Administration Of SP600125 Research Articles

Butyrate protects the intestinal barrier by upregulating Fut2 expression via MEK4-JNK signaling pathway activation.

Necrotizing enterocolitis (NEC) is a severe gastrointestinal inflammatory disease in neonates. Fucosyltransferase 2 (Fut2) regulates intestinal epithelial cell fucosylation. In this study, we aimed to investigate butyrate-mediated upregulation of Fut2 expression and the underlying mechanisms. In vivo and in vitro models were established. SP600125 was used to inhibit the MEK4-JNK pathway, and anisomycin was used to activate the MEK4-JNK pathway. Fut2, occludin, and ZO-1 expressions were assessed. Furthermore, intestinal permeability was analyzed by FITC-Dextran. The expression of proteins in the MEK-4-JNK pathway was examined by western blotting. In vivo, the addition of exogenous butyrate notably upregulated Fut2, occludin, and ZO-1 expressions and reduced intestinal permeability in mice with NEC. Butyrate may increase the phosphorylation of MEK4, JNK, and c-jun, which are key components of the MEK4-JNK pathway. Additionally, SP600125 inhibited their phosphorylation, which was reversed by anisomycin treatment. In vitro, butyrate substantially increased occludin and ZO-1 expressions. Butyrate considerably increased Fut2 expression and markedly upregulated p-MEK4, p-JNK, and p-c-jun expressions. SP600125 administration decreased their expressions, while anisomycin administration increased their expressions. Butyrate upregulated Fut2 expression via activation of the MEK4-JNK pathway, improved intestinal barrier integrity, and protected neonatal mice from NEC. We found that exogenous butyrate could improve intestinal barrier integrity and protect against NEC in neonatal mice. Our data showed that exogenous butyrate supplementation upregulated Fut2 expression by activating the MEK4-JNK pathway. Our study provides novel insights into the pathogenesis of NEC, thereby laying an experimental foundation for future clinical research on the use of butyrate in NEC treatment.

NCMP-18. NEUROLOGICAL SEQUELAE IN THE ID8 OVARIAN CANCER MOUSE MODEL: P38/JNK MAPK INHIBITION AS A POTENTIAL THERAPEUTIC FOR CANCER-RELATED COGNITIVE IMPAIRMENTS AND CHEMOTHERAPY-INDUCED PERIPHERAL NEUROPATHY

Abstract OBJECTIVES Chemotherapy-related cognitive impairment (CRCI) and chemotherapy-induced peripheral neuropathy (CIPN) are neurological sequelae of platinum-based chemotherapy. To examine the contribution of cancer itself and additional neurological impairment with chemotherapy, we used the ID8 syngeneic ovarian cancer mouse model and assessed cognition and hyperalgesia +/- cisplatin treatment. We examined the effect of p38 and c-JUN N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) inhibition on cisplatin-induced neuronal damage. METHODS B6 female mice were injected with 107 ID8 cells or 0.9% saline, i.p. Mice received cisplatin (2.3 m¬¬¬g/kg/day, i.p.) or 0.9% saline (OvT+CIS, OvT+VEH, respectively) for 5d, followed by 5d of rest for two cycles. Cognition was assessed longitudinally at 69d by the open field test (OFT), novel object recognition (NOR) at 75d and 104d, and novel place recognition (NPR) at 106d post-ID8 implantation. Hyperalgesia was assessed at 113d. Primary mouse and rat hippocampal neurons were pre-treated with VX-745 or SP600125, followed by cisplatin, and neuronal morphology was assessed. RESULTS OvT+VEH mice had detectable abdominal tumors 90d post-implantation. At 75d, OvT+VEH and OvT+CIS had impairments in NOR, with discrimination ratios (DR)= 0.52, 0.51, respectively. OvT+VEH showed trending differences vs. OvT+CIS (DR=0.39, DR=0.62, p=0.079) on NOR on 104d, and impairments on NPR on 106d (DR=0.41, DR=0.63, p=0.03). Cisplatin decreased mechanical (p< 0.05) and cold hyperalgesia (p< 0.07, n.s.) in OvT+CIS vs. OvT+VEH. VX-745 and SP600125 pre-treatment in mouse and rat hippocampal neurons prevented cisplatin-induced dendritic branching and spine density loss (p< 0.05). DISCUSSION While cisplatin transiently increased anxiogenic behavior, cognitive impairments, and hyperalgesia in OvT+CIS, these deficits persisted longitudinally in OvT+VEH, suggesting ovarian cancer may evoke sensory and progressive neurocognitive deficits in the absence of chemotherapy. Future studies will address hyperalgesia and cognitive differences between healthy control and ovarian cancer mice +/- cisplatin, and whether VX-745 and SP600125 administration ameliorates CRCI/CIPN in this model.

