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.

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.

Inhibition of p38 mitogen-activated protein kinase may decrease intestinal epithelial cell apoptosis and improve intestinal epithelial barrier function after ischemia- reperf usion injury

AIM: To investigate the role of p38 mitogen-activated protein kinase in rat small intestine after ischemia-reperfusion (I/R)insult and the relationship between activation of p38 MAPK and apoptotic cell death of intestine.METHODS: Ninety Wistar rats were divided randomly into three groups, namely sham-operated group (C), I/R vehicle group (R) and SB203580 pre-treated group(S).In groups R and S, the superior mesenteric artery(SMA)was separated and occluded for 45 min, then released for reperfusion for0.25, 0.5, 1, 2, 6, 12 and 24 h. In group C, SMA was separated without occlusion. Plasma D-lactate levels were examined and histological changes were observed under a light microscope. The activity of p38 MAPK was determined by Western immunoblotting and apoptotic cells were detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUDP-biotin nick end labeling (TUNEL).RESULTS: Intestinal ischemia followed by reperfusion activated p38 MAPK, and the maximal level of activation (7.3-fold vs sham-operated group) was reached 30 min after I/R. Treatment with SB 203580, a p38 MAPK inhibitor,reduced intestinal apoptosis (26.72±3.39% vs62.50±3.08%in I/R vehicle, P＜0.01) and decreased plasma D-lactate level (0.78±0.15 mmol/L in I/R vehicle vs0.42±0.17 mmol/L in SB-treated group) and improved post-ischemic intestinal histological damage.CONCLUSION: p38 MAPK plays a crucial role in the signal transduction pathway mediating post-ischemic intestinal apoptosis, and inhibition of p38 MAPK may attenuate ischemia-reperfusion injury.

INVOLVEMENT OF p38 MITOGEN-ACTIVATED PROTEIN KINASE IN E.coli-INDUCED U937 APOPTOSIS

Objective To investigate whether the effect of E.coli on U937 cell lines apoptosis is mediated via p38 mitogen-activated protein kinase (MAPK) activation. Methods The U937 cell lines were treated with E.coli at different time or together with SB203580, an inhibitor for p38. Cell apoptosis was analyzed by flow cytometry. p38 activities were detected by Western blotting. Results E.coli induced apoptosis in cultured U937 cell lines in a time-dependent manner. The phosphorylation of p38 was induced after 10 minutes infection, reached the peak after 20 minutes, and started to decline after 30 minutes. In contrast, the level of total p38 protein was not changed in whole experimental period. Inhibition of p38 with SB203580 significantly inhibited E.coli induced apoptosis in U937 cells. Conclusion The activation of the p38 MAPK in U937 cell lines by E.coli is a major pathway to mediate the apoptosis.

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.

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.

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.

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.

Role of Extracelluar Regulated Protein Kinases in FTY720-induced Apoptosis of Leukemia Cell Lines HL-60 and U937

The effects of a novel immunosuppressive agent FTY720 on proliferation inhibition and apoptosis of acute leukemia cell lines HL 60 and U937, and the role of extracelluar regulated protein kinase (ERK) in the course of proliferation inhibition and apoptosis induced by FTY720 were studied. The proliferation inhibition rate of HL 60 and U937 cells by various concentrations of FTY720 was detected by MTT assay. Cell apoptosis was detected by DNA fragment analysis and flow cytometry. The phosphorylated ERK1/2 protein expression was observed by Western blotting. The change of intracellular distribution of ERK1/2 protein was identified by SP immunohistochemical staining. The results showed that FTY720 could inhibit the growth of HL 60 and U937 cells effectively in a dose dependent manner. After incubation with FTY720 for 24 h, apoptosis was observed in HL 60 and U937 cells. The intracellular expression of phosphorylated ERK1/2 protein was also down regulated and the distribution of ERK1/2 protein in cell nuclear was reduced during FTY720 induced apoptosis. So, that FTY720 inhibited ERK1/2 phosphorylation might mediate the role of FTY720 induced apoptosis and proliferation inhibition of leukemia cells.

