Nuclear factor-κB Inhibitor Research Articles

Short-term salicylate treatment improves microvascular endothelium-dependent dilation in young adults with major depressive disorder.

Reactive oxygen species (ROS)-mediated reductions in nitric oxide (NO)-dependent dilation are evident in adults with major depressive disorder (MDD); however, the upstream mechanisms remain unclear. Here, we hypothesized that nuclear factor-κB (NF-κB) activation-induced ROS production contributes to microvascular endothelial dysfunction in MDD. Thirteen treatment-naive adults with MDD (6 women; 19-23 yr) and 10 healthy nondepressed adults (HAs; 5 women; 20-25 yr) were tested before and after (open-label design) systemic NF-κB knockdown (nonacetylated salicylate; 3,000-4,500 mg/day × 4 days). Red cell flux (laser Doppler flowmetry) was measured during graded intradermal microdialysis perfusion of the endothelium-dependent agonist acetylcholine (ACh), alone and in combination with NO synthase inhibition [NG-nitro-l-arginine methyl ester (l-NAME)] or ROS scavenging (apocynin). Serum salicylate concentrations following treatment were not different between groups (22.8 ± 7.4 HAs vs. 20.8 ± 4.3 mg/dL MDD; P = 0.46). When compared with HAs, the NO-dependent component of ACh-induced dilation was blunted in adults with MDD before (P = 0.023), but not after (P = 0.27), salsalate treatment. In adults with MDD, the magnitude of improvement in endothelium-dependent dilation following salsalate treatment was inversely related to the degree of functional impairment at baseline (R2 = 0.43; P = 0.025). Localized ROS scavenging improved NO-dependent dilation before (P < 0.01), but not after (P > 0.05), salsalate treatment. Salsalate did not alter systemic concentrations of pro- or anti-inflammatory cytokines (all P > 0.05). These data suggest that NF-κB activation, via increased vascular ROS production, contributes to blunted NO-dependent dilation in young adults with MDD but otherwise free of clinical disease. These data provide the first direct evidence for a mechanistic role of vascular inflammation-associated endothelial dysfunction in human depression.NEW & NOTEWORTHY Our data indicate that short-term treatment with therapeutic doses of the nuclear factor-κB (NF-κB) inhibitor salsalate improved nitric oxide (NO)-mediated endothelium-dependent dilation in adults with major depressive disorder (MDD). In adults with MDD, acute localized scavenging of reactive oxygen species (ROS) with apocynin improved NO-dependent dilation before, but not after, salsalate administration. These data suggest that activation of NF-κB, in part via stimulation of vascular ROS production, contributes to blunted NO-mediated endothelium-dependent dilation in young adults with MDD.

Hyperoxia Induced Bronchopulmonary Dysplasia-Like Inflammation via miR34a-TNIP2-IL-1β Pathway.

Lung injury induced by oxygen is a key contributor to the pathogenesis of preterm infant bronchopulmonary dysplasia (BPD). To date, there are comprehensive therapeutic strategy for this disease, but the underlying mechanism is still in progress. By using lentivirus, we constructed microRNA34a (miR34a)-overexpressing or knockdown A549 cell lines, and exposure to hyperoxia to mimic oxygen induce lung injury. In this study, we investigated 4 proinflammatory cytokines, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), angiopoietin-1 (Ang-1), and Cyclooxygenase-2 (COX-2) in the secreted sputum of infants who received mechanical ventilation, and found that IL-1β was substantially elevated in the first week after oxygen therapy and with no significant decrease until the fourth week, while TNF-α, Ang-1, and COX-2 were increased in the first week but decreased quickly in the following weeks. In addition, in vitro assay revealed that hyperoxia significantly increased the expression of miR-34a, which positively regulated the proinflammatory cytokine IL-1β in a time- and concentration-dependent manner in A549 cells. Overexpressing or knockdown miR34 would exacerbate or inhibit production of IL-1β and its upstream NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome signaling pathway. Mechanically, it’s found that TNFAIP3 interacting protein 2 (TNIP2), an inhibitor of nuclear factor κB (NF-κB), is a direct target of miR34a, negatively regulated activation of NLRP3 inflammasome and the production of IL-1β. Overexpressing TNIP2 ameliorated hyperoxia-induced production of IL-1β and cell apoptosis. Our findings suggest that TNIP2 may be a potential clinical marker in the diagnosis of BPD.

Electroacupuncture ameliorates neuroinflammation in animal models.

