Phosphorylation Of JAK2 Research Articles

Pectolinarigenin alleviated septic acute kidney injury via inhibiting Jak2/Stat3 signaling and mitochondria dysfunction

Sepsis is a systemic inflammatory response to infection, where sepsis-associated acute kidney injury (AKI) is a common morbid disease with a high morbidity and mortality, and however at present no effective therapy exists. Increasing evidence have shown that mitochondrial damage and inflammatory response are important initiating factors in pathogenesis of septic AKI. Natural flavonoid pectolinarigenin exerted anti-inflammatory properties in previous studies, while its role in septic AKI remains unknown. In the study, pectolinarigenin administration significantly ameliorated the dramatic rise of serum creatinine and blood urea nitrogen in lipopolysaccharide (LPS)- and cecal ligation/puncture (CLP)-induced septic mice, respectively. Consistently, LPS/CLP-induced renal damage as implied by histopathological score and the increased injury markers NGAL and KIM-1, which was attenuated by pectolinarigenin. Meanwhile, LPS/CLP triggered proinflammatory cytokine production and inflammation related proteins in the kidneys. However, pectolinarigenin inhibited renal expression of IL-6, IL-1β, TNF-α, and MCP-1 to improve inflammatory response. Furthermore, pectolinarigenin upregulated Bcl-2 protein expression and suppressed apoptotic protein of BAX and cleaved caspase-3 in the kidneys of CLP-induced septic AKI. Mechanistically, LPS could induce the high expression of IL-6 and trigger the phosphorylation of Jak2 and Stat3, while pectolinarigenin remarkably reduced their corresponding levels. Notably, CLP-induced kidney injury of mice significantly reduced the expression of PGC-1α, OPA1 and increased the expression of Drp1, Cyt-C, where pectolinarigenin pretreatment significantly restored their corresponding expression in mice. In summary, pectolinarigenin improved septic AKI via inhibiting JAK2/STAT3 signaling and mitochondria dysfunction.

Luteolin-rich fraction from Perilla frutescens seed meal inhibits spike glycoprotein S1 of SARS-CoV-2-induced NLRP3 inflammasome lung cell inflammation via regulation of JAK1/STAT3 pathway: A potential anti-inflammatory compound against inflammation-induced long-COVID.

The multi-systemic inflammation as a result of COVID-19 can persevere long after the initial symptoms of the illness have subsided. These effects are referred to as Long-COVID. Our research focused on the contribution of the Spike protein S1 subunit of SARS-CoV-2 (Spike S1) on the lung inflammation mediated by NLRP3 inflammasome machinery and the cytokine releases, interleukin 6 (IL-6), IL-1beta, and IL-18, in lung epithelial cells. This study has attempted to identify the naturally- occurring agents that act against inflammation-related long-COVID. The seed meal of Perilla frutescens (P. frutescens), which contains two major dietary polyphenols (rosmarinic acid and luteolin), has been reported to exhibit anti-inflammation activities. Therefore, we have established the ethyl acetate fraction of P. frutescens seed meal (PFEA) and determined its anti-inflammatory effects on Spike S1 exposure in A549 lung cells. PFEA was established using solvent-partitioned extraction. Rosmarinic acid (Ra) and luteolin (Lu) in PFEA were identified using the HPLC technique. The inhibitory effects of PFEA and its active compounds against Spike S1-induced inflammatory response in A549 cells were determined by RT-PCR and ELISA. The mechanistic study of anti-inflammatory properties of PFEA and Lu were determined using western blot technique. PFEA was found to contain Ra (388.70 ± 11.12 mg/g extract) and Lu (248.82 ± 12.34 mg/g extract) as its major polyphenols. Accordingly, A549 lung cells were pre-treated with PFEA (12.5-100 μg/mL) and its two major compounds (2.5-20 μg/mL) prior to the Spike S1 exposure at 100 ng/mL. PFEA dose-dependently exhibited anti-inflammatory properties upon Spike S1-exposed A549 cells through IL-6, IL-1β, IL-18, and NLRP3 gene suppressions, as well as IL-6, IL-1β, and IL-18 cytokine releases with statistical significance (p < 0.05). Importantly, Lu possesses superior anti-inflammatory properties when compared with Ra (p < 0.01). Mechanistically, PFEA and Lu effectively attenuated a Spike S1-induced inflammatory response through downregulation of the JAK1/STAT3-inflammasome-dependent inflammatory pathway as evidenced by the downregulation of NLRP3, ASC, and cleaved-caspase-1 of the NLRP3 inflammasome components and by modulating the phosphorylation of JAK1 and STAT3 proteins (p < 0.05). The findings suggested that luteolin and PFEA can modulate the signaling cascades that regulate Spike S1-induced lung inflammation during the incidence of Long-COVID. Consequently, luteolin and P. frutescens may be introduced as potential candidates in the preventive therapeutic strategy for inflammation-related post-acute sequelae of COVID-19.