Mechanism of stilbene glycosides on apoptosis of SH-SY5Y cells via regulating PI3K/AKT signaling pathway

Objective:To investigate the effects of stilbene glycoside(TSG)on okadaic acid-induced apoptosis in human neuroblastoma cells(SH-SY5Y)via the PI3K/AKT pathway.Methods:The optimal concentration of OA was screened by CCK-8 assay,and SH-SY5Y cells were divided into control group,model group,TSG group,LY294002 group and LY294002+TSG group.The proliferation and apoptosis in each group were detected by CCK-8 and TUNEL assays;Western blotting method and real-time fluorescence quantitative polymerase chain reaction was used to detect the expression of PI3K,P-PI3K(Y607),AKT,P-AKT(Ser473),Bcl-2 and Bax proteins.The relative protein expression was represented by P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax gray ratio.Results:CCK-8 screened the optimal concentration of OA as 40 nmol/L.Compared with the control group,the model group increased relative cell viability,decreased apoptosis rate,the pathway and apoptotic proteins expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were decreased,and the mRNA expression levels of PI3K,AKT and Bcl-2 were decreased.Bax mRNA expression level increased(P<0.05);Compared with model group,TSG group increased relative cell viability,decreased apoptosis rate,increased protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT,Bcl-2/Bax,and increased mRNA expression levels of PI3K,AKT,and Bcl-2.Bax mRNA expression decreased(P<0.05),LY294002 group decreased relative cell viability,increased apoptosis rate,P-PI3K(Y607)/PI3K protein expression levels were significantly decreased(P<0.05),P-AKT(Ser473)/AKT and Bcl-2/Bax protein expression levels were significantly decreased,but there was no statistical significance,PI3K,AKT and Bcl-2 mRNA expression levels were decreased,and Bax mRNA expression levels were increased(all P<0.05);Compared with LY294002 group,LY294002+TSG group increased relative cell viability,decreased apoptosis rate,and the protein expression levels of P-PI3K(Y607)/PI3K,P-AKT(Ser473)/AKT and Bcl-2/Bax were increased.The mRNA expression levels of PI3K,AKT,Bcl-2 were increased,Bax was decreased(all P<0.05).Conclusion:Stilbene glycoside may alleviate okadaic acid-induced apoptosis in SH-SY5Y cells by interfering with the PI3K/AKT signaling pathway,which in turn regulates the expression of apoptotic factors such as Bcl-2 and Bax.

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.

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway

BACKGROUND Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes,and leads to apoptosis.Apoptosis is very important in regulating the homeostasis of the hepatobiliary system.Endoplasmic reticulum(ER)stress is one of the signaling pathways that induce apoptosis.Moreover,the protein kinase RNA-like endoplasmic reticulum kinase(PERK)-induced apoptotic pathway is the main way;but its role in liver injury remains unclear.Yinchenhao decoction(YCHD)is a traditional Chinese medicine formula that alleviates liver injury and apoptosis,yet its mechanism is unknown.We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice(OJ).AIM To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein(CHOP)-growth arrest and DNA damage-inducible protein 34(GADD34)pathway and B cell lymphoma/leukemia-2 related X protein(Bax)/B cell lymphoma/leukemia-2(Bcl-2)ratio.METHODS For in vivo experiments,30 rats were divided into three groups:control group,OJ model group,and YCHD-treated group.Blood was collected to detect the indicators of liver function,and liver tissues were used for histological analysis.For in vitro experiments,30 rats were divided into three groups:G1,G2,and G3.The rats in group G1 had their bile duct exposed without ligation,the rats in group G2 underwent total bile duct ligation,and the rats in group G3 were given a gavage of YCHD.According to the serum pharmacology,serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells.Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling(TUNEL)assay was used to detect BRL-3A hepatocyte apoptosis.Alanine aminotransferase(ALT)and aspartate transaminase(AST)levels in the medium were detected.Western blot and quantitative real-time polymerase chain reaction(qRT-PCR)analyses were used to detect protein and gene expression levels of PERK,CHOP,GADD34,Bax,and Bcl-2 in the liver tissues and BRL-3A cells.RESULTS Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group.The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ.Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ.Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK,CHOP,and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ.The Bax and Bcl-2 levels were increased,and the Bax/Bcl-2 ratio was also increased.When YCHD was used,the PERK,CHOP,GADD34,and Bax levels quickly decreased,while the Bcl-2 levels increased,and the Bax/Bcl-2 ratio decreased.CONCLUSION OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio.YCHD can attenuate these changes.

