Inhibition Of JNK Signaling Pathway Research Articles

Zuogui Jiangtang Jieyu Formula ameliorating hippocampal neuronal apoptosis in diabetic rats with depression by inhibiting JNK signaling pathway

ObjectiveTo investigate the effect of Zuogui Jiangtang Jieyu Formula (左归降糖解郁方, ZJJF) on hippocampal neuron apoptosis in diabetic rats with depression and to ascertain whether its mechanism involves the regulation of JNK signaling pathway. Methods(i) A total of 72 specific pathogen-free (SPF) grade male Sprague Dawley (SD) rats were randomly divided into six groups, with 12 rats in each group: control, model, metformin (Met, 0.18 g/kg) + fluoxetine (Flu, 1.8 mg/kg), and the high-, medium-, and low-ZJJF dosages (ZJJF-H, 20.52 g/kg; ZJJF-M, 10.26 g/kg; ZJJF-L, 5.13 g/kg) groups. All groups except control group were injected once via the tail vein with streptozotocin (STZ, 38 mg/kg) combined with 28 d of chronic unpredictable mild stress (CUMS) to establish diabetic rat models with depression. During the CUMS modeling period, treatments were administered via gavage, with control and model groups receiving an equivalent volume of distilled water for 28 d. The efficacy of ZJJF in reducing blood sugar and alleviating depression was evaluated by measuring fasting blood glucose, insulin, and glycated hemoglobin levels, along with behavioral assessments, including the open field test (OFT), forced swim test (FST), and sucrose preference test (SPT). Hippocampal tissue damage and neuronal apoptosis were evaluated using hematoxylin-eosin (HE) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. Apoptosis-related proteins Bax, Bcl-2, caspase-3, and the expression levels of JNK/Elk-1/c-fos signaling pathway were detected using Western blot and real-time quantitative polymerase chain reaction (RT-qPCR). (ii) To further elucidate the role of JNK signaling pathway in hippocampal neuronal apoptosis and the pharmacological effects of ZJJF, an additional 50 SPF grade male SD rats were randomly divided into five groups, with 10 rats in each group: control, model, SP600125 (SP6, a JNK antagonist, 10 mg/kg), ZJJF (20.52 g/kg), and ZJJF (20.52 g/kg) + Anisomycin (Aniso, a JNK agonist, 15 mg/kg) groups. Except for control group, all groups were established as diabetic rat models with depression, and treatments were administered via gavage for ZJJF and intraperitoneal injection for SP6 and Aniso for 28 d during the CUMS modeling period. Behavioral changes in rats were evaluated through the OFT, FST, and SPT, and hippocampal neuron damage and apoptosis were observed using HE staining, Nissl staining, TUNEL staining, and transmission electron microscopy (TEM). Changes in apoptosis-related proteins and JNK signaling pathway in the hippocampal tissues of rats were also analyzed. Results(i) ZJJF significantly reduced the high blood glucose, insulin, and glycated hemoglobin levels in model rats (P < 0.01). It increased autonomous activity and decreased despair-like behaviors (P < 0.01), improved the pathological damage of hippocampal neurons, increased the number of neuronal nuclei (P < 0.01), and reduced the number of mechanocytes, vacuolar cells, and apoptotic neurons (P < 0.05, P < 0.01, and P < 0.01, respectively). ZJJF down-regulated the expression levels of pro-apoptotic proteins Bax and caspase-3 (P < 0.01), up-regulated the anti-apoptotic protein Bcl-2 (P < 0.01), and significantly inhibited the overexpression of phosphorylated JNK (p-JNK), Elk-1, and c-fos (P < 0.01). (ii) SP6 increased autonomous activity and reduced despair time in model rats (P < 0.05), although it had no significant effects on sucrose preference (P > 0.05). It increased the number of Nissl bodies in hippocampal neurons (P < 0.01), reduced the protein expression levels of Bax (P < 0.01) and caspase-3 (P < 0.05), and decreased the number of apoptotic neurons (P < 0.05). SP6 also increased the expression level of Bcl-2 (P < 0.01), and inhibited the high expression levels of p-JNK, Elk-1, and c-fos (P < 0.01, P < 0.01, and P < 0.05, respectively), suggesting that hippocampal neuronal apoptosis in diabetic rats with depression is associated with abnormal activation of JNK signaling pathway. Compared with ZJJF group, ZJJF + Aniso group showed a decrease in sucrose preference (P < 0.05) and an increase in despair time (P < 0.01) with more notable hippocampal neuronal damage. This group also exhibited a decrease in expression level (P < 0.01) Bcl-2 and an increase in expression levels of Bax, caspase-3, p-JNK, Elk-1, and c-fos (P < 0.01, P < 0.05, P < 0.05, P < 0.01, and P < 0.05, respectively), indicating that the antidepressant effects of ZJJF, its improvement of neuronal apoptosis, and regulation of JNK signaling molecules could all be reversed by a specific JNK agonist. ConclusionZJJF exerts a significant hypoglycemic effect and ameliorates the apoptosis of hippocampal neurons by inhibiting the activation of JNK signaling pathway, which is a promising formula for the treatment of diabetic depression in clinical settings.

