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Objective To investigate the role of c-Jun NH2-terminal kinase (c-JNK) signaling pathway on voltage-gated potassium channel (Kv) remodeling in left ventricular myocytes of diabetic rats,and explore the intrinsic regulatory mechanism.Methods Forty-five SD rats were randomly divided into DM group (n=25,modeling with streptozotocin induction) and control group (n=20,fed with normal diet).Transient outward potassium current (Ito) of rats' ventricular myocytes in DM group and control group was recorded by whole-cell patch-clamp method.The c-Jun activity was detected using a non-radioactive JNK kinase assay kit (Cell Signaling Technology).JNK inhibitor SP600125 was used to incubate the cardiomyocytes of diabetes rats in vitro,and then the changes of I,o in cardiomyocytes were observed.Thioredoxin reductase (TrxR) inhibitor--auranofin (AF) was used to treat the rats' cardiomyocytes incubated with SP600125,and then the changes of Ito in cardiomyocytes were observed.The content of Kv4.2 was tested using anti-Kv4.2 antibody,and the results were analyzed using a UVP bioimaging system.Results The JNK activity in DM group rose more than 1 times compared with control group,while the density of Ito decreased significantly (Control:30.2 ± 3.3pA/pF,n=16;DM:15.3 ± 2.1pA/pF,n=17;P＜0.05).The ventricular myocytes of DM rats were treated with SP600125 (10μmol/Lol/L) for 4 hours,then the Ito density increased to control group level (DM+SP600125:32.3 ± 3.7pA/pF,n=18;Control:30.2 ± 3.3pA/pF,n=16;P＜0.05).There was no significant difference in the maximum Ito density between the treated with SP600125 (Control+SP600125:31.6 ± 3.4pA/pF,n=18) and untreated control groups.The Ito density in DM myocardial cells significantly increased after treatment with the membrane permeable protein inhibitor JNKI-1 (10μmol/L),and no changes were found in control group after the same treatment.The augmentation effect of SP600125 on Ito current in DM myocytes was significantly inhibited by TrxR inhibitor auranofin (lμmol/L) (DM+SP600125+AF:15.7 ± 3.3pA/pF,n=15),while AF did not change the Ito density in control group.The expression of Kv4.2 protein was significantly increased in DM rats after administration of SP600125,which was consistent with the changes of Ito current observed in the myocardium of DM rats,although not fully restored to the level of control group myocardium.JNK inhibitor did not markedly alter the expression of Kv4.2 protein in control group myocardium.Conclusions Kv channel remodeling in DM rat's myocardium is redox-regulated,and the Ito remodeling might be assisted with the persistent activation of c-JNK signaling pathway.It has showed that c-JNK activity is significantly increased in DM rat heart and the current density of Kv channels is reduced.The inhibition of JNK signaling pathway can markedly improve Kv channel reconstruction and the process may be regulated by thioredoxin system.
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Glucagon like peptide-1 (GLP-1) stimulates glucose-dependent insulin secretion. Dipeptidyl peptidase-4 (DPP-4) inhibitors, which block inactivation of GLP-1, are currently in clinical use for type 2 diabetes mellitus. Recently, GLP-1 has also been reported to have neuroprotective effects in cases of cerebral ischemia. We therefore investigated the neuroprotective effects of GLP-1 receptor (GLP-1R) agonist, exendin-4 (ex-4), after cerebral ischemia-reperfusion injury. Transient middle cerebral artery occlusion (tMCAO) was induced in rats by intracerebroventricular (i.c.v.) administration of ex-4 or ex9-39. Oxygen-glucose deprivation was also induced in primary neurons, bEnd.3 cells, and BV-2. Ischemia-reperfusion injury reduced expression of GLP-1R. Additionally, higher oxidative stress in SOD2 KO mice decreased expression of GLP-1R. Downregulation of GLP-1R by ischemic injury was 70% restored by GLP-1R agonist, ex-4, which resulted in significant reduction of infarct volume. Levels of intracellular cyclic AMP, a second messenger of GLP-1R, were also increased by 2.7-fold as a result of high GLP-1R expression. Moreover, our results showed that ex-4 attenuated pro-inflammatory cyclooxygenase-2 (COX-2) and prostaglandin E₂ after MCAO. C-Jun NH₂ terminal kinase (JNK) signaling, which stimulates activation of COX-2, was 36% inhibited by i.c.v. injection of ex-4 at 24 h. Islet-brain 1 (IB1), a scaffold regulator of JNK, was 1.7-fold increased by ex-4. GLP-1R activation by ex-4 resulted in reduction of COX-2 through increasing IB1 expression, resulting in anti-inflammatory neuroprotection during stroke. Our study suggests that the anti-inflammatory action of GLP-1 could be used as a new strategy for the treatment of neuroinflammation after stroke accompanied by hyperglycemia.
