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.     

Koumine Attenuates Lipopolysaccaride-Stimulated Inflammation in RAW264.7 Macrophages,Coincidentally Associated with Inhibition of NF-κB,ERK and p38 Pathways

Medicinal herbal plants have been commonly used for intervention of different diseases and health enhancement worldwide. Koumine, an alkaloid monomer found abundantly in Gelsemium plants, can be effectively used as an anti-inflammatory medication. In this study, the mechanisms associated with the preventative effect of koumine on lipopolysaccharide (LPS)-mediated inflammation in RAW264.7 macrophages were investigated. Koumine induced a decrease in the level of inducible nitric oxide synthase (iNOS) protein, concomitant reduction in the production of nitric oxide (NO) and reduction of the levels of interleukin (IL)-6, tumor necrosis factor-α (TNF-α) and IL-1β. Furthermore, koumine decreased the phosphorylation of p65 and inhibited nuclear factor κ Bα (IκBα) proteins, resulting in lower production of nuclear factor (NF)-κB transactivation. Koumine also induced a decrease in the phosphorylation of extracellular-signal-regulated kinases (ERK) and p38 in RAW264 cells. In conclusion, these findings reveal that koumine decreases the productions of pro-inflammatory mediators though the suppression of p38 and ERK MAPK phosphorylation and the inhibition of NF-κB activation in RAW264.7 cells.     

Human Caspase 12 Enhances NF-κB Activity through Activation of IKK in Nasopharyngeal Carcinoma Cells

Human nasopharyngeal carcinoma (NPC) is a highly invasive cancer associated with proinflammation. Caspase-12 (Casp12), an inflammatory caspase, is implicated in the regulation of NF-κB-mediated cellular invasion via the modulation of the IκBα protein in NPC cells. However, the effect mechanisms of Casp12 need to be elucidated. NPC cells were transfected with the full length of human Casp12 cDNA (pC12) and the effect of human Casp12 (hCasp12) on the NF-κB activity was investigated. We found ectopic expression of hCasp12 increased the NF-κB activity accompanied by an increased p-IκBα expression and a decreased IκBα expression. Treatment of BMS, a specific IKK inhibitor, and pC12-transfected cells markedly decreased the NF-κB activity and ameliorated the expression level of IκBα reduced by hCasp12. Co-immunoprecipitation assays validated the physical interaction of hCasp12 with IKKα/β, but not with NEMO. Furthermore, the NF-κB activity of ΔCasp12-Q (a mutated catalytic of hCasp12) transfected cells was concentration-dependently induced, but lower than that of hCasp12-transfected cells. Importantly, the hCasp12-mediated NF-kB activity was enhanced by TNFα stimulation. That indicated a role of the catalytic motif of hCasp12 in the regulation of the NF-κB activity. This study indicated hCasp12 activated the NF-κB pathway through the activation of IKK in human NPC cells.     

Extract from the Coriolus versicolor Fungus as an Anti-Inflammatory Agent with Cytotoxic Properties against Endothelial Cells and Breast Cancer Cells

Chronic inflammation is a well-recognised tumour-enabling component, which includes bioactive molecules from cells infiltrating the tumour microenvironment and increases the risk of cancer progression. Since long-term use of the currently available anti-inflammatory drugs used in cancer therapy causes numerous side effects, the aim of this study was to investigate the effect of an extract isolated from the Coriolus versicolor fungus (CV extract) on HUVEC endothelial cells and MCF-7 breast cancer cells in a pro-inflammatory microenvironment mimicked by lipopolysaccharide (LPS). The cells were simultaneously stimulated with the LPS and CV extract. After co-treatment, the cell viability, generation of reactive oxygen species (ROS), wound-healing assay, production of the pro-inflammatory and pro-angiogenic factors (interleukin (IL) 6, IL-8, and metalloproteinase (MMP) 9)), as well as expression of Toll-like receptor (TLR) 4 and phosphorylated IκB (p-IκB) were evaluated. The results showed that the CV extract inhibited IL-6, IL-8, and MMP-9 production by the LPS-stimulated cells. This effect was accompanied by a decrease in TLR4 and p-IκB expression. The CV extract also had anti-migratory properties and induced a cytotoxic effect on the cells that was enhanced in the presence of LPS. The observed cytotoxicity was associated with an increase in ROS generation. We conclude that the CV extract possesses cytotoxic activity against cancer cells and endothelial cells and has the ability to inhibit the expression of the pro-tumorigenic factors associated with inflammation.     

