NF-κB Activation Research Articles

TRPML-1 Dysfunction and Renal Tubulopathy in Mucolipidosis Type IV.

Loss of function mutations in the lysosomal channel TRPML-1 cause mucolipidosis type IV (MLIV), a rare lysosomal storage disease characterized by neurological defects, progressive vision loss, and achlorhydria. Recent reports have highlighted kidney disease and kidney failure in patients with MLIV during the second to third decade of life; however, the molecular mechanisms driving kidney dysfunction remain poorly understood. A cross-sectional review of medical records from 21 MLIV patients (ages 3-43 years) was conducted to assess kidney function impairment. Additionally, we examined the kidney phenotype of MLIV mice at various ages, along with human kidney cells silenced for TRPML-1 and primary tubular cells from wild-type and MLIV mice. Immunohistology and cell biology approaches were used to phenotype nephron structure, the endolysosomal compartment, and inflammation. Kidney function was assessed through proteomic analysis of mouse urine and in vivo renal filtration measurements. Of the 21 MLIV patients only adults were diagnosed with stage 2-3 chronic kidney disease. Laboratory abnormalities included decreased eGFR, higher levels BUN/Creatine in bloodand proteinuria. In MLIV mice, we observed significant alterations in endolysosomal morphology, function, and impaired autophagy in proximal and distal tubules. This led to the accumulation of megalin (LRP2) in the subapical region of proximal tubular cells, indicating a block in apical receptor-mediated endocytosis. In vivo and in vitro experiments confirmed reduced fluid-phase endocytosis and impaired uptake of ligands, including β-lactoglobulin, transferrin, and albumin in MLIV proximal tubular cells. Urine analysis revealed tubular proteinuria and enzymuria in mice with MLIV. Additionally, early-stage disease was marked by increased inflammatory markers, fibrosis, and activation of the pro-inflammatory transcription factor NF-κB, coinciding with endolysosomal defects. Importantly, AAV-mediated TRPML-1 gene delivery reversed key pathological phenotypes in Mucolipidosis type IV mice, underscoring TRPML-1's critical role in kidney function. Our findings link TRPML-1 dysfunction to the development of kidney disease in MLIV, providing new insights into its pathogenesis and potential therapeutic targets.

Exogenous H2S targeting PI3K/AKT/mTOR pathway alleviates chronic intermittent hypoxia-induced myocardial damage through inhibiting oxidative stress and enhancing autophagy.

Hydrogen sulfide (H2S) is a novel gas signaling molecule that has been researched in several physiological and pathological conditions, indicating that strategies targeting H2S may provide clinical benefits in diseases such as chronic cardiomyopathy. Here, we reveal the effect of H2S on chronic intermittent hypoxia (CIH)-related myocardial damage and its mechanistic relevance to phosphoinositol-3 kinase (PI3K). Mice were subjected to a 4-week CIH process to induce myocardial damage, which was accompanied by daily administration of NaHS (a H2S donor) and LY294002 (an inhibitor of PI3K). Changes in heart function were evaluated via echocardiography. Histological examination was applied to assess heart tissue lesions. Myocardial apoptosis was detected by TUNEL staining and apoptosis-associated protein expression. Furthermore, the effects of NaHS on autophagy and the PI3K/AKT/mTOR pathway were investigated. Finally, the level of inflammation is also affected by related proteins. The CIH group presented increased myocardial dysfunction and heart tissue lesions. Echocardiography and histological analysis revealed that, compared with control mice, CIH-treated mice presented significantly more severe left ventricular remodeling and decreased myocardial contractile function. In addition, the apoptosis index and oxidative markers were significantly elevated in the CIH group compared with those in the control group. The autophagy marker Beclin-1 was decreased, while p62 was elevated by CIH treatment. H2S supplementation with NaHS significantly improved cardiac function and alleviated fibrosis, oxidative stress, and apoptosis but upregulated autophagy in CIH mice, and these effects were also observed in animals that underwent only PI3K blockade. Furthermore, PI3K/AKT pathway-mediated inhibition of the mammalian target of rapamycin (mTOR) pathway, the Nrf2/HO-1 pathway and proinflammatory NF-κB activity were shown to play a role in the therapeutic effect of NaHS after CIH stimulation.

