Administration Of SP600125 Research Articles

Protective effects of SP600125 in brain death-induced liver injury

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. All 40 Sprague-Dawley rats are anesthetized. Analysis of liver histology, function, JNK phosphorylation status, as well as apoptosis related protein was evaluated in this study. 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 the administration of SP600125. The results obtained from the present study suggested that targeting the JNK pathway provided a therapeutic target in liver injury induced by brain death.

JNK inhibitor SP600125 protects against lipopolysaccharide-induced acute lung injury via upregulation of claudin-4.

Although in vitro studies have previously demonstrated that mitogen-activated protein kinases are important for the activation of transcription factors and the regulation of proinflammatory mediators, the function of c-Jun NH2-terminal kinase (JNK) in acute lung injury (ALI) remains to be fully elucidated. The present study aimed to investigate the effect of the JNK selective inhibitor SP600125 on lipopolysaccharide (LPS)-induced ALI. Pulmonary edema, the expression of inflammatory cytokines and pathological alterations were found to be significantly attenuated in LPS-induced ALI following treatment with SP600125 in vivo. In vitro, it was demonstrated that SP600125 administration significantly improved A549 cell viability in a dose-dependent manner using the Cell Counting kit-8 and the 5-ethynyl-2′-deoxyuridine incorporation assay. Furthermore, flow cytometric analysis demonstrated that the apoptotic rate was significantly reduced in a concentration-dependent manner following SP600125 injection. At the molecular level, SP600125 treatment dose-dependently inhibited JNK activation and upregulated claudin-4 expression in vivo and in vitro. In combination, the results from the present study indicated that the JNK inhibitor SP600125 protected against LPS-induced ALI in vivo and in vitro, possibly by upregulating the expression of claudin-4.

The specific, reversible JNK inhibitor SP600125 improves survivability and attenuates neuronal cell death in experimental cerebral malaria (ECM)

Cerebral malaria (CM) is the most severe complication of Plasmodium falciparum in humans and major cause of death. SP600125 is a specific, small molecule inhibitor of JNK that prevents the phosphorylation of c-Jun and blocks the expression of proinflammatory cytokines and attenuates neuronal apoptosis in several neurodegenerative disorders. We evaluated the effect of SP600125 treatment on the survival of Plasmodium berghei ANKA (PbA)-infected C57BL/6J mice. Administration of SP600125 improved survival in PbA-infected C57BL6J mice but has no effect on parasitemia. Further, SP600125 administration resulted in attenuation of neuronal cell death along with inhibition of proinflammatory mediators TNF-α and COX-2 and proapoptotic mediators p-c-Jun and active caspase 3 in PbA-infected mice. The promising findings of this study make SP600125 a potential agent for supportive therapy to alleviate inflammation and neuronal cell death associated with CM.

Glycyrrhizin protects rat heart against ischemia-reperfusion injury through blockade of HMGB1-dependent phospho-JNK/Bax pathway

Glycyrrhizin (GL) has been found to inhibit extracellular HMGB1 cytokine's activity, and protect spinal cord, liver and brain against I/R-induced injury in experimental animals. The purpose of this study was to investigate the protective effect of GL in rat myocardial I/R-induced injury and to elucidate the underlying mechanisms. Male adult Sprague-Dawley rats underwent a 30-min left coronary artery occlusion followed by a 24-h reperfusion. The rats were treated with glycyrrhizin or glycyrrhizin plus recombinant HMGB1 after 30 min of ischemia and before reperfusion. Serum HMGB1, TNF-α and IL-6 levels, and hemodynamic parameters were measured at the onset and different time points of reperfusion. At the end of the experiment, the heart was excised, and the infarct size and histological changes were examined. The levels of Bcl2, Bax and cytochrome c, as well as phospho-ERK1/2, phospho-JNK and phospho-P38 in the heart tissue were evaluated using Western blot analysis, and myocardial caspase-3 activity was measured colorimetrically using BD pharmingen caspase 3 assay kit. Intravenous administration of GL (10 mg/kg) significantly reduced the infarct size, but did not change the hemodynamic parameters at different time points during reperfusion. GL significantly decreased the levels of serum HMGB1, TNF-α and IL-6. GL changed the distribution of Bax and cytochrome c expression between the mitochondrial and cytosolic fractions in the heart tissue, resulting in inhibition of myocardial apoptosis. Moreover, expression of phospho-JNK, but not ERK1/2 and P38 was decreased by GL in the heart tissue. All of the effects produced by GL treatment were reversed by co-administration with the recombinant HMGB1 (100 μg). Intravenous administration of SP600125, a selective phospho-JNK inhibitor (0.5 mg/kg), attenuated HMGB1-dependent Bax translocation and the subsequent apoptosis. These results demonstrate that GL alleviates rat myocardial I/R-induced injury via directly inhibiting extracellular HMGB1 cytokine activity and blocking the phospho-JNK/Bax pathway.

