JAK2 Protein Research Articles

Leptin activates the JAK/STAT pathway to promote angiogenesis in RF/6A cells in vitro.

To investigate the effect of leptin on the angiogenesis of RF/6A cells (monkey retinal choroidal endothelial cells) in vitro and test the cellular signaling in the mechanism. RF/6A cells were cultured in vitro and randomly divided into four groups: normal control, with leptin at 50, 100, 200 ng/mL for cell counting kit-8 (CCK8). RF/6A cell proliferation and migration were examined by Transwell assays, while RF/6A cell tube formation by Matrigel assay. JAK2, p-JAK2, STAT3, and p-STAT3 protein expression was measured by Western blotting. Cells were then divided into the following treatment groups: control, 100 ng/mL leptin and AG-490 (100 ng/mL leptin+10 µmol/L AG-490) for examinations of RF/6A cellular behaviour again. Analysis of differences was carried out using one-way ANOVA and least significant difference (LSD). RF/6A cell proliferation, migration and cell tube formation were promoted significantly by leptin in a dose-dependent manner (P<0.05). Western blotting showed that leptin up-regulated p-JAK2 and p-STAT3 expression levels. Treatment with the JAK/STAT pathway inhibitor, AG-490, decreased leptin-induced p-JAK2 and p-STAT3 expression, and inhibited cell proliferation, migration and cell tube formation induced by leptin (P<0.05). Leptin can promote RF/6A cell angiogenesis in vitro via activation of the JAK2/STAT3 signaling pathway.

Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition.

Non-communicable diseases are increasing and have an underlying low-grade inflammation in common, which may affect gut health. To maintain intestinal homeostasis, unwanted epithelial activation needs to be avoided. This study compared the efficacy of butyrate, propionate and acetate to suppress IFN-γ+/−TNF-α induced intestinal epithelial activation in association with their HDAC inhibitory capacity, while studying the canonical and non-canonical STAT1 pathway. HT-29 were activated with IFN-γ+/−TNF-α and treated with short chain fatty acids (SCFAs) or histone deacetylase (HDAC) inhibitors. CXCL10 release and protein and mRNA expression of proteins involved in the STAT1 pathway were determined. All SCFAs dose-dependently inhibited CXCL10 release of the cells after activation with IFN-γ or IFN-γ+TNF-α. Butyrate was the most effective, completely preventing CXCL10 induction. Butyrate did not affect phosphorylated STAT1, nor phosphorylated NFκB p65, but inhibited IRF9 and phosphorylated JAK2 protein expression in activated cells. Additionally, butyrate inhibited CXCL10, SOCS1, JAK2 and IRF9 mRNA in activated cells. The effect of butyrate was mimicked by class I HDAC inhibitors and a general HDAC inhibitor Trichostatin A. Butyrate is the most potent inhibitor of CXCL10 release compared to other SCFAs and acts via HDAC inhibition. This causes downregulation of CXCL10, JAK2 and IRF9 genes, resulting in a decreased IRF9 protein expression which inhibits the non-canonical pathway and CXCL10 transcription.

Periplocymarin alleviates pathological cardiac hypertrophy via inhibiting the JAK2/STAT3 signalling pathway.

Pathological cardiac hypertrophy is the most important risk factor for developing chronic heart failure. Therefore, the discovery of novel agents for treating pathological cardiac hypertrophy remains urgent. In the present study, we examined the therapeutic effect and mechanism of periplocymarin (PM)‐mediated protection against pathological cardiac hypertrophy using angiotensinII (AngII)‐stimulated cardiac hypertrophy in H9c2 cells and transverse aortic constriction (TAC)‐induced cardiac hypertrophy in mice. In vitro, PM treatment significantly reduced the surface area of H9c2 cells and expressions of hypertrophy‐related proteins. Meanwhile, PM markedly down‐regulated AngII‐induced translocation of p‐STAT3 into the nuclei and enhanced the phosphorylation levels of JAK2 and STAT3 proteins. The STAT3 specific inhibitor S3I‐201 or siRNA‐mediated depleted expression could alleviate AngII‐induced cardiac hypertrophy in H9c2 cells following PM treatment; however, PM failed to reduce the expressions of hypertrophy‐related proteins and phosphorylated STAT3 in STAT3‐overexpressing cells, indicating that PM protected against AngII‐induced cardiac hypertrophy by modulating STAT3 signalling. In vivo, PM reversed TAC‐induced cardiac hypertrophy, as determined by down‐regulating ratios of heart weight to body weight (HW/BW), heart weight to tibial length (HW/TL) and expressions of hypertrophy‐related proteins accompanied by the inhibition of the JAK2/STAT3 pathway. These results revealed that PM could effectively protect the cardiac structure and function in experimental models of pathological cardiac hypertrophy by inhibiting the JAK2/STAT3 signalling pathway. PM is expected to be a potential lead compound of the novel agents for treating pathological cardiac hypertrophy.

