NF-κB P65 Nuclear Translocation Research Articles

Liuwei Dihuang Decoction Drug-containing Serum Attenuates Transforming Growth Factor-β1-induced Epithelial-Mesenchymal Transition in HK-2 cells by Inhibiting NF-κB/Snail Signaling Pathway.

The nuclear factor-κB (NF-κB) signaling pathway plays an important role in regulating tubular epithelial-mesenchymal transition (EMT), an indispensable cellular programme for driving organ fibrosis and tumor progression. Liuwei Dihuang Decoction (LWD) is an effective Chinese formula for treating chronic renal failure. First, by using morphological examination, immunofluorescence staining assay, RT-qPCR, and Western blot analysis, in vitro experiments were designed to analyze NF-κB and EMT markers (including Snail, α-SMA, and E-cadherin) in transforming growth factor-β1 (TGF-β1) induced renal tubular epithelial cells (HK-2) and to detect the expression levels of LWD-CS co-treatment. Then, the recombinant lentiviral vector was overexpressed and knocked down by NF-κB and transfected into HK-2 cells. Cells were treated with TGF-β1 (10ng/ml) with blank serum or LWD-containing serum, respectively, and the expression of these molecules in the NF-κB/Snail signaling pathway was further evaluated. Our results confirmed that TGF-β1 could induce EMT, nuclear translocation of NF-κB p65, and activate the NF-κB/Snail signaling pathway in HK-2 cells. Furthermore, NF-κB knocked-down dramatically increases the TGF-β1-induced mRNA and protein expression level of E-cadherin and reduces the level of Snail and α-SMA; this is reversed by NF-κB overexpression. LWD can decrease the EMT levels through the NF-κB/Snail signaling activation in TGF-β1-induced EMT of HK-2 cells. The present study provides evidence suggesting a novel mechanism that LWD exerts anti-fibrosis effects through inhibiting activation of the NF-κB/Snail signaling pathway and consequently downregulating the TGF-β1-induced EMT in renal tubular epithelial cell.

Electroacupuncture stimulating Zhongji (CV3), Guanyuan (CV4), and bilateral Dahe (KI12) attenuates inflammation in rats with chronic nonbacterial prostatitis induced by estradiol through inhibiting toll-like receptor 4 pathway.

To investigate the anti-inflammatory effect of electroacupuncture (EA) on chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), also known as chronic nonbacterial prostatitis (CNP), and explore its underlying mechanism. A CNP rat was established by surgical castration combined with 17-β estradiol injection in male Sprague-Dawley rats for thirty consecutive days. The CNP rats received EA treatment once a day for eight days. Chronic pelvic pain was evaluated by mechanical withdrawal threshold measurement. The histological change was assessed by hematoxylin-eosin staining. The inflammatory cytokines in prostates were determined by enzyme-linked immunosorbent assays. The expressions of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), inhibitors of kappa-B alpha (IκBα), and nuclear factor-kappa B (NF-κB) were detected by Western blotting. The nuclear translocation of NF-κB and the location of TLR4 were observed with immunofluorescence staining. The results showed that EA decreased the prostate index, upregulated the mechanical withdrawal threshold, restored the histomorphology of the prostate, reduced the inflammatory factor levels, inhibited NF-κB p65 nuclear translocation, and downregulated the expression levels of critical proteins involved in the TLR4/NF-κB signaling pathway in prostates. Our findings suggested that EA could relieve pelvic pain and attenuate prostatic inflammation in estradiol-induced CNP rats. The underlying mechanism may be related to the inhibition of the TLR4/NF-κB signaling pathway.

A natural sesquiterpene lactone isolinderalactone attenuates lipopolysaccharide-induced inflammatory response and acute lung injury through inhibition of NF-κB pathway and activation Nrf2 pathway in macrophages