INHIBITION OF THE ACTIVITY OF POLY (ADP-RIBOSE) POLYMERASE REDUCES HEART ISCHAEMIA/REPERFUSION INJURY VIA SUPPRESSING JNK MEDIATED AIF TRANSLOCATION

ObjectivesPoly (ADP-ribose) polymerase (PARP) played an important role in the pathogenesis of heart ischaemia/reperfusion (I/R) injury. The mechanisms are still not thoroughly understood. Here we investigated the effect of PARP...

Different Roles of Peripheral Mitogen-Activated Protein Kinases in Carrageenan-Induced Arthritic Pain and Arthritis in Rats

Accumulating evidence suggests that extracellular signal-regulated protein kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) might be involved in hypersensitivity of various pain models. However, there is a lack of direct evidence for actual involvement of peripheral ERK, p38, and JNK in induction and maintenance of arthritic pain and the development of arthritis. We evaluated the effects of preemptive and therapeutic intra-articular administration of selective inhibitors of p38 (SB203580) and JNK (SP600125), and indirect inhibition of ERK with a blocker (PD98059) of the kinase that activates ERK (i.e., MEK, the mitogen-activated protein kinase [MAPK]/ERK kinase), on arthritic pain-related behavior such as reduction of weight load and the inflammatory responses such as neutrophil infiltration into the synovium and knee joint diameter in rats. In addition, arthritis-induced phosphorylation of ERK, p38, and JNK in synovium of knee joint was examined. Pretreatments with PD98059, SB203580, and SP600125 prevented the reduction of weight load induced by the carrageenan injected into the knee joint cavity, but their effects showed different time course patterns. Therapeutic administration of PD98059 and SB203580 partially reversed carrageen-induced reduction of weight load, and their effects showed a similar time course pattern. However, therapeutic administration of SP600125 had no effect on the reduction of weight load. Hematoxylin and eosin staining revealed that carrageenan-induced neutrophil infiltration into the synovium was inhibited by pretreatment with SB203580 or SP600125, but not PD98059. Western blot measurements showed distinct expression of phosphorylated ERK, p38, and JNK in the synovium at different time points after carrageenan injection. These results suggest that ERK, p38, and JNK signaling pathways at the peripheral level may play different roles in arthritic pain and arthritis of the knee joint.

The Sequential Combination of a JNK Inhibitor and Simvastatin Protects Porcine Islets from Peritransplant Apoptosis and Inflammation

Intraductal administration of a c-Jun NH(2)-terminal kinase (JNK) inhibitor enhances islet viability. However, its role in reducing the inflammatory response in islets is unknown. It is also unknown whether a JNK inhibitor could act in synergy with statins. We examined if the sequential combination of a JNK inhibitor and simvastatin would reduce islet inflammation and improve islet viability. We performed porcine islet isolation with or without intraductal administration of SP600125, a JNK inhibitor. This was followed by culture medium supplementation with either nicotinamide alone or nicotinamide plus simvastatin. We assessed the viability of islets by flow cytometry, islet loss during overnight culture, graft function in NOD/SCID mice, and expression of inflammation-related genes in islets. The sequential combination of a JNK inhibitor and simvastatin increased the β-cell viability index of porcine islets cultured overnight (p = 0.015) as well as islet viability as assessed by a DNA binding dye staining (p = 0.011). The combination of a JNK inhibitor and simvastatin significantly increased the islet survival rate (p = 0.027) when the histomorphometry of donor pancreas indicated a large islet proportion of greater than 50.55%. When we transplanted the same islet mass per recipient for each group, there was no difference in overall islet graft function. Intraductal administration of JNK inhibitor significantly suppressed mRNA expression levels of interleukin-1β (IL-1β), interferon-γ, tumor necrosis factor-α, IL-6, IL-8, and macrophage chemoattractant protein-1. It also decreased the concentration of IL-1β (p = 0.040) and IL-8 (p = 0.023) in the culture supernatant. In conclusion, the sequential combination of a JNK inhibitor and simvastatin protected porcine islets from peritransplant apoptosis. Inhibition of JNK reduced the inflammatory response and could be considered an alternative target for suppression of porcine islet inflammation.

