Administration Of AG490 Research Articles

Aloperine protects the testis against testicular ischemia/reperfusion injury in rats.

Testicular torsion/detorsion can cause testis loss and infertility. Aloperine is a major active alkaloid extracted from Sophora alopecuroides Linn. It has been shown to have organ-protective effects. However, the effects of aloperine on the testis and its underlying mechanisms remain unclear. This study investigated the effect of aloperine on testicular torsion/detorsion injury in rats. Male Sprague-Dawley rats were randomized to the sham-operated (sham), testicular I/R (TI/R), or aloperine preconditioning (ALOPre) or postconditioning (ALOPost) groups. All rats except for the sham-operated rats were subjected to 3h of right spermatic cord torsion (720°, clockwise), followed by 3h of detorsion. Aloperine (10mg/kg) was intravenously administered before testicular torsion (ALOPre) or at the onset of testicular detorsion (ALOPost). The therapeutic efficacy of aloperine was evaluated by histological analysis, oxidative stress evaluation, inflammatory response examination, apoptosis analysis, protein analysis, and immunohistological assessment. Compared with TI/R, aloperine protected both the ipsilateral and contralateral testes against unilateral testicular I/R, as evidenced by a reduced testicular weight to body weight (TW/BW) ratio (ALOPre: p=0.0037; ALOPost: p=0.0021) and volume (ALOPre: p=0.0020; ALOPost: p=0.0009), less structural damage with better Johnsen (ALOPre: p=0.0013; ALOPost: p=0.0021), and Cosentino scores (ALOPre: p<0.0001; ALOPost: p<0.0001), increased mean seminiferous tubule diameter and mean seminiferous tubule epithelial height, decreased testicular apoptosis, and less oxidative stress and inflammatory response. In addition, aloperine significantly stimulated the phosphorylation of signal transducer and activator of transcription (STAT)-3 in the ipsilateral testes following detorsion. Administration of Ag490 suppressed STAT-3 phosphorylation, thereby abrogating the protective effects exerted by aloperine on the ipsilateral testis. Aloperine has a strong testicular protective effect on the ipsilateral and contralateral testes after testicular torsion/detorsion. This aloperine-induced ipsilateral testicular protection is mediated via the STAT-3 signaling pathway.

Inhibition of CCR1 attenuates neuroinflammation via the JAK2/STAT3 signaling pathway after subarachnoid hemorrhage

Background and purposeNeuroinflammation is an important mechanism underlying brain injury caused by subarachnoid hemorrhage (SAH). C-C chemokine receptor type 1 (CCR1)-mediated inflammation is involved in the pathology of many central nervous system diseases. Herein, we investigated whether inhibition of CCR1 alleviated neuroinflammation after experimental SAH and aimed to elucidate the mechanisms of its potential protective effects. MethodsTo analyze SAH transcriptome data R studio was used, and a mouse model of SAH was established using endovascular perforations. In this model, the selective CCR1 antagonist Met-RANTES (Met-R) and the CCR1 agonist recombinant CCL5 (rCCL5) were administered 1 h after SAH induction. To investigate the possible downstream mechanisms of CCR1, the JAK2 inhibitor AG490 and the JAK2 activator coumermycin A1 (C-A1) were administered 1 h after SAH induction. Furthermore, post-SAH evaluation, including SAH grading, neurological function tests, Western blot, the terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and Fluoro-Jade B and fluorescent immunohistochemical staining were performed. Cerebrospinal fluid (CSF) samples were detected by ELISA. ResultsCCL5 and CCR1 expression levels increased significantly following SAH. Met-R significantly improved neurological deficits in mice, decreased apoptosis and degeneration of ipsilateral cerebral cortex neurons, reduced infiltrating neutrophils, and promoted microglial activation after SAH induction. Furthermore, Met-R inhibited the expression of p-JAK2, p-STAT3, interleukin-1β, and tumor necrosis factor-α. However, the protective effects of Met-R were abolished by C-A1 treatment. Furthermore, rCCL5 injection aggravated neurological dysfunction and increased the expression of p-JAK2, p-STAT3, interleukin-1β, and tumor necrosis factor-α in SAH mice, all of which were reversed by the administration of AG490. Finally, the levels of CCL5 and CCR1 were elevate in the CSF of SAH patient and high level of CCL5 and CCR1 levels were associated with poor outcome. ConclusionThe present results suggested that inhibition of CCR1 attenuates neuroinflammation after SAH via the JAK2/STAT3 signaling pathway, which may provide a new target for the treatment of SAH.

