IκBα Degradation Research Articles

Antibacterial and Anti-Inflammatory Properties of Peptide KN-17.

Peri-implantitis, an infectious disease originating from dental biofilm that forms around dental implants, which causes the loss of both osseointegration and bone tissue. KN-17, a truncated cecropin B peptide, demonstrated efficacy against certain bacterial strains associated with peri-implantitis. This study aimed to assess the antibacterial and anti-inflammatory properties and mechanisms of KN-17. The effects of KN-17 on oral pathogenic bacteria were assessed by measuring its minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Moreover, the cytotoxicity and anti-inflammatory effects of KN-17 were evaluated. KN-17 inhibited the growth of Streptococcus gordonii and Fusobacterium nucleatum during in vitro biofilm formation and possessed low toxicity to hBMSCs cells. KN-17 also caused RAW264.7 macrophages to transform from M1 to M2 by downregulating pro-inflammatory and upregulating anti-inflammatory factors. It inhibited the NF-κB signaling pathway by reducing IκBα and P65 protein phosphorylation while promoting IκBα degradation and nuclear P65 translocation. KN-17 might be an efficacious prophylaxis against peri-implant inflammation.

Hedychium flavum flower essential oil: Chemical composition, anti-inflammatory activities and related mechanisms in vitro and in vivo

Ethnopharmacological relevanceHedychium flavum, an ornamental, edible, and medicinal plant, is extensively cultivated as a source of aromatic essential oils (EO). Its flower is a traditional Chinese medicine for treating inflammation-related diseases like indigestion, diarrhea, and stomach pain. In particular, H. flavum flower EO has been used in cosmetics and as an aromatic stomachic to treat chronic gastritis in China. Aim of the studyThis research aimed to analyze H. flavum flower EO's chemical composition and explore its anti-inflammatory activities and related mechanisms in vitro and in vivo. Materials and methodsEO's chemical composition was determined by GC-FID/MS analysis. For in vitro test, the anti-inflammatory activity of EO was demonstrated by measuring the LPS-induced release of NO, PGE2, IL-1β, TNF-α, and IL-6 in RAW264.7 macrophages, and then its related mechanisms were explored using qRT-PCR, western blot, and immunofluorescent staining analysis. Next, EO's in vivo anti-inflammatory potential was further evaluated using a xylene-induced ear edema model, in which ear swelling and TNF-α, IL-6, and IL-1β levels in serum and tissue were examined. ResultsThe main components of EO were β-pinene (20.2%), α-pinene (9.3%), α-phellandrene (8.3%), 1,8-cineole (7.1%), E-nerolidol (5.4%), limonene (4.4%), borneol (4.1%), and β-caryophyllene (3.7%). For the anti-inflammatory activities in vitro, EO dramatically reduced the LPS-stimulated NO and PGE2 release by suppressing the mRNA and protein expression of iNOS and COX-2. Meanwhile, it remarkably decreased IL-6, TNF-α, and IL-1β production by inhibiting their mRNA levels. Related mechanism studies indicated that it not only inhibited IκBα phosphorylation and degradation, leading to blockade of NF-κB nuclear transfer but also suppressed MAPKs (ERK, p38, and JNK) phosphorylation in LPS-stimulated RAW264.7 cells. Further in vivo assay showed that EO ameliorated xylene-induced ear edema in mice and reduced TNF-α, IL-6, and IL-1β levels in serum and tissue. ConclusionsH. flavum EO exerted significant anti-inflammatory activity in vivo and in vitro, and its mechanism of action is related to the inhibition of MAPK and NF-κB activation. Thus, H. flavum EO could be considered a novel and promising anti-inflammatory agent and possess high potential for utilization in the pharmaceutical field.