Effect of lipoxin A4 methyl ester from arachidonic acid on JAK2/STAT3 pathway after cerebral ischemia-reperfusion injury

It was determined if lipoxin A4 methyl ester (LXA4 ME) affects JAK2/STAT3 pathway following transient focal cerebral ischemia-reperfusion injury (CIRI) in rats. Adult male SD rats were randomly assigned to sham-operated, CIRI group, DMSO + CIRI, AG490 + CIRI, and LXA4 ME + CIRI. Neurologic deficit was evaluated in rats after 24 h reperfusion. IL-6 levels of brain tissue were determined 6 and 24 h post-reperfusion using an ELISA assay. STAT3 and SOCS3 mRNA levels were analyzed via real-time quantitative PCR. P-JAK2, p-STAT3, and SOCS3 were measured via Western blotting, and localized by fluorescent immunohitochemistry. Transient cerebral ischemia in rats caused a significantly elevated concentration of IL-6, levels of STAT3 and SOCS3 mRNA, phosphorylation of JAK2 and STAT3, and immunoreactive glia of ipsilateral cortex 24 h post-reperfusion versus sham rats. The AG490 intervention downregulated cerebral ischemia-induced JAK2 and STAT3 phosphorylation, and their immunoreactive glia located in the ischemic cortex. LXA4 ME reduced the concentration of IL-6, increased SOCS3 expression, inhibited phosphorylation and immunoreactive glia of JAK2 and STAT3, as well as ameliorated neurologic dysfunction. In conclusions, LXA4 ME inhibited JAK2/STAT3 activation, perhaps via upregulating SOCS3, as well as suppressed IL-6 expression to ameliorate cerebral ischemic injury.

Mung bean peptides promote glucose uptake via Jak2 activation in L6 myotubes.

Mung beans are among the important edible legumes cultivated in Asia, Southern Europe, and Northern America. Mung beans contain 20-30% proteins with high digestibility and possess biological activities, but detailed health beneficial functions are not fully understood yet. In this study, we report the isolation and identification of active peptides from mung beans which promote glucose uptake and elucidate their mechanism in L6 myotubes. HTL, FLSSTEAQQSY, and TLVNPDGRDSY were isolated and identified as active peptides. These peptides promoted the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The tripeptide HTL promoted glucose uptake through the activation of adenosine monophosphate-activated protein kinase, while the oligopeptides FLSSTEAQQSY and TLVNPDGRDSY through the activation of the PI3K/Akt pathway. Furthermore, these peptides promoted the phosphorylation of Jak2 via interaction with the leptin receptor. Thus, mung bean is a promising functional food for the prevention of hyperglycemia and type 2 diabetes through promoting glucose uptake accompanied by JAK2 activation in the muscle cells.

Lotus Bee Pollen Extract Inhibits Isoproterenol-Induced Hypertrophy via JAK2/STAT3 Signaling Pathway in Rat H9c2 Cells.

Bee pollen possesses an anti-cardiomyocyte injury effect by reducing oxidative stress levels and inhibiting inflammatory response and apoptosis, but the possible effect mechanism has rarely been reported. This paper explores the effect of the extract of lotus bee pollen (LBPE) on cardiomyocyte hypertrophy (CH) and its mechanism. The main components of LBPE were identified via UPLC-QTOF MS. An isoproterenol-induced rat H9c2 CH model was subsequently used to evaluate the protection of LBPE on cells. LBPE (100, 250 and 500 μg∙mL-1) reduced the surface area, total protein content and MDA content, and increased SOD activity and GSH content in CH model in a dose-dependent manner. Meanwhile, quantitative real-time PCR trials confirmed that LBPE reduced the gene expression levels of CH markers, pro-inflammatory cytokines and pro-apoptosis factors, and increased the Bcl-2 mRNA expression and Bcl-2/Bax ratio in a dose-dependent manner. Furthermore, target fishing, bioinformatics analysis and molecular docking suggested JAK2 could be a pivotal target protein for the main active ingredients in the LBPE against CH. Ultimately, Western blot (WB) trials confirmed that LBPE can dose-dependently inhibit the phosphorylation of JAK2 and STAT3. The results show that LBPE can protect against ISO-induced CH, possibly via targeting the JAK2/STAT3 pathway, also suggesting that LBPE may be a promising candidate against CH.

