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 retinoi...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.展开更多
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(...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.展开更多
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(...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 μmol/L, respectively) and c-Jun N-terminal kinase(JNK) inhibitor SP600125(10, 20, and 40 μmol/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 Bax, Cyt C, Fas, Fas L, Caspase-3, p-JNK 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, Cyt C, Fas, Fas L, Caspase-3, p-JNK and p-c-Jun increased more remarkably as the juglone dosage increased(P<0.05). In cells treated with 40 μmol/L juglone and SP600125, the protein expressions of Bax, Cyt C, Fas, Fas L and Caspase-3 were significantly lower than those of cells treated with 40 μmol/L juglone(P<0.05), and the protein expressions of Bax, Cyt C, Fas, Fas L 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.展开更多
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 thermo...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.展开更多
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 positi...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.展开更多
AIM:To determine the role of c-Jun N-terminal kinase(JNK)activity in ethanol-induced apoptosis and themodulation of this signaling cascade by S-Adenosyl-methionine(AdoMet).METHODS:Primary hepatocyte cultures werepretr...AIM:To determine the role of c-Jun N-terminal kinase(JNK)activity in ethanol-induced apoptosis and themodulation of this signaling cascade by S-Adenosyl-methionine(AdoMet).METHODS:Primary hepatocyte cultures werepretreated with 100 μmol/L SP600125,a selective JNKinhibitor,1 mL/L DMSO or 4 mmol/L AdoMet and thenexposed to 100 mmo/L ethanol.Hepatocyte apoptosiswas determined by the TUNEL and DNA ladder assays.JNK activity and its inhibition by SP600125 and AdoMetwere determined by Western blot analysis of c-junphosphorylation and Bid fragmentation.SP600125 andAdoMet effects on the apoptotic signaling pathway weredetermined by Western blot analysis of cytochrome crelease and pro-caspase 3 fragmentation.The AdoMeteffect on glutathione levels was measured by Ellman'smethod and reactive oxygen species(ROS)generationby cell cytometry.RESULTS:The exposure of hepatocytes to ethanolinduced JNK activation,c-jun phosphorylation,Bidfragmentation,cytochrome c release and pro-caspase 3cleavage;these effects were diminished by SP600125,and caused a significant decrease in ethanol-inducedapoptosis(P<0.05).AdoMet exerted an antioxidanteffect maintaining glutathione levels and decreasing ROSgeneration,without a significant effect on JNK activity,and prevented cytochrome c release and pro-caspase 3cleavage. CONCLUSION:The JNK signaling cascade is a keycomponent of the proapoptotic signaling pathwayinduced by ethanol.JNK activation may be independentfrom ROS generation,since AdoMet which exertedantioxidant properties did not have a significant effect onJNK activity.JNK pathway modulator agents and AdoMetmay be components of promising therapies for alcoholicliver disease(ALD)treatment.展开更多
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...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.展开更多
c-Jun NH2-terminal kinase(JNK)-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B(Trk B) anterograde axonal transport. It remains unclear whether JNK-in...c-Jun NH2-terminal kinase(JNK)-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B(Trk B) anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.展开更多
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 phosp...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.展开更多
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, inc...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.展开更多
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 fourvesse...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.展开更多
The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off...The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramati- cally inhibited 24 h later. However, the expresion of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.展开更多
Pancreatic cancer is a dismal disease with high incidence and poor survival rates.With the aim to improve overall survival of pancreatic cancer patients,new therapeutic approaches are urgently needed.Protein kinases a...Pancreatic cancer is a dismal disease with high incidence and poor survival rates.With the aim to improve overall survival of pancreatic cancer patients,new therapeutic approaches are urgently needed.Protein kinases are key regulatory players in basically all stages of development,maintaining physiologic functions but also being involved in pathogenic processes.c-Jun N-terminal kinases(JNK)and p38 kinases,representatives of the mitogen-activated protein kinases,as well as the casein kinase 1(CK1)family of protein kinases are important mediators of adequate response to cellular stress following inflammatory and metabolic stressors,DNA damage,and others.In their physiologic roles,they are responsible for the regulation of cell cycle progression,cell proliferation and differentiation,and apoptosis.Dysregulation of the underlying pathways consequently has been identified in various cancer types,including pancreatic cancer.Pharmacological targeting of those pathways has been the field of interest for several years.While success in earlier studies was limited due to lacking specificity and off-target effects,more recent improvements in small molecule inhibitor design against stress-activated protein kinases and their use in combination therapies have shown promising in vitro results.Consequently,targeting of JNK,p38,and CK1 protein kinase family members may actually be of particular interest in the field of precision medicine in patients with highly deregulated kinase pathways related to these kinases.However,further studies are warranted,especially involving in vivo investigation and clinical trials,in order to advance inhibition of stress-activated kinases to the field of translational medicine.展开更多
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 cultu...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.展开更多
文摘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.
