Phosphorylation Of IκBα Research Articles

SALMONELLA INFECTION: INTERPLAY BETWEEN THE T3SSS EFFECTORS AND NF-ΚB SIGNALING PATHWAY

Salmonella is an important foodborne pathogen that can evade host immune defense by evolving unique mechanisms. Salmonella manipulate host cell various signaling pathways by delivering specific effectors into target cells to establish infection. The nuclear factor-κB (NF-κB) is an important nuclear transcription factor that regulates the host immune system in Salmonella infection. The Salmonella pathogenicity island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2) encode type III secretion systems (T3SSs), effectors that are associated with the NF-κB signaling pathway through regulate host inflammation response. SPI-1 effectors SipA, SopE, SopE2, and SopB all can activate NF-κB signaling pathway to facilitate Salmonella invasion and intracellular carriage. Studies have shown that T3SS1 and/or T3SS2 effectors such as GtgA, GogA and PipA contain two histidine residues and have metalloprotease activity to control Salmonella replication. These zinc metalloproteases redundantly target the NF-κB subunits p65, RelB, and c-Rel, whereas GogA and GtgA only inhibit NF-κB-dependent gene transcription. The T3SS2 effectors SseK1, SseK2, and SseK3 are death domain-containing proteins with N-linked glycosyltransferase characteristics that can inhibit NF-κB activity by inhibiting IκBα phosphorylation in TNF- α-treated 293ET cells. Among them, SseK1 and SseK3 also suppress Salmonella-induced NF-κB activity in macrophages. SseK3-mediated inhibition of the NF-B signaling pathway is not required for protein 32 containing a tripartite E3-ubiquitin ligase motif. In addition, the SPI-2 T3SS effector SpvD inhibits NF-κB activity by preventing nuclear translocation of p65 through interaction with Exportin-2, but this does not affect IκBα degradation, which ultimately leads to systemic Salmonella growth. However, other effectors SptP, AvrA, IpaJ, SspH1, GtgA, GogA, and SPI-2 encoded SseL, SpvB, SseK1, and GogB all can effectively inhibit NF-κB signaling pathway, and contribute to Salmonella intracellular replication and virulence. In this mini-review, we summarize the special mechanism how NF-κB signaling pathway is regulated by Salmonella T3SSs effectors in the persistent infection of Salmonella, which will further elucidate the pathogenesis of Salmonella.

Exosomes derived from human dermal fibroblasts (HDFn-Ex) alleviate DNCB-induced atopic dermatitis (AD) via PPARα.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Skin barrier dysfunction is the initial step in the development of AD. Recently, exosomes have been considered as potential cell-free medicine for skin defects such as aging, psoriasis and wounds. The aim of this study was to investigate the effects of human dermal fibroblast-neonatal-derived exosome (HDFn-Ex) on AD. HDFn-Ex increased the expression of peroxisome proliferator activated receptor α (PPARα) and alleviated the 1-chloro-2,4-dinitrobenzene (DNCB)-mediated downregulation of filaggrin, involucrin, loricrin, hyaluronic acid synthase 1 (HAS1) and HAS2 in human keratinocyte HaCaT cells. However, these effects were inhibited by the PPARα antagonist GW6471. In the artificial skin model, HDFn-Ex significantly inhibited DNCB-induced epidermal hyperplasia and the decrease in filaggrin and HAS1 levels via a PPARα. In the DNCB-induced AD-like mouse model, HDFn-Ex administration reduced epidermis thickening and mast cell infiltration into the dermis compared to DNCB treatment. Moreover, the decreases in PPARα, filaggrin and HAS1 expression, as well as the increases in IgE and IL4 levels induced by DNCB treatment were reversed by HDFn-Ex. These effects were blocked by pre-treatment with GW6471. Furthermore, HDFn-Ex exhibited an anti-inflammatory effect by inhibiting the DNCB-induced increases in IκBα phosphorylation and TNF-α expression. Collectively, HDFn-Ex exhibited a protective effect on AD. Notably, these effects were regulated by PPARα. Based on our results, we suggest that HDFn-Ex is a potential candidate for treating AD by recovering skin barrier dysfunction and exhibiting anti-inflammatory activity.