Administration of AG490 decreases the senescence of umbilical cord-mesenchymal stem cells and promotes the cytotherapeutic effect in liver fibrosis

The therapeutic potential of umbilical cord-mesenchymal stem cell (UC-MSC) transplantation in liver fibrosis has been highlighted. However, the fate of transplanted MSCs in the fibrotic microenvironment remains unclear. In this study, we aim to uncover the fate of transplanted MSCs and develop targeting strategies that could enhance the therapeutic efficacy of MSC therapy in liver fibrosis. We used human UC-MSCs as the study object. For in vitro experiments, we stimulated UC-MSCs with several fibrotic-related factors (Liver fibrotic Factors, LF), including TGFβ, TNFα and IFNγ for downstream investigations. We co-cultured LF-treated UC-MSCs with hepatic stellate cell line LX-2 to assess the anti-fibrotic effect. We showed that upon LF stimulation, UC-MSCs exhibited reduced anti-fibrotic activity and underwent rapid senescence. Pathway analysis showed that JAK/STAT3 signaling was highly activated upon LF stimulation, which significantly elevated senescence-associated secretory phenotype (SASP) and senescence in UC-MSCs and could be reversed by a specific JAK inhibitor AG490. Moreover, using both carbon tetrachloride (CCl4) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induce fibrosis models, we demonstrated that AG490 pretreatment promoted UC-MSCs survival within the fibrotic liver microenvironment and exhibited enhance therapeutic efficacy. Overall, we showed that targeting MSC senescence in vivo through AG490 pretreatment could enhance the anti-fibrotic activities of UC-MSCs.

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

BackgroundCerebral 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.MethodsIn 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.ResultsIn 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.ConclusionTherefore, 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.

Neuroprotective potential of imatinib in global ischemia-reperfusion-induced cerebral injury: possible role of Janus-activated kinase 2/signal transducer and activator of transcription 3 and connexin 43.

The present study was aimed to explore the neuroprotective role of imatinib in global ischemia-reperfusion-induced cerebral injury along with possible mechanisms. Global ischemia was induced in mice by bilateral carotid artery occlusion for 20 min, which was followed by reperfusion for 24 h by restoring the blood flow to the brain. The extent of cerebral injury was assessed after 24 h of global ischemia by measuring the locomotor activity (actophotometer test), motor coordination (inclined beam walking test), neurological severity score, learning and memory (object recognition test) and cerebral infarction (triphenyl tetrazolium chloride stain). Ischemia-reperfusion injury produced significant cerebral infarction, impaired the behavioral parameters and decreased the expression of connexin 43 and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the brain. A single dose administration of imatinib (20 and 40 mg/kg) attenuated ischemia-reperfusion-induced behavioral deficits and the extent of cerebral infarction along with the restoration of connexin 43 and p-STAT3 levels. However, administration of AG490, a selective Janus-activated kinase 2 (JAK2)/STAT3 inhibitor, abolished the neuroprotective actions of imatinib and decreased the expression of connexin 43 and p-STAT3. It is concluded that imatinib has the potential of attenuating global ischemia-reperfusion-induced cerebral injury, which may be possibly attributed to activation of JAK2/STAT3 signaling pathway along with the increase in the expression of connexin 43.

Effect of JAK2/STAT3 signaling pathway on liver injury associated with severe acute pancreatitis in rats.

Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling constitutes one of the major pathways for cytokine signal transduction. However, the role of the JAK2/STAT3 pathway in liver injury during severe acute pancreatitis (SAP) remains unclear. The aim of this study was to investigate the role of the JAK2/STAT3 signaling pathway in liver injury after SAP. In the present study 64 male Sprague-Dawley rats were randomly divided into four groups: Control, AG490 (inhibition of JAK2), SAP and SAP with AG490. SAP was induced by retrograde infusion of 4% sodium taurocholate into the biliopancreatic duct. The activities of amylase (AMY) and liver enzymes were measured in serum. Livers and pancreas were isolated for measurements of histological damage. Blood and liver samples were taken for the measurement of TNF-α, IL-6 and IL-18 concentrations. The expression levels of JAK2 and STAT3 in liver tissue were detected by immunohistochemical staining and western blotting. The results demonstrated that amylase and liver enzymes were higher in the SAP groups compared with the control, AG490 and AG490-treated groups. The serum levels of TNF-α, IL-6 and IL-18 were effectively increased in the SAP groups, whereas they were reduced by AG490. Interestingly, JAK2 and STAT3 protein expression levels were significantly increased following induction of SAP and were significantly decreased in the AG490-pretreated groups. Administration of AG490 decreased the activity of pro-inflammatory cytokines and significantly attenuated SAP associated-liver injury in the rats. These results suggested that the mechanism may relate to the inhibition of TNF-α, IL-6 and IL-18, and inhibiting excessive JAK2 and STAT3 activation, and may play a crucial role in the liver injury associated with SAP.

JAK2/STAT3 Pathway Was Associated with the Protective Effects of IL-22 On Aortic Dissection with Acute Lung Injury.

Patients with aortic dissection (AD) may present acute lung injury (ALI) that may affect the prognosis. In this study, we aim to investigate the roles and mechanism of IL-22 in the pathogenesis of AD complicated with ALI. Six hundred and twenty-one AD patients were included, and the incidence of ALI and pulmonary CT findings were analyzed. Mouse ALI model was established through AngII, and then IL-22 injection and AG490 were given. The pathological changes, infiltration of inflammatory cells, and expression of STAT3 were determined. For the in vitro experiment, cultivated pulmonary microvascular endothelial cells (PMVECs) were treated by angiotensin II (AngII), followed by treating with IL-22 and/or AG490. The expression and migration of STAT3 was determined. Flow cytometry was carried out to evaluate the apoptosis. IL-22 contributed to the expression of STAT3 in lung tissues and attenuation of ALI. IL-22 obviously inhibited the apoptosis of PMVECs mediated by AngII and downregulated the expression and intranuclear transmission of STAT3. Such phenomenon was completely inhibited upon administration of AG490, an inhibitor of JAK2. Our data showed IL-22 contributed to the inhibition of PMVEC apoptosis mediated by AngII through activating the JAK2/STAT3 signaling pathway, which may attenuate the ALI induced by AngII.

Janus kinase 2 activation participates in prostaglandin E2-induced hyperalgesia

Prostaglandin E2 (PGE2) is one of the major signaling molecules involved in hyperalgesia, acting directly on nociceptors and resulting in the activation of PKA and PKC. Once active, these kinases phosphorylate many cellular proteins, resulting in changes on nociceptors sensorial transduction properties. The Janus Kinases (JAKs) are a family of intracellular signaling molecules generally associated with cytokine signaling, and their activity can be increased in nociceptors after peripheral inflammation. However, there are no evidences of JAKs direct involvement in PGE2 mediated sensitization of nociceptors. Therefore, the aim of the present study was to explore a possible role for JAKs in PGE2 mediated sensitization. In cultured dorsal root ganglion (DRG) neurons, we observed that the administration of PGE2 increases capsaicin induced calcium transients, and a pre-incubation of DRG cells with the JAK inhibitor AG490 blocks this PGE2 in vitro effect. Intrathecal administration of AG490 to ten-weeks-old male Wistar rats reduces the hyperalgesia induced by the intraplantar administration of PGE2 or carrageenan in the right hind paw. We also observed that carrageenan administration in the right hind paw induced an increase in membrane associated PKCepsilon in the ipsilateral L5 DRG, and this increase was blocked by intrathecal AG490 administration. In conclusion the present study indicates that the JAKs expressed in the DRG and spinal cord may have a role in the sensitization of nociceptors by a peripheral inflammatory event. Moreover, the inhibition of JAKs may be a possible novel pharmacological target for the control of the inflammatory hyperalgesia.

