Phosphorylation Of Janus Kinase Research Articles

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

ObjectiveTo 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. ResultsRat 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). ConclusionThis 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.

Protective effects of Sphingosine-1-phosphate (S1P) on hypertrophic response in H9c2 cardiomyocytes

To investigate the effects of sphingosine-1-phosphate (S1P) on cardiac hypertrophic response in H9c2 cells. H9c2 cells were randomly divided into four groups: normal control group, S1P (1 μmol/L) treated group, Phenylephrine (PE) (100 μmol/L) treated group, PE (100 μmol/L) treated group combined with S1P (1 μmol/L) treatment. Each group has 3 duplicated wells. After 24 hours, the size of H9c2 cells in each group was detected by Actin-Trakcer Green immunofluorescence staining. Transcriptional levels of hypertrophic markers ( ANP, BNP and β-MHC) in H9c2 cells were determined by real-time PCR. Western blot was performed to examine the expression level of ANP in each group. Then H9c2 cells were randomly divided into five groups: normal control group, PE (100 μmol/L) treated group, PE (100 μmol/L) with S1P low-dose (0.1 μmol/L) treated group, PE (100 μmol/L) with S1P middle-dose (1 μmol/L) treated group and PE (100 μmol/L) with S1P high-dose (10 μmol/L) treated group. Each group has 3 duplicated wells. After 24 hours, Western blot was performed to examine the expressions of phosphorylated Janus kinase 2 (JAK2) and signal transducers and activators of transcription 3 (STAT3) under low, medium and high concentrations of S1P. Each experiment was repeated three times. Compared with normal control group, the surface area of H9c2 cells in PE group was increased significantly (P＜0.05), meanwhile, the transcription levels of ANP, BNP and β-MHC were increased significantly (all P＜0.05), and the expression of ANP was also increased significantly (P＜0.05) in PE group. While compared with PE group, the surface area of H9c2 cells in PE + S1P group was decreased significantly (P＜0.05), the transcription levels of ANP, BNP and β-MHC and the expression of ANP were also decreased significantly (all P＜0.05) in PE + S1P group. After treated with PE and different concentrations of S1P, the expressions of p-JAK2 and p-STAT3 were increased significantly compared with the normal control group and PE group (P＜0.05), in a dose-dependent manner. S1P could protect H9c2 cells against hypertrophic response induced by PE, which may be achieved by activating JAK2/STAT3 signal pathway.

Xueshuantong injection alleviates cerebral microcirculation disorder in middle cerebral artery occlusion/reperfusion rats by suppressing inflammation via JNK mediated JAK2/STAT3 and NF-κB signaling pathways

Ethnopharmacological relevanceIn the long history of traditional Chinese medicine, Panax notoginseng has been used as a key herb for the treatment of blood diseases. Brain microvessels support adequate blood circulation to maintain normal physiological function, therefore, brain microcirculation disorder is an important therapeutic target for various brain diseases.However, the role of Xueshuantong (XST) injection composed of saponins from P. Notoginseng (PNS) in the amelioration of cerebral microcirculation disorder is unclear. Aims of the studyCerebral microcirculation disorder and inflammation play a vital role in stroke. Capillary endothelial cells and adjacent tight junctions are fundamental to the structure and function of cerebrovascule. XST injection has been used clinically in the treatment of stroke, but no studies have reported its indication in cerebral microcirculation disorder. This study is to explore the action and mechanism of XST injection in the alleviation of cerebral microcirculation disorder in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Materials and methodsMCAO/R rats and LPS-induced bEnd.3 cells were employed for the investigation of effect and mechanism of XST injection. Brain damages were evaluated by neurobehavioral assessment, 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, hematoxylin and eosin staining (H&E), and Nissl staining. Morphology and density changes of cerebral microvessels were monitored by immunohistochemistry. Cell permeability was detected by measurement of trans-endothelial electrical resistance (TEER) and sodium fluorescein (NaF) leakage. The mRNA and protein expressions of inflammatory cytokines, tight junction proteins, adhesion molecules, Janus kinase 2 (JAK2), signal transducer and activator of transcription-3 (STAT3), inhibitor of NF-κB (IκB), nuclear factor-κB (NF-κB) and c-jun N-terminal kinase (JNK) in brain microvessels and lipopolysaccharide (LPS)-induced bEnd.3 cells were measured by real-time PCR and Western blot, respectively. ResultsXST injection at 48 mg/kg significantly improved the neurological damage, inflammatory infiltration, and microvessel morphology, and increased microvessel density in brain of MCAO/R rats. The endothelial permeability was significantly mitigated by XST injection in LPS-induced bEnd.3 cells. Meanwhile, the tight junction proteins such as zona occludens 1 (ZO-1) and occludin were elevated remarkably in brain microvessel of MCAO/R rats and LPS-induced bEnd.3 cells. Moreover, the expression of inflammatory mediators including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS), cycloocygenases 2 (COX-2), vascular cellular adhesion molecule-1 (VCAM-1), matrix metalloproteinase (MMP)-2, and MMP-9 were inhibited by XST injection. In addition, XST injection suppressed the phosphorylation of JAK2, STAT3, IκB, NF-κB and JNK, which could be abolished by anisomycin, the JNK agonist. ConclusionXST injection improved cerebral microvescular structure damage and dysfunction in MCAO/R rats through inhibiting inflammation activated by JNK mediated JAK2/STAT3 and NF-κB signaling pathways. The novel findings may provide theoretical basis for the clinical application in the treatment of cerebral microcirculation disorder.