Nebulized inhalation of LPAE-HDAC10 inhibits acetylation-mediated ROS/NF-κB pathway for silicosis treatment

Silicosis is a serious silica-induced respiratory disease for which there is currently no effective treatment. Irreversible pulmonary fibrosis caused by persistent inflammation is the main feature of silicosis. As an underlying mechanism, acetylation regulated by histone deacetylases (HDACs) are believed to be closely associated with persistent inflammation and pulmonary fibrosis. However, details of the mechanisms associated with the regulation of acetylated modification in silicosis have yet to be sufficiently established. Furthermore, studies on the efficient delivery of DNA to lung tissues by nebulized inhalation for the treatment of silicosis are limited. In this study, we established a mouse model of silicosis successfully. Differentially expressed genes (DEGs) between the lung tissues of silicosis and control mice were identified based on transcriptomic analysis, and HDAC10 was the only DEG among the HDACs. Acetylomic and combined acetylomic/proteomic analysis were performed and found that the differentially expressed acetylated proteins have diverse biological functions, among which 12 proteins were identified as the main targets of HDAC10. Subsequently, HDAC10 expression levels were confirmed to increase following nebulized inhalation of linear poly(β-amino ester) (LPAE)-HDAC10 nanocomplexes. The levels of oxidative stress, the phosphorylation of IKKβ, IκBα and p65, as well as inflammation were inhibited by HDAC10. Pulmonary fibrosis, and lung function in silicosis showed significant improvements in response to the upregulation of HDAC10. Similar results were obtained for the silica-treated macrophages in vitro. In conclusion, HDAC10 was identified as the main mediator of acetylation in silicosis. Nebulized inhalation of LPAE-HDAC10 nanocomplexes was confirmed to be a promising treatment option for silicosis. The ROS/NF-κB pathway was identified as an essential signaling pathway through which HDAC10 attenuates oxidative stress, inflammation, and pulmonary fibrosis in silicosis. This study provides a new theoretical basis for the treatment of silicosis.

Allograft inflammatory factor-1 enhances inflammation and oxidative stress via the NF-κB pathway of bladder urothelium in diabetic rat model

ObjectivesTo explore the role of allograft inflammatory factor-1 (AIF-1) both in diabetic rat bladder urothelium and in high-glucose-treated human urothelial cell line (SV-HUC-1). MethodsInflammation and oxidative stress (OS) promote diabetic cystopathy (DCP), but the mechanisms are not fully understood. The expression level of AIF-1 in diabetic rat bladder urothelium and in the SV-HUC-1 cells treated with high glucose was detected using tissue immunofluorescence, immunohistochemistry and western blot assays. AIF-1 was knocked down and NF-κB was suppressed with the specific inhibitor BAY 11–7082 in high-glucose-treated SV-HUC-1 cells. ResultsHigh-glucose condition induced AIF-1 upregulation in vivo and in vitro. The up-regulated AIF-1 induced the production of inflammatory factors IL-6 and TNF-α and elevation of ROS. Informatics analysis suggested that NF-κB pathway is implicated in DCP. Through knockdown of AIF-1, we confirmed that AIF-1 simulated NF-κB pathway by enhancing the phosphorylation of IκB (p-IκB) and promoting the translocation of NF-κB p65 from cytoplasm into nucleus. Additionally, High-glucose-induced inflammation in SV-HUC-1 cells was attenuated by the addition of NF-κB inhibitor. ConclusionsThis study provides novel information to understand the molecular regulation mechanisms of AIF-1 in DCP.

Carveol alleviates osteoarthritis progression by acting on synovial macrophage polarization transformation: An in vitro and in vivo study

Osteoarthritis (OA) is a heterogeneous disease that affects the entire joint. Its pathogenesis involves hypertrophy and hyperplasia of synovial cells and polarization infiltration of macrophages, in which macrophages, as a potential target, can delay the progression of the disease by improving the immune microenvironment in OA. To investigate the role and regulatory mechanism of Carveol in cartilage and synovial macrophage reprogramming and crosstalk during the development of OA. RAW264.7 mouse macrophage cell line was mainly used to stimulate macrophages to polarization towards M1 and M2 by LPS, IL4+IL13, respectively. Different concentrations of Carveol were given to intervene, and macrophage culture medium was collected to intervene mouse C57BL6J chondrocytes. ROS assay kit, western blotting, cellular immunofluorescence, scanning microscope and section histology were used to evaluate the effect of Carveol on anti-M1-polarization, M2-polarization promotion and cartilage protection. The mouse destabilization of medial meniscus (DMM) model was observed by micro-CT scan and histology. We found that CA could inhibit the increase of macrophage inflammation level under the intervention of LPS and promote the production of M2 anti-inflammatory substances under the intervention of IL-4+IL13. In addition, Carveol activated NRF2/HO-1/NQO1 pathway and enhanced ROS clearance in chondrocytes under the intervention of macrophage culture medium. The phosphorylation of I-κBα is inhibited, which further reduces the phosphorylation of P65 downstream of nuclear factor-κB (NF-κB) signaling pathway. In addition, Carveol inhibits mitogen activated protein kinase (MAPK) signaling molecules P-JNK, P-ERK and P–P38, and inhibits the production of inflammatory mediators. In vivo, Carveol can reduce osteophytes and bone spurs induced by DMM, reduce hypertrophy of synovial cells, reduce infiltration of macrophages, inhibit subchondral bone destruction, and reduce articular cartilage erosion. Our study suggests that synovial macrophages are potential targets for OA treatment, and Carveol is an effective candidate for OA treatment.