Lactylation-driven METTL3-mediated RNA m6A modification promotes immunosuppression of tumor-infiltrating myeloid cells

Tumor-infiltrating myeloid cells (TIMs) are crucial cell populations involved in tumor immune escape, andtheir functions are regulated by multiple epigenetic mechanisms. The precise regulation mode of RNA N6-methyladenosine (m6A) modification in controlling TIM function is still poorly understood. Our study revealed that the increased expression of methyltransferase-like 3 (METTL3) in TIMs was correlated with the poor prognosis of colon cancer patients, and myeloid deficiency of METTL3 attenuated tumor growth in mice. METTL3 mediated m6A modification on Jak1 mRNA in TIMs, the m6A-YTHDF1 axis enhanced JAK1 protein translation efficiency and subsequent phosphorylation of STAT3. Lactate accumulated in tumor microenvironment potently induced METTL3 upregulation in TIMs via H3K18 lactylation. Interestingly, we identified two lactylation modification sites in the zinc-finger domain of METTL3, which was essential forMETTL3 to capture target RNA. Our results emphasize the importance of lactylation-driven METTL3-mediated RNA m6A modification for promoting the immunosuppressive capacity of TIMs.

Triptolide inhibits inflammatory response and migration of fibroblast like synovial cells in rheumatoid arthritis through the circRNA 0003353/JAK2/STAT3 signaling pathway

To investigate the effect of triptolide (TPL) on inflammatory response and migration of fibroblast like synovial cells (FLS) in rheumatoid arthritis (RA-FLS) and the mechanism of circular noncoding RNA (circRNA) 0003353 for mediating this effect. We collected peripheral blood mononuclear cells (PBMCs) and serum samples from 50 hospitalized RA patients and 30 healthy individuals for detecting the expression of circRNA 0003353, immune and inflammatory indexes (ESR, CRP, RF, anti-CCP, IgA, IgG, IgM, C3, and C4) and DAS28 score. Cultured RA-FLS was treated with 10 ng/mL TPL and transfected with a circRNA 0003353 overexpression plasmid, and cell counting kit-8 (CCK-8) assay and Transwell assay were used to detect the changes in the viability and migration of the cells. Enzyme-linked immunosorbent assay (ELISA) was used to examine the cytokines IL-4, IL-6, and IL-17, and real-time fluorescence quantitative PCR (RT-qPCR) was performed to detect the expression of circRNA 003353; Western blotting was used to detect the expressions of p-JAK2, pSTAT3, JAK2 and STAT3 proteins in the treated cells. The expression of circRNA 0003353 was significantly increased in PBMCs from RA patients and showed a good performance in assisting the diagnosis of RA (AUC=90.5%, P < 0.001, 95% CI: 0.83-0.98). CircRNA 0003353 expression was positively correlated with ESR, RF and DAS28 (P < 0.05). Treatment with TPL significantly decreased the expression of circRNA 0003353, suppressed the viability and migration ability, decreased the expressions of IL-6 and IL-17, and increased the expression IL-4 in cultured RA-FLS in a time-dependent manner (P < 0.01). TNF-α stimulation of RA-FLS significantly increased the ratios of p-JAK2/JAK2 and p-STAT3/STAT3, which were obviously lowered by TPL treatment (P < 0.01). TPL-treated RA-FLS overexpressing circRNA 0003353 showed significantly increased cell viability and migration ability with decreased IL-4 expression and increased IL-6 and IL-17 expressions and ratios of p-JAK2/ JAK2 and p-STAT3/STAT3 (P < 0.01). The expression of circRNA 0003353 is increased in PBMCs in RA patients and in RA-FLS. TPL treatment can regulate JAK2/STAT3 signal pathway and inhibit the inflammatory response and migration of RA-FLS through circRNA 0003353.

Rhein Protects Against Severe Acute Pancreatitis In vitro and In vivo by Regulating the JAK2/STAT3 Pathway.