Neuroinflammation refers to a wide range of immune responses occurring in the brain or spinal cord. It is closely related to a variety of neurodegenerative diseases, for which it potentially represents a new direction for treatment. Electroacupuncture (EA) is one method of acupuncture treatment, which can be used as an adjuvant therapy for many diseases. This review focuses on molecular mechanisms of EA in the reduction of neuroinflammation, summarizes relevant basic research and outlines future directions for investigation. A growing body of basic research has shown that EA can ameliorate neuroinflammation centrally (in animal models of ischemic stroke, Alzheimer's disease, traumatic brain injury, spinal cord injury, Parkinson's disease and vascular dementia) and peripherally (e.g. after a surgical insult or injection of lipopolysaccharide) and that its effects involve different molecular mechanisms, including activation of the α7 nicotinic acetylcholine receptor signaling pathway and P2 type purinergic receptors, inhibition of nuclear factor κB, and mitigation of damage secondary to oxidative stress and NOD-like receptor protein 3 inflammasome activation. EA is capable of regulating multiple cell signal transduction pathways to alleviate neuroinflammation in animal models. Although the findings of animal studies are encouraging, further prospective clinical trials are needed to verify the efficacy of EA for the treatment of neuroinflammation.

The Antiviral Effects of Jasminin via Endogenous TNF-α and the Underlying TNF-α-Inducing Action.

Previous studies have reported that recombinant tumor necrosis factor (TNF)-α has powerful antiviral activity but severe systematic side effects. Jasminin is a common bioactive component found in Chinese herbal medicine beverage “Jasmine Tea”. Here, we report that jasminin-induced endogenous TNF-α showed antiviral activity in vitro. The underlying TNF-α-inducing action of jasminin was also investigated in RAW264.7 cells. The level of endogenous TNF-α stimulated by jasminin was first analyzed by an enzyme-linked immunosorbent assay (ELISA) from the cell culture supernatant of RAW264.7 cells. The supernatants were then collected to investigate the potential antiviral effect against herpes simplex virus 1 (HSV-1). The antiviral effects of jasminin alone or its supernatants were evaluated by a plaque reduction assay. The potential activation of the PI3K–Akt pathway, three main mitogen-activated protein kinases (MAPKs), and nuclear factor (NF)–κB signaling pathways that induce TNF-α production were also investigated. Jasminin induces TNF-α protein expression in RAW264.7 cells without additional stimuli 10-fold more than the control. No significant up-expression of type I, II, and III interferons; interleukins 2 and 10; nor TNF-β were observed by the jasminin stimuli. The supernatants, containing jasminin-induced-TNF-α, showed antiviral activity against HSV-1. The jasminin-stimulated cells caused the simultaneous activation of the Akt, MAPKs, and NF–κB signal pathways. Furthermore, the pretreatment of the cells with the Akt, MAPKs, and NF–κB inhibitors effectively suppressed jasminin-induced TNF-α production. Our research provides evidence that endogenous TNF-α can be used as a strategy to encounter viral infections. Additionally, the Akt, MAPKs, and NF–κB signaling pathways are involved in the TNF-α synthesis that induced by jasminin.

The alleviative effect of flavonol-type Nrf2 activator rhamnazin from Physalis alkekengi L. var. franchetii (Mast.) Makino on pulmonary disorders.

Rhamnazin (RN) is a flavonol isolated from the calyxes and fruits of Physalis alkekengi L. var. franchetii (Mast.) Makino, which has been used for treating pulmonary diseases in traditional Chinese medicine. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a therapeutic target for pulmonary diseases. In the present study, the underlying mechanism and pharmacological effect of RN against pulmonary disorders are investigated. Human lung epithelial Beas-2B cell and RAW 264.7 murine macrophage-based cell models, and a cigarette smoke (CS)-induced pulmonary impairment mice model are adopted for investigation in vitro and in vivo. RN is identified to be an Nrf2 activator, which promotes Nrf2 dissociation from Keap1 via reacting with the Cys151 cysteine residue of Keap1, and suppresses Nrf2 ubiquitination. In addition, RN is able to attenuate toxicant-stimulated oxidative stress and inflammatory response in vitro. Importantly, RN significantly relieves CS-induced oxidative insult and inflammation, and RN-induced inhibition of inflammation is related to inhibition of nuclear transcription factor-κB (NF-κB) and induction of cell autophagy. In conclusion, our data indicate that RN is an activator of the Nrf2 pathway and evidently alleviates pulmonary disorders via restricting NF-κB activation and promoting autophagy. RN is a promising candidate for the therapy of pulmonary disorders.