Rhamnus crenata leaf extracts exhibit anti-inflammatory activity via modulating the Nrf2/HO-1 and NF-κB/MAPK signaling pathways

Objective: To elucidate the potential anti-inflammatory mechanisms of Rhamnus crenata leaf extracts using RAW264.7 cells. Methods: We used 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay to measure cell viability. Nitric oxide (NO) production was measured using Griess reagent. Western blotting and RT-PCR assays were carried out for analyzing the protein and gene expressions of pro-inflammatory mediators, respectively. Moreover, PD98059 (ERK1/2 inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), and BAY11-7082 (NF-κB inhibitor) were used to evaluate the anti-inflammatory mechanism of Rhamnus crenata leaf extract. Results: Rhamnus crenata leaf extracts significantly inhibited the production of the pro-inflammatory mediators such as NO, iNOS, COX-2, IL-1β, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Rhamnus crenata leaf extracts also suppressed LPS-induced degradation of IκB-α and nuclear accumulation of p65, which resulted in the inhibition of NF-κB activation in RAW264.7 cells. Additionally, the extracts attenuated the phosphorylation of p38, ERK1/2, and JNK in LPS-stimulated RAW264.7 cells. Moreover, HO-1 expression induced by Rhamnus crenata leaf extracts was significantly downregulated by SB230580, PD98059, SP600125 and BAY11-7082. Conclusions: Rhamnus crenata leaf extract may upregulate HO-1 expression through inhibition of p38, ERK1/2, and NF-κB activation, which may contribute to the anti-inflammatory activity of the extracts. Rhamnus crenata leaf extracts may have great potential for the development of anti-inflammatory drugs to treat acute and chronic inflammatory diseases.

Protective effect of wogonin on inflammatory responses in BisGMA-treated macrophages through the inhibition of MAPK and NFκB pathways.

Composites, resins, and sealants that are commonly used in orthopedics and dentistry are based on 2,2-bis[p-(2'-hydroxy-3'-methacryloxypropoxy)phenylene]propane (BisGMA), which induces proinflammatory responses in macrophages. The present study aimed to explore the anti-inflammatory responses of wogonin, which is a natural dihydroxyl flavonoid compound, in BisGMA-treated macrophages. According to the findings, wogonin exhibits anti-inflammatory, antiallergic, anticancer, and antioxidative properties. The generation of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) were noted to be inhibited by wogonin in BisGMA-treated macrophages. Furthermore, the production of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 was reduced. In addition, BisGMA-induced nuclear factor (NF)-κB p65 phosphorylation and inhibitor of κB (IκB) degradation were inhibited. Finally, the BisGMA-induced phosphorylation of mitogen-activated protein kinases (MAPKs), including p38 MAPK, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) was inhibited. All these effects were induced by wogonin in the macrophages in a concentration-dependent manner. Similar inhibitory effects of wogonin were observed on the production of NO and proinflammatory cytokines, expression of iNOS, phosphorylation of NF-κB p65 and MAPK, and degradation of IκB. These results indicated that rutin is a potential anti-inflammatory agent for BisGMA-treated macrophages that undergo NFκB p65 phosphorylation and IκB degradation through upstream MAPK phosphorylation. Therefore, wogonin inhibits BisGMA-induced proinflammatory responses in macrophages through the regulation of the NFκB pathway and its upstream factor, MAPK.

Anti-oxidative and anti-inflammatory activities of the ethanol extract of edible flower from Chimonanthus praecox.

Chimonanthi Praecocis Flos, namely wintersweet flower, is the edible flower or flower bud of Chimonanthus praecox (L.) Link which is a deciduous shrub plant originated from China and is widely cultivated as a garden or ornamental plant all over the world. However, few studies focused on its anti-inflammatory property. In the present study, we explored the anti-inflammatory and anti-oxidative activities of ethanol extract of Chimonanthi Praecocis Flos (CPE) which contained 7.980% ± 0.176% total flavonoids and 1.461% ± 0.041% total alkaloids. In LPS-stimulated RAW264.7 macrophages, CPE significantly decreased the production of NO and prostaglandin E2 (PGE2) through reducing the expressions of their synthases—inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). It also suppressed the transcription and translation of pro-inflammatory cytokines interleukin-1β (IL-1β) and interleukin-6 (IL-6). Further research revealed that CPE impeded the phosphorylation and degradation of IκBα, thus restraining the nuclear translocation of p65, and consequently dampening NF-κB signaling. In endotoxemia mice, several pro-inflammatory cytokines in serum were also decreased after CPE treatment. Besides anti-inflammatory activity, anti-oxidative activity is another important capacity of wintersweet flower. Indeed, CPE reduced LPS-elevated intracellular total reactive oxygen species (ROS) level by weakening NADPH oxidase activity in cell system. Moreover, it directly scavenged DPPH radical and superoxide anion, and exerted ferric reducing ability in cell-free system. Our findings demonstrate that wintersweet flower can be used as a beneficial natural product or an additive by virtue of its anti-oxidative and anti-inflammatory properties.

