Signaling Pathway Molecules Research Articles

Analgesic mechanism of acupuncture on neuropathic pain

The research progress of acupuncture analgesia in recent years is analyzed to summarize the analgesic mechanism of acupuncture on neuropathic pain. The analgesic mechanism of acupuncture on neuropathic pain is discussed from peripheral level and central level, including peripheral sensitization and immune inflammatory response, changes of ion channel, central sensitization, regulation of cell signal pathway, activation of spinal glial cells, etc. It is suggested that the focus of future research should include conducting in-vitro studies with the help of multi-omics technology to detect the changes of metabolic substances and signal pathway molecules in patients with neuropathic pain before and after acupuncture to further clarify the mechanism of acupuncture analgesia.

Adoptive transfers of CD4+ CD25+ Tregs partially alleviate mouse premature ovarian insufficiency.

This study was designed to investigate the protective effect of CD4+ CD25+ regulatory T cells (Tregs) against zona pellucida glycoprotein 3 peptide (pZP3) immunization-induced premature ovarian insufficiency (POI) in mice. A mouse POI model was induced by two subcutaneous injections of pZP3 (50 nmol/L). Mice in the pZP3-Treg group were intraperitoneally injected with 5 × 105 CD4+ CD25+ Tregs after the POI model was established. Sex hormone levels, follicle numbers, apoptotic events, and the Akt/FOXO3a signaling pathway molecules in the ovaries were assessed. Compared with control group, the weight of ovaries in both pZP3 group and pZP3-Treg group was decreased and no difference was found between them. The number of follicles in the Treg transferred mice, like in pZP3 group, was significantly reduced compared to the control group, but showed a modest improvement when compared the pZP3 group alone. Significantly lower serum concentrations of follicle-stimulating hormone, luteinizing hormone, and anti-zona pellucida antibodies (AZPAbs) were found, while the concentrations of estradiol and anti-Mullerian hormone increased. In mechanism, Treg cell transfer to ZP3 treated mice restored the levels of Caspase3 to control levels, and partially restored Bax, however, had no effect on Bcl-2. Moreover, Treg cell transfer to ZP3 treated mice partially restored the levels of Akt and FOXO3a, and partially restored the ratios of p-Akt/Akt and p-FOXO3a/FOXO3a. In conclusion, Treg cells improved some aspects of ZP3-induced POI which may be mediate by suppressing ovarian cells apoptosis and involving the Akt/FOXO3a signaling pathway. Therefore, Treg cells may be protective against autoimmune POI.

TFAP2B, AP-1 and JAZF1 Expression in Tissues of Papillary Thyroid Carcinoma Patients; Clinical, Pathological and Prognostic Values

Objective:Transcription factor activating protein 2 B (TFAP2 B) is a transcription factor that regulates many steps of embryogenesis, cell growth, apoptosis and recently oncogenesis and cancer progression. AP-1 is a transcription factor that is a downstream molecule of the MAPK signaling pathway. Juxtaposed with zinc finger gene 1 (JAZF1) is a recently detected transforming growth factor which has a role in carcinogenesis. Hence the present study aimed to assess those markers expression in tissues from patients with such cancer correlation their expression with clinic-pathological findings of the tumor and prognostic and follow-up findings of patients. Methods:We have collected tissue samples from papillary thyroid cancer patients and adjacent non-neoplastic tissues from 80 patients. We assessed the expression of TFAP2B, AP-1 and JAZF1 using immunohistochemistry. Results:Expression of TFAP2B was positively associated with lymph nodes metastases (p=0.003), distant metastases (p=0.002), recurrence of the tumor (p=0.002), unfavourable disease-free survival rate (p=0.003). AP-1 expression is positively associated with advanced stage (p=0.002), presence of extra-thyroid invasion (p=0.005), recurrence of the tumor (p=0.005), unfavorable disease-free survival rate (p=0.01). JAZF1 expression is negatively associated with huge tumor size (0.023), vascular invasion (p=0.007) and unfavorable overall survival rate (p=.030). Conclusion:High expression levels of TFAP2B and AP-1 and low expression levels of JAZF1 were associated with unfavourable pathological, prognostic parameters and dismal patient’s outcome.