14-3-3γ Regulates Lipopolysaccharide-Induced Inflammatory Responses and Lactation in Dairy Cow Mammary Epithelial Cells by Inhibiting NF-κB and MAPKs and Up-Regulating mTOR Signaling

As a protective factor for lipopolysaccharide (LPS)-induced injury, 14-3-3γ has been the subject of recent research. Nevertheless, whether 14-3-3γ can regulate lactation in dairy cow mammary epithelial cells (DCMECs) induced by LPS remains unknown. Here, the anti-inflammatory effect and lactation regulating ability of 14-3-3γ in LPS-induced DCMECs are investigated for the first time, and the molecular mechanisms responsible for their effects are explored. The results of qRT-PCR showed that 14-3-3γ overexpression significantly inhibited the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and inducible nitric oxide synthase (iNOS). Enzyme-linked immunosorbent assay (ELISA) analysis revealed that 14-3-3γ overexpression also suppressed the production of TNF-α and IL-6 in cell culture supernatants. Meanwhile, CASY-TT Analyser System showed that 14-3-3γ overexpression clearly increased the viability and proliferation of cells. The results of kit methods and western blot analysis showed that 14-3-3γ overexpression promoted the secretion of triglycerides and lactose and the synthesis of β-casein. Furthermore, the expression of genes relevant to nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPKs) and lactation-associated proteins were assessed by western blot, and the results suggested that 14-3-3γ overexpression inactivated the NF-κB and MAPK signaling pathways by down-regulating extracellular signal regulated protein kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK) and inhibitor of NF-κB (IκB) phosphorylation levels, as well as by inhibiting NF-κB translocation. Meanwhile, 14-3-3γ overexpression enhanced the expression levels of β-casein, mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase 1 (S6K1), serine/threonine protein kinase Akt 1 (AKT1), sterol regulatory element binding protein 1 (SREBP1) and peroxisome proliferator-activated receptor gamma (PPARγ). These results suggest that 14-3-3γ was able to attenuate the LPS-induced inflammatory responses and promote proliferation and lactation in LPS-induced DCMECs by inhibiting the activation of the NF-κB and MAPK signaling pathways and up-regulating mTOR signaling pathways to protect against LPS-induced injury.     

A20 Overexpression Inhibits Lipopolysaccharide-Induced NF-κB Activation,TRAF6 and CD40 Expression in Rat Peritoneal Mesothelial Cells

Zinc finger protein A20 is a key negative regulator of inflammation. However, whether A20 may affect inflammation during peritoneal dialysis (PD)-associated peritonitis is still unclear. This study was aimed to investigate the effect of A20 overexpression on lipopolysaccharide (LPS)-induced inflammatory response in rat peritoneal mesothelial cells (RPMCs). Isolated and cultured RPMCs in vitro. Plasmid pGEM-T easy-A20 was transfected into RPMCs by Lipofectamine™2000. The protein expression of A20, phospho-IκBα, IκBα, TNF receptor-associated factor (TRAF) 6 and CD40 were analyzed by Western blot. The mRNA expression of TRAF6, CD40, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined by real time-PCR. NF-κB p65 DNA binding activity, IL-6 and TNF-α levels in cells culture supernatant were determined by ELISA. Our results revealed that RPMCs overexpression of A20 lead to significant decrease of LPS-induced IκBα phosphorylation and NF-κB DNA binding activity (all p < 0.01). In addition, A20 also attenuated the expression of TRAF6, CD40, IL-6 and TNF-α as well as levels of IL-6 and TNF-α in cells culture supernatant (all p < 0.05). However, A20 only partly inhibited CD40 expression. Our study indicated that A20 overexpression may depress the inflammatory response induced by LPS in cultured RPMCs through negatively regulated the relevant function of adaptors in LPS signaling pathway.     

Chrysin Suppressed Inflammatory Responses and the Inducible Nitric Oxide Synthase Pathway after Spinal Cord Injury in Rats

Chrysin (CH), a natural plant flavonoid, has shown a variety of beneficial effects. Our present study was conducted to evaluate the therapeutic potential of CH three days after spinal cord injury (SCI) in rats and to probe the underlying neuroprotective mechanisms. SCI was induced using the modified weight-drop method in Wistar rats. Then, they were treated with saline or CH by doses of 30 and 100 mg/kg for 26 days. Neuronal function was assessed with the Basso Beattle Bresnahan locomotor rating scale (BBB). The water content of spinal cord was determined after traumatic SCI. The NF-κB p65 unit, TNF-α, IL-1β and IL-6 in serums, as well as the apoptotic marker, caspase-3, of spinal cord tissues were measured using commercial kits. The protein level and activity of inducible nitric oxide synthase (iNOS) were detected by western blot and a commercial kit, respectively. NO (nitric oxide) production was evaluated by the determination of nitrite concentration. The rats with SCI showed marked reductions in BBB scores, coupled with increases in the water content of spinal cord, the NF-κB p65 unit, TNF-α, IL-1β, IL-6, iNOS, NO production and caspase-3. However, a CH supplement dramatically promoted the recovery of neuronal function and suppressed the inflammatory factors, as well as the iNOS pathway in rats with SCI. Our findings disclose that CH improved neural function after SCI in rats, which might be linked with suppressing inflammation and the iNOS pathway.     

