c-Jun NH2-terminal Protein Kinase Research Articles

JNK inhibition enhances cell-cell adhesion impaired by desmoglein3 gene disruption in keratinocytes.

c-Jun NH2-terminal protein kinase (JNK) and p38 are stress-activated mitogen-activated protein kinases (MAPK) that are phosphorylated by various stimuli. It has been reported that the loss of desmoglein (DSG) 3, a desmosomal transmembrane core molecule, in keratinocytes impairs cell-cell adhesion accompanied by p38 MAPK activation. To understand the biological role of DSG3 in desmosomes and its relationship with stress-activated MAPKs, we established DSG3 knockout keratinocytes (KO cells). Wild-type cells showed a linear localization of DSG1 to cell-cell contacts, whereas KO cells showed a remarkable reduction despite the increased protein levels of DSG1. Cell-cell adhesion in KO cells was impaired over time, as demonstrated by dispase-based dissociation assays. The linear localization of DSG1 to cell-cell contacts and the strength of cell-cell adhesion were promoted by the pharmacological inhibition of JNK. Conversely, pharmacological activation of JNK, but not p38 MAPK, in wild-type cells reduced the linear localization of DSG1 in cell-cell contacts. Our data indicate that DSG1 and DSG2 in KO cells cannot compensate for the attenuation of cell-cell adhesion strength caused by DSG3 deficiency and that JNK inhibition restores the strength of cell-cell adhesion by increasing the linear localization of DSG1 in cell-cell contacts in KO cells. Inhibition of JNK signaling may improve cell-cell adhesion in diseases in which DSG3 expression is impaired.

Stress-activated protein kinases are involved in the replication of porcine deltacoronavirus

This study was conducted to examine the role of stress-activated protein kinases (SAPKs), including c-Jun NH2-terminal kinases (JNK1/2) and p38 mitogen-activated protein kinase (MAPK), in porcine deltacoronavirus (PDCoV) infection. Results demonstrated the activation of JNK1/2 and p38 MAPK in PDCoV-infected cells, which occurred concomitant with viral biosynthesis and irrespective of cell type. Pharmacological inhibition or knockdown of either SAPK significantly attenuated PDCoV replication, whereas addition of a signaling activator augmented virus infectivity. Moreover, pharmacological inhibition of JNK1/2 or p38 MAPK activation was innocuous to viral entry but significantly detrimental to post uncoating stages of the replication cycle. Remarkably, cytokine gene expression in PDCoV-infected IPEC-J2 cells was modified by inhibiting the activation of either SAPK. Collectively, these data indicate that JNK1/2 and p38 MAPK signaling pathways contribute to viral biosynthesis and regulate immune responses, thereby favoring the replication of PDCoV.

Erythronium japonicum Alleviates Inflammatory Pain by Inhibiting MAPK Activation and by Suppressing NF-κB Activation via ERK/Nrf2/HO-1 Signaling Pathway.

Microglial activation-mediated neuroinflammation influences the development of inflammatory pain. The aim of this study was to investigate the anti-inflammatory effects and mechanisms of aqueous Erythronium japonicum extract (EJE) in microglia activation-mediated inflammatory pain. EJE was found to suppress lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), ionized calcium-binding adapter molecule 1 (IBA-1), and pro-inflammatory cytokines in BV2 microglial cells. In addition, LPS-induced c-Jun NH2 terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) phosphorylation were inhibited by EJE. Intriguingly, EJE also inhibited p65 phosphorylation by activating extracellular signal-regulated kinase-1/2 (ERK)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Furthermore, the effects of EJE treatment, such as HO-1 induction and the reduction of NF-ĸB activation, were reversed by ERK1/2 inhibition. In an inflammatory pain mouse model, Complete Freund’s Adjuvant (CFA)-induced mechanical allodynia and foot swelling were alleviated by the oral administration of EJE. Consistent with in vitro results, EJE increased HO-1, while decreasing CFA-induced COX-2, IBA-1, and pro-inflammatory cytokines in the spinal cord. Among the components of EJE, butanol most heavily suppressed LPS-induced microglial activation and increased HO-1 expression. These findings indicate that EJE can alleviate inflammatory pain by inhibiting p38 and JNK and by suppressing NF-ĸB via ERK/Nrf2/HO-1 signaling.

