Mechanism of Retinoic Acid and Mitogen-activated Protein Kinases Regulating Hyperoxia Lung Injury

To investigate the protective effect of retinoic acid （RA） on hyperoxic lung injury and the role of RA as a modulator on mitogen-activated protein kinases （MAPKs）, gastation 21 d Sprague- Dawley （SD） fetuses （term = 22 d） were delivered by hysterotomy. Within 12-24 h of birth, premature rat pups were randomly divided into 4 groups （n= 12 each） ： air-exposed control group （group Ⅰ ） ; hyperoxia-exposed group （ group Ⅱ ）, air-exposed plus RA group （group Ⅲ ）, hyperoxia-exposed plus RA group （group Ⅳ）. Group Ⅰ , Ⅲ were kept in room air, and group Ⅱ , Ⅳ were placed in 85 % oxygen. The pups in groups Ⅲ and Ⅳ were intraperitoneally injected with RA （500 μg/kg every day）. All lung tissues of premature rat pups were collected at the 4th day after birth. Terminal transferase d-UTP nick end labeling （TUNEL） staining was used for the detection of cell apoptosis. The expression of PCNA was immunohistochemically detected. Western blot analysis was employed for the determination of phosphorylated and total nonphosphorylated ERKs, JNKs or p38. Our results showed that lungs from the pups exposed to hyperoxia for 4 d exhibited TUNEL-positive nuclei increased markedly throughout the parenchyma （P〈0.01）, and decreased significantly after RA treatment （P〈0.01）. The index of PCNA-positive cells was significantly decreased （P〈0.01）, and was significantly increased by RA treatment （P〈0.01）. The air-space size was significantly enlarged, secondary crests were markedly decreased in hyperoxia-exposed animals. RA treatment improved lung air spaces and secondary crests in air-exposed pups, hut had no effect on hyperoxia-exposure pups. Western blotting showed that the amounts of JNK, p38 and ERK proteins in hyperoxia-exposure or RA-treated lung tissues were same as those in untreated lung tissues （P〈0.05）, whereas activation of these MAPKs was markedly altered by hyperoxia and RA. After hyperoxia exposure, p-ERK1/2, p-JNK1/2 and p-p38 were dramatically increased （P〈0.01）, whereas p-JNK1/2 and p-p38 were markedly declined and p-ERK1/2 was further elevated by RA treatment （P〈0.01）. It is concluded that RA could decrease cell apoptosis and stimulate cell proliferation under hyperoxic condition. The protection Of RA on hyperoxia-induced lung injury was related＇to the regulation of MAP kinase activation.

Arctigenin attenuates paraquat-induced human lung epithelial A549 cell injury by suppressing ROS/p38 mitogen-activated protein kinases-mediated apoptosis

BACKGROUND:Paraquat(PQ)-induced acute lung injury(ALI)and pulmonary fi brosis are common diseases with high mortality but without eff ective antidotes in emergency medicine.Our previous study has proved that arctigenin suppressed pulmonary fibrosis induced by PQ.We wondered whether arctigenin could also have a protective eff ect on PQ-induced ALI.METHODS:A PQ-induced A549 cell injury model was used,and the effect of arctigenin was determined by a cell counting kit-8(CCK-8)cell viability assay.In addition,terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labelling(TUNEL)staining assays and mitochondrial membrane potential assays were performed to evaluate the level of cell apoptosis.The generation of reactive oxygen species(ROS)was refl ected by dihydroethidium(DHE)staining and a 2’,7’-dichlorodihy drofluorescein diacetate(DCFH-DA)assay.Moreover,immunoblotting studies were used to assess the expression of mitogen-activated protein kinases(MAPKs)and p38 MAPK.RESULTS:Arctigenin attenuated PQ-induced inhibition of A549 cell viability in a dose-dependent manner.Arctigenin also significantly reduced PQ-induced A549 cell apoptosis,as refl ected by the TUNEL assay and mitochondrial membrane potential assay,which may result from suppressed ROS/p38 MAPK signaling because we found that arctigenin dramatically suppressed ROS generation and p38 MAPK phosphorylation.CONCLUSION:Arctigenin could attenuate PQ-induced lung epithelial A549 cell injury in vitro by suppressing ROS/p38 MAPK-mediated cell apoptosis,and arctigenin might be considered a potential candidate drug for PQ-induced ALI.