Blocking GSDME-mediated pyroptosis in renal tubular epithelial cells alleviates disease activity in lupus mice

An increase in apoptosis and/or defects in the clearance of apoptotic cells resulting in massive secondary necrosis have been recognized as the main causes of systemic lupus erythematosus (SLE). Recent findings have revealed that gasdermin E (GSDME)-mediated pyroptosis is a mechanism associated with secondary necrosis. We aimed to investigate the effects of GSDME-mediated pyroptosis on disease activity in lupus mice. In vivo, high levels of GSDME expression were observed in the renal tubules of pristane-induced lupus (PIL) mice and SLE patients. In lupus mice, GSDME knockout or SP600125 administration effectively ameliorated lupus-like features by inhibiting GSDME-mediated renal tubular epithelial cell pyroptosis. In vitro, treatment with tumour necrosis factor-α (TNF-α) plus cycloheximide (CHX) or SLE sera induced HK2 cells to undergo pyroptosis in a caspase-3- and GSDME-dependent manner. Likewise, SP600125 significantly reduced GSDME expression and decreased pyroptosis in HK2 cells. GSDME-mediated pyroptosis may be associated with SLE pathogenesis, and targeting GSDME may be a potential strategy for treating SLE.

JNK Inhibition Modulates the Cytoskeleton, Hypoxia, and Neurogenesis on the Protein Level in Glioblastoma Cells and Astrocytes: An Immunofluorescence Study.

To evaluate the effects of c-Jun N-terminal kinase (JNK) inhibition and signal blocking on hypoxia (hypoxia-inducible factor 1-alpha (HIF-1α)), differentiation and neurogenesis (bone morphogenetic protein (BMP4)), and the cytoskeleton (F-actin) in glioblastoma multiforme cells (GBMCs). We evaluated the differences between GBMCs and astrocytes in terms of the abovementioned parameters and assessed them with the aim of studying human GBMCs (U-87 MG) and astrocytes (SVG p12). The cells were exposed to different doses of the JNK inhibitor, SP600125, for 24, 48, and 72 hours. HIF-1α, BMP4, and F-actin expressions were evaluated using immunofluorescence image analysis. The half-maximal inhibitory concentration value for SP600125 was determined to be 10 μM at 24 hours of exposure. After SP600125 administration, elevated levels of HIF-1α and BMP4 were detected in GBMCs and astrocytes. F-actin level only increased in GBMCs after SP600125 administration. JNKs are important for cell proliferation, differentiation, survival, and death; thus, research on JNKs has become important for the treatment of many human diseases, especially brain tumors, Parkinson's disease, and Alzheimer's disease. The results of this study involving immunofluorescence techniques should be investigated and supported by studies that involve comprehensive molecular techniques.

NCMP-16. THE ROLE OF p38 AND JNK MAPK PATHWAYS IN CISPLATIN CHEMOTHERAPY-RELATED COGNITIVE IMPAIRMENT

Abstract OBJECTIVES Chemotherapy-related cognitive impairment (CRCI) is an adverse sequela of cancer treatment commonly reported in cancer survivors. Cisplatin is used for the treatment of various malignancies including ovarian, testicular, head and neck cancers, and pediatric brain tumors. More than 30% of advanced ovarian cancer patients develop CRCI during and after platinum-based chemotherapy. We examined the role of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) activation in cisplatin-induced CRCI, and whether the small molecule p38 MAPK inhibitor Neflamapimod and JNK inhibitor SP600125, can prevent cisplatin-induced neuronal damage. The p38 and JNK MAPK signaling pathways are involved in various stress response pathways in the CNS including oxidative stress. METHODS The effect of cisplatin on cognition in an ovarian cancer female rat model was assessed by novel object recognition (NOR). Hippocampal glutathione levels were measured post-behavioral testing. P38 and JNK MAPK signaling activation were assessed in the neural cell lines PC12 and SH-SY5Y by Western blot. Cultured hippocampal neurons were pretreated with Neflamapimod or SP600125 followed by cisplatin for 24 hours, and dendritic spine density and branch length were quantified. RESULTS Cisplatin increased phospho-p38 and phospho-JNK MAPK protein levels in PC12 and SH-SY5Y cells. Cisplatin reduced dendritic branching and spine density, which was prevented by Neflamapimod and SP600125 pre-treatment in hippocampal neurons, in vitro. Chronic cisplatin treatment decreased hippocampal glutathione levels and impaired cognitive function in the ovarian cancer rat model. DISCUSSION The cognitive deficits caused by cisplatin results in part from dendritic damage and neural apoptosis, which is mediated by oxidative stress and the p38 and JNK MAPK pathways. P38 and JNK MAPK inhibition mitigated cisplatin-induced dendritic spine loss and branching in vitro. Next, we will examine whether Neflamapimod and SP600125 administration in an ovarian cancer rat model is safe and if they can prevent cognitive impairment.