Chinese herbal component, Praeruptorin E, enhances anti-asthma efficacy and prevents toxicity of aminophylline by targeting the NF-κB/PXR/CYP3A4 pathway

BackgroundAminophylline is widely used for the treatment of asthma, but the therapeutic dose is very close to the toxic dose, which makes this drug prone to accumulation poisoning. In the present study, we explored whether the Chinese herbal component, Praeruptorin E (PE), enhances anti-asthma efficacy and prevents the toxicity of aminophylline.MethodsFirst, an ovalbumin (OVA)-induced mouse model of asthma, immunohistochemistry, pathological staining, and bronchoalveolar lavage fluid (BALF) were used to detect the lung condition of asthmatic mice. The content of Th2 cytokines in serum was measured by enzyme-linked immunosorbent assay (ELISA), and the expression of related proteins was detected by Western blotting and immunofluorescence. Concentrations of theophylline and its metabolites in rat serum were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). siRNA transfection and chromatin immunoprecipitation (ChIP) were used to investigate the mechanism of PE.ResultsPE was found to synergize with aminophylline to reduce the infiltration of inflammatory cells, collagen deposition, and mucus hyperplasia in the lungs of asthmatic mice. It inhibited the expression of Th2 cytokines, interleukin (IL)-4, IL-5, and IL-13; promoted lung tissue repair; and reduced the toxic effect of aminophylline on the heart. Moreover, LC-MS/MS analysis showed that PE reduced the plasma concentration of the parent theophylline and its metabolite 1,3-dimethyluric acid (1,3-DMU). PE facilitated aminophylline’s suppression of nuclear factor-κB (NF-κB), and increased the expression of the xenobiotic nuclear receptor pregnane X receptor (PXR) and its primary target gene, CYP3A11 [this is the mouse homolog of cytochrome P450 3A (CYP3A)] in the asthmatic mouse liver and in the L-02 human fetal hepatocyte cell culture model. In addition, the ChIP assay revealed that PE attenuated the binding of NF-κB to the promoter region of the PXR gene and prevented the suppression of PXR gene expression by NF-κB.ConclusionsPE has a dual function in enhancing the immune regulation and anti-inflammatory effects of theophylline, as well as preventing theophylline toxicity by targeting the NF-κB/PXR/CYP3A4 axis. PE is a promising herbal medicine that will benefit asthmatics taking theophylline.

Ubiquitin-specific peptidase 2 inhibits epithelial-mesenchymal transition in clear cell renal cell carcinoma metastasis by downregulating the NF-κB pathway

ABSTRACT Clear cell renal cell carcinoma, the most common type of renal cancer, is associated with poor survival. Ubiquitin-specific peptidase 2 regulates the molecular mechanisms of cancer cells. However, its mechanism in clear cell renal cell carcinoma remains unclear. Quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry were performed to assess ubiquitin-specific peptidase 2 expression in human clear cell renal cell carcinoma samples. Ubiquitin-specific peptidase 2 was weakly expressed in clear cell renal cell carcinoma samples and associated with poor patient outcomes. Ubiquitin-specific peptidase 2 inhibition promoted clear cell renal cell carcinoma cell proliferation, migration, and invasion. Ubiquitin-specific peptidase 2 overexpression inhibited clear cell renal cell carcinoma cell proliferation, migration, and invasion in vitro and in vivo. RNA-sequencing showed significant changes in the epithelial-mesenchymal transition-related pathways following ubiquitin-specific peptidase 2 knockdown. Western blotting was performed to detect the protein expression levels. Expression of p-nuclear factor-κB p65, N-cadherin, Vimentin, and Snail, which were markedly increased, as well as E-cadherin, which was decreased following ubiquitin-specific peptidase 2 knockdown. Rescue experiments using the nuclear factor-κB inhibitor BAY 11–7082 revealed that the migration and invasion abilities and the expression of epithelial-mesenchymal transition pathway proteins were inhibited in both the short hairpin RNA (shRNA) for ubiquitin-specific peptidase 2 and shRNA for negative control groups. Ubiquitin-specific peptidase 2 is a potential biomarker to distinguish clear cell renal cell carcinoma patients from healthy individuals. Ubiquitin-specific peptidase 2-mediated inhibition of epithelial-mesenchymal transition in clear cell renal cell carcinoma cells is dependent on the nuclear factor-κB pathway.

Interleukin-24 Limits Tumor-Infiltrating T Helper 17 Cell Response in Patients with Hepatitis B Virus-Related Hepatocellular Carcinoma.