Activation of the β‑TrCP/IκBα/inflammation axis limits the sensitivity of liver cancer cells to neddylation inhibition.

Upregulation of protein neddylation occurs in numerous types of human cancer, including liver cancer. MLN4924, a potent neddylation-inhibiting pharmacological agent, demonstrates anticancer ability in numerous cancers. However, the sensitivity of MLN4924 in liver cancer remains unsatisfactory due to factors causing resistance. RT-qPCR and western blotting were utilized to assess the mRNA and protein levels of genes, respectively. Cell Counting Kit-8 assay and colony formation assays were employed to assess cell viability and proliferation. The pathway of protein degradation and stability were determined by western blotting after treatment with MG132 and cycloheximide. An immunoprecipitation assay was utilized to detect the ubiquitination of protein. An in vitro ubiquitination assay was used to determine the ubiquitin linkage. To the best of our knowledge, the present study was the first to demonstrate that NF-κB inhibitor α (IκBα) downregulation and subsequent inflammation in response to MLN4924 limited the antitumor potential of MLN4924. Ectopic expression of IκBα enhanced the antitumor potential of MLN4924 in liver cancer cells. Moreover, the results of the present study demonstrated that MLN4924 decreased IκBα via promoting the K48 linkage of ubiquitin to IκBα. Mechanistic studies demonstrated that MLN4924 enhanced the protein stability of β-transducin repeat-containing protein (β-TrCP), promoting the ubiquitination of IκBα, which led to the ubiquitin-mediated degradation of IκBα. In addition, the results of the present study also demonstrated that β-TrCP knockdown markedly inhibited MLN4924 from suppressing the growth of liver cancer cells, via attenuating MLN4924-mediated IκBα downregulation and inflammation. Collectively, these results indicated that the β-TrCP/IκBα/inflammation pathway may act as a novel resistance factor of MLN4924, and targeting β-TrCP may be beneficial for the treatment of liver cancer.

Monotropein Protects against Inflammatory Bone Loss and Suppresses Osteoclast Formation and Bone Resorption by Inhibiting NFATc1 via NF-κB and Akt/GSK-3β Pathway.

Monotropein (Mon) is a kind of iridoid glycoside plant secondary metabolite primarily present in some edible and medicinal plants. The aim of this study was to investigate the effect of Mon on lipopolysaccharide (LPS)-induced inflammatory bone loss in mice and osteoclasts (OCs) derived from bone marrow-derived macrophages (BMMs), and explore the mechanisms underlying the effect of Mon on LPS-induced osteoclastogenesis. It was found that Mon markedly attenuated deterioration of the bone micro-architecture, enhanced tissue mineral content (TMC) and bone volume/total volume (BV/TV), reduced structure model index (SMI) and trabecular separation/spacing (Tb.Sp) in the bone tissue and decreased the activities of tartrate resistant acid phosphatase-5b (TRACP-5b), receptor activator NF-κB (RANK), and receptor activator NF-κB ligand (RANKL) as well as the serum levels of interleukin 6 (IL-6) and interleukin 1β (IL-1β) in LPS-treated mice. In addition, Mon treatment reduced the number of TRAP positive OCs in the bone tissue of LPS-treated mice and also exerted a stronger inhibitory effect on formation, differentiation, and F-actin ring construction of OCs derived from BMMs. Mon significantly inhibited the expression of the nuclear factor of activated T-cells c1 (NFATc1) and the immediate early gene (C-Fos) and nuclear translocation of NFATc1 in LPS-treated OCs, thereby inhibiting the expression of matrix metalloproteinase-9 (MMP-9), cathepsin K (CtsK), and TRAP. Mon significantly inhibited the expression of TRAF6, phosphorylation of P65, and degradation of IKBα, thus inhibiting the activation of NF-κB pathway in LPS-induced inflammatory mice and OCs derived from BMMs, and also inhibited LPS-induced phosphorylation of protein kinase B (Akt) and Glycogen synthase kinase 3β (GSK-3β) in OCs derived from BMMs. In conclusion, these results suggested that Mon could effectively inhibit osteoclastogenesis both in vitro and in vivo and therefore may prove to be potential option for prevention and treatment of osteoclastic bone resorption-related diseases.