Macrophage Dvl2 Deficiency Promotes NOD1-Driven Pyroptosis and Exacerbates Inflammatory Liver Injury

Dishevelled 2 (Dvl2) is a key mediator of the Wingless/Wnt signaling pathway that regulates cell proliferation, migration, and immune function. However, little is known about the role of macrophage Dvl2 in modulating NOD1-mediated pyroptosis and hepatocyte death in oxidative stress-induced inflammatory liver injury. In a mouse model of oxidative stress-induced liver inflammation, mice with myeloid-specific Dvl2 knockout (Dvl2M-KO) displayed exacerbated ischemia/reperfusion (IR) stress-induced hepatocellular damage with increased serum ALT levels, oxidative stress, and proinflammatory mediators. Unlike in Dvl2FL/FL controls, Dvl2M-KO enhanced NOD1, caspase-1, GSDMD, and NF-κB activation in liver macrophages after IR. Interestingly, IR stress enhanced YAP colocalized with HSF1 in Dvl2FL/FL macrophages, while macrophage Dvl2 deficiency reduced YAP and HSF1 colocalization in the nucleus under inflammatory conditions. Importantly, Dvl2 deletion diminished nuclear YAP interacted with HSF1 and augmented NOD1/caspase-1 and GSDMD activation in response to inflammatory stimulation. However, Dvl2 activation increased YAP interaction with HSF1 and activated HSF1 target gene eEF2, inhibiting NOD1/caspase-1, GSDMD, and NF-κB activity. Moreover, macrophage eEF2 deletion increased the NOD1-caspase-1 interaction, GSDMD activation, HMGB1 release, and hepatocyte LDH release after macrophage/hepatocyte co-culture. Adoptive transfer of eEF2-expressing macrophages in Dvl2M-KO mice alleviated IR-triggered liver inflammation and hepatocellular damage. Therefore, macrophage Dvl2 deficiency promotes NOD1-mediated pyroptosis and exacerbates IR-induced hepatocellular death by disrupting the YAP-HSF1 axis. eEF2 is crucial for modulating NOD1-driven pyroptosis, inflammatory response, and hepatocyte death. Our findings underscore a novel role of macrophage Dvl2 in modulating liver inflammatory injury and imply the therapeutic potential in organ IRI and transplant recipients.

Flavonoids analysis in citrus peels by UPLC-Q-TOF-MS/MS and its antioxidant and anti-inflammation activity

Citrus is the world's largest fruit, developed a rich variety resources in a long history of cultivation. Flavonoid is a natural product found widely in citrus plants. In this study, the major flavonoids and relative activities of 9 citrus peels were investigated by using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS) analysis and antioxidant and anti-inflammatory assays in A549 cell. A total of 51 flavonoids were identified, comprising a variety of subclasses. Notably, Citrus Reticulata Blanco (MJ) and Bu Zhi Huo (CJ) demonstrated distinctive flavonoid profiles. Flavonoid extracts from MJ and Citrus maxima (HY) peels significantly reduced reactive oxygen species (ROS) generation and decreased the levels of cytokines (IL-8, TNF-α, MCP-1, and IL-6) in A549 cells. Furthermore, these extracts up-regulated the expression of Nrf2 and HO-1 and inhibited the phosphorylation of p65 and NF-κB activity. Gray correlation analysis identified (-)-vestitone, dimefline, eupafolin, and trans-cinnamaldehyde as strongly associated with inflammatory markers. The findings suggest that flavonoids extracted from citrus peels possess significant antioxidant and anti-inflammatory activities, highlighting their potential as natural therapeutic agents for combating inflammation.

Anti-oxidative and immunological role of Cyclocarya paliurus polysaccharide on the liver injury of diabetic rats.