Interleukin (IL)-24 is a multifunction cytokine in infectious diseases and cancers. IL-17-secreting CD4+ T (Th17) and CD8+ T (Tc17) cells promotes pathogenesis of hepatitis B virus (HBV)-associated liver disorders. However, the regulatory role of IL-24 to Th17/Tc17 cell response was not fully elucidated during HBV infection and HBV-hepatocellular carcinoma (HCC). In this study, plasma and peripheral blood mononuclear cells were isolated from 27 chronic hepatitis B (CHB) patients, 42 HBV-HCC patients and 17 normal controls (NC). Liver-infiltrating lymphocytes (LILs) were prepared from tumor and para-tumor tissues of 17 HBV-HCC patients, whereas CD4+ T cells in LILs were purified. LILs were stimulated with recombinant human IL-24. CD3+CD4+IL-17+ Th17 cells and CD3+CD8+IL-17+ Tc17 cells were investigated by flow cytometry. IL-24 level was measured by enzyme-linked immunosorbent assay. Purified CD4+ T cells were polarized for Th17 cells, and the regulatory role of IL-24 to liver-infiltrating Th17 polarization was assessed. There were no significant differences of peripheral Th17 or Tc17 cell percentage among NC, CHB, and HBV-HCC patients. Liver-infiltrating Th17 and Tc17 cell proportion was reduced in tumor tissues compared with para-tumor tissues. In contrast, plasma IL-24 level was increased in CHB and HBV-HCC patients. Exogenous IL-24 stimulation (10 or 100 ng/mL) in vitro downregulated of Th17 frequency and IL-17 secretion in LILs from both para-tumor and tumor tissues without affecting cellular proliferation or Tc17 percentage. Only 100 ng/mL of IL-24 inhibited tumor-infiltrating Th17 polarization, and this process was accompanied by suppression of nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase phosphorylation. In conclusion, IL-24 might dampen Th17 cell response through NF-κB pathway in HBV-HCC tumor microenvironment. Elevated IL-24 might enhance anti-tumor immune response in HBV-HCC patients.

Inhibition of nuclear factor κB in the lungs protect bleomycin-induced lung fibrosis in mice

BackgroundPulmonary fibrosis is a debilitating condition with limited therapeutic avenues. The pathogenicity of pulmonary fibrosis constitutes involvement of cellular proliferation, activation, and transformational changes of fibroblast to myofibroblasts. It is a progressive lung disease and is primarily characterized by aberrant accumulation of extracellular matrix proteins in the lungs with poor prognosis. The inflammatory response in the pathogenesis of lung fibrosis is suggested because of release of several cytokines; however, the underlying mechanism remains undefined. A genetic model is the appropriate way to delineate the underlying mechanism of pulmonary fibrosis.Methods and resultsIn this report, we have used cc-10 promoter based IκBα mutant mice (IKBM, an inhibitor of NF-κB) which were challenged with bleomycin (BLM). Compared to wild-type (WT) mice, the IKBM mice showed significant reduction in several fibrotic, vascular, and inflammatory genes. Moreover, we have identified a new set of dysregulated microRNAs (miRNAs) by miRNA array analysis in BLM-induced WT mice. Among these miRNAs, let-7a-5p and miR-503-5p were further analyzed. Our data showed that these two miRNAs were upregulated in WT-BLM and were reduced in IKBM-BLM mice. Bioinformatic analyses showed that let-7a-5p and miR-503-5p target for endothelin1 and bone morphogenic receptor 1A (BMPR1A), respectively, and were downregulated in WT-BLM mice indicating a link in pulmonary fibrosis.ConclusionWe concluded that inhibition of NF-κB and modulation of let-7a-5p and miR-503-5p contribute a pivotal role in pulmonary fibrosis and may be considered as possible therapeutic target for the clinical management of lung fibrosis.

Nephrotoxicity of Calcineurin Inhibitors in Kidney Epithelial Cells is Independent of NFAT Signaling.