Apigenin alleviates resistance to doxorubicin in breast cancer cells by acting on the JAK/STAT signaling pathway.

Breast cancer is the most frequent cancer among women. Despite the effectiveness of Doxorubicin (DOX) as a chemotherapeutic for the treatment of breast cancer, the therapy-resistance remains unsolvable. Apigenin is a natural dietary flavonoid with potential anticancer activities. Our study's intention was to evaluate the effect of Apigenin on DOX resistance in MCF-7 cells. DOX-resistant MCF-7 cell line (MCF-7R) was developed by treating MCF-7 cells with increasing concentrations of DOX (0-100 µM). The viability of cell lines was assayed using MTT method. Quantitative polymerase chain reaction method was performed to measure multidrug-resistance 1 (MDR1) gene expression level. The expression of MDR1, Janus kinase 2 (JAK2) and Signal transducer and activator of transcription 3 (STAT3) proteins were determined by western blotting. MCF-7R cell line showed resistance to DOX in comparison to MCF-7 cells. Apigenin had a significant effect on the reduction of viability of both MCF-7 and MCF-7R cell lines. However, DOX-resistance in the MCF-7 cell line was considerably decreased due to the co-treatment of MCF-7R cells with Apigenin. This natural compound also downregulated the expression of MDR1 at mRNA and protein levels both in resistant and non-resistant cells. Apigenin significantly prohibited the phosphorylation and activation of JAK2 and STAT3 proteins both in MCF-7 and MCF-7R cell lines. The present results suggested, for the first time, Apigenin as an ideal therapeutic for ameliorating DOX resistance in breast cancer. These data also proposed a novel mechanism for the anti-resistance activity of Apigenin by regulating the JAK2/STAT3/MDR1 axis.

Impact of heat stress on prolactin-mediated ovarian JAK-STAT signaling in postpubertal gilts.

Heat stress (HS) compromises almost every aspect of animal agriculture including reproduction. In pigs, this infecundity is referred to as seasonal infertility (SI), a phenotype including ovarian dysfunction. In multiple species, HS-induced hyperprolactinemia has been described; hence, our study objectives were to characterize and compare HS effects on circulating prolactin (PRL) and ovarian Janus kinase/signal transducer and activator of transcription (JAK-STAT) signaling during the follicular (FOL) or luteal (LUT) phases of the estrous cycle in postpubertal gilts. Gilts were estrus synchronized using altrenogest and environmental treatments began immediately after altrenogest withdrawal. For the FOL study: postpubertal gilts were allocated to constant thermoneutral (TN; n = 6; 20 ± 1.2 °C) or cyclical HS (n = 6; 25 to 32 ± 1.2 °C) conditions for 5 d. In the LUT study: postpubertal gilts were assigned to either TN (n = 7; 20 ± 2.6 °C) or cyclical HS (n = 7; 32 to 35 ± 2.6 °C) conditions from 2 to 12 days postestrus (dpe). Blood was collected by jugular venipuncture for PRL quantification on day 5 in the FOL and on day 0 and day 12 in the LUT gilts. Ovaries and corpora lutea (CL) were obtained from euthanized FOL and LUT gilts on day 5 and day 12, respectively. Western blotting was performed to quantify prolactin receptor (PRLR) and JAK/STAT pathway protein abundance. In the FOL phase, no difference (P = 0.20) in circulating PRL between thermal groups was observed. There was no effect (P ≥ 0.34) of HS on PRLR, signal transducer and activator of transcription 3 (STAT3), signal transducer and activator of transcription 5α (STAT5α), and phosphorylated signal transducer and activator of transcription α/β tyrosine 694/699 (pSTAT5α/βTyr694/699) abundance and Janus kinase 2 (JAK2), phosphorylated janus kinase 2 tyrosine 1007/1008 (pJAK2Tyr1007/1008), STAT1, phosphorylated signal transducer and activator of transcription 1 tyrosine 701 (pSTAT1Tyr701), phosphorylated signal transducer and activator of transcription 1 serine 727 (pSTAT1Ser727), and phosphorylated signal transducer and activator of transcription 3 tyrosine 705 (pSTAT3Tyr705) were undetectable in FOL gilt ovaries. Ovarian pSTAT5α/βTyr694/699 abundance tended to moderately increase (4%; P = 0.07) in FOL gilts by HS. In the LUT phase, circulating PRL increased progressively from 2 to 12 dpe, but no thermal treatment-induced difference (P = 0.37) was noted. There was no effect (P ≥ 0.16) of HS on CL abundance of PRLR, pJAK2Tyr1007/1008, JAK2, STAT1, pSTAT1Tyr701, pSTAT1Ser727, pSTAT3Tyr705, STAT5α, or pSTAT5α/βTyr694/699. In LUT phase, CL STAT3 abundance was increased (11%; P < 0.03) by HS. There was no impact of HS (P ≥ 0.76) on levels of pJAK2Tyr1007/1008 and pSTAT5α/βTyr694/699 in LUT gilts; however, the CL pSTAT3Tyr705:STAT3 ratio tended to be decreased (P = 0.10) due to HS. These results indicate an HS-induced estrous cycle-stage-dependent effect on the ovarian JAK/STAT pathway, establishing a potential role for this signaling pathway as a potential contributor to SI.