Isolinderalactone is the main sesquiterpene lactone isolated from Lindera aggregata, a traditional Chinese medicine widely used to treat pain and inflammation. Although isolinderalactone has been demonstrated to possess anti-cancer effect, its anti-inflammatory activity and underlying mechanism has not been well characterized. Herein, isolinderalactone was able to significantly inhibit the production of NO and PGE2 by reducing the expressions of iNOS and COX2 in LPS-stimulated RAW264.7 macrophages and BMDMs, and decreased the mRNA levels of IL-1β, IL-6, and TNF-α in LPS-induced RAW264.7 cells. In vivo, isolinderalactone effectively alleviated LPS-induced acute lung injury (ALI), which manifested as reduction in pulmonary inflammatory infiltration, myeloperoxidase activity, and production of PGE2, IL-1β, IL-6, TNF-α, and malondialdehyde. Furthermore, isolinderalactone inhibited phosphorylation of IKKα/β, phosphorylation and degradation of IκBα, and nuclear translocation of NF-κB p65, thereby blocking NF-κB pro-inflammatory pathway. Meanwhile, isolinderalactone reduced the intracellular ROS through promoting the activation of Nrf2-HMOX1 antioxidant axis. By using drug affinity responsive target stability assay and molecular docking, isolinderalactone was found to covalently interact with IKKα/β and Keap1, which may contribute to its anti-inflammatory action. Additionally, a thiol donor β-mercaptoethanol significantly abolished isolinderalactone-mediated anti-inflammatory action in vitro, indicating the crucial role of the unsaturated lactone of isolinderalactone on its anti-inflammatory effects. Taken together, isolinderalactone protected against LPS-induced ALI in mice, which may be associated with its inhibition of NF-κB pathway and activation of Nrf2 signaling in macrophages.

The kinesin-14 family motor protein KIFC2 promotes prostate cancer progression by regulating p65

The kinesin-14 motor proteins play important roles in tumor development and drug resistance and have been reported as potential biomarkers or therapeutic targets for tumor treatment. However, kinesin family member C2 (KIFC2), one of the kinesin-14 motor family members, remains largely unknown in prostate cancer (PCa) progression. Here, we used the GEO and The Cancer Genome Atlas datasets, Western blotting, and immunohistochemistry analyses to detect KIFC2 expression in PCa tissues. Additionally, a series of invivo and invitro experiments were utilized to demonstrate the roles of KIFC2 in PCa cells. We found that KIFC2 was highly expressed and positively correlated with the clinicopathological characteristics in PCa. Functional experiments indicated that KIFC2 could promote PCa progression. Furthermore, we performed an analysis of the KEGG and GSEA databases, subcellular fractionation, and immunofluorescence to investigate the potential mechanisms of KIFC2 in PCa. We confirmed that KIFC2 could regulate the NF-κB pathway via mediating NF-κB p65 protein expression and nuclear translocation thereby promoting PCa progression and chemotherapeutic resistance. Together, our results suggest that KIFC2 is overexpressed in PCa. By regulating the NF-κB pathway, KIFC2 may play a crucial role in PCa.

Ethyl Lithospermate Reduces Lipopolysaccharide-Induced Inflammation through Inhibiting NF-κB and STAT3 Pathways in RAW 264.7 Cells and Zebrafish.

To explore the anti-inflammatory effects of ethyl lithospermate in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine-derived macrophages and zebrafish, and its underlying mechanisms. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) assays were performed to investigate the toxicity of ethyl lithospermate at different concentrations (12.5-100 µ mol/L) in RAW 264.7 cells. The cells were stimulated with LPS (100 ng/mL) for 12 h to establish an inflammation model in vitro, the production of pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor α (TNF-α) were assessed by enzyme linked immunosorbent assay (ELISA). Western blot was used to ascertain the protein expressions of signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NF-κB) p65, phospho-STAT3 (p-STAT3, Tyr705), inhibitor of NF-κB (IκB) α, and phospho-I κB α (p-IκB α, Ser32), and confocal imaging was used to identify the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Additionally, the yolk sacs of zebrafish (3 days post fertilization) were injected with 2 nL LPS (0.5 mg/mL) to induce an inflammation model in vivo. Survival analysis, hematoxylin-eosin (HE) staining, observation of neutrophil migration, and quantitative real-time polymerase chain reaction (qRT-PCR) were used to further study the anti-inflammatory effects of ethyl lithospermate and its probable mechanisms in vivo. The non-toxic concentrations of ethyl lithospermate have been found to range from 12.5 to 100 µ mol/L. Ethyl lithospermate inhibited the release of IL-6 and TNF-α(P<0.05 or P<0.01), decreased IκBα degradation and phosphorylation (P<0.05) as well as the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705) in LPS-induced RAW 264.7 cells (P<0.01). Ethyl lithospermate also decreased inflammatory cells infiltration and neutrophil migration while increasing the survival rate of LPS-stimulated zebrafish (P<0.05 or P<0.01). In addition, ethyl lithospermate also inhibited the mRNA expression levels of of IL-6, TNF-α, IκBα, STAT3, and NF-κB in LPS-stimulated zebrafish (P<0.01). Ethyl lithospermate exerts anti-Inflammatory effected by inhibiting the NF-κB and STAT3 signal pathways in RAW 264.7 macrophages and zebrafish.

