iNOS Protein Expression Research Articles

New formulation of ibuprofen-arginate reduces oxidative stress and prevents macrophage polarization toward M1 phenotype.

A hypertonic solution of Ibuprofen (Ibu) was designed to nebulize, associating a low concentration of Ibu with L-Arginine (AR), to increase solubility and serve as a nitric oxide donor. To provide preclinical research human bronchial epithelial cells derived from a cystic fibrosis patient homozygous for the ΔF508 CFTR mutation (CFBE41o-) and mouse RAW 264.7 macrophages were pre-treated with Ibu (10-100 μM), AR (20 and 200 μM), or the combination Ibu-AR (10-100 μM). After Angiotensin II (AngII) or LPS/Interferon ϒ (IFN) stimulation, Reactive Oxygen Species (ROS) generation, Nitric Oxide (NO) formation, and the expression of inflammatory markers were determined. Ibu-AR (10/20 μM) significantly reduced ROS generation stimulated by AngII (p < 0.01) in CFBE41o- cells preserved the NO pathway and inhibited LPS-stimulated nitrite generation (p < 0.001). In macrophages, the combination Ibu-Ar, in a ratio of 1:2-1:6, efficiently scavenged excessive ROS generated by LPS, and significantly induced NO generation (p < 0.001), but inhibited nitrite formation. In LPS/IFNϒ-activated Raw, gene signature of M1polarization including tumor necrosis factor (TNF-α), NADPH Oxidase 2 (NOX-2), MCP-1, and inducible nitric oxide synthase (iNOS) were significantly downregulated by Ibu-AR, as well TNF-α, IL-6, and iNOS protein expressions. The inhibitory effect produced by Ibu-AR on M1 macrophages was associated with the inhibition of p-ERK1/2 and p-STAT3. Ibu-AR represents an effective therapeutic strategy for reducing oxidative stress, preserving NO bioavailability, and modulating inflammation in chronic inflammatory diseases.

Mycoplasma pneumoniae MPN606 induces inflammation by activating MAPK and NF-κB signaling pathways.

Mycoplasma pneumoniae (M. pneumoniae) is one of the major pathogens causing community-acquired pneumonia (CAP), and its pathogenic mechanism is not fully understood. Inflammatory response is the most basic and common pathological phenomenon of CAP, but the specific mechanism needs further investigation. In this study, the inflammatory action of M. pneumoniae MPN606 protein was confirmed and its molecular mechanism was tentatively investigated. Compared with the control group treated with PBS, stimulation of RAW264.7 cells with rMPN606 can promote the release of NO, increase the expression level of TNF-α and IL⁃6 cytokines, and up-regulate the mRNA transcription levels of iNOS, IL-6 and TNF-α in RAW264.7 cells. In addition, rMPN606 also significantly induced the expression of iNOS protein in RAW264.7 cells, resulting in increased phosphorylation levels of p65, p38 and ERK proteins. The results of cellular immunofluorescence showed that NF-κB was transferred from cytoplasm to nucleus of RAW264.7 cells after stimulation with rMPN606, and nuclear translocation of NF-κB was significantly enhanced. These results indicate that Mycoplasma pneumoniae MPN606 induces M1-type activation of macrophages and secretes pro-inflammatory factors by activating NF-κB and MAPK pathways.

Synergistic Anti-Inflammatory Effects of Dibenzoylmethane and Silibinin: Insights From LPS-Induced RAW 264.7 Cells and TPA-Induced Mouse Model.

Inflammation is an important predisposing factor for many chronic diseases. The dietary flavonoid silibinin (SB) has excellent anti-inflammatory properties in cells, but its low bioavailability in the blood compromises its therapeutic potential. This study aims to investigate the potential of dibenzoylmethane (DBM) to synergistically enhance the anti-inflammatory benefits of SB. The synergistic effects of DBM and SB in combination were evaluated in lipopolysaccharide (LPS)-induced RAW264.7 cells and 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced mice. In addition, a network pharmacology approach and molecular docking were used to explore the key targets and signaling pathways of DBM and SB in combination. The results showed that DBM and SB synergistically inhibited the production of nitric oxide (NO), reactive oxygen species (ROS), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α) in a 1:1 concentration ratio. These two compounds may exert their synergistic effects by modulating the nuclear factor kappa-B (NF-κB) and HIF-1 signaling pathways, among others. Molecular docking revealed that both compounds exhibited high binding affinities to inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Compared with single-compound use, the two compounds in combination significantly reduced ear edema and inflammatory cell infiltration and inhibited the protein expression of iNOS and COX-2 in TPA-induced mice. This research provides a rationale for the combination of DBM and SB as an effective anti-inflammatory agent.

