Alpinetin inhibits neuroinflammation and neuronal apoptosis via targeting the JAK2/STAT3 signaling pathway in spinal cord injury.

BACKGROUND: A growing body of research shows that drug monomers from traditional Chinese herbal medicines have antineuroinflammatory and neuroprotective effects that can significantly improve the recovery of motor function after spinal cord injury (SCI). Here, we explore the role and molecular mechanisms of Alpinetin on activating microglia-mediated neuroinflammation and neuronal apoptosis after SCI. METHODS: Stimulation of microglia with lipopolysaccharide (LPS) to simulate neuroinflammation models in vitro, the effect of Alpinetin on the release of pro-inflammatory mediators in LPS-induced microglia and its mechanism were detected. In addition, a co-culture system of microglia and neuronal cells was constructed to assess the effect of Alpinetin on activating microglia-mediated neuronal apoptosis. Finally, rat spinal cord injury models were used to study the effects on inflammation, neuronal apoptosis, axonal regeneration, and motor function recovery in Alpinetin. RESULTS: Alpinetin inhibits microglia-mediated neuroinflammation and activity of the JAK2/STAT3 pathway. Alpinetin can also reverse activated microglia-mediated reactive oxygen species (ROS) production and decrease of mitochondrial membrane potential (MMP) in PC12 neuronal cells. In addition, in vivo Alpinetin significantly inhibits the inflammatory response and neuronal apoptosis, improves axonal regeneration, and recovery of motor function. CONCLUSION: Alpinetin can be used to treat neurodegenerative diseases and is a novel drug candidate for the treatment of microglia-mediated neuroinflammation.

Design and optimization of cranberry extract loaded bile salt augmented liposomes for targeting of MCP-1/STAT3/VEGF signaling pathway in DMN-intoxicated liver in rats.

Cranberry extract (CBE) is a major source of the antioxidant polyphenolics but suffers from limited bioavailability. The goal of this research was to encapsulate the nutraceutical (CBE), into bile salt augmented liposomes (BSALs) as a promising oral delivery system to potentiate its hepatoprotective impact against dimethylnitrosamine (DMN) induced liver injury in rats. The inclusion of bile salt in the liposomal structure can enhance their stability within the gastrointestinal tract and promote CBE permeability. CBE loaded BSALs formulations were fabricated utilizing a (23) factorial design to explore the impact of phospholipid type (X1), phospholipid amount (X2), and sodium glycocholate (SGC) amount (X3) on BSALs properties, namely; entrapment efficiency percent, (EE%); vesicle size, (VS); polydispersity index; (PDI); zeta potential, (ZP); and release efficiency percent, (RE%). The optimum formulation (F1) exhibited spherical vesicles with EE% of 71.27 ± 0.32%, VS; 148.60 ± 6.46 nm, PDI; 0.38 ± 0.02, ZP; -18.27 ± 0.67 mV and RE%; 61.96 ± 1.07%. Compared to CBE solution, F1 had attenuated DMN-induced hepatic injury, as evidenced by the significant decrease in serum level of ALT, AST, ALP, MDA, and elevation of GSH level, as well as SOD and GPX activities. Furthermore, F1 exhibited an anti-inflammatory character by suppressing TNF-α, MCP-1, and IL-6, as well as downregulation of VEGF-C, STAT-3, and IFN-γ mRNA levels. This study verified that when CBE was integrated into BSALs, F1, its hepatoprotective effect was significantly potentiated to protect the liver against DMN-induced damage. Therefore, F1 could be deliberated as an antioxidant, antiproliferative, and antifibrotic therapy to slow down the progression of hepatic damage.

Curcumin inhibits proliferation and invasion of papillary thyroid carcinoma cells by inhibiting the JAK2 / STAT3 pathway.

PURPOSE: The purpose of this study was to analyze the function of curcumin to suppress the proliferative and invasive abilities of papillary thyroid carcinoma (PTC) through inhibiting the JAK2/STAT3 pathway. METHODS: After treatment of different doses of curcumin in TPC-1 and SW1736 cells, changes in viability, clonality, cell cycle, apoptosis, wound healing and invasion were determined. Western blot analyses were performed to detect protein levels of apoptosis-associated genes, JAK2 and STAT3 in TPC-1 and SW1736 cells treated with different doses of curcumin. RESULTS: Curcumin treatment dose-dependently reduced viability, clonality and metastatic ability in TPC-1 and SW1736 cells. After treatment of 10 µM or 20 µM curcumin, PTC cells were blocked in G2/M phase, and their apoptotic rate increased. Curcumin treatment downregulated Bcl-2 and upregulated Bax in PTC cells. In addition, curcumin treatment downregulated p-JAK2 and p-STAT3 in TPC-1 and SW1736 cells. CONCLUSIONS: Curcumin treatment blocks PTC cells to proliferate and invade via inhibiting the JAK2/STAT3 pathway.

