JAK2/STAT3 Pathway Inhibition by AG490 Ameliorates Experimental Autoimmune Encephalomyelitis via Regulation of Th17 Cells and Autophagy.

Multiple sclerosis (MS) is an autoimmune inflammatory condition affecting the central nervous system, and experimental autoimmune encephalomyelitis (EAE) animal models have been extensively used to study it. T-helper 17 cells, which produce interleukin-17(IL-17), play crucial roles in MS pathogenesis, and the JAK2/STAT3 pathway has an essential function in their differentiation from naive CD4 + T cells. This study investigated the effects of the JAK2/STAT3 pathway inhibitor AG490 on EAE in vivo and in vitro, as well as the underlying mechanisms. AG490 ameliorated EAE severity and attenuated its typical symptoms by downregulating proteins associated with the JAK2/STAT3 pathway. Furthermore, it decreased T-helper 17 cell differentiation from naive CD4 + T cells by inactivating STAT3. In addition, it conferred protective effects against EAE by restoring autophagy. These findings indicate the potential of AG490 as a candidate anti-MS therapeutic.

Neurodegeneration and glial morphological changes are both prevented by TRPM2 inhibition during the progression of a Parkinson's disease mouse model.

Parkinson's disease (PD) is a neurodegenerative disease characterized by dopaminergic neuron death and neuroinflammation. Emerging evidence points to the involvement of the transient receptor potential melastatin 2 (TRPM2) channel in neuron death and glial activation in several neurodegenerative diseases. However, the involvement of TRPM2 in PD and specifically its relation to the neuroinflammation aspect of the disease remains poorly understood. Here, we hypothesized that AG490, a TRPM2 inhibitor, can be used as a treatment in a mouse model of PD. Mice underwent stereotaxic surgery for 6-hydroxydopamine (6-OHDA) administration in the right striatum. Motor behavioral tests (apomorphine, cylinder, and rotarod) were performed on day 3 post-injection to confirm the PD model induction. AG490 was then daily injected i.p. between days 3 to 6 after surgery. On day 6, motor behavior was assessed again. Substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry, immunoblotting, and RT-qPCR analysis on day 7. Our results revealed that AG490 post-treatment reduced motor behavior impairment and nigrostriatal neurodegeneration. In addition, the compound prevented TRPM2 upregulation and changes of the Akt/GSK-3ß/caspase-3 signaling pathway. The TRPM2 inhibition also avoids the glial morphology changes observed in the PD group. Remarkably, the morphometrical analysis revealed that the ameboid-shaped microglia, found in 6-OHDA-injected animals, were no longer present in the AG490-treated group. These results indicate that AG490 treatment can reduce dopaminergic neuronal death and suppress neuroinflammation in a PD mouse model. Inhibition of TRPM2 by AG490 could then represent a potential therapeutical strategy to be evaluated for PD treatment.

5-AZA Upregulates SOCS3 and PTPN6/SHP1, Inhibiting STAT3 and Potentiating the Effects of AG490 against Primary Effusion Lymphoma Cells.