JNK Signaling Pathway Involvement in Spinal Cord Neuron Development and Death.

The c-Jun NH2-terminal protein kinase (JNK) is a Janus-faced kinase, which, in the nervous system, plays important roles in a broad range of physiological and pathological processes. Three genes, encoding for 10 JNK isoforms, have been identified: jnk1, jnk2, and jnk3. In the developing spinal cord, JNK proteins control neuronal polarity, axon growth/pathfinding, and programmed cell death; in adulthood they can drive degeneration and regeneration, after pathological insults. Indeed, recent studies have highlighted a role for JNK in motor neuron (MN) diseases, such as amyotrophic lateral sclerosis and spinal muscular atrophy. In this review we discuss how JNK-dependent signaling regulates apparently contradictory functions in the spinal cord, in both the developmental and adult stages. In addition, we examine the evidence that the specific targeting of JNK signaling pathway may represent a promising therapeutic strategy for the treatment of MN diseases.

High glucose promotes annulus fibrosus cell apoptosis through activating the JNK and p38 MAPK pathways.

Diabetes mellitus (DM) is an important risk factor of intervertebral disc degeneration. A high glucose niche-mediated disc cell apoptosis is an implicate causative factor for the spine degenerative diseases related with DM. However, the effects of a high glucose niche on disc annulus fibrosus (AF) cell apoptosis and the potential signaling transduction pathway is unclear. The present study is to investigate the effects of high glucose on disc AF cell apoptosis and the role of two potential signaling pathways in this process. Rat AF cells were cultured in baseline medium or medium with different concentrations (0.1 and 0.2 M) of glucose for 3 days. Flow cytometry was used to assess the degree of apoptosis. Activity of caspase 3/9 was evaluated by chemical kit. Expression of pro-apoptotic and anti-apoptotic molecules was analyzed by real-time polymerase chain reaction and Western blot. In addition, activity of the C-Jun NH2-terminal kinases (JNK) pathway and p38 mitogen-activated protein kinase (MAPK) pathway was evaluated by Western blot. Compared with the control group, high glucose culture increased cell apoptosis ratio and caspase-3/9 activity, up-regulated expression of bax, caspase-3, cleaved caspase-3 and cleaved PARP, and down-regulated expression of bcl-2 in a glucose concentration-dependent manner. Additionally, high glucose culture increased expression of the p-JNK and p-p38 MAPK in a concentration-dependent manner. Further results showed that inhibition of the JNK or p38 MAPK pathway attenuated the effects of high glucose on AF cell apoptosis. Together, high glucose promoted disc AF cell apoptosis through regulating the JNK pathway and p38 MAPK pathway in a glucose concentration-dependent manner.

Cadmium Exposure Impairs Adult Hippocampal Neurogenesis.

Cadmium (Cd) is an environmental pollutant of considerable interest throughout the world and potentially a neurotoxicant. Our recent data indicate that Cd exposure induces impairment of hippocampus-dependent learning and memory in mice. However, the underlying mechanisms for this defect are not known. The goal of this study was to determine if Cd inhibits adult neurogenesis and to identify underlying signaling pathways responsible for this impairment. Adult hippocampal neurogenesis is a process in which adult neural progenitor/stem cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate functional new neurons in the hippocampus which contributes to hippocampus-dependent learning and memory. However, studies concerning the effects of neurotoxicants on adult hippocampal neurogenesis and the underlying signaling mechanisms are limited. Here, we report that Cd significantly induces apoptosis, inhibits proliferation, and impairs neuronal differentiation in primary cultured aNPCs derived from the SGZ. In addition, the c-Jun NH2-terminal kinase and p38 mitogen-activated protein kinase signaling pathways are activated by Cd and contribute to its toxicity. Furthermore, we exposed 8-week-old male C57BL/6 mice to Cd through drinking water for 13 weeks to assess the effects of Cd on adult hippocampal neurogenesis in vivo. Cd treatment reduced the number of 5-week-old adult-born cells in the DG and impaired the differentiation of adult-born hippocampal neurons. These results suggest that Cd exposure impairs adult hippocampal neurogenesis both in vitro and in vivo. This may contribute to Cd-mediated inhibition of hippocampus-dependent learning and memory.