Chemical Modification of Curcumin Increases Its Potency against Hypopharyngeal Carcinoma

Hypopharyngeal carcinoma is notorious for its poor prognosis among all head and neck cancers, posing a persistent challenge in clinical settings. The continuous hyperactivation of the NFκB signaling pathway has been noted in various cancer types, including hypopharyngeal carcinoma. In our quest to develop a novel drug that targets hypopharyngeal cancer via the NFκB pathway, we employed curcumin, a well-known lead compound, and performed chemical modifications to create a mono-carbonyl analog called L42H17. This compound exhibited exceptional stability and displayed an enhanced binding affinity to myeloid differentiation protein 2 (MD2). Consistent with expectations, L42H17 demonstrated the ability to inhibit TNF-α-induced phosphorylation of IKK, prevent IκB degradation, and subsequently impede NFκB-p65 nuclear translocation in hypopharyngeal cancer cells. Additionally, L42H17 exhibited a remarkable capacity to induce cell cycle arrest at the G2-M phase by inactivating the cdc2-cyclin B1 complex. Moreover, it facilitated cell apoptosis by reducing Bcl-2 levels and augmenting the expression of cle-PARP and cle-caspase3. Importantly, we observed a significant enhancement in the anti-cancer efficacy of L42H17 in a patient-derived tumor xenograft (PDTX) model of hypopharyngeal carcinoma. In conclusion, our findings strongly suggest that L42H17 holds promise as a potential candidate drug for the treatment of hypopharyngeal carcinoma in the future.

Role of interleukin-36γ induced by ultraviolet radiation in chronic actinic dermatitis.

Chronic actinic dermatitis (CAD) is an immune-mediated photodermatosis characterized by a high eosinophil count and total immunoglobulin E (IgE) in the peripheral blood of patients. At present, however, the reasons for their elevation remain unclear. The current study aimed to detect changes in inflammatory cytokines in CAD and explore their role in this disease. Enzyme-linked immunosorbent assay and Luminex assay were conducted to measure inflammatory factor levels. Immunohistochemical analysis and quantitative real-time polymerase chain reaction were performed to evaluate the expression levels of interleukin-36γ (IL-36γ), IL-8, chemokine (C-C motif) ligand 17 (CCL17), and CCL18. CCK8 kits were used to assess cell proliferation. Immunofluorescence was used to detect nuclear factor κB (NF-κB) p65 nuclear translocation. Western blot analysis was performed to detect the protein expression level of phosphorylated NF-κB (p-NF-κB) p65. Hematoxylin and eosin and Masson trichrome staining were applied to observe histological changes in a chronic photo-damaged mouse model. Eosinophils, total IgE, IL-36γ, IL-8, tumor necrosis factor α, CCL17, and CCL18 were elevated in CAD. Of note, IL-36γ promoted the proliferation of eosinophilic cells (EOL-1) and the production of IgE in peripheral blood mononuclear cells. IL-36γ also promoted the production of IL-8 and CCL18 in immortalized human keratinocytes (HaCaT cells), while ultraviolet radiation (UVR)-induced IL-36γ via activation of the NF-κB signaling pathway. IL-36γ was involved in the pathogenesis of CAD and UVR contributed to the production of IL-36γ, which may provide a novel therapeutic target for CAD.