Retinoic Aacid Diminished the Expression of MMP-2 in Hyperoxia-exposed Premature Rat Lung Fibroblasts through Regulating Mitogen-activated Protein Kinases

This study examined the effects of retinoic acid （RA）, PD98059, SP600125 and SB203580 on the hyperoxia-induced expression and regulation of matrix metalloproteinase-2 （MMP-2） and metalloproteinase-2 （TIMP-2） in premature rat lung fibroblasts （LFs）. LFs were exposed to hyperoxia or room air for 12 h in the presence of RA and the kinase inhibitors PD98059 （ERK1/2）, SP600125 （JNK1/2） and SB203580 （p38） respectively. The expression levels of MMP-2 and TIMP-2 mRNA were detected by semi-quantitative reverse transcription polymerase chain reaction （RT-PCR）. MMP-2 activity was measured by zymography. The amount of p-ERK1/2, REK1/2, p-JNK1/2, JNK1/2, p-p38 and p38 was determined by Western blotting. The results showed that： （1） PD98059, SP600125 and SB203580 significantly inhibited p-ERK1/2, p-JNK1/2 and p-p38 respectively in LFs; （2） The expression of MMP-2 mRNA in LFs exposed to hyperoxia was decreased after treatment with RA, SP600125 and SB203580 respectively （P0.01 or 0.05）, but did not change after treatment with PD98059 （P0.05）. Meanwhile, RA, PD98059, SP600125 and SB203580 had no effect on the expression of TIMP-2 mRNA in LFs exposed to room air or hyperoxia （P0.05）; （3） The expression of pro- and active MMP-2 experienced no change after treatment with RA or SP600125 in LFs exposed to room air （P0.05）, but decreased remarkably after hyperoxia （P0.01 or 0.05）. SB203580 inhibited the expression of pro- and active MMP-2 either in room air or under hyperoxia （P0.01）. PD98059 exerted no effect on the expression of pro- and active MMP-2 （P0.05）. It was suggested that RA had a protective effect on hyperoxia-induced lung injury by down-regulating the expression of MMP-2 through decreasing the JNK and p38 activation in hyperoxia.

Downregulation of cold-inducible RNA-binding protein activates mitogen-activated protein kinases and impairs spermatoRenic function in mouse testes

Cold-inducible RNA-binding protein （CIRP） is an RNA-binding protein that is expressed in normal testes and downregulated after heat stress caused by cryptorchidism, varicocele or environmental temperatures. The purpose of this study was to investigate the functions of CIRP in the testes. We employed RNAi technique to knock down the expression of CIRP in the testes, and performed haematoxylin and eosin staining to evaluate morphological changes following knockdown. Germ cell apoptosis was examined by terminal deoxynucleotidal transferase-mediated dUTP nick end labelling （TUNEL） assay, and mitogen-activated protein kinase （MAPK） signalling pathways were investigated by Western blotting to determine the possible mechanism of apoptosis. We found that using siRNA is a feasible and reliable method for knocking down gene expression in the testes. Compared to controls, the mean seminiferous tubule diameter （MSTD） and the thickness of the germ cell layers decreased following siRNA treatment, whereas the percentage of apoptotic seminiferous tubules increased. The p44/p42, p38 and SAPK/JNK MAPK pathways were activated after downregulation of CIRP. In conclusion, we discovered that downregulation of CIRP resulted in increased germ cell apoptosis, possibly viathe activation of the p44/p42, p38 and SAPK/JNK MAPK pathways.

Ceramide from sphingomyelin hydrolysis differentially mediates mitogen-activated protein kinases (MAPKs) activation following cerebral ischemia in rat hippocampal CA1 subregion

Objective： To explore the role that ceramide plays in the activation of mitogen-activated protein kinases （MAPKs） during cerebral ischemia and reperfusion. Methods： Rats were subjected to ischemia by the fourvessel occlusion （4-VO） method. The sphingomyelinase inhibitor TPCK was administered to the CA1 subregion of the rat hippocampus before inducing ischemia. Western blot was used to examine the activity of extracellular- signal regulated kinase （ERK） and c-Jun N-terminal protein kinase （JNK） using antibodies against ERK, JNK and diphosphorylated ERK and JNK. Results： At lh reperfusion post-ischemia, JNK reached its peak activity while ERK was undergoing a sharp inactivation （P 〈 0.05）. The level of diphosphorylated JNK was significantly reduced but the sharp inactivation of ERK was visibly reversed （P 〈 0.05） by the sphingomyelinase inhibitor. Conclusion： The ceramide signaling pathway is up-regulated through sphingomyelin hydrolysis in brain ischemia, promoting JNK activation and suppressing ERK activation, culminating in the ischemic lesion.