Inhibitors of Ceramide- and Sphingosine-Metabolizing Enzymes as Sensitizers in Radiotherapy and Chemotherapy for Head and Neck Squamous Cell Carcinoma.

In the treatment of advanced head and neck squamous cell carcinoma (HNSCC), including oral SCC, radiotherapy is a commonly performed therapeutic modality. The combined use of radiotherapy with chemotherapy improves therapeutic effects, but it also increases adverse events. Ceramide, a central molecule in sphingolipid metabolism and signaling pathways, mediates antiproliferative responses, and its level increases in response to radiotherapy and chemotherapy. However, when ceramide is metabolized, prosurvival factors, such as sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), and glucosylceramide, are produced, reducing the antitumor effects of ceramide. The activities of ceramide- and sphingosine-metabolizing enzymes are also associated with radio- and chemo-resistance. Ceramide analogs and low molecular-weight compounds targeting these enzymes exert anticancer effects. Synthetic ceramides and a therapeutic approach using ultrasound have also been developed. Inhibitors of ceramide- and sphingosine-metabolizing enzymes and synthetic ceramides can function as sensitizers of radiotherapy and chemotherapy for HNSCC.

Coptidis alkaloids extracted from Coptis chinensis Franch attenuate IFN-γ-induced destruction of bone marrow cells.

Coptidis alkaloids are the primary active components of Coptis chinensis Franch. Clinical and pharmacodynamic studies have confirmed that Coptidis alkaloids have multiple therapeutic effects including anti-inflammatory, antioxidant and antitumor effects, and they are usually used to treat various inflammatory disorders and related diseases. Mouse bone marrow cells (BMCs) were isolated from BALB/c mice. Immune-mediated destruction of BMCs was induced by interferon (IFN) -γ. High-performance liquid chromatography-electrospray ionization/ mass spectrometry was used to analyze the ingredients of the aqueous extract from Coptis chinensis Franch. The results confirmed that Coptidis alkaloids were the predominant ingredients in the aqueous extract from Coptis chinensis. The functional mechanism of Coptidis alkaloids in inhibiting immune-mediated destruction of BMCs was studied in vitro. After Coptidis alkaloid treatment, the percentages of apoptotic BMCs and the proliferation and differentiation of helper T (Th) cells and regulatory T (Treg) cells were measured by flow cytometry. The expression and distribution of T-bet in BMCs were observed by immunofluorescence. Western blotting analysis was used to assay the expression of key molecules in the Fas apoptosis and Jak/Stats signaling pathways in BMCs. We identified five alkaloids in the aqueous extract of Coptis chinensis. The apoptotic ratios of BMCs induced by IFN-γ were decreased significantly after Coptidis alkaloid treatment. The levels of key molecules (Fas, Caspase-3, cleaved Caspase-3, Caspase-8 and Caspase-8) in Fas apoptosis signaling pathways also decreased significantly after treatment with low concentrations of Coptidis alkaloids. Coptidis alkaloids were also found to inhibit the proliferation of Th1 and Th17 cells and induce the differentiation of Th2 and Treg cells; further, the distribution of T-bet in BMCs was decreased significantly. In addition, the levels of Stat-1, phospho-Stat-1 and phospho-Stat-3 were also reduced after Coptidis alkaloid treatment. These results indicate that Coptidis alkaloids extracted by water decoction from Coptis chinensis Franch could inhibit the proliferation and differentiation of T lymphocytes, attenuate the apoptosis of BMCs, and suppress the immune-mediated destruction of the BMCs induced by pro-inflammatory cytokines.

The Runx1/Notch1 Signaling Pathway Participates in M1/M2 Microglia Polarization in a Mouse Model of Temporal Lobe Epilepsy and in BV-2 Cells.