Modified rougan decoction attenuates hepatocyte apoptosis through ameliorating mitochondrial dysfunction by upregulated SIRT1/PGC-1α signaling pathway

The modified rougan decoction (MRGD) compound formula has been proven a certain ability to relieve lipopolysaccharide-enrofloxacin (LPS-ENR)-induced liver oxidant injury in chickens. Recent advances have shown that mitochondrial dysfunction affects the development of many diseases, leading to increased interest in exploring its effects. Using LPS-ENR-injured in vivo and in vitro to further evaluate the effects of MRGD on mitochondrial structure and function, and emphasized further investigation of its molecular mechanism. After LPS-ENR treatment, the levels of inflammation and apoptosis markers were increased, along with higher mitochondrial injury. Results showed that MRGD reduced inflammatory factors expression and inhibited the nuclear translocation of NF-κB P65, reducing the inflammatory response in vivo and in vitro. Additionally, MRGD pretreatment inhibited mitochondrial dysfunction, mitochondrial oxidative stress, and mitochondrial pathway apoptosis by maintaining mitochondrial structure and function. Moreover, treatment with the inhibitor EX527 showed that MRGD promoted mitochondrial biogenesis ability through the SIRT1/PGC-1α pathway and interfered with mitochondrial dynamics, and activate Nrf2. In summary, MRGD played a key role in promoting mitochondrial function and thus alleviating hepatocyte apoptosis in vivo and in vitro at least in part.

Equine induced pluripotent stem cells are responsive to inflammatory cytokines before and after differentiation into musculoskeletal cell types

Persistent inflammation is associated with the poor regeneration of musculoskeletal tissues. Embryonic stem cells (ESCs) have an attenuated response to inflammatory cytokines, but there are mixed reports on the response of induced pluripotent stem cells (iPSCs) to inflammation. Horses provide a relevant large animal model for studying musculoskeletal tissue diseases and the testing of novel therapies. The aim of this study was to determine if equine iPSCs are responsive to the inflammatory cytokines IL-1β, TNFα and IFN-γ in their undifferentiated state, or following differentiation into tendon and cartilage-like cells. We demonstrated that in undifferentiated iPSCs, the cytokines induce NF-κB P65 and STAT1 nuclear translocation which leads to cell death, decreased OCT4 expression and increased expression of inflammatory genes. Following differentiation towards cartilage-like cells exposure to the cytokines resulted in STAT1 nuclear translocation, changes in cartilage gene expression and increased expression of matrix metalloproteinases (MMPs) and inflammatory genes. Exposure of iPSC-derived tendon-like cells to the cytokines resulted nuclear translocation of NF-κB P65 and STAT1, altered tendon gene expression, increased MMP expression and increased expression of inflammatory genes. Equine iPSCs are therefore capable of responding to inflammatory stimulation and this may have relevance for their future clinical application.

Hepatoprotective effect of total flavonoids from Carthamus tinctorius L. leaves against carbon tetrachloride-induced chronic liver injury in mice

Carthamus tinctorius L. leaves, a waste product after Carthami flos production, are rich in flavonoids. Total flavonoids from C. tinctorius L. leaves (TFCTLL) exhibited the protective effect on acute liver injury in mice in previous studies. The aim of the present study was to evaluate the hepatoprotective effect of TFCTLL on chronic liver injury (CLI) and investigate the underlying mechanism. The chemical components of TFCTLL were identified by UPLC-Q-TOF/MS, and their migration into blood was evaluated. The protective effect of TFCTLL on CLI was evaluated by antioxidative and anti-inflammatory experiments in vitro, network pharmacology and a carbon tetrachloride (CCl4)-induced CLI mouse model. We indentified 18 chemical components in the TFCTLL samples and 4 components in plasma. TFCTLL showed significant anti-inflammatory activity and antioxidant capacity in vitro and in vivo. TFCTLL administration prominently improved the liver function and structure, decreased the mRNA expression levels of TLR2, TLR3, TLR4, NF-κB p65, IRF3, AKT1, TRIF, PI3K, MyD88, IL-1β and TNF-α and inhibited the protein expression and nuclear translocation of NF-κB p65 in mice with CLI. The molecular docking results showed that components in plasma had high binding affinity for the targets TLR4, PI3K and AKT1. Therefore, TFCTLL has a protective effect against CCl4-induced CLI, and the underlying mechanisms may be related to antioxidation, anti-inflammation and modulation of the TLRs/NF-κB and PI3K/AKT pathways.

The Extract of Scutellaria baicalensis Attenuates the Pattern Recognition Receptor Pathway Activation Induced by Influenza A Virus in Macrophages.

