Correlation between spina bifida manifesta in fetal rats and c-Jun N-terminal kinase signaling

Fetal rat models with neural tube defects were established by injection with retinoic acid at 10 days after conception. The immunofluorescence assay and western blot analysis showed that the number of caspase-3 positive cells in myeloid tissues for spina bifida manifesta was increased. There was also increased phosphorylation of c-Jun N-terminal kinase, a member of the mitogen activated protein kinase family. The c-Jun N-terminal kinase phosphorylation level was positively correlated with caspase-3 expression in myeloid tissues for spina bifida manifesta. Experimental findings indicate that abnormal apoptosis is involved in retinoic acid-induced dominant spina bifida formation in fetal rats, and may be associated with the c-Jun N-terminal kinase signal transduction pathway.

Experimental study on the apoptosis of cervical cancer Hela cells induced by juglone through c-Jun N-terminal kinase/c-Jun pathway

Objective: To study the regulatory effect and molecular mechanism of juglone on apoptosis of cervical cancer Hela cells. Methods: Cervical cancer Hela cells were cultured and treated with different dosages of juglone (10, 20, and 40 pmol/L, respectively) and c-Jun N-terminal kinase (JNK) inhibitor SP600125 (10, 20, and 40 mu mol/L. respectively). Then cellular proliferative activity and the expression of JNK/c-Jun pathway molecule and apoptotic molecule in the cells were detected. Results: After 6, 12. 18 and 24 h of treatment, the value for proliferative activity of cells treated with juglone was significantly lower than that of control group (p<0.05), and the anti-proliferative effect was more significant as the treatment period and juglone dosage increased (P<0.05). The protein expressions of Box, CytC, Fas, FasL, Caspase-3, and p-c-Jun in cells treated with juglone were significantly higher than those of control group (P<0.05), and the protein expressions of Bax, CytC, Fas. FasL, Caspase-3, p-JNK and p-c-Jun increased more remarkably as the juglone dosage increased (P<0.05). In cells treated with 40 pmol/L juglone and SP600125, the protein expressions of Bax, CytC, Fas. Fast.. and Caspase-3 were significantly lower than those of cells treated with 40 pmol/L juglone (J<0.05), and the protein expressions of Bax, CytC, Fas, FasL and Caspase-3 reduced more remarkably as the SP600125 dosage increased (P<0.05). Conclusion: Juglone can increase the expression of apoptotic molecules in mitochondrial pathway and death receptor pathway by activating JNK/c-Jun pathway, thus inducing apoptosis of cervical cancer cells.

All-trans Retinoic Acid Diminishes Collagen Production in a Hepatic Stellate Cell Line via Suppression of Active Protein-1 and c-Jun N-terminal Kinase Signal

Following acute and chronic liver injury,hepatic stellate cells (HSCs) become activated to undergo a phenotypic transformation into myofibroblast-like cells and lose their retinol content,but the mechanisms of retinoid loss and its potential roles in HSCs activation and liver fibrosis are not understood.The influence of retinoids on HSCs and hepatic fibrosis remains controversial.The purpose of this study was to evaluate the effects of all-trans retinoid acid (ATRA) on cell proliferation,mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),fibrolytic genes (MMP-3,MMP-13) and the upstream element (JNK and AP-1) in the rat hepatic stellate cell line (CFSC-2G).Cell proliferation was evaluated by measuring BrdU incorporation.The mRNA expression levels of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)],profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and fibrolytic genes (MMP-3,MMP-13) were quantitatively detected by using real-time PCR.The mRNA expression of JNK and AP-1 was quantified by RT-PCR.The results showed that ATRA inhibited HSCs proliferation and diminished the mRNA expression of collagen genes [procollagen α1 (Ⅰ),procollagen α1 (Ⅲ)] and profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly stimulated the mRNA expression of MMP-3 and MMP-13 in HSCs by suppressing the mRNA expression of JNK and AP-1.These findings suggested that ATRA could inhibit proliferation and collagen production of HSCs via the suppression of active protein-1 and c-Jun N-terminal kinase signal,then decrease the mRNAs expression of profibrogenic genes (TGF-β 1,CTGF,MMP-2,TIMP-1,TIMP-2,PAI-1),and significantly induce the mRNA expression of MMP-3 and MMP-13.

