The Development of a New Vagus Nerve Simulation Electroceutical to Improve the Signal Attenuation in a Living Implant Environment.

An electroceutical is a medical device that uses electrical signals to control biological functions. It can be inserted into the human body as an implant and has several crucial advantages over conventional medicines for certain diseases. This research develops a new vagus nerve simulation (VNS) electroceutical through an innovative approach to overcome the communication limitations of existing devices. A phased array antenna with a better communication performance was developed and applied to the electroceutical prototype. In order to effectively respond to changes in communication signals, we developed the steering algorithm and firmware, and designed the smart communication protocol that operates at a low power that is safe for the patients. This protocol is intended to improve a communication sensitivity related to the transmission and reception distance. Based on this technical approach, the heightened effectiveness and safety of the prototype have been ascertained, with the actual clinical tests using live animals. We confirmed the signal attenuation performance to be excellent, and a smooth communication was achieved even at a distance of 7 m. The prototype showed a much wider communication range than any other existing products. Through this, it is conceivable that various problems due to space constraints can be resolved, hence presenting many benefits to the patients whose last resort to the disease is the VNS electroceutical.

Anti-Inflammatory Effects of Chamaecyparis obtusa (Siebold & Zucc.) Endl. Leaf Extract Fermented by Ganoderma applanatum Mycelia.

Corticosteroids are commonly used anti-inflammatory agents. However, their prolonged use can lead to side effects. Therefore, the development of natural compounds with minimal side effects is necessary. This study was performed to investigate the anti-inflammatory effects and mechanisms of action of Chamaecyparis obtusa (Siebold & Zucc.) Endl. leaf (COL), bioconverted using Ganoderma applanatum (G. applanatum) in lipopolysaccharide (LPS)-induced RAW264.7 cells. The COL 70% EtOH extract fermented by G. applanatum (70COLGA) improved the high cytotoxicity of 70% EtOH extracts (70COL). When RAW264.7 cells were pre-treated with 100 and 200 µg/mL of 70COLGA for 2 h and then treated with LPS for 16 h, LPS induced the production of nitric oxide (NO), and the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) were significantly inhibited. When RAW264.7 cells were pre-treated with 100 and 200 µg/mL of 70COLGA for 2 h and then treated with LPS for 4 h, the phosphorylation of signal transducers and activators of transcription (STAT) was markedly decreased. In addition, 70COLGA markedly suppressed the production of the inflammatory cytokines interleukin (IL)-1ß and IL-6 in LPS-induced RAW264.7 cells. Analysis of pro-inflammatory molecules using cytokine arrays showed that macrophage inflammatory protein (MIP)-2, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and IL-27 expressions were also suppressed by 200 µg/mL of 70COLGA in LPS-induced RAW264.7 cells. These results demonstrate that 70COLGA significantly prevented inflammatory responses by inhibiting the secretion of pro-inflammatory molecules in LPS-induced RAW264.7 cells. When RAW264.7 cells were pre-treated with 100 and 200 µg/mL of 70COLGA for 2 h and then treated with LPS-conditioned medium (LPS-CM) for 30 min, 70COLGA directly inhibited STAT activation. In summary, our findings suggest that 70COLGA has therapeutic potential for the treatment of inflammatory diseases.

Anti-Inflammatory Effect of Chamaecyparis obtusa (Siebold & Zucc.) Endl. Leaf Essential Oil.

Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) belongs to the Cupressaceae family and is native to East Asian regions. Essential oils extracted from the leaves, bark, branches, and roots of C. obtusa have both aesthetic and medicinal properties and are thus widely used. However, detailed analyses of the active ingredients of C. obtusa extract are lacking. In this study, the sabinene content in the hydro-distillation of C. obtusa leaf essential oil (COD) was analyzed using GC-MS, and the anti-inflammatory effect of COD was compared with that of pure sabinene. Cell viability was evaluated by MTT assay, and nitric oxide (NO) production was measured using Griess reagent. Relative mRNA and protein levels were analyzed using RT-qPCR and western blot, and secreted cytokines were analyzed using a cytokine array kit. The results showed that both COD and sabinene inhibited the expression of inducible nitric oxide synthase (iNOS) and the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in lipopolysaccharide (LPS)-induced RAW 264.7 cells. COD and sabinene also reduced the production of pro-inflammatory cytokines interleukin (IL)-1ß, IL-6, IL-27, IL-1 receptor antagonist (IL-1ra), and granulocyte-macrophage colony-stimulating factor (GM-CSF). The anti-inflammatory mechanisms of COD and sabinene partially overlap, as COD was shown to inhibit MAPKs and the JAK/STAT axis, and sabinene inhibited MAPKs, thereby preventing LPS-induced macrophage activation.