HDAC6 regulates human erythroid differentiation through modulation of JAK2 signalling

Among histone deacetylases, HDAC6 is unusual in its cytoplasmic localization. Its inhibition leads to hyperacetylation of non-histone proteins, inhibiting cell cycle, proliferation and apoptosis. Ricolinostat (ACY-1215) is a selective inhibitor of the histone deacetylase HDAC6 with proven efficacy in the treatment of malignant diseases, but anaemia is one of the most frequent side effects. We investigated here the underlying mechanisms of this erythroid toxicity. We first confirmed that HDAC6 was strongly expressed at both RNA and protein levels in CD34+ -cells-derived erythroid progenitors. ACY-1215 exposure on CD34+ -cells driven in vitro towards the erythroid lineage led to a decreased cell count, an increased apoptotic rate and a delayed erythroid differentiation with accumulation of weakly hemoglobinized immature erythroblasts. This was accompanied by drastic changes in the transcriptomic profile of primary cells as shown by RNAseq. In erythroid cells, ACY-1215 and shRNA-mediated HDAC6 knockdown inhibited the EPO-dependent JAK2 phosphorylation. Using acetylome, we identified 14-3-3ζ, known to interact directly with the JAK2 negative regulator LNK, as a potential HDAC6 target in erythroid cells. We confirmed that 14-3-3ζ was hyperacetylated after ACY-1215 exposure, which decreased the 14-3-3ζ/LNK interaction while increased LNK ability to interact with JAK2. Thus, in addition to its previously described role in the enucleation of mouse fetal liver erythroblasts, we identified here a new mechanism of HDAC6-dependent control of erythropoiesis through 14-3-3ζ acetylation level, LNK availability and finally JAK2 activation in response to EPO, which is crucial downstream of EPO-R activation for human erythroid cell survival, proliferation and differentiation.

MST1 controls murine neutrophil homeostasis via the G-CSFR/STAT3 axis.

The release of neutrophils from the bone marrow into the blood circulation is essential for neutrophil homeostasis and the protection of the organism from invading microorganisms. Granulocyte colony-stimulating factor (G-CSF) plays a pivotal role in this process and guides granulopoiesis as well as the release of bone marrow neutrophils into the blood stream both during homeostasis and in case of infection through activation of the G-CSF receptor/signal transduction and activation of transcription 3 (STAT3) signaling pathway. Here, we investigated the role of the mammalian sterile 20-like kinase 1 (MST1) for neutrophil homeostasis and neutrophil mobilization. We found increased plasma levels of G-CSF in Mst1 -/- mice compared to wild type mice both under homeostatic conditions as well as after stimulation with the proinflammatory cytokine TNF-α. In addition, G-CSF-induced mobilization of neutrophils from the bone marrow into the blood circulation in vivo was markedly reduced in the absence of MST1. Interestingly, this was not accompanied by differences in the number of blood neutrophils. Addressing the underlying molecular mechanism of MST1-regulated neutrophil mobilization, we found reduced STAT3 phosphorylation and impaired upregulation of CXCR2 in Mst1 -/- bone marrow neutrophils compared to wild type cells, while JAK2 phosphorylation was not altered. Taken together, we identify MST1 as a critical modulator of neutrophil homeostasis and neutrophil mobilization from the bone marrow, which adds another important aspect to the complex role of MST1 in regulating innate immunity.

An Acute Bout of Exercise Suppresses Appetite via Central Lactate Metabolism

Exercise has been reported to elicit a transient suppression of appetite. Plasma lactate, which is produced by exercising muscle, is believed to have a critical effect on exercise-induced appetite suppression. However, the underlying mechanisms and signaling steps of central lactate metabolism remain unexplored. After central oxamate administration, C57BL/6J male mice performed 10 high-intensity interval running at 90% Vmax for 4 minutes each, which separated by 2 minutes at 12 m/min. Food intake and the expression of hypothalamic appetite-regulating neuropeptides including proopiomelanocortin (POMC) and neuropeptide Y (NPY) were investigated following exercise training. Janus kinase 2 (Jak2)-signal transducer and activator of transcription 3 (STAT3) signaling pathway was also determined by Western blot. In addition, hypoxia-inducible factor-1α (HIF-1α) was investigated to explore the effect of central lactate metabolism following exercise. We found that central oxamate administration reversed exercise-induced suppression of food intake, and as well as changes in the expression of POMC and NPY. Moreover, acute exercise led to an increase in the phosphorylation of Jak2 and STAT3 in the hypothalamus, while central lactate inhibition significantly blunted this effect. In addition, HIF-1α expression increased obviously after exercise, while it was attenuated by central oxamate administration. Collectively, our data reveal that central lactate metabolism mediates exercise-induced suppression of appetite and changes in neuropeptides, possibly through enhanced Jak2-STAT3 signaling.