TLR2 deficiency promotes IgE and inhibits IgG1 class-switching following ovalbumin sensitization.

BACKGROUND: To explore the roles of Toll-like receptor (TLR)2 in Th2 cytokine production and immunoglobulin (Ig) class switching following ovalbumin (OVA) sensitization. METHODS: TLR2-/- and wild-type C57BL/6 mice were sensitized by intraperitoneal injection with OVA. Lung pathology was assessed by hematoxylin and eosin staining. Abundance of interleukin (IL)4, IL5, IL13, and IL21 transcripts in the lungs was quantified by RT-PCR. OVA-specific IgG1, IgG2a, IgG2b, IgE and IgM were quantified by enzyme-linked immunosorbent assay. Phosphorylated signal transducer and activator of transcription (STAT)3 in lung tissue was detected by immunohistochemistry staining and nuclear factor (NF) κB activation was measured by immunofluorescence staining. STAT3 activation was inhibited using cryptotanshinone (CPT) treatment. Germline transcripts (Iµ-Cµ, Iγ-Cγ, Iα-Cα or Iε-Cε), post-recombination transcripts (Iµ-Cγ, Iµ-Cα or Iµ- Cε) and mature transcripts (VHDJH-Cγ, VHDJH-Cα or VHDJH-Cε) were analyzed from splenic B cells of OVA-sensitized wild-type mice (with or without CPT treatment) and TLR2-/- mice (with or without IL21 treatment). RESULTS: The lungs of TLR2-/- mice showed a lesser degree of inflammation than wild-type mice after OVA sensitization. Following OVA sensitization, levels of IL4, IL13, and IL21, but not IL5, were significantly lower in TLR2-/- compared with wild-type mice. Moreover, OVA-specific IgG1 and IgE titers were markedly lower and higher, respectively, in TLR2-/- mice. TLR2 deficiency inhibited STAT3 activation but not NF-κB p65 activation. CPT treatment reduced IgG1 titers via inhibition of Stat3 phosphorylation. Both TLR2 knockout and CPT treatment reduced the frequencies of Iγ1-Cγ1, Iγ3-Cγ3 and Iα-Cα transcripts, but IL21 treatment compensated for the effects of TLR2 deficiency. CONCLUSION: These results suggest a role of TLR2 in restricting OVA-sensitized lung inflammation via promotion of IgG1 and inhibition of IgE class switching regulated by IL21 and STAT3.

Network Pharmacology and Molecular Docking Study on the Potential Mechanism of Yi-Qi-Huo-Xue-Tong-Luo Formula in Treating Diabetic Peripheral Neuropathy.

OBJECTIVE: To investigate the potential mechanism of action of Yi-Qi-Huo-Xue-Tong-Luo formula (YQHXTLF) in the treatment of diabetic peripheral neuropathy (DPN). METHODS: Network pharmacology and molecular docking techniques were used in this study. Firstly, the active ingredients and the corresponding targets of YQHXTLF were retrieved using the Traditional Chinese Medicine Systems Pharmacology (TCMSP) platform; subsequently, the targets related to DPN were retrieved using GeneCards, Online Mendelian Inheritance in Man (OMIM), Pharmgkb, Therapeutic Target Database (TTD) and Drugbank databases; the common targets of YQHXTLF and DPN were obtained by Venn diagram; afterwards, the "YQHXTLF Pharmacodynamic Component-DPN Target" regulatory network was visualized using Cytoscape 3.6.1 software, and Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the potential targets using R 3.6.3 software. Finally, molecular docking of the main chemical components in the PPI network with the core targets was verified by Autodock Vina software. RESULTS: A total of 86 active ingredients and 229 targets in YQHXTLF were screened, and 81 active ingredients and 110 targets were identified to be closely related to diabetic peripheral neuropathy disease. PPI network mapping identified TP53, MAPK1, JUN, and STAT3 as possible core targets. KEGG pathway analysis showed that these targets are mostly involved in AGE-RAGE signaling pathway in diabetic complications, TNF signaling pathway, and MAPK signaling pathway. The molecular docking results showed that the main chemical components of YQHXTLF have a stable binding activity to the core pivotal targets. CONCLUSION: YQHXTLF may act on TP53, MAPK1, JUN, and STAT3 to regulate inflammatory response, apoptosis, or proliferation as a molecular mechanism for the treatment of diabetic peripheral neuropathy, reflecting its multitarget and multipathway action, and providing new ideas to further uncover its pharmacological basis and mechanism of action.