To investigate the effects of Cyclocarya paliurus (C. paliurus) polysaccharide on the liver injury of diabetic rats. Rats were divided into 6 groups, including normal group, model group, control group, low-dose group of C. paliurus polysaccharide treatment, middle-dose group of C. paliurus polysaccharide treatment and high-dose group of C. paliurus polysaccharide treatment. Histological analysis of liver was analyzed using hematoxilin and eosin. Levels of plasma biological parameters and anti-oxidative enzymes were determined by spectrophotometry. Nuclear factor kappa-B-p65 (NF-κB p65), tumor necrosis factor-α (TNF-α), interleukins 6 (IL-6), IL-1β were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Compared with that of model group, the glucose level of plasma decreased 62.32% (P < 0.01), but glycogen and insulin level increased 1.51 times and 1.27 times in the high-dose group of C. paliurus polysaccharide treatment, respectively. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) level decreased 47.47% (P < 0.05), 43.65% (P < 0.05) and 50.51% (P < 0.05) in the high-dose group of C. paliurus polysaccharide treatment, respectively. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase level increased 1.16 times (P < 0.01), 71.28% (P < 0.05), 1.29 times (P < 0.01) and 87.46% (P < 0.05) in the high-dose group of C. paliurus polysaccharide treatment, respectively. Enzyme activities of NF-κB, TNF-α, IL-1β and IL-6 were decreased 39.29% (P < 0.05), 51.11% (P < 0.05), 37.42% (P < 0.05) and 36.50% (P < 0.05), respectively. Administration of C. paliurus polysaccharide may play a protecting role for liver injury of diabetic rats through lowering glucose, ALT, AST, ALP level, increasing glycogen and insulin level, enhancing the anti-oxidative ability and down-regulating the inflammatory factors expression.

FABP4 Inhibitor Alleviates Lipopolysaccharide-Induced HUVEC Injury by Inactivating NF-κB and Activating PPARγ.

The study aims to examine the effect of FABP4 on inflammatory response and angiogenesis in the cell model of atherosclerosis and to explore its potential mechanism.Using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting, the mRNA and protein levels of FABP4 in human umbilical vein endothelial cells (HUVECs) treated with lipopolysaccharide (LPS) or oxidized low-density lipoprotein for 6, 12, 24, or 48 hours were measured. To silence FABP4 expression and NF-κB signaling in HUVECs, FABP4 inhibitor and NF-κB inhibitor were utilized. To assess cell survival rate and apoptosis, methyl thiazolyl tetrazolium assays and flow cytometry analysis were conducted. Genes related to cholesterol metabolism, including LOX-1, ABCA1, and ABCG1, were subjected to RT-qPCR and western blotting. Moreover, protein levels of apoptotic markers (Bcl-2 and Bax), PPARγ, the phosphorylated levels of NF-κB p65, and angiogenetic markers (ICAM1, VCAM1, and VEGF) were quantified via western blotting. Using an enzyme-linked immunosorbent assay, concentrations of inflammatory cytokines in HUVECs were measured.FABP4 expression was upregulated in LPS-stimulated HUVECs. The silencing of FABP4 lowered LOX-1 and p65 levels while upregulating ABCA1 and ABCG1 expression in the context of LPS. Furthermore, the inhibition of FABP4 or NF-κB signaling promoted the growth of HUVECs, upregulated Bcl-2 and PPARγ protein levels, and reduced Bax levels, angiogenetic markers, and inflammatory cytokines in the context of LPS. The combination of FABP4 inhibitor and NF-κB inhibitor treatment amplified the above-mentioned effects on cell growth, angiogenesis, and inflammation.Inhibition of FABP4 reduces LPS-induced HUVEC cell damage via the inactivation of NF-κB p65 and activation of PPARγ signaling.

Cinnamaldehyde-Mediated Suppression of MMP-13, COX-2, and IL-6 Through MAPK and NF-κB Signaling Inhibition in Chondrocytes and Synoviocytes Under Inflammatory Conditions