C-Jun N-terminal kinase induces axonal degeneration and limits motor recovery after spinal cord injury in mice

C-Jun N-terminal kinase (JNK) mediates neuronal death in response to stress and injury in the CNS and peripheral nervous system. Here, we show that JNK also regulates retrograde axonal degeneration (axonal dieback) after spinal cord injury (SCI) in mice. Activated phospho-JNK was highly expressed in damaged corticospinal tract (CST) axons after thoracic SCI by hemisection. Local administration of SP600125, a JNK inhibitor, prevented accumulation of amyloid-β precursor protein and retraction of the severed CST axons as well as preserved the axonal arbors rostral to the injury site. The treatment with SP600125 also improved functional recovery of the hindlimbs, assessed by Basso mouse scale open-field scores and the grid-walking test. In Jnk1 −/− and Jnk3 −/− mice, we observed prevention of axonal degeneration and enhancement of motor recovery after SCI. These results indicate that both JNK1 and JNK3 induce axonal degeneration and limit motor recovery after SCI. Thus, a JNK inhibitor may be a suitable therapeutic agent for SCI.

Regulation of JNK activity in the apoptotic response of intestinal epithelial cells

We have studied apoptosis of gastrointestinal epithelial cells by examining the receptor-mediated and DNA damage-induced pathways using TNF-α and camptothecin (CPT), respectively. TNF-α requires inhibition of antiapoptotic protein synthesis by cycloheximide (CHX). CHX also results in high levels of active JNK, which are necessary for TNF-induced apoptosis. While CPT induces apoptosis, the increase in JNK activity was not proportional to the degree of apoptosis. Thus the mechanism of activation of JNK and its role in apoptosis are unclear. We examined the course of JNK activation in response to a combination of TNF-α and CPT (TNF + CPT), which resulted in a three- to fourfold increase in apoptosis compared with CPT alone, indicating an amplification of apoptotic signaling pathways. TNF + CPT caused apoptosis by activating JNK, p38, and caspases-8, -9, and -3. TNF-α stimulated a transient phosphorylation of JNK1/2 and ERK1/2 at 15 min, which returned to basal by 60 min and remained low for 4 h. CPT increased JNK1/2 activity between 3 and 4 h. TNF + CPT caused a sustained and robust JNK1/2 and ERK1/2 phosphorylation by 2 h, which remained high at 4 h, suggesting involvement of MEKK4/7 and MEK1, respectively. When administered with TNF + CPT, SP-600125, a specific inhibitor of MEKK4/7, completely inhibited JNK1/2 and decreased apoptosis. However, administration of SP-600125 at 1 h after TNF + CPT failed to prevent JNK1/2 phosphorylation, and the protective effect of SP-600125 on apoptosis was abolished. These results indicate that the persistent activation of JNK might be due to inhibition of JNK-specific MAPK phosphatase 1 (MKP1). Small interfering RNA-mediated knockdown of MKP1 enhanced TNF + CPT-induced activity of JNK1/2 and caspases-9 and -3. Taken together, these results suggest that MKP1 activity determines the duration of JNK1/2 and p38 activation and, thereby, apoptosis in response to TNF + CPT.