Selective estrogen receptor modulators, acting as agonists of estrogen receptor α in osteoblasts, reduce the TGF-β-induced synthesis of macrophage colony-stimulating factor via inhibition of JNK signaling pathway.

Selective estrogen receptor modulator (SERM) binds to estrogen receptors (ERs) and acts as both an agonist or an antagonist, depending on the target tissue. Raloxifene and bazedoxifene as SERMs are currently used hormone replacement medicines for postmenopausal osteoporosis. Macrophage colony-stimulating factor (M-CSF) secreted from osteoblasts promotes osteoclastogenesis. We have previously demonstrated that transforming growth factor (TGF)-β induces the synthesis of M-CSF via SMAD2/3, p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and c-Jun N-terminal kinase (JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether SERM affects the M-CSF synthesis by TGF-β in MC3T3-E1 cells. Raloxifene and bazedoxifene significantly suppressed the synthesis of M-CSF. PPT, an ERα agonist, but not ERB041, an ERβ agonist, inhibited the release of M-CSF. MPP, an ERα antagonist, reversed the suppression by raloxifene of the M-CSF release. Raloxifene attenuated the TGF-β-induced phosphorylation of JNK but not SMAD3, p42 MAPK and p38 MAPK. Bazedoxifene and PPT also inhibited the phosphorylation of JNK. Furthermore, MPP, an ERα antagonist, reversed the suppression by both raloxifene and bazedoxifene of the phosphorylation of JNK. Our results strongly indicate that raloxifene and bazedoxifene, SERMs, suppress the TGF-β-induced synthesis of M-CSF through ERα-mediated inhibition of JNK pathway in osteoblasts.

JNK selective inhibitor, IQ-1S, protects the mice against lipopolysaccharides-induced sepsis

Sepsis is a severe systemic inflammatory response induced by infection. Innate immunity recognizes pathogen components such as lipopolysaccharides (LPS), and mediates the polarization of immune cells and the release of cytokines. However, this process is also crucial for triggering sepsis and septic shock. To investigate the potential therapeutic function of 11H-indeno [1,2–b] quinoxalin-11-one oxime (IQ-1S) to sepsis, LPS plus d-galactosamine was used to establish a sepsis mouse model. Flow cytometry was performed to catalyze T cells and macrophages in mouse spleen. ELISA assay and qRT-PCR assay were performed to estimate the expression levels of cytokines and related genes including TNF-α, IL-6, IL-1β, Nos2, Arg and Mrc. The protein levels of NF-κB, AP1, NF-Y, p-JNK2, JNK2, p-p38, p38, p-IκBα, IκBα, p-IKKβ and IKKβ were evaluated by Western blot assay. IQ-1S treatment significantly reduced mortality and lung inflammation in sepsis mice. IQ-1S treatment decreased the levels of inflammatory cytokines in sepsis mice. Polarization of M1 macrophages was suppressed by IQ-1S in vitro. IQ-1S significantly inhibited the activation of the JNK signaling pathway and reduced the phosphorylation level of JNK2 in sepsis mice. IQ-1S protected the mice against LPS-induced sepsis through inhibiting JNK signaling pathway.

D-serine Ameliorates Motor and Cognitive Impairments in β-amyloid 1-42 Injected Mice by Inhibiting JNK Signaling Pathway

The senile plaque formed by β-amyloid (Aβ) deposition in the brain is one of the main pathological features of Alzheimer’s disease (AD), and the c-Jun N-terminal kinase (JNK) signaling pathway plays an important role in the pathogenesis of AD. This study aimed to investigate that D-serine may ameliorate motor and cognitive impairment in Aβ injected mice by inhibiting JNK signaling pathway. Firstly, Kunming mice were injected intrahippocampally with Aβ1-42 to build AD model. The mice were injected intraperitoneally with saline, D-serine, D-amino acid oxidase (DAAO), and Sodium benzoate (BE) for 10 consecutive days, respectively. Subsequently, the motor and cognitive functions of mice were detected by behavioral tests. The silver staining and immunohistochemical methods were used to detect the distributions of Aβ in the hippocampus of mice. 18F-2-Fluro-D-deoxy-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) scans were performed to detected glucose metabolism of Aβ1-42 induced lesions. The expressions of relative JNK factors were detected by immunohistochemistry and Western blot methods. These results showed that Aβ severely impaired the motor and memory abilities of mice. The expressions of glial fibrillary acidic protein (GFAP), tumor necrosis factor (TNF-α), N-methyl-D-aspartate receptor 1 (NMDAR1), phospho-JNK (p-JNK), p-c-Jun and activating transcription factor 2 (ATF2) increased significantly. After D-serine treatment, the abilities of movement and memory of mice were improved, and the clearance rate of Aβ was accelerated. The expressions of GFAP, TNF-α, NMDAR1, p-JNK, p-c-Jun and ATF2 decreased significantly. DAAO and BE were administered to further validate these results. Therefore, this study showed that D-serine could alleviate the cognitive impairment of Aβ1-42 injected mice by inhibiting JNK signaling pathway. These results provide more evidences for the effect of D-serine on AD and relevant mechanism to treat AD.