Inhibition of inflammation by SP600125 in cholestatic liver injury is dependent on the administration‑based exposure profile.

SP600125 is a classic inhibitor of c‑Jun‑N‑terminal kinase (JNK) that is widely used in numerous medicinal studies, but its administration regimen has yet to be optimized. In the present study, intraperitoneal (i.p.) and intragastric (i.g.) injections of 15mg/kg SP600125 was performed in mice to compare the inhibitory effect against JNK signalling in cholestasis induced by α‑naphthylisothiocyanate (ANIT). SP600125 at a dose of 15mg/kg administered by i.p. substantially decreased ANIT‑induced liver injury as observed by biochemical and histopathological examinations. The adaptation of bile acid synthesis was inhibited in the A‑SP‑i.p. group compared with that in the A‑SP‑i.g. group, as indicated by the expression analysis of CYP7A1 and CYP8B1. The transcription of the pro‑inflammatory factors IL‑6, IL‑1β, ICAM‑1 and IL‑10 supported the differential toxic responses. Western blot analysis revealed that JNK signalling activated by ANIT was inhibited more markedly in the A‑SP‑i.p. group than in the A‑SP‑i.g. group. The peak concentration and the AUC0‑24 of SP600125 in the A‑SP‑i.p. group were 5‑fold and 1.56‑fold higher, respectively, compared with those in the A‑SP‑i.g. group. These data indicated that i.p. administration of SP600125 produced a high plasma exposure profile, which directly determined its efficacy of blocking the JNK signalling. This effect of SP600125 on the JNK pathway may provide an optimized design for future invivo investigations.

The JNK pathway represents a novel target in the treatment of rheumatoid arthritis through the suppression of MMP-3

Background and aimThe pathophysiology of rheumatoid arthritis (RA) is characterized by excess production of pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) by neutrophils and macrophages in synovium. Additionally, these cytokines promote the production of reactive oxygen species (ROS), and increased production of matrix metalloproteinases (MMPs), including MMP-3, in synoviocytes that result in joint destruction. There is limited information on how proteolytic enzymes such as MMP-3 can be regulated. We evaluated the effect of the antioxidant N-acetylcysteine (NAC) on RA and identified the relationship between the c-Jun N terminal kinase (JNK) pathway and MMP-3. We hypothesized that elucidating this relationship would lead to novel therapeutic approaches to RA treatment and management.MethodsWe investigated the effect of administering a low dose (1000 μM or less) of an antioxidant (NAC) to human rheumatoid fibroblast-like synoviocytes (MH7A cells). We also investigated the response of antioxidant genes such as nuclear factor erythroid -derived 2-related factor 2 (Nrf2) and Sequestosome 1 (p62). The influence of MMP-3 expression on the JNK pathway leading to joint destruction and the mechanisms underlying this relationship were investigated through primary dispersion culture cells collected from the synovial membranes of RA patients, consisting of rheumatoid arthritis-fibroblast-like synoviocytes (RA-FLS).ResultsLow-dose NAC (1000 μM) increased the expression of Nrf2 and phospho-p62 in MH7A cells, activating antioxidant genes, suppressing the expression of MMP-3, and inhibiting the phosphorylation of JNK. ROS, MMP-3 expression, and IL-6 was suppressed by administering 30 μM of SP600125 (a JNK inhibitor) in MH7A cells. Furthermore, the administration of SP600125 (30 μM) to RA-FLS suppressed MMP-3.ConclusionsWe demonstrated the existence of an MMP-3 suppression mechanism that utilizes the JNK pathway in RA-FLS. We consider that the JNK pathway could be a target for future RA therapies.

Inhibition of JNK Alleviates Chronic Hypoperfusion-Related Ischemia Induces Oxidative Stress and Brain Degeneration via Nrf2/HO-1 and NF-κB Signaling.