Phosphorylation of PBK/TOPK Tyr74 by JAK2 promotes Burkitt lymphoma tumor growth

Burkitt lymphoma (BL), which is characterized by high invasiveness, is a subgroup of non-Hodgkin lymphoma. Although BL is regarded as a highly curable disease, especially for children, some patients unfortunately still do not respond adequately. The understanding of the etiology and molecular mechanisms of BL is still limited, and targeted therapies are still lacking. Here, we found that T-LAK cell-derived protein kinase (TOPK) and phosphorylated Janus kinase 2 (p-JAK2) are highly expressed in the tissues of BL patients. We report that TOPK directly binds to and is phosphorylated at Tyr74 by JAK2. Histone H3, one of the downstream targets of TOPK, is also phosphorylated in vivo and in vitro. Furthermore, we report that the phosphorylation of TOPK at Tyr74 by JAK2 plays a vital role in the proliferation of BL cells and promotes BL tumorigenesis in vivo. Phosphorylation of TOPK at Tyr74 by JAK2 enhances the stability of TOPK. Collectively, our results suggest that the JAK2/TOPK/histone H3 axis plays a key role in the proliferation of BL cells and BL tumorigenesis in vivo.

Cell proliferation effects of S-allyl-L-cysteine are associated with phosphorylation of janus kinase 2, insulin-like growth factor type-I receptor tyrosine kinase, and extracellular signal-regulated kinase 2 in primary cultures of adult rat hepatocytes

The cell proliferation effect of S-allyl-L-cysteine (SAC) and its mechanisms were examined in primary cultures of adult rat hepatocytes. In serum-free cultivation, SAC (10−6 M)-stimulated hepatocytes showed significant proliferation compared to control at 5-h culture; the effect was dependent on the culture time and the dose of SAC (EC50 value 8.58 × 10−8 M). In addition, SAC-stimulated hepatocytes significantly increased mRNA expression levels of c-Myc and c-Fos at 1 h and cyclin B1 at 3.5 and 4 h, respectively. In contrast, alliin and allicin, structural analogs of SAC, did not show these effects observed with SAC. The SAC-induced hepatocyte proliferation effects were completely suppressed by monoclonal antibodies against growth hormone receptor and insulin-like growth factor type-I (IGF-I) receptor, respectively. Furthermore, the Janus kinase 2 (JAK2) inhibitor TG101209, phospholipase C (PLC) inhibitor U-73122, IGF-I receptor tyrosine kinase (RTK) inhibitor AG538, PI3 kinase inhibitor LY294002, MEK inhibitor PD98059, and mTOR inhibitor rapamycin completely suppressed the SAC-induced hepatocyte proliferation. JAK2 (p125 kDa) phosphorylation in cultured hepatocytes peaked 5 min after SAC stimulation. SAC-induced IGF-I RTK (p95 kDa) and ERK2 (p42 kDa) phosphorylation had slower rises than JAK2, peaking at 20 and 30 min, respectively. These results indicate that SAC promoted cell proliferation by growth hormone receptor/JAK2/PLC pathway activation followed by activation of the IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

Protective mechanisms of 10-gingerol against myocardial ischemia may involve activation of JAK2/STAT3 pathway and regulation of Ca2+ homeostasis

