c-Jun N-terminal Kinase Inhibitor Research Articles

Effects of butein on human osteosarcoma cell proliferation, apoptosis, and autophagy through oxidative stress.

Osteosarcoma (OS) is a primary malignant bone tumor, and the cure rate has stagnated in the past three decades. Butein, a plant polyphenol extracted from many herbs, has been proved to possess anti-tumor activity. However, the effect of butein on human OS and the underlying mechanisms remain to be elucidated. The OS cell line 143B was used. The effects of butein were evaluated through the cell proliferation assay, flow cytometry, florescence and transmission electron microscopy, and western blotting. All statistical analyses were performed using GraphPad Prism 7.0. Butein was found to inhibit cell proliferation by causing G2/ M phase arrest in the 143B cells. In addition, butein suppressed the invasion of 143B cells upon IL-6 treatment. Additionally, we found that butein inhibited the invasion of 143B cells stimulated with IL-6 via the p-STAT3-MMP9 signaling pathway. Remarkably, butein triggered extrinsic and intrinsic apoptosis and autophagy of 143B cells. The process of autophagy may have tumor-supporting effects. Furthermore, butein induced oxidative stress as evidenced by ROS generation, increase in malondialdehyde (MDA) level, and decrease in GSH/GSSH ratio and GPX4 expression. N-acetylcysteine can reverse the change of ROS. Further experiments indicated apoptosis and autophagy could be attenuated by the N-acetyl-L-cysteine and c-Jun N-terminal kinase (JNK) inhibitor SP600125. Additionally, butein inhibited the Akt/mammalian target of rapamycin (mTOR) signaling pathway, and suppressed the Akt kinase activity increased apoptosis and autophagy. Our results revealed butein induced apoptosis and autophagy by regulating oxidative stress, activating the JNK signaling pathway and blocking the Akt/mTOR signaling pathway in OS cells. Additionally, butein inhibited the invasion of 143B cells stimulated with IL-6 through the pSTAT3- MMP9 signaling pathway. In view of these results, butein may be a potential anti-tumor drug targeting osteosarcoma.

Pharmacological Actions of Myricetin in the Nervous System: A Comprehensive Review of Preclinical Studies in Animals and Cell Models.

Myricetin is a natural flavonoid extracted from a variety of plants, such as medicinal herbs, vegetables, berries, and tea leaves. A growing body of evidence has reported that myricetin supplementation display therapeutic activities in a lot of nervous system disorders, such as cerebral ischemia, Alzheimer’s disease, Parkinson’s disease, epilepsy, and glioblastoma. Myricetin supplementation can also protect against pathological changes and behavioral impairment induced by multiple sclerosis and chronic stress. On the basis of these pharmacological actions, myricetin could be developed as a potential drug for the prevention and/or treatment of nervous system disorders. Mechanistic studies have shown that inhibition of oxidative stress, cellular apoptosis, and neuroinflammatory response are common mechanisms for the neuroprotective actions of myricetin. Other mechanisms, including the activation of the nuclear factor E2-related factor 2 (Nrf2), extracellular signal-regulated kinase 1/2 (ERK1/2), protein kinase B (Akt), cyclic adenosine monophosphate-response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF) signaling, inhibition of intracellular Ca2+ increase, inhibition of c-Jun N-terminal kinase (JNK)-p38 activation, and suppression of mutant protein aggregation, may also mediate the neuroprotective effects of myricetin. Furthermore, myricetin treatment has been shown to promote the activation of the inhibitory neurons in the hypothalamic paraventricular nucleus, which subsequently produces anti-epilepsy effects. In this review, we make a comprehensive understanding about the pharmacological effects of myricetin in the nervous system, aiming to push the development of myricetin as a novel drug for the treatment of nervous system disorders.