The dual strategy of inhibiting the viral life cycle and reducing the host inflammatory response should be considered in the development of therapeutic drugs for influenza A virus (IAV). In this study, an extract of Scutellaria baicalinase (SBE) containing seven flavonoids was identified to exert both antiviral and anti-inflammatory effects in macrophages infected with IAV. We performed transcriptome analysis using high-throughput RNA sequencing and identified 315 genes whose transcription levels were increased after IAV infection but were able to be decreased after SBE intervention. Combined with Gene Ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis, these genes were mainly involved in TLR3/7/8, RIG-I/MDA5, NLRP3 and cGAS pattern recognition receptor (PRR)-mediated signaling pathways. SBE inhibited the transcription of essential genes in the above pathways and nuclear translocation of NF-κB p65 as confirmed by RT-qPCR and immunofluorescence, respectively, indicating that SBE reversed PR8-induced over-activation of the PRR signaling pathway and inflammation in macrophages. This study provides an experimental basis for applying Scutellaria baicalensis and its main effects in the clinical treatment of viral pneumonia. It also provides novel targets for screening and developing novel drugs to prevent and treat IAV infectious diseases.

Salvianolic acids from Salvia miltiorrhiza Bunge and their anti-inflammatory effects through the activation of α7nAchR signaling

Ethnopharmacological relevanceCardiovascular disease (CVD) is a serious disease with a high incidence rate and mortality. Inflammation is closely related to the occurrence of CVDs. As an essential medicine of promoting blood circulation and removing blood stasis in China, Salvia miltiorrhiza Bunge (Danshen) is widely used to treat CVDs due to its anti-inflammatory and cardiovascular protective effects. Salvianolic acids are the most abundant component in the water extract of S. miltiorrhiza, which has a significant effect on the treatment of CVDs. However, due to the complex composition of salvianolic acids, the active molecules and their underlying mechanisms have not been fully explored. Aim of this studyThe present study aims to isolate and identify salvianolic acids from Danshen with anti-inflammatory activity and explore the potential mechanisms of isolates. MethodsThe structures of isolated salvianolic acids were elucidated by UV, IR, NMR, MS and electronic circular dichroism (ECD) calculations. Then anti-inflammatory activities of isolates were screened out by the zebrafish inflammation models. The most active compound was further used to explore the anti-inflammatory mechanisms on LPS-stimulated RAW 264.7 cells. The key inflammatory cytokines IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay (ELISA). The protein expression levels of STAT3, p-STAT3 (Tyr705), NF-κB p65, IκBα, p-IκBα (Ser32) and α7nAchR were determined by Western blotting. The nuclear translocation of p-STAT3 (Tyr705) and NF-κB p65 was evaluated by immunofluorescence assays. Finally, the in vivo anti-inflammatory mechanisms were investigated by observation of neutrophil migration, H&E staining, survival analysis and quantitative PCR (Q-PCR) in LPS-microinjected zebrafish. ResultsTwo new and four known compounds were isolated from Danshen. Among them, isosalvianolic acid A-1 (C1) and ethyl lithospermate (C5) inhibited neutrophil migrations in three zebrafish inflammation models and C1 with the best activities decreased the secretion of IL-6 and TNF-α and inhibited the expression level of p-IκBα (Ser32) in LPS stimulated RAW 264.7 cells. In addition, C1 also reduced the nuclear translocation of NF-κB p65 and p-STAT3 (Tyr705). Moreover, C1 significantly upregulated the protein expression of α7nAchR, and the knockdown of α7nAchR counteracted the effects of C1 on the production of IL-6 and TNF-α and the expression levels of p-STAT3 (Tyr705), NF-κB p65 and p-IκBα (Ser32). In vivo experiments, C1 decreased the migration and infiltration of inflammatory cells, increased the survival ratio and inhibited the mRNA level of IL-6, TNF-α, STAT3, NF-κB and IκBα in LPS-microinjected zebrafish. ConclusionTwo new and four known compounds were isolated from Danshen. Among them, C1 exerted anti-inflammatory activities by activating α7nAchR signaling and subsequently inhibiting STAT3 and NF-κB pathways. This study provided evidence for the clinical application of Danshen and contributed to the development of C1 as a novel in the treatment of cardiovascular disease.