Rhein is widely used in inflammation treatment in China, but its effects on severe acute pancreatitis (SAP) have not been studied closely. This study investigated rhein’s protective effects against SAP using in vitro and in vivo models to determine whether its protective mechanism regulated the Janus kinase two and signal transducer and activator of transcription 3 (JAK2/STAT3) signalling pathway. Thirty-six male Sprague–Dawley rats were randomised into sham operation, SAP and rhein groups. The SAP model was induced by retrograde pancreatic bile duct injection of sodium taurocholate. Serum TNF-α and interleukin (IL)-6 levels were determined by ELISA, whereas serum amylase and lipase concentrations were measured using test kits. Western blot and/or immunohistochemistry quantified JAK2 and STAT3 expression. Furthermore, histopathological pancreatic changes were detected by haematoxylin and eosin staining. AR42J cells were randomly divided into the control, cerulein and rhein groups. Amylase activity was assessed using an amylase test kit; the tumour necrosis factor-α (TNF-α) expression was determined by enzyme-linked immunosorbent assay (ELISA). JAK2 and STAT3 protein expression were evaluated by western blot. SAP was concomitant with increased JAK2 and STAT3 expressions in vivo. Pre-treatment with rhein attenuated serum TNF–α and IL-6 levels effectively, and notably reduced p-JAK2, p-STAT3, JAK2 and STAT3 protein expression. Rhein significantly alleviated pancreatic histopathology. Compared to untreated groups, rhein significantly reduced amylase activity in supernatants of AR42J cells induced by cerulein in vitro. Furthermore, rhein altered JAK2 and STAT3 protein levels in AR42J cells after cerulein induction. Overall, rhein exerted protective effect on SAP in vitro and in vivo, possibly through the JAK2/STAT3 signalling pathway.

The Food Additive β-Caryophyllene Exerts Its Neuroprotective Effects Through the JAK2-STAT3-BACE1 Pathway.

Despite extensive research on Alzheimer’s disease (AD), its diagnosis and treatment remain challenging, and no effective therapies are currently available. Amyloid β (Aβ) extracellular plaques and intracellular neurofibrillary tangles are the histological characteristics of AD that have been directly linked to neuropathological events such as synaptic and neuronal cell loss. In this study, we explored whether the “JAK2-STAT3-BACE1” pathway is involved in neuroprotection conferred by the food flavouring agent β-caryophyllene (BCP). PC-12 cells with overexpressed amyloid-β protein precursor (APP) were utilised to construct an AD model in vitro, which was then split into four groups, namely control, empty vector, APP overexpression, and BCP (5, 10, and 20 μM). CCK-8 was used to evaluate cell viability, immunofluorescence was utilised to examine synaptic morphology, and quantitative real-time polymerase chain reaction and western blot were used to examine gene and protein expression levels. The relative expression levels of JAK2, STAT3, and BACE1 mRNA in the transfected PC-12 cells were found to be significantly upregulated. The cell morphology altered dramatically 72 h after transfection, becoming rounder, with a decrease in cell number. BCP exhibited the potential to dramatically increase PC-12 cell viability while protecting cell morphology. BCP inhibited APP, JAK2, STAT3, BACE1 mRNA and BACE1 protein overexpression, as well as JAK2 and STAT3 hyperphosphorylation. Molecular docking simulated the docking of BCP with JAK2, STAT3, BACE1, CB2. And JAK2 was found to be the most stable protein. In conclusion, inhibition of the “JAK2-STAT3-BACE1” signalling pathway may be one of the mechanisms through which BCP protects neurons and antagonises Aβ’s neurotoxicity.

JAK2-Mediated Phosphorylation of Stress-Induced Phosphoprotein-1 (STIP1) in Human Cells

Stress-induced phosphoprotein-1 (STIP1)—a heat shock protein (HSP)70/HSP90 adaptor protein—is commonly overexpressed in malignant cells, where it controls proliferation via multiple signaling pathways, including JAK2/STAT3. We have previously shown that STIP1 stabilizes the protein tyrosine kinase JAK2 in cancer cells via HSP90 binding. In this study, we demonstrate that STIP1 may act as a substrate for JAK2 and that phosphorylation of tyrosine residues 134 and 152 promoted STIP1 protein stability, induced its nuclear-cytoplasmic shuttling, and promoted its secretion into the extracellular space. We also found that JAK2-mediated STIP1 phosphorylation enhanced cell viability and increased resistance to cisplatin-induced cell death. Conversely, interference STIP1 with JAK2 interaction—attained either through site-directed mutagenesis or the use of cell-penetrating peptides—decreased JAK2 protein levels, ultimately leading to cell death. On analyzing human ovarian cancer specimens, JAK2 and STIP1 expression levels were found to be positively correlated with each other. Collectively, these results indicate that JAK2-mediated phosphorylation of STIP-1 is critical for sustaining the JAK2/STAT3 signaling pathway in cancer cells.