Discovery of Phenolic Glycoside from Hyssopus cuspidatus Attenuates LPS-Induced Inflammatory Responses by Inhibition of iNOS and COX-2 Expression through Suppression of NF-κB Activation

It seems quite necessary to obtain effective substances from natural products against inflammatory response (IR) as there are presently clinical problems regarding accompanying side effects and lowered quality of life. This work aimed to investigate the abilities of hyssopuside (HY), a novel phenolic glycoside isolated from Hyssopus cuspidatus (H. cuspidatus), against IR in lipopolysaccharide (LPS)-induced RAW 264.7 cells and mouse peritoneal macrophages. The results indicated that HY could reduce nitric oxide (NO) production and inhibit the production and secretion of pro-inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in LPS-stimulated macrophages. Moreover, data from the immunofluorescence study showed that HY suppressed nuclear translocation of nuclear factor-kappa B (NF-κB) upon LPS induction. The Western blot results suggested that HY reversed the LPS-induced degradation of IκB (inhibitor of NF-κB), which is normally required for the activation of NF-κB. Meanwhile, the overexpression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) diminished significantly with the presence of HY in response to LPS stimulation. On the other hand, HY had a negligible impact on the activation of mitogen-activated protein kinase (MAPK) pathways. Moreover, an in silico study of HY against four essential proteins/enzymes revealed that COX-2 was the most efficient enzyme for the interaction, and binding of residues Phe179, Asn351, and Ser424 with HY played crucial roles in the observed activity. The structure analysis indicated the typical characterizations with phenylethanoid glycoside contributed to the anti-inflammatory effects of HY. These results indicated that HY manipulated its anti-inflammatory effects mainly through blocking the NF-κB signal transduction pathways. Collectively, we believe that HY could be a potential alternative phenolic agent for alleviating excessive inflammation in many inflammation-associated diseases.     

The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma

Inhibition of the protein neddylation process by the small-molecule inhibitor MLN4924 has been recently indicated as a promising direction for cancer treatment. However, the knowledge of all biological consequences of MLN4924 for cancer cells is still incomplete. Here, we report that MLN4924 inhibits tumor necrosis factor-alpha (TNF-α)-induced matrix metalloproteinase 9 (MMP9)-driven cell migration. Using real-time polymerase chain reaction (PCR) and gelatin zymography, we found that MLN4924 inhibited expression and activity of MMP9 at the messenger RNA (mRNA) and protein levels in both resting cells and cells stimulated with TNF-α, and this inhibition was closely related to impaired cell migration. We also revealed that MLN4924, similar to TNF-α, induced phosphorylation of inhibitor of nuclear factor kappa B-alpha (IκB-α). However, contrary to TNF-α, MLN4924 did not induce IκB-α degradation in treated cells. In coimmunoprecipitation experiments, nuclear IκB-α which formed complexes with nuclear factor kappa B p65 subunit (NFκB/p65) was found to be highly phosphorylated at Ser32 in the cells treated with MLN4924, but not in the cells treated with TNF-α alone. Moreover, in the presence of MLN4924, nuclear NFκB/p65 complexes were found to be enriched in c-Jun and cyclin dependent kinase inhibitor 1 A (CDKN1A/p21) proteins. In these cells, NFκB/p65 was unable to bind to the MMP9 gene promoter, which was confirmed by the chromatin immunoprecipitation (ChIP) assay. Taken together, our findings identified MLN4924 as a suppressor of TNF-α-induced MMP9-driven cell migration in esophageal squamous cell carcinoma (ESCC), likely acting by affecting the nuclear ubiquitin–proteasome system that governs NFκB/p65 complex formation and its DNA binding activity in regard to the MMP9 promoter, suggesting that inhibition of neddylation might be a new therapeutic strategy to prevent invasion/metastasis in ESCC patients.     