10-Gingerol (10-Gin), an active ingredient extracted from ginger, has been reported to have beneficial effects on the cardiovascular system. However, its protective effects on myocardial ischemia (MI) and the underlying cellular mechanisms are still unclear. To investigate the protection conferred by 10-Gin against MI injury and its potential mechanisms in cardiomyocytes via patch-clamp and molecular biology techniques. A rat MI model was established using the subcutaneous injection of isoproterenol (85 mg/kg) administered on two consecutive days. 10-Gin was pre-administered to rats for seven days to assess its cardio-protection. The patch-clamp and IonOptix Myocam detection techniques were used to investigated 10-Gin’s effects on L-type Ca2+ channels (LTCCs), Ca2+ transients and cell contractility in isolated rat cardiomyocytes. 10-Gin administration alleviated MI injury, improved cardiac function and myocardial histopathology, reduced myocardial infarct area, downregulated oxidative stress and Ca2+ levels, and decreased the expression of apoptotic factors. Importantly, 10-Gin led to an increase in phosphorylated Janus kinase 2 and signal transducer and activator of transcription 3 (JAK2 and STAT3, respectively) expressions. Furthermore, 10-Gin inhibited LTCCs in a concentration-dependent manner with a half-maximal inhibitory concentration of 75.96 μM. Moreover, 10-Gin administration inhibited Ca2+ transients and cell contractility. Our results suggest that 10-Gin exerts cardioprotective effects on MI in vivo and in vitro in connection with the inhibition of oxidative stress and apoptosis via activation of the JAK2/STAT3 signalling pathway, and regulation of Ca2+ homeostasis by LTCCs.

S-Allyl-L-cysteine Promotes Cell Proliferation by Stimulating Growth Hormone Receptor/Janus Kinase 2/Phospholipase C Pathways and Promoting Insulin-Like Growth Factor Type-I Secretion in Primary Cultures of Adult Rat Hepatocytes.

The mechanism of insulin-like growth factor type-I (IGF-I) secretion stimulated by S-allyl-L-cysteine (SAC) was investigated as part of a study of SAC-induced DNA synthesis and cell proliferation in primary cultures of adult rat hepatocytes. When 10-6 M SAC was added to the culture, the amount of IGF-I in the medium was significantly increased at 10 min. The peak IGF-I level (140 pg/mL) was observed 20 min after SAC stimulation. The SAC-induced IGF-I secretion was completely suppressed by a selective Janus kinase 2 (JAK2) inhibitor (TG101209), a selective phospholipase C (PLC) inhibitor (U-73122), an intracellular Ca2+ chelating agent (BAPTA-AM), and a granule secretion inhibitor (somatostatin). On the other hand, 10-6 M SAC-stimulated hepatocytes showed increased intracellular Ca2+ concentration in a time-dependent manner from 0 to 10 min. Phosphorylation of SAC-induced JAK2 and IGF-I receptor tyrosine kinase (RTK) was completely suppressed by TG101209. In addition, U-73122, BAPTA-AM, and somatostatin did not suppress SAC-induced JAK2 phosphorylation, but significantly suppressed SAC-induced IGF-I RTK phosphorylation. Furthermore, binding of the monoclonal antibody against growth hormone (GH) to GH receptor was dose-dependently suppressed by SAC on immunofluorescence. These results showed that SAC promotes cell proliferation by stimulating GH receptor/JAK2/phospholipase C pathways and promoting autocrine secretion of IGF-I in primary cultures of adult rat hepatocytes.

Protective Role of Tripterygium wilfordii Polyglycosides in Rheumatoid Arthritis by Regulating Janus Kinase 2/Signal Transducer and Activator of Transcription 3 Pathway

To investigate the effect and mechanism of Tripterygium wilfordii polyglycosides on rheumatoid arthritis rats by regulating the signal transducer and activator of transcription 3 pathways. 45 specific-pathogen free male Sprague-Dawley rats were randomly divided into normal control group, model group and Tripterygium wilfordii polyglycosides group with 15 rats in each group. The histopathological changes of the synovium of the ankle joint of rats in each group, on the 12th, 20th and 28th d were compared. On the 12th d, the degree of tissue swelling in the model group was significantly higher than that in the normal control group (p<0.05); there was no significant difference in the degree of toe swelling between the Tripterygium wilfordii polyglycosides group and the model group (p>0.05). At 20 d and 28 d, the degree of toe swelling in the model group was significantly higher than that in the normal control group (p<0.05); the swelling degree of toes in Tripterygium wilfordii polyglycosides group was significantly lower than that in model group (p<0.05). The serum interleukin-6 level in model group was significantly higher than that in normal control group and interleukin-10 level was significantly lower than that in normal control group (p<0.05). The level of interleukin-6 in serum of rats in Tripterygium wilfordii polyglycosides group was significantly lower than that in model group and the level of interleukin-10 was significantly higher than that in model group (p<0.05). The expression levels of phosphorylated Janus kinase 2 and phosphorylated signal transducer and activator of transcription 3 protein in model group were significantly higher than those in normal control group; the expression levels of phosphorylated Janus kinase 2 and phosphorylated signal transducer and activator of transcription 3 protein in Tripterygium wilfordii polyglycosides group were significantly lower than those in the model group (p<0.05). Tripterygium wilfordii polyglycosides has the effect of anti-rheumatoid arthritis and its mechanism may be through regulating the Janus kinase 2/ signal transducer and activator of transcription 3 signal pathways by reducing the expression level of phosphorylated Janus kinase 2 and phosphorylated signal transducer and activator of transcription 3 proteins.