Notoginsenoside R1 counteracts mesenchymal stem cell-evoked oncogenesis and doxorubicin resistance in osteosarcoma cells by blocking IL-6 secretion-induced JAK2/STAT3 signaling.

Tumor microenvironment is a critical participant in the initiation, progression and drug resistance of carcinomas, including osteosarcoma. Notoginsenoside R1 (NGR1) is a proverbial active ingredient of the traditional Chinese medicine Panax notoginseng (PN) and possess undeniable roles in several cancers. Nevertheless, its function in osteosarcoma and tumor microenvironment remains elusive. In the current study, exposure to NGR1 dose-dependently inhibited osteosarcoma cell viability and migration, and induced apoptosis. Furthermore, osteosarcoma cells that were incubated with conditioned medium (CM) from bone marrow mesenchymal stem cells (BMSCs) exhibited greater proliferation, migration capacity and MMP-2 and MMP-9 expression relative to control cells, which was reversed when BMSCs were treated with NGR1. Notably, administration with NGR1 antagonized CM-evoked doxorubicin resistance in osteosarcoma cells by decreasing cell viability and increasing cell apoptosis and caspase-3/9 activity. Mechanically, NGR1 suppressed IL-6 secretion from BMSCs, as well as the subsequent activation of the JAK2/STAT3 signaling in osteosarcoma cells. In addition, blocking the JAK2 pathway by its antagonist AG490 reversed CM-induced osteosarcoma cell proliferation, migration and doxorubicin resistance. Moreover, exogenous supplementation with IL-6 engendered not only the reactivation of the JAK2/STAT3 signaling but also muted NGR1-mediated efficacy against osteosarcoma cell malignancy and doxorubicin resistance. Collectively, NGR1 may directly restrain osteosarcoma cell growth and migration, or indirectly antagonize MSC-evoked malignancy and drug resistance by interdicting IL-6 secretion-evoked activation of the JAK2/STAT3 pathway. Consequently, the current study may highlight a promising therapeutic strategy against osteosarcoma by regulating tumor cells and the tumor microenvironment.

Oxyresveratrol induces apoptosis and inhibits cell viability via inhibition of the STAT3 signaling pathway in Saos­2 cells.

Oxyresveratrol (ORES) is a natural phenolic compound with multiple biological functions including antioxidation, anti­inflammation and neuroprotection; however, the inhibitory effect of ORES on osteosarcoma remains largely unknown. The present study aimed to determine the effects of ORES on osteosarcoma cell Saos­2. Cell Counting Kit­8 assay was performed to detect Soas­2 cell viability. Annexin­FITC/PI staining and JC­1 staining were used to measure cell apoptosis and the change of mitochondrial membrane potential. In addition, western blotting was conducted to determine the expression levels of apoptotic proteins and the phosphorylation of STAT3. It was found that ORES inhibited cell viability and induced apoptosis of osteosarcoma Saos­2 cells in a concentration­dependent manner. In addition, ORES increased the expression levels of apoptotic proteases caspase­9 and caspase­3 and reduced mitochondrial membrane potential. In response to ORES treatment, the expression levels of pro­apoptotic proteins, Bad and Bax, were enhanced, whereas those of anti­apoptotic proteins, Bcl­2 and Bcl­xL, were reduced. In addition, the phosphorylation of STAT3 was attenuated in Saos­2 cells after treatment with ORES. Inhibition of cell viability and apoptosis induction by ORES were rescued by enhancement of STAT3 activation upon treatment with IL­6. Collectively, the present study indicated that ORES induced apoptosis and inhibited cell viability, which may be associated with the inhibition of STAT3 activation; thus, ORES represents a promising agent for treating osteosarcoma.

The Antitumor Efficacy of ß-Elemene by Changing Tumor Inflammatory Environment and Tumor Microenvironment.