c-Jun N-terminal kinase 3 deficiency protects axotomized retinal ganglion cells via affecting mitochondria involved apoptosis pathway

AIM: To illustrate the isoform-specific role and mechanism of c-Jun N-terminal kinases(JNKs) in mouse optic nerve axotomy induced neurotrauma. METHODS: We firstly investigated the expression of JNK1, JNK2, and JNK3 in the retinal ganglion cells(RGCs) by double-immunofluorescent staining. Then we created optic nerve axotomy model in wild type as well as JNK1, JNK2, JNK3, isoform specific gene deficiency mice. With that, we checked the protein expression profile of JNKs and its active form, and quantified the survival RGCs number by immunofluorescence staining. We further explored the molecules underlying isoform specific protective effect by real-time polymerase chain reaction(PCR) and Western blotting assay. RESULTS: We found that all the three isoforms of JNKs were expressed in the RGCs. Deficiency of JNK3, but not JNK1 or JNK2, significantly alleviated optic nerve axotomyinduced RGCs apoptosis. We further established that expression of Noxa, a pro-apoptotic member of BH3 family, was significantly suppressed only in JNK3 gene deficiency mice. But tumor necrosis factor receptor 1(TNFR1) and Fas, two key modulators of death receptor mediated apoptosis pathway, did not display obvious change in the expression. CONCLUSION: It is suggested that mitochondria mediated apoptosis, but not death receptor mediated apoptosis got involved in the JNK3 gene deficiency induced RGCs protection. Our study provides a novel insight into the isoform-specific role of JNKs in neurotrauma and indicates some cues for its therapeutics.

S-adenosyl-methionine decreases ethanol-induced apoptosis in primary hepatocyte cultures by a c-Jun N-terminal kinase activity-independent mechanism

AIM： To determine the role of c-Jun N-terminal kinase （JNK） activity in ethanol-induced apoptosis and the modulation of this signaling cascade by S-Adenosylmethionine （AdoMet）. METHODS： Primary hepatocyte cultures were pretreated with 100 IJmol/L SP600125, a selective JNK inhibitor, 1 mL/L DMSO or 4 mmol/L AdoMet and then exposed to 100 mmo/L ethanol. Hepatocyte apoptosis was determined by the TUNEL and DNA ladder assays. JNK activity and its inhibition by SP600125 and AdoMet were determined by Western blot analysis of c-jun phosphorylation and Bid fragmentation. SP600125 and AdoMet effects on the apoptotic signaling pathway were determined by Western blot analysis of cytochrome c release and pro-caspase 3 fragmentation. The AdoMet effect on glutathione levels was measured by EIIman＇s method and reactive oxygen species （ROS） generation by cell cytometry. RESULTS： The exposure of hepatocytes to ethanol induced JNK activation, c-jun phosphorylation, Bid fragmentation, cytochrome c release and pro-caspase 3 cleavage; these effects were diminished by SP600125, and caused a significant decrease in ethanol-induced apoptosis （P〈 0.05）. AdoMet exerted an antioxidant effect maintaining glutathione levels and decreasing ROS generation, without a significant effect on JNK activity, and prevented cytochrome c release and pro-caspase 3 cleavage.CONCLUSION： The JNK signaling cascade is a key component of the proapoptotic signaling pathway induced by ethanol. JNK activation may be independent from ROS generation, since AdoMet which exerted antioxidant properties did not have a significant effect on JNK activity. JNK pathway modulator agents and AdoMet may be components of promising therapies for alcoholic liver disease （ALD） treatment.

c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed "lipoapoptosis," in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet(HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12(AML12) cells treated with palmitate(PA) were used as an in vitro model. RESULTS: LC3-Ⅱ, p62, and Run domain Beclin-1 interacting and cysteine-rich containing(Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase(JNK) inhibition at both the m RNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-Ⅱ and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown.CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide antifibrosis in the NASH liver.

c-Jun N-terminal kinase is required for thermotherapyinduced apoptosis in human gastric cancer cells