Mutant p53 reactivator SLMP53-2 hinders ultraviolet B radiation-induced skin carcinogenesis

The growing incidence of skin cancer (SC) has prompted the search for additional preventive strategies to counteract this global health concern. Mutant p53 (mutp53), particularly with ultraviolet radiation (UVR) signature, has emerged as a promising target for SC prevention based on its key role in skin carcinogenesis. Herein, the preventive activity of our previously disclosed mutp53 reactivator SLMP53-2 against UVR-induced SC was investigated. The pre-treatment of keratinocyte HaCaT cells with SLMP53-2, before UVB exposure, depleted mutp53 protein levels with restoration of wild-type-like p53 DNA-binding ability and subsequent transcriptional activity. SLMP53-2 increased cell survival by promoting G1-phase cell cycle arrest, while reducing UVB-induced apoptosis through inhibition of c-Jun N-terminal kinase (JNK) activity. SLMP53-2 also protected cells from reactive oxygen species and oxidative damage induced by UVB. Moreover, it enhanced DNA repair through upregulation of nucleotide excision repair pathway and depletion of UVB-induced DNA damage, as evidenced by a reduction of DNA in comet tails, γH2AX staining and cyclobutane pyrimidine dimers (CPD) levels. SLMP53-2 further suppressed UVB-induced inflammation by inhibiting the nuclear translocation and DNA-binding ability of NF-κB, and promoted the expression of key players involved in keratinocytes differentiation. Consistently, the topical application of SLMP53-2 in mice skin, prior to UVB irradiation, reduced cell death and DNA damage. It also decreased the expression of inflammatory-related proteins and promoted cell differentiation, in UVB-exposed mice skin. Notably, SLMP53-2 did not show signs of skin toxicity for cumulative topical use. Overall, these results support a promising protective activity of SLMP53-2 against UVB-induced SC.

Assessment of drug-drug interactions of CC-90001, a potent and selective inhibitor of c-Jun N-terminal kinase

CC-90001 is predominantly metabolised via glucuronidation, while oxidative metabolism is a minor pathway in human hepatocytes and liver microsomes. In vitro, CC-90001 glucuronidation was catalysed by UGT1A9, UGT1A4, and UGT1A1, while oxidative metabolism was primarily mediated by CYP3A4/5 with minor contributions from CYP1A2, CYP2C9, CYP2B6, and CYP2D6. CC-90001 in vitro inhibits CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, and CYP3A4 ≤ 55% at 100 μM, and the inhibition was negligible at ≤30 μM. CC-90001 is not a time-dependent CYP inhibitor. In human hepatocytes CC-90001 is an inducer of CYP2B6 and CYP3A, with mRNA levels increased 34.4% to 52.8% relative to positive controls. In vitro CC-90001 is a substrate of P-gp, and an inhibitor of P-gp, BCRP, OAT3, OATP1B1, OATP1B3, OCT2, MATE1, and MATE2k with IC50 values of 30.3, 25.8, 17.7, 0.417, 19.9, 0.605, 4.17, and 20 μM, respectively. A clinical study demonstrated that CC-90001 has no or little impact on the exposure of warfarin (CYP2C9), omeprazole (CYP2C19), midazolam (CYP3A) or metformin (OCT2, MATE1/2k). CC-90001 co-administration increases the AUCt and Cmax 176% and 339% for rosuvastatin (BCRP/OATP1B1/3), 116% and 171% for digoxin (P-gp), and 266% and 321% for nintedanib (CYP3A & P-gp), respectively. In conclusion, CC-90001 in unlikely to be a victim or perpetrator of clinically relevant interactions involving CYPs or UGTs. Weak to moderate interactions are expected in clinic with substrates of P-gp and OATP1B1 due to CC-90001 inhibition of these transporters.

Inhibition of c-Jun N-terminal kinase signaling promotes osteoblastic differentiation of periodontal ligament stem cells and induces regeneration of periodontal tissues