4-methoxycinnamyl p-coumarate reduces neuroinflammation by blocking NF-κB, MAPK, and Akt/GSK-3β pathways and enhancing Nrf2/HO-1 signaling cascade in microglial cells

The active compound, 4-methoxycinnamyl p-coumarate (MCC), derived from the rhizome of Etlingera pavieana (Pierre ex Gagnep) R.M.Sm., has been shown to exert anti-inflammatory effects in several inflammatory models. However, its effects on microglial cells remain elusive. In the current study, we aimed to investigate the anti-neuroinflammatory activities of MCC and determine the potential mechanisms underlying its action on lipopolysaccharide (LPS)-induced BV2 microglial cells. Our results revealed that MCC significantly reduced the secretion of nitric oxide (NO) and prostaglandin E2, concomitantly inhibiting the expression levels of inducible NO synthase and cyclooxygenase-2 mRNA and proteins. Additionally, MCC effectively decreased the production of reactive oxygen species in LPS-induced BV2 microglial cells. MCC also attenuates the activation of NF-κB by suppressing the phosphorylation of IκBα and NF-κB p65 subunits and by blocking the nuclear translocation of NF-κB p65 subunits. Furthermore, MCC significantly reduced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β). In addition, MCC markedly increased the expression of heme oxygenase-1 (HO-1) by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Collectively, our findings suggest that the anti-inflammatory activities of MCC could be attributed to its ability to suppress the activation of NF-κB, MAPK, and Akt/GSK-3β while enhancing that of Nrf2-mediated HO-1. Accordingly, MCC has promising therapeutic potential to treat neuroinflammation-related diseases.

Xuelian injection ameliorates complete Freund's adjuvant-induced acute arthritis in rats via inhibiting TLR4 signaling

BackgroundXuelian injection (XI), a classic preparation extracted from Saussureae Involucratae Herba, has been clinically used to manage rheumatoid arthritis (RA) for nearly twenty years in China. However, the underlying anti-RA mechanism of XI remains unclear. In this study, complete Freund's adjuvant (CFA)-induced acute arthritic model was used to examine the anti-RA effects of XI in vivo. The molecular mechanisms of this action were further investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. MethodsXI and XI freeze dried powder were characterized by UPLC analysis. CD68 and TLR4 expression in the ankle joints was measured by immunohistochemistry. The secretion of inflammatory mediators was detected by ELISA. The expression levels of TLR4 involved components were measured by Western blotting. The localization of transcription factors was measured by immunofluorescence assay. ResultsXI treatment ameliorated arthritic symptoms induced by CFA in the ankle joints of rats. The serum levels of inflammatory mediators, including TNF-α, MCP-1, and Rantes were decreased by XI treatment. The elevation of CD68 and TLR4 levels in ankle joints caused by CFA was suppressed by XI treatment. Moreover, XI treatment inhibited the secretion of nitric oxide and prostaglandin E2 in LPS-treated RAW264.7 macrophages. The expression of their enzymes iNOS and COX-2 was also decreased after XI treatment. The production of inflammatory mediators, including TNF-α, IL-6, IL-1β, MCP-1, MIP-1α, and Rantes was reduced by XI treatment in LPS-stimulated RAW264.7 cells. The phosphorylation of p38, JNK, ERK, TBK1, IKKα/β, IκB, p65, c-Jun, and IRF3 was reduced after XI treatment. Additionally, the expression levels of nuclear proteins of p65, c-Jun, and IRF3 were inhibited by XI treatment. ConclusionsTaken together, XI possesses potential anti-RA effect and the underlying mechanism may be closely associated with the inhibition of TLR4 signaling. Our findings provide further pharmacological justifications for the clinical use of XI in RA treatment.