AIM:To investigate the role of c-Jun N-terminal kinase(JNK) in thermotherapy-induced apoptosis in human gastric cancer SGC-7901 cells.METHODS:Human gastric cancer SGC-7901 cells were cultured in vitro.Following thermotherapy at 43 ℃ for 0,0.5,1,2 or 3 h,the cells were cultured for a further 24 h with or without the JNK specific inhibitor,SP600125 for 2 h.Apoptosis was evaluated by immunohistochemistry [terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)] and flow cytometry(Annexin vs propidium iodide).Cell proliferation was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.The production of p-JNK,Bcl-2,Bax and caspase-3 proteins was evaluated by Western blotting.The expression of JNK at mRNA level was determined by reverse transcription polymerase chain reaction.RESULTS:The proliferation of gastric carcinoma SGC-7901 cells was significantly inhibited following thermotherapy,and was 32.7%,30.6%,43.8% and 52.9% at 0.5,1,2 and 3 h post-thermotherapy,respectively.Flow cytometry analysis revealed an increased population of SGC790l cells in G0/G1 phase,but a reduced population in S phase following thermotherapy for 1 or 2 h,compared to untreated cells(P < 0.05).The increased number of SGC-790l cells in G0/G1 phase was consistent with induced apoptosis(flow cytometry) following thermotherapy for 0.5,1,2 or 3 h,compared to the untreated group(46.5% ± 0.23%,39.9% ± 0.53%,56.6% ± 0.35% and 50.4% ± 0.29% vs 7.3% ± 0.10%,P < 0.01),respectively.This was supported by the TUNEL assay(48.2% ± 0.4%,40.1% ± 0.2%,61.2% ± 0.29% and 52.0% ± 0.42% vs 12.2% ± 0.22%,P < 0.01) respectively.More importantly,the expression of p-JNK protein and JNK mRNA levels were significantly higher at 0.5 h than at 0 h post-treatment(P < 0.01),and peaked at 2 h.A similar pattern was detected for Bax and caspase-3 proteins.Bcl-2 increased at 0.5 h,peaked at 1 h,and then decreased.Furthermore,the JNK specific inhibitor,SP600125,suppressed p-JNK,Bax and caspase-3 at the protein level in SGC790l cells following thermotherapy,compared to mock-inhibitor treatment,which was in line with the decreased rate of apoptosis.The expression of Bcl-2 was consistent with thermotherapy alone.CONCLUSION:Thermotherapy induced apoptosis in gastric cancer cells by promoting p-JNK at the mRNA and protein levels,and up-regulated the expression of Bax and caspase-3 proteins.Bcl-2 may play a protective role during thermotherapy.Activation of JNK via the Bax-caspase-3 pathway may be important in thermotherapy-induced apoptosis in gastric cancer cells.

Inhibition of c-Jun N-terminal Kinase Signaling Pathway Alleviates Lipopolysaccharide-induced Acute Respiratory Distress Syndrome in Rats

Background： An acute respiratory distress syndrome （ARDS） is still one of the major challenges in critically ill patients. This study aimed to investigate the effect of inhibiting c-Jun N-terminal kinase （JNK） on ARDS in a lipopolysaccharide （LPS）-induced ARDS rat model. Methods： Thirty-six rats were randomized into three groups： control, LPS, and LPS ＋ JNK inhibitor Rats were sacrificed 8 h alter LPS treatment. The lung edema was observed by measuring the wet-to-dry weight （W/D） ratio of the lung. The severity of ptdmonary inflammation was observed by measuring myeloperoxidase （MPO） activity of lung tissue. Moreover, the neutrophils in bronchoalveolar lavage fluid （BALF） were cotinted to observe the airway inflammation. In addition, lung collagen accumulation was quantified by Sircol Collagen Assay. At the same time, the pulmonary histologic examination was perlbrmed, and lung injury score was achieved in all three groups. Results： MPO activity in lung tissue was found increased in rats treated with LPS comparing with that in control （I.26 ＋ 0.15 U in LPS vs. 0.77 ± 0.27 U in control, P 〈 0.05）. Inhibiting ,INK attenuated LPS-induced MPO activity upregulation （0.52 ± 0. 12 U in LPS ＋ JNK inhibitor vs. 1.26 ± 0.15 U in LPS, P 〈 0.05）. Neutrophils in BALF were also found to be increased with LPS treatment, and inhibiting ,INK attenuated LPS-induced neutrophils increase in BALF （255.0 ± 164.4 in LPS vs. 53 （44.5-103） in control vs. 127.0 ± 44.3 in LPS JNK inhibitor, P 〈 0.05）. At the same time, the lung injury score showed a reduction in LPS ＋ JNK inhibitor group comparing with that in LPS group （ 13.42 ± 4.82 vs. 7.00 ± 1.83, P 0.001 ）. However, the lung W/D ratio and the collagen in BALF did not show any difl＇erences between LPS and LPS ＋ JNK inhibitor group.Conclusions： Inhibiting JNK alleviated LPS-induced acute lung inflammation and had no effects on pulmonary edema and fibrosis..INK inhibitor might be a potential therapeutic medication in ARDS, in the context of reducing lung inflammatory.