ObjectivesFew clinical treatments to regenerate periodontal tissue lost due to severe endodontic and periodontal disease have yet been developed. Therefore, the development of new treatment methods for the regeneration of periodontal tissue is expected. The purpose of this study was to investigate the effects of a c-Jun N-terminal kinase (JNK) inhibitor, SP600125, on the osteoblastic differentiation of periodontal ligament stem cells (PDLSCs) in vitro, and the function of SP600125 on the regeneration of alveolar bone in vivo. DesignAlizarin red S staining, quantitative RT-PCR, and western blotting analysis was performed to determine whether SP600125 affects osteoblastic differentiation of human PDLSCs (HPDLSCs) and bone-related intracellular signaling. The effect of SP600125 on the regeneration of alveolar bone was assessed by using a rat periodontal defect model. The healing of periodontal defects was evaluated using micro-CT scans and histological analysis. ResultsSP600125 promoted the osteoblastic differentiation such as Alizarin red S-positive mineralized nodule formation and the expression of osteoblast-related genes in HPDLSCs under osteogenic conditions. In addition, this inhibitor upregulated the BMP2 expression and the phosphorylation of Smad1/5/8 in HPDLSCs under the same conditions. The inhibition of Smad1/5/8 signaling by LDN193189 suppressed the SP600125-induced osteoblastic differentiation of HPDLSCs. Furthermore, the application of SP600125 promoted the regeneration of not only alveolar bone but also PDL tissue in periodontal defects. ConclusionThis study suggested that inhibition of JNK signaling promotes the osteoblastic differentiation of HPDLSCs through BMP2-Smad1/5/8 signaling, leading to the regeneration of periodontal tissues such as alveolar bone and PDL tissue.

A small molecule targeting CHI3L1 inhibits lung metastasis by blocking IL-13Rα2-mediated JNK-AP-1 signals.

Our previous big data analyses showed a high level of association between chitinase 3 like1 (CHI3L1) expression and lung tumor development. In the present study, we investigated whether a CHI3L1‐inhibiting chemical, 2‐({3‐[2‐(1‐cyclohexen‐1‐yl)ethyl]‐6,7‐dimethoxy‐4‐oxo‐3,4‐dihydro‐2‐quinazolinyl}sulfanyl)‐N‐(4‐ethylphenyl)butanamide (K284), could inhibit lung metastasis and studied its mechanism of action. We investigated the antitumor effect of K284 both in vitro and in vivo. K284 (0.5 mg·kg−1 body weight) significantly inhibited lung metastasis in in vivo models after injection of murine melanoma cells (B16F10) or adenocarcinomic human alveolar basal epithelial cells (A549). K284 significantly and concentration‐dependently also inhibited cancer cell proliferation and migration in the A549 and H460 lung cancer cell lines. We found that the binding of K284 to the chitin‐binding domain (CBD) of CHI3L1 prevented the binding of CHI3L1 to its receptor, interleukin‐13 receptor subunit alpha‐2 (IL‐13Rα2), thereby suppressing the CHI3L1 signal. This blocking of the CHI3L1‐IL‐13Rα2 signal caused the inhibition of c‐Jun N‐terminal kinase (JNK)‐activator protein 1 (AP‐1) signals, resulting in the prevention of lung metastasis and cancer cell growth. Our data demonstrate that K284 may serve as a potential candidate anticancer compound targeting CHI3L1.

A Perspective on the Development of c-Jun N-terminal Kinase Inhibitors as Therapeutics for Alzheimer's Disease: Investigating Structure through Docking Studies.

c-Jun N-terminal kinase (JNK) plays an important role in cell death caused by various stimuli. Because the isoform JNK3 is mainly expressed in the brain, it is believed to play a pivotal role in various neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD), which still lack plausible therapeutics. To develop a novel and selective JNK3 inhibitor, we conducted a decadal review (2011 to 2021) of published articles on JNK inhibitors, particularly those focusing on a structural perspective and docking insights. We observed the structures of three isoforms of JNK, namely holo-proteins and co-crystal structures, with JNK3 inhibitors and summarized the significant structural aspects of selective JNK3 inhibitors as AD therapeutics.

Induction of Apoptosis by Isoalantolactone in Human Hepatocellular Carcinoma Hep3B Cells through Activation of the ROS-Dependent JNK Signaling Pathway.