Protective Effects of Repetitive Transcranial Magnetic Stimulation Against Streptozotocin-Induced Alzheimer's Disease.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation under investigation for treatment of a wide range of neurological disorders. In particular, the therapeutic application of rTMS for neurodegenerative diseases such as Alzheimer's disease (AD) is attracting attention. However, the mechanisms underlying the therapeutic efficacy of rTMS have not yet been elucidated, and few studies have systematically analyzed the stimulation parameters. In this study, we found that treatment with rTMS contributed to restoration of memory deficits by activating genes involved in synaptic plasticity and long-term memory. We evaluated changes in several intracellular signaling pathways in response to rTMS stimulation; rTMS treatment activated STAT, MAPK, Akt/p70S6K, and CREB signaling. We also systematically investigated the influence of rTMS parameters. We found an effective range of applications for rTMS and determined the optimal combination to achieve the highest efficiency. Moreover, application of rTMS inhibited the increase in cell death induced by hydrogen peroxide. These results suggest that rTMS treatment exerts a neuroprotective effect on cellular damage induced by oxidative stress, which plays an important role in the pathogenesis of neurological disorders. rTMS treatment attenuated streptozotocin (STZ)-mediated cell death and AD-like pathology in neuronal cells. In an animal model of sporadic AD caused by intracerebroventricular STZ injection, rTMS application improved cognitive decline and showed neuroprotective effects on hippocampal histology. Overall, this study will help in the design of stimulation protocols for rTMS application and presents a novel mechanism that may explain the therapeutic effects of rTMS in neurodegenerative diseases, including AD.

Anti-inflammatory effects of Allium cepa L. peel extracts via inhibition of JAK-STAT pathway in LPS-stimulated RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Allium cepa L. (A. cepa) is one of the oldest cultivated plants in the world. A. cepa has been used in traditional folk medicine to treat inflammatory disease in several regions, such as Palestine and Serbia. A. cepa peel has a higher content of flavonoids, such as quercetin, than the edible parts. These flavonoids alleviate inflammatory diseases. However, the anti-inflammatory effects of A. cepa peel extract-obtained using various extraction methods-and their underlying mechanisms require further investigation. AIM OF THE STUDY: Although research to find safe anti-inflammatory substances in various natural products has been actively conducted for many years, it is important to continue identifying potential anti-inflammatory effects in natural materials. The purpose of this study was to investigate the ethnopharmacological properties of the A. cepa peel extract, whose efficacy when obtained through different extraction methods and underlying action mechanisms is not well known. The present study specifically aimed to observe the anti-inflammatory effects of the A. cepa peel extracts obtained using various extraction methods and the related detailed mechanisms of A. cepa peel extracts in lipopolysaccharide (LPS)-induced RAW264.7 cells. MATERIALS AND METHODS: The total flavonoid content of the A. cepa peel extracts was determined the diethylene glycol colorimetric method and measured using a calibration curve prepared using quercetin as a standard solution. The antioxidant activity was evaluated using the ABTS assay, and cytotoxicity was measured using the MTT assay. NO production was measured using Griess reagent. Protein levels were measured by western blotting, and mRNA expression was measured by RT-qPCR. Secreted cytokines were analyzed using ELISA or cytokine arrays. In the GSE160086 dataset, we calculated Z-scores for individual genes of interest and displayed using a heat map. RESULTS: Of the three A. cepa peel extracts obtained using different extraction methods, the A. cepa peel 50% EtOH extract (AP50E) was the most effective at inhibiting LPS-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Furthermore, AP50E significantly reduced the levels of pro-inflammation cytokines interleukin (IL)-1α, IL-1ß, IL-6, and IL-27. Additionally, AP50E directly inhibited the Janus kinase-signaling transducer and activator of transcription (JAK-STAT) pathway. CONCLUSIONS: These results showed that AP50E exhibited an anti-inflammatory effect in LPS-induced RAW264.7 mouse macrophages by directly inhibiting JAK-STAT signaling. Based on these findings, we propose AP50E as a potential candidate for the development of preventive or therapeutic agents against inflammatory diseases.