Background: Calcineurin inhibitors (CNIs) such as cyclosporine A and tacrolimus are commonly used after renal transplantation to suppress the immune system. In lymphoid cells, cyclosporine A acts via the calcineurin/nuclear factor of activated T-cell (NFAT) axis. In non-lymphoid cells, such as kidney epithelial cells, cyclosporine A induces calcineurin inhibitor toxicity. It is unknown via which off-targets cyclosporine A induces calcineurin inhibitor toxicity in kidney epithelial cells. Methods: To measure a compound’s potential to induce nephrotoxicity, the expression of the surrogate marker Fn14 was measured by flow cytometry. Compounds were tested for their potential to induce Fn14 either chemically or plasmid-mediated. Mice were injected with various compounds, and changes in nephrotoxic gene expression levels of the kidney epithelial cells were then analyzed. Results: Fn14 is specifically upregulated due to calcineurin inhibitor toxicity inducing agents. Inhibition of the NFAT axis showed no increase of the Fn14 expression on the surface of kidney cells. However, inhibition of p38 MAPK, phosphoinositide-3-kinase (PI3K)/Akt, protein kinase C (PKC), and inhibitor of nuclear factor-κB (IκB) kinase (IKK) showed clear induction of Fn14 and increased expressions of nephrotoxic, inflammatory, and fibrotic genes in vitro and in vivo. Conclusions: These findings show that cyclosporine A acts independently of NFAT on kidney epithelial cells. Moreover, inhibition of serine/threonine protein kinases mimics cyclosporine A’s activity on kidney epithelial cells. This mimicking effect indicates that these protein kinases are off-targets of cyclosporine A and damage structural renal cells when inhibited and therefore contributes likely to the development and progression of calcineurin inhibitor toxicity.

Preventive effects of cristacarpin on experimentally induced uveitis by targeting NF-κB.

Cristacarpin is a novel prenylated pterocarpan that reportedly exhibits broad anti-cancer activity by enhancing endoplasmic reticulum stress. However, whether and how cristacarpin affects in-flammatory processes remain largely unknown. In the present study, the anti-inflammatory effect of cristacarpin on lipopolysaccharide (LPS)-induced inflammation was investigated using zebrafish embryos, RAW 264.7 macrophages, and mouse uveitis models. In the non-toxic concentration range (from 20 to 100μM), cristacarpin suppressed pro-inflammatory mediators such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while stimulating anti-inflammatory mediators such as IL-4 and IL-10 in LPS-stimulated RAW 264.7 cells and uveitis mouse models. Cristacarpin decreased cell adhesion of macrophages through downregulation of the expression of Ninjurin1 and matrix metalloproteinases. Furthermore, cristacarpin reduced macrophage migration in zebrafish embryos in vivo. Cristacarpin also increased cytosolic levels of inhibitor of nuclear factor-κB and suppressed the nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells. Collectively, our results suggest that cristacarpin is a potential therapeutic candidate for developing ocular anti-inflammatory drugs.

Tianxiangdan Improves Coronary Microvascular Dysfunction in Rats by Inhibiting Microvascular Inflammation via Nrf2 Activation.

Background Tianxiangdan (TXD) is used in traditional Chinese medicine because of its therapeutic and preventive effects in the treatment of coronary heart disease. However, the underlying mechanism of TXD in coronary microvascular disease (CMD) remains unclear. Methods A rat model of CMD was developed to study the mechanism of TXD activity. Sodium laurate was injected into the left ventricle of Sprague–Dawley rats to induce CMD. The rats were divided into six groups: a sham-operated (sham) group, an untreated CMD group, a low-dose TXD group (0.81 g·kg−1·d−1), a mid-dose TXD (TXD-M) group (1.62 g·kg−1·d−1), a high-dose TXD (TXD-H) group (3.24 g·kg−1·d−1), and a nicorandil (NCR) group (1.35 mg·kg−1·d−1). The effect of TXD on rats with CMD was observed after four weeks, and the mechanism of TXD in lipopolysaccharide (LPS)-induced cardiac microvascular endothelial cells (CMECs) was explored through treatment with 50 μg/mL TXD. Results Compared with the rats in the untreated CMD group, rats in the TXD-M and TXD-H groups showed higher left ventricular ejection fraction values, improved pathological structures, decreased expressions of interleukin (IL)-1β, tumor necrosis factor-alpha (TNF-α), phosphorylated nuclear factor-κB inhibitor α (IκBα) and phosphorylated p65, and increased expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (P < 0.05). These effects were more pronounced in the TXD-H group than in the TXD-M group. In vitro experiments showed that TXD treatment increased the viability of LPS-induced CMECs and decreased the expression of IL-1β, TNF-α, phosphorylated IκBα, and phosphorylated p65 (P < 0.05). However, the effects of TXD on CMECs were markedly reversed upon treatment with ML385 (Nrf2 inhibitor). Conclusion The results showed that TXD exerts a protective effect on rats with CMD and related inflammatory injuries, and its anti-inflammatory mechanism is related to the activation of Nrf2 signalling.