Isoalantolactone (IALT) is one of the isomeric sesquiterpene lactones isolated from the roots of Inula helenium L. IALT is known to possess various biological and pharmacological activities, but its anti-cancer mechanisms are not well understood. The aim of the present study was to investigate the anti-proliferative effects of IALT in human hepatocellular carcinoma (HCC) cells and to evaluate the potential anti-cancer mechanisms. Our results demonstrated that IALT treatment concentration-dependently suppressed the cell survival of HCC Hep3B cells, which was associated with the induction of apoptosis. IALT increased the expression of death-receptor-related proteins, activated caspases, and induced Bid truncation, subsequently leading to cleavage of poly (ADP-ribose) polymerase. In addition, IALT contributed to the cytosolic release of cytochrome c by destroying mitochondrial integrity, following an increase in the Bax/Bcl-2 expression ratio. However, IALT-mediated growth inhibition and apoptosis were significantly attenuated in the presence of a pan-caspase inhibitor, suggesting that IALT induced caspase-dependent apoptosis in Hep3B cells. Moreover, IALT activated the mitogen-activated protein kinases signaling pathway, and the anti-cancer effect of IALT was significantly diminished in the presence of a potent c-Jun N-terminal kinase (JNK) inhibitor. IALT also improved the generation of intracellular reactive oxygen species (ROS), whereas the ROS inhibitor significantly abrogated IALT-induced growth reduction, apoptosis, and JNK activation. Furthermore, ROS-dependent apoptosis was revealed as a mechanism involved in the anti-cancer activity of IALT in a 3D multicellular tumor spheroid model of Hep3B cells. Taken together, our findings indicate that IALT exhibited anti-cancer activity in HCC Hep3B cells by inducing ROS-dependent activation of the JNK signaling pathway.

Remote limb ischaemic conditioning produces cardioprotection in rats with testicular ischaemia-reperfusion injury.

What is the central question of this study? Can remote limb ischaemic conditioning produce cardioprotection in rats with testicular ischaemia-reperfusion injury? What is the main finding and its importance? Testicular ischaemia-reperfusion (TI/R)-injured rats were predisposed to myocardial reperfusion-induced atrioventricular block. Remote limb ischaemia preconditioning and postconditioning protected TI/R hearts against ischaemia-provoked ventricular arrhythmia and ultimately reduced the incidence of sudden cardiac death, with a possible role of c-Jun N-terminal kinase inhibition and connexin 43 activation. Remote ischaemic conditioning can protect hearts against arrhythmia. Testicularischaemia-reperfusion (TI/R) injury is associated with electrocardiographic abnormalities. We investigated the effect of remote limb ischaemia preconditioning (RIPre) and postconditioning (RIPost) on arrhythmogenesis in TI/R rats, and determined the potential role of c-Jun N-terminal kinase (JNK)/connexin 43 (Cx43) signalling. Rats were randomized to sham-operated, control, TI/R, RIPre and RIPost groups. TI/R rats were more predisposed to myocardial reperfusion-induced atrioventricular block (AVB). RIPre and RIPost reduced the incidence of sudden cardiac death (SCD) or AVB, and duration of ventricular tachyarrhythmias during myocardial reperfusion. RIPre and RIPost decreased myocardial I/R-induced phosphorylation level of JNK, while preserving myocardial Cx43 expression in TI/R rats. Taken together, TI/R rats were predisposed to myocardial reperfusion-induced AVB. RIPre and RIPost protected TI/R hearts against ischaemia-provoked ventricular arrhythmia and ultimately reduced the incidence of SCD by suppressing JNK activation and restoring Cx43 expression.

Design and synthesis of naphthylchalcones as novel anti-leukaemia agents

A series of new hydroxylated chalcone derivatives with different substitution patterns on a phenyl ring A and B, were prepared by Claisen–Schmidt condensation in an aqueous alkaline base. The antiproliferative activity of the studied compounds was evaluated against the human leukaemia cell line U-937. The structure–activity relationship of these naphthylchalcones was investigated by the introduction of one methoxy or two methyl groups on the A ring, the introduction of a methoxy group on the naphthyl ring or by varying the position of the methoxy group on the A ring. The results revealed that the naphthylchalcone containing a methoxy group in position 6́ of the A ring was the most cytotoxic compound, with an IC50 value of 4.7 ± 0.5 μM against U-937 cells. This synthetic chalcone induced S and G2-M cell cycle arrest, a time-dependent increase in sub-G1 ratio and annexin-V positive cells, caspase activation and poly(ADP-ribose) polymerase cleavage. Apoptosis induction was blocked by a pan-caspase inhibitor and by the selective caspase-3/7 inhibitor and attenuated by the inhibition of c-jun N-terminal kinases / stress-activated protein kinases (JNK/SAPK) and phosphoinositide 3-kinase. The structure–activity relationship of naphthylchalcones against human leukaemia cells reveals that the major determining in cytotoxicity is the presence of a methoxy group in position 6́ of the A ring that suggest the potential of this compound or derivatives in the development of new anti-leukaemia drugs.

Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II.

Studies on neural development and neuronal regeneration after injury are mainly based on animal models. The establishment of pluripotent stem cell (PSC) technology, however, opened new perspectives for better understanding these processes in human models by providing unlimited cell source for hard-to-obtain human tissues. Here, we aimed at identifying the molecular factors that confine and modulate an early step of neural regeneration, the formation of neurites in human neural progenitor cells (NPCs). Enhanced green fluorescent protein (eGFP) was stably expressed in NPCs differentiated from human embryonic and induced PSC lines, and the neurite outgrowth was investigated under normal and injury-related conditions using a high-content screening system. We found that inhibitors of the non-muscle myosin II (NMII), blebbistatin and its novel, non-toxic derivatives, initiated extensive neurite outgrowth in human NPCs. The extracellular matrix components strongly influenced the rate of neurite formation but NMII inhibitors were able to override the inhibitory effect of a restrictive environment. Non-additive stimulatory effect on neurite generation was also detected by the inhibition of Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), the upstream regulator of NMII. In contrast, inhibition of c-Jun N-terminal kinases (JNKs) had only a negligible effect, suggesting that the ROCK1 signal is dominantly manifested by actomyosin activity. In addition to providing a reliable cell-based in vitro model for identifying intrinsic mechanisms and environmental factors responsible for impeded axonal regeneration in humans, our results demonstrate that NMII and ROCK1 are important pharmacological targets for the augmentation of neural regeneration at the progenitor level. These studies may open novel perspectives for development of more effective pharmacological treatments and cell therapies for various neurodegenerative disorders.

Sex and dose-dependent antinociceptive effects of the JNK (c-Jun N-terminal kinase) inhibitor SU 3327 are mediated by CB2 receptors in female, and CB1/CB2 receptors in male mice in an inflammatory pain model

Activation of c-Jun N-terminal kinases (JNKs) has been implicated in the development and persistence of inflammatory and neuropathic pain in animal models. Moreover, JNKs have been involved in the maintenance of chronic pain, as well as development of tolerance to antinociceptive agents in the opioid and cannabinoid class of compounds. In this study, we evaluated the antinociceptive effects of the JNK inhibitor SU 3327 (0.3–30 mg/kg) in the formalin pain model with an emphasis on the sex-specific actions of this compound. In wild-type C57BL6J mice, SU 3327 produced strong antinociceptive effects in the formalin pain model which were mediated by CB2 receptors in females, and both CB1 and CB2 receptors in males. SU 3327 at a dose of 10 mg/kg produced antinociception, hypothermia, motor impairment, and hypolocomotion to a similar extent in both males and females. The antinociceptive effects of SU 3327 were more potent in males at lower doses (1 and 3 mg/kg), while females were more sensitive to the hypothermic, and motor-suppression effects at lower (3 mg/kg) doses versus males. Analysis of spinal cords, using qPCR following SU 3327 administration in the formalin test, revealed changes in cannabinoid, tolerance and inflammatory markers in females only, and only in the high (10–30 mg/kg) dose conditions. Indeed, females showed an increase in mRNA levels of cannabinoid (CB2), but a decrease in tolerance (β-arrestin 1) and inflammatory (TNF-α, IL-1β, IL-6)-associated markers. The differences between males and females, in this study, support sex as an important factor in nociception and antinociceptive responses mediated by JNK and the endocannabinoid system.