Microglial activation and phenotypic shift play vital roles in many neurological diseases. Runt-related transcription factor-1 (Runx1), which is localized on microglia, inhibits amoeboid microglial proliferation. Preliminary data have indicated that the interaction of Runx1 with the Notch1 pathway affects the hemogenic endothelial cell shift. However, little is known about the effect of Runx1 and the Notch1 signaling pathway on the phenotypic shift of microglia during neuroinflammation, especially in temporal lobe epilepsy (TLE). A mouse model of TLE induced by pilocarpine and the murine microglia cell line BV-2 were used in this study. The proportion of microglia was analyzed using flow cytometry. Western blot (WB) analysis and quantitative real-time polymerase chain reaction were used to analyze protein and gene transcript levels, respectively. Immunohistochemistry was used to show the distribution of Runx1. In the present study, we first found that in a male mouse model of TLE induced by pilocarpine, flow cytometry revealed a time-dependent M2-to-M1 microglial transition after status epilepticus. The dynamic expression patterns of Runx1 and the downstream Notch1/Jagged1/Hes5 signaling pathway molecules in the epileptic hippocampus were determined. Next, Runx1 knockdown by small interfering RNA in BV-2 cells strongly promoted an M2-to-M1 microglial phenotype shift and inhibited Notch1/Jagged1/Hes5 pathway expression. In conclusion, Runx1 may play a critical role in the M2-to-M1 microglial phenotype shift via the Notch1 signaling pathway during epileptogenesis in a TLE mouse model and in BV-2 cells.

Central cellular signaling pathways involved with the regulation of lipid metabolism in the liver: a review

The liver is primarily responsible for energy homeostasis and the regulation of lipid, carbohydrate and protein metabolism. Lipid metabolism consists of distributing lipids to peripheral tissues or ensuring their return to the liver to be reprocessed. Additionally, cellular metabolism is regulated by several molecules in different signaling pathways. Lipid homeostasis in the liver is mainly regulated by AKT, AMPK, SREBP, PPAR, and JNK. The PI3K/AKT/mTOR signaling pathway results in the biosynthesis of macromolecules and regulates lipogenesis and the expression of lipogenic genes. AMPK is an energy sensor that regulates metabolism and is activated when stored ATP is depleted, and it is responsible for the suppression of several key lipogenic factors in the liver related to cholesterol and fatty acid synthesis. SREBPs control lipogenic gene expression and cholesterol metabolism and act in the nutritional regulation of fatty acids and triglycerides. The continued activation of SREBPs is associated with cellular stress, inflammation and ultimately steatosis. PPARs are intrinsically important regulators of lipid metabolism. These genes are essential to various metabolic processes, especially lipid and glucose homeostasis, and can play a role in cell differentiation. JNK signaling is related to insulin resistance and its activation results in decreased mitochondrial activity and fat accumulation. Therefore, the study of cell signaling pathways related to lipid metabolism and liver function may help to identify abnormalities and develop strategies to manage and regulate metabolic disorders and resulting complications.

Expression of Angiopoietins and Angiogenic Signaling Pathway Molecules in Chronic Subdural Hematomas.

Chronic subdural hematoma (CSDH) is an angiogenic disease that is involved with many inflammatory mediators. Tie2 is predominantly expressed in the embryonic endothelium and plays an important role in the maturation and stabilization of the vasculature. Angiopoietin (Ang)1 and Ang2 are well-known ligands of the Tie2 receptor. We examined the expression of Ang1 and Ang2 in CSDH fluid and the expression of Tie-2 receptor and components of the angiogenic signaling pathways in the outer membrane of CSDH. Twenty-five samples of CSDH fluid and eight samples of outer membrane of CSDH were included. The concentrations of Ang1 and Ang2 in the CSDH fluid were measured using enzyme-linked immunosorbent assay (ELISA) kits. The expression of Tie2, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt) mechanistic target of rapamycin (mTOR), GβL, 70 kDa ribosomal protein S6 kinase (p70S6K), eukaryotic initiation factor 4E (eIF-4E), and β-actin was examined by a Western blot analysis. The expression of Tie2, Akt, and mTOR was also examined by immunohistochemistry. The concentration of Ang2 in CSDH fluid was significantly higher than that in the serum or cerebrospinal fluid (CSF), and also higher than that of Ang1 in CSDH fluid. Tie2, PI3K, Akt, mTOR, GβL, p70S6K, and eIF-4E were detected in all cases. In addition, Tie2, Akt, and mTOR were localized in the endothelial cells of vessels in the CSDH outer membrane. Our data suggest that Ang2, although not Ang1, in CSDH fluid promotes angiogenesis in endothelial cells through the Tie2 receptor. The Ang2/Tie2 signaling pathway might therefore be a useful therapeutic target for treating the growth of intractable CSDH.