Cerebral ischemia is one of the leading causes of neurological disorders. The exact molecular mechanism related to chronic unilateral cerebral ischemia-induced neurodegeneration and memory deficit has not been precisely elucidated. In this study, we examined the effect of chronic ischemia on the induction of oxidative stress and c-Jun N-terminal kinase-associated detrimental effects and unveiled the inhibitory effect of specific JNK inhibitor (SP600125) on JNK-mediated brain degeneration in adult mice. Our behavioral, biochemical, and immunofluorescence studies revealed that chronic ischemic injuries sustained increased levels of oxidative stress-induced active JNK for a long time, whereas SP600125 significantly reduced the elevated level of active JNK and further regulated Nrf2/HO-1 and NF-κB signaling, which have been confirmed in vivo. Neuroinflammatory mediators and loss of neuronal cells was significantly reduced with the administration of SP600125. Ischemic brain injury caused synaptic dysfunction and memory impairment in mice. However, these were significantly improved with SP600125. On the whole, these findings suggest that elevated ROS-mediated JNK is a key mediator in chronic ischemic conditions and has a crucial role in neuroinflammation, neurodegeneration, and memory dysfunction. Our findings suggest that chronic oxidative stress associated JNK would be a potential target in time-dependent studies of chronic ischemic conditions induced brain degeneration.

Protective properties of heme oxygenase-1 expressed in umbilical cord mesenchymal stem cells help restore the ovarian function of premature ovarian failure mice through activating the JNK/Bcl-2 signal pathway-regulated autophagy and upregulating the circulating of CD8+CD28\u2212 T cells

BackgroundUmbilical cord-derived mesenchymal stem cell (UCMSCs) transplantation has been widely studied in premature ovarian failure (POF). However, the underlying mechanism remains elusive. This study aims to investigate the protective properties and mechanisms of heme oxygenase-1 (HO-1) expressed in UCMSCs in restoring the ovarian function of POF mice.MethodsIn in vitro and in vivo experiments, mice were treated with the presence or absence of the HO-1/shHO-1-transfected UCMSCs, and the administration of SP600125 or anisomycin, the inhibitor or activator of JNK. The viability and apoptosis of granulosa cells (GCs) at different time points of co-cultivation were assessed in vitro. In in vivo experiments, mouse ovarian function was assessed by detecting the serum levels of hormone and observing the ovarian morphological changes. Multiple molecular indices of JNK/Bcl-2 signal pathway were performed. And the autophagy changes in GCs were assessed by detecting the associated cytokines and observing the intracellular autophagosome accumulation. Additionally, the spleen levels of CD8+CD28− T cells and serum levels of interleukin 10 (IL-10) were tested to evaluate the immune mechanisms involved.ResultsUCMSCs transfected with shHO-1 or treated with SP600125 inhibited GCs’ viability and promoted its apoptosis in a time-dependent manner in vitro. In in vivo experiments, mice in both groups showed little therapeutic efficiency which presented as the increased extent of ovarian fibrosis with decreased number of functional follicles, and disordered hormone production. Additionally, the JNK/Bcl-2-associated cytokines were obviously declined. The inhibited autophagy-related cytokines, the chromatin condensation and abound vacuolar autophagosome in GCs, and weakened fluorescence intensity by MDC were observed. The downregulated levels of CD8+CD28− T cells and serum levels of IL-10 were also detected. The damages above can be alleviated with HO-1-MSCs treatment or anisomycin administration.ConclusionsHO-1 expressed in UCMSCs is critical in restoring the ovarian function in POF mice with UCMSC transplantation, which is mediated by the activation of JNK/Bcl-2 signal pathway-regulated autophagy and upregulating the circulating of CD8+CD28− T cells.

Roles of SP600125 in expression of JNK, RANKL and OPG in cultured dental follicle cells.

To investigate the expression of C-JNK, RANKL and OPG after SP600125 administration in cultured dental follicle cells (DFCs). TRAP staining and electron microscope were carried out on day 7 and 9 after coculture of BMMs and DFCs with a ratio of 5:1 in different groups. To determine the effects of SP600125 on the expression of C-JNK, RANKL and OPG mRNA and protein, cultured DFCs were divided into control group, DMSO group and SP600125 groups. Real-time PCR and Western blot analysis were performed to investigate the expression of the mRNA and protein, respectively. TRAP assay indicated that the number of multinucleated osteoclasts in the SP600125 group showed significant decrease compared with that of control (P < 0.05). The expression of JNK protein in the SP600125 groups showed significant decline compared with that of the control group and blank control (P < 0.05). Significant decrease was noticed in the RANKL protein expression with the elevation of SP600125. SP600125 could inhibit the formation of osteoclast in the coculture system of DFCs and BMMs. After SP600125 treatment, the expression of RANKL and JNK showed a trend of decrease, and the expression of OPG showed gradual increase followed by gradual decrease.