Dehydromiltirone inhibits osteoclast differentiation in RAW264.7 and bone marrow macrophages by modulating MAPK and NF-κB activity.

Background: Osteoporosis is a type of systematic metabolic bone disease caused by the decrease in osteogenic activity or excessive resorption of bone with the relative enhancement of osteoclast function. As osteoporosis seriously affects the quality of patients’ life, effective drugs are needed to treat this disease. Based on the combination of network pharmacology and cellular studies, this study aimed to investigate the probable mechanism of Dehydromiltirone (DHT) in the treatment of osteoporosis. Method: The targets of DHT in osteoporosis were searched using the PharmGKB, OMIM, and Genecard platforms. The PPI core targets, and the GO and KEGG enrichment analysis results were obtained using Cytoscape software, and the David and Metascape databases, respectively. The network pharmacology results were also verified via in vitro cellular experiments. Results: Through network pharmacology and docking analysis, we found DHT was involved in peptide tyrosine phosphorylation, cell surface receptor tyrosine kinase signaling pathways, and MAPK signaling pathways. According to the molecular docking results, the binding of DHT to MAPK14 was more stable than other proteins, which suggests that DHT may affect osteoclast formation through the MAPK signaling pathway. Moreover, DHT was found to inhibit the expression of osteoclast-associated genes, including NFATc1, CTSK, c-Fos, Acp5, and MMP9; as well as the phosphorylation of P38, ERK, and JNK of the MAPK signaling pathway; and the degradation of IκB-α of NF-κB signaling pathway. Conclusion: DHT exhibited an anti-osteoclastogenesis effect by reducing the expression of related genes, ultimately inhibiting bone resorption in vitro.

Polyhydric Stigmastane-Type Steroids Derivative from Vernonia amygdalina and Their Anti-Neuroinflammatory Activity.

Vernonia amygdalina Del. is a traditional medicinal plant and vegetable originating from tropical Africa. The phytochemical investigation of V. amygdalina led to eight undescribed polyhydric stigmastane-type steroids, vernonin M–T (1–8). Their gross structures and stereochemistry were elucidated by HR-ESI-MS, 1D and 2D NMR spectra, X-ray diffraction, quantum chemical computation of the ECD spectrum, and the in situ dimolybdenum CD method. The anti-neuroinflammatory activity of the isolated compounds was performed in BV-2 microglia cells. As a result, compound 1 displayed a notable anti-neuroinflammatory effect via suppressing the LPS-induced IκB degradation and restricting the activation of the PI3K/AKT and p38 MAPK pathways.

MiR-1246-overexpressing exosomes suppress UVB-induced photoaging via regulation of TGF-β/Smad and attenuation of MAPK/AP-1 pathway.