Effect of Caesalpinia bonduc Polyphenol Extract on Alloxan-Induced Diabetic Rats in Attenuating Hyperglycemia by Upregulating Insulin Secretion and Inhibiting JNK Signaling Pathway.

Caesalpinia bonduc has been used in herbal medicines for the treatment of a wide range of diseases from decades. The present study has explored the remedial potential and underlying mechanism of polyphenol extract of Caesalpinia bonduc in alloxanized diabetic rats. HPLC/MS analysis confirmed the presence of phenolics in considerable concentrations in Caesalpinia bonduc extract. Administration of different doses (250 and 500 mg/kg) of CPP extract to hyperglycemic rats for 8 weeks restored blood and serum glucose, insulin, glycosylated hemoglobin, leptin, amylin, and carbohydrate metabolizing enzymes level towards normal compared to alloxanized diabetic group. The effect of CPP extract on various genes such as Pdx-1, Ins-1, ngn-3, GLUT-4, and IRS-1 in insulin signaling pathway and Traf-4, Traf-6, and Mapk-8 in MAPK downstream JNK cascade was examined through qRT-PCR to access the core molecular mechanism involved in CPP-induced recovery of diabetes. Results have revealed that CPP extract reduced oxidative stress in pancreatic β cells by restoring free radical scavenging potential, reducing the mRNA expression of Mapk-8, Traf-4, and Traf-6, and increasing the Pdx-1, Ins-1, ngn-3, GLUT-4, and IRS-1 expression ensuing regeneration of β cells and subsequent insulin release from pancreas. The results obtained in this study recommend that CPP extract may be a promising therapeutic restorative agent in the treatment of diabetes mellitus.

MiR-122 Exerts Inhibitory Effects on Osteoblast Proliferation/Differentiation in Osteoporosis by Activating the PCP4-Mediated JNK Pathway

Osteoporosis is characterized by the reduction of bone mineral density and deterioration of bone quality which leads to high risk of fractures. Some microRNAs (miRNAs) have been confirmed as potential modulators of osteoblast differentiation to maintain bone mass maintenance. We aimed to clarify whether miR-122 could regulate osteoblast differentiation in ovariectomized rats with osteoporosis. miR-122 was upregulated and Purkinje cell protein 4 (PCP4) was downregulated in ovariectomized rats. PCP4 was identified as a target of miR-122 by dual-luciferase reporter gene assay. We transfected isolated osteoblasts from ovariectomized rats with miR-122 mimic or inhibitor or PCP4 overexpression vectors. Proliferation and differentiation of osteoblasts were repressed by the overexpression of miR-122 but enhanced by overexpression of PCP4. miR-122 could induce the activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway, while PCP4 blocked this pathway. Rescue experiments further demonstrated that the inhibiting effects of miR-122 on osteoblast differentiation could be compensated by activation of the PCP4 or inhibition of JNK signaling pathway. Collectively, our data imply that miR-122 inhibits osteoblast proliferation and differentiation in rats with osteoporosis, highlighting a novel therapeutic target for osteoporotic patients.

C-Jun N terminal kinase signaling pathways mediate cannabinoid tolerance in an agonist-specific manner