Mutated JAK2 signal transduction in human induced pluripotent stem cell (iPSC)-derived megakaryocytes

Janus kinase 2 (JAK2) gene mutations are the main drivers for polycythemia vera (PV) and essential thrombocythemia (ET). The mechanisms of single altered gene causing two different diseases are unclear. Additionally, novel treatments specifically targeting mutated JAK2 proteins are needed. In this study, the induced pluripotent stem cells (iPSCs) were virally transduced to express wild-type JAK2 (JAK2WT), JAK2p.V617F (JAK2V617F) or JAK2p.N542_E543del (JAK2exon12) under a doxycycline-inducible system. The modified iPSCs which were differentiated into megakaryocytes in the presence vs. absence of doxycycline were compared to ensure that the differences were solely from mutated JAK2 expressions. The JAK2V617-expressing iPSCs yielded significantly higher numbers of megakaryocytes consistent with the ET phenotype, while there was no enhancement by JAK2exon12 expression compatible with the pure erythrocytosis in humans. Capillary Western analyses revealed significantly greater JAK2 phosphorylation in iPSCs carrying JAK2V617F but not in JAK2WT and JAK2exon12 iPSCs. Activation of STAT3, STAT5 and AKT was increased by JAK2V617F, while they were decreased in JAK2exon12 iPSCs. Notably, interferon alpha and/or arsenic trioxide inhibited megakaryocytes proliferation and reduced JAK2, STAT3, STAT5 and AKT phosphorylation in mutant JAK2-expressing iPSCs compared with those without induction. In conclusion, JAK2V617F expression in iPSCs preferentially promoted megakaryocytes with a signaling profile distinctive from JAK2exon12 expression. Treatments with interferon alpha or arsenic trioxide preferentially suppressed the mutated over wild-type JAK2 signaling. This iPSC model is helpful in mechanistic studies and novel therapy screen for myeloproliferative neoplasm.

Estrogen receptor β alleviates inflammatory lesions in a rat model of inflammatory bowel disease via down-regulating P2X7R expression in macrophages.

Estrogen receptor beta (ERβ) agonists could inhibit inflammation in animal models of inflammatory bowel disease (IBD). However, the mechanism underlying such effect and the potential role of P2 × 7 receptor (P2X7R) remains unclear. In the present study, we examined whether the effect of ERβ activation in IBD rats was related to P2X7R. Overexpression of ERβ using a recombinant lentivirus in IBD rats improved the IBD-like symptoms, including weight loss, disease activity index (DAI) scores, and inflammatory responses. ERβ agonists DPN and ERB-041 attenuated P2X7R expression in macrophages from colitis rats and in a murine macrophage cell line (RAW264.7) in response to either lipopolysaccharide (LPS) or adenosine triphosphate (ATP). DPN and ERB-041 also blocked increased production of TNF-α, IL-6, and IL-1β in the rectocolon of colitis rats. The two ERβ agonists reversed LPS- and ATP-induced up-regulation of P2X7R and its downstream proteins, including NLRP3, ASC, caspase-1, and pro-IL-1β, in RAW264.7 cells. Also, in both the rectocolon of colitis rats and RAW264.7 cells, ERβ agonists reversed the up-regulation of extracellular regulated protein kinases (ERK), the Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3 (STAT3), but not up-regulation of serine threonine kinase or cAMP-response element binding protein (CREB). Blockade of JAK2 or STAT3 phosphorylation significantly reduced the ability of DPN to down-regulate P2X7R expression and the ability of ERB-041 and DPN to inhibit IL-1β release from RAW264.7 cells. We found that ERβ and P2X7R co-localized in the macrophages of rat rectocolon and in RAW264.7 cells. Deletion of macrophages from colitis rats with clodronate abolished the inhibitory effect of DPN. These results suggest that ERβ plays an important anti-inflammatory role in IBD rats by down-regulating P2X7R expression and inhibiting IL-1β release from macrophages through the JAK2/STAT3 signaling pathway.

SDF-1/CXCR4 axis promotes osteogenic differentiation of BMSCs through the JAK2/STAT3 pathway.