Pharmacological anti-tumor effects of natural Chamaecyparis obtusa (siebold & zucc.) endl. Leaf extracts on breast cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (C. obtusa, cypress species) is a plant that grows mainly in the temperate Northern Hemisphere and has long been used as a traditional anti-inflammatory treatment in East Asia. C. obtusa contains phytoncides, flavonoids, and terpenes, which have excellent anti-cancer effects and have been reported to prevent the progression of various cancers. However, the detailed mechanisms underlying the anti-cancer effects of C. obtusa extracts are unknown. AIM OF THE STUDY: We sought to confirm the anti-cancer effects of C. obtusa leaf extracts and to reveal the mechanism of action, with the possibility of its application in the treatment or prevention of cancer. MATERIAL &METHODS: The cytotoxicity of C. obtusa leaf extracts was confirmed using an MTT assay. Intracellular changes in protein levels were measured by immunoblotting, and mRNA levels were measured with qRT-PCR. Wound healing assay and transwell migration assay were used to evaluate the metastatic potential of breast cancer cells. The extract-induced apoptosis was observed using IncuCyte Annexin V Red staining analysis. A syngeneic breast cancer mouse model was established by injecting 4T1-Luc mouse breast cancer cells into the fat pad of female BALB/c mice, and the extract was administered orally. Luciferin solution was injected intraperitoneally to assess primary tumor development and metastasis by bioluminescence. RESULTS: C. obtusa leaf extracts were extracted with boiling water, 70% EtOH, and 99% EtOH. Among the extracts, the 99% EtOH extract of C. obtusa leaf (CO99EL) most clearly inhibited the tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (pY-STAT3) in MDA-MB-231 breast cancer cells at a concentration of 25 and 50 µg/mL. In addition, CO99EL strongly inhibited not only endogenous pY-STAT3 levels but also IL-6-induced STAT3 activation in various types of cancer cells, including breast cancer. CO99EL inhibited metastatic potential by downregulating the expression of N-cadherin, fibronectin, TWIST, MMP2, and MMP9 in MDA-MB-231 breast cancer cells. CO99EL also induced apoptotic cell death by increasing cleaved caspase-3 and decreasing anti-apoptotic proteins Bcl-2 and Bcl-xL. In an in vivo syngeneic breast cancer mouse model, 100 mg/kg CO99EL suppressed tumor growth and induced apoptosis of cancer cells. Moreover, CO99EL significantly inhibited lung metastasis from primary breast cancer. CONCLUSIONS: Our study demonstrated that 100 mg/kg CO99EL has potent anti-tumor effects against breast cancer, thus suggesting that 100 mg/kg CO99EL has potential applications in the treatment and prevention of breast cancer.

High-harmonic generation from a flat liquid-sheet plasma mirror.

High-harmonic radiation can be generated when an ultra-intense laser beam is reflected from an over-dense plasma, known as a plasma mirror. It is considered a promising technique for generating intense attosecond pulses in the extreme ultraviolet and X-ray wavelength ranges. However, a solid target used for the formation of the over-dense plasma is completely damaged by the interaction. Thus, it is challenging to use a solid target for applications such as time-resolved studies and attosecond streaking experiments that require a large amount of data. Here we demonstrate that high-harmonic radiation can be continuously generated from a liquid plasma mirror in both the coherent wake emission and relativistic oscillating mirror regimes. These results will pave the way for the development of bright, stable, and high-repetition-rate attosecond light sources, which can greatly benefit the study of ultrafast laser-matter interactions.

Loss of phospholipase Cγ1 suppresses hepatocellular carcinogenesis through blockade of STAT3-mediated cancer development.

Phospholipase C gamma 1 (PLCγ1) plays an oncogenic role in several cancers, alongside its usual physiological roles. Despite studies aimed at identifying the effect of PLCγ1 on tumors, the pathogenic role of PLCγ1 in the tumorigenesis and development of hepatocellular carcinoma (HCC) remains unknown. To investigate the function of PLCγ1 in HCC, we generated hepatocyte-specific PLCγ1 conditional knockout (PLCγ1f/f ; Alb-Cre) mice and induced HCC with diethylnitrosamine (DEN). Here, we identified that hepatocyte-specific PLCγ1 deletion effectively prevented DEN-induced HCC in mice. PLCγ1f/f ; Alb-Cre mice showed reduced tumor burden and tumor progression, as well as a decreased incidence of HCC and less marked proliferative and inflammatory responses. We also showed that oncogenic phenotypes such as repressed apoptosis, and promoted proliferation, cell cycle progression and migration, were induced by PLCγ1. In terms of molecular mechanism, PLCγ1 regulated the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Moreover, PLCγ1 expression is elevated in human HCC and correlates with a poor prognosis in patients with HCC. Our results suggest that PLCγ1 promotes the pathogenic progression of HCC, and PLCγ1/STAT3 axis was identified as a potential therapeutic target pathway for HCC.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

Targeted drug release system based on pH-responsive PAA-POSS nanoparticles.