Inflammatory mediators and inflammatory cells in the inflammatory microenvironment promote the transformation of normal cells to cancer cells in the early stage of cancer, promote the growth and development of cancer cells, and induce tumor immune escape. The monomeric active ingredient ß-elemene is extracted from the traditional Chinese medicine Curcuma wenyujin and has been proven to have good anti-inflammatory and antitumor activities in clinical applications for more than 20 years in China. Recent studies have found that this traditional Chinese medicine plays a vital role in macrophage infiltration and M2 polarization, as well as in regulating immune disorders, and it even regulates the transcription factors NF-κB and STAT3 to alter inflammation, tumorigenesis, and development. In addition, ß-elemene regulates not only different inflammatory factors (such as TNF-α, IFN, TGF-ß, and IL-6/10) but also oxidative stress in vivo and in vitro. The excellent anti-inflammatory and antitumor effects of ß-elemene and its ability to alter the inflammatory microenvironment of tumors have been gradually elaborated. Although the study of monomeric active ingredients in traditional Chinese medicines is insufficient in terms of quality and quantity, the pharmacological effects of more active ingredients of traditional Chinese medicines will be revealed after ß-elemene.

Arctiin is a pharmacological inhibitor of STAT3 phosphorylation at tyrosine 705 residue and potentiates bortezomib-induced apoptotic and anti-angiogenic effects in human multiple myeloma cells.

BACKGROUND: Arctiin is a main component from the fruits of Arctium lappa L., that can be prescribed for cold or flu in East Asian countries; it has also been found to exert chemopreventive actions against various tumor cells. HYPOTHESIS: In view of this evidence, we examined arctiin for its ability to trigger apoptosis and inhibit the activation of signal transducer and activator of transcription 3 (STAT3) in human multiple myeloma (MM) cells. METHODS: We evaluated the effect of arctiin on STAT3 signaling cascades and its regulated functional responses in MM cells. RESULTS: Arctiin effectively blocked the constitutive activation of STAT3 phosphorylation in the residue of tyrosine 705. Arctiin also abrogated the constitutive activation of Src phosphorylation and Janus-activated kinases (JAKs) 1/2. Furthermore, it was found that arctiin treatment clearly enhanced the mRNA and protein levels of protein tyrosine phosphatase ε (PTPε), and the silencing of PTPε caused a reversal of the arctiin-induced PTPε expression and the blockadge of STAT3 phosphorylation. Interestingly, arctiin could not repress IL-6-induced STAT3 activation in serum-starved U266 cells and when arctiin was incubated with a complete culture medium in RPMI 8226 and MM.1S cells. Arctiin suppressed cell proliferation, accumulated cells in the G2/M cell-cycle phase, and induced apoptosis within U266 cells, although the knockdown of PTPε prevented PARP cleavage and caspase-3 activation induced by the arctiin. In addition, arctiin exerted cytotoxicity in MM cells, but did not do so in peripheral blood mononuclear cells. Arctiin down-modulated diverse oncogenic gene products regulated by STAT3, although the induction of apoptosis by arctiin was abrogated upon transfection with pMXs-STAT3C in mouse embryonic fibroblast (MEF) cells. Arctiin also potentiated bortezomib-induced antitumor effects in U266 cells. CONCLUSION: On the whole, our results indicate that arctiin is a potentially new inhibitor of constitutive STAT3 activation through the induction of PTPε in MM, cells and therefore has great value in treating various tumors sheltering constitutively activated STAT3.

Antiproliferative Effects of Cynaropicrin on Anaplastic Thyroid Cancer Cells.

BACKGROUND: The sesquiterpene lactone cynaropicrin, a major constituent of the artichoke leaves extracts, has shown several biologic activities in many preclinical experimental models, including anti-proliferative effects. OBJECTIVE: Herein we evaluated the effects of cynaropicrin on the growth of three human anaplastic thyroid carcinoma cell lines, investigating the molecular mechanism underlying its action. METHOD: MTT assay was used to evaluate the viability of CAL-62, 8505C and SW1736 cells, and flow cytometry to analyse cell cycle distribution. Western blot was performed to detect the levels of STAT3 phosphorylation and NFkB activation. Antioxidant effects were analyzed by measuring the reactive oxygen species and malonyldialdehyde dosage was used to check the presence of lipid peroxidation. RESULTS: Viability of CAL-62, 8505C and SW1736 cells was significantly reduced by cynaropicrin in a dose- and time-dependent way, with an EC50 of about 5 µM observed after 48 h of treatment with the compound. Cellular growth inhibition was accompanied both by an arrest of the cell cycle, mainly in the G2/M phase, and the presence of a significant percentage of necrotic cells. After 48 h of treatment with 10 µM of cynaropicrin, a reduced nuclear expression of NFkB and STAT3 phosphorylation were also revealed. Moreover, we observed an increase in lipid peroxidation, without any significant effect on the reactive oxygen species production. CONCLUSION: These results demonstrate that cynaropicrin reduces the viability and promotes cytotoxic effects in anaplastic thyroid cancer cells associated with reduced NFkB expression, STAT3 phosphorylation and increased lipid peroxidation. Further characterization of the properties of this natural compound may open the way for using cynaropicrin as an adjuvant in the treatment of thyroid cancer.