Spinal leptin contributes to the development of morphine antinociceptive tolerance by activating the STAT3-NMDA receptor pathway in rats.

Leptin, an adipokine synthesized mainly by non‑neuronal tissues, has been reported to contribute to the pathogenesis of neuropathic pain. It has been hypothesized that morphine tolerance and neuropathic pain share some common pathological mechanisms. The present study was designed to examine whether spinal leptin is implicated in the development of morphine antinociceptive tolerance, and whether spinal leptin induces the activation of signal transducer and activator of transcription 3 (STAT3) signaling pathway and the NR1 subunit of N‑methyl‑D‑aspartate (NMDA) receptor, in morphine antinociceptive tolerance in rats. The results demonstrated that intrathecal (i.t.) administration of a leptin antagonist (LA) prevented the development of morphine antinociceptive tolerance in rats. Further studies revealed that the levels of the spinal leptin and the leptin receptor (Ob‑R) were time‑dependently increased following chronic morphine treatment. Mechanistic examination indicated that chronic morphine triggered activation of the STAT3 pathway and an increase in the expression of the NR1 subunit of the NMDA receptor, which was ameliorated by i.t. administration of AG490 [a Janus kinase (JAK)‑STAT inhibitor]. The increased activation of STAT3 and the NR1 subunit was markedly attenuated by i.t. treatment with LA. In addition, the spinal administration of AG490 or MK‑801 (a non‑competitive NMDA receptor inhibitor) blocked the development of morphine antinociceptive tolerance. Taken together, these results have demonstrated, for the first time, to the best of our knowledge, that spinal leptin contributes to the development of morphine antinociceptive tolerance by activating the spinal STAT3‑NMDA receptor pathway.

Tyrosine Kinase Inhibitor Tyrphostin AG490 Inhibits Osteoclast Differentiation in Collagenase-Induced Osteoarthritis

The janus kinase (JAK)-signal transducer and activator of transcription (STAT) cascade plays a principal role in the signaling of a vast array of cytokines and growth factors which stimulates diverse cellular functions and immune responses. Osteoarthritis (OA) is the most common joint disease in the adult population. The present study was designed to evaluate the effects of tyrosine kinase inhibitor, tyrphostin AG490 in a mouse model of collagenase-induced osteoarthritis (CIOA). CIOA was provoked by two intraarticular (i.a.) injections of collagenase in mice and intraperitoneally (i.p.) treated with AG490 at a dose of 5 mg/kg at days 0, 5 and 10 and at a dose of 8 mg/kg at day 18. The administration of AG490 in CIOA mice inhibited osteoclast generation in bone and the loss of glycosaminoglycans and proteoglycans in cartilage. Tyrphostin decreased the levels of IFN-γ, IL1, IL-6 and IL-17 in the synovial fluid (SF) dependant on the time post AG490 administration. Limited numbers of CD11b positive Ly6G neutrophils in blood and SF along with a decrease of F4/80 positive cells in synovial fluid (SF) were observed in tyrphostin AG490-treated arthritic mice. AG490 inhibited M-CSF+RANKL-induced cytokine production by bone marrow (BM) cells and the differentiation of BM cells in vitro. Because of the findings presented, we argue that tyrphostin AG490 may hold promising therapeutic potential against important clinical conditions such as osteoarthritis (OA).

Combination of AG490, a Jak2 inhibitor, and methylsulfonylmethane synergistically suppresses bladder tumor growth via the Jak2/STAT3 pathway