文摘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.
基金supported by the Hainan Health Department(2002lx-12)
文摘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 μmol/L, respectively) and c-Jun N-terminal kinase(JNK) inhibitor SP600125(10, 20, and 40 μmol/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 Bax, Cyt C, Fas, Fas L, Caspase-3, p-JNK 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, Cyt C, Fas, Fas L, Caspase-3, p-JNK and p-c-Jun increased more remarkably as the juglone dosage increased(P<0.05). In cells treated with 40 μmol/L juglone and SP600125, the protein expressions of Bax, Cyt C, Fas, Fas L and Caspase-3 were significantly lower than those of cells treated with 40 μmol/L juglone(P<0.05), and the protein expressions of Bax, Cyt C, Fas, Fas L 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.
基金Supported by A grant from the National Eleventh Five-Year Technology Support Project of China,No. 2008 BAI68B01
文摘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.
基金supported by the National Natural Science Foundation of China, No.30872705/HD426 and No.81070538/HD429
文摘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.
文摘AIM:To determine the role of c-Jun N-terminal kinase(JNK)activity in ethanol-induced apoptosis and themodulation of this signaling cascade by S-Adenosyl-methionine(AdoMet).METHODS:Primary hepatocyte cultures werepretreated with 100 μmol/L SP600125,a selective JNKinhibitor,1 mL/L DMSO or 4 mmol/L AdoMet and thenexposed to 100 mmo/L ethanol.Hepatocyte apoptosiswas determined by the TUNEL and DNA ladder assays.JNK activity and its inhibition by SP600125 and AdoMetwere determined by Western blot analysis of c-junphosphorylation and Bid fragmentation.SP600125 andAdoMet effects on the apoptotic signaling pathway weredetermined by Western blot analysis of cytochrome crelease and pro-caspase 3 fragmentation.The AdoMeteffect on glutathione levels was measured by Ellman'smethod and reactive oxygen species(ROS)generationby cell cytometry.RESULTS:The exposure of hepatocytes to ethanolinduced JNK activation,c-jun phosphorylation,Bidfragmentation,cytochrome c release and pro-caspase 3cleavage;these effects were diminished by SP600125,and caused a significant decrease in ethanol-inducedapoptosis(P<0.05).AdoMet exerted an antioxidanteffect maintaining glutathione levels and decreasing ROSgeneration,without a significant effect on JNK activity,and prevented cytochrome c release and pro-caspase 3cleavage. CONCLUSION:The JNK signaling cascade is a keycomponent of the proapoptotic signaling pathwayinduced by ethanol.JNK activation may be independentfrom ROS generation,since AdoMet which exertedantioxidant properties did not have a significant effect onJNK activity.JNK pathway modulator agents and AdoMetmay be components of promising therapies for alcoholicliver disease(ALD)treatment.
文摘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.