Diarylheptanoids from Curcuma phaeocaulis Suppress IL-6-Induced STAT3 Activation.

Three undescribed diarylheptanoids (3: -5: ) and six known curcuminoids (1, 2: , and 6: -9: ) were obtained from the ethyl acetate-soluble fraction of an ethanolic extract of Curcuma phaeocaulis. Their chemical structures and absolute configurations were elucidated by high-resolution electrospray ionization mass spectrometry, nuclear magnetic resonance spectroscopy, circular dichroism spectroscopy, and the modified Mosher's method. Previous studies constructed Hep3B cells stably transfected with pSTAT3-Luc plasmid containing STAT3 binding site to discover STAT3 inhibitors from natural products. The STAT3 inhibitory activities of all isolates were measured in transfected Hep3B cells after treatment with IL-6. Compound 5: ((5R)-1,7-Bis(3,4-dimethoxyphenyl)-3-methoxy-1-hepten-5-ol), demethoxycurcumin (7: ), and curcumin (8: ) exhibited significant inhibitory activity (IC50 values: 11.1, 1.9, and 1.6 µM, respectively). Furthermore, IL-6-induced phosphorylation of STAT3, and the mRNA expression levels of inflammation-related genes such as CRP, IL-1ß, ICAM-1, and SOCS3 were significantly reduced by exposure to compound 5: . These data suggested that the inhibitory activity of 5: is associated with the suppression of STAT3 phosphorylation. Thus, compound 5: may be a promising candidate for the treatment of cancer or inflammatory diseases related to the IL-6/STAT3 signaling pathway.

Xuesaitong May Protect Against Ischemic Stroke by Modulating Microglial Phenotypes and Inhibiting Neuronal Cell Apoptosis via the STAT3 Signaling Pathway.

BACKGROUND: Xuesaitong mainly comprises Panax notoginseng saponins and has shown a promising feature in an acute ischemic stroke model; however, its effect on long-term recovery following stroke, and the related mechanisms, are unknown. METHODS: The objective of this study was to investigate the long-term protective effects of xuesaitong against ischemic stroke and its effect on microglial polarization. Experimental cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) for 45 min, and C57BL/6 mice were immediately injected with xuesaitong or vehicle through the caudal vein at the onset of cerebral reperfusion consecutively for 14 days. The animals were randomly divided into three groups: a sham-operated group, vehicle-treated group and xuesaitong-treated group at a dose of 15µg/g. Subsequently, 2,3,5-triphenyltetrazolium chloride staining was used to assess infarct volume, and adhesive removal tests and balance beam tests were used to evaluate neurological deficits at days 1, 3, 7 and 14 following ischemia. Reverse-transcriptase polymerase chain reaction and immunofluorescence staining for M1 markers (CD16, iNOS) and M2 markers (CD206, arginase-1) were performed to characterize phenotypic changes in microglia. Elisa was used to determine the release of pro-inflammatory and anti-inflammatory cytokines. TUNEL staining was conducted to detect neuronal cell apoptosis, and western blots were used to determine the activation of signal transducer and activator of transcription 3 (STAT3). RESULTS: Our results revealed that xuesaitong treatment, compared with vehicle treatment, significantly reduced cerebral infarct volume 1 and 3 days after MCAO and resulted in significant improvements in long-term neurological outcomes. Furthermore, xuesaitong treatment, compared with vehicle treatment, significantly reduced M1 markers and elevated M2 markers 7 and 14 days after MCAO at both the mRNA and protein level in ipsilateral brain tissue. This finding was also accompanied by a reduction in neuronal cell apoptosis and p-STAT3 transcription factor levels in the xuesaitong-treated group compared with the vehicle-treated group. CONCLUSION: We demonstrated that xuesaitong has long-term neuroprotective effects against ischemic stroke, possibly by promoting the polarization of microglia to an M2 phenotype and by inhibiting neuronal cell death via down-regulation of the STAT3 signaling pathway, providing new evidence that xuesaitong might be a promising therapeutic strategy for ischemic stroke.