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Animals , Mice , Rats , Brain Ischemia , Cyclic AMP , Cyclooxygenase 2 , Diabetes Mellitus, Type 2 , Down-Regulation , Glucagon , Glucagon-Like Peptide 1 , Glucagon-Like Peptide-1 Receptor , Hyperglycemia , Infarction, Middle Cerebral Artery , Insulin , Neurons , Neuroprotection , Neuroprotective Agents , Oxidative Stress , Phosphotransferases , Reperfusion Injury , Second Messenger Systems , StrokeABSTRACT
Objective To explore the protective effect and probable mechanism of JNK inhibitor SP600125 on hippocampal neurons in rats with status epilepsy following lithium?pilocarpine. Methods 48 Wistar rats,in accordance with the random number table,were divided into control,status epilepticus ( SE) and JNK in?hibitor SP600125 group ( SP ) . HE staining and fluorescent TUNEL method were used to observe pathological changes and neuronal apoptosis in the hippocampal area of rats in each group. Western blot was applied to detect the phosphorylation expression of JNK and its downstream effector molecule c?JUN in hippocampal tissues of rats in each group. Results Compared with control group,neuronal loss and apoptosis in CA3 area of hippocampus in SE group were significant (percentage of TUNEL positive cells (26.34±3.04)%, P<0.05). The mortality of rats was significantly decreased and neuronal loss and apoptosis were obviously reduced in SP group than in SE group ( mor?tality in SP and SE group :6.25%,37.5% respectively, P<0.05). Meanwhile,the expression levels of phospho?JNK and phospho?c?JUN were significantly increased in hippocampus of rats in SE group ( The relative OD values respectively 0.447±0.025,0.552±0.035, P<0.05 compared with Control group). After treated with SP600125 in SP group,the phosphorylation levels of JNK and c?JUN were obviously decreased ( The relative OD values respec?tively 0.211±0.016,0.237±0.028, P<0.05 compared with SE group). Conclusion JNK inhibitor SP600125 may play an important protective effect on neurons in the rat hippocampus after status epilepticus through inhibition of JNK and c?JUN phosphorylation.
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Objective To examine the role of c-Jun NH2-terminal kinase(JNK) 1 in cytolytic T lymphocyte (CTL) responses against virus infection.Methods Wild-type (WT) and JNK1-knockout (JNK1-/-) mice were infected with ectromelia virus(ECTV) through hind footpads.Survival and virus titers in the target organs (liver and spleen) were analyzed.Effector T cells in the spleen and popliteal lymph nodes were determined on day 3 and 7 post-infection.Proliferation and INF-γ production of CTL were also detected.Results JNK1 deficiency caused an increased susceptibility to ECTV infection in mice,indicated by higher case fatality and viral burden in target organs.The decrease in CTL response correlated with a defect in CTL proliferation and INF-γproduction.Conclusion The data suggest that JNK1 is involved in expansion of activated CTL during ECTV infection,and plays an important antiviral role in regulating the proliferation and effector function of CTL.