Carvacrol may alleviate vascular inflammation in diabetic db/db mice.

Type 2 diabetes mellitus (T2DM) is associated with chronic low-grade inflammation. Carvacrol has been confirmed to possess anti-inflammatory properties, but its effect on diabetic vasculature remains unknown. The aim of the present study was to investigate the possible protective effects of carvacrol against vascular endothelial inflammation. The mice were divided into four groups (n=15 per group) as follows: Non-diabetic control mice, db/db mice, db/db mice + carvacrol (low) and db/db mice + carvacrol (high) groups. The effects of carvacrol on the pathomorphism of the thoracoab-dominal aorta in db/db mice were evaluated using hematoxylin and eosin and Masson's trichrome staining. The serum levels of insulin signaling molecules, such as phosphorylated insulin receptor, phosphorylated insulin receptor substrate-1, insulin, triglyceride (TG) and inflammatory cytokines [tumor necrosis factor-α, interleukin (IL)-1β, IL-6 and IL-8] were measured by ELISA. Furthermore, the protein levels of the toll-like receptor (TLR)4/nuclear factor (NF)-κB inflammatory signaling pathway molecules were investigated in the thoracoabdominal aorta of db/db mice and in high glucose-induced endothelial cells. Vascular endothelial cell apoptosis and viability were assessed by using flow cytometry and Cell Counting Kit-8 assays, respectively. The results demonstrated that carva-crol alleviated vascular endothelial cell injury. Carvacrol reduced the expression levels of insulin signaling molecules, insulin, TG and inflammatory cytokines in the serum of db/db mice. Moreover, carvacrol reduced the activation of the TLR4/NF-κB signaling pathway in vivo and in vitro. In vitro, carvacrol inhibited high glucose-induced endothelial cell function by promoting vascular endothelial cell apoptosis and suppressing cell viability. These findings demonstrated that carvacrol could alleviate endothelial dysfunction and vascular inflammation in T2DM.

Mitochondrial DNA Damage: Prevalence, Biological Consequence, and Emerging Pathways.

Mitochondria have a plethora of functions within a eukaryotic cell, ranging from energy production, cell signaling, and protein cofactor synthesis to various aspects of metabolism. Mitochondrial dysfunction is known to cause over 200 named disorders and has been implicated in many human diseases and aging. Mitochondria have their own genetic material, mitochondrial DNA (mtDNA), which encodes 13 protein subunits in the oxidative phosphorylation system and a full set of transfer and rRNAs. Although more than 99% of the proteins in mitochondria are nuclear DNA (nDNA)-encoded, the integrity of mtDNA is critical for mitochondrial functions, as evidenced by mitochondrial diseases sourced from mtDNA mutations and depletions and the vital role of fragmented mtDNA molecules in cell signaling pathways. Previous research has shown that mtDNA is an important target of genotoxic assaults by a variety of chemical and physical factors. This Perspective discusses the prevalence of mtDNA damage by comparing the abundance of lesions in mDNA and nDNA and summarizes current knowledge on the biological pathways to cope with mtDNA damage, including mtDNA repair, mtDNA degradation, and mitochondrial fission and fusion. Also, emerging roles of mtDNA damage in mutagenesis and immune responses are reviewed.