Modified Citrus Pectin Prevents Blood-Brain Barrier Disruption in Mouse Subarachnoid Hemorrhage by Inhibiting Galectin-3.

Background and Purpose- Plasma levels of galectin-3-a matricellular protein-are increased after aneurysmal subarachnoid hemorrhage (SAH), but the functional significance remains undetermined. This study was conducted to evaluate whether modified citrus pectin (MCP; galectin-3 inhibitor) prevents post-SAH early brain injury, focusing on blood-brain barrier disruption. Methods- C57BL/6 male adult mice (n=251) underwent sham or filament perforation SAH modeling, followed by a random intracerebroventricular injection of vehicle or drug at 30 minutes post-modeling. First, vehicle-treated and 0.8, 4, 16, or 32 µg MCP-treated mice were assessed by neuroscore and brain water content at 24 and 48 hours post-modeling. Second, Evans blue extravasation, Western blotting, coimmunoprecipitation and immunostaining were performed in vehicle-treated or 4 µg MCP-treated mice at 24 hours post-modeling. Third, vehicle or R-galectin-3 (recombinant galectin-3) was administered to SAH mice simultaneously with vehicle or MCP, and neuroscore and Evans blue extravasation were evaluated at 24 hours post-modeling. Fourth, vehicle or R-galectin-3 was administered to MCP-treated SAH mice at 24 hours, and neuroscore and IgG immunostaining were evaluated at 48 hours post-SAH. Results- Among tested dosages, 4 µg MCP showed the best neuroprotective effects as to preventing neurological impairments and brain edema at 24 to 48 hours post-SAH. Four micrograms MCP attenuated post-SAH blood-brain barrier disruption and galectin-3 upregulation in brain capillary endothelial cells, associated with inactivation of ERK (extracellular signal-related kinase) 1/2, STAT (signal transducer and activator of transcription)-3, and MMP (matrix metalloproteinase)-9, and the consequent preservation of a tight junction protein ZO-1 (zonula occludens-1). Coimmunoprecipitation assay demonstrated physical interactions between galectin-3 and TLR (Toll-like receptor) 4. R-galectin-3 blocked the neuroprotective effects of MCP. Conclusions- MCP prevents post-SAH blood-brain barrier disruption possibly by inhibiting galectin-3, of which the mechanisms may include binding to TLR4 and activating ERK1/2, STAT-3, and MMP-9. This study suggests galectin-3 to be a novel therapeutic target against post-SAH early brain injury.

Inhibitory Effects of Compounds and Extracts from Ampelopsis brevipedunculata on IL-6-Induced STAT3 Activation.

Ampelopsis brevipedunculata (Maxim.) Trautv. (AB), a traditional East Asian medicine, exhibits protective effects against several inflammatory diseases. Our search for an inhibitor of IL-6-induced JAK2/STAT3 activation revealed that AB ethanolic extract (ABE) had a significant inhibitory effect on IL-6-induced STAT3 expression in Hep3B cells. The isolation and purification of an EtOAc-soluble fraction of ABE (ABEA) using reversed-phase high-performance liquid chromatography (RP-HPLC) afforded 17 compounds. The structures of these compounds (1-17) were elucidated based on 1H and 13C nuclear magnetic resonance (NMR) spectroscopy as well as electrospray-ionization mass spectrometry (ESI-MS) data. ABE and ABEA were screened by a luciferase assay using Hep3B cells transfected with the STAT3 reporter gene. ABEA exhibited potent inhibitory effects on IL-6-induced STAT3 expression; moreover, these effects arose from the inhibition of the phosphorylation of the STAT3, JAK2, and ERK proteins in U266 cells. In addition, the compounds isolated from ABEA were measured for their inhibitory effects on IL-6-stimulated STAT3 expression. Of the compounds isolated, betulin showed the greatest inhibitory effects on IL-6-induced STAT3 activation in the luciferase assay (IC50 value: 3.12 µM). Because of its potential for inhibiting STAT3 activation, A. brevipedunculata could be considered a source of compounds of pharmaceutical interest.

Zeylenone inhibits proliferation and promotes apoptosis in ovarian carcinoma cells via Janus kinase 2 / signal transducers and activators of transcription 3 pathways.