CHD1L accelated the progression of cutaneous squamous cell carcinoma via promoting PI3K/PD-L1 signaling pathway induced M2 polarization of TAMs

To investigate CHD1L’s impacts and molecular processes in hypoxic cutaneous squamous cell carcinoma. Monoclonal proliferation assays and CCK-8 were used to detect the proliferation capacity of A431 cells and Colon16 cells; wound healing experiments and Transwell assays were used to examine the migration and invasion capacity of A431 cells and Colon16 cells; angiogenesis experiments were conducted to assess the influence of A431 cells on angiogenesis; a nude mouse tumor xenograft experiment and HE staining were utilized to evaluate the impact of CHD1L on the progression of cutaneous squamous cell carcinoma; western blot analysis was performed to detect the expression of p-PI3K, p-AKT, and PD-L1 in A431 cells, as well as CD9, TSG101, PD-L1 in exosomes, and CD206, Arginase-1, iNOS, IL-1β, p-AKT, p-mTOR, VEGF, COX-2, MMP2, MMP9, p-ERK1/2 in tumor-associated macrophages. Under hypoxic conditions, CHD1L promoted the proliferation, migration, invasion, and angiogenesis of cutaneous squamous cell carcinoma. Furthermore, CHD1L facilitated the progression of cutaneous squamous cell carcinoma. CHD1L also increased the relative protein expression of p-PI3K, p-AKT, and PD-L1 in A431 cells, as well as CD9, TSG101, PD-L1 in exosomes, CD206, Arginase-1, p-AKT, p-mTOR, VEGF, COX-2, MMP2, MMP9, and p-ERK1/2 in tumor-associated macrophages, while inhibiting the relative protein expression of iNOS and IL-1β. Under hypoxic conditions, CHD1L can promote the proliferation and migration of cutaneous squamous cell carcinoma.

Polyphenols-rich Portulaca oleracea L. (purslane) alleviates ulcerative colitis through restiring the intestinal barrier, gut microbiota and metabolites.

Ulcerative colitis (UC) is a recurrent intestinal disease caused by a complex of factors, and there are serious adverse effects and tolerance problems associated with the current long-term use of therapeutic drugs. The development of natural food sources and multi-targeted drugs for the treatment of UC is imminent. Portulaca oleracea L. (PO), as a vegetable, has been shown in studies to have an anti-UC effects. However, the relationship between the abundant active ingredients contained in Portulaca oleracea L. and the improvement of intestinal barrier, gut microbiota and metabolites is unclear. In the present study, Portulaca oleracea L. which was found to be rich in phenolic acid-based active ingredients, were effective in alleviating dextran sulfate sodium (DSS)-induced body weight loss, disease activity index (DAI) score and colon length in mice. It also decreased C-reactive protein (CRP) and myeloperoxidase (MPO) responses, reduced the permeation of fluorescein isothiocyanate (FITC)-dextran, lipopolysaccharide (LPS) and evans blue (EB), and improved histopathological scores. Meanwhile, in vitro and in vivo validation revealed the protective effects of purslane on the intestinal barrier indicators ZO-1, Occludin and Claudin-1, and inhibited the expression of inflammation-associated iNOS and NLRP3 proteins through the NF-κB signaling pathway. In addition, purslane increased the diversity of the intestinal flora, enhancing the proportion of the genera Butyricoccus, Dorea and Bifidobacterium and decreasing the percentage of Bacteroides, Turicibacter and Parabacteroides. Serum metabolomics analysis showed that the imbalance of 39 metabolites was significantly reversed after PO deployment. Enrichment analysis showed that Pentose phosphate pathway and Pyruvate metabolism pathway were the key pathways of PO against UC. Overall, purslane effectively improved the intestinal barrier disruption and intestinal inflammation by inhibiting the NF-κB signaling pathway, and adjusted the disorder of gut microbiota and metabolites to exert anti-UC effects.

Th22 cells promote the transition from homeostatic to reactive microglia in diabetic encephalopathy.