Sucralose Promotes Colitis-Associated Colorectal Cancer Risk in a Murine Model Along With Changes in Microbiota.

Sucralose is a calorie-free high-intensity artificial sweetener that is widely used in thousands of foods and beverages all over the world. Although it was initially regarded as a safe, inert food additive, its adverse effect on gut microbiota and health has drawn more and more attention as evidence accumulates. Studies by us and others revealed that sucralose exacerbated gut damage and inflammation in animal models for inflammatory bowel disease (IBD), including those for both ulcerative colitis, and Crohn's disease. Our study demonstrated that sucralose greatly aggravated dextran sulfate sodium (DSS)-induced colitis along with causing changes in gut microbiota, the gut barrier and impaired inactivation of digestive proteases mediated by deconjugated bilirubin. It is well-documented that IBD greatly increases the risk of colorectal cancer (CRC), the globally third-most-common cancer, which, like IBD, has a high rate in the developed countries. Azoxymethane (AOM)/DSS has been the most commonly used animal model for CRC. In this study, we further explored the effect of sucralose on tumorigenesis and the possible mechanism involved using the AOM/DSS mouse model. First, 1.5 mg/ml sucralose was included in the drinking water for 6 weeks to reach a relatively stable phase of impact on gut microbiota. Then, 10 mg/kg AOM was administered through intraperitoneal injection. Seven days later, 2.5% DSS was put in the drinking water for 5 days, followed by 2 weeks without DSS. The 5 days of DSS was then repeated, and the mice were sacrificed 6 weeks after AOM injection. The results showed that sucralose caused significant increases in the number and size of AOM/DSS-induced colorectal tumors along with changes in other parameters such as body and spleen weight, pathological scores, mortality, fecal β-glucuronidase and digestive proteases, gut barrier molecules, gut microbiota, inflammatory cytokines and pathways (TNFα, IL-1β, IL-6, IL-10, and TLR4/Myd88/NF-κB signaling), and STAT3/VEGF tumor-associated signaling pathway molecules. These results suggest that sucralose may increase tumorigenesis along with dysbiosis of gut microbiota, impaired inactivation of digestive protease, damage to the gut barrier, and exacerbated inflammation.

PDE3 Inhibition Reduces Epithelial Mast Cell Numbers in Allergic Airway Inflammation and Attenuates Degranulation of Basophils and Mast Cells

Epithelial mast cells are generally present in the airways of patients with allergic asthma that are inadequately controlled. Airway mast cells (MCs) are critically involved in allergic airway inflammation and contribute directly to the main symptoms of allergic patients. Phosphodiesterase 3 (PDE3) tailors signaling of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), which are critical intracellular second messenger molecules in various signaling pathways. This paper investigates the pathophysiological role and disease-modifying effects of PDE3 in mouse bone marrow-derived MCs (bmMCs), human LAD2- and HMC1 mast cell lines, human blood basophils, and peripheral blood-derived primary human MCs (HuMCs). In a chronic house dust mite (HDM)-driven allergic airway inflammation mouse model, we observed that PDE3 deficiency or PDE3 inhibition (PDE3i) therapy reduced the numbers of epithelial MCs, when compared to control mice. Mouse bone marrow-derived MCs (bmMCs) and the human HMC1 and LAD2 cell lines predominantly expressed PDE3B and PDE4A. BmMCs from Pde3−/− mice showed reduced loss of the degranulation marker CD107b compared with wild-type BmMCs, when stimulated in an immunoglobulin E (IgE)-dependent manner. Following both IgE-mediated and substance P-mediated activation, PDE3i-pretreated basophils, LAD2 cells, and HuMCs, showed less degranulation than diluent controls, as measured by surface CD63 expression. MCs lacking PDE3 or treated with the PDE3i enoximone exhibited a lower calcium flux upon stimulation with ionomycine. In conclusion PDE3 plays a critical role in basophil and mast cell degranulation and therefore its inhibition may be a treatment option in allergic disease.