AIM: Ovarian cancer is the fifth common cancer in females. The aim of our study was to determine function of Zeylenone on cell viability and apoptosis of ovarian carcinoma SKOV3 cells. METHODS: Cell viability was measured by Cell counting kit-8 (CCK8) assay; Mitochondrial membrane potential (MMP) and apoptosis were detected by flow cytometry. The mRNA and protein levels of related factors were determined by Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot, respectively. RESULTS: Cell viability was decreased by Zeylenone in a dose-dependent manner. Zeylenone with concentrations of 2.5, 5 and 10 µmol/L was used to treat ovarian carcinoma SKOV3 cells for 24 h in the following study. The loss of MMP and apoptosis were both significantly increased by Zeylenone. The mRNA and protein levels of cytochrome c (cyto c) and apoptosis inducing factor (AIF) in cytosol were increased by Zeylenone. The mRNA and protein levels of Caspase-3, Fas, Fasl and Bax were increased; while the expression of Bcl-2 was decreased by Zeylenone. The expression of (Janus family of tyrosine kinase) p-JAK and signal transducer and activator of transcription (p-STAT) was decreased significantly by Zeylenone. CONCLUSION: Zeylenone inhibited cell proliferation and promoted apoptosis in ovarian carcinoma cells. The JAK-STAT pathway was involved in this progress.

Characterizations of mitochondrial uncoupling induced by chemical mitochondrial uncouplers in cardiomyocytes.

Induction of mild mitochondrial uncoupling is protective in a variety of disorders; however, it is unclear how to recognize the mild mitochondrial uncoupling induced by chemical mitochondrial uncouplers. The aim of the present study is to identify the pharmacological properties of mitochondrial uncoupling induced by mitochondrial uncouplers in cardiomyocytes. Neonatal rat cardiomyocytes were cultured. Protein levels were measured by using western blot technique. The whole cell respiratory function was determined by using high-resolution respirometry. The typical types of chemical mitochondrial uncouplers, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), niclosamide, and BAM15, induced biphasic change of STAT3 activity in cardiomyocytes, activating STAT3 at low dose and inhibiting STAT3 at high dose, though the dose range of these drugs was distinct. Low-dose uncouplers induced STAT3 activation through the mild increase of mitochondrial ROS (mitoROS) generation and the subsequent JAK/STAT3 activation in cardiomyocytes. However, high-dose uncouplers induced inhibition of STAT3, decrease of ATP production, and cardiomyocyte death. High-dose uncouplers induced STAT3 inhibition through the excessive mitoROS generation and the decreased ATP -induced AMPK activation. Low-dose mitochondrial uncouplers attenuated doxorubicin (DOX)-induced STAT3 inhibition and cardiomyocyte death, and activated STAT3 contributed to the cardioprotection of low-dose mitochondrial uncouplers. Uncoupler-induced mild mitochondrial uncoupling in cardiomyocytes is characterized by STAT3 activation and ATP increase whereas excessive mitochondrial uncoupling is characterized by STAT3 inhibition, ATP decrease and cell injury. Development of mitochondrial uncoupler with optimal dose window of inducing mild uncoupling is a promising strategy for heart protection.

Shikonin enhances sensitization of gefitinib against wild-type EGFR non-small cell lung cancer via inhibition PKM2/stat3/cyclinD1 signal pathway.

AIMS: Mutant EGFR Non-small cell lung cancer has benefit from gefitinib, but it has limited effect for wild-type EGFR tumors. Shikonin, a natural naphthoquinone isolated from a traditional Chinese medicine, the plant Lithospermum erythrorhizon (zicao), not only can inhibit the tumor growth, but also overcome cancer drug resistance. Our aim is to investigate whether shikonin can enhance antitumor effect of gefitinib in EGFR wild-type lung cancer cells in vitro and in vivo. MATERIALS AND METHODS: CCK-8 was used to determine the proliferation of EGFR wild-type non-small cell lung cancer. Apoptosis and cell cycle were detected by flow cytometry. PKM2, STAT3, p-STAT3 and cyclinD1 were detected by Western blot. A549 tumor model was established to observe the antitumor effect of shikonin combination with gefitinib in vivo. KEY FINDINGS: The results showed that combination of shikonin with gefitinib exhibited synergistic antitumor effect in vitro and in vivo. Its potential molecular mechanisms may be associated with inhibition of PKM2/STAT3/cyclinD1. SIGNIFICANCE: These results provide a promising therapeutic approach for the treatment of wild-type EGFR non-small cell lung cancer.

Tea Polysaccharides Inhibit Colitis-Associated Colorectal Cancer via Interleukin-6/STAT3 Pathway.