Tolerance to the antinociceptive effects of cannabinoids represents a significant limitation to their clinical use in managing chronic pain. Tolerance likely results from desensitization and down-regulation of the cannabinoid type 1 receptor (CB1R), with CB1R desensitization occurring via phosphorylation of CB1Rs by a G protein-coupled receptor kinase and subsequent association with an arrestin protein. Previous studies have shown that (1) desensitization-resistant S426A/S430A mice exhibit a modest delay in tolerance for Δ9-THC and (-)-CP55,940 but a more pronounced disruption in tolerance for WIN 55,212-2 and (2) that c-Jun N-terminal kinase (JNK) signaling may selectively mediate antinociceptive tolerance to morphine compared to other opioid analgesics. In the current study, we found that pretreatment with the JNK inhibitor SP600125 (3 mg/kg) attenuates tolerance to the antinociceptive in the formalin test and to the anti-allodynic effects of Δ9-THC (6 mg/kg) in cisplatin-evoked neuropathic pain using wild-type mice. We also find that SP600125 causes an especially robust reduction in tolerance to the antinociceptive effects of Δ9-THC (30 mg/kg), but not WIN 55,212-2 (10 mg/kg) in the tail-flick assay using S426A/S430A mice. Interestingly, SP600125 pretreatment accelerated tolerance to the antinociceptive and anti-allodynic effects of (-)-CP55,940 (0.3 mg/kg) in mice with acute and neuropathic pain. These results demonstrate that inhibition of JNK signaling pathways delay tolerance to Δ9-THC, but not to CP55,940 or WIN55,212-2, demonstrating that the mechanisms of cannabinoid tolerance are agonist-specific.

Yes-associated protein reduces neuroinflammation through upregulation of Sirt3 and inhibition of JNK signaling pathway

Objective: Neuroinflammation is linked to a series of neurodegenerative diseases through the unknown mechanisms.Aim: The aim of this study was to investigate the role of Yes-associated protein (Yap) in the regulation of neuroinflammation.Methods: BV-2 neuroglia cells were treated with TNFα in vitro. Then, western blots, qPCR, immunofluorescence, and ELISA were used to verify the influence of Yap in BV-2 cells neuroinflammation response.Results: After exposure to TNFα, viability of BV-2 cells decreased whereas apoptosis index was increased. Of note, Yap expression in BV-2 cells was significantly reduced, when compared to the normal cells. Interestingly, adenovirus-induced Yap overexpression was capable to reverse cell viability and thus reduce apoptotic index in TNFα-treated BV-2 cells. Molecular investigation demonstrated that Yap overexpression was linked to Sirt3 upregulation. Increased Sirt3 reduced endoplasmic reticulum (ER) stress, attenuated mitochondrial damage, and blocked JNK pro-apoptotic pathway. Interestingly, loss of Sirt3 abolished the protective effects induced by Yap overexpression in TNFα-treated BV-2 cells.Conclusions: Altogether, our results demonstrated that neuroinflammation could be caused by Yap downregulation, possible driven through Sirt3 inhibition and JNK activation. However, overexpression of Yap could protect BV-2 cells against TNFα-mediated apoptosis through modulating Sirt3-JNK signaling pathways.

Astragalus polysaccharide improves anti-tumor immunity mediated by macrophages and natural killer cells in mice

Objective To evaluate the regulatory effects of Astragalus polysaccharide (APS) on macrophage polarization and NK cell-mediated anti-tumor responses in mice. Methods C57BL/6 mice were injected intraperitoneally with APS once a day for seven consecutive days. Activation of immune cells was then induced by intraperitoneal injection of polyinosinic-polycytidylic acid (Poly I∶C) 24 h after the APS intervention. Peritoneal macrophages were collected 24 h after induction to analyze the status of polarization and the production of nitric oxide (NO). Cytotoxicity and exocytosis of activated NK cells were measured to assess the effector functions of these cells. NK cell activities induced by NKG2D were studied in the absence of the whole JNK or JNK2 signaling pathway. Results Intraperitoneal injection of APS promoted the polarization of macrophages induced by tumor cells in mice, and enhanced the cytotoxicity of NK cells to tumor cells. However, APS was in need of the involvement of appropriate stimulatory factors to have regulatory effects. Complete inhibition of JNK signaling pathway dramatically reduced the effector functions of NK cells, which could not be recovered by APS administration. Conclusions APS was involved in the regulation of anti-tumor innate immunity through enhancing the M1-polarization of macrophages and improving the effector functions of NK cells. This study might to some extent elucidate the mechanism of APS in immune regulation and anti-tumor immunity. Key words: Astragalus polysaccharide; Innate immunity; Anti-tumor immunity; Immune regulation

Mechanism of effective components of Mori Folium in alleviating insulin resistance based on JNK signaling pathway