Molecular Mechanisms of Luteolin Against Atopic Dermatitis Based on Network Pharmacology and in vivo Experimental Validation.

To undercover the underlying mechanisms of luteolin against atopic dermatitis (AD), clinically characterized by recurrenteczematous lesionsandintenseitching, based on network pharmacology, molecular docking and in vivo experimental validation. TCMSP, STITCH and SwissTargetPrediction databases were utilized to screen the corresponding targets of luteolin. Targets related to AD werecollected from DisGeNET, GeneCards and TTD databases. PPI network of intersection targets was constructed through STRING 11.0 database andCytoscape3.9.0 software. GO and KEGG enrichment analysis were performed to investigate the critical pathways of luteolin against AD. Further, the therapeutic effects and candidate targets/signaling pathways predicted from network pharmacology analysis were experimentally validated in a mouse model of AD induced by 2, 4-dinitrofluorobenzene (DNFB). A total of 31 intersection targets were obtained by matching 151 targets of luteolin with 553 targets of AD. Among all, 20 core targets were identified by PPI network topology analysis, including IL-6, TNF, IL-10, VEGFA, IL-4, etc., and molecular dockingindicated that luteolin binds strongly to these core targets. KEGGpathway enrichmentanalysissuggested that the intersected targets were significantly enriched in IL-17 signaling pathway, Th17 cell differentiation, Th1 and Th2 cell differentiation, JAK/STAT signaling pathway, etc. The in vivo experiment validated that luteolin could alleviate AD-like skin symptoms, as evidenced by the lower SCORADscore, the reduced infiltration of mast cells and the recovery of skin barrier function. Furthermore,luteolin restored immune balance by regulating the production ofTh1/Th2/Th17-mediated cytokines, which were both the predicted core targets. Moreover, luteolin inhibited the phosphorylation of JAK2 and STAT3 in the lesional skin. Together, the present study systematically clarifies the ameliorative effects and possible molecular mechanisms of luteolin against AD through the combination of network pharmacology and experimental validation, shedding light on the future developmentand clinical application of luteolin.

Angelica sinensis Polysaccharide and Astragalus membranaceus Polysaccharide Accelerate Liver Regeneration by Enhanced Glycolysis via Activation of JAK2/STAT3/HK2 Pathway.

The promotion of liver regeneration is crucial to avoid liver failure after hepatectomy. Angelica sinensis polysaccharide (ASP) and Astragalus membranaceus polysaccharide (AMP) have been identified as being associated with hepatoprotective effects. However, their roles and specific mechanisms in liver regeneration remain to be elucidated. In the present study, it suggested that the respective use of ASP or AMP strikingly promoted hepatocyte proliferation in vitro with a wide range of concentrations (from 12.5 μg/mL to 3200 μg/mL), and a stronger promoting effect was observed in combined interventions. A significantly enhanced liver/body weight ratio (4.20%) on day 7 and reduced serum transaminase (ALT 243.53 IU/L and AST 423.74 IU/L) and total bilirubin (52.61 IU/L) levels on day 3 were achieved by means of ASP-AMP administration after partial hepatectomy in mice. Metabonomics showed that differential metabolites were enriched in glycolysis with high expression of beta-d-fructose 6-phosphate and lactate, followed by significantly strengthened lactate secretion in the supernatant (0.54) and serum (0.43) normalized to control. Upon ASP-AMP treatment, the knockdown of hexokinase 2 (HK2) or inhibited glycolysis caused by 2-deoxy-d-glucose decreased hepatocyte proliferation in vitro and in vivo. Furthermore, pathway analysis predicted the role of JAK2/STAT3 pathway in ASP-AMP-regulated liver regeneration, and phosphorylation of JAK2 and STAT3 was proven to be elevated in this promoting process. Finally, downregulated expression of HK2, an attenuated level of lactate secretion, and reduced hepatocyte proliferation were displayed when STAT3 was knocked out in vitro. Therefore, it can be concluded that ASP-AMP accelerated liver regeneration and exerted a hepatoprotective effect after hepatectomy, in which the JAK2/STAT3/HK2 pathway was actively involved in activating glycolysis.