Inhibition of JNK phosphorylation reverses memory deficit induced by β-amyloid (1–42) associated with decrease of apoptotic factors

Alzheimer's disease (AD) is the most common form of dementia that is degenerative and terminal disease. The main reason of the disease is still unknown. β-amyloid (Aβ) plaques are the important hallmarks of memory impairment in patients suffering from AD. Aggregation of these plaques in the hippocampus appears during the development of the disease. One of the prominent factors having crucial impact in this process is MAPK. JNK, as a member of MAPK family has a pivotal role, especially in cell survival. We hypothesized that JNK may have beneficial effect on the process of memory improvement. Hence, we performed Morris water maze to investigate the possible impact of JNK inhibitor on spatial memory in Aβ-injected rats. Our data indicated that intracerebroventricular administration of SP600125, a JNK inhibitor, could significantly decrease escape latency and increase time spent in target quadrant, in treatment group. Furthermore, we evaluated some of the apoptotic factors in the hippocampus of the treated rats. Based on our data, the inhibitor led to the significant decrease in the amount of caspase-3, TUNEL positive cells, cyclooxygenase-2 and increase in Bcl-2/Bax ratio. Given the possible neuroprotective effects of SP600125 on Aβ-induced memory impairment and apoptosis, our results may open a new avenue for the treatment of AD.

The c-Jun N-terminal kinase inhibitor SP600125 is neuroprotective in amygdala kindled rats

c-Jun N-terminal kinase (JNK) is implicated in cell death in neurodegenerative disorders and has been taken as a critical point between the physiological and pathological status. JNK specific inhibitor SP600125 has been found to have a protective effect against transient brain ischemia/reperfusion-induced neuronal death in rat hippocampal CA1 region. Former studies have shown the relation between JNK phosphorylation and neuronal damage in models of brain ischemia or in chemically or acute electrically kindled animal in which the stimulus-induced JNK phosphorylation was temporary or transient. In this study, the effect of repeated activation of JNK was examined in the amygdala kindled rats, under or without intraventrical infusion of SP600125, compared with the sham control. JNK phosphorylation was detected by Western blot and immunofluorescent staining in hippocampal extracts and in slices respectively. Nissl staining was performed to detect the neuronal defect. We found that the level of JNK phosphorylation (46 KD) in the hippocampus increased in the amygdala kindled rats, whereas such JNK phosphorylation could be inhibited by SP600125. Expression of total JNK in the hippocampus remained unchanged in kindled rats compared with the sham control, and was not affected by SP600125. Neuronal defect was marked in the kindling group and in the vehicle DMSO group, and was alleviated under the administration of SP600125. These findings suggest that JNK phosphorylation is involved in the process of hippocampal sclerosis in the mesial temporal lobe epilepsy (TLE). JNK signaling pathway may be a new target to interfere with the development of hippocampal sclerosis in the temporal lobe epilepsy. The JNK inhibitor SP600125 can show a protective effect on hippocampal neurons in TLE by inhibiting JNK activation.

Effect of intrathecal administration of SP600125 on MWT and TWL of rats after chronic constriction injury

Objective To observe the effect of intrathecal administration of SP600125 on both MWT and TWL of rats after chronic constriction injury (CCI) of the sciatic nerve. Methods 40 male SD rats were randomized to deride into 5 groups (n=8). Rats in group SP5 received SP600125 5 μg after CCI; rats in group SP25 received SP600125 25 μg after CCI; rats in group SP50 received SP600125 50 μg after CCI; rats in group DMSO received 2% DMSO 10 μl after CCI; rats in group Naive received SP600125 50 μg without sciatic nerve injury. SP600125 was dissolved in 10 μl 2%DMSO solvent. On the 7th day after CCI, MWT and TWL were determined with yon Frey filaments and thermal radiation apparatus repectively after intrathecal administration of SP600125. Results Intrathecal administration certain dosage of SP600125 could attenuate the established mechanical allodynia and thermal hyperalgesia induced by CCI rather than normal rats. Conclusion Intrathecal administration certain dosage of SP600125 could attenuate the established mechanical allodynia and thermal hyperalgesia induced by CCI. Key words: lntrathecal administration; SP600125; CCI; Mechanical allodynia; Thermal hyperalgesia

Inhibition of c‐Jun NH2‐terminal kinase switches Smad3 signaling from oncogenesis to tumor‐ suppression in rat hepatocellular carcinoma†