Evidence for a role of mitogen-activated protein kinases in the treatment of experimental acute pancreatitis

Acute pancreatitis(AP) is an inflammatory disease characterized by acute inflammation and necrosis of the pancreatic parenchyma. AP is often associated with organ failure, sepsis, and high mortality. The pathogenesis of AP is still not well understood. In recent years several papers have highlighted the cellular and molecular events of acute pancreatitis. Pancreatitis is initiated by activation of digestive enzymes within the acinar cells that are involved in autodigestion of the gland, followed by a massive infiltration of neutrophils and macrophages and release of inflammatory mediators, responsible for the local and systemic inflammatory response. The hallmark of AP is parenchymal cell necrosis that represents the cause of the high morbidity and mortality, so that new potential therapeutic approaches are indispensable for the treatment of patients at high risk of complications. However, not all factors that determine the onset and course of the disease have been explained. Aim of this article is to review the role of mitogen-activated protein kinases in pathogenesis of acute pancreatitis.

Differential activation of mitogen-activated protein kinases by γ-irradi-ation in IEC-6 cells: Role of intracellular Ca^(2+)

Abstract Objective:To explore the effects of γ-irradiation on mitogen-activatedprotein kinases(MAPKs) and role intracellular calcium in this event in intestinal epithelial cell line 6(IEC-6 cells).Methods:After cultured rat IIEC-6 cells with or without the pretreatment of intracellular Ca^2+ chelator were exposed to γ-ir-radiation of 6 Gy, the total and phosphorylated MAPKs in the cells were determined with Western blotting and apoptosis was examined with flow cytometry.Activities of Extracellular signal-regulated protein kinase (ERK) and p38 MAPK were determined by using immuoprecipitation followed by Western blotting.Results:In response to γ-irradiation,phosphorylation of ERK was not significantly observed ,while the levels of phos-phorylated c-Jun NH2-terminal kinase(JNK) and p38 MAPK were increased in 30 min and reached the peak 2h after exposure to 6Gy γ-irradiation,though the cell viability was significantly lowered 12h.On the other hand, no obvious changes were seen in the total protein levels of ERK, JNK and p38 MAPK.Chelation of in-tracellular Ca^2+ almost completely suppressed the JNK and p38 MAPK phosphorylation induced by γ-irradiation is a potent activator for JNK and p38 MAPK, and Ca^2+ mobilized from intracellular stores plays an important role in the activation of MAPKs and the induction of apoptosis in IEC-6 cells.

Genome-wide identification of the mitogen-activated protein kinase kinases in pear and their functional analysis in response to black spot

The mitogen-activated protein kinase(MAPK)cascade is crucial to plant growth,development,and stress responses.MAPK kinases(MAPKK)play a vital role in linking upstream MAPKK kinases(MAPKKK)with the downstream MAPK.Black spot is one of the most serious fungal diseases of pear which is an important part of the fruit industry in China.The MAPKK genes have been identified in many plants,however,none has been reported in pear(Pyrus bretschneideri).In order to explore whether MAPK gene of pear is related to black spot disease,we designed this experiment.The present study investigated eight putative PbrMAPKK genes obtained from the Chinese white pear genome.The phylogenetic analysis revealed that PbrMAPKK genes were divided into A,B,C,and D groups.These PbrMAPKK genes are randomly distributed on 7 out of 17 chromosomes and mainly originated from the whole-genome duplication(WGD)event.The expression analysis of PbrMAPKK genes in seven pear tissues and the leaves of susceptible and resistant varieties after Alternaria alternata infection by quantitative real-time PCR(qRT-PCR)identified seven candidate genes associated with resistance.Furthermore,virus-induced gene silencing(VIGS)indicated that PbrMAPKK6 gene enhanced resistance to pear black spot disease in pear.