Biallelic Deleterious Germline SH2B3 Variants Cause a Novel Clinical Syndrome of Myeloproliferation and Multi-Organ Autoimmunity

SH2B3 is a negative regulator of cytokine receptor signalling pathways in hematopoietic tissue. Acquired deleterious SH2B3 variants resulting in increased JAK/STAT signalling are observed in myeloproliferative neoplasms (MPN) however the clinical implications and phenotype of deleterious germline variants in SH2B3 are less clear. A single consanguineous kindred has been previously described with homozygous deleterious germline SH2B3 variants presenting with hepatosplenomegaly, autoimmune hepatitis, developmental delay, autoimmune thyroiditis and acute lymphoblastic leukemia (Perez-Garcia, A., et al, Blood, 2013), however a clinically valid gene-disease pair according to formal criteria has not been met to date for SH2B3. We identified two probands from unrelated kindred with germline homozygous deleterious SH2B3 variants. The first patient presented with splenomegaly, thrombocytosis, neutrophilia and a leukoerythroblastic blood film at 3 months old. He developed alopecia areata at 2 years and autoimmune hypothyroidism at 6 years. He was otherwise developmentally normal without any detected liver abnormalities or thrombotic episodes. Whilst his splenomegaly and neutrophil count gradually improved throughout life, his platelet count remained markedly elevated (~1000x109/L at 18 years of age). Targeted NGS identified a homozygous frameshift variant in SH2B3 (NM_005475.3:c.441_468del; p.(Arg148Profs*40)). Both parents were heterozygous carriers of this variant. The second patient was a male born to consanguineous parents and noted shortly after birth to have isolated splenomegaly. He was diagnosed with autoimmune hypothyroidism and Raynaud syndrome at 12 years and was noted to have a persistent thrombocytosis ranging from 550 - 780 x109/L. He developed autoimmune hepatitis and insulin dependent diabetes at 17 years and around the same time he also suffered a right-sided middle cerebral artery territory infarct with a right carotid artery thrombus. Whole genome sequencing detected a homozygous missense variant in SH2B3 (NM_005475.3:c.1204G>A; p.(Val402Met)) in the patient with both parents being heterozygous carriers. In order to understand these variants further we modelled the human p.(Arg148Profs*40) variant from patient 1 in zebrafish. CRISPR-Cas9 gene editing was targeted to the analogous region of zebrafish sh2b3 to create assorted carboxyl-truncating mutations in F0 crispants. On-target gene-editing was verified by Sanger sequencing and NGS. F0 crispants had a significantly increased number of macrophages and CD41-expressing cells (HSCs and thrombocytes), replicating the patient phenotype. Treatment of the sh2b3 crispant fish with the JAK inhibitor ruxolitinib intercepted the myeloproliferative phenotype. The myeloproliferative phenotype was not observed in stable deletion mutants, likely reflecting genetic compensation not present in crispants. Skin-derived fibroblasts from patient 2 who carried the Val402Met homozygous variant, showed increased phosphorylation of JAK2, STAT5 and STAT3 by immunoblotting after stimulation with IL3, GH, GM-CSF and EPO compared to healthy controls. In conclusion, these newly described kindreds showing striking phenotypic similarity to each other as well as the previously described kindred and in conjunction with our functional data provide sufficient evidence for biallelic homozygous deleterious variants in SH2B3 to be considered a valid gene-disease association with a clinical syndrome of bone marrow myeloproliferation and multi-organ autoimmune manifestations. Notably, neither of our patients had evidence of acute lymphoblastic leukemia or overt hematological malignancy and further affected individuals will need to be described in order to fully understand the breadth of the phenotype. Moreover, our data supports ruxolitinib as being a potential therapeutic option for patients with this rare novel immune dysregulatory syndrome.

NIK/MAP3K14 in hepatocytes orchestrates NASH to hepatocellular carcinoma progression via JAK2/STAT5 inhibition

ObjectiveNonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis (NASH), which often progresses to hepatocellular carcinoma (HCC) through a largely undefined mechanism. NASH and HCC depend on inflammatory signaling, whose master regulator is the NFκB transcription factor family, activated by canonical and non-canonical pathways. MethodsHere, we investigated non-canonical NFκB-inducing kinase (NIK/MAP3K14) in metabolic NASH, NASH to HCC transition, and DEN-induced HCC. To this end, we performed dietary and chemical interventions in mice that were analyzed via single nucleus sequencing, gene expression and histochemical methods. Ultimately, we verified our mouse results in human patient samples. ResultsWe revealed that hepatocyte-specific NIK deficiency (NIKLKO) ameliorated metabolic NASH complications and reduced hepatocarcinogenesis, independent of its role in the NFκB pathway. Instead, hepatic NIK attenuated hepatoprotective JAK2/STAT5 signaling that is a prerequisite for NASH and NASH to HCC progression in mice and humans. ConclusionsOur data suggest NIK-mediated inhibitory JAK2 phosphorylation at serine 633 that might be amenable for future therapeutic interventions in patients.

Hydroxyzine Induces Cell Death in Triple-Negative Breast Cancer Cells via Mitochondrial Superoxide and Modulation of Jak2/STAT3 Signaling.