Epigenetic modifications, including aberrant DNA methylation occurring at the promoters of oncogenes and oncosuppressor genes and histone modifications, can contribute to carcinogenesis. Aberrant methylation mediated by histone methylatransferases, alongside histones, can affect methylation of proteins involved in the regulation of pro-survival pathways such as JAK/STAT and contribute to their activation. In this study, we used DNA or histone demethylating agents, 5-Azacytidine (5-AZA) or DS-3201 (valemetostat), respectively, to treat primary effusion lymphoma (PEL) cells, alone or in combination with AG490, a Signal transducer and activator of transcription 3 (STAT3) inhibitor. Cell viability was investigated by trypan blue assay and FACS analysis. The molecular changes induced by 5-AZA and/or AG490 treatments were investigated by Western blot analysis, while cytokine release by PEL cells treated by these drugs was evaluated by Luminex. Statistical analyses were performed with Graphpad Prism® software (version 9) and analyzed by Student's t test or a nonparametric one-way ANOVA test. The results obtained in this study suggest that 5-AZA upregulated molecules that inhibit STAT3 tyrosine phosphorylation, namely Suppressor of Cytokine Signaling 3 (SOCS3) and tyrosine-protein phosphatase non-receptor type (PTPN) 6/Src homology region 2 domain-containing phosphatase-1 (SHP-1), reducing STAT3 activation and downregulating several STAT3 pro-survival targets in PEL cells. As this lymphoma is highly dependent on the constitutive activation of STAT3, 5-AZA impaired PEL cell survival, and when used in combination with AG490 JAK2/STAT3 inhibitor, it potentiated its cytotoxic effect. Differently from 5-AZA, the inhibition of the EZH1/2 histone methyltransferase by DS-3201, reported to contribute to STAT3 activation in other cancers, slightly affected STAT3 phosphorylation or survival in PEL cells, either alone or in combination with AG490. This study suggests that 5-AZA, by upregulating the expression level of SOCS3 and PTPN6/SHP1, reduced STAT3 activation and improved the outcome of treatment targeting this transcription factor in PEL cells.

The transient receptor potential melastatin 2: a new therapeutical target for Parkinson's disease?

The transient receptor potential melastatin 2 is a calcium-permeable cation channel member of the TRP family. Also known as an oxidative stress-activated channel, the transient receptor potential melastatin 2 gating mechanism is dependent on reactive oxygen species. In pathological conditions, transient receptor potential melastatin 2 is overactivated, leading to a Ca2+ influx that alters cell homeostasis and promotes cell death. The role of transient receptor potential melastatin 2 in neurodegenerative diseases, including Alzheimer's disease and ischemia, has already been described and reviewed. However, data on transient receptor potential melastatin 2 involvement in Parkinson's disease pathology has emerged only in recent years and the issue lacks review studies that focus specifically on this topic. The present review aims to elucidate the role of the transient receptor potential melastatin 2 channel in Parkinson's disease by reviewing, summarizing, and discussing the in vitro, in vivo, and human studies published until August 2022. Here we describe fourteen studies that evaluated the transient receptor potential melastatin 2 channel in Parkinson's disease. The Parkinson's disease model used, transient receptor potential melastatin 2 antagonist and genetic approaches, and the main outcomes reported were discussed. The studies described transient receptor potential melastatin 2 activation and enhanced expression in different Parkinson's disease models. They also evidenced protective and restorative effects when using transient receptor potential melastatin 2 antagonists, knockout, or silencing. This review provides a literature overview and suggests where there is a need for more research. As a perspective point, this review shows evidence that supports transient receptor potential melastatin 2 as a pharmacological target for Parkinson's disease in the future.

Role and mechanism of the p-JAK2/p-STAT3 signaling pathway in follicular development in PCOS rats.

OBJECTIVE: To identify the association between the phosphorylated Janus kinase 2/phosphorylated signal transducer and activator of transcription (p-JAK2/p-STAT3) signaling pathway and follicular development in polycystic ovary syndrome (PCOS) rats, and explore the underlying mechanism. To evaluate the role of exogenous JAK2 inhibitor AG490 in the model and the associations among luteinizing hormone/choriogonadotropin receptor (LHCGR), follicle-stimulating hormone receptor (FSHR), cytochrome P450 17α (CYP17a), cytochrome P450 19 (CYP19), and PCOS. RESULTS: Rat models of PCOS was established. PCOS rats were intraperitoneally treated with double-distilled water (ddH2O)/DMSO/AG490. The rate of ovarian morphological recovery in the AG490 group was significantly higher compared with the DMSO group (83.3 % vs 9.1 %, X2 = 12.68, P < 0.001). Moreover, the short in the time the estrous cycle was resumed in the AG490 group (hazard ratio = 16.32, P < 0.001) compared with the DMSO group. Compared with the controls, p-JAK2, p-STAT3, LHCGR, and CYP17a expression levels were increased whereas that of FSHR and CYP19 were decreased in the ovaries of PCOS rats. However, an opposite trend was observed after treatment with AG490. Software prediction revealed that the p-STAT3 bound to the promoter regions of LHCGR, FSHR, CYP17a, and CYP19 genes. This finding was confirmed by results of correlation analysis (R = 0.834, -0.836, 0.875 and -0.712, respectively, all P < 0.001). CONCLUSION: This study demonstrated that the p-JAK2/p-STAT3 signaling pathway was involved in follicular development in PCOS rats by upregulating LHCGR and CYP17a expression, and downregulating that of FSHR and CYP19. AG490 treatment exerted beneficial effects. LHCGR, FSHR, CYP17a, and CYP19 are candidate genes associated with follicular development in PCOS rats.