Role of mitogen-activated protein kinases in the regulation of paraventricular nucleus to gastric ischemia-reperfusion injuries

Background We investigated the role in electrical stimulations of paraventricular nucleus （PVN） on gastric mucosal cells and the activity of mitogen-activated protein kinases （MAPKs） family members induced by gastric ischemia-reperfusion （GI-R）. And we elucidated the molecular mechanisms of the protection of PVN from GI-R injuries. Methods Sprague-Dawley rats were divided randomly into 4 groups： Group I, the sham-operated GI-R control group; Group II, the sham-operated electrical stimulations to PVN ＋ sham-operated GI-R control group; Group III, the GI-R group; and Group IV, the electrical stimulations to PVN ＋ GI-R group. In all of the experiments, the PVN was stimulated prior to the induction of GI-R. The GI-R model was established by clamping the celiac artery for 30 minutes to induce ischemia and then was released to allow reperfusion for 30 minutes, 1 hour, 3 hours and 6 hours, respectively. The gastric mucosal cellular apoptosis, proliferation, and the expression and activity of MAPKs protein were observed by immunohistochemistry and Western blotting, respectively. Results Compared with the GI-R group, the application of electrical stimulations in the PVN significantly depressed gastric mucosal cellular apoptosis and enhanced gastric mucosal cellular proliferation following the 30-minute, 1-hour and 3-hour intervals of reperfusion; it also promoted the activation of p-ERK during the early phase of reperfusion but inhibited the activation of p-JNK1/2 and p-p38 following the 30-minute, 1-hour and 3-hour intervals of reperfusion. Conclusions The protection of PVN against GI-R injuries may attribute to the inhibition of apoptosis and the promotion of the proliferation of gastric mucosal cells during GI-R. This protective effect is mediated by activating the ERK pathway and depressing the JNK, the JNK. p38 MAPK oathwavs of the oastric mucosal cells.

Stimulatory effect of puerarin on bone formation through co-activation of nitric oxide and bone morphogenetic protein-2/ mitogen-activated protein kinases pathways in mice

Background Estrogen deficiency results in loss of bone mass compounds with estrogen-like activity that bind to estrogen receptors effect of the phytoestrogen puerarin on adult mouse osteoblasts. Methods Osteoblast cells were harvested from 8-month old female Phytoestrogens are plant-derived non-steroidal The main aim of this study was to investigate the mprinting control region （ICR） mice. The effects of puerarin stimulation on the proliferation, differentiation and maturation of osteoblasts were examined. The production of nitric oxide （NO） and the expression of bone morphogenetic protein-2 （BMP-2）, SMAD4, mitogen-activated protein kinases （MAPK）, core binding factor all runt-related transcription factor 2 （Cbfal/Runx2）, osteoprotegerin （OPG）, and receptor activator of NF-KB ligand （RANKL） genes were analyzed. The activation of signal pathways was further confirmed by specific pathway inhibitors. Results The osteoblast viability reached its maximum at 10-8 mol/L puerarin. At this concentration, puerarin increases the proliferation and matrix mineralization of osteoblasts and promotes NO synthesis. With 108 mol/L puerarin treatment, BMP-2, SMAD4, Cbfal/Runx2, and OPG gene expression were up-regulated, while the RANKL gene expression is down-regulated. Concurrent treatment involving the （bone morphogenetic protein） BMP antagonist Noggin or the NOS inhibitor L-NAME diminishes puerarin induced cell proliferation, Alkaline phosphatase （ALP） activity, NO production, as well as the BMP-2, SMAD4, Cbfal/Runx2, OPG, and RANKL gene expression. Conclusions In this in vitro study, we demonstrate that puerarin is a bone anabolic agent that exerts its osteogenic effects through the induction of BMP-2 and NO synthesis, subsequently regulating Cbfal/Runx2, OPG, and RANKL gene expression. This effect may contribute to its induction of osteoblast proliferation and differentiation, resulting in bone formation.