Galectin-9 Induces Mitochondria-Mediated Apoptosis of Esophageal Cancer In Vitro and In Vivo in a Xenograft Mouse Model.

Galectin-9 (Gal-9) enhances tumor immunity mediated by T cells, macrophages, and dendritic cells. Its expression level in various cancers correlates with prognosis. Furthermore, Gal-9 directly induces apoptosis in various cancers; however, its mechanism of action and bioactivity has not been clarified. We evaluated Gal-9 antitumor effect against esophageal squamous cell carcinoma (ESCC) to analyze the dynamics of apoptosis-related molecules, elucidate its mechanism of action, and identify relevant changes in miRNA expressions. KYSE-150 and KYSE-180 cells were treated with Gal-9 and their proliferation was evaluated. Gal-9 inhibited cell proliferation in a concentration-dependent manner. The xenograft mouse model established with KYSE-150 cells was administered with Gal-9 and significant suppression in the tumor growth observed. Gal-9 treatment of KYSE-150 cells increased the number of Annexin V-positive cells, activation of caspase-3, and collapse of mitochondrial potential, indicating apoptosis induction. c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) phosphorylation were activated and could be involved in apoptosis. Therefore, Gal-9 induces mitochondria-mediated apoptosis of ESCC and inhibits cell proliferation in vitro and in vivo with JNK and p38 activation.

MKK7, the essential regulator of JNK signaling involved in cancer cell survival: a newly emerging anticancer therapeutic target.

One of the mitogen-activated protein kinases (MAPKs), c-Jun NH2-terminal protein kinase (JNK) plays an important role in regulating cell fate, such as proliferation, differentiation, development, transformation, and apoptosis. Its activity is induced through the interaction of MAPK kinase kinases (MAP3Ks), MAPK kinases (MAP2Ks), and various scaffolding proteins. Because of the importance of the JNK cascade to intracellular bioactivity, many studies have been conducted to reveal its precise intracellular functions and mechanisms, but its regulatory mechanisms remain elusive. In this review, we discuss the molecular characterization, activation process, and physiological functions of mitogen-activated protein kinase kinase 7 (MKK7), the MAP2K that most specifically controls the activity of JNK. Understanding the role of MKK7/JNK signaling in physiological conditions could spark new hypotheses for targeted anticancer therapies.

MicroRNA-21 contributes to high glucose-induced fibrosis in peritoneal mesothelial cells in rat models by activation of the Ras-MAPK signaling pathway via Sprouty-1.

Peritoneal fibrosis remains to be one of the most severe causes of failure in continuous peritoneal dialysis. The current study cultured peritoneal mesothelial cells in high glucose to stimulate the environment of peritoneal fibrosis model in rats, and investigate whether microRNA-21 (miR-21) targeting Sprouty-1 affects high glucose-induced fibrosis in peritoneal mesothelial cells via the rennin angiotensin system (Ras)-mitogen-activated protein kinase (MAPK) signaling pathway, as well as potential mechanisms. Peritoneal tissues in fibrosis rats were collected to extract peritoneal mesothelial cells, which, after in vitro culture, were transfected with a series of mimic or inhibitor of miR-21, or the small interfering RNA (siRNA) against Sprouty-1. Reverse-transcription quantitative polymerase chain reaction and western blot analyses were performed to determine the levels of related genes or proteins. MTT assay and flow cytometry were conducted to observe the cell viability and cell apoptosis of peritoneal mesothelial cells. Dual-luciferase reporter gene assay revealed that Sprouty-1 is the target gene of miR-21. Peritoneal fibrosis manifestedwith elevated miR-21, extracellular-signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), RAS and p38MAPK but reduced Sprouty-1. Cells transfected with miR-21 mimic exhibited decreased Sprouty-1 expressions, but increased levels of ERK, JNK, RAS, and p38MAPK. As for cellular process, miR-21 mimic or siRNA against Sprouty-1 exposure reduced cell viability, which resulted in more cells arrested at the G1 stage, and induced apoptosis. In contrast, miR-21 inhibitor exposure was observed to have induced effects on peritoneal mesothelial cells. These key findings provide evidence that miR-21 inhibits Sprouty-1 to promote the progression of fibrosis in peritoneal mesothelial cells by activating the Ras-MAPK signaling pathway.