Natural Compound Resveratrol Attenuates TNF-Alpha-Induced Vascular Dysfunction in Mice and Human Endothelial Cells: The Involvement of the NF-κB Signaling Pathway

Resveratrol, a natural compound in grapes and red wine, has drawn attention due to potential cardiovascular-related health benefits. However, its effect on vascular inflammation at physiologically achievable concentrations is largely unknown. In this study, resveratrol in concentrations as low as 1 μm suppressed TNF-α-induced monocyte adhesion to human EA.hy926 endothelial cells (ECs), a key event in the initiation and development of atherosclerosis. Low concentrations of resveratrol (0.25–2 μm) also significantly attenuated TNF-α-stimulated mRNA expressions of MCP-1/CCL2 and ICAM-1, which are vital mediators of EC-monocyte adhesion molecules and cytokines for cardiovascular plaque formation. Additionally, resveratrol diminished TNF-α-induced IκB-α degradation and subsequent nuclear translocation of NF-κB p65 in ECs. In the animal study, resveratrol supplementation in diet significantly diminished TNF-α-induced increases in circulating levels of adhesion molecules and cytokines, monocyte adhesion to mouse aortic ECs, F4/80-positive macrophages and VCAM-1 expression in mice aortas and restored the disruption in aortic elastin fiber caused by TNF-α treatment. The animal study also confirmed that resveratrol blocks the activation of NF-κB In Vivo. In conclusion, resveratrol at physiologically achievable concentrations displayed protective effects against TNF-α-induced vascular endothelial inflammation in vitro and In Vivo. The ability of resveratrol in reducing inflammation may be associated with its role as a down-regulator of the NF-κB pathway.     

Linagliptin Protects against Endotoxin-Induced Acute Kidney Injury in Rats by Decreasing Inflammatory Cytokines and Reactive Oxygen Species

Septic shock can increase pro-inflammatory cytokines, reactive oxygen species (ROS), and multiple organ dysfunction syndrome (MODs) and even lead to death. Dipeptidyl peptidase-4 (DPP-4) inhibitors have been proven to exert potential antioxidant and anti-inflammatory effects. We investigated the effects of linagliptin on endotoxic shock and acute kidney injury (AKI) in animal and cell models. In the cell model, linagliptin attenuated ROS by activating the AMP-activated protein kinase (AMPK) pathway, restoring nuclear-factor-erythroid-2-related factor (Nrf2) and heme oxygenase 1 (HO-1) protein, and decreasing pro-inflammatory cytokines (tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β)). In the animal model, 14-week-old conscious Wistar–Kyoto rats were randomly divided into three groups (n = 8 in each group). Endotoxin shock with MODs was induced by the intravenous injection of Klebsiella pneumoniae lipopolysaccharide (LPS, 20 mg/kg). Linagliptin improved animal survival without affecting hemodynamic profiles. In the histopathology and immunohistochemistry examinations of the rat kidneys, linagliptin (10 mg/kg) suppressed nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and inducible nitric oxide synthase (iNOS), decreased injury scores, and preserved E-cadherin expression from LPS damage. In conclusion, linagliptin ameliorated endotoxin-shock-induced AKI by reducing ROS via AMPK pathway activation and suppressing the release of TNF-α and IL-1β in conscious rats.     

Moracin C,A Phenolic Compound Isolated from Artocarpus heterophyllus,Suppresses Lipopolysaccharide-Activated Inflammatory Responses in Murine Raw264.7 Macrophages

Artocarpus heterophyllus, a popular tropical fruit commonly known as the jackfruit tree, is normally planted in subtropical or tropical areas. Since a variety of phytochemicals isolated from A. heterophyllus have been found to possess potently anti-inflammatory, antiviral and antimalarial activities, researchers have devoted much interest to its potential pharmaceutical value. However, the exact mechanism underlying its anti-inflammatory activity is not well characterized. In this study, seven natural products isolated from A. heterophyllus, including 25-Hydroxycycloart-23-en-3-one (HY), Artocarpin (AR), Dadahol A (DA), Morachalcone A (MA), Artoheterophyllin B (AB), Cycloheterophyllin (CY) and Moracin C (MC) were collected. Lipopolysaccharide (LPS)-stimulated inflammatory response in RAW264.7 macrophages were used in this study. Among these compounds, MC significantly inhibited LPS-activated reactive oxygen species (ROS) and nitric oxide (NO) release without marked cytotoxicity. Furthermore, MC effectively reduced LPS stimulated up-regulation of mRNA and protein expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and serval pro-inflammatory cytokines (interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α)). Mechanistic studies revealed that the anti-inflammatory effect of MC was associated with the activation of the mitogen activated protein kinases (MAPKs) (including p38, ERK and JNK) and nuclear factor-κB (NF-κB) pathways, especially reducing the nuclear translocation of NF-κB p65 subunit as revealed by nuclear separation experiment and confocal microscopy.     