Efficacy of quercetin, oleanolic acid, icariin on apoptosis and mitogen-activated protein kinases signaling pathways in hippocampal neurons of Sprague-Dawley rats cultured with high glucose medium

OBJECTIVE:To investigate the effect of quercetin,oleanolic acid,icariin and their compatibility on the apoptosisofhippocampalneuronsof Sprague-Dawley(SD)rats cultured with high glucose medium and the possible mechanism.METHODS:The extracts were purchased from China Food and Drug Control Institute and Sellect.Hippocampus was obtained from newborn 24 h SD rats.After culturing the hippocampus in different medium for 72 h,flow cytometry was used to detect the apoptosis of hippocampal neurons,and Western blot was utilized to test the expressions of p-p38,p38,p-c-Jun N-terminal kinase(JNK)and JNK.RESULTS:Compared with the control group(CG),the neuronal apoptosis rate and the ratios of p-p38/p38 and p-JNK/JNK were significantly increased in the high glucose group(GG)(P<0.01);Compared with the GG,the apoptosis rate and the ratios ofp-p38/p38 and p-JNK/JNK were significantly decreased in other drug groups(P<0.01);Compared with the monomer groups respectively,the apoptosis rate and the ratios of p-p38/p38 and p-JNK/JNK in the two-drug groups and the three-drug group all decreased(P<0.01);Compared with the two-drug groups,the neuronal apoptosis rate and the ratio of p-JNK/JNK of the three-drug group decreased(P<0.05).CONCLUSION:Under the condition of high glucose,the quercetin,oleanolic acid and icariin can alleviate the apoptosis of hippocampus neurons,reduce the phosphorylation of p38 and JNK in p38 mitogen-activated protein kinases and JNK signaling pathway.And the efficacy of the three drugs in combination with each other can be strengthened.

MicroRNA-370-5p inhibits pigmentation and cell proliferation by downregulating mitogen-activated protein kinase kinase kinase 8 expression in sheep melanocytes

In mammals,microRNAs(miRNAs)play key roles in multiple biological processes by regulating the expression of target genes.Studies have found that the levels of miR-370-5p expression differ significantly in the skins of sheep with different hair colors;however,its function remains unclear.In this study,we investigated the roles of miR-370-5p in sheep melanocytes and found that the overexpression of miR-370-5p significantly inhibited cell proliferation(P<0.01),tyrosinase activity(P=0.001)and significantly reduced(P<0.001)melanin production.Functional prediction revealed that the 3′-untranslated region(UTR)of MAP3K8 has a putative miR-370-5p binding site,and the interaction between these two molecules was confirmed using luciferase reporter assays.In situ hybridization assays revealed that MAP3K8 is expressed in the cytoplasm of melanocytes.The results of quantitative RT-PCR and Western blotting analyses revealed that overexpression of miR-370-5p in melanocytes significantly inhibits(P<0.01)MAP3K8 expression via direct targeting of its 3′UTR.Inhibition of MAP3K8 expression by siRNA-MAP3K8 transfection induced a significant inhibition(P<0.01)of melanocyte proliferation and significant reduction(P<0.001)in melanin production,which is consistent with our observations for miR-370-5p.Target gene rescue experiments indicated that the expression of MAP3K8 in melanocytes co-transfected with miR-370-5p and MAP3K8-cDNA(containing sites for the targeted binding to miR-370-5p)was significantly rescued(P≤0.001),which subsequently promoted significant increases in cell proliferation(P<0.001)and melanin production(P<0.01).Collectively,these findings indicate that miR-370-5p plays a functional role in inhibiting sheep melanocyte proliferation and melanogenesis by downregulating the expression of MAP3K8.

Effects of U0126 on growth and activation of mitogen-activated protein kinases in Aspergillus fumigatus

Background Invasive aspergillosis （IA）, which is mainly caused by Aspergillus fumigatus （A. fumigatus）, is a major cause of morbidity and mortality in immunocompromised patients. Despite considerable progress in currently available antifungals the mortality still remains high in critically ill patients. U0126 which is a highly selective inhibitor of MEK1 and MEK2 in the RAF/MEK/ERK pathway in mammalian cells has been demonstrated to have an anti-proliferative role in cancer cells. The purpose of this study was to explore the role of U0126 on growth inhibition and activation of mitogen-activated protein kinases （MAPKs） in A. fumigatus. Methods Germination percentage and hyphae growth in A. fumigatus treated with U0126 were observed and compared with untreated controls. Western blotting analysis was used to detect changes in activation of SakA, MpkA and MpkB. Results U0126 inhibited germination and hyphae growth in A. fumigatus and enhanced the phosphorylation of SakA and MpkA under oxidative stress. U0126 at 10 pmol/L did not block the activation of MpkB during nitrogen starvation stress. Conclusion U0126 shows promise as an antifungal candidate and the MAPK pathway may be a possible antifungal drug target for A. fumigatus.