Histone lactylation-derived LINC01127 promotes the self-renewal of glioblastoma stem cells via the cis-regulating the MAP4K4 to activate JNK pathway

Gliomas are the most prevalent and aggressive brain tumors, exhibiting high proliferation, abnormal glycolysis, and poor prognosis. LncRNAs act as regulatory molecules and play crucial roles in the malignant behaviors of GBM cells, including cell self-renewal. However, the regulatory mechanisms involved are largely unknown. In this study, we performed bioinformatics analysis to explore NF-κB pathway-related lncRNAs. ECAR and qRT-PCR were used to measure the relationship between glycolytic activity and lncRNA expression. Assays such as RIP-PCR and ChIP-PCR were employed to reveal the regulatory mechanisms of the lncRNA. Neurosphere formation and limiting dilution assays were performed to evaluate the self-renewal capacity of GBM cells. In our study, we identified an NF-κB pathway-related lncRNA named LINC01127 in GBM, which was found to be associated with poor progression of GBM. Functionally, the NF-κB pathway promoted warburg effect, which, in turn, induced the lactylation of H3 histone and increased the expression of LINC01127. Consequently, this enhancement of LINC01127 expression led to the promotion of self-renewal in GBM cells. Furthermore, LINC01127 regulated MAP4K4 expression in cis by directly guiding POLR2A to the MAP4K4 promoter regions, thereby leading to JNK pathway activation, and ultimately modulating the self-renewal of GBM cells. Moreover, the activated JNK pathway promoted the phosphorylation of IκBα. Overall, targeting LINC01127-mediated axis impeded orthotopic tumor growth in GBM xenografts. Taken together these results revealed that warburg effect-induced histone lactylation drives NF-κB-related LINC01127 expression, thereby promoting the self-renewal of GBM cells through the MAP4K4/JNK/NF-κB axis, and providing substantial evidence that LINC01127 might provide a novel therapeutic strategy for GBM patients.

Quinoa protein and its hydrolysate ameliorated DSS-induced colitis in mice by modulating intestinal microbiota and inhibiting inflammatory response

The objective of this study was to investigate the protective effects of quinoa protein (QPro) and its derived peptides (QPep) in dextran sulfate sodium (DSS)-induced colitis in mice. The results demonstrated that oral administration of QPro and QPep significantly alleviated colitis symptoms, including diarrhea, abdominal pain, bloody stools, weight loss, as well as reduced colonic shortening, inflammatory factor release, and intestinal barrier injury. Short-chain fatty acids (SCFAs) production rose as QPro and QPep modulated the composition of the intestinal microbiota. Western blotting results revealed that QPro and QPep also suppressed TLR4 levels and inhibited IκB-α and NF-κB phosphorylation in colon tissue, implying that the TLR4/IκB-α/NF-κB signaling pathway may be involved in the amelioration of QPro and QPep in DSS-induced colitis.These results indicate the potential of quinoa protein and its hydrolysate to serve as bioactive components in functional diets for intestinal health and to significantly lower intestinal inflammation.

Improvement of Cyclophosphamide-Induced Immunosuppression by Miaoyao Fanggan Sachet Extract

Chemotherapy drugs, such as cyclophosphamide, often produce side effects of immunosuppression in the treatment of tumors. This study investigated whether the Miaoyao Fanggan sachet extracts—diammonium glycyrrhizinate and magnesium isoglycyrrhizinate—can regulate respiratory immunity in mice. A cyclophosphamide-induced immunosuppressive mice model was treated with diammonium glycyrrhizinate and magnesium isoglycyrrhizinate. Interferon (IFN)-α, interferon-β, interleukin (IL)-6, and interleukin-18 levels in the bronchoalveolar lavage fluid and lung tissue were detected using enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction, respectively. The p-(phospho) transforming growth factor beta-activated kinase 1 (TAK1)/TAK1 and p-IkappaB (IκB)/IκB in the lung tissue and bronchial mucosa were detected by Western blot. Compared with the control group, the IFN-α and IL-6 levels decreased, while IFN-βand IL-18 levels increased in the mice model. However, these changes were reversed with the treatment with diammonium glycyrrhizinate and magnesium isoglycyrrhizinate; p-TAK1/TAK1 and p-IκB/IκB in the model group decreased significantly and increased with the increasing treatment dose of diammonium glycyrrhizinate and magnesium isoglycyrrhizinate. Diammonium glycyrrhizinate and magnesium isoglycyrrhizinate had a certain effect on cyclophosphamide-induced immunosuppression in mice, likely mediated through the phosphorylation of TAK1 and IκB, activating nuclear factor kappa-B and subsequently stimulating the Janus kinase/signal transducer and activator of transcription/mitogen-activated protein kinase pathway.