Human urinary bladder cancer is the fifth most common cancer, with a worldwide estimate of about two million patients. Recurrence after complete transurethral prostatic resection is the most important problem in therapy. Combination therapy is a new approach in the treatment of cancers that do not respond to current therapies. These therapies have many advantages over conventional therapies, such as fewer side-effects and greater efficiency. Research efforts using natural compounds for the elimination or growth suppression of the cancer arise from studies on methylsulfonylmethane (MSM). MSM is a natural sulfur compound with no side-effects. AG490 is a tyrosine kinase inhibitor that has been extensively used for inhibiting Jak2 invitro and invivo. In our study, the combinatorial effect of these two agents on human bladder cancer cell lines and xenografts was analyzed. We observed that the combination of AG490 and MSM inhibited cancer cell viability and cell migration invitro. This combination inhibited VEGF mRNA expression in bladder cancer cell lines. Invivo experiments showed that oral administration of AG490 and MSM combination significantly inhibited the growth of tumor xenografts in mice. Our study clearly demonstrates that the predominant effect of this combination is the reduction of signaling molecules including STAT3, STAT5b, IGF-1R, VEGF and VEGF-R2 which are involved in the growth, progression and metastasis of human bladder cancer. The anti-metastatic ability of this drug combination is confirmed using metastatic animal models. Therefore, this combination could have the effect of genesistasis and powerful anticancer effects against bladder cancer.

Postconditioning inhibits myocardial apoptosis during prolonged reperfusion via a JAK2-STAT3-Bcl-2 pathway

BackgroundPostconditioning (PostC) inhibits myocardial apoptosis after ischemia-reperfusion (I/R) injury. The JAK2-STAT3 pathway has anti-apoptotic effects and plays an essential role in the late protection of preconditioning. Our aim was to investigate the anti-apoptotic effect of PostC after prolonged reperfusion and the role of the JAK2-STAT3 pathway in the anti-apoptotic effect of PostC.MethodsWistar rats were subjected to 30 minutes ischemia and 2 or 24 hours (h) reperfusion, with or without PostC (three cycles of 10 seconds reperfusion and 10 seconds reocclusion at the onset of reperfusion). Separate groups of rats were treated with a JAK2 inhibitor (AG490) or a PI3K inhibitor (wortmannin) 5 minutes before PostC. Immunohistochemistry was used to analyze Bcl-2 protein levels after reperfusion. mRNA levels of Bcl-2 were detected by qRT-PCR. TTC staining was used to detect myocardial infarction size. Myocardial apoptosis was evaluated by TUNEL staining. Western-blot was used to detect p-STAT3 and p-Akt levels after reperfusion.ResultsThere was more myocardial apoptosis at 24 h vs 2 h after reperfusion in all groups. PostC significantly reduced myocardial apoptosis and elevated Bcl-2 levels at both 2 and 24 hours after reperfusion. PostC increased p-STAT3 and p-Akt levels after reperfusion. Administration of AG490 reduced p-STAT3 and p-Akt levels and attenuated the anti-apoptotic effect of PostC. Wortmannin also reduced p-Akt levels and attenuated the anti-apoptotic effect of PostC but had no effect on p-STAT3 levels. AG490 abrogated the up-regulation of Bcl-2 by PostC.ConclusionPostC may reduce myocardial apoptosis during prolonged reperfusion via a JAK2-STAT3-Bcl-2 pathway. As a downstream target of JAK2 signaling, activation of PI3K/Akt pathway may be necessary in the protection of PostC.

The effect of Janus kinase 2 inhibitor AG490 on renal tubular epithelial-myofibroblast transdifferentiation induced by interleukin-1β

To investigate the effect of AG490, a Janus kinase 2 inhibitor, on epithelial-myofibroblast transdifferentiation induced by interleukin-1β (IL-1β). Cultured human renal tubular epithelial cell line (HKCs) were divided into three groups: blank control group, IL-1β (5 ng/ml) group and AG490 group (IL-1β 5 ng/ml+AG490 10 μmol/L). The cells in all groups were collected at 24, 48, 72 hours after intervention. Immunocytochemistry and Western blotting analysis were used to determine the expressions of cytokeratin-18 (CK-18) and α-smooth muscle actin (α-SMA). The higher expression of CK-18 (1.25±0.08) and mild expression of α-SMA (0.17±0.01) were found in blank control group. In IL-1β group, the protein level of CK-18 was gradually decreased with prolongation of stimulus (24 hours : 0.69±0.04, 48 hours: 0.52±0.03, 72 hours: 0.30±0.01), while the expression level of α-SMA was gradually increased (24 hours: 0.56±0.04, 48 hours: 1.05±0.07, 72 hours: 1.43±0.07), and the difference between blank control group and IL-1β group was statistically significant (all P<0.05). The administration of AG490 could restore the expression of CK-18 (24 hours: 1.07±0.07, 48 hours: 0.93±0.06, 72 hours: 0.83±0.06), and inhibit the expression of α-SMA induced by IL-1β (24 hours: 0.33± 0.01, 48 hours: 0.52±0.01, 72 hours: 0.61±0.04). There was significant difference between AG490 group and IL-1β group (all P<0.05). The results of immunocytochemistry and that of Western blotting were identical. IL-1β can induce the transdifferentiation of renal tubular epithelial cells, up-regulate the expression of α-SMA, induce the renal tubular epithelial cells to transform to myofibroblast, while AG490 can inhibit the effect of IL-1β.