Blockade of JAK2 retards cartilage degeneration and IL-6-induced pain amplification in osteoarthritis.

Osteoarthritis (OA) is a complex chronic inflammatory disease characterized by articular degeneration and pain. Recent studies have identified interleukin 6 (IL-6) as a potential mediator leading to OA, but the therapeutic effects of inhibiting IL-6 signaling in intreating OA need to be further clarified. Here, we identified the intracellular signal transduction induced by recombinant IL-6 and focused on the impact of tyrphostin AG490 (a JAK2 inhibitor) on cartilage degeneration and OA pain. We found that IL-6 increased the inflammatory cytokines production and hypertrophic markers expression of primary mouse chondrocytes by activating JAK2/STAT3. Meanwhile, tyrphostin AG490 significantly attenuated articular degeneration and osteophyte formation in experimental mice with anterior cruciate ligament transection (ACLT) surgery. In vivo electrophysiological experiments showed that articular stimulation of IL-6 induced spinal hyperexcitability, which was prevented by coinjection of tyrphostin AG490. Specifically, compared with DMSO-treated ACLT mice, tyrphostin AG490 improved ambulate activity of mice and abolished the enhancement of serum bradykinin induced by IL-6. Together, we suggest that tyrphostin AG490 protected against progression of OA and improved OA prognosis by reducing cartilage degeneration and arthritis pain. Our findings provide further evidence for targeting IL-6 signaling in the treatment of OA.

JAK-STAT signaling mediates the senescence of cartilage-derived stem/progenitor cells.

Aging is a major risk factor for degenerative joint diseases, such as osteoarthritis (OA). Previous studies have confirmed the link between senescent mesenchymal stem cells (MSCs) and OA. Cartilage-derived stem/progenitor cells (CSPCs) with MSCs properties have been extracted from a variety of species. We inferred that the senescence of CSPCs may promote the development of osteoarthritis. However, the cellular and molecular mechanisms of CSPCs senescence remains unknown. In this study, we investigated the role of JAK-STAT signaling pathway in a replicative senescence model of CSPCs. We showed that the late CSPCs (> 15th passage) exhibited distinct senescent phenotypes, including increased proportion of ß-gal positive senescent cells and F-actin content, as well as cell cycle arrest. In late CSPCs, the activity of JAK-STAT signaling pathway was significantly increased. Activation of JAK-STAT signaling pathway promoted cell senescence in early CSPCs (< 6th passage). Conversely, pharmacological inhibition or genetic knockdown of JAK-STAT signaling pathway attenuated cell senescence in late CSPCs. In conclusion, our results demonstrated the critical role of JAK-STAT signaling pathway in CSPCs senescence.

Inhibition of TRPM2 by AG490 Is Neuroprotective in a Parkinson's Disease Animal Model.