Electrical acupuncture moderately decreases cardiac ischemiareperfusion injury in rats

Restoration of blood flow to the ischemic myocardium termed reperfusion unavoidably leads to many heart complications (such as rhythm abnormalities, and sequentially heart failure), together with the induced damage of cardiomyocytes, which can worsen infarct size and is termed myocardial ischemia-reperfusion (I/R) injury. Electroacupuncture plays an important role in attenuating I/R injury. High mobility group box-1 protein (HMGB1) is upregulated in myocardial I/R injury and activates the inflammatory response which enhances the myocardial injury. In our study, we aimed to detect the roles of electrical acupuncture at Neiguan acupoints (PC6) in myocardial I/R injury and investigate the relationship between electrical acupuncture and HMGB1-related pathway. We found that electrical acupuncture pretreatment protected against myocardial I/R injury through inhibiting cell apoptosis. Electroacupuncture also decreased the expression levels of HMGB1 and inflammatory cytokines induced by I/R injury. In addition, the phosphorylation levels of JNK (c-Jun N-terminal kinase) was decreased by electroacupuncture in heart tissues. Furthermore, the effects of electrical acupuncture pretreatment were abrogated by the additional administration of recombinant HMGB1. Finally, the SP600125 administration, a selective JNK inhibitor, attenuated HMGB1-dependent cell apoptosis and cytokines release in myocardial I/R injury. In conclusion, our data demonstrate that electrical acupuncture at PC6 plays a protective role in myocardial I/R injury, and this effect is associated with inhibited HMGB1-JNK pathway, which provides a beneficial therapeutic strategy for patients with myocardial I/R injury.

Astrocyte contributes to pain development via MMP2-JNK1/2 signaling in a mouse model of complex regional pain syndrome

BackgroundThe activation of spinal glial cells (astrocyte and microglia) is reported in patient with complex regional pain syndrome (CRPS). However, the roles of spinal glial activities in the pathophysiology of CRPS are unclear. Here, we explored the roles of spinal astrocyte and microglia and the molecular mechanisms underlying CRPS using a mouse model of chronic post-ischemia pain (CPIP). ResultsCPIP injury increased the level of glial fibrillary acidic protein (GFAP, reactive astrocyte biomarker), but had no significant impact on ionized calcium binding adaptor molecule 1 (IBA1, reactive microglia biomarker), in the ipsilateral dorsal horn on post-injury day (PID) 3 when the pain threshold started to reduce significantly. Astrocytic inhibition with fluorocitrate but not microglial inhibition with minocycline attenuated the development of allodynia in CPIP-injured mice, which was concomitant with increased spinal levels of phosphorylated c-jun N-terminal kinase 1/2 (pJNK1/2) on PID 3. Furthermore, the intrathecal administration of SP600125 (JNK inhibitor) prevented the development of allodynia in CPIP-injured mice. Double immunofluorescence staining showed that pJNK1/2 was mainly co-localized with GFAP. Subsequently, increased levels of pJNK1/2 were reversed by intrathecal fluorocitrate. Furthermore, the level of spinal matrix metalloproteinase-2 (MMP2) was increased and mainly expressed in NeuN (neuron biomarker) on PID 3 in the CPIP-injured mice, while intrathecal APR 100 (MMP2 inhibitor) delayed the development of allodynia and decreased spinal levels of GFAP and pJNK1/2 on PID 3. ConclusionThis study shows that activation of astrocyte MMP2/JNK1/2 signaling pathway contributes to the pathogenesis of pain hypersensitivity in the CPIP model.

Thrombin-induced apoptosis in neurons through activation of c-Jun-N-terminal kinase

Context: Studies have shown that thrombin activation played a central role in cell injuries associated with intracerebral hemorrhage (ICH).Objective: Here, our study investigated the cytotoxicity of thrombin on neurons, and determined the involvement of JNK pathways in thrombin-induced neuronal apoptosis.Materials and methods: Primary cultured neurons were treated with different doses of thrombin. Some neurons were given either SP600125 or vehicle. LDH release assay and flow cytometry were used to measure neuronal apoptosis caused by thrombin. The activation of JNK and capases-3 were measured by Western blot.Results: Our results showed large doses of thrombin that increased the LDH release, the level of cleaved caspase-3 and apoptosis rate of neurons. JNK was activated by thrombin in a time-dependent manner. Administration of SP600125 protects neurons from thrombin-induced apoptosis.Conclusion: These data indicate that the activation of JNK is crucial for thrombin-induced neuronal apoptosis, and inhibition of JNK may be a potential therapeutic target for ICH.

