Sonidegib Suppresses Production of Inflammatory Mediators and Cell Migration in BV2 Microglial Cells and Mice Treated with Lipopolysaccharide via JNK and NF-κB Inhibition

Our structure-based virtual screening of the FDA-approved drug library has revealed that sonidegib, a smoothened antagonist clinically used to treat basal cell carcinoma, is a potential c-Jun N-terminal kinase 3 (JNK3) inhibitor. This study investigated the binding of sonidegib to JNK3 via ¹⁹F NMR and its inhibitory effect on JNK phosphorylation in BV2 cells. Pharmacological properties of sonidegib to exert anti-inflammatory and anti-migratory effects were also characterized. We found that sonidegib bound to the ATP binding site of JNK3 and inhibited JNK phosphorylation in BV2 cells, confirming our virtual screening results. Sonidegib also inhibited the phosphorylation of MKK4 and c-Jun, the upstream and downstream signals of JNK, respectively. It reduced the lipopolysaccharide (LPS)-induced production of pro-inflammatory factors, including interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), and nitric oxide (NO), and the expression of inducible NO synthase and cyclooxygenase-2. The LPS-induced cell migration was suppressed by sonidegib. Sonidegib inhibited the LPS-induced IκBα phosphorylation, thereby blocking NF-κB nuclear translocation. Consistent with these findings, orally administered sonidegib attenuated IL-6 and TNF-α levels in the brains of LPS-treated mice. Collectively, our results indicate that sonidegib suppresses inflammation and cell migration in LPS-treated BV2 cells and mice by inhibiting JNK and NF-κB signaling. Therefore, sonidegib may be implicated for drug repurposing to alleviate neuroinflammation associated with microglial activation.     

Protective Effect of Resveratrol against IL-1β-Induced Inflammatory Response on Human Osteoarthritic Chondrocytes Partly via the TLR4/MyD88/NF-κB Signaling Pathway: An “in Vitro Study”

Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1β-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-κB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1β before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1β were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-κB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1β-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1β-induced reduction in cell viability. Stimulation of chondrocytes with IL-1β caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-κB activation associated with the synthesis of IL-1β and TNFα. These IL-1β-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-κB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-κB translocation. These data suggested that resveratrol prevented IL-1β-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms.     

Suppression of LPS-Induced Inflammation and Cell Migration by Azelastine through Inhibition of JNK/NF-κB Pathway in BV2 Microglial Cells

The c-Jun N-terminal kinases (JNKs) are implicated in many neuropathological conditions, including neurodegenerative diseases. To explore potential JNK3 inhibitors from the U.S. Food and Drug Administration-approved drug library, we performed structure-based virtual screening and identified azelastine (Aze) as one of the candidates. NMR spectroscopy indicated its direct binding to the ATP-binding site of JNK3, validating our observations. Although the antihistamine effect of Aze is well documented, the involvement of the JNK pathway in its action remains to be elucidated. This study investigated the effects of Aze on lipopolysaccharide (LPS)-induced JNK phosphorylation, pro-inflammatory mediators, and cell migration in BV2 microglial cells. Aze was found to inhibit the LPS-induced phosphorylation of JNK and c-Jun. It also inhibited the LPS-induced production of pro-inflammatory mediators, including interleukin-6, tumor necrosis factor-α, and nitric oxide. Wound healing and transwell migration assays indicated that Aze attenuated LPS-induced BV2 cell migration. Furthermore, Aze inhibited LPS-induced IκB phosphorylation, thereby suppressing nuclear translocation of NF-κB. Collectively, our data demonstrate that Aze exerts anti-inflammatory and anti-migratory effects through inhibition of the JNK/NF-κB pathway in BV2 cells. Based on our findings, Aze may be a potential candidate for drug repurposing to mitigate neuroinflammation in various neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases.     