Tyrphostin AG1024 Suppresses Coronaviral Replication by Downregulating JAK1 via an IR/IGF-1R Independent Proteolysis Mediated by Ndfip1/2_NEDD4-like E3 Ligase Itch.

JAK1 depletion or downregulation was previously reported to account for coronavirus inhibition. Here, we found that AG1024, an IR (insulin receptor) and IGF-1R (insulin-like growth factor 1 receptor) inhibitor, diminishes JAK1 protein levels and exerts anti-coronaviral activities with EC50 values of 5.2 ± 0.3 μM against transmissible gastroenteritis coronavirus (TGEV) and 4.3 ± 0.3 μM against human flu coronavirus OC43. However, although the IR and IGF-1R signaling pathways are activated by insulin or IGF-1 in swine testis cells, they are not triggered upon TGEV infection. AG1024, therefore, inhibits coronaviral replication and downregulates JAK1 protein levels independently of IR and IGF-1R. Moreover, JAK1 proteolysis caused by AG1024 was found through activation of upstream Ndfip1/2 and its effector NEDD4-like E3 ligase Itch. In addition, ouabain, which was reported to mediate JAK1 proteolysis causing anti-coronaviral activity by activation of Ndfip1/2 and NEDD4 E3 ligase, additively inhibited anti-coronaviral activity and JAK1 diminishment in combination with AG1024. This study provides novel insights into the pharmacological effects of AG1024 and Itch E3 ligase mediated JAK1 proteolysis and identified Ndfip1/2 as a cognate effector for JAK1 proteolysis via the diversified E3 ligases NEDD4 and NEDD4-like Itch. These findings are expected to provide valued information for the future development of anti-viral agents.

Inhibition of adult T-cell leukemia cell proliferation by polymerized proanthocyanidin from blueberry leaves through JAK proteolysis.

We have previously reported that the proanthocyanidin (PAC) fraction of blueberry leaf extract (BB‐PAC) inhibits the proliferation of HTLV‐1‐infected adult T‐cell leukemia (ATL) by inducing apoptosis. In the present study, we further analyzed the structure of BB‐PAC and elucidated the molecular mechanism underlying the inhibitory function of HTLV‐1‐infected and ATL cells. After hot water extraction with fractionation with methanol‐acetone, BB‐PAC was found to be concentrated in fractions 4 to 7 (Fr7). The strongest inhibition of ATL cell growth was observed with Fr7, which contained the highest BB‐PAC polymerization degree of 14. The basic structure of BB‐PAC is mainly B‐type bonds, with A‐type bonds (7.1%) and cinchonain I units as the terminal unit (6.1%). The molecular mechanism of cytotoxicity observed around Fr7 against ATL cells was the degradation of JAK1 to 3 and the dephosphorylation of STAT3/5, which occurs by proteasome‐dependent proteolysis, confirming that PAC directly binds to heat shock protein 90 (HSP90). JAK degradation was caused by proteasome‐dependent proteolysis, and we identified the direct binding of PAC to HSP90. In addition, the binding of cochaperone ATPase homolog 1 (AHA1) to HSP90, which is required for activation of the cofactor HSP90, was inhibited by BB‐PAC treatment. Therefore, BB‐PAC inhibited the formation of the HSP90/AHA1 complex and promoted the degradation of JAK protein due to HSP90 dysfunction. These results suggest that the highly polymerized PAC component from blueberry leaves has great potential as a preventive and therapeutic agent against HTLV‐1‐infected and ATL cells.

P043 3-oxo-C12:2, a Quorum Sensing molecule from the gut, exerts anti-inflammatory effects through a bitter taste receptor