Transforming growth factor beta (TGF-beta) signaling involves both tumor-suppression and oncogenesis. TGF-beta activates the TGF-beta type I receptor (TbetaRI) and c-Jun N-terminal kinase (JNK), which differentially phosphorylate the mediator Smad3 to become COOH-terminally phosphorylated Smad3 (pSmad3C) and linker-phosphorylated Smad3 (pSmad3L). TbetaRI-dependent pSmad3C transmits a tumor-suppressive TGF-beta signal, while JNK-dependent pSmad3L promotes carcinogenesis in human chronic liver disorders. The aim of this study is to elucidate how SP600125, a JNK inhibitor, affected rat hepatocellular carcinoma (HCC) development, while focusing on the domain-specific phosphorylation of Smad3. The rats received subcutaneous injections of either SP600125 or vehicle 11 times weekly together with 100 ppm N-diethylnitrosamine (DEN) administration for 56 days and were sacrificed in order to evaluate HCC development 28 days after the last DEN administration. The number of tumor nodules greater than 3 mm in diameter and the liver weight/body weight ratio were significantly lower in the SP600125-treated rats than those in the vehicle-treated rats (7.9 +/- 0.8 versus 17.7 +/- 0.9: P < 0.001; 6.3 +/- 1.2 versus 7.1 +/- 0.2%: P < 0.05). SP600125 significantly prolonged the median survival time in rats with DEN-induced HCC (113 versus 97 days: log-rank P = 0.0018). JNK/pSmad3L/c-Myc was enhanced in the rat hepatocytes exposed to DEN. However, TbetaRI/pSmad3C/p21(WAF1) was impaired as DEN-induced HCC developed and progressed. The specific inhibition of JNK activity by SP600125 suppressed pSmad3L/c-Myc in the damaged hepatocytes and enhanced pSmad3C/p21(WAF1), acting as a tumor suppressor in normal hepatocytes. Administration of SP600125 to DEN-treated rats shifted hepatocytic Smad3-mediated signal from oncogenesis to tumor suppression, thus suggesting that JNK could be a therapeutic target of human HCC development and progression.

Inhibition of the activity of Rho-kinase reduces cardiomyocyte apoptosis in heart ischemia/reperfusion via suppressing JNK-mediated AIF translocation

Background Recent studies have demonstrated that Rho-kinase has been proposed to play an important role in the pathogenesis of heart ischemia/reperfusion (I/R) injury. However, the mechanism of Rho-kinase mediated cardiomyocyte apoptosis in I/R is still not thoroughly understood. Method Studies were performed with female Wistar rats. Results Ischemia followed by reperfusion caused a significant increase in Rho-kinase, c-Jun NH2-terminal kinase (JNK) and apoptosis-inducing factor (AIF) activity. Administration of fasudil, an inhibitor of Rho-kinase, decreased myocardial infarction size from 59.89 ± 3.83% to 38.62 ± 2.66% ( P < 0.05) and cell apoptosis from 32.78 ± 5.1% to 17.05 ± 4.2% ( P < 0.05). Western blot analysis showed that administration of fasudil reduced the activation of JNK and attenuated mitochondrial-nuclear translocation of AIF. Additionally, administration of SP600125, an inhibitor of JNK, attenuated mitochondrial-nuclear translocation of AIF. Conclusion The inhibition of Rho-kinase reduced cell apoptosis in I/R in vivo via suppression of JNK-mediated AIF translocation.

C-Jun N-Terminal Kinase Mediates Lactacystin-Induced Dopamine Neuron Degeneration

Parkinson disease is characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. It has been proposed that dysfunction of the ubiquitin proteasome system plays an important role in the pathogenesis of Parkinson disease, but the mechanisms underlying ubiquitin proteasome system-related neuron degeneration are unknown. Here, we demonstrate that the proteasome inhibitor lactacystin induces phosphorylation of c-Jun N-terminal kinase (JNK) and c-Jun, the release of cytochrome c, activation of both caspase-9 and caspase-3, and sequential apoptosis of dopaminergic neurons in vitro. Most of these effects can be attenuated by the JNK inhibitor SP600125. Furthermore, infusion of lactacystin in rats in vivo also leads to phosphorylation of JNK before nigral neuron loss; chronic administration of SP600125 also blocks this loss. These results indicate that JNK is involved in proteasome inhibition-induced dopaminergic neuron degeneration through caspase-3-mediated apoptotic pathways, suggesting that this kinase may be a therapeutic target for the prevention of substantia nigra pars compacta degeneration in Parkinson disease patients.