Diabetic encephalopathy (DE) is one of the most serious complications of diabetes mellitus (DM), and its pathogenesis has not yet been clarified. Th22 cells are a newly discovered class of CD4+ T cells that play important roles in inflammatory, autoimmune and infectious diseases. However, it is unclear whether Th22 cells are involved in the pathogenesis of DE. We established a T2DM mouse model in vivo and cocultured Th22 cells with microglia under high glucose (HG) conditions in vitro. Cognitive dysfunction was evaluated using the Morris water maze (MWM) test; blood‒brain barrier (BBB) integrity was evaluated using the Evans blue (EB) extravasation assay; Th22 cells and IL-22 receptors were detected by immunofluorescence; and IL-1β, TNF-α, iNOS, CD86, Arg-1, and CD206 protein expression was measured by Western Blot (WB) analysis. Th22 cells passed through the BBB into the hippocampus and secreted interleukin-22 (IL-22), and the mice subsequently exhibited decreased learning and memory abilities. In the DE model, IL-22 promoted the transformation of homeostatic microglia into reactive microglia as well as the inflammatory response. Additionally, coculture of Th22 cells with BV2 microglia cultured under HG conditions increased the production of proinflammatory cytokines, and the microglia showed reactive changes. Mechanistically, IL-22Rα1 acted as a ligand, and IL-22 bound to IL-22Rα1 on microglia to drive primary microglia-induced inflammatory responses. Interestingly, interleukin-22 binding protein (IL-22BP) directly binds to IL-22Rα1 on microglia to inhibit the proinflammatory effects of IL-22. Th22 cells secrete IL-22 after passing through the BBB into the hippocampus and promote the transformation of homeostatic microglia into reactive microglia, which induces an inflammatory response, exacerbates learning and memory impairment and cognitive deficits, and contributes to and accelerates the development of DE.

Role of inducible nitric oxide (iNOS) and nitrosative stress in regulating sex differences in secondary lymphedema.

Secondary lymphedema is a common complication following surgical treatment of solid tumors. Although more prevalent in women due to higher breast cancer rates, men also develop lymphedema, often with more severe manifestations. Despite these differences in clinical presentation, the cellular mechanisms underlying sex differences are poorly understood. Previous studies have shown that inducible nitric oxide synthase (iNOS) expression by inflammatory cells is an important regulator of lymphatic pumping and leakiness in lymphedema and that lymphatic endothelial cells are highly sensitive to nitrosative stress. Based on this rationale, we used a mouse tail model of lymphedema to study the role of nitric oxide in sex-related differences in disease severity. Consistent with clinical findings, we found that male mice have significantly worse tail edema and higher rates of tail necrosis compared with female mice following tail skin/lymphatic excision (p = 0.001). Our findings correlated with increased tissue infiltration of iNOS + inflammatory cells, increased iNOS protein expression, and increased nitrosative stress in male mouse lymphedematous skin tissues (p < 0.05). Importantly, transgenic male mice lacking the iNOS gene (iNOS-KO) displayed markedly reduced swelling, inflammation, and tissue necrosis rates, whereas no differences were observed between wild-type and iNOS-KO female mice. Overall, our results indicate that iNOS-mediated nitric oxide production contributes to sex-based differences in secondary lymphedema severity, emphasizing the need to consider sex as a biological variable in lymphedema research.

Soufeng sanjie formula alleviates osteoarthritis by inhibiting macrophage M1 polarization and modulating intestinal metabolites