基金supported by the Henan Province Education Department Key Project of Science and Technology Research in China,No.12A350006
文摘c-Jun NH2-terminal kinase(JNK)-interacting protein 3 plays an important role in brain-derived neurotrophic factor/tropomyosin-related kinase B(Trk B) anterograde axonal transport. It remains unclear whether JNK-interacting protein 1 mediates similar effects, or whether JNK-interacting protein 1 affects the regulation of Trk B anterograde axonal transport. In this study, we isolated rat embryonic hippocampus and cultured hippocampal neurons in vitro. Coimmunoprecipitation results demonstrated that JNK-interacting protein 1 formed Trk B complexes in vitro and in vivo. Immunocytochemistry results showed that when JNK-interacting protein 1 was highly expressed, the distribution of Trk B gradually increased in axon terminals. However, the distribution of Trk B reduced in axon terminals after knocking out JNK-interacting protein 1. In addition, there were differences in distribution of Trk B after JNK-interacting protein 1 was knocked out compared with not. However, knockout of JNK-interacting protein 1 did not affect the distribution of Trk B in dendrites. These findings confirm that JNK-interacting protein 1 can interact with Trk B in neuronal cells, and can regulate the transport of Trk B in axons, but not in dendrites.
文摘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.
基金supported by the National Natural Science Foundation of ChinaNo.81271387+3 种基金the Research Special Fund of Public Welfare and Health Department of ChinaNo.201402009the National Key Technology R&D Program in ChinaNo.Z141107002514031
文摘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.
基金supported by grants from the National Natural Science Foundation of China (No.30871200)the Practice and Innovation Training Program for Students in Colleges and Universities of Jiangsu Province (NO.20090370)
文摘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.
基金supported by National Natural Science Foundation of China(No.30270556)The National Basic Research Program(No.2002CB513004).
文摘The basal activity of JNK is low in normal growing cells and inactivated JNK targets p53 for ubiquitination. To elucidate if the C-terminal part of JNK is responsible for its binding to p53, the low background tet-off inducible NIH3T3 cell line was selected by luciferase reporter gene and a double stable C-JNK Aa (203-424) cell line was established. After withdrawing tetracycline, the C-JNK fragment expression was induced and cell growth was dramati- cally inhibited 24 h later. However, the expresion of p53 was found to be increased after the induction of C-JNK fragment, evaluated by transfecting p21waf-luciferase reporter genes. Our further studies showed that C-JNK fragment could form complex with p53 both in vivo and in vitro. Induction of C-JNK fragment in vivo can increase p53 stability by inhibiting p53 ubiquitination.
基金German Research Foundation(DFG),No.TR1663/1-1 and No.KN356/9-1and Else Kröner-Fresenius-Stiftung,No.2017_A142.
文摘Pancreatic cancer is a dismal disease with high incidence and poor survival rates.With the aim to improve overall survival of pancreatic cancer patients,new therapeutic approaches are urgently needed.Protein kinases are key regulatory players in basically all stages of development,maintaining physiologic functions but also being involved in pathogenic processes.c-Jun N-terminal kinases(JNK)and p38 kinases,representatives of the mitogen-activated protein kinases,as well as the casein kinase 1(CK1)family of protein kinases are important mediators of adequate response to cellular stress following inflammatory and metabolic stressors,DNA damage,and others.In their physiologic roles,they are responsible for the regulation of cell cycle progression,cell proliferation and differentiation,and apoptosis.Dysregulation of the underlying pathways consequently has been identified in various cancer types,including pancreatic cancer.Pharmacological targeting of those pathways has been the field of interest for several years.While success in earlier studies was limited due to lacking specificity and off-target effects,more recent improvements in small molecule inhibitor design against stress-activated protein kinases and their use in combination therapies have shown promising in vitro results.Consequently,targeting of JNK,p38,and CK1 protein kinase family members may actually be of particular interest in the field of precision medicine in patients with highly deregulated kinase pathways related to these kinases.However,further studies are warranted,especially involving in vivo investigation and clinical trials,in order to advance inhibition of stress-activated kinases to the field of translational medicine.
基金in part by Natural Sciences Foundation of China (No. 39870239)by the Sasagawa Fellowship,Japan.
文摘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.