Extracellular SOD modulates canonical TNFα signaling and α5β1 integrin transactivation in vascular smooth muscle cells

TNFα activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O2•-) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O2•- to signal transduction remain obscure. Extracellular superoxide dismutase (ecSOD, SOD3 gene) is a secreted protein that binds to cell surface heparin sulfate proteoglycans or to Fibulin-5 (Fib-5, FBLN5 gene), an extracellular matrix protein that also associates with elastin and integrins. ecSOD converts O2•- to hydrogen peroxide (H2O2) which prevents NO• inactivation, limits generation of hydroxyl radical (OH•), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNFα signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNFα signals including MAPK (JNK and ERK phosphorylation) and NF-κB activation (luciferase reporter and IκB phosphorylation), interleukin-6 (IL-6) secretion, iNOS and VCAM expression, and proliferation (Sulforhodamine B assay, PCNA western blot). These effects were associated with significant reductions in the expression of both Type1 and 2 TNFα receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-κB activation by TNFα, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNFα-induced phosphorylation of FAK and of glycogen synthase kinase-3β (GSK3β), a downstream target of integrin linked kinase (ILK). These effects were dependent upon α5β1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNFα-induced increases in sulfenylation. To determine how ecSOD modified TNFα-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNFα (10 ng/ml) in culture for 48 h. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNFα signaling while tonically inhibiting α5β1 integrin activation.

Total Flavonoids from Camellia oleifera Alleviated Mycoplasma pneumoniae-Induced Lung Injury via Inhibition of the TLR2-Mediated NF-κB and MAPK Pathways.

Mycoplasma pneumoniae (M. pneumoniae) is an atypical bacterial pathogen responsible for community-acquired pneumonia primarily among school-aged children and young adults. Camellia oleifera (C. oleifera) has been used as a medicinal and edible plant in China for centuries, the constituents from which possessed various bioactivities. Notably, flavonoids existing in residues of C. oleifera defatted seeds exhibited significant anti-inflammatory activities. In the present study, we investigated the impact of total flavonoids from C. oleifera (TFCO) seed extract on M. pneumoniae pneumonia. TFCO was obtained using multiple column chromatography methods and identified as kaempferol glycosides via UPLC-HRESIMS. In a M. pneumoniae pneumonia mouse model, TFCO significantly reduced the lung damage, suppressed IL-1β, IL-6, and TNF-α production, and curbed TLR2 activation triggered by M. pneumoniae. Similarly, in RAW264.7 macrophage cells stimulated by lipid-associated membrane proteins (LAMPs), TFCO suppressed the generation of proinflammatory cytokines and TLR2 expression. Moreover, TFCO diminished the phosphorylation of IκBα, JNK, ERK, p38, and p65 nuclear translocation in vitro. In conclusion, TFCO alleviated M. pneumoniae-induced lung damage via inhibition of TLR2-mediated NF-κB and MAPK pathways, suggesting its potential therapeutic application in M. pneumoniae-triggered lung inflammation.

Caspase 6 promotes innate immune activation by functional crosstalk between RIPK1-IκBα axis in liver inflammation