Stem cell therapy is widely employed for the treatment of skin diseases, especially in skin rejuvenation. Exosomes derived from stem cells have been demonstrated to possess anti-photoaging effects; however, the precise components within exosomes that are responsible for this effect remain unknown. Previously, miR-1246 was found to be one of the most abundant nucleic acids in adipose-derived stem cells (ADSCs)-derived exosomes. This study examined whether miR-1246 was the major therapeutic agent employed by ADSCs to protect against UVB-induced photoaging. Lentivirus infection was used to obtain miR-1246-overexpressing ADSCs and exosomes. We then determined the anti-photoaging effects of miR-1246-overexpressing exosomes (OE-EX) on both UVB-irradiated humanskinfibroblasts (HSFs) and Kunming mice. The results showed that OE-EX could significantly decrease MMP-1 by inhibiting the MAPK/AP-1 signaling pathway. Meanwhile, OE-EX markedly increased procollagen type I secretion by activating the TGF-β/Smad pathway. OE-EX also exhibited an anti-inflammatory effect by preventing the UVB-induced degradation of IκB-α and NF-κB overexpression. Animal experiments demonstrated that OE-EX could reduce UVB-induced wrinkle formation, epidermis thickening, and the loss of collagen fibers reduction in Kunming mice. The combined results suggested that miR-1246 is the key component within ADSCs-derived exosomes that protects against UVB-induced skin photoaging.

Betulinic acid improves TNF-α-induced insulin resistance by inhibiting negative regulator of insulin signalling and inflammation-activated protein kinase in 3T3-L1 adipocytes

Context Obesity is related to insulin resistance, and adipose tissue-secreted TNF-α may play a role in inducing obesity. TNF-α activates inflammatory protein kinase and impairs insulin signalling. Objectives We investigated the effect of betulinic acid on insulin resistance caused by TNF-α treatment in 3T3-L1 adipocytes. Material and methods 3T3-L1 was exposed to TNF-α in the presence and absence of betulinic acid. Various parameters such as glucose uptake assay, cell viability, expression of proteins involved in insulin resistance were studied. Results Betulinic acid increased glucose uptake in TNF-α pre-treated cells and inhibited the activation of PTP1B and JNK and reduced IκBα degradation. Tyrosine phosphorylation was increased, and serine phosphorylation was decreased in IRS-1. Discussion Betulinic acid restored TNF-α impaired insulin signalling and increased PI3K activation and phosphorylation of Akt and increased plasma membrane expression of GLUT 4, which stimulated glucose uptake concentration-dependently. Conclusion These results suggest that betulinic acid is effective at improving TNF-α-induced insulin resistance in adipocytes via inhibiting the activation of negative regulator of insulin signalling and inflammation-activated protein kinase and may potentially improve insulin resistance.

TRIM46 aggravated high glucose-induced hyper permeability and inflammatory response in human retinal capillary endothelial cells by promoting IκBα ubiquitination

BackgroundDiabetic retinopathy (DR) as a severe diabetic complication contributes to blindness. The increased permeability of retinal capillary endothelial cells (RCECs) as well as the production of inflammatory markers are closely related to DR occurrence. We recently revealed that TRIM46 promotes high glucose (HG)-caused ferroptosis in human RCECs (HRCECs). The current study aims to explore the molecular mechanism of how TRIM46 plays its role in DR progression.MethodsWestern blot was utilized to determine protein expression. The cell counting kit-8 assay was used to observe cell viability. The permeability of the cell layer was determined by measuring the transepithelial electrical resistance and fluorescein isothiocyanate (FITC)-dextran leak. Enzyme-linked immunosorbent assay was used to quantify the protein level of pro-inflammatory cytokines and co-immunoprecipitation was employed to verify the relationship between TRIM46 and IκBα.ResultsHG dramatically upregulated TRIM46 protein expression in a dose-dependent way. Silencing TRIM46 effectively reversed HG-induced cell growth inhibition, cell cycle arrest, hyper permeability and pro-inflammatory cytokines secretion in HRCECs, while overexpression of TRIM46 exhibited an opposite effect. Furthermore, TRIM46 was able to interact with IκBα and promote the ubiquitination and degradation of IκBα. IκBα overexpression recovered the effects of TRIM46 overexpression in HRCECs. Furthermore, inhibiting the activation of NF-κB partially recovered HG-induced HRCEC injury, whereas TRIM46 overexpression reversed these effects.ConclusionThis study demonstrates that TRIM46 interacts with IκBα to activate the NF-κB signaling pathway, thereby enhancing cell proliferation inhibition, hyper permeability and the inflammatory response of HRCECs in a HG state.

Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Anti-inflammatory Effects of Myrrh Ethanol Extract on Particulate Matter-induced Skin Injury

Objectives: Myrrh have been used as a traditional remedy to treat infectious and inflammatory diseases. However, it is largely unknown whether myrrh ethanol extract could exhibit the inhibitory activities against particulate matter (PM)-induced skin injury on human keratinocytes, HaCaT cells. Therefore, this study was aimed to investigate the inhibitory activity of myrrh ethanol extract on PM-induced skin injury in HaCaT cells.Methods: To investigate the inhibitory effects of myrrh ethanol extract in HaCaT cells, the skin injury model of HaCaT cells was established under PM treatment. HaCaT keratinocyte cells were pre-treated with myrrh ethanol extract for 1 h, and then stimulated with PM. Then, the cells were harvested to measure the cell viability, reactive oxygen species (ROS), pro-inflammatory cytokines including interleukin (IL) 1-beta, IL-6, and tumor necrosis factor (TNF)-α, hyaluronidase, collagen, MMPs. In addition, we examined the mitogen activated protein kinases (MAPKs) and inhibitory kappa B alpha (Iκ-Bα) as inhibitory mechanisms of myrrh ethanol extract.Results: The treatment of myrrh ethanol extract inhibited the PM-induced cell death and ROS production in HaCaT cells. In addition, myrrh ethanol extract treatment inhibited the PM-induced elevation of IL-1beta, IL-6, and TNF-α. Also, myrrh ethanol extract treatment inhibited the increase of hyaluronidase, MMP and decrease of collagen. Furthermore, myrrh ethanol extract treatment inhibited the activation of MAPKs and the degradation of Iκ-Bα.Conclusions: Our result suggest that treatment of myrrh ethanol extract could inhibit the PM-induced skin injury via deactivation of MAPKs and nuclear factor (NF)-κB in HaCaT cells. This study could suggest that myrrh ethanol extract could be a beneficial agent to prevent skin damage or inflammation.

Carbon monoxide-releasing molecule-401, a water-soluble manganese-based metal carbonyl, suppresses Prevotella intermedia lipopolysaccharide-induced production of nitric oxide in murine macrophages

Context Many reports in the literature have suggested the therapeutic value of carbon monoxide-releasing molecules (CORMs) against various diseases. However, to date, little is known about their possible influence on periodontal disease. Objective This study was performed to investigate the influence of CORM-401 on the generation of nitric oxide (NO) in murine macrophage cells activated with lipopolysaccharide (LPS) derived from Prevotella intermedia, a pathogen associated with periodontal disease. Materials and methods LPS was isolated by the hot phenol-water method. Culture supernatants were analyzed for NO. Real-time PCR and immunoblotting were conducted to quantify mRNA and protein expression, respectively. NF-κB-dependent SEAP levels were estimated by reporter assay. DNA-binding of NF-κB was also analyzed. Results CORM-401 caused an apparent suppression of NO production through inhibition of iNOS at both the mRNA and protein levels in RAW264.7 cells stimulated with P. intermedia LPS. CORM-401 upregulated the expression of both the HO-1 gene and its protein in LPS-activated cells, and treatment with the HO-1 inhibitor significantly reversed the attenuating influence of CORM-401 against LPS-induced generation of NO. CORM-401 caused an apparent attenuation of NF-κB-dependent SEAP release induced by LPS. IκB-α degradation and nuclear translocation of NF-κB p50 subunit induced by LPS were significantly reduced by CORM-401. Additionally, CORM-401 significantly attenuated DNA-binding of p65 and p50 induced by LPS. CORM-401 attenuated NO generation induced by P. intermedia LPS independently of PPAR-γ, JNK, p38 and STAT1/3. Conclusion The modulation of host inflammatory response by CORM-401 might be of help in the therapy of periodontal disease.