Artemisinin Attenuates Amyloid-Induced Brain Inflammation and Memory Impairments by Modulating TLR4/NF-κB Signaling

The abnormal immune response is an early change in the pathogenesis of Alzheimer’s disease (AD). Microglial activation is a crucial regulator of the immune response, which contributes to progressive neuronal injury by releasing neurotoxic products. Therefore, finding effective drugs to regulate microglial homeostasis and neuroinflammation has become a new AD treatment strategy. Artemisinin has potent anti-inflammatory and immune activities. However, it is unclear whether Artemisinin contributes to the regulation of microglial activation, thereby improving AD pathology. This study found that Artemisinin significantly reduced amyloid beta-peptide 1–42 (Aβ_1–42)-induced increases in nitric oxide and reactive oxygen species and inflammatory factors in BV2 cells. In addition, Artemisinin inhibited the migration of microglia and prevented the expansion of the inflammatory cascade. The mechanical studies showed Artemisinin inhibited neuroinflammation and exerted neuroprotective effects by regulating the Toll-like receptor 4 (TLR4)/Nuclear factor-kappa B (NF-κB) signaling pathway. Similar results were obtained in AD model mice, in which Artemisinin administration attenuated Aβ_1–42-induced neuroinflammation and neuronal injury, reversing spatial learning and memory deficits. The anti-inflammatory effect of Artemisinin is also accompanied by the activation of the TLR4/NF-κB signaling pathway in the animal model. Our results indicate that Artemisinin attenuated Aβ_1–42-induced neuroinflammation and neuronal injury by stimulating the TLR4/NF-κB signaling pathway. These findings suggest that Artemisinin is a potential therapeutic agent for AD.     

Resveratrol Attenuates Acute Inflammatory Injury in Experimental Subarachnoid Hemorrhage in Rats via Inhibition of TLR4 Pathway

Toll-like receptor 4 (TLR4) has been proven to play a critical role in neuroinflammation and to represent an important therapeutic target following subarachnoid hemorrhage (SAH). Resveratrol (RSV), a natural occurring polyphenolic compound, has a powerful anti-inflammatory property. However, the underlying molecular mechanisms of RSV in protecting against early brain injury (EBI) after SAH remain obscure. The purpose of this study was to investigate the effects of RSV on the TLR4-related inflammatory signaling pathway and EBI in rats after SAH. A prechiasmatic cistern SAH model was used in our experiment. The expressions of TLR4, high-mobility group box 1 (HMGB1), myeloid differentiation factor 88 (MyD88), and nuclear factor-κB (NF-κB) were evaluated by Western blot and immunohistochemistry. The expressions of Iba-1 and pro-inflammatory cytokines in brain cortex were determined by Western blot, immunofluorescence staining, or enzyme-linked immunosorbent assay. Neural apoptosis, brain edema, and neurological function were further evaluated to investigate the development of EBI. We found that post-SAH treatment with RSV could markedly inhibit the expressions of TLR4, HMGB1, MyD88, and NF-κB. Meanwhile, RSV significantly reduced microglia activation, as well as inflammatory cytokines leading to the amelioration of neural apoptosis, brain edema, and neurological behavior impairment at 24 h after SAH. However, RSV treatment failed to alleviate brain edema and neurological deficits at 72 h after SAH. These results indicated that RSV treatment could alleviate EBI after SAH, at least in part, via inhibition of TLR4-mediated inflammatory signaling pathway.     

Ophiopogon Polysaccharide Liposome Regulated the Immune Activity of Kupffer Cell through miR-4796

The purpose of this article is to study the effects and mechanism of miR-4796 in the process of ophiopogon polysaccharide liposome (OPL) regulation of the immune activity of Kupffer cells (KCs). In this study, KCs were used as cell models, and were treated with OPL in different concentrations after being transfected with miR-4796 mimic or miR-4796 inhibitor. Firstly, the secretion of NO and iNOS, phagocytic activity, the expression of surface molecules CD14 and MHC II, apoptosis and ROS secretion were measured by Griess, flow cytometry, fluorescence staining and ELISA. Then, real-time PCR and Western blot were used to measure the expression of TLR4, IKKβ, MyD88 and NF-κB in the TLR4-NF-κB signaling pathway. The results showed that after transfection with miR-4796 mimic, the secretion of NO and iNOS, cell migration, cell phagocytosis and expression levels of CD14 and MHC II in the OPL group were significantly higher than those in the miR-4796 mimic control group (p < 0.05; p < 0.01). In addition, the mRNA and protein expression levels of TLR4, MyD88 and NF-κB were significantly higher than those in miR-4796 mimic control group (p < 0.05; p < 0.01). After transfection with miR-4796 inhibitor, the secretion of NO and iNOS, cell migration, cell phagocytosis, expression of CD14 and MHCII in OPL group were significantly higher than those in the miR-4796 inhibitor control group (p < 0.05; p < 0.01). These results indicated that OPL could regulate the immune activity of KCs by regulating miR-4796 and activating the TLR4-NF-κB signaling pathway.     