2,5-dimethyl celecoxib induces apoptosis and autophagy via activation of ROS/JNK axis in nasopharyngeal carcinoma cells

2,5-dimethyl celecoxib (DMC), a close derivative of celecoxib, has also been reported to have anticancer effects. However, the effects and underlying molecular mechanisms of DMC with respect to nasopharyngeal carcinoma are still largely unknown. In this study, we present that DMC has displayed anticancer potency in nasopharyngeal carcinoma in vitro and in vivo. Mechanistically, we found DMC induced apoptosis and autophagy for anticancer therapy against nasopharyngeal carcinoma. Furthermore, DMC-induced autophagy could remarkably attenuate after the treatment of reactive oxygen species (ROS) scavenger N-acetyl cysteine (NAC) and c-Jun N-terminal kinase (JNK) inhibitor SP600125 (SP). Taken together, these results suggested DMC induced apoptosis and autophagic death via activation of ROS/JNK axis in NPC cells, which providing us new insights into developing potential therapeutic agents for nasopharyngeal carcinoma patients.

The molecular mechanism of GADD153 in apoptosis of keloid fibroblasts exposed to botulinum toxin type A

Apoptosis plays a key role in keloids. Growth arrest and DNA damage‐inducible gene 153 (GADD153) is regulated by apoptosis. Botulinum toxin type A (BTXA) can induce apoptosis in keloid fibroblasts. This research aimed to explore the hypothesis that GADD153 mediates apoptosis in keloid fibroblasts exposed to BTXA. BTXA significantly induced GADD153 protein and mRNA expression in keloid fibroblasts. Treatment with c‐Jun N‐terminal kinase (JNK) inhibitor SP600125, JNK small interfering RNA (siRNA) and tumour necrosis factor‐alpha (TNF‐α) antibodies reversed the BTXA‐induced GADD153 expression. BTXA enhanced the transcriptional activity of GADD153, whereas the GADD153 mutant plasmid, JNK siRNA and anti‐TNF‐α antibody treatment abolished the BTXA‐induced transcriptional activity of GADD153. The addition of TNF‐α to keloid fibroblasts markedly increased GADD153 protein expression. The addition of GADD153 siRNA, SP600125 and anti‐TNF‐α antibodies reversed cell death and caspase 3 and 9 activity induced by BTXA.

Curcumin attenuates renal ischemia reperfusion injury via JNK pathway with the involvement of p300/CBP-mediated histone acetylation.

Apoptosis is proved responsible for renal damage during ischemia/reperfusion. The regulation for renal apoptosis induced by ischemia/reperfusion injury (IRI) has still been unclearly characterized to date. In the present study, we investigated the regulation of histone acetylation on IRI-induced renal apoptosis and the molecular mechanisms in rats with the application of curcumin possessing a variety of biological activities involving inhibition of apoptosis. Sprague–Dawley rats were randomized into four experimental groups (SHAM, IRI, curcumin, SP600125). Results showed that curcumin significantly decreased renal apoptosis and caspase-3/-9 expression and enhanced renal function in IRI rats. Treatment with curcumin in IRI rats also led to the decrease in expression of p300/cyclic AMP response element-binding protein (CBP) and activity of histone acetyltransferases (HATs). Reduced histone H3 lysine 9 (H3K9) acetylation was found near the promoter region of caspase-3/-9 after curcumin treatment. In a similar way, SP600125, an inhibitor of c-Jun N-terminal kinase (JNK), also attenuated renal apoptosis and enhanced renal function in IRI rats. In addition, SP600125 suppressed the binding level of p300/CBP and H3K9 acetylation near the promoter region of caspase-3/-9, and curcumin could inhibit JNK phosphorylation like SP600125. These results indicate that curcumin could attenuate renal IRI via JNK/p300/CBP-mediated anti-apoptosis signaling.

Contributions of Sodium-Hydrogen Exchanger 1 and Mitogen-Activated Protein Kinases to Enhanced Retinal Venular Constriction to Endothelin-1 in Diabetes.