Signaling interactions among neurons impact cell fitness and death in Alzheimer's disease

The pathology of Alzheimer’s disease involves a long preclinical period,where the characteristic clinical symptoms of the changes in the brain are undetectable.During the preclinical period,homeostatic mechanisms may help prevent widespread cell death.Evidence has pointed towards selective cell death of diseased neurons playing a potentially protective role.As the disease progresses,dysregulation of signaling pathways that govern cell death contributes to neurodegeneration.Aberrant activation of the c-Jun N-terminal kinase pathway has been established in human and animal models of Alzheimer’s disease caused by amyloid-beta 42-or tau-mediated neurodegeneration.Clonal mosaic studies in Drosophila that examine amyloid-beta 42 in a subset of neurons suggest complex interplay between amyloid-beta 42-expressing and wild-type cells.This review examines the role of c-Jun N-terminal kinase signaling in the context of cell competition and short-range signaling interactions between amyloid-beta 42-expressing and wild-type neurons.Cell competition is a conserved phenomenon regulating tissue integrity by assessing the fitness of cells relative to their neighbors and eliminating suboptimal cells.Somatic clones of amyloid-beta 42 that juxtapose genetically distinct neuronal cell populations show promise for studying neurodegeneration.Generating genetic mosaics with labeled clones of amyloid-beta 42-or tau-expressing and wild-type neurons will allow us to understand how short-range signaling alterations trigger cell death in neurons and thereby contribute to the progression of Alzheimer’s disease.These approaches have the potential to uncover biomarkers for early Alzheimer’s disease detection and new therapeutic targets for intervention.

HER2-induced metastasis is mediated by AKT/JNK/EMT signaling pathway in gastric cancer

AIM To investigated the relationships between HER2, c-Jun N-terminal kinase(JNK) and protein kinase B(AKT) with respect to metastatic potential of HER2-positive gastric cancer(GC) cells.METHODS Immunohistochemistry was performed on tissue array slides containing 423 human GC specimens. Using HER2-positve GC cell lines SNU-216 and NCI-N87, HER2 expression was silenced by RNA interference, and the activations of JNK and AKT were suppressed by SP600125 and LY294002, respectively. Transwell assay, Western blot, semi-quantitative reverse transcriptionpolymerase chain reaction and immunofluorescence staining were used in cell culture experiments. RESULTS In GC specimens, HER2, JNK, and AKT activations were positively correlated with each other. In vitro analysis revealed a positive regulatory feedback loop between HER2 and JNK in GC cell lines and the role of JNK as a downstream effector of AKT in the HER2/AKT signaling pathway. JNK inhibition suppressed migratory capacity through reversing EMT and dual inhibition of JNK and AKT induced a more profound effect on cancer cell motility.CONCLUSION HER2, JNK and AKT in human GC specimens are positively associated with each other. JNK and AKT, downstream effectors of HER2, co-operatively contribute to the metastatic potential of HER2-positive GC cells. Thus, targeting of these two molecules in combination with HER2 downregulation may be a good approach to combat HER2-positive GC.