Ethnopharmacological relevanceOsteoarthritis (OA) is defined as “bone bi” disease based on clinical symptoms in Chinese medicine. Soufeng sanjie formula (SF) is a traditional formula for treating “bone bi” disease, which consists of Scolopendra (dried body of Scolopendra subspinipes mutilans L. Koch) (0.5 g), Scorpions (dried body of Buthus martensii Karsch) (0.5 g), Astragali radix (dried root of Astragalus membranaceus (Fisch.) Bge) (20 g) and Black soybean seed coats (seed coats of Glycine max (L.) Merr) (30 g), and it can be used to treat rheumatoid arthritis. Nonetheless, the potential of SF to postpone the advancement of OA and its underlying mechanisms remain unexplored. Aim of the studyThis study investigated whether SF could alleviate OA and the underlying mechanisms. Materials and methodsAnterior cruciate ligament transection (ACLT) was performed to establish an OA mice model. Mechanical pain and cold pain were assessed to evaluate changes in pain sensitivity in OA mice. Micro-CT was used to observe the microstructure and quantify the bone morphological parameters of knee joints. Safranin O-fast green staining was used to evaluate cartilage damage, and Osteoarthritis Research Society International (OARSI) scores were calculated. Immunohistochemistry was used to assess the expression of inflammatory factors in the synovium of OA mice following SF administration. Immunofluorescence analyzed the fraction of CD80 and iNOS positive regions in the synovium of knee joints. The effect of SF on macrophage M1 polarization was investigated using flow cytometry, western blot and quantitative PCR (qPCR) in vitro. Untargeted metabolomics was used to identify the differential metabolites associated with OA. ResultsSF-treatment markedly reduced the cartilage damage, lowered the OARSI score and downregulated the pain sensitivity in the OA mice. Secondly, SF decreased the expression of IL-6, IL-1β, and TNF-α in the OA synovium. SF also reduced the percentage of CD80 and iNOS in the synovium of the knee joint after ACLT surgery by immunofluorescence. Thirdly, SF inhibited the protein expression of iNOS and COX-2, decreased the percentage of CD80, and reduced the mRNA levels of IL-6, IL-1β, and TNF-α in BMDM cells. Furthermore, SF inhibited the macrophage M1 polarization-related AKT/NF-κB signaling pathway. Finally, untargeted metabolomics showed that SF effectively reduced the levels of intestinal metabolite 18-hydroxyoleic acid in OA mice. ConclusionOur results suggested that SF reduced pain symptoms and joint inflammation in mice with OA. Furthermore, SF inhibited synovial macrophage M1 polarization and modified the levels of the pro-inflammatory intestinal metabolite 18-hydroxyoleic acid in OA mice. Therefore, SF may be act as a potential Chinese medicine for the treatment of OA.

Amelioration of Toll-like Receptor-4 Signaling and Promotion of Mitochondrial Function by Mature Silkworm Extracts in Ex Vivo and in Vitro Macrophages.

The unknown immune-enhancing effects of steamed mature silkworms (Bombyx mori L.), known as HongJam (HJ), were investigated. Supercritical fluid extracts from the White Jade variety of HJ (WJ-SCEs) were applied to in vitro RAW264.7 macrophages (RAWMs) and ex vivo bone marrow-derived macrophages (BMDMs). WJ-SCE enhanced the proliferation and viability of both RAWMs and BMDMs. Supplementation with WJ-SCE significantly reduced the lipopolysaccharide (LPS)-induced expression of iNOS mRNA and protein, resulting in decreased nitric oxide (NO) production. Additionally, WJ-SCE lowered the mRNA and protein expression of COX-2 and reduced the levels of pro-inflammatory cytokines. The mitochondrial function, ATP levels, and reactive oxygen species levels in LPS-treated macrophages were restored following WJ-SCE treatment. WJ-SCE modulated LPS-Toll-like receptor-4 (TLR-4) signaling by reducing the levels of phosphorylated (p)-p38, p-ERK1/2, and p-p65. WJ-SCE also restored gene expression related to cytokines, chemokines, glucose transport, mitochondrial metabolism, and TLR-4 signaling, suggesting the inhibition of pro-inflammatory M1 macrophage polarization. Furthermore, WJ-SCE enhanced macrophage phagocytic and pinocytotic activity. WJ-SCE demonstrated anti-inflammatory effects by inhibiting LPS-induced M1 polarization in both macrophage types, potentially suppressing chronic inflammation while enhancing phagocytosis and pinocytosis.

Ammopiptanthus nanus (M. Pop.) Cheng f. stem ethanolic extract ameliorates rheumatoid arthritis by inhibiting PI3K/AKT/NF-κB pathway-mediated macrophage infiltration