Spectasterols, Aromatic Ergosterols with 6/6/6/5/5, 6/6/6/6, and 6/6/6/5 Ring Systems from Aspergillus spectabilis.

Spectasterols A-E (1-5), aromatic ergosterols with unique ring systems, were isolated from Aspergillus spectabilis. Compounds 1 and 2 possess a 6/6/6/5/5 ring system with an additional cyclopentene, while 3 and 4 have an uncommon 6/6/6/6 ring system generated by the D-ring expansion via 1,2-alkyl shifts. Compound 3 exhibited cytotoxic activity (IC50 6.9 μM) and induced cell cycle arrest and apoptosis in HL60 cells. Compound 3 was anti-inflammatory; it decreased COX-2 levels at the transcription and protein levels and inhibited the nuclear translocation of NF-κB p65.

Interleukin-33 promotes the epithelial-mesenchymal transition of renal tubular epithelial cells via the NF-κB/Twist1 signalling pathway

Objectives: Epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells (RTECs) is a pathogenic factor for renal interstitial fibrosis (RIF). Interleukin-33 (IL-33) is related to the occurrence and development of RIF, but the underlying mechanism remains unclear. Here, we investigated whether IL-33 mediates the EMT of RTECs by activating the NF-κB/Twist1 signalling pathway. Methods: In vivo, the RIF animal model induced by unilateral ureteral obstruction (UUO) was established. The effects of exogenous IL-33 and anti-IL-33 antibodies were evaluated. In vitro, the EMT of RTECs was induced by IL-33. The inhibition of the nuclear factor kappa-B (NF-κB) by the pyrrolidine dithiocarbamate (PDTC) and the knockdown of the suppression of tumorigenicity 2 (ST2) by small interference RNA were used to observe whether IL-33 mediates the EMT of RTECs through the NF-κB/Twist1 signaling pathway. Results: In vivo, exogenous IL-33 significantly aggravated UUO-induced pathological damage and collagen deposition, down-regulated E-cadherin expression, and up-regulated α-smooth muscle actin and Vimentin expressions. Moreover, exogenous IL-33 increased the levels of phospho-IκB-α (p-IκB-α) and phospho-NF-κB p65 (p-NF-κB p65), NF-κB p65 nuclear translocation, and Twist1 nuclear expression. However, these effects were reversed by the anti-IL-33 antibody. In vitro , the increases in the levels of p-IκB-α, p-NF-κB p65, NF-κB p65 nuclear translocation, and Twist1 nuclear expression induced by IL-33 were inhibited by the knockdown of PDTC or ST2. IL-33-mediated EMT of RTECs was also significantly reversed. However, NF-κB inhibitor PDTC had no significant effect on ST2 expression. Conclusions: The IL-33/ST2 axis may up-regulate the expression of Twist1 through the NF-κB signalling pathway, thereby inducing the EMT of RTECs and leading to RIF.

Regulation of osteoclast differentiation and inflammatory signaling by TCF8 in periodontitis.

The aim of this study was to explore the potential role of zinc-finger homeodomain transcription factor (TCF8) in osteoclastogenesis and inflammation during periodontitis. Rats with periodontitis were induced via Porphyromonas gingivalis-lipopolysaccharide (Pg-LPS) injection. The recombinant lentivirus delivering short hairpin RNA (shRNA) against TCF8 was used to downregulate TCF8 in vivo. Alveolar bone loss in rats was determined by micro-computed tomography (Micro-CT). Typical pathological changes, periodontal tissue inflammation, and osteoclastogenesis were evaluated via histological analyses. The RAW264.7-derived osteoclasts were induced by RANKL stimulation. TCF8 downregulation in vitro was achieved by lentivirus infection. The osteoclast differentiation and inflammatory signaling in RANKL-induced cells were measured via immunofluorescence methods and molecular biology approaches. Porphyromonas gingivalis-lipopolysaccharide induced rats exhibited overexpressed TCF8 in their periodontal tissues, while TCF8 knockdown attenuated the bone loss, tissue inflammation, and osteoclastogenesis in LPS-induced rats. Besides, TCF8 silencing inhibited RANKL-induced osteoclast differentiation in RAW264.7 cells, as evidenced by the reduced numbers of TRAP-positive osteoclasts, less formation of F-actin rings, and downregulated expressions of osteoclast-specific markers. It also exerted an inhibitory effect on the NF-κB signaling in RANKL-induced cells via blocking NF-κB p65 phosphorylation and nuclear translocation. TCF8 silencing inhibited alveolar bone loss, osteoclast differentiation, and inflammation in periodontitis.