Jianpi Gushen Huayu decoction ameliorated diabetic nephropathy through modulating metabolites in kidney,and inhibiting TLR4/NF-κB/NLRP3 and JNK/P38 pathways

BACKGROUND Jianpi Gushen Huayu Decoction(JPGS)has been used to clinically treat diabetic nephropathy(DN)for many years.However,the protective mechanism of JPGS in treating DN remains unclear.AIM To evaluate the therapeutic effects and the possible mechanism of JPGS on DN.METHODS We first evaluated the therapeutic potential of JPGS on a DN mouse model.We then investigated the effect of JPGS on the renal metabolite levels of DN mice using non-targeted metabolomics.Furthermore,we examined the effects of JPGS on c-Jun N-terminal kinase(JNK)/P38-mediated apoptosis and the inflammatory responses mediated by toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB)/NOD-like receptor family pyrin domain containing 3(NLRP3).RESULTS The ameliorative effects of JPGS on DN mice included the alleviation of renal injury and the control of inflammation and oxidative stress.Untargeted metabolomic analysis revealed that JPGS altered the metabolites of the kidneys in DN mice.A total of 51 differential metabolites were screened.Pathway analysis results indicated that nine pathways significantly changed between the control and model groups,while six pathways significantly altered between the model and JPGS groups.Pathways related to cysteine and methionine metabolism;alanine,tryptophan metabolism;aspartate and glutamate metabolism;and riboflavin metabolism were identified as the key pathways through which JPGS affects DN.Further experimental validation showed that JPGS treatment reduced the expression of TLR4/NF-κB/NLRP3 pathways and JNK/P38 pathway-mediated apoptosis related factors.CONCLUSION JPGS could markedly treat mice with streptozotocin(STZ)-induced DN,which is possibly related to the regulation of several metabolic pathways found in kidneys.Furthermore,JPGS could improve kidney inflammatory responses and ameliorate kidney injuries in DN mice via the TLR4/NF-κB/NLRP3 pathway and inhibit JNK/P38 pathwaymediated apoptosis in DN mice.

Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways

Background:G-protein coupled receptors(GPCRs)are recognized as attractive targets for drug therapy.However,it remains poorly understood how GPCRs,except for a few chemokine receptors,regulate the progression of liver fibrosis.Here,we aimed to reveal the role of GPR65,a proton-sensing receptor,in liver fibrosis and to elucidate the underlying mechanism.Methods:The expression level of GPR65 was evaluated in both human and mouse fibrotic livers.Furthermore,Gpr65-deficient mice were treated with either bile duct ligation(BDL)for 21 d or carbon tetrachloride(CCl4)for 8 weeks to investigate the role of GPR65 in liver fibrosis.A combination of experimental approaches,including Western blotting,quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR),and enzyme-linked immunosorbent assay(ELISA),confocal microscopy and rescue studies,were used to explore the underlying mechanisms of GPR65’s action in liver fibrosis.Additionally,the therapeutic potential of GPR65 inhibitor in the development of liver fibrosis was investigated.Results:We found that hepatic macrophage(HM)-enriched GPR65 was upregulated in both human and mouse fibrotic livers.Moreover,knockout of Gpr65 significantly alleviated BDL-and CCl4-induced liver inflammation,injury and fibrosis in vivo,and mouse bone marrow transplantation(BMT)experiments further demonstrated that the protective effect of Gpr65knockout is primarily mediated by bone marrow-derived macrophages(BMMs).Additionally,in vitro data demonstrated that Gpr65 silencing and GPR65 antagonist inhibited,while GPR65 overexpression and application of GPR65 endogenous and exogenous agonists enhanced the expression and release of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and transforming growth factor-β(TGF-β),all of which subsequently promoted the activation of hepatic stellate cells(HSCs)and the damage of hepatocytes(HCs).Mechanistically,GPR65 overexpression,the acidic pH and GPR65 exogenous agonist induced up-regulation of TNF-αand IL-6 via the Gαq-Ca^(2+)-JNK/NF-κB pathways,while promoted the expression of TGF-βthrough the Gαq-Ca^(2+)-MLK3-MKK7-JNK pathway.Notably,pharmacological GPR65 inhibition retarded the development of inflammation,HCs injury and fibrosis invivo.Conclusions:GPR65 is a major regulator that modulates the progression of liver fibrosis.Thus,targeting GPR65 could be an effective therapeutic strategy for the prevention of liver fibrosis.