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Objective: To explore the role of c-Jun NH2 terminal kinase (JNK) activation and JNK-mediated apoptotic signal pathway in intestinal ischemia/reperfusion (II/R) in mice. Methods: C57BL/6 mice were randomly divided into sham-operated group (n = 6) and II/R groups (n = 36); the latter was further divided according to time after perfusion (0,0. 5,1,4,6 and 12 h). Animal II/R model was established by clamping the superior mesenteric artery (SMA) for 40 min followed by reperfusion. Animals in the sham-operated group received no clamping. Animals in the two groups were sacrificed at defined time points, and the expression of JNK, phosphorylation (phospho-) JNK, cleaved caspase-3,Bcl-2 and Bax protein in the intestinal tissue was examined by Western blotting analysis, and the pathological changes of ileum tissue were observed under optical microscope. Results: Most severe intestinal injury was found at the early stage of reperfusion, and the intestinal tissues almost recovered 12 h later. The phospho-JNK in the intestine was significantly elevated within 1 h after II/R compared with sham group (P<0. 01). Cleaved caspase-3 was significantly increased in II/R group at 0. 5 h, 1 h after reperfusion compared to sham group (P<0. 01); the expression of Bcl-2 protein in II/R group was significantly decreased compared with the sham-operated group (P<0. 01), and there was no significant difference in Bax expression between different groups. Conclusion: JNK phosphorylation plays an essential role in the intestinal damages induced by II/R,possibly through down-regulating Bcl-2 protein expression and caspase-3 dependent apoptosis pathway.
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AIM: To study the role of c-Jun NH_2-terminal kinase (JNK) in the development of insulin resistance induced by tumor necrosis factor-α (TNF-α) or H_2O_2 in 3T3-L1 adipocytes. METHODS: Differentiated 3T3-L1 adipocytes were pretreated with JNK1 small interfering RNA (siRNA) or JNK inhibitor SP600125, then exposed to 1 nmol/L of TNF-α or micromolar H_2O_2 generated by adding glucose oxidase (50 U/L) to the medium for 12 h. The cellular glucose uptake was determined by radioactive method. RESULTS: Compared to control adipocytes, 12 h incubation with TNF-α or H_2O_2 led to 50%-55% reduction (P<0.01) of the insulin-dependent glucose uptake. JNK1 siRNA transfection significantly inhibited JNK1 expression and blocked the TNF-α or H_2O_2-induced impairments of cellular glucose uptake. Pretreatment with SP600125 (20 μmol/L) resulted in significant increases in insulin-stimulated glucose uptakes in both TNF-α (66%) and H_2O_2 (62%) treated adipocytes (P<0.01). CONCLUSION: JNK plays a key role in TNF-α or H_2O_2 induced insulin resistance in 3T3-L1 adipocytes, and inhibition of JNK over-activation may be a new therapeutic target for insulin resistance.
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Objective To study the expression of JNK/SAPK(c-Jun NH2-terminal kinase/stress activated protein kinase)in lung and bronchus of radon-exposed mice.Methods Male BALB/c mice were exposed to radon and its progeny with the cumulative dose of 0.02,30 or 60 working level month(WLM),respectively.The expression levels of JNK/SAPK in lung and bronchus were determined with Real-Time PCR,Western blot and immunohistochemistry methods.Results The JNK mRNA levels in lung tissues of mice exposed to radon of 30 and 60 WLM were higher than those of the control by 3.56 and 2.96 times,respectively.The relative expression levels of JNK and phospho-JNK proteins were higher than those of the control by using Western blot and immunohistochemistry methods.Condusiom Expose to the radon and its progeny might activate the JNK/SAPK intracellular signaling pathway.