In vitro and in vivo neuroprotective effects of cJun N-terminal kinase inhibitors on retinal ganglion cells.

BackgroundThe c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in neuronal pathophysiology. Using JNK inhibitors, we examined involvement of the JNK pathway in cultured rat retinal ganglion cell (RGC) death and in mouse retinal ischemia/reperfusion (I/R) injury of the visual axis. The in vitro effects of JNK inhibitors were evaluated in cultured adult rat retinal cells enriched in RGCs. Retinal I/R was induced in C57BL/6J mice through elevation of intraocular pressure to 120 mmHg for 60 min followed by reperfusion. SP600125 was administered intraperitoneally once daily for 28 days. Phosphorylation of JNK and c-Jun in the retina was examined by immunoblotting and immunohistochemistry. The thickness of retinal layers and cell numbers in the ganglion cell layer (GCL) were examined using H&E stained retinal cross sections and spectral domain optical coherence tomography (SD-OCT). Retinal function was measured by scotopic flash electroretinography (ERG). Volumetric measurement of the superior colliculus (SC) as well as VGLUT2 and PSD95 expression were studied.ResultsJNK inhibitors SP600125 and TAT-JNK-III, dose-dependently and significantly (p < 0.05) protected against glutamate excitotoxicity and trophic factor withdrawal induced RGC death in culture. In the I/R model, phosphorylation of JNK (pJNK) in the retina was significantly (p < 0.05) increased after injury. I/R injury significantly (p < 0.05) decreased the thickness of retinal layers, including the whole retina, inner plexiform layer, and inner nuclear layer and cell numbers in the GCL. Administration of SP600125 for 28 days protected against all these degenerative morphological changes (p < 0.05). In addition, SP600125 significantly (p < 0.05) protected against I/R-induced reduction in scotopic ERG b-wave amplitude at 3, 7, 14, 21 and 28 days after injury. SP600125 also protected against the I/R-induced losses in volume and levels of synaptic markers in the SC. Moreover, the protective effects of SP600125 in the retina and SC were also detected even with only 7 days (Days 1–7 after I/R) of SP600125 treatment.ConclusionsOur results demonstrate the important role the JNK pathway plays in retinal degeneration in both in vitro and in vivo models and suggest that JNK inhibitors may be a useful therapeutic strategy for neuroprotection of RGCs in the retina.Electronic supplementary materialThe online version of this article (doi:10.1186/s13024-016-0093-4) contains supplementary material, which is available to authorized users.

Reduction of autophagy markers mediated protective effects of JNK inhibitor and bucladesine on memory deficit induced by Aβ in rats.

Autophagy, the process of self-degradation of cellular components, has an important role in neurodegenerative diseases, such as Alzheimer's disease. In this study, we investigated the effects of SP600125 as c-Jun N-terminal kinase (JNK) inhibitor and bucladesine as a cyclic adenosine 3',5'-monophosphate (cAMP) analog on spatial memory and expression of autophagic factors in Aβ-injected rats. Male Wistar rats were used. Rats were randomly allocated into five groups as following: amyloid beta (Aβ)-only group, Aβ + SP600125 (30 μg/1 μ/side, n = 7) and/or bucladesine (100 μM/1 μl/side, n = 7), and the normal control (vehicle only) group. The treatments were administered bilaterally to the CA1 sub-region of the hippocampus stereotaxically. Spatial reference memory was performed using Morris Water Maze 21 days later. The expression of authophagy markers (beclin1, Atg7, Atg12, and LC3 II/LC3 I) in the hippocampus was evaluated using western blotting. Compared to the vehicle group, Aβ administration reduced spatial reference learning (P < 0.001) and memory (P < 0.01) and upregulated the expression of beclin1, Atg7, Atg12, and LC3 II/I (P < 0.0001). Compare to Aβ-only group, the administration of SP600125 and/or bucladesine improved spatial reference learning (P < 0.001) and memory (P < 0.01). Compared to the Aβ-only group, the treatment with SP600125 and/or bucladesine also reduced beclin1, Atg7, Atg12, and LC3 II/I (P < 0.0001) which was similar to amount of normal rats. In summary, it seems that the improvement of spatial memory by SP600125 and/or bucladesine in Aβ-injected rats is in relation with normalizing of autophagy to the physiologic level, possibly through neuroprotection and/or neuroplasticity.