Lobohedleolide suppresses hepatitis C virus replication via JNK/c-Jun-C/EBP-mediated down-regulation of cyclooxygenase-2 expression

Hepatitis C virus (HCV) chronically infects 2–3% people of the global population, which leads to liver cirrhosis and hepatocellular carcinoma. Drug resistance remains a serious problem that limits the effectiveness of US Food and Drug Administration (FDA)-approved direct-acting antiviral (DAA) drugs against HCV proteins. The objective of our study was to discover new antivirals from natural products to supplement current therapeutics. We demonstrated that lobohedleolide, isolated from the Formosan soft coral Lobophytum crassum, significantly reduced HCV replication in replicon cells and JFH-1 infection system, with EC50 values of 10 ± 0.56 and 22 ± 0.75 μM, respectively, at non-toxic concentrations. We further observed that the inhibitory effect of lobohedleolide on HCV replication is due to suppression of HCV-induced cyclooxygenase-2 (COX-2) expression. Based on deletion-mutant analysis of the COX-2 promoter, we identified CCAAT/enhancer-binding protein (C/EBP) as a key transcription factor for the down-regulation of COX-2 by lobohedleolide, through which lobohedleolide decreased the phosphorylation of c-Jun NH2-terminal protein kinase and c-Jun to suppress HCV-induced C/EBP expression. The combination treatment of lobohedleolide with clinically used HCV drugs synergistically reduced HCV RNA replication, indicating that lobohedleolide exhibited a high biomedical potential to be used as a supplementary therapeutic agent to control HCV infection.

Ochratoxin A, citrinin and deoxynivalenol decrease claudin-2 expression in mouse rectum CMT93-II cells

Intestinal epithelial cells are the first targets of ingested mycotoxins, such as ochratoxin A, citrinin and deoxynivalenol. It has been reported that paracellular permeability regulated by tight junctions is modulated by several mycotoxins by reducing the expression of specific claudins and integral membrane proteins in cell-cell contacts, accompanied by increase in phosphorylation of mitogen-activated protein kinases, including extracellular signal-related kinase (ERK) 1/2, p38 and c-Jun NH2-terminal protein kinase. Claudin-2 is expressed in the deep crypt cells, but not in the villus/surface cells in vivo. While Caco-2, T84 and IPEC-J2 cells, which are widely used intestinal epithelial cell lines to assess the influence of mycotoxins, do not express claudin-2, CMT93-II cells express claudin-2. We previously reported that inhibition of the ERK pathway reduced claudin-2 levels in cell-cell contacts in CMT93-II cells. In this study, we examined whether ochratoxin A, citrinin and deoxynivalenol affect claudin-2 expression and ERK1/2 phosphorylation in CMT93-II cells. We found that all mycotoxins reduced claudin-2 expression in cell-cell contacts, with reduction (by citrinin and deoxynivalenol) or no change (by ochratoxin A) in phosphorylated ERK1/2. All mycotoxins increased transepithelial electrical resistance, but did not affect flux of fluorescein. While ochratoxin A and citrinin are known to be nephrotoxic, only deoxynivalenol reduced claudin-2 expression in MDCK II cells derived from the renal tubule. These results suggest that claudin-2 expression is regulated not only by the ERK pathway, but also by other pathways in an organ-specific manner.

Electric Stimulation of Ear Reduces the Effect of Toll-Like Receptor 4 Signaling Pathway on Kainic Acid-Induced Epileptic Seizures in Rats.