Isoamericanin A improves lipopolysaccharide-induced memory impairment in mice through suppression of the nicotinamide adenine dinucleotide phosphateoxidase-dependent nuclear factor kappa B signaling pathway.

Alzheimer's disease (AD) is the most frequent cause of dementia in the elderly. Isoamericanin A (ISOA) is a natural lignan possessing great potential for AD treatment. This study investigated the efficacy of ISOA on memory impairments in the mice intrahippocampal injected with lipopolysaccharide (LPS) and the underlying mechanism. Y-maze and Morris Water Maze data suggested that ISOA (5 and 10 mg/kg) ameliorated short- and long-term memory impairments, and attenuated neuronal loss and lactate dehydrogenase activity. ISOA exerted anti-inflammatory effect demonstrating by the reduction of ionized calcium-binding adapter molecule 1 positive cells and suppression of marker protein and pro-inflammation cytokines expressions induced by LPS. ISOA suppressed the nuclear factor kappa B (NF-κB) signaling pathway by inhibiting IκBα phosphorylation and NF-κB p65 phosphorylation and nuclear translocation. ISOA inhibited superoxide and intracellular reactive oxygen species accumulation by reducing nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, demonstrating by suppressing NADP+ and NADPH contents, gp91phox expression, and p47phox expression and membrane translocation. These effects were enhanced in combination with NADPH oxidase inhibitor apocynin. The neuroprotective effect of ISOA was further proved in the in vitro models. Overall, our data revealed a novel pharmacological activity of ISOA: ameliorating memory impairment in AD via inhibiting neuroinflammation.

Recombinant human KAI1/CD82 attenuates M1 macrophage polarization on LPS-stimulated RAW264.7 cells via blocking TLR4/JNK/NF-κB signal pathway

Therefore, the aim of the present study was to investigate the effect of recombinant protein for second extracellular domain of human KAI1 (Gly 111-Leu 228, rhKAI1) on LPS-stimulated RAW264.7 macrophage-like cells and mouse bone marrow-derived macrophages (BMDM), and to identify the its underlying mechanism. Our data showed that rhKAI1 suppressed the expression of active macrophages (M1) phenotype-related surface markers F4/80+CD86+ in LPS-stimulated BMDM and RAW264.7 cells. In addition, LPS markedly up-regulated the mRNA expression and release levels of pro-inflammatory cytokines and mediators, such as interleukin (IL)-1β, IL-6, tumor necrosis factor-α, cyclooxygenase-2, nitric oxide and prostaglandin E2, whereas this increase was substantially down-regulated by rhKAI1. Furthermore, LPS strongly promoted the fluorescence expression of NF-κB p65 in the nuclei, and increased the phosphorylation of ERK, JNK, and p38 MAPK. However, the nuclear translocation of NF-κB p65 and the phosphorylation of JNK were greatly reversed in the presence of the rhKAI1. Especially, rhKAI1 markedly suppressed the expression of -like receptor (TLR4) and it prevent the binding of LPS with TLR4 through molecular docking predict analysis. Importantly, Glu 214 of rhKAI1 residue strongly interact with Lys 360 of TLR4 residue, and the distance between the bound was 2.9 Å. Taken together, all these findings suggested that rhKAI1 had anti-inflammatory effect in LPS-polarized macrophages by interacting with TLR4 and down-regulating the JNK/NF-κB signaling pathway.