Involvement of the JAK‐STAT pathway and SOCS3 in the regulation of adiponectin‐generated reactive oxygen species in murine macrophage RAW 264 cells

Adiponectin is a protein hormone produced by differentiating adipocytes and has been proposed to have anti-diabetic and immunosuppressive properties. We previously reported that the globular form of adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO), followed by caspase-dependent apoptotic cell death in RAW 264 cells. Here, we demonstrate that gAd-induced ROS generation and apoptosis were diminished by suppressor of cytokine signaling 3 (SOCS3). The phosphorylation level of signal transducer and activator of transcription (STAT) 3 detected by Western blotting was highest at 20 min in gAd-treated RAW 264 cells. This phosphorylation was inhibited by AG490, a specific inhibitor of janus-activator kinase (JAK). The gAd-induced ROS and NO were reduced by administration of AG490 and Jak-2-specific siRNA in RAW 264 cells. The gAd stimulation transiently induced SOCS3 mRNA expression and protein production. We examined SOCS3-overexpressing RAW 264 cells to investigate the role of the JAK-STAT pathway in gAd-induced ROS and NO generation. SOCS3 overexpression significantly reduced both ROS and NO generation. Additionally, gAd-induced caspase activation and apoptotic cell death were reduced in SOCS3 transfectants compared with vector control transfectants. These results suggest that the JAK-STAT pathway, which can be suppressed by SOCS3 expression, is involved in gAd-induced ROS and NO generation followed by apoptotic cell death.

STAT3 Does Not Regulate Acute Liver Injury After Ischemia/Reperfusion

Hepatic ischemia/reperfusion (I/R) injury is a serious complication of liver surgery and transplantation. Regulation of this injury response occurs at the cellular and molecular levels. Previous studies have shown that interleukin-6 (IL-6) is a negative regulator of the acute inflammatory injury occurring as a result of hepatic I/R. The signal transducer and activator of transcription-3 (STAT3) is a key target of receptor signaling for IL-6. Both IL-6 and STAT3 have been implicated in the protective effects of ischemic preconditioning of the liver. However, there have been no studies that have directly addressed the potential role of STAT3 in regulating acute inflammatory liver injury induced by I/R. In the current study, we investigated whether blockade of STAT3 phosphorylation altered the injury response to hepatic I/R injury. Male Balb/c mice were subjected to 90 min of partial hepatic ischemia followed by reperfusion with or without treatment with specific inhibitors of STAT3 activation, AG490 (selective JAK2 inhibitor), or STATTIC (direct inhibitor of STAT3 phosphorylation). Mice were sacrificed at 8 and 24 h after reperfusion. STAT3 activation was induced by I/R. This activation was partially inhibited by administration of AG490 and almost completely abrogated by treatment with STATTIC. Despite the blockade of STAT3, neither AG490 nor STATTIC had any effect on acute liver injury induced by I/R. Treatment with STATTIC did reduce hepatic neutrophil accumulation. The data suggest that STAT3 is not a central regulator of acute liver injury induced by I/R.