An amphipathic PAA-POSS@DOX drug delivery system that responds sensitively to pH changes in the cancer microenvironment has been developed using a nanoparticle based on inorganic polyhedral oligomeric silsesquioxane (POSS). POSS was introduced to the carboxylic acid group of polyacrylic acid to which doxorubicin anticancer drug was loaded to prepare 480 ± 192 nm self-assembled nanoparticles. PAA-POSS had a high loading efficiency of over 75% and doxorubicin was quickly released to the target area responding sensitively to weakly acidic conditions. The possibility of employing PAA-POSS as a targeted drug delivery system has been confirmed by observing the death of cells of the MDA-MB-231 breast cancer line.

Chamaecyparis obtusa (Siebold & Zucc.) Endl. leaf extracts prevent inflammatory responses via inhibition of the JAK/STAT axis in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) has been used as folk medicine in East Asia and has been reported to alleviate inflammatory diseases. However, the detailed mechanisms for the anti-inflammatory effects of C. obtusa remain unclear. AIM OF THE STUDY: Although the anti-inflammatory mechanisms of natural products have been studied for decades, it is still important to identify the potential anti-inflammatory effects of natural sources. In this study, we investigated the anti-inflammatory effects and underlying mechanism of C. obtusa leaf extracts. MATERIAL &METHODS: The cell viability was determined by MTT and crystal violet staining. NO production in the supernatant was measured using Griess reagent. The cell lysates were analyzed by immunoblotting and RT-qPCR. Secreted cytokines were analyzed using ELISA kit and cytokine array kit. mRNA expression from the GSE9632 database set. Z-scores were calculated for each gene and visualized by heat map. RESULTS: Among the extracts of C. obtusa obtained with different extraction methods, the 99% ethanol leaf extract (CO99EL) strongly inhibited lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and Janus kinase/signaling transducer and activator of transcription (JAK/STAT) phosphorylation in RAW264.7 cells. In addition, CO99EL strongly inhibited LPS-induced interleukin (IL)-1ß, IL-6, IL-27, and C-C motif chemokine ligand (CCL)-1 production and directly inhibited LPS-induced JAK/STAT phosphorylation in RAW264.7 cells. CONCLUSIONS: These findings demonstrate that CO99EL significantly prevents LPS-induced macrophage activation by inhibiting the JAK/STAT axis. Therefore, we suggest the use of C. obtusa extracts as therapeutic approach for inflammatory diseases.

3D Vehicle Trajectory Extraction Using DCNN in an Overlapping Multi-Camera Crossroad Scene.

The 3D vehicle trajectory in complex traffic conditions such as crossroads and heavy traffic is practically very useful in autonomous driving. In order to accurately extract the 3D vehicle trajectory from a perspective camera in a crossroad where the vehicle has an angular range of 360 degrees, problems such as the narrow visual angle in single-camera scene, vehicle occlusion under conditions of low camera perspective, and lack of vehicle physical information must be solved. In this paper, we propose a method for estimating the 3D bounding boxes of vehicles and extracting trajectories using a deep convolutional neural network (DCNN) in an overlapping multi-camera crossroad scene. First, traffic data were collected using overlapping multi-cameras to obtain a wide range of trajectories around the crossroad. Then, 3D bounding boxes of vehicles were estimated and tracked in each single-camera scene through DCNN models (YOLOv4, multi-branch CNN) combined with camera calibration. Using the abovementioned information, the 3D vehicle trajectory could be extracted on the ground plane of the crossroad by calculating results obtained from the overlapping multi-camera with a homography matrix. Finally, in experiments, the errors of extracted trajectories were corrected through a simple linear interpolation and regression, and the accuracy of the proposed method was verified by calculating the difference with ground-truth data. Compared with other previously reported methods, our approach is shown to be more accurate and more practical.

Insulin smart drug delivery nanoparticles of aminophenylboronic acid-POSS molecule at neutral pH.