JNK in spinal cord facilitates bone cancer pain in rats through modulation of CXCL1.

In patients with advanced cancer, cancer-induced bone pain (CIBP) is a severe and common problem that is difficult to manage and explain. As c-Jun N-terminal kinase (JNK) and chemokine (C-X-C motif) ligand 1 (CXCL1) have been shown to participate in several chronic pain processes, we investigated the role of JNK and CXCL1 in CIBP and the relationship between them. A rat bone cancer pain model was established by intramedullary injection of Walker 256 rat gland mammary carcinoma cells into the left tibia of Sprague-Dawley rats. As a result, intramedullary injection of Walker 256 carcinoma cells induced significant bone destruction and persistent pain. Both phosphorylated JNK1 (pJNK1) and pJNK2 showed time-dependent increases in the ipsilateral spinal cord from day 7 to day 18 after tumor injection. Inhibition of JNK activation by intrathecal administration of SP600125, a selective pJNK inhibitor, attenuated mechanical allodynia and heat hyperalgesia caused by tumor inoculation. Tumor cell inoculation also induced robust CXCL1 upregulation in the ipsilateral spinal cord on day 18 after tumor injection. Inhibition of CXCL1 by intrathecal administration of CXCL1 neutralizing antibody showed a stable analgesic effect. Intrathecal administration of SP600125 reduced CXCL1 increase in the spinal cord, whereas inhibition of CXCL1 in the spinal cord showed no influence on JNK activation. Taken together, these results suggested that JNK activation in spinal cord contributed to the maintenance of CIBP, which may act through modulation of CXCL1. Inhibition of the pJNK/CXCL1 pathway may provide a new choice for treatment of CIBP.

Combined Activities of JNK1 and JNK2 in Hepatocytes Protect Against Toxic Liver Injury

c-Jun N-terminal kinase (JNK) 1 and JNK2 are expressed in hepatocytes and have overlapping and distinct functions. JNK proteins are activated via phosphorylation in response to acetaminophen- or carbon tetrachloride (CCl4)-induced liver damage; the level of activation correlates with the degree of injury. SP600125, a JNK inhibitor, has been reported to block acetaminophen-induced liver injury. We investigated the role of JNK in drug-induced liver injury (DILI) in liver tissue from patients and in mice with genetic deletion of JNK in hepatocytes. We studied liver sections from patients with DILI (dueto acetaminophen, phenprocoumon, nonsteroidal anti-inflammatory drugs, or autoimmune hepatitis) or patients without acute liver failure (controls) collected from a DILI Biobank in Germany. Levels of total and activated (phosphorylated) JNK were measured by immunohistochemistry and Western blotting. Mice with hepatocyte-specific deletion of Jnk1 (Jnk1(Δhepa)) or combination of Jnk1 and Jnk2 (Jnk(Δhepa)), as well as Jnk1-floxed C57BL/6 (control) mice, were given injections of CCl4 (to induce fibrosis) or acetaminophen (to induce toxic liver injury). We performed gene expression microarray and phosphoproteomic analyses to determine mechanisms of JNK activity in hepatocytes. Liver samples from DILI patients contained more activated JNK, predominantly in nuclei of hepatocytes and in immune cells, than healthy tissue. Administration of acetaminophen to Jnk(Δhepa) mice produced a greater level of liver injury than that observed in Jnk1(Δhepa) or control mice, based on levels of serum markers and microscopic and histologic analysis of liver tissues. Administration of CCl4 also induced stronger hepatic injury in Jnk(Δhepa) mice, based on increased inflammation, cell proliferation, and fibrosis progression, compared with Jnk1(Δhepa) or control mice. Hepatocytes from Jnk(Δhepa) mice given acetaminophen had an increased oxidative stress response, leading to decreased activation of adenosine monophosphate-activated protein kinase, total protein adenosine monophosphate-activated protein kinase levels, and pJunD and subsequent necrosis. Administration of SP600125 before or with acetaminophen protected Jnk(Δhepa) and control mice from liver injury. In hepatocytes, JNK1 and JNK2 appear to have combined effects in protecting mice from CCl4- and acetaminophen-induced liver injury. It is important to study the tissue-specific functions of both proteins, rather than just JNK1, in the onset of toxic liver injury. JNK inhibition with SP600125 shows off-target effects.

Activation of JNK pathway in spinal astrocytes contributes to acute ultra-low-dose morphine thermal hyperalgesia.