Hepato-protective effect of thymoquinone against acetaminophen induced liver injury is associated with regulation of JNK and AMPK signaling pathway

OBJECTIVE To investigate the hepato-protective mechanism of thymoquinone(TQ) on the development of acetaminophen(APAP)-induced liver injury.METHODS In vivo,male kunming mice were injected with a single dose of 300 mg·kg^(-1) APAP.Some mice were pretreated with TQ(5 or 20 mg·kg^(-1))and N-acetylcysteine(NAC,300 mg·kg^(-1))2 h before APAP injection.Mice were euthanized at 2 h,6 h,12 h after APAP treatment.In vitro,human Chang liver cells were incubated with 3.125,6.25 or 12.5μmol·L^(-1) TQ,10μmol·L^(-1) SP600125 and 500μmol·L^(-1) AICAR in the presence of APAP for 24 h.Cell viability were analyzed by MTT assay,protein expressions were assessed by Western blot.RESULTS TQ pretreatment significantly reduced serum aminotransferase and increased hepatic glutathione(GSH)and glutathione peroxidase(GSH-PX)activities,while significantly inhibited interleukin-1β(IL^(-1)β)levels.TQ significantly inhibited c-Jun N-terminal kinase(JNK),extracellular signal regulated kinase(ERK)and P38 phosphorylation induced by APAP.Moreover,TQ inhibited phosphatidylinositol 3-kinase(PI3K)/mammalian target of rapamycin(m TOR)signaling activation and activated AMPK phosphorylation induced by APAP.In addition,TQ inhibited signal transducer and activator of transcription 3(STAT3)phosphorylation on APAP-induced liver injury.In vitro,APAP enhanced JNK phosphorylation and attenuated AMPK phosphorylation in Chang liver cel s,and these effects were blocked by pretreatment with TQ,SP600125(JNK inhibitor)and AICAR(AMPK activator).CONCLUSION Our findings suggest that TQ may actively prevent APAP-induced liver injury,and this effect may be mediated by JNK and AMPK signaling pathways.

Role of JNK signaling pathway in sensitivity to radiotherapy of nasopharyngeal carcinoma

Objective:The aim of the study was to investigate the effect of c-Jun N-terminal protein kinase(JNK) signaling pathway on influencing the sensitivity to radiotherapy of human nasopharyngeal carcinoma CNE cells.Methods:Human nasopharyngeal carcinoma CNE multicellular spheroids(MCS) were constructed with three dimensional cell culture methods.Western blot was employed to analyze the activity of JNK signaling pathway in MCS after X-ray irradiation,and the expression of caspase-3 protein before and after using SP600125(a special inhibitor of JNK).X-ray induced cell apoptosis in MCS before and after treated with SP600125 were detected by TUNEL.Results:The level of JNK phosphorylation in MCS was a dynamic course after radiation,and there was a phosphorylation peaks at 2 h later,the apoptotic rate of MCS(P < 0.05) and the expression of caspase-3 protein(P < 0.05) were significantly increased after treated with SP600125.Conclusion:The transient activation of JNK played a important role in sensitivity to radiotherapy of CNE MCS via mediating survival signals,blocking this pathway accelerate cell apoptosis,which may be related to the increased expression of caspase-3.

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.

Effects of Different Therapeutic Methods and Typical Recipes of Chinese Medicine on Activation of c-Jun N-terminal Kinase in Kupffer Cells of Rats with Fatty Liver Disease