Diabetes elevates endothelin-1 (ET-1) in the vitreous and enhances constriction of retinal venules to this peptide. However, mechanisms contributing to ET-1-induced constriction of retinal venules are incompletely understood. We examined roles of sodium-hydrogen exchanger 1 (NHE1), protein kinase C (PKC), mitogen-activated protein kinases (MAPKs), and extracellular calcium (Ca2+) in retinal venular constriction to ET-1 and the impact of diabetes on these signaling molecules. Retinal venules were isolated from control pigs and pigs with streptozocin-induced diabetes for in vitro studies. ET-1-induced vasoconstriction was abolished in the absence of extracellular Ca2+ and sensitive to c-Jun N-terminal kinase (JNK) inhibitor SP600125 but unaffected by extracellular signal-regulated kinase (ERK) inhibitor PD98059, p38 kinase inhibitor SB203580, or broad-spectrum PKC inhibitor Gö 6983. Diabetes (after 2 weeks) enhanced venular constriction to ET-1, which was insensitive to PD98059 and Gö 6983 but was prevented by NHE1 inhibitor cariporide, SB203580, and SP600125. In conclusion, extracellular Ca2+ entry and activation of JNK, independent of ERK and PKC, mediate constriction of retinal venules to ET-1. Diabetes activates p38 MAPK and NHE1, which cause enhanced venular constriction to ET-1. Treatments targeting these vascular molecules may lessen retinal complications in early diabetes.

ZnT8 Deficiency Protects From APAP-Induced Acute Liver Injury by Reducing Oxidative Stress Through Upregulating Hepatic Zinc and Metallothioneins.

Zinc transporter 8 (ZnT8) is an important zinc transporter highly expressed in pancreatic islets. Deficiency of ZnT8 leads to a marked decrease in islet zinc, which is thought to prevent liver diseases associated with oxidative stress. Herein, we aimed to investigate whether loss of islet zinc affects the antioxidant capacity of the liver and acute drug-induced liver injury. To address this question, we treated ZnT8 knockout (KO) or wild-type control mice with 300 mg/ kg acetaminophen (APAP) or phosphate-buffered saline (PBS). Unexpectedly, we found that loss of ZnT8 in mice ameliorated APAP-induced injury and was accompanied by inhibition of c-Jun N-terminal kinase (JNK) activation, reduced hepatocyte death, and decreased serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). An increase in hepatic glutathione (GSH) was observed, corresponding to a decrease in malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) levels. APAP-induced inflammation and glycogen depletion were alleviated. In contrast, no significant changes were observed in cytochrome P450 family 2 subfamily E member 1 (CYP2E1), the main enzyme responsible for drug metabolism. Elevated levels of hepatic zinc and metallothionein (MT) were also observed, which may contribute to the hepatoprotective effect in ZnT8 KO mice. Taken together, these results suggest that ZnT8 deficiency protects the liver from APAP toxicity by attenuating oxidative stress and promoting hepatocyte proliferation. This study provides new insights into the functions of ZnT8 and zinc as key mediators linking pancreatic and hepatic functions.

Exendin-4 inhibits high glucose-induced oxidative stress in retinal pigment epithelial cells by modulating the expression and activation of p66Shc

Purpose Activation of p66Sch, an adaptor protein, is associated with oxidative stress and apoptosis and has been implicated in the pathogenesis of diabetes-induced retinal pigment epithelial cell damage and diabetic retinopathy. Exendin-4 is a glucagon-like protein that protects against diabetic retinopathy, but the mechanism of action is not well understood. This study aimed to investigate whether Exendin-4 could protect against high glucose-induced oxidative stress and apoptosis in the adult human retinal pigment epithelial-19 cell line by modulating levels and activation of p66Shc and to study the underlying mechanisms. Materials and Methods Adult human retinal pigment epithelial-19 cells were cultured under low (5 µM) or high glucose (100 µM) conditions in the presence or absence of Exendin-4 and with or without pre-incubation with Exendin-9-39, a glucagon-like peptide-1 receptor antagonist. Results In a dose-dependent manner, Exendin-4 inhibited high glucose-induced cell death and decreased levels of reactive oxygen species, lactate dehydrogenase release, and single single-stranded DNA. At the most effective concentration (100 µM), Exendin-4 reduced mitochondrial levels of phospho-p66Shc (Ser36), cytoplasmic levels of cleaved caspase-3 and cytochrome-c, and NADPH oxidase levels in high glucose-treated cells. It also increased levels of glutathione and magnesium superoxide dismutase and protein levels of magnesium superoxide dismutase but downregulated total protein levels of protein kinase-β and p66Shc and inhibited c-Jun N-terminal kinase phosphorylation in both low- and high glucose-treated cells. All these Exendin-4 effects, however, were inhibited by Exendin-9-39. Conclusions Exendin-4 protects against high glucose-induced adult human retinal pigment epithelial-19 cell damage by increasing antioxidants, downregulating NADPH, and inhibiting mitochondria-mediated apoptosis, effects that are associated with the inhibition of c-Jun N-terminal kinase and downregulation of protein kinase-β and p66Shc.