A stable hepatoma cell line(Hep G2 cell) insulin resistance model was established and used to analyze the effect of effective components of Mori Folium in alleviating insulin resistance,and preliminary explore the mechanism for alleviating insulin resistance. The Hep G2 insulin action concentration and the duration of action were investigated using the glucose oxidase method(GOD-POD method) to establish a stable Hep G2 insulin resistance model. Normal control group,model group,Mori Folium polysaccharide group,Mori Folium flavonoid group and rosiglitazone group were divided to determine the glucose consumption. The effect of Mori Folium effective components on Hep G2 insulin resistance was analyzed. The mRNA expressions of JNK,IRS-1 and PDX-1 in each group were detected by Real-time quantitative PCR(qRT-PCR). The protein expressions of p-JNK,IRS-1 and PDX-1 were detected by Western blot. And the mechanism of effective components of Mori Folium in alleviating insulin resistance was investigated. The results showed that the glucose consumption was significantly decreased in the insulin resistance cells after incubation with 25. 0 mg·L-1 insulin for 36 h(P<0. 01),and the model was relatively stable within 36 h. Mori Folium polysaccharides and flavonoids all alleviated insulin resistance,among which Mori Folium flavonoids had better effect in alleviating Hep G2 insulin resistance(P<0. 05). The qRT-PCR analysis showed that Mori Folium polysaccharides and flavonoids could inhibit JNK and IRS-1 mRNA expressions,while enhancing PDX-1 mRNA expression. Western blot analysis displayed that Mori Folium polysaccharides and flavonoids could inhibit p-JNK and IRS-1 protein expressions,while enhancing PDX-1 protein expression. Mori Folium polysaccharides and flavonoids can alleviate insulin resistance in Hep G2 cells,and its mechanism may be the alleviation of insulin resistance by inhibiting JNK signaling pathway.

MicroRNA-125b-5p improves pancreatic β-cell function through inhibiting JNK signaling pathway by targeting DACT1 in mice with type 2 diabetes mellitus

Type 2 diabetes mellitus (T2DM) is a progressive disease, accompanied by increased insulin resistance and deteriorating β-cell function. Previous studies have revealed that microRNA (miRNA) plays a crucial role in the treatment of T2DM. Hence, we aim to investigate the role of microRNA-125b-5p (miR-125b-5p) in pancreatic β-cell function and insulin sensitivity of mice with T2DM with the involvement of Dishevelled antagonist Dapper1 (DACT1) and the c-Jun NH2-terminal kinases (JNK) signaling pathway. Firstly, a mouse model of T2DM was established by administering a high-fat diet plus low dosage of streptozotocin, and function of pancreatic β-cell and insulin sensitivity in the normal and T2DM mice were detected. Then, the pancreatic β-cells were collected from pancreatic islet tissues and treated with different mimics, inhibitors and siRNAs. After that, the relationship among miR-125b-5p, DACT1, and the JNK signaling-related factors in T2DM mice was determined. Finally, cell proliferation and apoptosis were determined. Mice with T2DM had lower pancreatic β-cell function and insulin sensitivity, as well as diminished expression of miR-125b-5p but enhanced expressions of DACT1, JNK and c-Jun. miR-125b-5p inhibited DACT1 expression and the activation of the JNK signaling pathway, as well as restrained cell proliferation and promoted cell apoptosis. The current results suggest that up-regulated miR-125b-5p promotes insulin sensitivity and enhances pancreatic β-cell function through inhibiting the JNK signaling pathway by negatively mediating DACT1.

Acinetobacter baumannii outer membrane protein A induces HeLa cell autophagy via MAPK/JNK signaling pathway

Autophagy is an evolutionary conserved self-balancing process that plays an important role in maintaining cellular homeostasis via the clearance of damaged organelles and misfolded proteins. Infection-triggered autophagy specifically inhibits the invasion of intracellular bacterial replication and hence protects the cells from microbial infections. It has been reported that Acinetobacter baumannii trigger cell autophagy. However, the role of its virulence protein OmpA remains unclear. Therefore, this study aimed to explore the effects of Acinetobacter baumannii OmpA on cell autophagy and its underlying molecular mechanisms. The results showed that OmpA induced autophagy in HeLa and RAW264.7 cells, increased LC3BII expression, and hindered p62 degradation. Moreover, OmpA triggered incomplete autophagy by interfering the fusion of autophagosomes with lysosomes. Besides, OmpA activated MAPK/JNK signaling pathway and enhanced the phosphorylation levels of JNK, p38, and ERK, c-Jun. Inhibition of JNK signaling pathway suppressed OmpA-induced autophagy in HeLa cells. Ab wild-type strains carrying OmpA triggered incomplete autophagy and resulted in a large number of IL-1β production. Ab-△OmpA strain (OmpA gene mutation) restored autophagic flux and reduced the accumulation of p62 and the release of IL-1β in HeLa cells. Rapamycin activated autophagy to inhibit OmpA-induced IL-1β secretion and protect HeLa cells from inflammatory damage. Collectively, these results suggest that OmpA can induce autophagy in HeLa cells through MAPK/JNK signaling pathway. Pre-treatment with Rapamycin activates autophagy and protects against cell death.