Parkinson's disease (PD) is characterized by motor impairment and dopaminergic neuronal loss. There is no cure for the disease, and treatments have several limitations. The transient receptor potential melastatin 2 (TRPM2), a calcium-permeable non-selective cation channel, has been reported to be upregulated in neuronal death. However, there are no in vivo studies evaluating TRPM2's role and neuroprotective effects in PD. Here, we test the hypothesis that TRPM2 is upregulated in the 6-hydroxydopamine (6-OHDA) mouse model of PD and that its inhibition, by the AG490, is neuroprotective. For that, AG490 or vehicle were intraperitoneally administered into C57BL/6 mice. Mice then received 6-OHDA into the right striatum. Motor behavior assessments were evaluated 6, 13, and 20 days after surgery using the cylinder and apomorphine-induced rotational testes, and 7, 14, and 21 days after surgery using rotarod test. Brain samples of substantia nigra (SNc) and striatum (CPu) were collected for immunohistochemistry and immunoblotting on days 7 and 21. We showed that TRPM2 protein expression was upregulated in 6-OHDA-treated animals. In addition, AG490 prevented dopaminergic neuron loss, microglial activation, and astrocyte reactivity in 6-OHDA-treated animals. The compound improved motor behaviors and Akt/GSK-3ß/caspase-3 signaling. We conclude that TRPM2 inhibition by AG490 is neuroprotective in the 6-OHDA model and that the TRPM2 channel may represent a potential therapeutic target for PD.

Electroacupuncture Improves Cerebral Ischemic Injury by Enhancing the EPO-JAK2-STAT5 Pathway in Rats.

OBJECTIVE: Clinically, electroacupuncture (EA) improves cerebral ischemic injury, but its mechanism remains unknown. The aim of this study was to confirm the protective effects of EA on focal cerebral ischemia (FCI)-induced injury and the possible mechanism. METHODS: Sprague-Dawley (SD) rats served as the FCI model and were divided into the sham, model, EA, AG490 and EA+AG490 groups. Rats in the EA and EA+AG490 groups were acupunctured at the Baihui (GV20) and Dazhui (GV14) acupoints, and those in the AG490 and EA+AG490 groups were administered an intracerebroventricular injection of AG490 (a Janus-tyrosine kinase-2 (JAK-2) phosphorylation inhibitor). Neurological deficits and morphological changes in the ischemic cortex were observed through neurological deficit scoring and HE staining, respectively, and neuronal apoptosis was examined using the TUNEL assay. Transmission electron microscopy was used to observe neuronal ultrastructure, and HIF-1α, erythropoietin (EPO), phosphorylated (p)-JAK2, p-STAT5, HSP70, Bax and Bcl-2 expression was measured by RT-PCR and immunohistochemistry. RESULTS: FCI model rats showed obvious neurological deficits and neuronal apoptosis compared with sham rats. EA alleviated FCI-induced neurological deficits, improved neuronal ultrastructure, reduced neuronal apoptosis, and induced HIF-1α, EPO, p-JAK2, p-STAT5, HSP70 and Bcl-2 expression in a time-dependent manner. In contrast, AG490 treatment impaired the effects of EA on neurological deficits, neuronal apoptosis and HIF-1α, EPO, p-JAK2, p-STAT5, HSP70, Bax and Bcl-2 expression. CONCLUSION: EA at GV20 and GV14 could improve neurological deficits and reduce neuronal apoptosis, thereby improving FCI-induced injury, which may be related to enhancing the EPO-JAK2-STAT5 pathway.

Central Nervous System Inflammation Induced by Lipopolysaccharide Up-Regulates Hepatic Hepcidin Expression by Activating the IL-6/JAK2/STAT3 Pathway in Mice.

It is known that lipopolysaccharide (LPS) triggers inflammatory response after intracerebroventricular (ICV) injection and elevates the expression of hepcidin through the interleukin 6/janus kinase 2/transducer and activator of the transcription 3 (IL-6/JAK2/STAT3) signaling pathway in the brain. This study was conducted to determine whether LPS ICV injection can regulate peripheral hepatic hepcidin expression and iron metabolism. Here, we studied the hepcidin expression in the liver, as well as serum iron and transferrin saturation, after LPS ICV injection. We also demonstrated the role of the IL-6/JAK2/STAT3 pathway in hepcidin expression in the livers of IL-6 knockout (IL-6-/- mice) and IL-6+/+ mice. AG490 was used to verify the effect of the IL-6/JAK2/STAT3 pathway on hepatic hepcidin expression. Our present study demonstrated that LPS ICV injection up-regulated hepatic hepcidin expression. This finding provides further evidence for highlighting the importance of the central inflammation on hepatic hepcidin expression and peripheral iron metabolism.