Treatment of triple-negative breast cancer (TNBC) has been limited due to the lack of molecular targets. In this study, we evaluated the cytotoxicity of hydroxyzine, a histamine H1 receptor antagonist in human triple-negative breast cancer BT-20 and HCC-70 cells. Hydroxyzine inhibited the growth of cells in dose- and time-dependent manners. The annexin V/propidium iodide double staining assay showed that hydroxyzine induced apoptosis. The hydroxyzine-induced apoptosis was accompanied down-regulation of cyclins and CDKs, as well as the generation of reactive oxygen species (ROS) without cell cycle arrest. The effect of hydroxyzine on the induction of ROS and apoptosis on TNBC cells was prevented by pre-treatment with ROS scavengers, N-acetyl cysteine or Mito-TEMPO, a mitochondria-targeted antioxidant, indicating that an increase in the generation of ROS mediated the apoptosis induced by hydroxyzine. Western blot analysis showed that hydroxyzine-induced apoptosis was through down-regulation of the phosphorylation of JAK2 and STAT3 by hydroxyzine treatment. In addition, hydroxyzine induced the phosphorylation of JNK and p38 MAPK. Our results indicate that hydroxyzine induced apoptosis via mitochondrial superoxide generation and the suppression of JAK2/STAT3 signaling.

Ketoprofen suppresses triple negative breast cancer cell growth by inducing apoptosis and inhibiting autophagy.

Triple-negative breast cancer (TNBC) is an invasive phenotype with undesirable clinical features, poor prognosis, and therapy resistance. Ketoprofen is a Non-steroidal anti-inflammatory drug (NSAID) with anti-tumor properties. To investigate the effects of Ketoprofen on apoptosis and autophagy in TNBC cell line MDA-MB-231. The cytotoxic activity of Ketoprofen was assayed by the MTS method. Flowcytometry was utilized to measure the number of apoptotic MDA-MB-231 cells. The expression levels of apoptosis and autophagy markers, JAK2 and STAT3 were determined using quantitative real time-PCR (qRT-PCR) and western blotting methods. Ketoprofen significantly decreased the proliferation of MDA-MB-231 cells compared to control cells. It also considerably induced apoptosis and apoptotic markers in these cells in comparison to controls. Treating the MADA-MB-231 cell line with Ketoprofen had an inhibitory effect on autophagy markers in this cell line. The use of FasL, as a death ligand, and ZB4, as an antibody that blocks the extrinsic pathway of apoptosis, revealed the involvement of the extrinsic pathway in the apoptosis-stimulating effect of Ketoprofen in the MADA-MB-231 cell line. Ketoprofen also hindered the phosphorylation and activation of JAK2 and STAT molecules leading to the inhibition of the JAK/STAT pathway in this TNBC cell line. The outcomes of this study uncovered the anti-TNBC activity of Ketoprofen by inducing apoptosis and inhibiting viability and autophagy in MADA-MB-231 cells. Our data also suggested that Ketoprofen impedes apoptosis in TNBC cells by two different mechanisms including the induction of the extrinsic apoptotic pathway and inhibition of the JAK/STAT signaling.

Cachrys spp. from Southern Italy: Phytochemical Characterization and JAK/STAT Signaling Pathway Inhibition.

Different phytochemical compounds have been demonstrated to modulate the JAK/STAT signaling pathway. Here, three Cachrys species from Southern Italy were investigated for both the phytochemical profile and the potential anti-inflammatory properties. The aerial parts were extracted with methanol through Naviglio Extractor®, an innovative solid-liquid extraction technique that allows to obtain high quality extracts by working with gradient pressure. Extracts were analyzed with GC-MS and standardized in furanocoumarin content, resulting rich in xanthotoxin, bergapten and isopimpinellin. Given the known ability of bergapten to inhibit the JAK/STAT signaling pathway by decreasing the levels of pro-inflammatory cytokines (TNF-α, IL-6) and inflammatory mediators (NO) in RAW 264.7 cells activated by LPS, Cachrys extracts were investigated for their biological properties. The results obtained in this study showed that Cachrys pungens extract, presenting the highest content in furanocoumarins (7.48 ± 0.48 and 2.94 ± 0.16 mg/50 mg of extract for xanthotoxin and bergapten, respectively), significantly decreased STAT3 protein levels, pro-inflammatory cytokines (TNF-α, IL-6) and increased IL-10 anti-inflammatory cytokine. Cachrys ferulacea significantly decreased JAK2 phosphorylation, being even more effective than bergapten. In conclusion, investigated extracts could be potential candidates for the search of novel anti-inflammatory agents acting via inhibiting the JAK/STAT signaling pathway.