Retracted: Knockdown of long noncoding RNA GAS5 attenuates H2 O2 -induced damage in retinal ganglion cells through upregulating miR-124: Potential role in traumatic brain injury.

Optic nerve injury is one of the most common and serious complications in traumatic brain injury (TBI). Alleviating degree of optic nerve injury is important to cure of TBI. This study explored the role of long noncoding RNA (lncRNA) GAS5 in mice retinal ganglion cells (RGCs) suffered to H2 O 2 injury. Primary RGC (PRGCs) were treated with H2 O 2 to simulate an in vitro oxidation stress model. LncRNA GAS5 and miR-124 expressions were knocked down by cell transfection with short-hairpin RNA against GAS5 and miR-124 inhibitor, and the transfection efficiency was determined by qRT-PCR. Cell viability, apoptotic cell rate, and production of reactive oxygen species (ROS) was analyzed by CCK-8 assay, PI/FITC-Annexin V method, and DCFH-DA fluorometric assay. Cell apoptosis-associated proteins as well as activations of JAK/STAT3 signaling and JNK signaling were analyzed by Western blot. H2 O 2 treatment-induced cell injury was inhibited by lncRNA GAS5 silence. Specifically, knockdown of GAS5 improved viability of primary PRGCs, inhibited apoptosis, decreased ROS expression, increased antiapoptosis proteins' expressions, and decreased proapoptosis proteins' expressions. It was also found that miR-124 inhibitor treatment impaired the cell protective effect of GAS5 silence, indicating low level of GAS5 protected PRGCs via upregulating miR-124. GAS5 silence might exert cytoprotection effect via activating JAK/STAT3 signaling pathway and inhibiting activation of JNK signaling pathway. Knocking down lncRNA GAS5 alleviated H2 O 2 -induced injury in PRGCs via upregulation of miR-124, which might dependent on activation of JAK/STAT3 signaling pathway and inhibition of JNK signaling pathway.

PINK1 Protects Auditory Hair Cells and Spiral Ganglion Neurons from Cisplatin-induced Ototoxicity via Inducing Autophagy and Inhibiting JNK Signaling Pathway.

Phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) gene encodes a serine/threonine kinase, which acts as a molecular sensor of mitochondrial health necessary for mitochondrial quality control. The present study was designed to examine whether PINK1 expressed in C57BL/6 murine cochlea and HEI-OC1 cells and, if so, to investigate the possible mechanisms underlying the action of PINK1 in cisplatin-induced death of sensory hair cells (HCs) and spiral ganglion neurons (SGNs) in vitro. The expression pattern of PINK1, formation of parkin particles, and autophagy were determined by immunofluorescent staining. The expressions of PINK1, LC3B, cleaved-caspase 3 and p-JNK were measured by Western blotting. The levels of reactive oxygen species (ROS) were evaluated by DCFH-DA and Mito-Sox Red staining. The mitochondrial membrane potential was detected by Tetramethylrhodamine methyl ester perchlorate (TMRM) and Rhodamine 123. Cell viability and apoptosis were examined by CCK8 assay, TUNEL staining and Annexin V Apoptosis Detection Kit, respectively. We found that PINK1 was widely expressed in the cytoplasm in HCs, SGNs, stria vascularis of C57BL/6 cochlea and HEI-OC1 cells and, notably, the expression level in cochlear HCs and SGNs of postnatal day 4 (P4) mice was higher than that in adult mice. Moreover, treatment with 30 μM cisplatin elicited the formation of ROS, which, in turn, led to PINK1 activation, parkin recruitment, autophagy formation and JNK pathway relevant to apoptosis in HEI-OC1 cells, HCs, and SGNs. Meanwhile, co-treatment with ROS scavenger N-acetyl-L-cysteine (NAC) or H2O2 consumer catalase-polyethylene glycol (PEG-catalase) inhibited parkin recruitment, alleviated autophagy formation, and mitigated JNK pathway related apoptosis. In addition, PINK1 silencing resulted in a lower level of autophagy, but, a higher mortality in HEI-OC1 cells treated with cisplatin. Taken together, data from this work reveal that PINK1 possesses the protective effect via induction of autophagy and resistance of apoptosis under cisplatin stimulus in sensory HCs and SGNs, implying that PINK1 might serve as an important regulator of cisplatin-elicited ototoxicity.

Ethyl Pyruvate Attenuates Early Brain Injury Following Subarachnoid Hemorrhage in the Endovascular Perforation Rabbit Model Possibly Via Anti-inflammation and Inhibition of JNK Signaling Pathway.