Inhibition of the activity of poly (ADP‐ribose) polymerase reduces heart ischaemia/reperfusion injury via suppressing JNK‐mediated AIF translocation

Poly (ADP-ribose) polymerase (PARP) has been proposed to play an important role in the pathogenesis of heart ischaemia/reperfusion (I/R) injury. However, the mechanisms of PARP-mediated heart I/R injury in vivo are still not thoroughly understood. Therefore, in this study, we investigate the effect of PARP inhibition on heart I/R injury and try to elucidate the underlying mechanisms. Studies were performed with I/R rats' hearts in vivo. Ischaemia followed by reperfusion caused a significant increase in Poly (ADP-ribose) (PAR), c-Jun NH2-terminal kinase (JNK) and apoptosis-inducing factor (AIF) activity. Administration of 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (DPQ), an inhibitor of PARP, decreased myocardial infarction size from 61.11±7.46%[0] to 38.83±5.67% (P<0.05) and cells apoptosis from 35±5.3% to 20±4.1% (P<0.05) and simultaneously improved the cardiac function. Western blot analysis showed that administration of DPQ reduced the activation of JNK and attenuated mitochondrial-nuclear translocation of AIF. Additionally, administration of SP600125, an inhibitor of JNK, attenuated mitochondrial-nuclear translocation of AIF. The results of the present study demonstrated that the inhibition of PARP was able to reduce heart I/R injury in vivo. Our results also suggested that JNK may be downstream of PARP activation and be required for PARP-mediated AIF translocation. Inhibition of the activity of PARP may reduce heart I/R injury via suppressing AIF translocation mediated by JNK.

Involvement of c-Jun NH2-terminal kinase and nitric oxide-mediated mitochondria-dependent intrinsic pathway signaling in cardiotoxin-induced muscle cell death: role of testosterone

To test the hypothesis that c-Jun NH2-terminal kinase (JNK) and nitric oxide (NO)-mediated signaling plays an important role in muscle cell apoptosis, we examined the contribution of these molecules in muscle cell apoptosis during cardiotoxin (ctx)-induced muscle injury in mice. Compared to controls, where no apoptosis was detected, the percent of muscle cell apoptosis rose significantly (P < 0.05) at 4 h (27%) after ctx-treatment and increased further progressively up to 16 h posttreatment (80%), before it fell again at 24 h posttreatment (38%). Initiation of apoptosis was preceded by JNK activation and elevated levels of B-cell lymphoma-2 (BCL-2) in the mitochondrial fractions (BAX levels remained unaffected). Ctx treatment also resulted in the inactivation of BCL-2 through phosphorylation at serine 70, thereby perturbing the BAX/BCL-2 rheostat, and the subsequent activation of the cytochrome c-mediated death pathway. Concomitant administration of SP600125, a selective JNK inhibitor, or aminoguanidine (AG), a selected inducible nitric oxide synthase (iNOS) inhibitor, effectively diminished BCL-2 phosphorylation, suppressed cytochrome c release from mitochondria and caspase activation, and significantly prevented ctx-induced muscle cell apoptosis. In additional studies, we examined the role of testosterone in preventing such ctx-induced muscle cell apoptosis. Collectively, the present study emphasizes the role of a new signal transduction pathway involving JNK and iNOS that promotes ctx-induced myocyte apoptosis by provoking BCL-2 phosphorylation, leading to its inactivation, and subsequent activation of the intrinsic pathway signaling. Testosterone therapy has no protective effect in acute muscle injury associated with increased muscle cell death after ctx-treatment.

An inhibitor of c- Jun NH2-terminal kinase, SP600125, protects mice from d-galactosamine/lipopolysaccharide-induced hepatic failure by modulating BH3-only proteins