Prenylated Flavonoids from Cudrania tricuspidata Suppress Lipopolysaccharide-Induced Neuroinflammatory Activities in BV2 Microglial Cells

In Korea and China, Cudrania tricuspidata Bureau (Moraceae) is an important traditional medicinal plant used to treat lumbago, hemoptysis, and contusions. The C. tricuspidata methanol extract suppressed both production of NO and PGE₂ in BV2 microglial cells. Cudraflavanone D (1), isolated from this extract, remarkably suppressed the protein expression of inducible NO synthase and cyclooxygenase-2, and decreased the levels of NO and PGE₂ in BV2 microglial cells exposed to lipopolysaccharide. Cudraflavanone D (1) also decreased IL-6, TNF-α, IL-12, and IL-1β production, blocked nuclear translocation of NF-κB heterodimers (p50 and p65) by interrupting the degradation and phosphorylation of inhibitor of IκB-α, and inhibited NF-κB binding. In addition, cudraflavanone D (1) suppressed the phosphorylation of c-Jun N-terminal kinase (JNK) and p38 MAPK pathways. This study indicated that cudraflavanone D (1) can be a potential drug candidate for the cure of neuroinflammation.     

Advanced Glycation End-Products Enhance Lung Cancer Cell Invasion and Migration

Effects of carboxymethyllysine (CML) and pentosidine, two advanced glycation end-products (AGEs), upon invasion and migration in A549 and Calu-6 cells, two non-small cell lung cancer (NSCLC) cell lines were examined. CML or pentosidine at 1, 2, 4, 8 or 16 μmol/L were added into cells. Proliferation, invasion and migration were measured. CML or pentosidine at 4–16 μmol/L promoted invasion and migration in both cell lines, and increased the production of reactive oxygen species, tumor necrosis factor-α, interleukin-6 and transforming growth factor-β1. CML or pentosidine at 2–16 μmol/L up-regulated the protein expression of AGE receptor, p47^phox, intercellular adhesion molecule-1 and fibronectin in test NSCLC cells. Matrix metalloproteinase-2 protein expression in A549 and Calu-6 cells was increased by CML or pentosidine at 4–16 μmol/L. These two AGEs at 2–16 μmol/L enhanced nuclear factor κ-B (NF-κ B) p65 protein expression and p38 phosphorylation in A549 cells. However, CML or pentosidine at 4–16 μmol/L up-regulated NF-κB p65 and p-p38 protein expression in Calu-6 cells. These findings suggest that CML and pentosidine, by promoting the invasion, migration and production of associated factors, benefit NSCLC metastasis.     

Anti-Inflammatory Effect of Ginsenoside Rg5 in Lipopolysaccharide-Stimulated BV2 Microglial Cells

Microglia are resident immune cells in the central nervous system. They play a role in normal brain development and neuronal recovery. However, overactivation of microglia causes neuronal death, which is associated with neurodegenerative diseases, such as Parkinson’s disease and Alzheimer’s disease. Therefore, controlling microglial activation has been suggested as an important target for treatment of neurodegenerative diseases. In the present study, we investigated the anti-inflammatory effect of ginsenoside Rg5 in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and rat primary microglia. The data showed that Rg5 suppressed LPS-induced nitric oxide (NO) production and proinflammatory TNF-α secretion. In addition, Rg5 inhibited the mRNA expressions of iNOS, TNF-α, IL-1β, COX-2 and MMP-9 induced by LPS. Further mechanistic studies revealed that Rg5 inhibited the phophorylations of PI3K/Akt and MAPKs and the DNA binding activities of NF-κB and AP-1, which are upstream molecules controlling inflammatory reactions. Moreover, Rg5 suppressed ROS production with upregulation of hemeoxygenase-1 (HO-1) expression in LPS-stimulated BV2 cells. Overall, microglial inactivation by ginsenoside Rg5 may provide a therapeutic potential for various neuroinflammatory disorders.     