Inhibition of JAK-STAT Signaling Pathway Alleviates Age-Related Phenotypes in Tendon Stem/Progenitor Cells.

Diminished regeneration or healing capacity of tendon occurs during aging. It has been well demonstrated that tendon stem/progenitor cells (TSPCs) play a vital role in tendon maintenance and repair. Here, we identified an accumulation of senescent TSPCs in tendon tissue with aging. In aged TSPCs, the activity of JAK-STAT signaling pathway was increased. Besides, genetic knockdown of JAK2 or STAT3 significantly attenuated TSPC senescence in aged TSPCs. Pharmacological inhibition of JAK-STAT signaling pathway with AG490 similarly attenuated cellular senescence and senescence-associated secretory phenotype (SASP) of aged TSPCs. In addition, inhibition of JAK-STAT signaling pathway also restored the age-related dysfunctions of TSPCs, including self-renewal, migration, actin dynamics, and stemness. Together, our findings reveal the critical role of JAK-STAT signaling pathway in the regulation of TSPC aging and suggest an ideal therapeutic target for the age-related tendon disorders.

Functional metabolomics reveal the role of AHR/GPR35 mediated kynurenic acid gradient sensing in chemotherapy-induced intestinal damage.

Intestinal toxicity induced by chemotherapeutics has become an important reason for the interruption of therapy and withdrawal of approved agents. In this study, we demonstrated that chemotherapeutics-induced intestinal damage were commonly characterized by the sharp upregulation of tryptophan (Trp)-kynurenine (KYN)-kynurenic acid (KA) axis metabolism. Mechanistically, chemotherapy-induced intestinal damage triggered the formation of an interleukin-6 (IL-6)-indoleamine 2,3-dioxygenase 1 (IDO1)-aryl hydrocarbon receptor (AHR) positive feedback loop, which accelerated kynurenine pathway metabolism in gut. Besides, AHR and G protein-coupled receptor 35 (GPR35) negative feedback regulates intestinal damage and inflammation to maintain intestinal integrity and homeostasis through gradually sensing kynurenic acid level in gut and macrophage, respectively. Moreover, based on virtual screening and biological verification, vardenafil and linagliptin as GPR35 and AHR agonists respectively were discovered from 2388 approved drugs. Importantly, the results that vardenafil and linagliptin significantly alleviated chemotherapy-induced intestinal toxicity in vivo suggests that chemotherapeutics combined with the two could be a promising therapeutic strategy for cancer patients in clinic. This work highlights GPR35 and AHR as the guardian of kynurenine pathway metabolism and core component of defense responses against intestinal damage.

Hiltonol Cocktail Kills Lung Cancer Cells by Activating Cancer-Suppressors, PKR/OAS, and Restraining the Tumor Microenvironment.