Self-regulated "smart" insulin administration system that mimic pancreatic endocrine function would be highly desirable for diabetes management. Here, a glucose-responsive continuous insulin delivery system is developed, where novel polyhedral oligosilsesquioxane (POSS) modified with 3-aminophenylboronic acid (APBA) were used to encapsulate insulin (insulin entrapment efficiency: 73.2%) to prepare a fast response, high stability, good distribution, and excellent biocompatible system. Due to the strong hydrophobicity of POSS, the POSS moiety is located at the core in aqueous solution and combines with the boronic group of APBA and the diol generated in PEG-insulin to form a nanomicelle structure, that is, nanoparticles naturally. Micelles self-assembled from these molecules possess glucose-responsiveness at varying glucose concentrations. The interaction of the PBA and diol containing insulin via boronate ester bond and its interchange with glucose was investigated by FT-IR, 1H NMR and XPS. Furthermore, the successful glucose-triggered release of insulin from the POSS-APBA micelles was investigated at neutral pH. A linear graph was plotted with the measured released insulin vs glucose concentrations, with a linear correlation coefficient (R2) value close to 1. Circular dichroism (CD) spectroscopy analysis was performed to measure insulin activity by comparing secondary structures of insulin, PEG-Insulin, and POSS-APBA@insulin. When confirming intracellular apoptosis signaling, cleaved caspase 3 and caspase 9 were not increased by 640 µg/ml POSS-APBA and POSS-APBA@insulin in HeLa, HDF and HUVE cells. Application in the biomedical field for controlled delivery of insulin appear to be promising.

Extracellular Acidosis Promotes Metastatic Potency via Decrease of the BMAL1 Circadian Clock Gene in Breast Cancer.

Circadian oscillation is an essential process that influences many physiological and biological mechanisms and a decrease of circadian genes is associated with many diseases such as cancer. Despite many efforts to identify the detailed mechanism for decreasing circadian genes and recovering reduced circadian genes in cancer, it is still largely unknown. We found that BMAL1 was reduced in tumor hypoxia-induced acidosis, and recovered by selectively targeting acidic pH in breast cancer cell lines. Surprisingly, BMAL1 was reduced by decrease of protein stability as well as inhibition of transcription under acidosis. In addition, melatonin significantly prevented acidosis-mediated decrease of BMAL1 by inhibiting lactate dehydrogenase-A during hypoxia. Remarkably, acidosis-mediated metastasis was significantly alleviated by BMAL1 overexpression in breast cancer cells. We therefore suggest that tumor hypoxia-induced acidosis promotes metastatic potency by decreasing BMAL1, and that tumor acidosis could be a target for preventing breast cancer metastasis by sustaining BMAL1.

Successful management of proteinuria and systemic hypertension in a dog with renal cell carcinoma with surgery, telmisartan, and amlodipine.

An 11-year-old neutered male Yorkshire terrier dog was presented with a 3-week history of hematuria and anorexia. A unilateral renal mass was detected and surgically removed. The renal mass was diagnosed on histopathologic examination as a renal carcinoma. Supportive medical therapy was carried out and persistent systemic hypertension was managed using telmisartan.

Gestion réussie de la protéinurie et de l'hypertension systémique chez un chien atteint d'un carcinome rénal à l'aide d'une chirurgie, de telmisartan et d'amlodipine. Un chien Yorkshire terrier mâle stérilisé âgé de 11 ans a été présenté avec une anamnèse de 3 semaines d'hématurie et d'anorexie. Une masse rénale unilatérale a été détectée et excisée par chirurgie. La masse rénale a été diagnostiquée à l'examen histopathologique comme étant un carcinome rénal. Une thérapie médicale de soutien a été réalisée et l'hypertension systémique persistante a été gérée à l'aide de telmisartan.(Traduit par Isabelle Vallières).

Dysfunction of Microglial STAT3 Alleviates Depressive Behavior via Neuron-Microglia Interactions.

Neuron-microglia interactions have a crucial role in maintaining the neuroimmune system. The balance of neuroimmune system has emerged as an important process in the pathophysiology of depression. However, how neuron-microglia interactions contribute to major depressive disorders has been poorly understood. Herein, we demonstrated that microglia-derived synaptic changes induced antidepressive-like behavior by using microglia-specific signal transducer and activator of transcription 3 (STAT3) knockout (KO) (STAT3fl/fl;LysM-Cre+/-) mice. We found that microglia-specific STAT3 KO mice showed antidepressive-like behavior in the forced swim, tail suspension, sucrose preference, and open-field tests. Surprisingly, the secretion of macrophage colony-stimulating factor (M-CSF) was increased from neuronal cells in the brains of STAT3fl/fl;LysM-Cre+/- mice. Moreover, the phosphorylation of antidepressant-targeting mediators and brain-derived neurotrophic factor expression were increased in the brains of STAT3fl/fl;LysM-Cre+/- mice as well as in neuronal cells in response to M-CSF stimulation. Importantly, the miniature excitatory postsynaptic current frequency in the medial prefrontal cortex was increased in STAT3fl/fl;LysM-Cre+/- mice and in the M-CSF treatment group. Collectively, microglial STAT3 regulates depression-related behaviors via neuronal M-CSF-mediated synaptic activity, suggesting that inhibition of microglial STAT3 might be a new therapeutic strategy for depression.