The Acute Lymphoblastic Leukemia-associated JAK2 L611S Mutant Induces Tumorigenesis in Nude Mice

JAK2 plays important roles in the regulation of a variety of cellular processes including cell migration, proliferation, and protection from apoptosis. Recently the L611S point mutation in JAK2 has been identified in a child with acute lymphoblastic leukemia. Here we analyzed the mechanism by which JAK2 exhibits its oncogenicity. In BaF3 murine hematopoietic cells, L611S mutant increased the expression of antiapoptotic proteins including X chromosome-linked inhibitor of apoptosis protein, inhibitor of apoptosis protein, and Bcl-XL. We also showed that JAK2 L611S mutant protects BaF3 cells from cytokine withdrawal-induced apoptotic cell death and leads to cytokine-independent cell growth. Furthermore BaF3 cells expressing JAK2 L611S mutant gained the ability to induce tumorigenesis in nude mice. The L611S mutant also exhibited malignancy, including prompt invasion and spreading into various organs, leading to rapid lethality of the mice. Finally we showed that a specific JAK2 inhibitor, AG490, potently inhibited cytokine-independent cell growth induced by JAK2 L611S mutant via the induction of apoptotic cell death. In addition, treatment with AG490 significantly inhibited the JAK2 L611S mutant-induced tumorigenesis in nude mice. Thus, our results both in vitro and in vivo strongly suggest that L611S mutant of JAK2 harbors potent oncogenic activity, and this probably requires the antiapoptotic signaling pathway.

Inhibition of Janus Kinase 2 and Signal Transduction and Activator of Transcription 3 Protect Against Cecal Ligation and Puncture-Induced Multiple Organ Damage and Mortality

Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway plays an important role in sepsis, transducing a multitude of inflammatory signals. To date, knowledge of JAK/STAT pathway in sepsis is limited. This study was to investigate the potential role of JAK/STAT pathway in mediating multiple organ damage and mortality in septic rats. Our data showed that inhibition of JAK2/STAT3 attenuated cecal ligation and puncture-induced multiple organ damage and mortality in 48 hours in rats. A total of 98 male Wistar rats were randomly divided into 4 groups as follows: (1) normal control group (n = 10); (2) cecal ligation and puncture (CLP) group (n = 40), which was further divided into 2, 6, 24, 48-hour post-CLP groups; (3) AG490 (8.0 mg/kg, Calbiochem, La Jolla, CA) treatment group (n = 24), which was further divided into 2, 6, 24, 48-hour post-CLP groups; (4) rapamycin (0.4 mg/kg, Calbiochem, Calbiochem, La Jolla, CA) treatment group (n = 24), which was further divided into 2, 6, 24, 48-hour post-CLP groups; CLP was performed to induce experimental sepsis. AG490 (8 mg/kg) or rapamycin (0.4 mg/kg) was injected subcutaneously 0.5 hour before CLP in respective group. Animals were killed at destined time after CLP (not including the death rate observation group), and specimens of serum, liver, and lungs were harvested and stored in liquid nitrogen for subsequent analyses. In an additional experiment, 88 animals were randomly divided into three groups to compare the survival rate, including CLP group (n = 40), AG490 treatment group (8 mg/kg, n = 24), and rapamycin treatment group (0.4 mg/kg, n = 24). Mortality of rats in each group was recorded up to 48 hours after the procedure. After CLP challenge, myeloperoxidase (MPO), aspartate transaminase, and alanine aminotransferase levels, as well as activation of JAK2 and STAT3, were markedly increased. Administration of AG490 or rapamycin significantly decreased activation of JAK2 and STAT3, as well as high mobility group box-1 protein, MPO, alanine aminotransferase levels (p < 0.05 or p < 0.01). In addition, treatment with AG490 or rapamycin significantly improved the 48-hour survival rate from 37.5% (15 of 40) to 66.7% (16 of 24) and 70.8% (17 of 24), respectively (both p < 0.05). JAK2/STAT3 pathway might play a role in the development of multiple organ damage in septic rats, which suggested a potential strategy to control sepsis.