The liver X receptor agonist T0901317 reduces the inflammation of alveolar epithelial cells induced by polyhexamethylene guanidine through inhibition of the NFκB signaling pathway

BackgroundAs a kind of disinfectant, polyhexamethylene guanidine (PHMG) can cause pulmonary inflammation. In addition to liver X receptors (LXRs) playing an important role in cholesterol and lipid metabolism, it has also been found to be involved in inflammation in recent years. This article explores the role of LXRs agonist T0901317 in the inflammation of alveolar epithelial cells induced by PHMG.MethodsThe A549 human alveolar basal epithelial cell line was exposed to PHMG, T0901317, or the nuclear factor (NF)κB inhibitor BAY11-7082. The cell survival rate was used to determine the cytotoxicity of PHMG and T0901317 to A549 cells. Western blot analysis was used to determine the expression of proteins related to the LXRs and the NFκB signaling pathway. Enzyme-linked immunosorbent assay (ELISA) was conducted to examine the expression of inflammatory cytokines such as interleukin (IL)-8 and interleukin (IL)-6.ResultsIncubation of A549 cells with PHMG decreased the expression of LXRs-related proteins, reduced the expression of cellular IκB, increased the expression of nuclear NFκB, and increased the levels of the inflammatory cytokineIL-8 and IL-6. However, pretreatment with the LXR agonist T0901317 partially reversed the effects of PHMG. The effects of T0901317on NFκB signaling pathway was similar to that observed with the NFκB inhibitor BAY11-7082.ConclusionsThe LXRs agonist T0901317 may reduce the inflammation of alveolar epithelial cells induced by PHMG by inhibiting the NFκB signaling pathway.

Physalin A Inhibits MAPK and NF-κB Signal Transduction Through Integrin αVβ3 and Exerts Chondroprotective Effect.

Osteoarthritis (OA) is a common articular ailment presented with cartilage loss and destruction that is common observed in the elderly population. Physalin A (PA), a natural bioactive withanolide, exerts anti-inflammatory residences in more than a few diseases; however, little is known about its efficacy for OA treatment. Here, we explored the therapeutic effects and potential mechanism of PA in mouse OA. After the in vitro administration of PA, the expression of inflammation indicators including inducible nitric oxide synthase and cyclooxygenase-2 was low, indicating that PA could alleviate the IL-1β-induced chondrocyte inflammation response. Moreover, PA reduced IL-1β-induced destruction of the extracellular matrix by upregulating the gene expression of anabolism factors, including collagen II, aggrecan, and sry-box transcription factor 9, and downregulating the gene expression of catabolic factors, including thrombospondin motif 5 and matrix metalloproteinases. In addition, the chondroprotective effect of PA was credited to the inhibition of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) signaling pathways. Furthermore, in vivo experiments showed that intra-articular injection of PA could alleviate cartilage destruction in a mouse OA model. However, the anti-inflammatory, anabolism enhancing, catabolism inhibiting, and MAPK and NF-κB signaling pathway inhibiting properties of PA on IL-1β-induced chondrocytes could be reversed when integrin αVβ3 is knocked down by siRNA. In conclusion, our work demonstrates that PA exhibits a chondroprotective effect that may be mediated by integrin αVβ3. Thus, PA or integrin αVβ3 might be a promising agent or molecular target for the treatment of OA.

Epididymal embryonic development harbors TLR4/NFKB signaling pathway as a morphogenetic player

The Wolffian duct (WD) is an embryonic tissue that undergoes androgen-induced morphological changes to become the epididymis. Toll-like receptor 4 (TLR4)- and nuclear factor kB (NFKB)-induced effectors are expressed in the adult epididymis and represent important players in epididymal innate immune responses. TLR4/NFKB signaling pathway is evolutionarily conserved and plays a critical morphogenetic role in several species; however, its function during WD morphogenesis is unknown. We hypothesized that TLR4/NFKB pathway plays a role during WD development. Here we examined TLR4 expression and regulation of TLR4-target genes during rat WD morphogenesis between embryonic days (e) 17.5−20.5. The functionality of TLR4/NFKB signaling was examined using WD organotypic cultures treated with lipopolysaccharide (LPS) from E. coli (TLR4 agonist) and PDTC (NFKB inhibitor). TLR4 was detected at mRNA level in e17.5 (uncoiled duct) and e20.5 (coiled duct) WDs, and spatio-temporal changes in TLR4 immunoreactivity were observed between these two time points. Expression level analysis of a subset of TLR4-regulated genes showed that TLR4/NFKB pathway was activated after exposure of cultured WD to LPS (4 h), an event that was abrogated by PDTC. Long-term exposure of cultured WDs to LPS (96 h) resulted in dysregulations of morphogenetic events and LAMA1 immunodistribution changes, suggesting the extracellular matrix at the intersection between WD morphogenesis and balance of innate immune components. Our results unveil the epididymal morphogenesis as an event equipped with TLR4/NFKB signaling components that may serve developmental functions, and eventually transition to host defense function when the fetus is exposed to an infectious or noninfectious threat.

