cancer cells and the underlying mechanisms by which TMPyP4 exerted its actions.ResultsAfter human cervical cancer cells were treated with different doses of TMPyP4, cell viability was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) method, the apoptosis was observed by flow cytometry (FCM), and the expression of p38 mitogen-activated protein kinase (MAPK), phosphated p38 MAPK (p-p38 MAPK), capase-3, MAPKAPK2 (MK-2) and poly ADP-ribose polymerase (PARP) was measured by Western blot analysis. The analysis revealed that TMPyP4 potently suppressed cell viability and induced the apoptosis of human cervical cancer cells in a dose-dependent manner. In addition, the up-regulation of p-p38 MAPK expression levels was detected in TMPyP4-treated human cervical cancer cells. However, followed by the block of p38 MAPK signaling pathway using the inhibitor SB203580, the effects of TMPyP4 on proliferation and apoptosis of human cervical cancer cells were significantly changed.ConclusionsIt was indicated that TMPyP4-inhibited proliferation and -induced apoptosis in human cervical cancer cells was accompanied by activating the p38 MAPK signaling pathway. Taken together, our study demonstrates that TMPyP4 may represent a potential therapeutic method for the treatment of cervical carcinoma.

Notch1-MAPK Signaling Axis Regulates CD133+ Cancer Stem Cell-Mediated Melanoma Growth and Angiogenesis

Functional characterization and understanding of the intricate signaling mechanisms in stem-like cells is crucial for the development of effective therapies in melanoma. We have studied whether melanoma cells are phenotypically distinct and hierarchically organized according to their tumorigenic nature. We report that melanoma-specific CD133+ cancer stem cells exhibit increased tumor-initiating potential, tumor-endothelial cell interaction, and lung metastasis. These cells are able to transdifferentiate into an endothelial-like phenotype when cultured under endothelial differentiation-promoting conditions. Mechanistically, Notch1 upregulates mitogen-activated protein kinase activation through CD133, which ultimately controls vascular endothelial growth factor and matrix metalloproteinase expression in CD133+ stem cells leading to melanoma growth, angiogenesis, and lung metastasis. Blockade or genetic ablation of Notch1 and mitogen-activated protein kinase pathways abolishes melanoma cell migration and angiogenesis. Chromatin immunoprecipitation and reporter assays revealed that Notch1 intracellular domain regulates CD133 expression at the transcriptional level. Andrographolide inhibits Notch1 intracellular domain expression, Notch1 intracellular domain-dependent CD133-mediated mitogen-activated protein kinase and activator protein-1 activation, and epithelial to mesenchymal-specific gene expression, ultimately attenuating melanoma growth and lung metastasis. Human malignant melanoma specimen analyses revealed a strong correlation between Notch1 intracellular domain, CD133, and p-p38 mitogen-activated protein kinase expression and malignant melanoma progression. Thus, targeting Notch1 and its regulated signaling network may have potential therapeutic implications for the management of cancer stem cell-mediated melanoma progression.

Clonidine, an alpha-2 adrenoceptor agonist relieves mechanical allodynia in oxaliplatin-induced neuropathic mice; potentiation by spinal p38 MAPK inhibition without motor dysfunction and hypotension.

Cancer chemotherapy with platinum-based antineoplastic agents including oxaliplatin frequently results in a debilitating and painful peripheral neuropathy. We evaluated the antinociceptive effects of the alpha-2 adrenoceptor agonist, clonidine on oxaliplatin-induced neuropathic pain. Specifically, we determined if (i) the intraperitoneal (i.p.) injection of clonidine reduces mechanical allodynia in mice with an oxaliplatin-induced neuropathy and (ii) concurrent inhibition of p38 mitogen-activated protein kinase (MAPK) activity by the p38 MAPK inhibitor SB203580 enhances clonidine's antiallodynic effect. Clonidine (0.01-0.1 mg kg(-1), i.p.), with or without SB203580(1-10 nmol, intrathecal) was administered two weeks after oxaliplatin injection(10 mg kg(-1), i.p.) to mice. Mechanical withdrawal threshold, motor coordination and blood pressure were measured. Postmortem expression of p38 MAPK and ERK as well as their phosphorylated forms(p-p38 and p-ERK) were quantified 30 min or 4 hr after drug injection in the spinal cord dorsal horn of treated and control mice. Clonidine dose-dependently reduced oxaliplatin-induced mechanical allodynia and spinal p-p38 MAPK expression, but not p-ERK. At 0.1 mg kg(-1), clonidine also impaired motor coordination and decreased blood pressure. A 10 nmol dose of SB203580 alone significantly reduced mechanical allodynia and p-p38 MAPK expression, while a subeffective dose(3 nmol) potentiated the antiallodynic effect of 0.03 mg kg(-1) clonidine and reduced the increased p-p38 MAPK. Coadministration of SB203580 and 0.03 mg kg(-1) clonidine decreased allodynia similar to that of 0.10 mg kg(-1) clonidine, but without significant motor or vascular effects. These findings demonstrate that clonidine treatment reduces oxaliplatin-induced mechanical allodynia. The concurrent administration of SB203580 reduces the dosage requirements for clonidine, thereby alleviating allodynia without producing undesirable motor or cardiovascular effects.

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.

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.