Objective： To observe the effects of different therapeutic methods and the recipes of Chinese medicine （CM） on the activation of c-Jun N-terminal kinase （JNK） in Kupffer cells of rats with fatty liver disease and to explore the mechanisms of these therapeutic methods. Methods： By using a random number table, 98 rats were randomly divided into 7 groups： control group, model group, and 5 treatment groups, including soothing Liver （Gan） recipe group, invigorating Spleen （Pi） recipe group, dispelling dampness recipe group, promoting blood recipe group, and complex recipe group. Rats in the control group were fed with normal food and distilled water by gastric perfusion, while rats in the model group were fed with high-fat food and distilled spirits by gastric perfusion. Rats in the 5 treatment groups were fed with high-fat food and corresponding recipes by gastric perfusion. Twelve weeks later, all rats were sacrificed and liver tissues were stained for pathohistological observation. Kupffer cells were isolated from livers of rats to evaluate JNK and phospho-JNK expressions by Western blotting. Results： The grade of hepatic steatosis was higher in the model group than the control group （P〈0.05）. Compared with the model group, the grade of fatty degeneration in soothing Liver recipe group and invigorating Spleen recipe group were significantly ameliorated （P〈0.05）. Expressions of JNK and phospho-JNK in Kupffer cells were significantly higher in the model group than those in the control group （P〈0.05, P〈0.01）. Compared with the model group, expressions of JNK in all treatment groups decreased, especially in invigorating Spleen recipe group and promoting blood recipe group （P〈0.05）. Compared with the model group, expressions of phospho-JNK in all treatment groups declined significantly （P〈0.01）, especially in soothing Live recipe group and invigorating Spleen recipe group. Conclusions： The high expressions of JNK and phospho-JNK in Kupffer cells might play an important role in the pathogenesis of fatty liver disease in rats. The recipes of CM, especially invigorating Spleen recipe and soothing Liver recipe, might protect liver against injury by reducing the total JNK protein content and inhibiting the activation of JNK protein in Kupffer cells of fatty liver model rats, which showed beneficial effects on fatty liver disease.

Activation of c-Jun N-terminal kinase 1/2 regulated by nitric oxide is associated with neuronal survival in hippocampal neurons in a rat model of ischemia

Background C-Jun N-terminal kinase （JNK） signaling pathway plays a critical role in cerebral ischemia. Although the mechanistic basis for this activation of JNK1/2 is uncertain, oxidative stress may play a role. The purpose of this study was to investigate whether the activation of JNK1/2 is associated with the production of endogenous nitric oxide （NO）. Methods Ischemia and reperfusion （I/R） was induced by cerebral four-vessel occlusion. Sprague-Dawley （SD） rats were divided into 6 groups： sham group, I/R group, neuronal nitric oxide synthase （nNOS） inhibitor （7-nitroindazole, 7-NI） given group, inducible nitric oxide synthase （iNOS） inhibitor （2-amino-5,6-dihydro-methylthiazine, AMT） given group, sodium chloride control group, and 1% dimethyl sulfoxide （DMSO） control group. The levels of protein expression and phospho-JNK1/2 were detected by Western blotting and the survival hippocampus neurons in CA1 zone were observed by cresyl violet staining. Results The study illustrated two peaks of JNK1/2 activation occurred at 30 minutes and 3 days during reperfusion. 7-NI inhibited JNK1/2 activation during the early reperfusion, whereas AMT preferably attenuated JNK1/2 activation during the later reperfusion. Administration of 7-NI and AMT can decrease I/R-induced neuronal loss in hippocampal CA1 region. Conclusion JNK1/2 activation is associated with endogenous NO in response to ischemic insult.

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.

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.

Smad3 phospho-isoform signaling in hepatitis C virus-related chronic liver diseases

The risk of hepatocellular carcinoma (HCC) development increases as hepatitis virus C (HCV)-related liver diseases progress, especially in patients with active inflammation. Insight into hepatic carcinogenesis have emerged from recent detailed analyses of transforming growth factor-&#x003b2; and c-Jun-N-terminal kinase signaling processes directed by multiple phosphorylated (phospho)-isoforms of a Smad3 mediator. In the course of HCV-related chronic liver diseases, chronic inflammation and host genetic/epigenetic alterations additively shift the hepatocytic Smad3 phospho-isoform signaling from tumor suppression to carcinogenesis, increasing the risk of HCC. Chronic inflammation represents an early carcinogenic step that provides a nonmutagenic tumor-promoting stimulus. After undergoing successful antiviral therapy, patients with chronic hepatitis C could experience a lower risk of HCC as Smad3 phospho-isoform signaling reverses from potential carcinogenesis to tumor suppression. Even after HCV clearance, however, patients with cirrhosis could still develop HCC because of sustained, intense carcinogenic Smad3 phospho-isoform signaling that is possibly caused by genetic or epigenetic alterations. Smad3 phospho-isoforms should assist with evaluating the effectiveness of interventions aimed at reducing human HCC.