The interleukin-6 (IL-6)/signal transducer and activator of transcription (STAT)-3 signaling pathway regulates proliferation and survival of intestinal epithelial cells and has profound impact on the tumorigenesis of colitis-associated cancer (CAC). Tea polysaccharides (TPS) are the major nutraceutical component isolated from tea-leaves and are known to possess antioxidant, anti-inflammatory, and antitumor bioactivities. Here, we investigated the antitumor activities of TPS on CAC using the azoxymethane/dextran sulfate sodium (AOM/DSS) mouse model and IL-6-induced colorectal cancer cell line (CT26) and determined whether TPS exerted its antitumor effects through the IL-6/STAT3 pathway. Results demonstrated that TPS significantly decreased the tumor incidence, tumor size, and markedly inhibited the infiltration of pro-inflammatory cells and the secretion of pro-inflammatory cytokines via balancing cellular microenvironment. Furthermore, we found that TPS suppressed the activation of STAT3 and transcriptionally regulated the expressions of downstream genes including MMP2, cyclin Dl, survivin, and VEGF both in vivo and in vitro. Thus, it was concluded that TPS attenuated the progress of CAC via suppressing IL-6/STAT3 pathway and downstream genes' expressions, which indicated that TPS may be a hopeful antitumor agent for the prevention and treatment of colon cancer.

Ex vivo immunomodulatory effect of ethanolic extract of propolis during Celiac Disease: involvement of nitric oxide pathway.

Celiac Disease (CeD) is a chronic immune-mediated enteropathy, in which dietary gluten induces an inflammatory reaction, predominantly in the duodenum. Propolis is a resinous hive product, collected by honeybees from various plant sources. Propolis is well-known for its anti-inflammatory, anti-oxidant and immunomodulatory effects, due to its major compounds, polyphenols and flavonoids. The aim of our study was to assess the ex vivo effect of ethanolic extract of propolis (EEP) upon the activity and expression of iNOS, along with IFN-γ and IL-10 production in Algerian Celiac patients. In this context, PBMCs isolated from peripheral blood of Celiac patients and healthy controls were cultured with different concentrations of EEP. NO production was measured using the Griess method, whereas quantitation of IFN-γ and IL-10 levels was performed by ELISA. Inducible nitric oxide synthase (iNOS) expression, NFκB and pSTAT-3 activity were analyzed by immunofluorescence assay. Our results showed that PBMCs from Celiac patients produced high levels of NO and IFN-γ compared with healthy controls (HC). Interestingly, EEP reduced significantly, NO and IFN-γ levels and significantly increased IL-10 levels at a concentration of 50 µg/mL. Importantly, EEP downmodulated the iNOS expression as well as the activity of NFκB and pSTAT-3 transcription factors. Altogether, our results highlight the immunomodulatory effect of propolis on NO pathway and on pro-inflammatory cytokines. Therefore, we suggest that propolis may constitute a potential candidate to modulate inflammation during Celiac Disease and has a potential therapeutic value.

Silibinin from Silybum marianum Stimulates Embryonic Stem Cell Vascular Differentiation via the STAT3/PI3-K/AKT Axis and Nitric Oxide.

Silibinin, the bioactive compound of milk thistle (Silybum marianum), exerts tissue protective and regenerative effects that may include stem cell differentiation toward vascular cells. The purpose of the present study was to investigate whether silibinin stimulates blood vessel formation from mouse embryonic stem (ES) cells and to unravel the underlying signaling cascade. Vascular branching points were assessed by confocal laser scanning microscopy and computer-assisted image analysis of CD31-positive cell structures. Protein expression of vascular markers and activation of protein kinases were determined by western blot. Nitric oxide (NO) generation was investigated by use of the fluorescent dye 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate. Silibinin dose-dependently increased CD31-positive vascular branching points in embryoid bodies cultivated from ES cells. This was paralleled by increase of protein expression levels for the endothelial-specific markers vascular endothelial cadherin (VE-cadherin), vascular endothelial growth factor receptor 2, and hypoxia-inducible factor-1α. Moreover, silibinin increased activation of endothelial nitric oxide synthase (eNOS), which boosted generation of NO in embryoid bodies and enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphoinositide 3-kinase (PI3-K) and AKT. Vasculogenesis, VE-cadherin expression, STAT3 and AKT phosphorylation, NO generation, and eNOS phosphorylation were inhibited by the small molecule STAT3 inhibitor Stattic, AKT inhibitor VIII, the PI3-K inhibitor LY294002, or the NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride. In conclusion, our findings indicate that silibinin induces vasculogenesis of ES cells via activation of STAT3, PI3-K, and AKT, which regulate NO generation by eNOS.