Overexpression of mitogen-activated protein kinase phosphatase-1 in endothelial cells reduces blood-brain barrier injury in a mouse model of ischemic stroke

Ischemic stroke can cause blood-brain barrier(BBB)injury,which worsens brain damage induced by stroke.Abnormal expression of tight junction proteins in endothelial cells(ECs)can increase intracellular space and BBB leakage.Selective inhibition of mitogen-activated protein kinase,the negative regulatory substrate of mitogen-activated protein kinase phosphatase(MKP)-1,improves tight junction protein function in ECs,and genetic deletion of MKP-1 aggravates ischemic brain injury.However,whether the latter affects BBB integrity,and the cell type-specific mechanism underlying this process,remain unclear.In this study,we established an adult male mouse model of ischemic stroke by occluding the middle cerebral artery for 60 minutes and overexpressed MKP-1 in ECs on the injured side via lentiviral transfection before stroke.We found that overexpression of MKP-1 in ECs reduced infarct volume,reduced the level of inflammatory factors interleukin-1β,interleukin-6,and chemokine C-C motif ligand-2,inhibited vascular injury,and promoted the recovery of sensorimotor and memory/cognitive function.Overexpression of MKP-1 in ECs also inhibited the activation of cerebral ischemia-induced extracellular signal-regulated kinase(ERK)1/2 and the downregulation of occludin expression.Finally,to investigate the mechanism by which MKP-1 exerted these functions in ECs,we established an ischemic stroke model in vitro by depriving the primary endothelial cell of oxygen and glucose,and pharmacologically inhibited the activity of MKP-1 and ERK1/2.Our findings suggest that MKP-1 inhibition aggravates oxygen and glucose deprivation-induced cell death,cell monolayer leakage,and downregulation of occludin expression,and that inhibiting ERK1/2 can reverse these effects.In addition,co-inhibition of MKP-1 and ERK1/2 exhibited similar effects to inhibition of ERK1/2.These findings suggest that overexpression of MKP-1 in ECs can prevent ischemia-induced occludin downregulation and cell death via deactivating ERK1/2,thereby protecting the integrity of BBB,alleviating brain injury,and improving post-stroke prognosis.

Effect of oxymatrine on the p38 mitogen-activated protein kinases signalling pathway in rats with CCl4 induced hepatic fibrosis

Background Recent studies have suggested that p38 mitogen-activated protein kinases （MAPK） signalling pathway plays an important role in hepatic fibrosis. This study explored the antifibrotic effect of oxymatrine on tetrachloromethane induced liver fibrosis in rats and its modulation on the p38 MAPK signalling pathway. Methods One hundred and twenty healthy male Sprague-Dawley rats were randomly assigned to six groups： normal （n=20）, induced fibrosis （n=20）, colchicine （n=20） and three treatment groups of oxymatrine （n=20x3）. We obesrved changes in deposition of collagen, hyaluronic acid （HA）, laminin （LN）, collagen type IV （CIV）, procollagen III （PCIll） and hydroxyproline （Hyp）, a-smooth muscle actin （α-SMA） and phosphor-p38 （pp38）. Results The relative indicators of changes in histopathology, HA, LN, CIV, PCIII, Hyp, a-SMA and pp38 were raised significantly in the induced fibrosis group （P〈0.01 vs normal group）. The semiquantitative hepatic fibrosis staging scores of middle dose group and high dose group were decreased （P 〈0.05 and P 〈0.01 respectively vs the induced fibrosis group）, as was the average area of collagen in rats＇ liver, the concentrations of serum HA, LN, CIV, PCIII and liver tissue homogenate Hyp. The gene expression of α-SMA mRNA was considerably decreased in the treated animals, as was the protein espression of pp38 protein. Conclusions Oxymatrine is effective in reducing the production and deposition of collagen in the liver tissue of experimental rats in ways which relate to modulating the fibrogenic signal transduction via p38 MAPK signalling pathway.