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Objective:To investigate the effects of Chlamydia pneumoniae(Cpn) on SR-A1 and CD36 expression in THP-1-derived macrophages and role of c-Jun NH_2-terminal signal transduction pathway in the process.Methods:Cpn was propagated in Hep-2 cells.THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate(PMA)for 48h,and were randomly allocated into four groups to be incubated continually: control group;Cpn infection group;Cpn and SP600125(a JNK inhibiter)group and SP600125 group.Lipid droplets in cytoplasm were observed by oil red O staining.The contents of intracellular cholesterol ester were detected by enzyme-fluorescence.The expression of SR-A1 and CD36mRNA and protein were determined by RT-PCR and Western blot, respectively. Results:THP-1-derived macrophages infected with Cpn resulted in large accumulation of lipid droplets and foam cell formation when co-cultured with LDL.Meanwhile,the expression of SR-A1 mRNA and protein were up-regulated by Cpn infection (P<0.05).However,the expressions of CD36 mRNA and protein in THP-1-derived macrophages infected with Cpn were unchanged.Moreover,the up-regulation of SR-A1 and foam cell formation induced by Cpn could be restrained by the JNK inhibiter SP600125 in a dose-dependent manner,and SP600125 had little impact on the expression of CD36 in THP-1-derived macrophages infected with Cpn.Conclusion:The up-regulation of SR-A1 but not CD36 expression is involved in mechanisms of Cpn inducing foam cell formation.And Chlamydia pneumoniae up-regulates the expression of SR-A1 via the JNK signal transduction pathway.This may be a novel mechanism for the foam cell formation induced by Cpn.
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Objective:To investigate survival and apoptotic responses of alveolar epithelial typeⅡcells(ATⅡ cell)under oxidative stress and to discuss the regulation mechanism mediated by c-jun NH2-terminal kinase(JNK).Methods:Primary rat alveolar epithelial typeⅡcells were attacked by Hydrogen peroxide(H2O2)and cells were pretreated with SP600125 in some groups.Cell viability,apoptotic rate and the expression of phosphorylated JNK(p-JNK)were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,flow cytometry and western blot analysis,respectively.Results:Compared with control group,decreased cell viability and increased apoptotic rate in ATⅡ cells occurred in time-dependent manner when treated with 500?mol/L H2O2 in different time.H2O2 induces phosphorylation of JNK.SP600125 enhanced cell viability and decreased apoptotic rate after H2O2-exposure.Conclusion:Apoptosis could be induced by H2O2 in ATⅡ cells in time-dependent manner.JNK signaling pathway may play a proapoptotic role in the regulation of apoptosis induced by H2O2.
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PURPOSE: In a developing kidney, the renin-angiotensin system(RAS) is markedly activated and is thought to play an important role in postnatal renal growth and maturation. We previously demonstrated that angiotensin converting enzyme(ACE) inhibition in a developing rat kidney increases apoptosis and decreases its related gene expressions, which may account for renal growth impairment. Among the mitogen-activated protein kinases(MAPK) family members, c-jun N terminal kinase(JNK) and p38 MAPK(p38) are thought to inhibit cell growth and induce apoptosis. This study was designed to investigate the relationship between the RAS and the MAPK family during neonatal renal development. METHODS: Forty-nine neonatal Spargue-Dawley rats were separated into two groups. The enalapril group was treated with ACE inhibitor(enalapril:30 mg/kg/day) and the control group with normal saline for seven days. Their kidneys were removed for immunohistochemical stain and western blot analysis of JNK-2 and p38. RESULTS: In the enalapril group, JNK-2 expression was strongly detected in the dilated cortical tubular epithelial cells, and JNK-2 protein expression was significantly increased compared to the control group. p38 expression was noted in the dilated tubular epithelial cells by ACE inhibitor and also p38 protein expression was significantly increased. CONCLUSION: These results suggest that the expressions of the MAPK family, especially JNK and p38, are modulated by ACE inhibition in the neonatal rat kidney. In regard of the correlation between MAPK activations and the occurrence of apoptosis in renal growth impairment by ACE inhibition, JNK and p38 may be implicated to participate in angiotensin II related intracellular signaling pathways of renal apoptosis in developing kidney.