NSCLC (non-small cell lung cancer) is a leading cause of cancer-related deaths worldwide. Clinical trials showed that Hiltonol, a stable dsRNA representing an advanced form of polyI:C (polyinosinic-polycytidilic acid), is an adjuvant cancer-immunomodulator. However, its mechanisms of action and effect on lung cancer have not been explored pre-clinically. Here, we examined, for the first time, how a novel Hiltonol cocktail kills NSCLC cells. By retrospective analysis of NSCLC patient tissues obtained from the tumor biobank; pre-clinical studies with Hiltonol alone or Hiltonol+++ cocktail [Hiltonol+anti-IL6+AG490 (JAK2 inhibitor)+Stattic (STAT3 inhibitor)]; cytokine analysis; gene knockdown and gain/loss-of-function studies, we uncovered the mechanisms of action of Hiltonol+++. We demonstrated that Hiltonol+++ kills the cancer cells and suppresses the metastatic potential of NSCLC through: (i) upregulation of pro-apoptotic Caspase-9 and Caspase-3, (ii) induction of cytosolic cytochrome c, (iii) modulation of pro-inflammatory cytokines (GRO, MCP-1, IL-8, and IL-6) and anticancer IL-24 in NSCLC subtypes, and (iv) upregulation of tumor suppressors, PKR (protein kinase R) and OAS (2'5' oligoadenylate synthetase). In silico analysis showed that Lys296 of PKR and Lys66 of OAS interact with Hiltonol. These Lys residues are purportedly involved in the catalytic/signaling activity of the tumor suppressors. Furthermore, knockdown of PKR/OAS abrogated the anticancer action of Hiltonol, provoking survival of cancer cells. Ex vivo analysis of NSCLC patient tissues corroborated that loss of PKR and OAS is associated with cancer advancement. Altogether, our findings unraveled the significance of studying tumor biobank tissues, which suggests PKR and OAS as precision oncological suppressor candidates to be targeted by this novel Hiltonol+++ cocktail which represents a prospective drug for development into a potent and tailored therapy for NSCLC subtypes.

AG490 protects cerebral ischemia/reperfusion injury via inhibiting the JAK2/3 signaling pathway.

BACKGROUND: Cerebral ischemia/reperfusion injury is a severe problem in patients with brain ischemia. Brain injury caused by the immune response is important in the pathogenesis of cerebral ischemia/reperfusion injury and immune pathways. It is important to investigate potential targets for the treatment of cerebral ischemia/reperfusion injury. METHODS: In this experiment, we evaluated the effect of an exogenous JAK antagonist AG490 in the cerebral ischemia/reperfusion injury model, which was established by middle cerebral artery occlusion (MCAO). Histology study, TUNEL staining, Western blot, and RT-PCR were employed to examine the effects of AG490 in cerebral ischemia/reperfusion injury. RESULTS: In the brain tissue of MCAO mice, JAK2 was highly expressed. AG490 is an inhibitor of JAK2, which reduced the phosphorylation level of JAK2. AG490 downregulated the phosphorylated activation of JAK3 and their downstream STAT3. The antiapoptotic activity of AG490 on cerebral ischemia/reperfusion injury mice was consistent with in vitro data. It reduced the phosphorylation of JAK2/JAK3/STAT3 and the apoptosis rate in cultured neurons upon apoptosis induction. Besides, we also observed the neuroprotective effects of AG490 on cerebral ischemia/reperfusion injury. Administration of AG490 could further enhance the expression of neurotrophins including BNDF, NT3, and the neurotrophin receptor TrkB. CONCLUSION: Therefore, AG490 is pluripotent for cerebral ischemia/reperfusion injury through both antiapoptosis and neuroprotective activities. The antiapoptosis effect is dependent on its regulation of the JAK-STAT pathway.

A small-molecule screen reveals novel modulators of MeCP2 and X-chromosome inactivation maintenance.

BACKGROUND: Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. While MeCP2 mutations are lethal in most males, females survive birth but show severe neurological defects. Because X-chromosome inactivation (XCI) is a random process, approximately 50% of the cells silence the wild-type (WT) copy of the MeCP2 gene. Thus, reactivating the silent WT copy of MeCP2 could provide therapeutic intervention for RTT. METHODS: Toward this goal, we screened ~ 28,000 small-molecule compounds from several libraries using a MeCP2-luciferase reporter cell line and cortical neurons from a MeCP2-EGFP mouse model. We used gain/increase of luminescence or fluorescence as a readout of MeCP2 reactivation and tested the efficacy of these drugs under different drug regimens, conditions, and cellular contexts. RESULTS: We identified inhibitors of the JAK/STAT pathway as XCI-reactivating agents, both by in vitro and ex vivo assays. In particular, we show that AG-490, a Janus Kinase 2 (JAK2) kinase inhibitor, and Jaki, a pan JAK/STAT inhibitor, are capable of reactivating MeCP2 from the inactive X chromosome, in different cellular contexts. CONCLUSIONS: Our results suggest that inhibition of the JAK/STAT pathway is a new potential pathway to reinstate MeCP2 gene expression as an efficient RTT treatment.