Abstract Background Acyl-Homoserine Lactones (AHLs) are Quorum Sensing molecules involved in the communication network of bacteria and can also have an impact on the host’s cells. We recently showed, in the human gut ecosystem, the presence of AHLs. Among them, we identified one that has never been described: 3-oxo-C12:2. This molecule was decreased in Inflammatory Bowel Disease (IBD) patients, and its presence was correlated to normobiosis. Interestingly, 3-oxo-C12:2 is structurally close to an AHL well described and synthesized by P. aeruginosa, 3-oxo-C12. We intent to describe 3-oxo-C12:2 effects on gut inflammation and to identified which signalling pathways are involved. Given its analogous structure to 3-oxo-C12, we hypothesized that 3-oxo-C12:2 can interact with the same cellular partners, in particular a bitter taste receptor (BTR), called T2R138, which is a GPCR expressed by immune and epithelial gut cells. Methods To test our hypothesis, we used murine macrophages cell line RAW264.7, stimulated by interferon-γ (IFN-γ, 20U/mL) and lipopolysaccharide (LPS, 10ng/mL). Inflammatory response was monitored by measuring cytokine secretion via ELISA We performed a transcriptome analysis to identify inflammatory pathways involved in the effects and analyse pathways by capillary Western blot. Probenecid, a known allosteric inhibitor for T2R138, was used to study T2R138 role in AHL signalling. BTR screening assay was performed to extend search for 3-oxo-C12:2 receptors. Results After LPS/IFN-γ activation, we observed a decrease of secreted TNFα when cells were exposed to 3-oxo-C12:2, in a dose dependent manner: 15μM (-30%, p&amp;lt;0.05), 25μM (-50%, p&amp;lt;0.001) et 50μM (-65%, p&amp;lt;0.0001) while no change were observed in steady state. This reflects an anti-inflammatory effect, in absence of cytotoxicity. By transcriptomic analysis, we identified the JAK-STAT pathway as differentially down-regulated. Exposing cells to 3-oxo-C12:2 prevented JAK1 and STAT1 protein phosphorylation. In addition, the observed anti-inflammatory effects were lost in presence of Probenecid, a T2R138 inhibitor. In a BTR screening assay, we confirmed that 3-oxo-C12:2 activates T2R38, but also five other BTR (T2R13, T2R8, T2R14, T2R1, T2R10). Conclusion 3-oxo-C12:2 exerts a dose dependent anti-inflammatory effect on murine immune cells by preventing the activation of the JAK-STAT pathway. This response is partly mediated by the bitter taste receptor T2R138. This receptor is a potential target of our AHL of interest. Studying the signalling between the receptor and the anti-inflammatory response would allow us to better understand the inter-kingdom dialogue between microbiota involving AHL in IBD.

Immunohistochemical Expression Patterns of CD45RO, p105/p50, JAK3, TOX, and IL-17 in Early-Stage Mycosis Fungoides.

The morphologic changes in early-stage mycosis fungoides (MF) might overlap with benign inflammatory dermatitis (BID). Previous studies have described altered expression patterns of several proteins in MF, but their diagnostic significance is uncertain. This study aims at examining the frequency of expression of CD45RO, NFkB-p105/p50, JAK3, TOX, and IL-17 proteins by immunohistochemistry. The cohorts included 21 patients of early-stage MF and 19 with benign BID as a control group. CD45RO was positive in all patients of MF and BID. NFkB-p105/p50 showed normal cytoplasmic staining, indicating an inactive status in all patients of both groups. JAK3 was positive in 3 (14%) MF and in 17 (89%) BID patients (p = 0.003). TOX was expressed in 19 (90%) and 13 (68%) patients of MF and BID, respectively (p = 0.120). IL-17 was detected in 13 (62%) MF and in 7 (37%) BID patients (p = 0.056). Co-expression of TOX and IL-17 was seen in 11 (52%) MF patients but in only 3 (16%) BID patients, which was statistically significant (p = 0.021). We conclude that a double expression of TOX and IL-17 may support the diagnosis of MF in the right clinicopathologic setting, while none of the immunohistochemical stains alone provided a significant discrimination between MF and BID.

Study on the mechanism of rhynchophylline in improving obese hypertension through JAK2 / STAT3 pathway

Objective The paper studied the effects of Rhynchophylline on the leptin-mediated JAK2/STAT3 pathway in hypothalamus. Using the leptin / leptin receptor and leptin-mediated JAK2/STAT3 pathway as an entry point, the research evaluated the effect of the hypotensive drug, and clarified the preliminary mechanism, expecting to provide new therapeutic ideas for essentially improving obese hypertension, balancing the body's neuro-endocrine function, and preventing the occurrence of cardiovascular diseases. Methods: A model of obese hypertensive rats was established, and the serum lipids, Lep, AngII, NPY and NO contents in each group of rats were measured. Immunohistochemical staining was used to observe the distribution of leptin receptor (LepR) and angiotensin II type 1 receptor (AT1R) in hypothalamus and aorta of rats. Western Blot method was employed to detect JAK2, STAT3, SOCS3, LepR and AT1R protein expressions in the hypothalamus of rats. After the third ventricle injection of AG490, Western blot was applied to detect the expressions of various proteins. Results: Rhynchophylline can significantly reduce AngII, Lep, and NPY levels in serum circulation. Immunohistochemistry showed that the hypothalamus and aorta AT1R distribution was significantly reduced, the hypothalamic LepR distribution increased, and the aortic LepR distribution decreased. The expressions of JAK2, LepR, and STAT3 proteins in the hypothalamus increased significantly, and the expressions of SOCS3 and AT1R proteins significantly decreased. After injection of AG490 in the third ventricle of Rhynchophylline rats, the expressions of JAK2 and STAT3 in the hypothalamus decreased significantly, while the SOCS3expression increased remarkably. Conclusion: Rhynchophylline can reduce blood lipid, as well as serum AngII, Lep, and NPY levels, increase serum NO levels in obese hypertension rats. Rhynchophylline can effectively inhibit the expression of SOCS3 by activating the JAK2/STAT3 pathway in the hypothalamus. Therefore, it is speculated that the JAK2/STAT3 signaling pathway is the main pathway through which Rhynchophylline plays a role in improving selective leptin resistance and reducing blood pressure and weight.