Discovery of novel NF-кB inhibitor based on scaffold hopping: 1,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidine

NF-κB is a key signaling pathway molecule linking hepatoma and chronic inflammation. Inhibition of NF-κB activation can alleviate inflammation, and promote hepatoma cell apoptosis. In this study, a series of fluoro-substituted 1,4,5,6,7,8-hexahydropyrido[4,3-d]pyrimidines (PPMs, 31–57) were synthesized from 3,5-bis(arylidene)-4-piperidones (BAPs, 4–30) based on scaffold hopping. We successfully discovered the most potent 43 substituted by electron-withdrawing substitutes (3-F and 4-CF3) exhibited less toxicity and higher anti-inflammatory activity. Preliminary mechanistic studies revealed that 43 induced dose-dependent cell apoptosis at cell and protein level, while inhibited NF-κB activation by suppressing LPS-induced phosphorylation levels of p65, IκBα and Akt, and by indirectly suppressing MAPK signaling, and by inhibiting the nuclear translocation of NF-κB induced by TNF-α or LPS. Docking analysis verified simulated 43 could reasonably bind to the active site of Bcl-2, p65 and p38 proteins. This compound, as a novel NF-κB inhibitor, also demonstrated both anti-inflammatory and anti-hepatoma activities, warranting its further development as a potential multifunctional agent for the clinical treatment of liver cancers and inflammatory diseases.

Myeloid-derived suppressor cells promote epithelial ovarian cancer cell stemness by inducing the CSF2/p-STAT3 signalling pathway.

Myeloid‐derived suppressor cells (MDSCs) are known to contribute to tumour immune evasion, and studies have verified that MDSCs can induce cancer stem cells (CSCs) and promote tumour immune evasion in breast cancers, cervical cancers and glioblastoma. However, the potential function of MDSCs in regulating CSCs in epithelial ovarian cancer (EOC) progression is unknown. Our results indicated that compared to nonmalignant ovarian patients, EOC patients showed a significantly increased proportion of MDSCs in the peripheral blood. In addition, MDSCs dramatically promoted tumour sphere formation, cell colony formation and CSC accumulation, and MDSCs enhanced the expression of the stemness biomarkers NANOG and c‐MYC in EOC cells during coculture. Moreover, the mechanisms by which MDSCs enhance EOC stemness were further explored, and 586 differentially expressed genes were found in EOC cells cocultured with or without MDSCs; during coculture, the expression level of colony‐stimulating factor 2 (CSF2) was significantly increased in EOC cells cocultured with MDSCs. Furthermore, the depletion of CSF2 in EOC cells was successfully performed, the promotive effects of MDSCs on EOC cell stemness could be markedly reversed by downregulating CSF2 expression, p‐STAT3 signalling pathway molecules were also altered, and the p‐STAT3 inhibitor could markedly reverse the promotive effects of MDSCs on EOC cell stemness. In addition, the CSF2 expression level was correlated with EOC clinical staging. Therefore, MDSCs enhance the stemness of EOC cells by inducing the CSF2/p‐STAT3 signalling pathway. Targeting MDSCs or CSF2 may be a reasonable strategy for enhancing the efficacy of conventional treatments.DatabaseGene expression data files are available in the GEO databases under the accession number(s) GSE145374.