BackgroundCaspase 6 is an essential regulator in innate immunity, inflammasome activation and host defense. We aimed to characterize the causal mechanism of Caspase 6 in liver sterile inflammatory injury.MethodsHuman liver tissues were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. Subsequently, we created Caspase 6-knockout (Caspase 6KO) mice to analyze roles and molecular mechanisms of macrophage Caspase 6 in murine models of liver ischemia/reperfusion (IR) injury.ResultsIn human liver biopsies, Caspase 6 expression was positively correlated with more severe histopathological injury and higher serum ALT/AST level at one day postoperatively. Moreover, Caspase 6 was mainly elevated in macrophages but not hepatocytes in ischemic livers. Unlike in controls, the Caspase 6-deficient livers were protected against IR injury, as evidenced by inhibition of inflammation, oxidative stress and iron overload. Disruption of macrophage NF-κB essential modulator (NEMO) in Caspase 6-deficient livers deteriorated liver inflammation and ferroptosis. Mechanistically, Caspase 6 deficiency spurred NEMO-mediated IκBα phosphorylation in macrophage. Then phosphorylated-inhibitor of NF-κBα (p-IκBα) co-localized with receptor-interacting serine/ threonine-protein kinase 1 (RIPK1) in the cytoplasm to degradate RIPK1 under inflammatory conditions. The disruption of RIPK1-IκBα interaction preserved RIPK1 degradation, triggering downstream apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and inciting NIMA-related kinase 7/NOD-like receptor family pyrin domain containing 3 (NEK7/NLRP3) activation in macrophages. Moreover, ablation of macrophage RIPK1 or ASK1 diminished NEK7/NLRP3-driven inflammatory response and dampened hepatocyte ferroptosis by reducing HMGB1 release from macrophages.ConclusionsOur findings underscore a novel mechanism of Caspase 6 mediated RIPK1-IκBα interaction in regulating macrophage NEK7/NLRP3 function and hepatocytes ferroptosis, which provides therapeutic targets for clinical liver IR injury.Graphical DRFRodggEVQV_MBi1PPYnNVideo

Quercetin improves cerebral ischemia/reperfusion injury by promoting microglia/macrophages M2 polarization via regulating PI3K/Akt/NF-κB signaling pathway

The modulation of microglial polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype shows promise as a therapeutic strategy for ischemic stroke. Quercetin, a natural flavonoid abundant in various plants, possesses anti-inflammatory, anti-apoptotic, and antioxidant properties. Nevertheless, its effect and underlying mechanism on microglia/macrophages M1/M2 polarization in the treatment of cerebral ischemia/reperfusion injury (CI/RI) remain poorly explored. In the current study, we observed that quercetin ameliorated neurological deficits, reduced infarct volume, decreased the number of M1 microglia/macrophages (CD16/32+/Iba1+), and enhanced the number of M2 microglia/macrophages (CD206+/Iba1+) after establishing the CI/RI model in rats. Subsequent in vivo and in vitro experiments indicated that quercetin downregulated M1 markers (CD86, iNOS, TNF-α, IL-1β, and IL-6) and upregulated M2 markers (CD206, Arg-1, IL-10, and TGF-β). Network pharmacology analysis and molecular docking revealed that the PI3K/Akt/NF-κB signaling pathway emerged as the core pathway. Western blot confirmed that quercetin upregulated the phosphorylation of PI3K and Akt, while alleviating the phosphorylation of IκBα and NF-κB both in vivo and in vitro. However, the PI3K inhibitor LY294002 reversed the effects of quercetin on M2 polarization and the expression of key proteins in the PI3K/Akt/NF-κB pathway in primary microglia after oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Collectively, our findings demonstrate that quercetin facilitates microglia/macrophages M2 polarization by modulating the PI3K/Akt/NF-κB signaling pathway in the treatment of CI/RI. These findings provide novel insights into the therapeutic mechanisms of quercetin in ischemic stroke.

Dimethyl Fumarate Protects against Lipopolysaccharide- (LPS-) Induced Sepsis through Inhibition of NF-κB Pathway in Mice.

Sepsis is one of the most severe complications and causes of mortality in the clinic. It remains a great challenge with no effective treatment for clinicians worldwide. Inhibiting the release of proinflammatory cytokines during sepsis is considered as an important strategy for treating sepsis and improving survival. In the present study, we have observed the effect of dimethyl fumarate (DMF) on lipopolysaccharide- (LPS-) induced sepsis and investigated the possible mechanism. By screening a subset of the Johns Hopkins Drug Library, we identified DMF as a novel inhibitor of nitric oxide synthesis in LPS-stimulated RAW264.7 cells, suggesting that DMF could be a potential drug to treat sepsis. To further characterize the effect of DMF on LPS signaling, TNF-α, MCP-1, G-CMF, and IL-6 expression levels were determined by using cytokine array panels. In addition, an endotoxemia model with C57BL/6 mice was used to assess the in vivo efficacy of DMF on sepsis. The survival rate was assessed, and HE staining was performed to investigate histopathological damage to the organs. DMF was found to increase the survival of septic mice by 50% and attenuate organ damage, consistent with the reduction in IL-10, IL-6, and TNF-α (inflammatory cytokines) in serum. In vitro experiments revealed DMF's inhibitory effect on the phosphorylation of p65, IκB, and IKK, suggesting that the primary inhibitory effects of DMF can be attributed, at least in part, to the inhibition of phosphorylation of IκBα, IKK as well as nuclear factor-κB (NF-κB) upon LPS stimulation. The findings demonstrate that DMF dramatically inhibits NO and proinflammatory cytokine production in response to LPS and improves survival in septic mice, raising the possibility that DMF has the potential to be repurposed as a new treatment of sepsis.