Ginsenoside Rb1 promotes angiogenesis potentially by activating the JAK-STAT3 signalling pathway

Conventional revascularization strategies for ischemic heart disease (IHD) are designed to prompt reperfusion of the coronary artery to the salvaged cardiomyocytes. However, these strategies may cause myocardial reperfusion injuries. Therefore, a safe and effective strategy needs to be developed to improve the conventional strategies. Here, we investigated the pro-angiogenic effect of Ginsenoside Rb1 (Rb1) to provide the experimental basis for angiogenesis-mediated drug therapy of IHD. Thus, Human umbilical vein endothelial cells (HUVECs) were treated with either a vehicle or Rb1 at 4, 8, 12 or 16 μM for 24 h. A model of hindlimb ischemia was established using C57BL/6J mice. In sham-operated mice, only the femoral artery was isolated without ligation whereas the other operations and supplementation control group were consistent. The mice in the supplementation group were injected with Rb1 (50 mg/kg body wt./day) for 7 days. The results indicated that Rb1 promotes cell proliferation, adhesion, migration and tube formation in the HUVECs in a dose-dependent manner. The ED50 of Rb1 to improve cell adhesion is 8 μM. In mice, Rb1 promoted angiogenesis after the ligation of the femoral artery and ameliorated the ischemic conditions. Intriguingly, more blood flow recovery was observed in the Rb1 supplemented mice than in the vehicle-treated mice (0.85 ± 0.05 vs. 0.71±0.10 on day 3; 0.94±0.10 vs. 0.75±0.08 on day 7). In HUVECs, Rb1 increased the phosphorylation of STAT3 and JAK, which may be the mechanism through which Rb1 mitigates IHD. Moreover, our results confirmed that Rb1mitigates IHD potentially by activating the JAK-STAT3 pathway. Further clinical trials are warranted to verify the clinical implications of Rb1.

Quercetin-ferrum nanoparticles enhance photothermal therapy by modulating the tumor immunosuppressive microenvironment

Photothermal therapy (PTT) was reported to induce synergistic immunogenic cell death (ICD) which may convert tumor cells into “therapeutic vaccines”. However, this is often insufficient to prevent tumor recurrence, in part because of the immunosuppressive microenvironment in tumors. Therefore, remodeling tumor microenvironment is of great importance to enhance the therapeutic efficacy of PTT. We herein fabricated a versatile nano-photosensitizer by assembling quercetin and Ferrum ion (QFN). The released quercetin from QFN could reduce programmed death ligand 1 (PD-L1) in tumor cells by inhibiting the phosphorylation of JAK2 and STAT3, and reshape extracellular matrix (ECM) by down-regulating α-SMA+ fibroblast in tumors. Moreover, QFN could capture tumor antigen and deliver it to the tumor-draining lymph nodes after PTT, which further enhanced the activation of antigen-presenting cells. As a result, QFN-based PTT eliminated melanoma and induced long-term immune memory to prevent tumor metastasis and recurrence. This study provides an effective and translationally feasible photothermic agent for photothermal/immunotherapy. Statement of significanceThe efficacy of photothermal therapy (PTT) in cancer treatment is often limited by the immunosuppressive microenvironment in tumors. Herein, we prepared a versatile photosensitizer by assembling quercetin and Ferrum ion (QFN). Upon near-infrared light irradiation, QFN-PTT induced cancer cells destruction and tumor antigen release. QFN then captured antigen and delivered it to the tumor-draining lymph nodes, thus promoting dendritic cell maturation and T cells activation. Quercetin released from QFN in tumors improved T cells infiltration and activation in tumor by regulating immunosuppressive microenvironment. The QFN-PTT-treated mice exhibited significantly elongated survival time, and gained strong anti-tumor immune memory to prevent tumor metastasis and recurrence. Thus, this work provided a simple and versatile photothermic agent, and it has important implications for designing effective and translationally feasible photosensitizers for PTT.

Targeted pharmacokinetics and bioinformatics screening strategy reveals JAK2 as the main target for Xin-Ji-Er-Kang in treatment of MIR injury