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is the main cause to poor outcomes of SAH patients, and early inflammation plays an important role in the acute pathophysiological events. It has been demonstrated that ethyl pyruvate (EP) has anti-inflammatory and neuroprotective effects in various critical diseases, however, the role of EP on EBI following SAH remains to be elucidated. Our study aimed to evaluate the effects of EP on EBI following SAH in the endovascular perforation rabbit model. All rabbits were randomly divided into three groups: sham, SAH + Vehicle (equal volume) and SAH + EP (30 mg/kg/day). MRI was performed to estimate the reliability of the EBI at 24 and 72 h after SAH. Neurological scores were recorded to evaluate the neurological deficit, ELISA kit was used to measure the level of tumor necrosis factor-α (TNF-α), and western blot was used to detect the expression of TNF-α, tJNK, pJNK, bax and bcl-2 at 24 and 72 h after SAH. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and Fluoro-jade B (FJB) staining were used to detect neuronal apoptosis and neurodegeneration respectively, meanwhile hematoxylin and eosin (H&E) staining was used to assess the degree of vasospasm. Our results demonstrated that EP alleviated brain tissue injury (characterized by diffusion weighted imaging and T2 sequence in MRI scan), and significantly improved neurological scores at 72 h after SAH. EP decreased the level of TNF-α and downregulated pJNK/tJNK and bax/bcl-2 in cerebral cortex and hippocampus effectively both at 24 and 72 h after SAH. Furthermore, EP reduced TUNEL and FJB positive cells significantly. In conclusion, the present study supported that EP afforded neuroprotective effects possibly via reducing TNF-α expression and inhibition of the JNK signaling pathway. Therefore, EP may be a potent therapeutic agent to attenuate EBI following SAH.

P054 Metformin protects against intestinal barrier disruption via AMPKα1-dependent inhibition of JNK signaling pathway

P054 Metformin protects against intestinal barrier disruption via AMPKα1-dependent inhibition of JNK signaling pathway

Docosahexaenoic acid confers analgesic effects after median nerve injury via inhibition of c-Jun N-terminal kinase activation in microglia

The c-Jun N-terminal kinase (JNK) in the central nervous system plays a critical role in the processing of neuropathic pain. Docosahexaenoic acid (DHA), a predominant omega-3 polyunsaturated fatty acid in the central nervous system, has a neuroprotective efficacy. In this study, we examined the relationships between JNK activation in the cuneate nucleus (CN) and behavioral hypersensitivity after chronic constriction injury (CCI) of the median nerve. We further investigated the effects of DHA administration on JNK activation and development of hypersensitivity. Using immunohistochemistry and immunoblotting, low levels of phosphorylated JNK (p-JNK) were detected in the CN of sham-operated rats. As early as 1day after CCI, p-JNK levels in the ipsilateral CN were significantly increased and peaked at 7days. Double-immunofluorescence labeling with cell-specific markers showed that p-JNK immunoreactive cells coexpressed OX-42, a microglia activation marker, suggesting the expression of p-JNK in the microglia. Microinjection of SP600125, a JNK inhibitor, into the CN 1day after CCI attenuated injury-induced behavioral hypersensitivity in a dose-dependent manner. Furthermore, animals received intravenous injection of DHA at doses of 100, 250 or 500nmol/kg 30min after median nerve CCI. DHA treatment decreased p-JNK and OX-42 levels, diminished the release of proinflammatory cytokines and improved behavioral hypersensitivity following CCI. In conclusion, median nerve injury-induced microglial JNK activation in the CN modulated development of behavioral hypersensitivity. DHA has analgesic effects on neuropathic pain, at least in part, by means of suppressing a microglia-mediated inflammatory response through the inhibition of JNK signaling pathway.

Tolerance to the antinociceptive effects of chronic morphine requires c-Jun N-terminal kinase.