Distribution profiles of phenolic compounds in a cultivar of wampee (Clausena lansium (Lour.) Skeels) fruits and in vitro anti-inflammatory activity

Ethnopharmacological relevanceThe fruits of wampee (Clausena lansium (Lour.) Skeels), which contain significant amounts of phenolics, are frequently applied as traditional medications to prevent or relieve inflammatory symptoms. Green honey wampee (GHW) is a local cultivar specially cultivated in Lianjiang City, Guangdong Province. Aim of the studyThis study aimed to investigate phenolic distribution profiles in the peels, seeds and pulp of GHW as well as elucidate the underlying molecular mechanisms of the effective compounds for anti-inflammatory activity. Materials and methodPhenolic compounds in the extract were identified through UPLC-MS/MS and their ability to alleviate inflammation was assessed using RAW 264.7 macrophages exposed to lipopolysaccharide. ResultsAmong the three parts of GHW fruits, the total phenolic contents followed a descending order of peels > pulp > seeds. Additionally, eighty-six phenols were tentatively determined from the three parts, of which flavonoids accounted for the highest proportion. Furthermore, the phenolic extract of peels, seeds and pulp exhibited potential anti-inflammatory activity through the suppression effect on different pro-inflammatory mediators (NO, IL-6 and TNF-α). Among the three principal phenolic compounds (rutin, quercitrin, isorhamnetin-3-O-neohesperidoside) detected in GHW fruits, quercitrin was proved to be a more important anti-inflammatory compound inhibiting the iNOS and TNF-α mRNA expressions through the suppression effect on the phosphorylation of IκBα and ERK, belonging to the NF-κB and MAPK signaling pathway respectively. ConclusionsNot only wampee pulp but also its by-products like peels and seeds are able to be comprehensively utilized as immunomodulatory supplements for daily diets due to their rich phenolic contents.

Honey gold nanoparticles attenuate the secretion of IL-6 by LPS-activated macrophages.

Interleukin-6 (IL-6) is a pleiotropic cytokine that coordinates host immune responses to infection. Though essential to the acute phase response, prolonged IL-6-mediated recruitment of mononuclear cells has been implicated in chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and Crohn's disease. Accordingly, identifying novel therapeutics that diminish circulating IL-6 levels could benefit individuals suffering from chronic inflammation. In immunocompetent hosts, bacterial lipopolysaccharide (LPS) recognition by toll-like receptor 4 (TLR4) activates the transcription factor NF-κB, driving macrophage production of IL-6. Interestingly, both citrate-stabilized and 'green' synthesized gold nanoparticles (AuNPs) have been shown to modulate the cytokine responses of LPS-activated macrophages. Here we demonstrate that AuNPs, synthesized with commercial and locally sourced honey, downregulate LPS-induced macrophage secretion of IL-6. Compared to LPS-only controls, inhibition of IL-6 levels was observed for all three types of honey AuNPs. The effect was likely driven by honey AuNP-mediated perturbation of the TLR4/NF-κB signaling pathway, as evidenced by a reduction in the phosphorylation of IκB. Further investigation into the anti-inflammatory properties of honey AuNPs may yield novel therapeutics for the treatment of chronic inflammation.

Camellia oil alleviates DSS-induced colitis in mice by regulating the abundance of intestinal flora and suppressing the NF-κB signaling pathway