Background and purposeXin-Ji-Er-Kang (XJEK) is traditional Chinese formula presented excellent protective effects on several heart diseases, but the potential components and targets are still unclear. The aim of this study is to elucidate the effective components of XJEK and reveal its potential mechanism of cardioprotective effect in myocardial ischemia-reperfusion (MIR) injury. Experimental approachFirstly, the key compounds in XJEK, plasma and heart tissue were analyzed by high resolution mass spectrometry. Bioinformatics studies were also involved to disclose the potential targets and the binding sites for the key compounds. Secondly, to study the protective effect of XJEK on MIR injury and related mechanism, mice subjected to MIR surgery and gavage administered with XJEK for 6 weeks. Cardiac function parameters and apoptosis level of cardiac tissue were assessed. The potential mechanism was further verified by knock down of target protein in vitro. ResultsPharmacokinetics studies showed that Sophora flavescens alkaloids, primarily composed with matrine, are the key component of XJEK. And, through bioinformatic analysis, we speculated JAK2 could be the potential target for XJEK, and could form stable hydrogen bonds with matrine. Administration of XJEK and matrine significantly improved heart function and reduced apoptosis of cardiomyocytes by increasing the phosphorylation of JAK2 and STAT3. The anti-apoptosis effect of XJEK and matrine was also observed on AC16 cells, and could be reversed by co-treatment with JAK2 inhibitor AG490 or knock-down of JAK2. ConclusionXJEK exerts cardioprotective effect on MIR injury, which may be associated with the activation of JAK2/STAT3 signaling pathway.

Candesartan Attenuates Cisplatin-Induced Lung Injury by Modulating Oxidative Stress, Inflammation, and TLR-4/NF-κB, JAK1/STAT3, and Nrf2/HO-1 Signaling.

Cisplatin (CIS) is an effective chemotherapeutic agent against different cancers. The use of CIS is associated with acute lung injury (ALI) and other adverse effects, and oxidative stress and inflammation were implicated in its toxic effects. Candesartan (CAN), an angiotensin II (Ang II) receptor blocker, showed beneficial effects against oxidative stress and inflammation. Therefore, this study investigated the potential of CAN to prevent CIS-induced oxidative stress, inflammation, and lung injury in rats, pointing to the involvement of TLR4/NF-κB, JAK1/STAT3, PPARγ, and Nrf2/HO-1 signaling. The rats received CAN (5 mg/kg) for 10 days and were challenged with a single dose of CIS (7 mg/kg) on day 7. CIS caused injury to the alveoli and the bronchial tree, increased lipid peroxidation, nitric oxide, myeloperoxidase, TLR-4, NF-κB p65, iNOS, TNF-α, IL-6, IL-1β, and caspase-3, and decreased cellular antioxidants and IL-6 in the lungs of rats. CAN effectively prevented tissue injury, suppressed TLR-4/ NF-κB signaling, and ameliorated oxidative stress, inflammatory markers, and caspase-3 in CIS-administered rats. CAN enhanced antioxidants and IL-10, decreased Ang II, increased Ang (1–7), suppressed the phosphorylation of JAK1 and STAT3, and upregulated SOCS3 in CIS-administered rats. These effects were associated with the downregulation of Keap1 and enhanced Nrf2, GCLC, HO-1, and PPARγ. In conclusion, CAN prevented CIS-induced lung injury by attenuating oxidative stress, suppressing TLR-4/NF-κB and JAK1/STAT3 signaling, Ang II, and pro-inflammatory mediators, and upregulating PPARγ, and Nrf2/HO-1 signaling.

Curculigoside Represses the Proliferation and Metastasis of Osteosarcoma via the JAK/STAT and NF-κB Signaling Pathways.

Curculigoside (Cur) is a natural component from Curculigo orchioides Gaertn, with various bioactivities. The function of Cur in the nervous system and osteoarthritis has been reported. However, its role in osteosarcoma (OS) needs to be investigated. Hence, we focus on probing the impact of Cur on OS. In vitro, cell counting kit 8 (CCK-8), flow cytometry and Transwell assay were used to investigate the effects of Cur on OS cell proliferation, apoptosis, migration and invasion. In vivo, we developed a xenograft model to figure out the effect of Cur on tumor growth in nude mice. Western blotting (WB) was conducted to compare the levels of Cur on apoptosis-related proteins (C-caspase-3, Bax, and Bcl-2), epithelial-mesenchymal transition (EMT)-related proteins (N-cadherin, Snail, and E-cadherin) and the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) and nuclear factor-κB (NF-κB) pathways in vitro and in vivo. In-vitro data testified that Cur treatment markedly hampered OS cells' growth, migration and invasion and intensified their apoptosis compared to that of the control group. In vivo, Cur treatment notably hampered the growth of OS tumors in mice. In addition, both in vitro and in vivo experiments demonstrated that the phosphorylation of JAK2, STAT3, and NF-κB were inhibited through Cur treatment. Furthermore, the inhibition of Cur in OS cells was demonstrated by up-regulating the expression of JAK/STAT and NF-κB pathways protein levels. In summary, the data suggest that Cur curbs OS growth by down-regulating the JAK/STAT and NF-κB pathways, which is an underlying therapeutic option for OS treatment.