Ethnopharmacological relevanceAmmopiptanthus nanus (M. Pop.) Cheng f. (A. nanus), a traditional Kirgiz medicinal plant, its stem has shown potential in treating rheumatoid arthritis (RA) in China, either through oral medication or by topical application directly to the affected joints, but its underlying mechanism of action remains unexplored. Aim of the studyThe purpose of this study is to elucidate pharmacological mechanism of A. nanus in ameliorating RA using a comprehensive approach that combines network pharmacology, molecular docking and experimental evaluations. Materials and methodsFirstly, the major constituents of A. nanus stem ethanolic extract were identified and quantified by High-Performance Liquid Chromatography (HPLC). Disease target data from Gene Cards database was then used to define RA-associated targets. A protein-protein interaction (PPI) network was created via STRING database. The DAVID database powered gene ontology (GO) function and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to gain functional insights. In vitro, RAW264.7 cells were treated with A. nanus to investigate the roles of target proteins and pathways during lipopolysaccharide (LPS) - induced inflammation. Immunofluorescence assays were performed to assess the effects of A. nanus on macrophage infiltration. The key targets and signalling pathways were validated using enzyme-linked immunosorbent assay (ELISA), real-time quantitative polymerase chain reaction (RT-qPCR), molecular docking, immunohistochemical analysis, western blotting and immunofluorescence. Finally, the therapeutic potential of A. nanus in RA was evaluated in a carrageenan-induced rat model. ResultsNetwork analysis identified 31 potential targets of A. nanus associated with RA, including 10 hub targets. KEGG analysis highlighted the involvement of PI3K/AKT signaling pathway. In vivo experiments demonstrated that A. nanus treatment significantly protected against carrageenan-induced inflammatory paw tissue and attenuated macrophage infiltration. Both in vivo and in vitro experiments confirmed that A. nanus significantly downregulated the protein expression of COX-2, iNOS and IL-1β, and inhibited PI3K/AKT/NFκB pathway, which are closely linked to RA. Furthermore, molecular docking and cellular thermal shift assay revealed that licoflavanone showed a strong binding affinity with key targets. ConclusionIn summary, this study provides the first evidence of the potent anti-inflammatory activity of A. nanus in experimental RA. The mechanism of action appears to involve inactivation of the PI3K/AKT/NF-κB pathway-mediated macrophage infiltration. These findings indicate that A. nanus has significant potential as a therapeutic potential agent for RA treatment and offer novel insights for future research and drug development in this field.

Acute Chikungunya Infection Induces Vascular Dysfunction by Directly Disrupting Redox Signaling in Endothelial Cells.

Chikungunya virus (CHIKV) infection is characterized by febrile illness, severe joint pain, myalgia, and cardiovascular complications. Given that CHIKV stimulates reactive oxygen species (ROS) and pro- and anti-inflammatory cytokines, events that disrupt vascular homeostasis, we hypothesized that CHIKV induces arterial dysfunction by directly impacting redox-related mechanisms in vascular cells. Wild-type (WT) and iNOS knockout (iNOS-/-) mice were administered either CHIKV (1.0 × 106 PFU/µL) or Mock vehicle via the intracaudal route. In vivo, CHIKV infection induced vascular dysfunction (assessed by a wire myograph), decreased systolic blood pressure (tail-cuff plethysmography), increased IL-6 and IFN-γ, but not TNF-α levels (determined by ELISA), and increased protein content by Western blot. Marked contractile hyporesponsiveness to phenylephrine was observed 48 h post-infection, which was restored by endothelium removal. L-NAME, 1400W, Tiron, and iNOS gene deletion prevented phenylephrine hyporesponsiveness. CHIKV infection increased vascular nitrite concentration (Griess reaction) and superoxide anion (O2•-) generation (lucigenin chemiluminescence), and decreased hydrogen peroxide (H2O2, by Amplex Red) levels 48 h post-infection, alongside increased TBARS levels. In vitro, CHIKV infected endothelial cells (EA.hy926) and upregulated ICAM-1 and iNOS protein expression (determined by Western blot). These data support the conclusion that CHIKV-induced alterations in vascular ROS/NF-kB/iNOS/NO signaling potentially contribute to cardiovascular events associated with Chikungunya infection.

Evaluation of antioxidant and anti-inflammatory properties, bioactive compound profiling, and molecular mechanisms of a multicomponent Thai herbal formulation