Epilepsy is a common clinical syndrome with recurrent neuronal discharges in the temporal lobe, cerebral cortex, and hippocampus. Clinical antiepileptic medicines are often ineffective or of little benefit in 30% of epileptic patients and usually cause severe side effects. Emerging evidence indicates the crucial role of inflammatory mediators in epilepsy. The current study investigates the role of toll-like receptor 4 (TLR4) and its underlying mechanisms in kainic acid- (KA-) induced epileptic seizures in rats. Experimental KA injection successfully initiated an epileptic seizure accompanied by increased expression of TLR4 in the prefrontal cortex, hippocampus, and somatosensory cortex. In addition, calcium-sensitive phosphorylated Ca2+/calmodulin-dependent protein kinase II (pCaMKIIα) increased after the initiation of the epileptic seizure. Furthermore, downstream-phosphorylated signal-regulated kinase (ERK), c-Jun NH2-terminal protein kinase (JNK), and p38 kinase simultaneously increased in these brain areas. Moreover, the transcriptional factor phosphorylated nuclear factor-κB (pNF-κB) increased, suggesting that nucleus transcription was affected. Furthermore, the aforementioned molecules decreased by an electric stimulation (ES) of either 2 Hz or 15 Hz of the ear in the three brain areas. Accordingly, we suggest that ES of the ear can successfully control epileptic seizures by regulating the TLR4 signaling pathway and has a therapeutic benefit in reducing epileptic seizures.

Amelioration of Endurance Training on Cardiac Mitochondrial Quality Control System in the Post-infarction Rats

Aerobic interval training (AIT) has been reported to exert beneficial effects on various cardiovascular diseases. However, effects of AIT on the post-myocardial infarction (MI)–associated mitochondrial dysfunction was still unclear. In this study, we investigated the favorable protection of AIT on myocardial mitochondria in the post-MI rats by focusing on mitochondrial dynamics and biogenesis. Mitochondrial ultrastructure and respiratory functions (respiratory control ratio [RCR], value of P/O), complex activities, dynamic proteins (mitofusin [mfn] 1/2, type 1 optic atrophy [OPA1] and dynamin-related protein1 [Drp1]), biogenesis, oxidative signaling of extracellular signal-regulated kinase [ERK] 1/2, (c-Jun NH2-terminal protein kinase [JNK] and P53 were determined. Post-MI rats exhibited adverse mitochondrial ultrastructure changes and dysfunctions. Also, reduced fusion and increased fission accidents were observed, which were revealed to associate with activated ERK1/2-JNK-P53 signaling. Furthermore, mitochondrial biogenesis was decreased. After AIT, not only did mitochondria appeared more normal in structure, but also improved MI-associated mitochondrial dysfunction (elevated RCR and P/O, enhanced activities of complex I, III and IV), which were accompanied by increased fusion (mfn2 and OPA1) and biogenesis, decreased fission (Drp1) and inactivated ERK1/2-JNK-P53 signal pathway. These data showed that AIT may restore mitochondrial functions by protecting structural integrity, inhibiting dynamics pathological remodelling and increasing biogenesis.

GLP-1 Receptor Activation Inhibits Palmitate-Induced Apoptosis via Ceramide in Human Cardiac Progenitor Cells.

Increased apoptosis of cardiomyocytes and cardiac progenitor cells (CPCs) in response to saturated fatty acids (SFAs) can lead to myocardial damage and dysfunction. Ceramides mediate lipotoxicity-induced apoptosis. Glucagonlike peptide-1 receptor (GLP1R) agonists exert beneficial effects on cardiac cells in experimental models. To investigate the protective effects of GLP1R activation on SFA-mediated apoptotic death of human CPCs. Human CPCs were isolated from cardiac appendages of nondiabetic donors and then exposed to palmitate with or without pretreatment with the GLP1R agonist exendin-4. Ceramide accumulation was evaluated by immunofluorescence. Expression of key enzymes in de novo ceramide biosynthesis was studied by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Apoptosis was evaluated by measuring release of oligonucleosomes, caspase-3 cleavage, caspase activity, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. Exposure of the CPCs to palmitate resulted in 2.3- and 1.9-fold higher expression of ceramide synthase 5 (CERS5) and ceramide desaturase-1, respectively (P < 0.05). This was associated with intracellular accumulation of ceramide and activation of c-Jun NH2-terminal protein kinase (JNK) signaling and apoptosis (P < 0.05). Both coincubation with fumonisin B1, a specific ceramide synthase inhibitor, and CERS5 knockdown prevented ceramide accumulation, JNK activation, and apoptosis in response to palmitate (P < 0.05). Exendin-4 also prevented the activation of the ceramide biosynthesis and JNK in response to palmitate, inhibiting apoptosis (P < 0.05). Excess palmitate results in activation of ceramide biosynthesis, JNK signaling, and apoptosis in human CPCs. GLP1R activation counteracts this lipotoxic damage via inhibition of ceramide generation, and this may represent a cardioprotective mechanism.