This study aimed to investigate the effects of stromal cell-derived factor-1 (SDF-1) and activation of its receptor, chemokine receptor 4 (CXCR4), on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), and the key signaling mechanisms involved in these effects. BMSCs were treated with 100 μg/L SDF-1 and cultured in osteogenic medium for 7 days. RT-qPCR and western blotting were used to detect the protein and mRNA levels of Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), Runt-related transcription factor 2 (Runx2), and osteocalcin (OCN). Alizarin-red staining was used to detect the mineralization-inducing ability of the cells. After BMSCs were treated with SDF-1, the levels of JAK2 mRNA, STAT3 mRNA, and protein phosphorylation increased, the number of mineralized nodules of BMSCs increased, and the osteogenic-differentiation ability was enhanced. In addition, after BMSCs were treated with an inhibitor of JAK2 phosphorylation, the levels of JAK2, STAT3, Runx2, and OCN decreased significantly, the number of mineralized nodules of BMSCs also decreased, and the osteogenic-differentiation ability decreased. The inhibition of CXCR4-treated BMSCs further confirmed that SDF-1/CXCR4 activated JAK2/STAT3 to regulate the osteogenic differentiation of BMSCs. SDF-1/CXCR4 promoted the osteogenic differentiation of BMSCs through JAK2/STAT3 activation.

The Antimicrobial Peptide Human β-Defensin-3 Accelerates Wound Healing by Promoting Angiogenesis, Cell Migration, and Proliferation Through the FGFR/JAK2/STAT3 Signaling Pathway.

In addition to its antimicrobial activity, the skin-derived antimicrobial peptide human β-defensin-3 (hBD-3) promotes keratinocyte proliferation and migration to initiate the wound healing process; however, its effects on fibroblasts, which are the major cell type responsible for wound healing, remain unclear. We investigated the role of hBD-3 in cell migration, proliferation and production of angiogenic growth factors in human fibroblasts and evaluated the in vivo effect of hBD-3 on promoting wound healing and angiogenesis. Following hBD-3 treatment, the mouse wounds healed faster and showed accumulation of neutrophils and macrophages in the early phase of wound healing and reduction of these phagocytes 4 days later. hBD-3-treated wounds also displayed an increased number of fibroblasts and newly formed vessels compared to those of the control mice. Furthermore, the expression of various angiogenic growth factors was increased in the hBD-3-treated wounds. Additionally, in vitro studies demonstrated that hBD-3 enhanced the secretion of angiogenic growth factors such as fibroblast growth factor, platelet-derived growth factor and vascular endothelial growth factor and induced the migration and proliferation of human fibroblasts. The hBD-3-mediated activation of fibroblasts involves the fibroblast growth factor receptor 1 (FGFR1)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathways, as evidenced by the inhibitory effects of pathway-specific inhibitors. We indeed confirmed that hBD-3 enhanced the phosphorylation of FGFR1, JAK2 and STAT3. Collectively, the current study provides novel evidence that hBD-3 might be a potential candidate for the treatment of wounds through its ability to promote wound healing, angiogenesis and fibroblast activation.

Water extract of medicinal ink (WEMI) attenuates lipopolysaccharide-induced NO production of Raw264.7 cells via downregulating JAK2/STAT3-mediated iNOS expression

Ethnopharmacological relevanceMedicinal ink is used as a traditional topical medicine for treating inflammatory diseases via detoxification, relieving pain, hemostasis, and reducing swelling. However, the effect of medicinal ink on the inhibition of inflammatory responses and the underlying molecular mechanism remain unclear. Aim of the studyThe present study aimed to investigate the anti-inflammatory function of water extract of medical ink (WEMI) and elucidate its active mechanisms. Materials and methodsCell viability was assessed using crystal violet staining assay. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected by ELISA. Nitric oxide (NO) production was measured by Griess assay. The activation of inflammatory signaling molecules stimulated by lipopolysaccharide (LPS) was evaluated by assessing levels of inducible nitric oxide synthase (iNOS), phosphorylated Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) using Western blot assay. ResultsWater extract of medical ink (WEMI) did not present cytotoxic effect on murine macrophage Raw264.7 cells. High dosage of WEMI slightly rescued LPS-suppressed cell viability of Raw264.7 cells. WEMI did not induce NO production or IL-6 secretion, though WEMI significantly induced secretion of TNF-α on Raw264.7 cells not stimulated with LPS. On the other hand, LPS effectively stimulated inflammation on Raw264.7 cells; however, WEMI dramatically reduced LPS-induced NO production. WEMI alleviated LPS-stimulated IL-6 secretion but did not affect the content of TNF-α. In addition, WEMI effectively reduced expression of iNOS by abolishing LPS-mediated phosphorylation of JAK2 and STAT3 but not TLR4-mediated NF-κB and MAPK molecules. ConclusionsOur findings suggest that WEMI targets of the JAK2/STAT3-mediated iNOS expression play a key role in alleviating LPS-induced inflammatory responses in RAW264.7 macrophages. Therefore, medicinal ink may be a potential topical agent for treating fasciitis or synovitis via regulating the immune system.