BackgroundMedicinal plants have long been used as dietary supplements to reduce the risk of chronic diseases, inflammation, cancer, and metabolic syndromes. Traditional remedies possess therapeutic properties, making them promising candidates for further investigation and potential pharmaceutical development. Aim of the studyThis study aimed to evaluate the efficacy of Belog Plus (BP), a polyherbal product containing five Thai medicinal plants: Citrus aurantifolia, Cannabis sativa, Tiliacora triandra, Alpinia galanga, and Piper nigrum, combined with safflower seed oil. This study focused on quantifying the total phenolic content (TPC) and total flavonoid content (TFC), and identifying key herbal biomarkers that contribute to its therapeutic properties. In addition to assessing the antioxidant and anti-inflammatory potential of the plant extracts, the study explored the molecular mechanisms underlying their anti-inflammatory activity, offering a comprehensive understanding of BP's efficacy. MethodsThe ethanolic extract of BP and its components were assessed for antioxidant potential using DPPH, FRAP, and H2O2 induced oxidative stress. The anti-inflammatory activity was evaluated using the Griess reaction assay, and the mechanisms were explored through mRNA and protein expression analysis. The TPC and TFC were measured using the Folin–Ciocalteu (F–C) assay and the aluminium chloride colorimetric assay, respectively, while herbal biomarkers were identified using LC-MS/MS analysis. ResultsThe results revealed that the ethanolic extract of BP (6.25–100 µg/ml) exhibited a potent anti-inflammatory effect through nitric oxide inhibition (IC50 24.4 μg/ml) and significantly reducing the expression of inflammatory genes iNOS, COX-2, IL-6, and TNF-α. Additionally, it significantly reduced the expression of inflammatory proteins iNOS and COX-2 at concentrations of 6.25–25 µg/ml. Among the individual components, C. aurantifolia, A. galanga, and P. nigrum showed potent anti-inflammatory effects with IC50 values of 35.7, 6.5, and 23.3 μg/ml, respectively. The TPC and TFC results demonstrated significant levels of phenolic (80.6 µg GAE/mg) and flavonoid (47.4 µg catechin/mg) compounds. Additionally, the LC-MS/MS analysis identified a variety of bioactive compounds known for their anti-inflammatory properties. ConclusionThese findings support BP's potential use as a dietary supplement to mitigate the risk of chronic inflammatory diseases, with its ethanolic extract containing bioactive phenolic and flavonoid compounds that significantly suppress iNOS and COX-2 protein expressions.

Biotransformation of Glaucic Acid Isolated from the Root of Lindera glauca by the Endophytic Fungi Penicillium Chrysogenum SHJ-07.

Glaucic acid isolated from the root of Lindera glauca, was investigated by the biotransformation methods via the endophytic fungi, resulting in the production of five new glausesquiterpenes A-E (1-5), along with a known analogue 6. Their structures were elucidated based on spectroscopic methods and electronic circular dichroism (ECD) calculations. In the bioassays, glausesquiterpene A (1) showed good inhibitory activity of NO production in LPS-activated RAW 264.7 macrophages with an IC50 value of 20.1 μM than positive control (Indomethacin, IC50 24.1 μM). Further in vitro studies demonstrated that glausesquiterpene A significantly suppressed the protein expression of iNOS and COX-2 at the concentration of 25.0 μM.

MTORC2 inhibition by JR-AB2-011 improves IL-1β-induced inflammation, catabolic response, and apoptosis in human chondrocytes through IκB-α/NF-κB p65.

Osteoarthritis (OA) is a very common chronic joint condition marked by inflammation and cartilage loss. mTOR is a well-known mediator of inflammation, cell survival, and aging; however, its role in OA has not been determined. To explore the role of mTORC2 in OA-and associated pathological changes, we examined the contribution of mTORC2-mediated Akt, rictor and IκB-α/NF-κB p65 pathway in interleukin (IL)-1β-treated human chondrocytes. We focused on the protein expression of proinflammatory cytokines and catabolic and apoptotic factors, including TNF-α, IL-6, iNOS, MMP13, Bax, and caspase3, which may occur through this signalling pathway in IL-1β-treated chondrocytes. Chondrocytes were cultured and treated with either 2 ng/mL IL‑1β alone or in combination with increasing concentrations of JR-AB2-011 (50, 100, or 250 µM), a selective mTORC2 inhibitor. The protein levels of phosphorylated (p)‑Akt, Akt, rictor, p-NF-κB p65, NF-κB p65, IκB-α, p-IκB-α, iNOS, MMP13, Bax, and caspase3 were evaluated by Western blotting. In IL-1β-stimulated chondrocytes, mTORC2 activity was increased with increased phosphorylation of Akt and expression of rictor. IL-1β increased the expression of p-IκBα, p-NF-κB p65, NF-κB p65, IL-6, TNF-α, iNOS, Bax, and caspase3 proteins and decreased the expression of IκB-α. All of these IL-1β-induced alterations were prevented by JR-AB2-011. The main novel finding in the present study is that selective mTORC2 inhibition by JR-AB2-011 prevents the inflammatory, catabolic, and apoptotic responses induced by IL-1β via modulation of IκB-α/NF-κB activity. Therefore, we demonstrated a previously unknown function of mTORC2 inhibition that seems to be a potential therapeutic target for OA.