Anti-Lung Cancer Activities of 1,2,3-Triazole Curcumin Derivatives via Regulation of the MAPK/NF-κB/STAT3 Signaling Pathways.

In this study, a series of curcumin derivatives containing 1,2,3-triazole were designed and synthesized, and their inhibitory activities against the proliferation of lung cancer cells were studied. Compound 5 k (3,4-dichlorobenzyltriazole methyl curcumin) had the best activity against A549 cells, with a half-maximal inhibitory concentration (IC50 ) of 2.27 μM, which was approximately 10 times higher than that of the lead curcumin and higher than that of gefitinib (IC50 =8.64 μM). Western blotting revealed that 5 k increased the phosphorylation levels of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Compound 5 k also promoted the expression of the inhibitor of nuclear factor-κB (IκBα) and decreased that of nuclear factor-κB (NF-κB), signal transducer and activator of transcription 3 (STAT3), and β-catenin. Therefore, 5 k suppresses A549 cell proliferation by activating the mitogen-activated protein kinases and suppressing NF-κB/STAT3 signaling pathways. So, 5 k can potentially be used for treating non-small cell lung cancer.

Tolerance induction by liposomes targeting a single CD8 epitope IGRP206-214 in a model of type 1 diabetes is impeded by co-targeting a CD4+ islet epitope.

The autoimmune disease type 1 diabetes is predominantly mediated by CD8+ cytotoxic T-cell destruction of islet beta cells, of which islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206-214 is a dominant target antigen specificity. Previously, we found that a liposome-based antigen-specific immunotherapy encapsulating the CD4+ T-cell islet epitope 2.5mim together with the nuclear factor-κB inhibitor calcitriol induced regulatory T cells and protected from diabetes in NOD mice. Here we investigated whether the same system delivering IGRP206-214 could induce antigen-specific CD8+ T-cell-targeted immune regulation and delay diabetes. Subcutaneous administration of IGRP206-214 /calcitriol liposomes transiently activated and expanded IGRP-specific T-cell receptor transgenic 8.3 CD8+ T cells. Liposomal co-delivery of calcitriol was required to optimally suppress endogenous IGRP-specific CD8+ T-cell interferon-γ production and cytotoxicity. Concordantly, a short course of IGRP206-214 /calcitriol liposomes delayed diabetes progression and reduced insulitis. However, when IGRP206-214 /calcitriol liposomes were delivered together with 2.5mim /calcitriol liposomes, disease protection was not observed and the regulatory effect of 2.5mim /calcitriol liposomes was abrogated. Thus, tolerogenic liposomes that target either a dominant CD8+ or a CD4+ T-cell islet epitope can delay diabetes progression but combining multiple epitopes does not enhance protection.

Effect and mechanism of TNF-α and etanercept on the invasion ability of extravillous trophoblast cell in URSA patients

Objective: To investigate the effect and mechanism of tumor necrosis factor α (TNF-α) and its inhibitor etanercept (ETA) on the invasion ability of extravillous trophoblast in patients with unexplained recurrent spontaneous abortion (URSA). Methods: (1) Patients were collected from March to June in 2019. They were divided into the URSA group (n=15) and the normal control group (n=15), according to whether diagnosed with URSA or not. The mRNA expression levels of TNF-α in villi tissue of patients in the two groups were detected by quantitative real-time PCR (qRT-PCR). (2) The mRNA and protein expression levels of matrix metalloproteinase-2 (MMP-2), Slug and CXC chemokine rceptor 4 (CXCR4) in HTR-8/SVneo cells were detected by qRT-PCR or western blot after being stimulated by exogenous TNF-α (0.2, 2, 20 ng/ml) alone or TNF-α along with ETA, or phosphate buffered saline (PBS) as control. (3) The invasion ability of HTR-8/SVneo cells was investigated by transwell test after stimulating by TNF-α alone or TNF-α along with ETA. (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells, which were stimulated by TNF-α (2 ng/ml) alone after nuclear factor-κB (NF-κB) inhibitor, BAY 11-7028, preconditioning, were detected by qRT-PCR or western blot. Results: (1) The mRNA expression level of TNF-α in villi tissue of URSA group (4.10±0.49) was 4.1 times as much as the normal control group (t=10.51, P<0.05). (2) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells of TNF-α group were significantly lower than those in PBS control group (P<0.05) and those in TNF-α along with ETA group (P<0.05). (3) The invasion ability of HTR-8/SVneo cells in TNF-α group was significantly decreased than PBS group and TNF-α along with ETA group (78±14 vs 373±26 vs 227±44, P<0.05). (4) The mRNA and protein expression levels of MMP-2, Slug and CXCR4 in HTR-8/SVneo cells with BAY 11-7028 preconditioning (mRNA: 1.03±0.10, 1.03±0.06, 1.09±0.08; protein: 1.09±0.03, 1.49±0.03, 1.12±0.03) were significantly higher than without preconditioning after being stimulated by TNF-α (all P<0.05). Conclusions: The expression of TNF-α in the villi of URSA patients is much higher than normal early pregnant women. TNF-α could decrease the capacity of invasion by suppressing the expression of MMP-2, Slug and CXCR4 through NF-κB signaling pathway in extravillous trophoblast cells. While ETA could improve the invasiveness capability of extravillous trophoblast cells through inhibiting the negative effect of TNF-α.