Role of p38 mitogen-activated protein kinases in cardioprotection of morphine preconditioning

Background p38 mitogen-activated protein kinases （MAPK） in ischemic preconditioning （IPC） may be essential to cardioprotection. We assessed whether protective effect of morphine-induced preconditioning （MPC） on myocardial ischemia and reperfusion injury in rat hearts involved p38 MAPK activation.Methods Male Spargue-Dawley rats （weighing 300--350 g） were randomly assigned to 1 of the following 8 groups： control （CON, saline vehicle, n=9）, SB 203580 （SB, a p38 MAPK inhibitor, n=6）, MPC （n=6）, IPC （n=9）, SB＋MPC, SB＋IPC, MPC＋SB, and IPC＋SB （n=6）. Infarct sizes （IS）, a percentage of the area at risk （AAR）, were determined by triphenyltetrazolium （TTC） staining. Hssue samples were processed from the entire AAR of left ventricle for the determination of p38 MAPK protein expression （5 hearts/group）. The bands representing the proteins were visualized using an enhanced chemiluminescence detection system. Results The IS/AAR was significantly reduced by I PC （12.9±1.6）% or MPC （25.3±2.9）% compared to the control （52.7±5.5）%. SB 203580 administered prior to preconditioning abolished the effect of IPC （SB＋IPC： （43.8±2.6）%, P〉0.05 vs CON, P〈0.01 vs IPC）, but not MPC （SB＋MPC： （30.7±0.9）%, P〈0.01 vs CON, P〉0.05 vs MPC）. Treatment with SB 203580 prior to sustained ischemia diminished the protective effect of both MPC （MPC＋SB： （42.4±2.9）%, P〉0.05 vs CON） and IPC （IPC＋SB： （52.0±2.5）%, P〉0.05 vs CON） on IS/AAR. In the IPC group, phospho-p38 MAPK protein increased significantly within 5 minutes into ischemia and remained elevated at 30 minutes into reperfusion, while phospho-p38 MAPK protein in the MPC group only increased significantly at 30 minutes into reperfusion.Conclusion The activation of p38 MAPK just acts as a mediator of MPC,whereas it acts as both a trigger and a mediator in IPC.

Mitogen-activated protein kinases mediate the oxidative burst and saponin synthesis induced by chitosan in cell cultures of Panax ginseng

Chitosan (CHN) specially induced the activities of 39 kD and 42 kD protein kinases in ginseng cells, which could be suppressed by an inhibitor of mitogen-activated protein kinase (MAPK) pathway, PD98059. The immunoprecipitation (IP) using MAPK antibody or kinase assay in vitro also showed that CHN-induced 42 kD and 39 kD protein kinases belonged to the MAPK family. PD98059 suppressed CHN-induced transcriptions of ginseng squalene synthase and ginseng squalene epoxidase genes (gss and gse), CHN-induced accumulation of b-Amyrin synthase (b-AS) and synthesis of saponin. These results showed that CHN-induced activities of MAPKs were necessary for the CHN-induced saponin synthesis. EGTA and LaCl3 suppressed CHN-induced 39 kD and 42 kD MAPK activities. Ruthenium red (RR) could suppress CHN-induced 39 kD activity. All of them suppressed CHN-induced saponin synthesis. These results indicated that CHN-induced increment of cytosolic calcium was necessary for CHN-induced saponin synthesis. PD98059 also suppressed CHN-induced oxidative burst (in-cluding the increment of activity of plasma membrane NADPH oxidase and production of H2O2), but diphenylene iodonium (DPI), dimethylthiourea (DMTU) and 2,5-dihydroxycinnamic acid methyl ester (DHC) could not suppress CHN-induced MAPK activities, which indicated that MAPK was possibly function upstream of CHN-induced oxidative burst.

Electroacupuncture reduces apoptotic index and inhibits p38 mitogen-activated protein kinase signaling pathway in the hippocampus of rats with cerebral ischemia/reperfusion injury