Combination of Melatonin and Small Molecules Improved Reprogramming Neural Cell Fates via Autophagy Activation.

Reprogramming cell fates towards mature cell types are a promising cell supply for treating degenerative diseases. Recently, transcription factors and some small molecules have turned into impressive modulating elements for reprogramming cell fates. Melatonin, a pineal hormone, has neuroprotective functions including neural stem cell (NSC) proliferative and differentiative modulation in both embryonic and adult brain. We developed a protocol that could be implemented in the direct reprogramming of human skin fibroblast towards neural cells by using histone deacetylase (HDAC) inhibitor, glycogen synthase kinase-3 (GSK3) inhibitor (CHIR99021), c-Jun N-terminal kinase(JNK) inhibitor, rho-associated protein kinase inhibitor (Y-27632), cAMP activator, and melatonin treatment. We found that melatonin enhanced neural-transcription factor genes expressions, including brain-specific homeobox/POU domain protein 2 (BRN2), Achaete-Scute Family BHLH transcription Factor 1 (ASCL1), and Myelin Transcription Factor 1 Like (MYT1L). Melatonin also increased the expression of different neural-specific proteins such as doublecortin (DCX), Sex determining region Y-box 2 (Sox2), and neuronal nuclei (NeuN) compared with other five small molecules (valproic acid (VPA), CHIR99021, Forskolin, 1,9 pyrazoloanthrone (SP600125), and Y-27632) combination in the presence and absence of melatonin. A noticeable upregulation of autophagy proteins (microtubule-associated protein 1A/1B-light chain 3 (LC3) and Beclin-1) were seen in the melatonin treatment during the induction period while these were reverted in the presence of L-leucine, an autophagy inhibitor. In addition, the expression of NeuN was also significantly reduced by L-leucine. Collectively, our findings revealed an activation of autophagy during neural induction; melatonin enhanced reprogramming efficiency for neuron induction through the modulation of autophagy activation.

High fructose and streptozotocin induced diabetic impairments are mitigated by Indirubin-3-hydrazone via downregulation of PKR pathway in Wistar rats

Metabolic disorders are becoming more common in young population due to increased consumption of carbohydrate rich diet, lack of physical activity and stress. Fructose is used as a sweetener in many carbonated beverages and is a known inducer of oxidative stress and hypertension. Up-regulation of the double-stranded RNA-dependent protein kinase (PKR) causes impairment in insulin signaling pathway and metabolic dysfunctions in type 2 diabetes mellitus. In the present study we investigated the role of PKR and associated pathways in high fructose (HF) and streptozotocin (STZ) induced diabetes and whether indirubin-3-hydrazone (IHZ), a novel PKR inhibitor can reverse the HF and STZ induced diabetic impairments in Wistar rats. Diabetes was induced by feeding rats 20% high fructose in drinking water for 6 weeks and by giving a single dose of STZ (35 mg/kg., i.p) at the end of week 5. Glucose and lipid levels were measured by using assay kits. Expression of PKR and its downstream genes were determined by immunohistochemistry, qRT-PCR and western blotting techniques. Histo-pathological studies were performed using H&E staining. Fibrosis was detected in insulin sensitive tissues and organs using Sirius red and Masson’s trichrome staining and apoptosis by TUNEL assay. HF and STZ induced hyperglycemia, fibrosis, oxidative stress, and inflammation in liver, pancreas, skeletal muscle and adipose tissue are mediated via PKR pathway and its downstream effectors, and these effects were attenuated by PKR inhibitor IHZ. Thus, inhibition of PKR can protect insulin sensitive organs and tissues from HF induced diabetic impairments via the inhibition of c-Jun N-terminal kinase (JNK) pathway.