Dimethylaminoparthenolide (DMAPT) as an alternative approach for treatment of Familial Mediterranean Fever (FMF).

Objective(s): Familial Mediterranean Fever (FMF) is a hereditary auto-inflammatory disorder that is caused by mutations in the Mediterranean fever (MEFV) gene and is associated with an increase in pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and interleukin-18 (IL-18), leading to excess inflammation. Colchicine is a common drug widely used for treatment of FMF attacks, but about 5–15% of the patients show resistance to the regular colchicine treatment. In this study, we used dimethylamino-parthenolide (DMAPT), as a small molecule inhibitor of Nuclear factor-κB (NF-κB), NLR family Pyrin domain containing 3 (NLRP3), and cysteine-aspartic acid protease 1(Caspase-1) on FMF-derived peripheral blood mononuclear cells (PBMCs).Materials and Methods:The effects of DMAPT and colchicine on metabolic activity and apoptosis of FMF-derived PBMCs were evaluated by MTT and Annexin V/PI assays, respectively. Also, the expression levels of NF-κB, NLRP3, MEFV, CASP1, and IL-1β mRNA were investigated using a TaqMan real-time PCR, and the protein levels of IL-1β, IL-18, and IL-37 were assessed via an enzyme-linked immunosorbent assay (ELISA) in LPS/ ATP-stimulated PBMCs.Results:DMAPT decreased the expression levels of NFκB (0.38±0.096, P<0.0001), NLRP3 (0.39±0.12, P<0.001), MEFV (0.384±0.145, P<0.001), CASP1 (0.48±0.13, P=0.0023), and IL-1β (0.09±0.09, P<0.0001) and reduced the secretion levels of IL-1β (8.92±5.3 vs. 149.85±20.92, P<0.0001), IL-18 (135±32.1 vs. 192±22.18, P=0.01), and IL-37 (27.5±6.3 vs. 78.19±14.3, P<0.0001) as compared to untreated cells.Conclusion:Given the obtained results in comparison with previous research, the future clinical development of DMAPT could result in the expansion of new anti-inflammatory therapeutics for FMF disorder.

Gut microbiota-mediated secondary bile acids regulate dendritic cells to attenuate autoimmune uveitis through TGR5 signaling.

Gut microbiota-mediated secondary bile acids (BAs) play an important role in energy balance and host metabolism via G protein-coupled receptors and/or nuclear receptors. Emerging evidence suggests that BAs are important for maintaining innate immune responses via these receptors. However, the effect of BAs on autoimmune uveitis is still unknown. Here, we demonstrate decreased microbiota-related secondary BA concentration in feces and serum of animals with experimental autoimmune uveitis (EAU). Restoration of the gut BAs pool attenuates severity of EAU in association with inhibition of nuclear factor κB (NF-κB)-related pro-inflammatory cytokines in dendritic cells (DCs). TGR5 deficiency partially reverses the inhibitory effect of deoxycholic acid (DCA) on DCs. TGR5 signaling also inhibits NF-κB activation via the cyclic AMP (cAMP)-protein kinase A (PKA) pathway in DCs. Additionally, both DCA and TGR5 agonists inhibit human monocyte-derived DC activation. Taken together, our results suggest that BA metabolism plays an important role in adaptive immune responses and might be a therapeutic target in autoimmune uveitis.