Electroacupuncture attenuates cerebral hypoxia and neuronal apoptosis induced by cerebral ischemia/reperfusion injury.To further identify the involved mechanisms,we assumed that electroacupuncture used to treat cerebral ischemia/reperfusion injury was associated with the p38 mitogen-activated protein kinase（MAPK） signaling pathway.We established rat models of cerebral ischemia/reperfusion injury using the modified Zea-Longa＇s method.At 30 minutes before model establishment,p38 MAPK blocker SB20358 was injected into the left lateral ventricles.At 1.5 hours after model establishment,electroacupuncture was administered at acupoints of Chize（LU5）,Hegu（LI4）,Zusanli（ST36）,and Sanyinjiao（SP6） for 20 minutes in the affected side.Results showed that the combination of EA and SB20358 injection significantly decreased neurologic impairment scores,but no significant differences were determined among different interventional groups.Hematoxylin-eosin staining also showed reduced brain tissue injuries.Compared with the SB20358 group,the cells were regularly arranged,the structures were complete,and the number of viable neurons was higher in the SB20358 ＋ electroacupuncture group.Terminal deoxynucleotidyl transferase（Td T）-mediated d UTP nick-end labeling assay showed a decreased apoptotic index in each group,with a significant decrease in the SB20358 ＋ electroacupuncture group.Immunohistochemistry revealed reduced phosphorylated p38 expression at 3 days in the electroacupuncture group and SB20358 ＋ electroacupuncture group compared with the ischemia/reperfusion group.There was no significant difference in phosphorylated p38 expression between the ischemia/reperfusion group and SB20358 group.These findings confirmed that the electroacupuncture effects on mitigating cerebral ischemia/reperfusion injury are possibly associated with the p38 MAPK signaling pathway.A time period of 3 days could promote the repair of ischemic cerebral nerves.

Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-,mitogen-activated protein kinase-,and Nrf2-dependent pathway

BACKGROUND Enterotoxigenic Bacteroides fragilis(ETBF)causes colitis and diarrhea,and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers.These diseases are dependent on ETBF-secreted toxin(BFT).Dendritic cells(DCs)play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1(HO-1)is involved in the regulation of DC function.AIM To investigate the role of BFT in HO-1 expression in DCs.METHODS Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2−/−knockout mice.DCs were exposed to BFT,after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR,EMSA,fluorescent microscopy,immunoblot,and ELISA.RESULTS HO-1 expression was upregulated in DCs stimulated with BFT.Although BFT activated transcription factors such as NF-κB,AP-1,and Nrf2,activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs.Instead,upregulation of HO-1 expression was dependent on Nrf2 activation in DCs.Moreover,HO-1 expression via Nrf2 in DCs was regulated by mitogenactivated protein kinases such as ERK and p38.Furthermore,BFT enhanced the production of reactive oxygen species(ROS)and inhibition of ROS production resulted in a significant decrease of phospho-ERK,phospho-p38,Nrf2,and HO-1 CONCLUSION These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.

Mitogen-activated protein kinase phosphatase 1 protects PC12 cells from amyloid beta-induced neurotoxicity

The mitogen-activated protein kinase（MAPK） signaling pathway plays an important role in the regulation of cell growth, proliferation, differentiation, transformation and death. Mitogen-activated protein kinase phosphatase 1（MKP1） has an inhibitory effect on the p38 MAPK and JNK pathways, but it is unknown whether it plays a role in Aβ-induced oxidative stress and neuronal inflammation. In this study, PC12 cells were infected with MKP1 sh RNA, MKP1 lentivirus or control lentivirus for 12 hours, and then treated with 0.1, 1, 10 or 100 μM amyloid beta 42（Aβ42）. The cell survival rate was measured using the cell counting kit-8 assay. MKP1, tumor necrosis factor-alpha（TNF-α） and interleukin-1β（IL-1β） m RNA expression levels were analyzed using quantitative real time-polymerase chain reaction. MKP1 and phospho-c-Jun N-terminal kinase（JNK） expression levels were assessed using western blot assay. Reactive oxygen species（ROS） levels were detected using 2′,7′-dichlorofluorescein diacetate. Mitochondrial membrane potential was measured using flow cytometry. Superoxide dismutase activity and malondialdehyde levels were evaluated using the colorimetric method. Lactate dehydrogenase activity was measured using a microplate reader. Caspase-3 expression levels were assessed by enzyme-linked immunosorbent assay. Apoptosis was evaluated using the terminal deoxynucleotidyl transferase d UTP nick end labeling method. MKP1 overexpression inhibited Aβ-induced JNK phosphorylation and the increase in ROS levels. It also suppressed the Aβ-induced increase in TNF-α and IL-1β levels as well as apoptosis in PC12 cells. In contrast, MKP1 knockdown by RNA interference aggravated Aβ-induced oxidative stress, inflammation and cell damage in PC12 cells. Furthermore, the JNK-specific inhibitor SP600125 abolished this effect of MKP1 knockdown on Aβ-induced neurotoxicity. Collectively, these results show that MKP1 mitigates Aβ-induced apoptosis, oxidative stress and neuroinflammation by inhibiting the JNK signaling pathway, thereby playing a neuroprotective role.