The Dynamics of Pseudophosphatase MK‐STYX and HDAC6 Ubiquitination

MK‐STYX [mitogen‐activated protein kinase phospho‐serine/threonine/tyrosine binding protein] is a pseudophosphatase and a member of the MAP kinase phosphatase family. Due to the absence of cysteine and histidine residues in the signature active site motif (HCX5R), MK‐STYX is catalytically inactive. Even so, it maintains the ability to bind phosphorylated residues. MK‐STYX is an important molecule in various signaling pathways, including the stress response pathway. Our previous research showed that the expression of MK‐STYX decreases stress granules [SG], which are aggregates of untranslated mRNA and misfolded proteins caused by cellular stress. However, the molecular mechanism by which MK‐STYX decreases SG remains elusive. Histone deacetylase isoform 6 (HDAC6) serves as a major component of SG. Therefore, we investigated whether MK‐STYX affects the dynamics of HDAC6. Our findings showed that MK‐STYX alters the subcellular localization of HDC6, which became nuclear, and decreased and altered the location of HDAC6‐aggregates. Because HDAC6 contains a ubiquitin binding domain, we investigated whether MK‐STYX affects the ubiquitination of HDAC6 and/or the ubiquitin proteasome system. Ubiquitin assays in stressed and unstressed cells showed that ubiquitinated proteins increased in the presence of MK‐STYX. Immunoprecipitation of HDAC6 and ubiquitin assays show that HDAC6‐ubiquitin interactions decreased in the presence of MK‐STYX, when HEK‐293 cells were exposed to MG132, whereas HDAC6‐ubiquitin interaction increased in cells expressing the active mutant of MK‐STYX (MK‐STYX(active)), which dephosphorylates phosphorylated residues. Overall, our research suggests that MK‐STYX affects ubiquitination; however, further studies are important to determine whether the effects of MK‐STYX on HDAC6 are linked to the decrease in stress granules.Support or Funding InformationDepartment of Biology, Integrated Science Center, William and Mary, Williamsburg, VA 23187

Activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting siRNA.

BackgroundFibroblast-like synoviocytes (FLSs), resident mesenchymal cells of synovial joints, play an important role in the pathogenesis of rheumatoid arthritis (RA). Dickkopf-1 (DKK-1) has been proposed to be a master regulator of bone remodeling in inflammatory arthritis. Here, potential impairation on the activity of FLSs derived from RA to small interfering RNAs (siRNAs) targeting DKK-1 was investigated.MethodssiRNAs targeting DKK-1 were transfected into FLSs of patients with RA. Interleukin (IL)-1β, IL-6, IL-8, matrix metalloproteinase (MMP) 2, MMP3, MMP9, transforming growth factor (TGF)-β1, TGF-β2 and monocyte chemoattractant protein (MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Invasion assay and 3H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation, respectively. Western blotting was performed to analyze the expression of nuclear factor (NF)-κB, interleukin-1 receptor-associated kinase (IRAK)1, extracellular regulated protein kinases (ERK)1, Jun N-terminal kinase (JNK) and β-catenin in FLSs.ResultsDKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs (P < 0.01). siRNAs induced a significant reduction of the levels of IL-6, IL-8, MMP2, MMP3 and MMP9 in FLSs compared to the control group (P < 0.05). DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs (P < 0.05). Important molecules of pro-inflammatory signaling in FLSs, including IRAK1 and ERK1, were decreased by the inhibition of DKK-1 in FLSs. In contrast, β-catenin, a pivotal downstream molecule of the Wnt signaling pathway was increased.ConclusionsBy inhibiting DKK-1, we were able to inhibit the proliferation, invasion and pro-inflammatory cytokine secretion of FLSs derived from RA, which was mediated by the ERK or the IRAK-1 signaling pathway. These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

TLR2 Signaling Pathway Combats Streptococcus uberis Infection by Inducing Mitochondrial Reactive Oxygen Species Production.

Mastitis caused by Streptococcus uberis (S. uberis) is a common and difficult-to-cure clinical disease in dairy cows. In this study, the role of Toll-like receptors (TLRs) and TLR-mediated signaling pathways in mastitis caused by S. uberis was investigated using mouse models and mammary epithelial cells (MECs). We used S. uberis to infect mammary glands of wild type, TLR2−/− and TLR4−/− mice and quantified the adaptor molecules in TLR signaling pathways, proinflammatory cytokines, tissue damage, and bacterial count. When compared with TLR4 deficiency, TLR2 deficiency induced more severe pathological changes through myeloid differentiation primary response 88 (MyD88)-mediated signaling pathways during S. uberis infection. In MECs, TLR2 detected S. uberis infection and induced mitochondrial reactive oxygen species (mROS) to assist host in controlling the secretion of inflammatory factors and the elimination of intracellular S. uberis. Our results demonstrated that TLR2-mediated mROS has a significant effect on S. uberis-induced host defense responses in mammary glands as well as in MECs.