Krüppel-like factor 7 attenuates hippocampal neuronal injury after traumatic brain injury.

Our previous study has shown that the transcription factor Krüppel-like factor 7 (KLF7) promotes peripheral nerve regeneration and motor function recovery after spinal cord injury. KLF7 also participates in traumatic brain injury, but its regulatory mechanisms remain poorly understood. In the present study, an HT22 cell model of traumatic brain injury was established by stretch injury and oxygen-glucose deprivation. These cells were then transfected with an adeno-associated virus carrying KLF7 (AAV-KLF7). The results revealed that, after stretch injury and oxygen-glucose deprivation, KLF7 greatly reduced apoptosis, activated caspase-3 and lactate dehydrogenase, downregulated the expression of the apoptotic markers B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and cleaved caspase-3, and increased the expression of βIII-tubulin and the antiapoptotic marker Bcl-2. Furthermore, KLF7 overexpression upregulated Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in HT22 cells treated by stretch injury and oxygen-glucose deprivation. Immunoprecipitation assays revealed that KLF7 directly participated in the phosphorylation of STAT3. In addition, treatment with AG490, a selective inhibitor of JAK2/STAT3, weakened the protective effects of KLF7. A mouse controlled cortical impact model of traumatic brain injury was then established. At 30 minutes before modeling, AAV-KLF7 was injected into the ipsilateral lateral ventricle. The protein and mRNA levels of KLF7 in the hippocampus were increased at 1 day after injury and recovered to normal levels at 3 days after injury. KLF7 reduced ipsilateral hippocampal atrophy, decreased the injured cortex volume, downregulated Bax and cleaved caspase-3 expression, and increased the number of 5-bromo-2'-deoxyuridine-positive neurons and Bcl-2 protein expression. Moreover, KLF7 transfection greatly enhanced the phosphorylation of JAK2 and STAT3 in the ipsilateral hippocampus. These results suggest that KLF7 may protect hippocampal neurons after traumatic brain injury through activation of the JAK2/STAT3 signaling pathway. The study was approved by the Institutional Review Board of Mudanjiang Medical University, China (approval No. mdjyxy-2018-0012) on March 6, 2018.

JAK2 Phosphorylation Signals and Their Associated Cytokines Involved in Chronic Rhinosinusitis with Nasal Polyps and Correlated with Disease Severity.

Janus kinase 2 (JAK2) is a member of the JAK family that transduces cytokine-mediated signals via the JAKs/STATs (signal transducer and activator of transcription proteins) pathway, which plays an important role in many inflammatory diseases. This study investigates the association of p-JAK2 and JAK2-associated cytokines from nasal polyp (NP) tissue with disease severity, and evaluates the p-JAK2-mediated STATs in chronic rhinosinusitis (CRS) with NP. Sixty-one CRSwNP patients with nasal polyps undergoing endoscopic sinus surgery were enrolled, while the turbinate tissues from 26 nasal obstruction patients were examined as the control group. Elevated levels of p-JAK2 were detected in CRSwNP, and significantly correlated with scores of disease severity (LMK-CT, TPS, and SNOT-22). Expressions of the JAK2-associated cytokines, such as IL-5, IL-6, IL-13, G-CSF, and IFN-γ were significantly higher in CRSwNP than in the controls, while the levels of IL-5, IL-6, IL-13, or G-CSF had positive correlation with scores of disease severity. Moreover, markedly increased expression of p-STAT3 in CRSwNP was observed relative to the control. Taken together, these data showed that the JAK2-associated cytokines including IL-6 and G-CSF may stimulate JAK2 phosphorylation to activate p-STAT3, indicating an association with disease severity and supporting its development of JAK2 inhibitor as a potential therapeutic agent for CRS.

PERK-Dependent Activation of the JAK2/STAT3 Pathway Contributes to High Glucose-Induced Extracellular Matrix Deposition in Renal Tubular Epithelial Cells.