Anti-Inflammatory Effect of Allicin Associated with Fibrosis in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling. Recent evidence supports that inflammation plays a key role in triggering and maintaining pulmonary vascular remodeling. Recent studies have shown that garlic extract has protective effects in PAH, but the precise role of allicin, a compound derived from garlic, is unknown. Thus, we used allicin to evaluate its effects on inflammation and fibrosis in PAH. Male Wistar rats were divided into three groups: control (CON), monocrotaline (60 mg/kg) (MCT), and MCT plus allicin (16 mg/kg/oral gavage) (MCT + A). Right ventricle (RV) hypertrophy and pulmonary arterial medial wall thickness were determined. IL-1β, IL-6, TNF-α, NFκB p65, Iκβ, TGF-β, and α-SMA were determined by Western blot analysis. In addition, TNF-α and TGF-β were determined by immunohistochemistry, and miR-21-5p and mRNA expressions of Cd68, Bmpr2, and Smad5 were determined by RT-qPCR. Results: Allicin prevented increases in vessel wall thickness due to TNF-α, IL-6, IL-1β, and Cd68 in the lung. In addition, TGF-β, α-SMA, and fibrosis were lower in the MCT + A group compared with the MCT group. In the RV, allicin prevented increases in TNF-α, IL-6, and TGF-β. These observations suggest that, through the modulation of proinflammatory and profibrotic markers in the lung and heart, allicin delays the progression of PAH.     

Anti-Inflammatory and Analgesic Effects of Pyeongwisan on LPS-Stimulated Murine Macrophages and Mouse Models of Acetic Acid-Induced Writhing Response and Xylene-Induced Ear Edema

Pyeongwisan (PW) is an herbal medication used in traditional East Asian medicine to treat anorexia, abdominal distension, borborygmus and diarrhea caused by gastric catarrh, atony and dilatation. However, its effects on inflammation-related diseases are unknown. In this study, we investigated the biological effects of PW on lipopolysaccharide (LPS)-mediated inflammation in macrophages and on local inflammation in vivo. We investigated the biological effects of PW on the production of inflammatory mediators, pro-inflammatory cytokines and related products as well as the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated macrophages. Additionally, we evaluated the analgesic effect on the acetic acid-induced writhing response and the inhibitory activity on xylene-induced ear edema in mice. PW showed anti-inflammatory effects by inhibiting the production of nitric oxide (NO), tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and interleukin-1β (IL-1β). In addition, PW strongly suppressed inducible nitric oxide synthase (iNOS), a NO synthesis enzyme, induced heme oxygenase-1 (HO-1) expression and inhibited NF-κB activation and MAPK phosphorylation. Also, PW suppressed TNF-α, IL-6 and IL-1β cytokine production in LPS-stimulated peritoneal macrophage cells. Furthermore, PW showed an analgesic effect on the writhing response and an inhibitory effect on mice ear edema. We demonstrated the anti-inflammatory effects and inhibitory mechanism in macrophages as well as inhibitory activity of PW in vivo for the first time. Our results suggest the potential value of PW as an inflammatory therapeutic agent developed from a natural substance.     

Isoleucilactucin Ameliorates Coal Fly Ash-Induced Inflammation through the NF-κB and MAPK Pathways in MH-S Cells

We investigated whether isoleucilactucin, an active constituent of Ixeridium dentatum, reduces inflammation caused by coal fly ash (CFA) in alveolar macrophages (MH-S). The anti-inflammatory effects of isoleucilactucin were assessed by measuring the concentration of nitric oxide (NO) and the expression of pro-inflammatory mediators in MH-S cells exposed to CFA-induced inflammation. We found that isoleucilactucin reduced CFA-induced NO generation dose-dependently in MH-S cells. Moreover, isoleucilactucin suppressed CFA-activated proinflammatory mediators, including cyclooxygenase-2 (COX2) and inducible NO synthase (iNOS), and the proinflammatory cytokines such as interleukin-(IL)-1β, IL-6, and tumor necrosis factor (TNF-α). The inhibiting properties of isoleucilactucin on the nuclear translocation of phosphorylated nuclear factor-kappa B (p-NF-κB) were observed. The effects of isoleucilactucin on the NF-κB and mitogen-activated protein kinase (MAPK) pathways were also measured in CFA-stimulated MH-S cells. These results indicate that isoleucilactucin suppressed CFA-stimulated inflammation in MH-S cells by inhibiting the NF-κB and MAPK pathways, which suggest it might exert anti-inflammatory properties in the lung.