Facile constructed meroterpenoids with novel hexadecahydroacephenanthrylene carbon skeleton using the biotransformation and chemical synthesis method

Bioaspermeroterpenoid A (1), the first meroterpenoid with an unprecedented hexadecahydroacephenanthrylene carbon skeleton, together with two analogues bioaspermeroterpenoids B and C (2 and 3) were co-isolated from the biotransformation extract of aspermeroterpene C by the fungus Penicillium herquei GZU-31-6. On the other hand, bioaspermeroterpenoid Aa (1a) featuring the same hexadecahydroacephenanthrylene carbon skeleton was synthesized from the precursor aspermeroterpene C by the nucleophilic addition reaction in the presence of CH3ONa. Furthermore, bioaspermeroterpenoids A and C showed good inhibitory activities against lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7 cells with IC50 values of 26.08 and 7.50 µM, respectively, compared to the positive control (Indomethacin, IC50 24.1 µM). Especially, bioaspermeroterpenoids A and C also significantly suppressed the protein expression of iNOS and COX-2 at the concentration of 12.5 μM.

Demethylzeylasteral alleviates inflammation and colitis via dual suppression of NF-κB and STAT3/5 by targeting IKKα/β and JAK2

BackgroundUlcerative colitis (UC) is a common inflammatory bowel disease and a risk factor of colorectal cancer. Demethylzeylasteral (DZT), a bioactive component mainly isolated from Tripterygium wilfordii, has been shown to inhibit inflammation and cancer. However, its anti-UC function and molecular mechanisms have not been well characterized. This study aims to explore the therapeutic effect and functional targets of demethylzeylasteral against UC. MethodsRT-qPCR, Western blot and ELISA were used to detect the generation of pro-inflammatory cytokines and chemokines in murine macrophage cells. Luciferase reporter gene, Western blot, pull-down, CETSA, DARTS, and virtual docking were employed to detect the anti-inflammatory targets and molecular mechanisms of demethylzeylasteral. The anti-inflammatory and anti-colitis effects of demethylzeylasteral were further determined in DSS-challenged mice. ResultsIn vitro, demethylzeylasteral inhibited NO and PGE2 production by suppressing the mRNA and protein expression of iNOS and COX-2, and suppressed the mRNA expression of TNF-α, IL-1β, IL-6, MCP-1, CXCL9, and CXCL10 in RAW264.7 macrophages stimulated by LPS/IFNγ. Furthermore, demethylzeylasteral was not only capable of inhibiting IKKα/β-NF-κB activation, but also able to block JAKs-STAT3/5 activation in LPS/INFγ-incubated RAW264.7 cells or DSS-exposed colon tissues of mice. Mechanistically, demethylzeylasteral was found to directly bind to IKKα/β and JAK2 kinases, leading to inactivation of pro-inflammatory signaling cascades and reduced generation of cytokines and chemokines. In vivo, oral administration of demethylzeylasteral significantly attenuated DSS-induced colitis, which was mainly manifested as mitigated symptoms of colitis, colonic mucosal barrier damage, and colonic inflammation. ConclusionWe demonstrated that demethylzeylasteral alleviated UC pathology by blocking NF-κB and STAT3/5 pathways via targeting IKKα/β and JAK2 kinases, raising the possibility that demethylzeylasteral could act as a candidate for the treatment of UC.

ARL11 knockdown alleviates spinal cord injury by inhibiting neuroinflammation and M1 activation of microglia in mice

Spinal cord injury (SCI) is a severe central nervous system injury and microglia are major participants in neuroinflammation after injury. ADP-ribosylation factor-like GTPase 11 (ARL11) is a GTP-binding protein. Whether ARL11 is involved in the SCI progression is unknown. In the impactor-induced moderate SCI mouse model, ARL11 protein and mRNA expression were significantly increased in the injury site. LPS (100 ng/mL) and IFN-γ (20 ng/mL) were incubated with BV2 cells (immortalized mouse microglial cell line) to drive them into an M1-like phenotype. ARL11 up-regulation was also observed in activated microglia in SCI mice and LPS and IFN-γ treated BV2 cells. Basso Mouse Scale scores and inclined plate test revealed that ARL11 deletion promoted motor function recovery in SCI mice. Pathological examination showed ARL11 knockdown reduced spinal cord tissue damage, increased neuron numbers, and inhibited neuronal apoptosis in SCI mice. ARL11 knockdown notably inhibited IL-1β and IL-6 production in vivo and in vitro. Furthermore, ARL11 deletion significantly inhibited iNOS protein and mRNA expression in vivo and in vitro, and COX-2 expression in vivo. Mechanism studies revealed that ARL11 silencing decreased phosphorylated ERK1/2 protein expression. Additionally, ELF1 knockdown significantly inhibited ARL11 protein and mRNA expression in vitro. ELF1 acted as a transcription activator in regulating ARL11 expression by binding to the promoter. In conclusion, ARL11 knockdown protects neurons by inhibiting M1 microglia-induced neuroinflammation, thereby promoting motor functional recovery in SCI mice. This may occur in part under the regulation of ELF1. Our study provides a new molecular target for SCI treatment.