Colitis is characterized by colonic inflammation and impaired gut health, and alterations in the gut microbiota may lead to the development of inflammatory bowel disease (IBD). Camellia oil (CO) possesses high nutritional value and offers various health benefits for human metabolic disorders and diseases. However, it is unclear whether CO ameliorates IBD and plays a role in the gut microbiota. This study aimed to investigate the effect of CO on colonic inflammation and gut microbiota using a mice model of dextran sodium sulfate (Dss)-induced ulcerative colitis (UC). Our findings demonstrated that CO inhibited weight loss and colon shortening and improved intestinal barrier function. Additionally, CO treatment reduced oxidative stress and inflammatory responses in Dss-induced colitis. Furthermore, CO significantly inhibited p65 and IκBα phosphorylation and alleviated colonic inflammation. Moreover, we observed that CO treatment increased the abundance of Bacteroides, Lactobacillus, and Odoribacter, while decreasing the abundance of Alistipes, Lachnospiraceae NK4A136 group, Ruminococcaceae UCG-014, uncultured Bacteroidales bacterium, and Prevotellaceae_UCG-001. Additionally, CO treatment promoted the production of SCFAs. These data indicated the promising potential of CO to prevent UC by maintaining gut barrier function, inhibiting the NF-κB signaling pathway, and modulating the gut microbiota.

Modified Curcuminoid-Rich Extract Liposomal CRE-SDInhibits Osteoclastogenesis via the Canonical NF-κB Signaling Pathway.

Curcuminoids, namely curcumin, demethoxycurcumin, and bisdemethoxycurcumin, are the major active compounds found in Curcuma longa L. (turmeric). Although their suppressive effects on bone resorption have been demonstrated, their pharmacokinetic disadvantages remain a concern. Herein, we utilized solid dispersion of a curcuminoid-rich extract (CRE), comprising such curcuminoids, to prepare CRE-SD; subsequently, we performed liposome encapsulation of the CRE-SD to yield liposomal CRE-SD. In vitro release assessment revealed that a lower cumulative mass percentage of CRE-SD was released from liposomal CRE-SD than from CRE-SD samples. After culture of murine RANKL-stimulated RAW 264.7 macrophages, our in vitro examinations confirmed that liposomal CRE-SD may impede osteoclastogenesis by suppressing p65 and IκBα phosphorylation, together with nuclear translocation and transcriptional activity of phosphorylated p65. Blind docking simulations showed the high binding affinity between curcuminoids and the IκBα/p50/p65 protein complex, along with many intermolecular interactions, which corroborated our in vitro findings. Therefore, liposomal CRE-SD can inhibit osteoclastogenesis via the canonical NF-κB signaling pathway, suggesting its pharmacological potential for treating bone diseases with excessive osteoclastogenesis.

Nitric oxide synthase regulates coelomocytes apoptosis through the NF-κB signaling pathway in the sea cucumber Apostichopus japonicus

Nitric oxide synthase (NOS) was initially discovered to participate in the generation of nitric oxide as a defense mechanism against pathogenic infections. In recent years, it has been found that NOS plays a pivotal role in regulating apoptosis and inflammation in mammals. However, the mechanisms underlying NOS-mediated apoptosis in invertebrates remain largely unclear. In this study, we found that the Apostichopus japonicus NOS (AjNOS) expression levels were upregulated by 2.20-fold and 3.46-fold after being challenged with Vibrio splendidus at concentrations of 107 CFU mL−1 and 108 CFU mL−1 for 12 h compared to the control group, respectively. Under these conditions, the rates of coelomocytes apoptosis were increased from 14.7% to 32.7% and 45.4%, respectively. Treatment with NOS inhibitor (l-NAME) resulted in a reduction of coelomocytes apoptosis rates from 32.6% to 26.5% in V. splendidus (107 CFU mL−1) groups and from 42.3% to 33.3% in V. splendidus (108 CFU mL−1) groups, respectively. NOS has been reported to regulate apoptosis through IκBα phosphorylation. Simultaneously, exposure to V. splendidus in conjunction with l-NAME resulted in down-regulation of AjIκBα phosphorylation levels compared to the group infected solely with V. splendidus. Furthermore, immunofluorescence analysis revealed that treatment with l-NAME or interference of AjNOS using siRNA inhibited translocation of AjNF-κB/p65 (RelA) into the nucleus. Previous studies have shown that NF-κB can down-regulate expression levels of Bcl-2 family members, which is an important pathway for regulating apoptosis. In the present study, treatment with l-NAME was found to promote anti-apoptotic AjBcl-2 mRNA increase to 1.41-fold and protein expression increase to 1.86-fold at 12 h post V. splendidus challenge. However, these effects were suppressed by PMA (an NF-κB activator). Overall, our findings demonstrate that AjNOS regulates coelomocytes apoptosis induced by V. splendidus through activation of the AjNF-κB signaling pathway and down-regulation of AjBcl-2 in A. japonicus.