Downregulation of miR-885-5p Promotes NF-κB Pathway Activation and Immune Recruitment in Cutaneous Lupus Erythematosus

Cutaneous lupus erythematosus (CLE) has a specific microRNA expression profile. MiR-885-5p has been found to be downregulated in the epidermis of CLE lesions; however, its biological role in the disease has not been studied. In this study, we show that miR-885-5p is markedly reduced in CLE keratinocytes (KCs) with IFN-α and UVB being strong miR-885-5p regulators invitro. Microarray expression profiling of anti‒miR-885-5p‒transfected KCs identified PSMB5 as a direct target. Specific inhibition of miR-885-5p increased epidermal proliferation by modulating keratin 16 gene K16, BIRC5, TP63, and CDK4 proliferative genes and promoted NF-κB signaling pathway in human primary KCs by increasing IκBα degradation. Silencing PSMB5 rescued the effect of miR-885-5p inhibition, indicating that miR-885-5p regulates proliferation and NF-κB activation by targeting PSMB5 in KCs. In addition, inhibition of miR-885-5p increased the ability of KCs to attract leukocytes in a PSMB5-independent manner. We identified TRAF1 as another direct target, and its silencing reduced leukocyte migration. Collectively, our findings suggest that UVB and IFN-ɑ downregulate miR-885-5p in CLE KCs, leading to epidermal inflammation by NF-κB activity enhancement and proliferation through PSMB5 and immune recruitment through TRAF1. Our data indicate that miR-885-5p is a potential therapeutic target in CLE.

Pharmacologic inhibition of PARP5, but not that of PARP1 or 2, promotes cytokine production and osteoclastogenesis through different pathways.

PARPs, which are members of the poly(ADP-ribose) polymerase superfamily, promote tumorigenesis and tumour-associated inflammation and are thus therapeutic targets for several cancers. The aim of the present study is to investigate the mechanistic insight into the roles PARPs for inflammation. Primary murine macrophages were cultured in the presence or absence of the PARP5 inhibitor NVP-TNKS656 to examine the role of PARP5 for cytokine production. In contrast to the roles of other PARPs for induction of inflammation, we found in the present study that pharmacologic inhibition of PARP5 induces production of inflammatory cytokines in primary murine macrophages. We found that treatment with the PARP5 inhibitor NVP-TNKS656 in macrophages enhanced steady-state and LPS-mediated cytokine production through degradation of IκBα and subsequent nuclear translocation of NF-κB. We also found that pharmacologic inhibition of PARP5 stabilises the adaptor protein 3BP2, a substrate of PARP5, and that accelerated cytokine production induced by PARP5 inhibition was rescued in 3BP2-deleted macrophages. Additionally, we found that LPS increases the expression of 3BP2 and AXIN1, a negative regulator of β-catenin, through suppression of PARP5 transcripts in macrophages, leading to further activation of cytokine production and inhibition of β-catenin-mediated cell proliferation, respectively. Lastly, we found that PARP5 inhibition in macrophages promotes osteoclastogenesis through stabilisation of 3BP2 and AXIN1, leading to activation of SRC and suppression of β-catenin, respectively. Our results show that pharmacologic inhibition of PARP5 against cancers unexpectedly induces adverse autoinflammatory side effects through activation of innate immunity, unlike inhibition of other PARPs.

Aerobic glycolysis promotes tumor immune evasion by hexokinase2-mediated phosphorylation of IκBα

High expression of PD-L1 in tumor cells contributes to tumor immune evasion. However, whether PD-L1 expression in tumor cells is regulated by the availability of nutrients is unknown. Here, we show that in human glioblastoma cells, high glucose promotes hexokinase (HK) 2 dissociation from mitochondria and its subsequent binding and phosphorylation of IκBα at T291. This leads to increased interaction between IκBα and μ-calpain protease and subsequent μ-calpain-mediated IκBα degradation and NF-κB activation-dependent transcriptional upregulation of PD-L1 expression. Expression of IκBα T291A in glioblastoma cells blocked high glucose-induced PD-L1 expression and promoted CD8+ Tcell activation and infiltration into the tumor tissue, reducing brain tumor growth. Combined treatment with an HK inhibitor and an anti-PD-1 antibody eliminates tumor immune evasion and remarkably enhances the anti-tumor effect of immune checkpoint blockade. These findings elucidate a novel mechanism underlying the upregulation of PD-L1 expression mediated by aerobic glycolysis and underscore the roles of HK2 as a glucose sensor and a protein kinase in regulation of tumor immune evasion.