Pharmacological Effects of Polyphenol Phytochemicals on the Intestinal Inflammation via Targeting TLR4/NF-κB Signaling Pathway

TLR4/NF-κB is a key inflammatory signaling transduction pathway, closely involved in cell differentiation, proliferation, apoptosis, and pro-inflammatory response. Toll like receptor 4 (TLR4), the first mammalian TLR to be characterized, is the innate immune receptor that plays a key role in inflammatory signal transductions. Nuclear factor kappa B (NF-κB), the TLR4 downstream, is the key to accounting for the expression of multiple genes involved in inflammatory responses, such as pro-inflammatory cytokines. Inflammatory bowel disease (IBD) in humans is a chronic inflammatory disease with high incidence and prevalence worldwide. Targeting the TLR4/NF-κB signaling pathway might be an effective strategy to alleviate intestinal inflammation. Polyphenol phytochemicals have shown noticeable alleviative effects by acting on the TLR4/NF-κB signaling pathway in intestinal inflammation. This review summarizes the pharmacological effects of more than 20 kinds of polyphenols on intestinal inflammation via targeting the TLR4/NF-κB signaling pathway. We expected that polyphenol phytochemicals targeting the TLR4/NF-κB signaling pathway might be an effective approach to treat IBD in future clinical research applications.     

Epigallocatechin Gallate Protects against Hypoxia-Induced Inflammation in Microglia via NF-κB Suppression and Nrf-2/HO-1 Activation

Hypoxia-induced neuroinflammation in stroke, neonatal hypoxic encephalopathy, and other diseases subsequently contributes to neurological damage and neuronal diseases. Microglia are the primary neuroimmune cells that play a crucial role in cerebral inflammation. Epigallocatechin gallate (EGCG) has a protective antioxidant and anti-inflammatory effects against neuroinflammation. However, the effects of EGCG on hypoxia-induced inflammation in microglia and the underlying mechanism remain unclear. In this study, we investigated whether EGCG might have a protective effect against hypoxia injury in microglia by treatment with CoCl₂ to establish a hypoxic model of BV2 microglia cells following EGCG pre-treatment. An exposure of cells to CoCl₂ caused an increase in inflammatory mediator interleukin (IL)-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2 expression, which were significantly ameliorated by EGCG via inhibition of NF-κB pathway. In addition, EGCG attenuated the expression of hypoxia-inducible factor (HIF)-1α and the generation of ROS in hypoxic BV2 cells. Furthermore, the suppression of hypoxia-induced IL-6 production by EGCG was mediated via the inhibition of HIF-1α expression and the suppression of ROS generation in BV2 cells. Notably, EGCG increased the Nrf-2 levels and HO-1 levels in the presence of CoCl₂. Additionally, EGCG suppressed hypoxia-induced apoptosis of BV2 microglia with cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3. In summary, EGCG protects microglia from hypoxia-induced inflammation and oxidative stress via abrogating the NF-κB pathway as well as activating the Nrf-2/HO-1 pathway.     

Geranylgeraniol Inhibits Lipopolysaccharide-Induced Inflammation in Mouse-Derived MG6 Microglial Cells via NF-κB Signaling Modulation

Persistent inflammatory reactions in microglial cells are strongly associated with neurodegenerative pathogenesis. Additionally, geranylgeraniol (GGOH), a plant-derived isoprenoid, has been found to improve inflammatory conditions in several animal models. It has also been observed that its chemical structure is similar to that of the side chain of menaquinone-4, which is a vitamin K₂ sub-type that suppresses inflammation in mouse-derived microglial cells. In this study, we investigated whether GGOH has a similar anti-inflammatory effect in activated microglial cells. Particularly, mouse-derived MG6 cells pre-treated with GGOH were exposed to lipopolysaccharide (LPS). Thereafter, the mRNA levels of pro-inflammatory cytokines were determined via qRT-PCR, while protein expression levels, especially the expression of NF-κB signaling cascade-related proteins, were determined via Western blot analysis. The distribution of NF-κB p65 protein was also analyzed via fluorescence microscopy. Thus, it was observed that GGOH dose-dependently suppressed the LPS-induced increase in the mRNA levels of Il-1β, Tnf-α, Il-6, and Cox-2. Furthermore, GGOH inhibited the phosphorylation of TAK1, IKKα/β, and NF-κB p65 proteins as well as NF-κB nuclear translocation induced by LPS while maintaining IκBα expression. We showed that GGOH, similar to menaquinone-4, could alleviate LPS-induced microglial inflammation by targeting the NF-kB signaling pathway.     