ESTAT3 Inhibitor AG-490 Inhibits the Growth of Prostate Cancer by miR-503-5p Both In Vivo and In Vitro.

OBJECTIVE: To explore the effect and the related mechanism of STAT3 inhibitor AG-490 on inhibiting the proliferation of prostate cancer cells. METHODS: PC3 cells and DU145 cells were cultured stably and treated with AG-490 to detect the changes in the activity of PC3 cells and DU145 cells. Thirty 6-8 weeks male BALB/c nude mouse were randomly divided into a control group, a DMSO group, and an AG-490 group to detect differences in various indexes . RESULTS: The overexpression of miR-503-5p depends on the activation of STAT3. After treatment with AG-490, The proliferation and invasion of PC3 cells and DU145 cells and the expression of miR-503-5p were all reduced. Luciferase reporter assay demonstrated that the target proteins of miR-503-5p include PDCD4, TIMP-3, and PTEN. After treatment with AG-490, the expression of PDCD4, TIMP-3, and PTEN in cells was significantly up-regulated. IL-6-induced overexpression of miR-503-5p and restored the expression of STAT3, demonstrating the correlation between STAT3 and miR-503-5p. AG-490 can inhibit tumor growth and induce tumor cell apoptosis in the PC3 BALB/c nude mouse xenograft model. Western blotting and immunohistochemical staining showed that the expression levels of STAT3, Ki67, Bcl-2 and MMP-2 in the AG-490 group were significantly reduced, and the expression of PDCD4, TIMP-3 and PTEN increased. CONCLUSION: AG-490 can inhibit the growth of prostate cancer cells in a miR-503-5p-dependent manner by targeting STAT3. AG-490 is expected to become a new candidate drug for the treatment of prostate cancer.

Edaravone attenuates myocyte apoptosis through the JAK2/STAT3 pathway in acute myocardial infarction.

Basic and clinical studies have demonstrated that the free radical scavenger edaravone has cytoprotective effects on acute myocardial infarction (AMI) but the underlying mechanism is not fully understood. The aim of this research is to explore the effect of edaravone on the apoptotic process involving the JAK2/STAT3 signaling pathway. AMI in rats was established by left anterior descending coronary artery ligation. Two hours after AMI model established rats were treated with edaravone, edaravone plus AG490, physiological saline, respectively. We detected antioxidant effects by reduced glutathione (GSH), glutathione S-transferase (GST), and Glutathione Peroxidase (GSHPx) Activity. The expressions of t-JAK2, p-JAK2, t-STAT3, p-STAT3 and cleaved caspase-3 were examined by western blot. The mRNA levels for Bcl-2, Bax, Fas, and FasL were measured by RT-PCR and apoptosis was assessed by TUNEL. Edaravone significantly improved hemodynamics after AMI (p < 0.05) and reduced the total infarct volumes (p < 0.05). Compared with Sham rats, the mRNA of Bax, Fas, and FasL increased in different degrees in the AMI group, however, the mRNA of Bcl-2 and the ratio of Bcl-2/Bax decreased, especially the myocardial apoptosis index significantly increased in AMI hearts (all p < 0.05). After treatment with edaravone, the mRNA levels of Bcl-2 and the ratio of Bcl-2/Bax significantly upregulated whereas Bax, Fas, FasL apparently decreased, and the protein expressions of p-JAK2 and p-STAT3 dramatically increased (p < 0.05). In addition, cotreatment with JAK2 inhibitor AG490 abolished the effects of edaravone. We conclude that edaravone attenuated myocardial apoptosis induced by AMI via JAK2/STAT3 signaling pathway.

Human Heme Oxygenase-1 Induced by Interleukin-6 via JAK/STAT3 Pathways Is a Tumor Suppressor Gene in Hepatoma Cells.