Inflammatory disorders encompass a range of conditions, including osteoarthritis (OA), characterized by the body’s heightened immune response to diverse stimuli. OA is a prevalent degenerative joint disease characterized by the progressive deterioration of joint cartilage and subchondral bone, leading to pain, limited mobility, and physical disability. Synovitis, the inflammation of the synovial membrane, is increasingly recognized as a critical factor in OA pathogenesis and progression. This study evaluates the therapeutic potential of cinnamaldehyde (CA), a bioactive compound derived from cinnamon, on synovial and articular inflammation in OA. Given CA’s established anti-inflammatory, antioxidant, and antibacterial properties, this research explores its specific impact on OA and synovitis. The cytotoxicity of CA was assessed using a CCK-8 assay in human IL-1β pretreated chondrocytes and synoviocytes, which serve as in vitro models of OA and synovitis. The study further examined the effects of CA on the expression of proinflammatory cytokines, including IL-6, COX-2, and TNF-α, utilizing multiple analytical techniques. Additionally, the production of matrix metalloproteinases (MMP-3 and MMP-13) and the activation of the NF-κB signaling pathway, particularly the phosphorylation of p65 (pp65), were investigated. The role of the NF-κB inhibitor 5HPP-33 and its downstream effects on gene expression, including COX-2 and IL-6, as well as the MAPK pathway components (p38, ERK, and JNK), were also explored. An MEK inhibitor (U0126) was employed to assess its downstream impact on COX-2 and IL-6 expressions. The results demonstrated that CA significantly inhibited the expression of proinflammatory cytokines and suppressed NF-κB activation in IL-1β pretreated chondrocytes and synoviocytes. These findings suggest that CA, in a dose-dependent manner, may serve as an effective therapeutic agent for preventing OA and synovitis, offering valuable insights into its potential role in managing synovial inflammation and OA.

Training Status Influences Regulation of Muscle and PBMC TLR4 Expression and Systemic Cytokine Responses to Vigorous Endurance Exercise.

A bout of vigorous endurance exercise transiently activates Toll-like receptor 4 (TLR4) and reduces TLR4 protein expressed on peripheral blood mononuclear cells (PBMCs). Endurance training, on the other hand, reduces TLR4-mediated signaling and minimizes the physiological stress imposed by exercise. Less is known about what occurs in skeletal muscle regarding TLR4 regulation and signaling. Therefore, this study aimed to investigate the regulation of TLR4 expressed in different tissue types (PBMCs and skeletal muscle samples) between endurance-trained and untrained men following vigorous endurance exercise and determine the effect of training status on cytokine responses associated with TLR4 activation. Endurance-trained (n = 7) and untrained (n = 5) men cycled for 1-hr at their respiratory compensation point, with blood and skeletal muscle samples collected pre- and 3-hours post-exercise. In response to vigorous exercise, untrained men experienced a decrease in inhibitor of κBα (IκBα) protein (suggesting IκB degradation and the activation of TLR4-associated transcription factor NF-κB) and TLR4 protein levels, along with a simultaneous increase in TLR4 mRNA expression in both skeletal muscle and PBMCs. Moreover, this exercise session led to elevated levels of circulating interleukin-6, tumor necrosis factor-α, and interleukin-1β. Collectively, these results suggest a heightened TLR4-mediated signaling pathway in untrained men. However, no changes in these targets were observed in endurance-trained men, possibly indicating a potential mechanism by which regular endurance training blunts systemic inflammation. These findings highlight the potential of endurance training to mitigate TLR4-mediated signaling, such as systemic inflammation, and shed light on the effects of exercise on TLR4 expression in PBMCs and skeletal muscle.

Triptolide induces apoptosis of glioma cells by inhibiting NF-κB activation during oxidative stress

Glioma is a common and fatal malignant primary brain tumor. Radiotherapy and first-line chemotherapy have little effect on the survival rate of patients, requiring alternative therapies. The main active ingredient of Tripterygium wilfordii Hook. F. triptolide (TP) has been shown to have anti-inflammatory and anti-proliferative properties, along with a wide range of anticancer activities. This study aimed to investigate the molecular mechanisms of triptolide in glioma treatment through network pharmacology and experimental validation. Cell viability was first assessed using Cell Counting Kit-8 (CCK8), followed by cell scratch assay and cell migration ability. Apoptosis-related markers, including TUNEL staining, Bcl-2-associated X protein (Bax), and B-cell lymphoma-2 (Bcl-2), were detected. Network pharmacology was used to predict the key targets of glioma, detect its signal pathways, screen the key components and targets for molecular docking, and explore the signaling pathways of TP. Lastly, immunofluorescence assays and ELISA were performed to elucidate the underlying mechanistic pathways. The network pharmacology data suggested that TP may inhibit glioma proliferation by regulating the signaling pathway of the nuclear factor kappa-B (NF-κB). The results showed that the underlying mechanism involved the regulation of the NF-κB signaling pathway to promote the generation of reactive oxygen species, thereby enhancing oxidative stress response and promoting cell apoptosis.