BackgroundMorphine and fentanyl are opioid analgesics in wide clinical use that act through the μ-opioid receptor (MOR). However, one limitation of their long-term effectiveness is the development of tolerance. Receptor desensitization has been proposed as a putative mechanism driving tolerance to G protein-coupled receptor (GPCR) agonists. Recent studies have found that tolerance to morphine is mediated by the c-Jun N-terminal Kinase (JNK) signaling pathway. The goal of the present study was to test the hypotheses that: 1) JNK inhibition will be antinociceptive on its own; 2) JNK inhibition will augment morphine antinociception and; 3) JNK mediates chronic tolerance for the antinociceptive effects of morphine using acute (hotplate and tail-flick), inflammatory (10 μl of formalin 2.5 %) and chemotherapy (cisplatin 5 mg/kg ip once weekly)-induced neuropathic pain assays.ResultsWe found that JNK inhibition by SP600125 (3 mg/kg) produces a greater antinociceptive effect than morphine (6 mg/kg) alone in the formalin test. Moreover, co-administration of morphine (6 mg/kg) with SP600125 (3 mg/kg) produced a sub-additive antinociceptive effect in the formalin test. We also show that pre-treatment with SP600125 (3 or 10 mg/kg), attenuates tolerance to the antinociceptive effects of morphine (10 mg/kg), but not fentanyl (0.3 mg/kg), in the tail-flick and hotplate tests. Pre-treatment with SP600125 also attenuates tolerance to the hypothermic effects of both morphine and fentanyl. We also examined the role of JNK in morphine tolerance in a cisplatin-induced model of neuropathic pain. Interestingly, treatment with SP600125 (3 mg/kg) alone attenuated mechanical and cold allodynia in a chemotherapy-induced pain model using cisplatin. Strikingly, SP600125 (3 mg/kg) pre-treatment prolonged the anti-allodynic effect of morphine by several days (5 and 7 days for mechanical and cold, respectively).ConclusionsThese results demonstrate that JNK signaling plays a crucial role in mediating antinociception as well as chronic tolerance to the antinociceptive effects of morphine in acute, inflammatory, and neuropathic pain states. Thus, inhibition of JNK signaling pathway, via SP600125, represents an efficacious pharmacological approach to delay tolerance to the antinociceptive effects of chronic morphine in diverse pain models.

Effect of Vascular Cell Adhesion Molecule -1 Overexpression on Adipogenic Differentiation of Murine Mesenchymal Stem Cells and Its Mechanism

To investigate the effect of vascular cell adhesion molecule-1 (VCAM-1) gene overexpression on adipogenic differentiation of mouse mesenchymal stem cells(MSC) and explore its molecular mechanism. VCAM-1 overexpression MSC (MIGR1-VCAM-1/MSC) and the empty plasmid transfection MSC (MIGR1/MSC) were induced to adipogenic differentiation, oil-red-O staining and real-time PCR were used to detect the adipogenic differentiation ability and the mRNA expression level of key transcription factors C/EBP α and PPAR γ. The activation of P38, ERK and JNK pathways were analyzed by Western blot. Furthermore, the specific chemical inhibitors of MAPK pathway (SB203580, PD98059 and JNK inhibitor II) were added to the induced culture system and the alteration of the MSC adipogenic differentiation ability were evaluated. no matter in self or induced differentiation groups, the lipid droplets in MIGR1-VCAM-1/MSC became larger, the amount of adipocyte increased than that in MIGR1/MSC (P<0.01), the mRNA expression level of C/EBPα and PPARγ were upregulated, and JNK pathway were down-regulated while the P38 and ERK pathway were significantly up-regulated. The inhibition of JNK pathway of MIGR1-VCAM-1/MSC could lead to increased mRNA expression level of C/EBP α and PPAR γ, the amount of adipocytes increased (P<0.01), however, the inhibition of the P38 and ERK pathway of MIGR1-VCAM-1/MSC could lead to decreased mRNA expression level of C/EBP α and PPAR γ, and the lipid droplets and the number of adipocytes became smaller and less. The overexpression of VCAM-1 may promote MSC to differentiate into adipocytes through inhibiting JNK signaling pathway, activating P38 and ERK pathways.

Pyrroloquinoline quinone protects mouse brain endothelial cells from high glucose-induced damage in vitro.

To investigate the effects of pyrroloquinoline quinone (PQQ), an oxidoreductase cofactor, on high glucose-induced mouse endothelial cell damage in vitro. Mouse brain microvascular endothelial bEND.3 cells were exposed to different glucose concentrations (5.56, 25 and 40 mmol/L) for 24 or 48 h. The cell viability was examined using MTT assay. Flow cytometry was used to analyze the apoptosis and ROS levels in the cells. MitoTracker Green staining was used to examine the mitochondria numbers in the cells. Western blot analysis was used to analyze the expression of HIF-1α and the proteins in JNK pathway. Treatment of bEND.3 cells with high glucose significantly decreased the cell viability, while addition of PQQ (1 and 10 μmol/L) reversed the high glucose-induced cell damage in a concentration-dependent manner. Furthermore, PQQ (100 μmol/L) significantly suppressed the high glucose-induced apoptosis and ROS production in the cells. PQQ significantly reversed the high glucose-induced reduction in both the mitochondrial membrane potential and mitochondria number in the cells. The high glucose treatment significantly increased the expression of HIF-1α and JNK phosphorylation in the cells, and addition of PQQ led to a further increase of HIF-1α level and a decrease of JNK phosphorylation. Addition of JNK inhibitor SP600125 (10 μmol/L) also significantly suppressed high glucose-induced apoptosis and JNK phosphorylation in bEND.3 cells. PQQ protects mouse brain endothelial cells from high glucose damage in vitro by suppressing intracellular ROS and apoptosis via inhibiting JNK signaling pathway.