Heme oxygenase-1 (HO-1) has several important roles in hepatocytes in terms of anti-inflammation, anti-apoptosis, and antioxidant properties. Interleukin-6 (IL-6) is a pleiotropic cytokine associated with liver regeneration and protection against injury. The aim of this study was to determine the potential crosstalk between HO-1 and IL-6, and to elucidate the signaling pathways involved in the induction of HO-1 by IL-6 in human hepatoma cells. Ectopic overexpression of HO-1 not only attenuated cell proliferation in vitro and in vivo, but also blocked the reactive oxygen species (ROS) induced by H2O2 and the pyocyanin in HepG2 or Hep3B cells. IL-6 expression was negatively regulated by HO-1, while IL-6 induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and HO-1 gene expression in HepG2 cells. The co-transfected HO-1 reporter vector and a protein inhibitor of the activated STAT3 (PIAS3) expression vector blocked the IL-6-induced HO-1 reporter activity. Both interferon γ and interleukin-1ß treatments induced STAT1 but not STAT3 phosphorylation, which had no effects on the HO-1 expression. Treatments of AG490 and luteolin blocked the JAK/STAT3 signaling pathways which attenuated IL-6 activation on the HO-1 expression. Our results indicated that HO-1 is the antitumor gene induced by IL-6 through the IL-6/JAK/STAT3 pathways; moreover, a feedback circuit may exist between IL-6 and HO-1 in hepatoma cells.

The JAK2 inhibitor AG490 regulates the Treg/Th17 balance and alleviates DSS-induced intestinal damage in IBD rats.

The pathogenesis of inflammatory bowel disease (IBD) remains unclear, and it is currently believed that an imbalance in regulatory T (Treg) cells/T helper 17 cells (Th17 cells) is related to the occurrence and development of IBD. Recently, the JAK2 inhibitor AG490 has been used in animal models such as rheumatoid arthritis and bronchial asthma models and shown to exert immunoregulatory functions that improve disorder in the Treg/Th17 cell balance. This study aimed to evaluate the effect of AG490 on the intestinal inflammatory process in an IBD rat model. A dextran sulfate sodium (DSS)-induced IBD rat model was established, and disease activity index (DAI) scores were calculated. The histopathological damage score was determined by haematoxylin-eosin (H&E) staining. Treg/Th17 cells in the spleen were detected by flow cytometry. The levels of interleukin (IL)-10, IL-6 and IL-17A were detected by enzyme-linked immunosorbent assay (ELISA). AG490 attenuated DSS-induced IBD injury by regulating the Treg/Th17 balance and related cytokine secretion to reduce the DAI and colonic tissue damage. Thus, AG490 may be a new method for effective treatment of IBD.

JAK2/STAT3 involves oxidative stress-induced cell injury in N2a cells and a rat MCAO model.

Purpose: In this study, we sought to test the hypothesis that oxidative stress injury in ischemic brains and H2O2-treated mouse neuroblastoma Neuro-2a cells (N2a) was related to STAT3 activation.Materials and methods: Rat middle cerebral artery occlusion (MCAO) model and H2O2-treated mouse neuroblastoma Neuro-2a cells (N2a) were used to investigate the relationship between oxidative stress injury and STAT3 activation.Results: 8-Hydroxy-2'-deoxyguanosine (8-OHdG) content and STAT3 protein phosphorylation level were significantly increased after cerebral ischemia-reperfusion. H2O2 treatment inhibited the cell viability, induced the apoptosis, and further raised pSTAT3 protein level in N2a cells. Moreover, the addition of AG490, the protein inhibitor of JAK2, significantly alleviated cerebral ischemic damage in vivo and H2O2-induced injury in vitro, and JAK2 siRNA also alleviated H2O2-induced injury in N2a cell.Conclusions: JAK2/STAT3 pathway may play a crucial role in mediating reactive oxidative species (ROS)-induced cell injury in rat middle cerebral artery occlusion (MCAO) model and N2a cells. ROS scavenging and down-regulation of STAT3 activation might be a candidate design of therapeutic strategies against oxidative stress-related neurological diseases.