Minocycline modulates NF\u03baB phosphorylation and enhances antimicrobial activity against Staphylococcus aureus in mesenchymal stromal/stem cells

BackgroundMesenchymal stromal/stem cells (MSCs) have demonstrated pro-healing properties due to their anti-inflammatory, angiogenic, and even antibacterial properties. We have shown previously that minocycline enhances the wound healing phenotype of MSCs, and MSCs encapsulated in poly(ethylene glycol) and gelatin-based hydrogels with minocycline have antibacterial properties against Staphylococcus aureus (SA). Here, we investigated the signaling pathway that minocycline modulates in MSCs which results in their enhanced wound healing phenotype and determined whether preconditioning MSCs with minocycline has an effect on antimicrobial activity. We further investigated the in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels in inoculated full-thickness cutaneous wounds.MethodsModulation of cell signaling pathways in MSCs with minocycline was analyzed via western blot, immunofluorescence, and ELISA. Antimicrobial efficacy of MSCs pretreated with minocycline was determined by direct and transwell coculture with SA. MSC viability after SA coculture was determined via a LIVE/DEAD® stain. Internalization of SA by MSCs pretreated with minocycline was determined via confocal imaging. All protein and cytokine analysis was done via ELISA. The in-vivo antimicrobial efficacy of MSC and antibiotic-loaded hydrogels was determined in Sprague–Dawley rats inoculated with SA. Two-way ANOVA for multiple comparisons was used with Bonferroni test assessment and an unpaired two-tailed Student’s t test was used to determine p values for all assays with multiple or two conditions, respectively.ResultsMinocycline leads to the phosphorylation of transcriptional nuclear factor-κB (NFκB), but not c-Jun NH2-terminal kinase (JNK) or mitogen-activated protein kinase (ERK). Inhibition of NFκB activation prevented the minocycline-induced increase in VEGF secretion. Preconditioning of MSCs with minocycline led to a reduced production of the antimicrobial peptide LL-37, but enhanced antimicrobial activity against SA via an increased production of IL-6 and SA internalization. MSC and antibiotic-loaded hydrogels reduced SA bioburden in inoculated wounds over 3 days and accelerated reepithelialization.ConclusionsMinocycline modulates the NFκB pathway in MSCs that leads to an enhanced production of IL-6 and internalization of SA. This mechanism may have contributed to the in-vivo antibacterial efficacy of MSC and antibiotic-loaded hydrogels.

Synthesis and anti-diabetic activity of new N,N-dimethylphenylenediamine-derivatized nitrilotriacetic acid vanadyl complexes

Vanadium compounds are promising anti-diabetic agents. However, reducing the metal toxicity while keeping/improving the hypoglycemic effect is still a big challenge towards the success of anti-diabetic vanadium drugs. To improve the therapeutic potency using the anti-oxidative strategy, we synthesized new N,N-dimethylphenylenediamine (DMPD)-derivatized nitrilotriacetic acid vanadyl complexes ([VO(dmada)]). The in vitro biological evaluations revealed that the DMPD-derivatized complexes showed improved antioxidant capacity and lowered cytotoxicity on HK-2 cells than bis(maltolato)oxidovanadium (IV) (BMOV). In type II diabetic mice, [VO(p-dmada)] (0.15mmolkg−1/day) exhibited better hypoglycemic effects than BMOV especially on improving glucose tolerance and alleviating the hyperglycemia-induced liver damage. These insulin enhancement effects were associated with increased expression of peroxisome proliferator-activated receptor α and γ (PPARα/γ) in fat, activation of Akt (v-Akt murine thymoma viral oncogene)/PKB (protein kinase-B) in fat and liver, and inactivation of c-Jun NH2-terminal protein kinases (JNK) in liver. Moreover, [VO(p-dmada)] showed no tissue toxicity at the therapeutic dose in diabetic mice and the oral acute toxicity (LD50) was determined to be 1640mgkg−1. Overall, the experimental results indicated that [VO(p-dmada)] can be a potent insulin enhancement agent with improved efficacy-over- toxicity index for further drug development. In addition, the results on brain Tau phosphorylation suggested necessary investigation on the effects of vanadyl complexes on the pathology of the Alzheimer's disease in the future.