Mitogen-activated protein kinase signaling pathway and invasion and metastasis of gastric cancer

The mortality rate of gastric cancer worldwide is as high as 70%, despite the development of novel therapeutic strategies. One reason for the high mortality is the rapid and uninhibited spread of the disease, such that the majority of patients are diagnosed at a stage when efficient therapeutic treatment is not available. Therefore, in-depth research is needed to investigate the mechanism of gastric cancer metastasis and invasion to improve outcomes and provide biomarkers for early diagnosis. The mitogen-activated protein kinase(MAPK) signaling pathway is widely expressed in multicellular organisms, with critical roles in multiple biological processes, such as cell proliferation, death, differentiation, migration, and invasion. The MAPK pathway typically responds to extracellular stimulation. However, the MAPK pathway is often involved in the occurrence and progression of cancer when abnormally regulated. Many studies have researched the relationship between the MAPK signaling pathway and cancer metastasis and invasion, but little is known about the important roles that the MAPK signaling pathway plays in gastric cancer. Based on an analysis of published data, this review aims to summarize the important role that the MAP kinases play in the invasion and metastasis of gastric cancer and attempts to provide potential directions for further research and clinical treatment.

Prostaglandin E1 protects hepatocytes against endoplasmic reticulum stress-induced apoptosis via protein kinase A-dependent induction of glucose-regulated protein 78 expression

AIM To investigate the protective effect of prostaglandin E1(PGE1) against endoplasmic reticulum(ER) stressinduced hepatocyte apoptosis, and to explore its underlying mechanisms.METHODS Thapsigargin(TG) was used to induce ER stress in the human hepatic cell line L02 and hepatocarcinomaderived cell line Hep G2. To evaluate the effects of PGE1 on TG-induced apoptosis, PGE1 was used an hour prior to TG treatment. Activation of unfolded protein response signaling pathways were detected by western blotting and quantitative real-time RTPCR. Apoptotic index and cell viability of L02 cells and Hep G2 cells were determined with flow cytometry and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium] assay. RESULTS Pretreatment with 1 μmol/L PGE1 protected against TG-induced apoptosis in both L02 cells and Hep G2 cells. PGE1 enhanced the TG-induced expression of C/EBP homologous protein(CHOP), glucose-regulated protein(GRP) 78 and spliced X box-binding protein 1 at 6 h. However, it attenuated their expressions after 24 h. PGE1 alone induced protein and m RNA expressions of GRP78; PGE1 also induced protein expression of DNA damage-inducible gene 34 and inhibited the expressions of phospho-PKR-like ER kinase, phosphoeukaryotic initiation factor 2α and CHOP. Treatment with protein kinase A(PKA)-inhibitor H89 or KT5720 blocked PGE1-induced up-regulation of GRP78. Further, the cytoprotective effect of PGE1 on hepatocytes was not observed after blockade of GRP78 expression by H89 or small interfering RNA specifically targeted against human GRP78.CONCLUSION Our study demonstrates that PGE1 protects against ER stress-induced hepatocyte apoptosis via PKA pathwaydependent induction of GRP78 expression.

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.

Physiological roles of mitogen-activated-protein-kinase-activated p38-regulated/activated protein kinase

Mitogen-activated protein kinases(MAPKs)are a family of proteins that constitute signaling pathways involved in processes that control gene expression,cell division, cell survival,apoptosis,metabolism,differentiation and motility.The MAPK pathways can be divided into conventional and atypical MAPK pathways.The first group converts a signal into a cellular response through a relay of three consecutive phosphorylation events exerted by MAPK kinase kinases,MAPK kinase,and MAPK.Atypical MAPK pathways are not organized into this three-tiered cascade.MAPK that belongs to both conventional and atypical MAPK pathways can phosphorylate both non-protein kinase substrates and other protein kinases.The latter are referred to as MAPK-activated protein kinases.This review focuses on one such MAPK-activated protein kinase,MAPK-activated protein kinase 5(MK5)or p38-regulated/activated protein kinase(PRAK).This protein is highly conserved throughout the animal kingdom and seems to be the target of both conventional and atypical MAPK pathways.Recent findings on the regulation of the activity and subcellular localization,bona fide interaction partners and physiological roles of MK5/PRAK are discussed.