Minimally modified low-density lipoprotein upregulates mouse mesenteric arterial 5-HT1B receptor in vivo via activation of the JAK2/STAT3 pathway.

To explore whether minimally modified low-density lipoprotein (mmLDL) upregulates mesenteric arterial 5-hydroxytryptamine 1B (5-HT1B) receptor expression by activating the JAK2/STAT3 signaling pathway. Mice were randomly divided into the following groups: the normal saline (NS), LDL, mmLDL, mmLDL+galiellactone (GL, a JAK2/STAT3 pathway inhibitor), and mmLDL+DMSO groups. The dose-response curve of mesenteric arterial ring constriction after administration of 5-carboxamidotryptamine (5-CT), an agonist of 5-HT1B, was recorded with a microvascular tensiometer. JAK2, p-JAK2, STAT3, p-STAT3, and 5-HT1B receptor protein expression levels were determined by Western blotting. 5-HT1B receptor mRNA levels were measured by RT-PCR. 5-HT1B receptor protein expression was determined by immunofluorescence. Injection of mmLDL into the tail vein significantly increased the contractile dose-response curve after 5-CT stimulation, as the Emax was 82.15 ± 6.15% in the NS group and 171.88 ± 5.78% in the mmLDL group (P < 0.01); significantly elevated 5-HT1B receptor mRNA and protein expression levels; and significantly increased p-JAK2 and p-STAT3 protein expression levels. After intraperitoneal injection of GL, the vasoconstrictive response was significantly reduced compared with that in the mmLDL group, as the Emax was decreased to 97.14 ± 1.20% (P < 0.01); 5-HT1B receptor mRNA and protein expression levels were significantly reduced; STAT3 phosphorylation and p-JAK2 and p-STAT3 protein expression were not significantly changed; and 5-HT1B receptor expression was altered via inhibition of p-STAT3 binding to DNA, which suppressed transcription. mmLDL can upregulate 5-HT1B receptor expression in mouse mesenteric arteries by activating the JAK2/STAT3 signaling pathway.

Ginkgolic Acid (GA) Inhibits the Growth of OCa by Inhibiting lncRNA MALAT1/JAK2 Axis

Objective We aimed to observe the impact of ginkgolic acid (GA) on the proliferation and metastasis ability of ovarian cancer (OCa) cells and to further explore whether GA affects the malignant progress of OCa via regulating the lncRNA MALAT1/JAK2 axis. Methods OCa cells SKOV3 and CAOV3 were administered with 1 ng/ml GA, 5 ng/ml GA, 10 ng/ml GA, 20 ng/ml GA, and DSMO as control, respectively. The cell proliferation and migration ability of the abovementioned cells in each group were measured by CCK-8 test and Transwell experiments. The expression levels of lncRNA MALAT1 and JAK2 protein were examined by qRT-PCR and western blot, respectively. Subsequently, in OCa cells treated with GA, lncRNA MALAT1 overexpression vector was transfected to continue to detect the proliferation activity and migration ability of each treatment group. Finally, the regulation of GA on activity of lncRNA MALAT1/JAK2 axis in OCa cells was further explored in nude mice. Results Our data showed that the proliferation inhibition rate of cells at each ginkgolic acid concentration was higher than that of the control group (P < 0.05), suggesting that GA has an inhibitory influence on the proliferation of OCa cells, in a dose-dependent way. GA was able to inhibit the proliferation rate and migration ability of OCa cells. Administration of ginkgolic acid downregulated the levels of lncRNA MALAT1 and JAK2 protein. Overexpression of lncRNA MALAT1 partially reversed the inhibited OCa proliferative capacity caused by GA treatment. Consistent with the results observed in vitro, we also found that the OCa tumor weight and volume of nude mice injected with lncRNA MALAT1 overexpression vector were enhanced and JAK2 protein level increased remarkably in comparison to the ginkgolic acid group. Conclusions In summary, GA may exert its inhibitory effect on the proliferative and migratory capacities of OCa cells through suppressing the activity of lncRNA MALAT1/JAK2 axis.