Fulminant hepatic failure (FHF) is a dramatic clinical syndrome characterized by massive hepatocyte apoptosis and very high mortality. The c- Jun-N-terminal kinase (JNK) pathway is an important stress-responsive kinase activated by several forms of liver injury. The aim of this study is to assess the role of JNK during d-galactosamine (GalN)/lipopolysaccharide (LPS)-induced liver injury, an experimental model of FHF, using SP600125, a small molecule JNK-specific inhibitor. Mice were given an intraperitoneal dose of GalN (800 μg/g body weight)/LPS (100 ng/g body weight) with and without subcutaneous SP600125 (50 mg/kg body weight) treatment (at 6 and 2 h before and 2 h after GalN/LPS administration). GalN/LPS treatment induced sustained JNK activation. Administration of SP600125 diminished JNK activity, suppressed lethality and the elevation of both serum alanine aminotransferase and aspartate aminotransferase, but had no effect on serum tumor necrosis factor-α, and reduced hepatocyte apoptosis after GalN/LPS administration. In support of the role of JNK in promoting the mitochondria-mediated apoptosis pathway, SP600125 prevented cytochrome c release, caspase-9 and caspase-3 activity. Moreover, SP600125 downregulated the mRNA and protein expression of Bad in the early periods following GalN/LPS injection and prevented Bid cleavage in the late periods. These results confirm the role of JNK as a critical apoptotic mediator in GalN/LPS-induced FHF. SP600125 has the potential to protect FHF by downregulating Bad and inhibiting Bid cleavage.

Bad as a Converging Signaling Molecule between Survival PI3-K/Akt and Death JNK in Neurons after Transient Focal Cerebral Ischemia in Rats

Bad, a proapoptotic Bcl-2 family protein, plays a critical role in determining cell death/survival. The phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the c-Jun N-terminal kinase (JNK) pathway are thought to be involved in regulation of Bad. Therefore, the present study was performed to clarify the role of Bad as a common target of the PI3-K/Akt and JNK pathways after transient focal cerebral ischemia (tFCI) in rats. We found that Akt activity increased at 3 h and then decreased, whereas JNK activity increased 7 to 24 h in the peripheral area after tFCI. Administration of LY294002, a PI3-K-specific inhibitor, exacerbated DNA fragmentation, whereas administration of SP600125, a JNK-specific inhibitor, attenuated it. Inhibited by LY294002, phospho-Bad (Ser136) expression increased in the peripheral area 3 h after tFCI, with suppression of Akt activity. Furthermore, phospho-Bad (Ser136) and phospho-Akt (Ser473) were colocalized. Decreases in phospho-Bad (Ser136) and Bad/14-3-3 dimerization and increases in Bcl-X(L)/Bad or Bcl-2/Bad dimerization observed 7 to 24 h after tFCI, were prevented by SP600125 administration, with inhibition of JNK activity. The present study indicates that signal predominance varies from PI3-K/Akt-mediated survival signaling to JNK-mediated death signaling with the development of neuronal damage in the peripheral area after tFCI. This study also suggests that PI3-K/Akt has a role in Bad inactivation, whereas the JNK pathway is involved in Bad activation. We conclude that Bad may be an integrated checkpoint of PI3-K/Akt-mediated survival signaling and JNK-mediated death signaling and that it contributes to cell fate in the peripheral area after cerebral ischemia.

Neuroprotection against ischemic brain injury by SP600125 via suppressing the extrinsic and intrinsic pathways of apoptosis

Our previous studies and the others have strongly suggested that JNK signaling pathway plays a critical role in ischemic brain injury. Here, we reported that SP600125, a potent, cell-permeable, selective, and reversible inhibitor of c-Jun N-terminal kinase (JNK), potently decrease neuronal apoptosis induced by global ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. As a result, SP600125 diminished the increased phosphorylation of c-Jun and the increased expression of FasL induced by ischemia/reperfusion in the vulnerable hippocampal CA1 subregion. At the same time, through inhibiting phosphorylation of Bcl-2 and the release of Bax from Bcl-2/Bax dimers, SP600125 attenuated Bax translocation to mitochondria and the release of cytochrome c induced by ischemia/reperfusion (I/R). Furthermore, the activation of caspase-3 induced by ischemia/reperfusion was also significantly suppressed by preinfusion of SP600125. Importantly, the same neuropotective effect was showed by administration of SP600125 both before and after ischemia. Thus, our findings imply that SP600125 can inhibit the activation of JNK signaling pathway and induce neuroprotection against ischemia/reperfusion in rat hippocampal CA1 region via suppressing the extrinsic and intrinsic pathways of apoptosis. Taken together, these results indicate that targeting the JNK pathway provides a promising therapeutic approach for ischemic brain injury.