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Animals , Humans , Rats , Angiotensin II , Angiotensins , Apoptosis , Blotting, Western , Enalapril , Epithelial Cells , Gene Expression , Kidney , Mitogen-Activated Protein Kinases , Models, Animal , p38 Mitogen-Activated Protein Kinases , Peptidyl-Dipeptidase A , Protein Kinases , Renin-Angiotensin SystemABSTRACT
Objective To explore the effect of c-Jun NH2-terminal kinase(JNK)signal transduction pathway on hyperoxia-induced lung injury in rats.Methods Twenty-four Wistar rats aged 3 weeks were randomly divided into 3 groups(n=8):room-air control group,7 d hyperoxia exposure group,and 7 d hyperoxia exposure with inhibitor of JNK intervention group.The rats in hyperoxia exposure group were exposed to high concentration of oxygen [fractional concentration of inspired oxygen(FiO2)≥950 mL?L-1] at normal pressure.The rats in room-air control group were placed in room air(FiO2=210 mL?L-1)at normal pressure.The rats in JNK inhibitor intervention group were intraperitoneally injected 30 mg?kg-1 SP600125 and exposed to hyperoxia 2 h later.The histopathological changes of lung tissues were observed by means of light microscope,therefore the changes of lung W/D weight ratio,total protein in bronchoalveolar lavage fluid(BALF)and lung permeation index were detected.The extent of lung cells apoptosis was analyzed by terminal deoxynucleotidyltrans-ferase-mediated dUTP nick end labeling assay.The protein level of p-JNK was measured by Western blotting analysis.Results Compared with room-air control group,conspicuous hyperaemia,edema,hemorrhage and extensive inflammation cells infiltration in the lung tissues were significantly observed in 7 d hyperoxia exposure group.The lung W/D weight ratio,total protein in BALF,lung permeation index,cell apoptotic index and the p-JNK protein levels of lung tissues all significantly increased in 7 d hyperoxia exposure group compared with those in room-air control group(Pa
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AIM:To study the role of c-Jun NH2-terminal kinase (JNK) in the development of insulin resistance induced by tumor necrosis factor-? (TNF-?) or H2O2 in 3T3-L1 adipocytes. METHODS:Differentiated 3T3-L1 adipocytes were pretreated with JNK1 small interfering RNA (siRNA) or JNK inhibitor SP600125,then exposed to 1 nmol/L of TNF-? or micromolar H2O2 generated by adding glucose oxidase (50 U/L) to the medium for 12 h. The cellular glucose uptake was determined by radioactive method. RESULTS:Compared to control adipocytes,12 h incubation with TNF-? or H2O2 led to 50%-55% reduction (P
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AIM: To find out the mechanism of focal adhesion kinase(FAK) facilitating human pulmonary artery smooth muscle cells(HPASMCs) proliferation.METHODS: HPASMCs were isolated from normal part of lungs of two carcinoma patients who undergone lung partial resection.Cultured HPASMCs stimulated by fibronection(40 mg/L) were passively transfected with ODNs,sense focal adhesion kinase(FAK),mismatch sense and antisense-FAK,respectively.Expression of FAK,Jun NH2-terminal kinase(JNK) and cyclin-dependent kinase2(CDK2) proteins were detected by immunoprecipitation and Western blotting.Cell cycle and cell apoptosis were analyzed by flow cytometry.In addition,cytoplasma FAK expression was detected by immunohistochemistry staining.RESULTS: The protein expressions of FAK,JNK and CDK2 in HPASMCs decreased in FAK ASODNs group and increased in FAK SODNs group.Meanwhile,the proportion of cells at G1 phase decreased significantly in FAK SODNs group,while the cells at S phase increased significantly.In contrast,the proportion of cells at G1 phase was increased significantly in FAK ASODNs group.The level of cell apoptosis in FAK ASODNs group was higher.FAK expression in FAK SODNs group was strongly stained by immunocytochemistry,whereas that in FAK ASODNs group was weakly stained.CONCLUSION: The results suggest that FAK via JNK,CDK2 signaling pathway enhances HPASMCs proliferation.