Potential immune regulatory role of miR-146a upon Aeromonas hydrophila and Edwardsiella piscicida infections in zebrafish.

This study was aimed to understand the expression of miR-146a in zebrafish (Danio rerio) and its role in regulating immune responses during Aeromonas hydrophila and Edwardsiella piscicida infections. The miR-146a expression was observed from the 1-h post fertilization (hpf) stage and gradually increased up to the early larval stage of zebrafish. The ubiquitous expression of miR-146a was detected in all tested tissues, with the highest level in gills. The expression of miR-146a was significantly increased in larvae when exposed to E. piscicida infection at 24 and 48h post exposure (hpe). Intraperitoneally (i.p.) injected A. hydrophila and E. piscicida into adult zebrafish showed significant upregulation of miR-146a in gills. Furthermore, immune-related genes, toll-like receptor,tlr-4, transducing signaling pathway molecules, traf-6 and myd88 (bacteria-infected larvae and adults), transcription factor relA and mcp-1b (bacteria-infected adults), pro-inflammatory, il-6 (A. hydrophila-exposed larvae) and mmp-9 (bacteria-exposed larvae) were significantly repressed. In contrast, il-1β, tnf-α, cxcl-18b, and ccl-34a.4 were induced in both bacteria-challenged larvae and adults. Based on the results, it is suggested that endogenous miR-146a could act as an infection inducible miRNA in zebrafish upon A. hydrophila and E. piscicida infections; also, it could potentially regulate the immune responses in zebrafish.

Targeting BKCa Channels in Migraine: Rationale and Perspectives.

Large (big)-conductance calcium-activated potassium (BKCa) channels are expressed in migraine-related structures such as the cranial arteries, trigeminal ganglion and trigeminal spinal nucleus, and they play a substantial role in vascular tonus and neuronal excitability. Using synthetic BKCa channels openers was associated with headache as a frequent adverse effect in healthy volunteers. Additionally, BKCa channels are downstream molecules in migraine signalling pathways that are activated by several compounds known to provoke migraine, including calcitonin gene-related peptide (CGRP), pituitary adenylate cyclase-activating polypeptide (PACAP) and glyceryl trinitrate (GTN). Also, there is a high affinity and a close coupling between BKCa channels and ATP-sensitive potassium (KATP) channels, the role of which has recently been established in migraine pathophysiology. These observations raise the question as to whether direct BKCa channel activation can provoke migraine in migraine patients, and whether the BKCa channel could be a potential novel anti-migraine target. Hence, randomized and placebo-controlled clinical studies on BKCa channel openers or blockers in migraine patients are needed.

Bioactive compounds from dietary whole grape seed meal improved colonic inflammation via inhibition of MAPKs and NF-kB signaling in pigs with DSS induced colitis

Abstract The management of Inflammatory Bowel Diseases symptoms is focused to find new alternative strategies which could have beneficial effects on intestinal inflammation including the nutritional interventions. Using an in vivo approach together with gene and protein analysis, we investigated the effect of a diet with 8% grape seed meal on inflammatory markers and signaling pathway molecules. DSS-challenged pigs were used as IBD animal model. Our results showed that the DSS treatment induced a widespread inflammation at colon level by increasing a wide range of pro-inflammatory molecules via activation of pro-inflammatory p38/ERK/JNK MAPK signaling cascades followed by activation of NF-kB and other nuclear receptors. Diet with grape seed meal was efficient in counteracting inflammatory disorders by inhibiting MAPKs, attenuating NF-kB gene and protein expression and diminishing pro-inflammatory cytokines and chemokines production toward the control level. Grape seed meal could be used as alternative food supplements to reduce inflammation at intestinal level.