The Novel Mechanisms Concerning the Inhibitions of Palmitate-Induced Proinflammatory Factor Releases and Endogenous Cellular Stress with Astaxanthin on MIN6 β-Cells.

Astaxanthin, an antioxidant agent, can protect pancreatic β-cells of db/db mice from glucotoxicity and resolve chronic inflammation in adipose tissue. Nonetheless, the effects of astaxanthin on free-fatty-acid-induced inflammation and cellular stress in β-cells remain to be demonstrated. Meanwhile, palmitate enhances the secretion of pro-inflammatory adipokines monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF120). We therefore investigated the influence of astaxanthin on palmitate-stimulated MCP-1 and VEGF120 secretion in mouse insulinoma (MIN6) pancreatic β-cells. Furthermore, whether astaxanthin prevents cellular stress in MIN6 cells was also assessed. Pre-treatment with astaxanthin or with N-acetyl-cysteine (NAC) which is an antioxidant drug, significantly attenuated the palmitate-induced MCP-1 release through downregulation of phosphorylated c-Jun NH2-terminal protein kinase (JNK) pathways, and suppressed VEGF120 through the PI3K/Akt pathways relative to the cells stimulated with palmitate alone. In addition, palmitate significantly upregulated homologous protein (CHOP) and anti-glucose-regulated protein (GRP78), which are endoplasmic reticulum (ER) stress markers, in MIN6 cells. On the other hand, astaxanthin attenuated the increased CHOP content, but further up-regulated palmitate-stimulated GRP78 protein expression. By contrast, NAC had no effects on either CHOP or GRP78 enhancement induced by palmitate in MIN6 cells. In conclusion, astaxanthin diminishes the palmitate-stimulated increase in MCP-1 secretion via the downregulation of JNK pathways in MIN6 cells, and affects VEGF120 secretion through PI3K/Akt pathways. Moreover, astaxanthin can prevent not only oxidative stress caused endogenously by palmitate but also ER stress, which NAC fails to attenuate, via upregulation of GRP78, an ER chaperon.

Fisetin Protects PC12 Cells from Tunicamycin-Mediated Cell Death via Reactive Oxygen Species Scavenging and Modulation of Nrf2-Driven Gene Expression, SIRT1 and MAPK Signaling in PC12 Cells.