Differential expressions of NOD-like receptors and their associations with inflammatory responses in rheumatoid arthritis.

OBJECTIVES: To determine the differential expressions of nucleotide oligomerisation domain (NOD)-like receptors (NLRs) and to investigate their association with inflammatory responses in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). METHODS: Gene expression and protein levels of various NLRs, including NOD1, NOD2, NLRP1, NLRP3, NLRP12, NLRX1, and NLRC3, were determined in FLS and synovial tissues from patients with RA and patients with osteoarthritis (OA) using quantitative real-time PCR and immunohistochemistry. After transfection of NOD2 RNAi plasmids or a pcDNA3.1-NLRX1 vector, gene expression levels of pro-inflammatory cytokines in RA FLS and the protein levels of these cytokines in culture supernatants were determined using quantitative real-time PCR and enzyme-linked immunosorbent assays. The effects of NLR gene regulation on NF-κB and caspase-1 were evaluated using Western blot analysis. RESULTS: Gene expression levels of NOD1, NLRP1, NLRP3, NLRP12, and NLRC3 were not different between RA and OA samples. NOD2 gene expression and protein levels were significantly increased in RA samples, whereas the levels of NLRX1 were significantly decreased. Downregulation of NOD2 gene expression by transfection with NOD2 RNAi plasmid significantly reduced pro-inflammatory cytokine levels in RA FLS, while transfection with adenoviral vectors encoding NLRX1 had no effect on pro-inflammatory cytokine levels. Downregulation of NOD2 gene expression significantly decreased NF-κB, TRAF6, and IKK levels, but not caspase-1 levels, in RA FLS. CONCLUSIONS: NOD2 is upregulated in RA FLS; moreover, downregulation of NOD2 gene expression reduces pro-inflammatory cytokine and NF-κB levels in RA FLS. These findings provide evidence that NOD2 exerts pro-inflammatory effects in RA.

Secreted frizzled-related protein 5 suppresses inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes through down-regulation of c-Jun N-terminal kinase.

OBJECTIVE: This study was performed to investigate the effect of secreted frizzled-related protein 5 (Sfrp5), a novel anti-inflammatory adipokine that competes with the frizzled proteins for Wnt binding, on inflammatory response and the c-Jun N-terminal kinase (JNK) signalling pathway in RA. METHODS: Expression of Sfrp5 mRNA in peripheral blood mononuclear cells (PBMCs) and fibroblast-like synoviocytes (FLSs) from patients with RA and OA was determined using real-time quantitative PCR (qPCR). Sfrp5 RNA interference (RNAi) plasmids were transfected to abrogate Sfrp5 expression in RA FLSs, and adenovirus containing the Sfrp5 transcript was delivered into RA FLSs to strengthen Sfrp5 expression. Levels of pro-inflammatory genes and their protein products were determined using real-time qPCR and ELISA in RA FLSs. Production of mitogen-activated protein kinase kinase 7 (MKK-7), JNK and c-Jun were assessed by Western blot analysis. RESULTS: Expression of Sfrp5 mRNA was decreased in PMBCs and FLSs from patients with RA compared with patients with OA. Gene expression and production of IL-1ß, IL-6, chemokine ligand 2 (CCL-2), CCL-7, cyclooxygenase 2 and MMP-9 were markedly increased in Sfrp5 RNAi plasmid-transfected RA FLSs, while transfection with adenoviral vectors encoding Sfrp5 induced reductions in those levels. Phosphorylated forms of MKK-7, JNK and c-Jun were increased by Sfrp5 RNAi plasmids and were decreased by adenoviral vectors encoding Sfrp5. CONCLUSION: Sfrp5 suppressed the inflammatory response and down-regulated JNK signalling in RA FLSs. These findings provide evidence for the anti-inflammatory effect of Sfrp5 in RA.