Thymoquinone Induces Downregulation of BCR-ABL/JAK/STAT Pathway and Apoptosis in K562 Leukemia Cells.

Objective:BCR ABL oncogene encodes the BCR-ABL chimeric protein, which is a constitutively activated non-receptor tyrosine kinase. The BCR-ABL oncoprotein is a key molecular basis for the pathogenesis of chronic myeloid leukemia (CML) via activation of several downstream signaling pathways including JAK/STAT pathway. Development of leukemia involves constitutive activation of signaling molecules including, JAK2, STAT3, STAT5A and STAT5B. Thymoquinone (TQ) is a bioactive constituent of Nigella sativa that has shown anticancer properties in various cancers. The present study aimed to evaluate the effect of TQ on the expression of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes and their consequences on the cell proliferation and apoptosis in K562 CML cells. Methods:BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis. Results:TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001). Conclusion:our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.

Isolation and identification of the components in Cybister chinensis Motschulsky against inflammation and their mechanisms of action based on network pharmacology and molecular docking

Cybister chinensis Motschulsky belongs to the family Dytiscidae. As a traditional Chinese medicine, the insect is called Longshi in the folk and is commonly used to treat enuresis in children and frequent urination in the elderly. Inflammation is involved in chronic kidney disease. The previous study proved ethanol extract of C. chinensis exhibited anti-inflammation effects in the Doxorubicin-induced kidney disease. However, the material basis and their possible mechanism of the insect were still unclear. Thus, we aimed to separate the active compounds of the ethanol extract from C. chinensis and to investigate their possible mechanism of anti-inflammation by network pharmacology and molecular docking. The insect was extracted with 75% ethanol to produce ethanol extracts and then were extracted by petroleum ether, ethyl acetate and n-butanol respectively. Silica gel column chromatography and preparative HPLC were applied to separate the compounds of the extract. The compounds were characterized and identified by NMR and mass. The compound associated genes were collected by BATMAN-TCM database and the inflammation associated genes were obtained through DigSee database. The protein-protein interaction (PPI) network was carried out via Search Tool for the Retrieval of Interacting Genes/Protein (STRING) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) target pathway analysis was performed in Database for Annotation, Visualization and Integrated Discovery (DAVID). The possible mechanism of compounds against inflammation was investigated by molecular docking. Finally, the anti-inflammatory effect of the representative compound was verified by the LPS-induced Raw 264.7cell inflammatory model. TNF-α, IL-1β and IL-6 of the cell supernatants were analyzed via using ELISA kits and the key proteins in JAK2/STAT3 signaling pathway were verified via the Western blot assays. Among crude extracts from C. chinensis, ethyl acetate extract showed the obvious anti-inflammatory effects. Nine compounds were isolated from ethyl acetate extract of Cybister chinensis for the first time, including benzoic acid (1), hydroxytyrosol (2), protocatechualdehyde (3), N-[2-(4-hydroxyphenyl)ethyl]acetamide (4), (2E)-3-phenylprop-2-enoic acid (5), 3-phenylpropionic acid (6), methyl 3,4-dihydroxybenzoate (7), 1,4-diphenyl butane-2,3-diol (8) and p-N,N-dimethylaminobenzaldehyde (9). After searching in the database, 1079 compound associated genes and 467 inflammation associated genes were found. The 137 common targets covered 77 signaling pathways, in which HIF-1 signaling pathway, TNF signaling pathway, influenza A, PI3K/Akt signaling pathway, NOD-like receptor signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway and Jak-STAT signaling pathway were important for inflammation. Molecular docking studies showed compound 1, 4, 5, 6, 7 and 8 were the potential inhibitors of JAK2 protein. In addition, the in vitro test showed compound 5 reduced the expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in lipopolysaccharide (LPS)-stimulated RAW264.7cells in a dose-dependent manner. Furthermore, it was found that compound 5 inhibited the expression of p-JAK2 and p-STAT3 in LPS-induced RAW264.7cells in a dose-dependent manner. Based on the network pharmacology and molecular docking, the study suggested that C. chinensis could relieve the inflammation based on the multi-compounds and multi-pathways, which provided the foundation for the medicinal application of C. chinensis.

Nifuroxazide Mitigates Angiogenesis in Ehlrich's Solid Carcinoma: Molecular Docking, Bioinformatic and Experimental Studies on Inhibition of Il-6/Jak2/Stat3 Signaling.