TREM1 promotes neuroinflammation after traumatic brain injury in rats: Possible involvement of ERK/cPLA2 signalling pathway

The neuroinflammatory response promotes secondary brain injury after traumatic brain injury (TBI). Triggering receptor expressed on myeloid cells 1 (TREM1) is a key regulator of inflammation. However, the role of TREM1 in TBI is poorly studied. The purpose of this study was to investigate the role of TREM1 in TBI and the possible underlying mechanism. We found that the protein expression of TREM1 significantly increased after TBI in rats, and the TREM1 protein localized to microglia. Inhibition of the TREM1 protein with LP17 significantly blocked ERK phosphorylation and reduced cytoplasmic phospholipase A2 (cPLA2) protein expression and phosphorylation. In addition, LP17-mediated TREM1 inhibition significantly reduced the protein expression of iNOS and increased the protein expression of Arg1. Moreover, after TREM1 was inhibited, the secretion of the proinflammatory factors TNF-α and IL-1β was significantly reduced, while the secretion of the anti-inflammatory factors IL-4 and IL-10 was significantly increased. Additionally, inhibition of TREM1 by LP17 significantly reduced neuronal apoptosis and ameliorated nerve dysfunction in TBI model rats. In conclusion, our findings suggest that TREM1 enhances neuroinflammation and promotes neuronal apoptosis after TBI, and these effects may be partly mediated via the ERK/cPLA2 signalling pathway.

Eudesmane-like sesquiterpenoids from the roots of Anisodus tanguticus and their anti-inflammatory effects

Eleven eudesmane-like sesquiterpenoids including eight novel compounds (1–4 and 6–9) were isolated from the roots of Anisodus tanguticus. Interestingly, compound 1 belongs to an unprecedented sesquiterpenoid type which was named isoeudesmane-type. Compounds 6–10 were unusual 15-nor-eudesmanes. Their structures were elucidated by comprehensive spectroscopic techniques, including NMR, HR-ESI-MS, ECD, and NMR calculations. Compounds 1, 5, and 7–10 exhibited inhibitory effects against LPS-induced nitric oxide (NO) production in RAW264.7 macrophages with IC50 values of 20.92, 21.98, 34.45, 10.56, 14.39, and 5.20 μM, respectively. Compound 9 showed a significant anti-inflammatory effect by reducing the secretion of TNF-α and IL-6, as well as the protein expression of iNOS and COX-2.

Sulfate-Reducing Bacteria Induce Pro-Inflammatory TNF-α and iNOS via PI3K/Akt Pathway in a TLR 2-Dependent Manner.

Desulfovibrio, resident gut sulfate-reducing bacteria (SRB), are found to overgrow in diseases such as inflammatory bowel disease and Parkinson's disease. They activate a pro-inflammatory response, suggesting that Desulfovibrio may play a causal role in inflammation. Class I phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway regulates key events in the inflammatory response to infection. Dysfunctional PI3K/Akt signaling is linked to numerous diseases. Bacterial-induced PI3K/Akt pathway may be activated downstream of toll-like receptor (TLR) signaling. Here, we tested the hypothesis that Desulfovibrio vulgaris (DSV) may induce tumor necrosis factor alpha (TNF-α) and inducible nitric oxide synthase (iNOS) expression via PI3K/Akt in a TLR 2-dependent manner. RAW 264.7 macrophages were infected with DSV, and protein expression of p-Akt, p-p70S6K, p-NF-κB, p-IkB, TNF-α, and iNOS was measured. We found that DSV induced these proteins in a time-dependent manner. Heat-killed and live DSV, but not bacterial culture supernatant or a probiotic Lactobacillus plantarum, significantly caused PI3K/AKT/TNF/iNOS activation. LY294002, a PI3K/Akt signaling inhibitor, and TL2-C29, a TLR 2 antagonist, inhibited DSV-induced PI3K/AKT pathway. Thus, DSV induces pro-inflammatory TNF-α and iNOS via PI3K/Akt pathway in a TLR 2-dependent manner. Taken together, our study identifies a novel mechanism by which SRB such as Desulfovibrio may trigger inflammation in diseases associated with SRB overgrowth.