Serine-threonine protein kinase activation may be an effective target for reducing neuronal apoptosis after spinal cord injury

The signaling mechanisms underlying ischemia-induced nerve cell apoptosis are poorly understood. We investigated the effects of apoptosis-related signal transduction pathways following ischemic spinal cord injury, including extracellular signal-regulated kinase（ERK）, serine-threonine protein kinase（Akt） and c-Jun N-terminal kinase（JNK） signaling pathways. We established a rat model of acute spinal cord injury by inserting a catheter balloon in the left subclavian artery for 25 minutes. Rat models exhibited notable hindlimb dysfunction. Apoptotic cells were abundant in the anterior horn and central canal of the spinal cord. The number of apoptotic neurons was highest 48 hours post injury. The expression of phosphorylated Akt（pAkt） and phosphorylated ERK（p-ERK） increased immediately after reperfusion, peaked at 4 hours（p-Akt） or 2 hours（p-ERK）, decreased at 12 hours, and then increased at 24 hours. Phosphorylated JNK expression reduced after reperfusion, increased at 12 hours to near normal levels, and then showed a downward trend at 24 hours. Pearson linear correlation analysis also demonstrated that the number of apoptotic cells negatively correlated with p-Akt expression. These findings suggest that activation of Akt may be a key contributing factor in the delay of neuronal apoptosis after spinal cord ischemia, particularly at the stage of reperfusion, and thus may be a target for neuronal protection and reduction of neuronal apoptosis after spinal cord injury.

Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis- mutase 1 mutant （SOD1G93A） and wild-type （SOD1wv） mouse models were exposed to H202. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by flow cytometry. Moreover, we evaluated the expression of the adenos- ine monophosphate-activated protein kinase （AMPK） ct-subunit, paired box 3 （Pax3） protein, and p53 in western blot analyses. Compared with SOD1wr cells, SOD1~93A embryonic neural stem cells were more likely to undergo H202-induced apoptosis. Phosphorylation of AMPKct in SOD1G93A cells was higher than that in SOD1wr cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKct. p53 protein levels were also correlated with AMPKct phosphorylation levels. Compound C, an inhibitor of AMPKa, attenuated the effects of H20~. These results suggest that embryonic neural stem cells from SOD1C93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKa pathway.

Jiawei Wendan decoction affects mitogen-activated protein kinase signal pathway in the hippocampus of depression rats

A previous study from our group showed that Jiawei Wendan decoction inhibits protein expression of interleukin-1β, 2, and 6, as well as plasma neuropeptide Y, P substance and somatostatin in the hippocampus of depression rat models. The present study analyzed the influence of Jiawei Wendan decoction on the mitogen-activated protein kinase signal transduction pathway in the hippocampus. Results demonstrated that Jiawei Wendan decoction effectively upregulated expression of small molecular G proteins, extracellular regulated kinase 1/2, and activated ribosomal S6 kinase protein in the rat hippocampus. In addition, Jiawei Wendan decoction exhibits antidepressant effects similar to fluoxetine. The underlying mechanisms were shown to be dependent on increased mitogen-activated protein kinase signal transduction pathway activity.

Cathelicidin stimulates colonic mucus synthesis by up-regulating MUC1 and MUC2 expression through a mitogen-activated protein kinase pathway

Objective Mucus forms the physical barrier along the gastrointestinal(GI)tract.It plays an important role to prevent mucosal damage and inflammation.Our previous finding showed that antibacterial peptide 'cathelicidin' increased mucus thickness and prevented inflammation in the colon.In the current study,we examined the protective mechanisms by which the peptide increased mucus synthesis in vitro.Methods Human colonic cell line(HT-29)was used to assess the stimulatory action of cathelicidin on mucus synthesis which was measured by the D-[6-3H] glucosamine incorporation assay.Results Human cathelicidin(LL-37)dose-dependently(10-40 μg·mL-1)and significantly stimulated mucus synthesis.Real-time PCR data showed that addition of LL-37 induced more than 50% increase in MUC1 and MUC2 mRNA levels.Treatment with MUC1 and MUC2 siRNAs normalized the stimulatory action of LL-37 on mucus synthesis.LL-37 also activated the phosphorylation of mitogen-activated protein(MAP)kinase in the cells.A specific inhibitor of the MAP kinase pathway,U0126,completely blocked the increase of MUC1 and MUC2 expression as well as mucus synthesis by LL-37.Conclusions Taken together LL-37 stimulates mucus synthesis through the activation of MUC1 and MUC2 expression and the MAP kinase pathway in human colonic cells.