Accumulating evidence suggests that opioid analgesics can lead to paradoxical sensitization to pain when delivered in different administration patterns. Although opioid tolerance-induced hyperalgesia is largely studied, little is known about the mechanisms underlying acute ultra-low-dose morphine hyperalgesia. Activation of spinal glial cells is reported to regulate pain hypersensitivity. To elucidate the mechanism involved in acute ultra-low-dose morphine hyperalgesia, we tested whether an opioid agonist promoted the activation of spinal astrocytes and microglia and investigated the cellular pathways involved. Ultra-low-dose morphine activated spinal astrocytes with no effect on microglia. The astrocyte activation was selectively prevented by the opioid antagonist naloxone, the μ-opioid receptor (MOR) silencing and the JNK inhibitor SP600125. Morphine elevated spinal JNK1, JNK2, and c-Jun phosphorylation. Conversely, phosphorylation of cAMP response element-binding protein (CREB) and signal transducer and activator of transcription-1 (STAT-1) was not elevated, and nuclear factor kappa B (NF-κB) levels remained unmodified. Administration of SP600125 and the N-methyl-D-aspartate (NMDA) antagonist MK801 prevented morphine hyperalgesia. Ultra-low-dose morphine increased protein kinase C (PKC) γ phosphorylation. Pretreatment with a PKC inhibitor prevented morphine hyperalgesia and JNK and c-Jun overphosphorylation, indicating PKC is a JNK upstream modulator and illustrating the presence of a pathway involving PKC, NMDA, and JNK activated by morphine. Immunofluorescence experiments indicated the neuronal localization of spinal MOR. However, JNK was not detected in MOR-expressing cells, showing the presence of a neuron-astrocyte signaling pathway. These results illustrate the selective activation of an astrocyte JNK pathway after the stimulation of neuronal MOR, which contributes to ultra-low-dose morphine hyperalgesia.

Inhibition of c-Jun N-terminal kinase activation reverses Alzheimer disease phenotypes in APPswe/PS1dE9 mice.

Growing evidence indicates that the activation of c-Jun N-terminal kinase (JNK) is implicated in the multiple major pathological features of Alzheimer disease (AD). However, whether specific inhibition of JNK activation could prevent disease progression in adult transgenic AD models at moderate stage remains unknown. Here we first investigated the potential disease-modifying therapeutic effect of systemic administration of SP600125, a small-molecule JNK-specific inhibitor, in middle-aged APPswe/PS1dE9 mice. Using behavioral, histological, and biochemical methods, outcomes of SP600125 treatment on neuropathology and cognitive deficits were studied in APPswe/PS1dE9 mice. Compared with vehicle-treated APPswe/PS1dE9 mice, chronic treatment of SP600125 for 12 weeks potently inhibited JNK activation, which resulted in a marked improvement of behavioral measures of cognitive deficits and a dramatic reduction in amyloid plaque burden, β-amyloid production, tau hyperphosphorylation, inflammatory responses, and synaptic loss in these transgenic animals. In particular, we found that SP600125 treatment strongly promoted nonamyloidogenic amyloid precursor protein (APP) processing and inhibited amyloidogenic APP processing via regulating APP-cleavage secretase expression (ie, ADAM10, BACE1, and PS1) in APPswe/PS1dE9 mice. Our findings demonstrate that chronic SP600125 treatment is powerfully effective in slowing down disease progression by markedly reducing multiple pathological features and ameliorating cognitive deficits associated with AD. This study highlights the concept that active JNK actually contributes to the development of the disease, and provides critical preclinical evidence that specific inhibition of JNK activation by SP600125 treatment may be a novel promising disease-modifying therapeutic strategy for the treatment of AD.

Protective Effects of SP600125 in Brain Death Induced Liver Injury.

Objectives: The aim of the present study was to investigate the protective effect of SP600125, a selective c-Jun N-terminal kinase inhibitor, in brain death induced liver injury. Methods: All 40 Sprague-Dawley rats are anesthetized and brain death model is established. Analysis of liver histology, function, JNK phosphorylation status, as well as apoptosis related protein was evaluated in this study. Results: As a result,SP600125 diminished the increased phosphorylation of JNK, whereas, expression of total JNK in the liver remained unchanged compared with the sham control and was not affected by SP600125. At the same time, SP600125 attenuated Bax translocation to mitochondria and the release of cytochrome c induced by brain death. Furthermore, the activation of caspase-3 and apoptosis induced by brain death was also significantly suppressed by administration of SP600125. Conclusions: The results obtained from the present study suggested that targeting the JNK pathway provided a therapeutic target in liver injury induced by brain death.