Background Although the deposition of extracellular matrix (ECM) is critical leading to tubular damage in diabetic kidney disease (DKD), the mechanism still remains unclear. The purpose of this study was to demonstrate a role for protein kinase R-like endoplasmic reticulum kinase (PERK) (a protein located in the endoplasmic reticulum membrane) in this pathologic process. Methods NRK-52E cells were grown in the media containing different concentrations of glucose or thapsigargin for different durations. Cells were subsequently incubated with or without AG490, a selective inhibitor of Janus kinase 2 (JAK2) or GSK2606414 (a selective PERK inhibitor). We evaluated the production of TGF-β1, fibronectin, and collagen I proteins by ELISA. The levels of 78 kD-glucose-regulated protein (GRP78) and PERK, as well as the phosphorylation statues of PERK and JAK2/signal transducer and activator of transcription (STAT3), were determined by western blotting analysis. Results We showed that the increased phosphorylation of JAK2 and STAT3 was accompanied by overexpression of TGF-β1 and ECM deposition in high glucose medium. Disruption of the JAK2/STAT3 pathway with AG490 significantly prevents the high glucose-induced increase in TGF-β1, fibronectin, and collagen I. High glucose induced the overproduction of GRP78 and phosphorylation of PERK, which indicated that endoplasmic reticulum stress (ERS) was triggered in NRK-52E cells cultured under high glucose condition. Inhibition of PERK phosphorylation with GSK2606414, however, blocked the effect of JAK2/STAT3 on the production of TGF-β1 and ECM components in NRK-52E cells. Conclusion Our data indicated that the ECM accumulation induced by high glucose arouse via the PERK-dependent JAK2/STAT3-signaling pathway in renal tubular epithelial cells.

Retardant effect of dihydroartemisinin on ulcerative colitis in a JAK2/STAT3-dependent manner.

Dihydroartemisinin (DHA) is a semi-synthetic derivative and the main active metabolite of artemisinin. The purpose of this study was to investigate the effect of DHA on the ulcerative colitis (UC) in both in vivo and in vitro models. Weight, survival rate, colon length, and Disease Activity Index score were used to evaluate the severity of colitis. Reverse transcription quantitative polymerase chain reaction and enzyme-linked immunosorbent assay were used to detect the expressions of cytokines interleukin (IL)-1, IL-1β, IL-4, IL-6, IL-10, IL-12, and tumor necrosis factor-α (TNF-α). The expressions of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3), and the phosphorylation of JAK2 (p-JAK2) and STAT3 (p-STAT3), were measured by western blot analysis. Western blot analysis and immunohistochemistry were used to detect the expressions of tight junction proteins. We found that the weights and colon lengths of mice in dextran sodium sulfate (DSS)+DHA group were significantly lower and longer than those in the DSS group, respectively. Compared with those in the DSS group, the expressions of IL-1β, IL-6, IL-17, and TNF-α in the DSS+DHA and DSS+5-aminosalicylic acid (5-ASA) groups were decreased, while the expressions of IL-4 and IL-10 were significantly upregulated. DHA largely increased the expressions of zonula occludens-1 and occludin. Western blot analysis and/or immunohistochemical staining analysis showed that the expressions of JAK2, STAT3, p-JAK2, and p-STAT3 in DSS+DHA and DSS+5-ASA groups were significantly lower than those in DSS group. DHA has a specific therapeutic effect on UC. The anti-inflammatory mechanism of DHA is related to the blockage of the JAK2/STAT3 signaling pathway. These findings provide evidence that DHA may be a useful drug and is expected to become a promising new treatment for human UC.

Cinnamic Acid Reduces Inflammation and Apoptosis in Necrotizing Enterocolitis

Necrotizing enterocolitis is characterized by an inflammatory condition in the intestine that could result in intestinal necrosis and cell death. Cinnamic acid, an unsaturated carboxylic acid, possesses anti-inflammatory capacity. However, the regulatory role of cinnamic acid on necrotizing enterocolitis has not been investigated yet. To this end, human fetal colon cells were incubated with increasing concentrations of lipopolysaccharides to establish a necrotizing enterocolitis cell model. Data from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis showed that lipopolysaccharides, in a dosage-dependent manner, reduced cell viability of fetal human colon cells. Also, cinnamic acid prevented the cytotoxic effect of lipopolysaccharides on fetal human colon cells and increased the cell viability. Furthermore, cinnamic acid attenuated lipopolysaccharides-induced decrease in interleukin-10 and increase in interleukin-6 and tumor necrosis factor-α caused by lipopolysaccharides. The lipopolysaccharides-induced increase in cell apoptosis in fetal human colon cells was accompanied with upregulated B-cell lymphoma 2 protein-associated X protein and downregulated B-cell lymphoma 2 protein. These changes were reversed by cinnamic acid treatment. Lastly, expression of protein for suppressor of cytokine signaling 3 was reduced, while phosphorylation of Janus kinase 2 and signal transducers and activators of transcription 3 were enhanced in lipopolysaccharides-induced fetal human colon cells. Once again, cinnamic acid reversed the expression of suppressor of cytokine signaling 3, phospho- Janus kinase 2, and phospho-signal transducers and activators of transcription 3 in lipopolysaccharides-induced fetal human colon cells. In conclusion, cinnamic acid exerted antiapoptotic and anti-inflammatory effects and protected enterocytes against necrotizing enterocolitis through regulation of signal transducers and activators of transcription-mediated Janus kinase 2/signal transducers and activators of transcription 3 pathway.