Nifuroxazide is an antidiarrheal medication that has promising anticancer activity against diverse types of tumors. The present study tested the anticancer activity of nifuroxazide against Ehrlich’s mammary carcinoma grown in vivo. Furthermore, we investigated the effect of nifuroxazide on IL-6/jak2/STAT3 signaling and the possible impact on tumor angiogenesis. The biological study was supported by molecular docking and bioinformatic predictions for the possible effect of nifuroxazide on this signaling pathway. Female albino mice were injected with Ehrlich carcinoma cells to produce Ehrlich’s solid tumors (ESTs). The experimental groups were as follows: EST control, EST + nifuroxazide (5 mg/kg), and EST + nifuroxazide (10 mg/kg). Nifuroxazide was found to reduce tumor masses (730.83 ± 73.19 and 381.42 ± 109.69 mg vs. 1099.5 ± 310.83) and lessen tumor pathologies. Furthermore, nifuroxazide downregulated IL-6, TNF-α, NFk-β, angiostatin, and Jak2 proteins, and it also reduced tumoral VEGF, as indicated by ELISA and immunohistochemical analysis. Furthermore, nifuroxazide dose-dependently downregulated STAT3 phosphorylation (60% and 30% reductions, respectively). Collectively, the current experiment shed light on the antitumor activity of nifuroxazide against mammary solid carcinoma grown in vivo. The antitumor activity was at least partly mediated by inhibition of IL-6/Jak2/STAT3 signaling that affected angiogenesis (low VEGF and high angiostatin) in the EST. Therefore, nifuroxazide might be a promising antitumor medication if appropriate human studies will be conducted.

JAK2/STAT3‑mediated dose‑dependent cytostatic and cytotoxic effects of sesquiterpene lactones from Gymnanthemum extensum on A549 human lung carcinoma cells.

Due to drug resistance and disease recurrence, lung cancer remains one of the primary cancer-related causes of death in both men and women worldwide. In addition, lung cancer is clinically silent and thus most patients are at an advanced stage at the time of diagnosis. The limited efficiency of current conventional chemotherapies necessitates the search for novel effective anticancer agents. The present study demonstrated the anti-proliferative effect and apoptosis-inducing activity of three sesquiterpene lactones isolated from Gymnanthemum extensum, vernodalin (VDa), vernolepin (VLe) and vernolide (VLi), on A549 human lung cancer cells. Treatment with sub-cytotoxic doses (cell viability remaining >75%) of VDa, VLe and VLi, arrested progression of the A549 cell cycle at the G0/G1 phase, while cytotoxic doses of the three compounds induced G2/M phase arrest and apoptosis. Mechanistic studies revealed that VDa, VLe and VLi may exert their anti-tumor activity through the JAK2/STAT3 pathway. Molecular docking analysis confirmed that VDa, VLe and VLi formed hydrogen bonds with the FERM domain of JAK2 protein. Overall, the present study highlighted the potential therapeutic value of VDa, VLe and VLi to be further developed as anticancer agents for the treatment of lung cancer.

Effect of combination therapy with neural stem cell transplantation and teramethylpyrazine in rats following acute spinal cord injury.

This study was to explore the effects of teramethylpyrazine (TMP) administered in conjunction with neural stem cell transplantation on motor function, pathological lesions and the Janus kinase (JAK)2/signal transducer and activator of transcription 3 signal transduction pathway in rats following acute spinal cord injury (SCI). Female Sprague-Dawley rats were randomly divided into sham, model, neural stem cells (NSCs) and NSCs+TMP groups. Motor function was evaluated using the Basso, Beattie, Bresnahan scale. Spinal cord neuropathies and neuron apoptosis were observed by HE and TUNEL staining. The brain-derived neurotrophic factor (BDNF), Nogo-A, JAK2 and p-JAK2 protein levels were measured by western blot analysis. NSCs+TMP significantly improved rat motor function, attenuated impaired spinal cords, and decreased cellular apoptosis, compared with NSCs therapy alone (P < 0.05). In addition, expression of BDNF protein was significantly higher in NSCs+TMP rats compared with other groups regardless of time postinjury (P < 0.05). The highest expression levels of Nogo-A protein were observed in the model group. The expression of p-JAK2 in the NSCs+TMP group was relatively lower than the model and NSCs groups (P < 0.05). In rats with SCI, NSCs+TMP effectively improved motor function and offered spinal cord protection by increasing BDNF and decreasing Nogo-A levels, as well as inhibiting the JAK2/STAT3 signal transduction pathway, suggesting that TMP could be a useful agent in NSCs transplantation in the treatment of SCI.