ROLE OF JANUS KINASE/SIGNAL TRANSDUCER AND ACTIVATOR OF TRANSCRIPTION PATHWAY IN REGULATION OF EXPRESSION AND INFLAMMATION-PROMOTING ACTIVITY OF HIGH MOBILITY GROUP BOX PROTEIN 1 IN RAT PERITONEAL MACROPHAGES

Signal transduction mechanism in the regulation of high mobility group box protein 1 (HMGB1) has not yet been well elucidated. Our data showed for the first time that Janus kinase-signal transduction and activator of transcription (JAK/STAT) pathway played a major role in the regulation of expression and inflammatory effect of HMGB1. The study was carried out in the following sequence. Firstly, the role of JAK/STAT pathway in the regulation of expression of HMGB1 was examined. After stimulation with 75 ng/mL LPS in vitro, significant increases in HMGB1 expression and prompt activation of JAK/STAT pathway were demonstrated in cultured macrophages. On the other hand, administration of AG490 (specific inhibitor for JAK2), fludarabine (specific inhibitor for STAT1) or rapamycin (specific inhibitor for STAT3) markedly suppressed HMGB1 expression. Secondly, the role of JAK/STAT pathway in the regulation of TNF-alpha expression induced by HMGB1 was examined. When macrophages were stimulated with 10 microg/mL HMGB1 in vitro, significant increases in TNF-alpha expression and prompt activation of JAK/STAT pathway were demonstrated, whereas inhibitors of JAK/STAT pathway significantly suppressed TNF-alpha expression. Taken together, our data strongly indicated that expression and inflammatory effect of HMGB1 could be mediated by JAK/STAT pathway and suggested a possible clinical strategy to control an inflammatory effect of HMGB1 in sepsis.

Direct Effects of Prolactin on Adrenal Steroid Release in Male Hatano High-Avoidance (HAA) Rats May be Mediated Through Janus Kinase 2 (Jak2) Activity

Prolactin (PRL) has been proposed to directly stimulate corticosterone release. However, the role of PRL on adrenocortical function in male HAA rats has not been fully clarified. The aim of this study was to investigate the effect of PRL on the secretion of corticosterone and progesterone using an in vitro cell culture system in male rats. Administration of PRL (10(-7) and 10(-6) M) resulted in dose-dependent increases in corticosterone and progesterone release. Cotreatment with PRL produced an increase in the stimulatory effects of ACTH-induced corticosterone and progesterone secretion. However, the PRL-induced corticosterone and progesterone releases were significantly reduced by treatment with AG490, a specific Janus kinase 2 (Jak2) inhibitor. In addition, administration of AG490 blunted the significant inhibition of ACTH-induced corticosterone and progesterone secretion by PRL. These results demonstrated that PRL could act directly on the adrenal gland to drive corticosterone and progesterone secretion in male rats. Additionally, the results emphasize that PRL stimulation of adrenal steroid release may be mediated through Jak2 activity.

The role of Jak 2 and Stat 5 in antigen-induced eosinophil recruitment into the airways

We studied the role of Jak 2 and Stat 5 in antigen-induced eosinophil recruitment into the airways of sensitized mice. The in vivo administration of a Jak 2 inhibitor AG-490 prevented antigen induced eosinophil recruitment into the airways of sensitized mice in a dose-dependent manner, whereas the administration of AG-490 did not affect antigen induced IL-5 production in the airways. On the other hand, both antigen induced IL -5 production and antigen induced airway eosinophilia was diminished in Stat 5 a-deficient (Stat 5 a-/-) mice and Stat 5 b-deficient (Stat 5 b-/-) mice. In addition, eosinophilopoiesis induced by the administration of recombinant IL-5 was diminished in Stat 5 a-/- mice and Stat 5 b-/- mice. Interestingly, antigen-induced Th 2 cell differentiation was decreased but antigen-induced Th 1 cell differentiation was increased in Stat 5 a-/- mice. Together, these results indicate that Jak 2 and Stat 5 activation are essential for the induction of IL-5-dependent, antigen-induced eosinophil recruitment into the airways and that the defects in antigen induced eosinophil recruitment in Stat 5 a-/- mice and Stat 5 b-/- mice result from both impaired IL -5 production in the airways and diminished IL -5 responsiveness of eosinophils.