SIRT2 regulates extracellular vesicle-mediated liver–bone communication

The interplay between liver and bone metabolism remains largely uncharacterized. Here, we uncover a mechanism of liver-bone crosstalk regulated by hepatocyte SIRT2. We demonstrate that hepatocyte SIRT2 expression is increased in aged mice and elderly humans. Liver-specific SIRT2 deficiency inhibits osteoclastogenesis and alleviates bone loss in mouse models of osteoporosis. We identify leucine-rich α-2-glycoprotein 1 (LRG1) as a functional cargo in hepatocyte-derived small extracellular vesicles (sEVs). In SIRT2-deficient hepatocytes, LRG1 levels in sEVs are upregulated, leading to increased transfer of LRG1 to bone-marrow-derived monocytes (BMDMs), and in turn, to inhibition of osteoclast differentiation via reduced nuclear translocation of NF-κB p65. Treatment with sEVs carrying high levels of LRG1 inhibits osteoclast differentiation in human BMDMs and in mice with osteoporosis, resulting in attenuated bone loss in mice. Furthermore, the plasma level of sEVs carrying LRG1 is positively correlated with bone mineral density in humans. Thus, drugs targeting hepatocyte-osteoclast communication may constitute a promising therapeutic strategy for primary osteoporosis.

Anti-rheumatic effect of KMU-11361 in various rheumatoid arthritis-related cells targeting multiple protein kinase

Abstract Protein kinases important for regulating protein phosphorylation, which is a promising therapeutic target for inflammatory disease, such as rheumatoid arthritis (RA). In this study, we synthesized a novel multi-protein kinase inhibitor, KMU-11361, a derivative of indolin-2-one and investigated the mechanisms of anti-inflammatory and anti-rheumatic effects using THP-1 cells, RAW264.7 cells, Jurkat T cells, and human RA fibroblast-like synoviocytes (RA-FLS). Our results revealed that KMU-11361 inhibited LPS-induced upregulation of inducible iNOS, and COX-2, and suppressed LPS-induced phosphorylation of TAK1, JNK, inhibitor of NF-κB kinase α/β (IKKα/β), IκBα, and NF-κB p65 as well as nuclear translocation of NF-κB p65 in THP-1 cells. Furthermore, KMU-11361 attenuated proinflammatory cytokines such as IL-1β, TNF-α, and IL-6, and, remarkably, inhibited LPS-induced activation of the NLRP3 inflammasome in THP-1 cells. Additionally, in activated Jurkat T cells, KMU-11361 inhibited the TAK1-IKKα-mediated NF-κB pathway as well as the phosphorylation of MAPKs. Moreover, KMU-11361 inhibited anti-CD3/CD28 antibodies-induced upregulation of proinflammatory cytokine IL-2. Moreover, it suppressed LPS-induced upregulation of proinflammatory cytokines, p-TAK1, p-IKKα/β, and p-NF-κB p65 in RA-FLS. Surprisingly, KMU-11361 also inhibited the receptor activator of NF-κB-induced osteoclast differentiation and up-regulation of nuclear factor of activated T-cells, NFATc-1 and c-Fos in RAW264.7 cells. Taken together, our results suggest that KMU-11361 has anti-inflammatory and anti-rheumatic effects in various RA-related cells and could be developed as a potent anti-rheumatic drug.

The pro-inflammatory phenotype of human macrophages is restricted by the activity of the orphan nuclear receptor Nurr1

Abstract The nuclear receptor related 1 protein (Nurr1) is a member of the orphan nuclear hormone receptor family whose expression has been associated with several inflammatory diseases. Macrophages are key regulators of the inflammatory processes, yet information about the role of Nurr1 in human macrophages is limited. In the present study we examined the expression and activity of Nurr1 in steady state and activated human monocyte-derived macrophages. Pro- and anti-inflammatory macrophages were derived in vitro by culture of blood monocytes with the hematopoietic cytokines GM-CSF and M-CSF, respectively. Nurr1 expression was predominant in macrophages with a pro-inflammatory phenotype. Accordingly, its expression was induced by a variety of inflammatory stimuli such as TLR-2, -3 and -4 ligands, TNF, IFN-β, and IL-4. Pro-inflammatory macrophages exposed to the Nurr1 agonist C-DIM12 decreased their production of IL-1β, IL-6, and reactive oxygen species. Conversely, Nurr1 deficient macrophages produced enhanced levels of IL-6. Mechanistically, Nurr1 agonists partially abrogated the activation of the NF-κB signaling pathway in pro-inflammatory macrophages exposed to LPS, rendering cells with a lower rate of NF-κB p65 nuclear translocation. These results suggest that Nurr1 expression is linked with the pro-inflammatory phenotype of human macrophages where it may constitute a brake to attenuate the synthesis of inflammatory mediators. Supported by grants from Secretaria de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)