Binding of Gamma-Glutamyl Transferase to TLR4 Signalling Allows Tissue Factor Activation in Monocytes

Gamma-glutamyl transferase (GGT) is involved in the progression of atherosclerosis, since its enzymatic activity promotes the generation of reactive oxygen species (ROS). Besides, GGT may act as a prothrombotic factor by inducing tissue factor (TF) expression, independently of its enzymatic activity. The aim of this study was to assess whether GGT-induced TF stimulation was a consequence of binding to toll-like receptor 4 (TLR4) expressed on monocytes, the precursors of macrophages and foam cells which colocalize with GGT activity within atherosclerotic plaques. Experiments were performed in human peripheral blood mononuclear cells (PBMCs), THP-1 cells (a monocytic cellular model), and HEK293 cells, which were genetically modified to study the activation of TLR4. TF procoagulant activity was assessed by a one-stage clotting time test, and TF protein expression was estimated by western blot. Human recombinant (hr) GGT protein increased TF procoagulant activity and protein expression in both PBMCs and THP-1 cells. The GGT-induced TF stimulation was prevented by cellular pretreatment with TLR4/NF-κB inhibitors (LPS-Rs, CLI-095, and BAY-11-7082), and HEK293 cells lacking TLR4 confirmed that TLR4 is essential for GGT-induced activation of NF-κB. In conclusion, hrGGT induced TF expression in monocytes through a cytokine-like mechanism that involved the activation of TLR4/NF-κB signaling.     

Biglycan Is a Novel Mineralocorticoid Receptor Target Involved in Aldosterone/Salt-Induced Glomerular Injury

The beneficial effects of mineralocorticoid receptor (MR) antagonists (MRAs) for various kidney diseases are established. However, the underlying mechanisms of kidney injury induced by MR activation remain to be elucidated. We recently reported aldosterone-induced enhancement of proteoglycan expression in mitral valve interstitial cells and its association with fibromyxomatous valvular disorder. As the expression of certain proteoglycans is elevated in several kidney diseases, we hypothesized that proteoglycans mediate kidney injury in the context of aldosterone/MR pathway activation. We evaluated the proteoglycan expression and tissue injury in the kidney and isolated glomeruli of uninephrectomy/aldosterone/salt (NAS) mice. The MRA eplerenone was administered to assess the role of the MR pathway. We investigated the direct effects of biglycan, one of the proteoglycans, on macrophages using isolated macrophages. The kidney samples from NAS-treated mice showed enhanced fibrosis and increased expression of biglycan accompanying glomerular macrophage infiltration and enhanced expression of TNF-α, iNOS, Nox2, CCL3 (C-C motif chemokine ligand 3), and phosphorylated NF-κB. Eplerenone blunted these changes. Purified biglycan stimulated macrophages to express TNF-α, iNOS, Nox2, and CCL3. This was prevented by a toll-like receptor 4 (TLR4) or NF-κB inhibitor, indicating that biglycan stimulation is dependent on the TLR4/NF-κB pathway. We identified the proteoglycan biglycan as a novel target of MR involved in MR-induced glomerular injury and macrophage infiltration via a biglycan/TLR4/NF-κB/CCL3 cascade.     

CK2 Down-Regulation Increases the Expression of Senescence-Associated Secretory Phenotype Factors through NF-κB Activation

Senescent cells secrete pro-inflammatory factors, and a hallmark feature of senescence is senescence-associated secretory phenotype (SASP). The aim of this study is to investigate the protein kinase CK2 (CK2) effects on SASP factors expression in cellular senescence and organism aging. Here CK2 down-regulation induced the expression of SASP factors, including interleukin (IL)-1β, IL-6, and matrix metalloproteinase (MMP) 3, through the activation of nuclear factor-κB (NF-κB) signaling in MCF-7 and HCT116 cells. CK2 down-regulation-mediated SIRT1 inactivation promoted the degradation of inhibitors of NF-κB (IκB) by activating the AKT-IκB kinase (IKK) axis and increased the acetylation of lysine 310 on RelA/p65, an important site for the activity of NF-κB. kin-10 (the ortholog of CK2β) knockdown increased zmp-1, -2, and -3 (the orthologs of MMP) expression in nematodes, but AKT inhibitor triciribine and SIRT activator resveratrol significantly abrogated the increased expression of these genes. Finally, antisense inhibitors of miR-186, miR-216b, miR-337-3p, and miR-760 suppressed CK2α down-regulation, activation of the AKT-IKK-NF-κB axis, RelA/p65 acetylation, and expression of SASP genes in cells treated with lipopolysaccharide. Therefore, this study indicated that CK2 down-regulation induces the expression of SASP factors through NF-κB activation, which is mediated by both activation of the SIRT1-AKT-IKK axis and RelA/p65 acetylation, suggesting that the mixture of the four miRNA inhibitors can be used as anti-inflammatory agents.