Background: Fisetin (3,7,3′,4′-tetrahydroxyflavone) is a dietary flavonol and exhibits antioxidant, anti-inflammatory, and neuroprotective activities. However, high concentration of fisetin is reported to produce reactive oxygen species (ROS), induce endoplasmic reticulum (ER) stress and cause cytotoxicity in cancer cells. The aim of this study is to investigate the cytoprotective effects of low concentration of fisetin against tunicamycin (Tm)-mediated cytotoxicity in neuronal-like catecholaminergic PC12 cells. Methods: Cell viability was assayed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and apoptotic and autophagic markers were analyzed by Western blot. Gene expression of unfolded protein response (UPR) and Phase II enzymes was further investigated using RT-Q-PCR or Western blotting. Intracellular ROS level was measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) by a fluorometer. The effects of fisetin on mitogen activated protein kinases (MAPKs) and SIRT1 (Sirtuin 1) signaling pathways were examined using Western blotting and specific inhibitors. Results: Fisetin (<20 µM) restored cell viability and repressed apoptosis, autophagy and ROS production in Tm-treated cells. Fisetin attenuated Tm-mediated expression of ER stress genes, such as glucose-regulated proteins 78 (GRP78), C/EBP homologous protein (CHOP also known as GADD153) and Tribbles homolog 3 (TRB3), but induced the expression of nuclear E2 related factor (Nrf)2-targeted heme oxygenase (HO)-1, glutamate cysteine ligase (GCL) and cystine/glutamate transporter (xCT/SLC7A11), in both the presence and absence of Tm. Moreover, fisetin enhanced phosphorylation of ERK (extracellular signal-regulated kinase), JNK (c-JUN NH2-terminal protein kinase), and p38 MAPK. Addition of JNK and p38 MAPK inhibitor significantly antagonized its cytoprotective activity and modulatory effects on UPR. Fisetin also restored Tm-inhibited SIRT1 expression and addition of sirtinol (SIRT1 activation inhibitor) significantly blocked fisetin-mediated cytoprotection. In conclusion, this result shows that fisetin activates Nrf2, MAPK and SIRT1, which may elicit adaptive cellular stress response pathways so as to protect cells from Tm-induced cytotoxicity.

Immunostimulatory Activity of Opuntia ficus-indica var. Saboten Cladodes Fermented by Lactobacillus plantarum and Bacillus subtilis in RAW 264.7 Macrophages.

To increase the functionality of Opuntia ficus-indica var. saboten cladodes, it was fermented by Lactobacillus plantarum and Bacillus subtilis. Eighty percent methanol extracts were investigated for their effects on nitric oxide (NO) production, cytokine secretion, nuclear factor-κB (NF-κB) activity, and mitogen-activated protein kinase (MAPK) phosphorylation in RAW 264.7 cells. Methanol extracts of L. plantarum culture medium (LPCME) and B. subtilis culture medium (BSCME) did not affect lipopolysaccharide (LPS)-induced NO production but, at 500 μg/mL, increased interferon (IFN)-γ-induced NO production by 55.2 and 66.5 μM, respectively, in RAW 264.7 cells. In RAW 264.7 cells not treated with LPS and IFN-γ, LPCME did not affect NO production, but BSCME increased NO production significantly in a dose-dependent manner. In addition, BSCME induced the expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW 264.7 cells in a dose-dependent manner. BSCME at 500 μg/mL increased TNF-α and IL-1β mRNA levels by 83.8% and 82.2%, respectively. BSCME increased NF-κB-dependent luciferase activity in a dose-dependent manner; 500 μg/mL BSCME increased activity 9.1-fold compared with the control. BSCME induced the phosphorylation of p38, c-JUN NH2-terminal protein kinase (JNK), and extracellular signal-regulated kinase (ERK) in a dose-dependent manner, but did not affect total ERK levels. In conclusion, BSCME exerted immunostimulatory effects, which were mediated by MAPK phosphorylation and NF-κB activation, resulting in increased TNF-α and IL-1β gene expression in RAW 264.7 macrophages. Therefore, BSCM shows promise for use as an immunostimulatory therapeutic.

Adenosine 5'-monophosphate blocks acetaminophen toxicity by increasing ubiquitination-mediated ASK1 degradation.

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced liver failure in the world. Hepatic c-jun NH2-terminal protein kinase (JNK) activation is thought to be a consequence of oxidative stress produced during APAP metabolism. Activation of JNK signals causes hepatocellular damage with necrotic and apoptotic cell death. Here we found that APAP caused a feedback increase in plasma adenosine 5′-monophsphate (5′-AMP). We demonstrated that co-administration of APAP and 5′-AMP significantly ameliorated APAP-induced hepatotoxicity in mice, without influences on APAP metabolism and its analgesic function. The mechanism of protection by 5′-AMP was through inhibiting APAP-induced activation of JNK, and attenuating downstream c-jun and c-fos gene expression. This was triggered by attenuating apoptosis signal-regulated kinase 1(ASK1) methylation and increasing ubiquitination-mediated ASK1 protein degradation. Our findings indicate that replacing the current APAP with a safe and functional APAP/5′-AMP formulation could prevent APAP-induced hepatotoxicity.