Vitex trifolia L. modulates inflammatory mediators via down-regulation of the NF-κB signaling pathway in carrageenan-induced acute inflammation in experimental rats

Ethnopharmacological relevanceVitex trifolia L. (V. trifolia L.), commonly known as the three-leaved chaste tree, is extensively employed in traditional Chinese medicine (TCM) to treat various conditions associated with inflammation. Aim of the studyThe present study aimed to delineate the molecular mechanisms responsible for the anti-inflammatory effect of V. trifolia L. in carrageenan (CA)-induced acute inflammation in experimental rats. Materials and methodsCA-induced rat paw edema model was adopted to investigate the anti-inflammatory effect of methanolic extract from leaves of V. trifolia L. (VTME) in vivo. Leukocyte infiltration into the site of inflammation was determined by histopathological analysis. Further, the effect of VTME on CA-induced local and systemic levels of specific cytokines was quantified by enzyme-linked immunosorbent assay (ELISA). Moreover, its impact on the nuclear translocation of nuclear factor Kappa B (NF-κB) was analyzed by employing the western blotting technique. ResultsVTME at the doses of 100 mg/kg and 200 mg/kg significantly inhibited the paw edema induced by CA (p < 0.05) and effectively reduced the inflammatory leukocyte infiltration. Further, VTME markedly inhibited the CA-induced levels of Interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α in tissue, and that of cytokine-induced neutrophil chemoattractant (CINC)-2/C-X-C motif chemokine (CXCL)3 and CINC-3/CXCL2 in tissue as well as in serum. On the other hand, VTME significantly upregulated the tissue concentration of anti-inflammatory cytokine IL-10. Moreover, VTME significantly attenuated the CA-induced IκBα degradation and nuclear translocation of NF-κB p65. ConclusionsOur results demonstrate the potent anti-inflammatory effect of V. trifolia L. in vivo, providing insight into its molecular mechanism, which is mediated through down-regulation of NF-κB signal transduction.

Imperatorin Inhibits Proliferation, Migration, and Inflammation via Blocking the NF-κB and MAPK Pathways in Rheumatoid Fibroblast-like Synoviocytes

Rheumatoid arthritis (RA) is a chronic joint inflammatory disease associated with the aberrant activation of fibroblast-like synoviocytes (FLSs). Searching for natural compounds that may suppress the activation of FLSs has become a complementary approach for RA treatment. Here, we investigated the effects and mechanisms of imperatorin (IPT) on proliferation, migration, and inflammation in primary cultured arthritic FLSs. We found that IPT significantly suppressed TNFα-induced proliferation and migration of arthritic FLSs, but showed little effect on survival and apoptosis. In addition, IPT treatment significantly reduced the TNFα-induced expression of pro-inflammatory cytokines (IL-1β, TNFα, IL-6, and IL-8) in arthritic FLSs. Further mechanism studies suggested that IPT inhibited the activations of p38 and extracellular signal-regulated kinase (ERK). Also, IPT blocked the nuclear factor of κB (NF-κB) activation by suppressing the phosphorylation and degradation of IκBα, thereby preventing the translocation of p65. Collectively, our results demonstrated that IPT could inhibit the over-activated phenotypes of arthritic FLSs via the mitogen-activated protein kinase (MAPK) (p38 and ERK) and NF-κB pathways leading to the down-regulation of pro-inflammatory cytokines, which might be beneficial to the anti-proliferative and anti-migratory activities of FLS cells. These findings suggest that IPT has the potential to be developed as a novel agent for RA treatment.