Norepinephrine stimulates M2 macrophage polarization via β2-adrenergic receptor-mediated IL-6 production in breast cancer cells

Previous studies have demonstrated that norepinephrine (NE) released during chronic stress promotes breast cancer (BC) metastasis via adrenergic receptors (ARs). However, the effect of NE on tumor-associated macrophage polarization and the underlying mechanisms remain largely unknown. In this study, we aimed to investigate the influence of NE on M2 macrophage polarization, with a particular focus on the crosstalk between macrophages and BC cells. Our results demonstrated that, although NE alone did not directly induce the expression of M2 macrophage markers, conditioned medium from NE-treated MDA-MB-231 human BC cells (NE CM) significantly promoted M2 macrophage polarization in THP-1 macrophages. We found that NE stimulated IL-6 production in MDA-MB-231 cells via β2-AR/NF-κB pathway, which activated STAT3 in THP-1 cells to induce M2 macrophage polarization. NE failed to induce IL-6 production and NF-κB activation when ADRB2 was knocked down in MDA-MB-231 cells. Furthermore, ADRB2 knockdown in cancer cells suppressed NE CM-induced M2 macrophage polarization, as well as M2 macrophage-induced cancer cell migration. Taken together, our results suggest that NE stimulates M2 macrophage polarization by inducing IL-6 secretion from BC cells through a β2-AR-dependent mechanism, which subsequently promotes cancer cell migration. Targeting β2-AR may represent a promising strategy to prevent chronic stress-induced BC metastasis.

Molecular Mechanisms of Curdlan-Induced Suppression of NFATc1 Expression in Osteoclasts.

Osteoclasts derived from hematopoietic stem cells express immunoreceptors on their cell surface. Previously, we showed that the β-glucan curdlan suppressed osteoclastogenesis via binding to dectin-1, a pattern recognition receptor. Curdlan negatively regulates osteoclast differentiation and bone resorption capacity by suppressing the expression of nuclear factor of activated T cells 1 (NFATc1), a master factor for osteoclast differentiation, in a dectin-1-dependent manner; however, the mechanism involved in this process has not yet been fully elucidated. In this study, we aimed to elucidate the molecular mechanism involved in the suppression of RANKL-induced osteoclast differentiation by curdlan. Real-time RT-qPCR results showed that curdlan suppressed the expression of NFATc1 in cells of the osteoclast progenitor cell line RAW264.7 overexpressing dectin-1 (d-RAW cells), without altering the expression of negative regulators. Therefore, we examined the effect of curdlan on the NF-κB pathway, which is important for the induction of NFATc1 expression. Western blot analysis results showed that curdlan addition suppressed RANKL-induced NF-κB activation in the vector control line (c-RAW) cells with low expression of dectin-1, in d-RAW cells, and the parental RAW264.7 (RAW) cells. The results of tartrate-resistant alkaline phosphatase staining and real-time RT-qPCR showed that curdlan addition suppressed osteoclast differentiation in RAW cells, suggesting the presence of a dectin-1-independent modification system. Finally, we focused on the complement receptor 3 (CR3), which binds β-glucan, and revealed that blocking the binding of β-glucan to the CD11b molecule, a component of CR3, by neutralizing antibody, recovered the suppression of IκBα degradation by curdlan. These results suggest that the suppression of osteoclast differentiation by curdlan involves not only the dectin-1-dependent pathway but also the negative regulation of NFATc1 via modification of the NF-κB pathway via CR3 recognition. The results of this study may aid to establish treatment methods for metabolic bone diseases and inflammatory bone destruction and to clarify their pathogenesis.

Chronic Nf-κB Inhibition Prevents Experimental Aging Nephropathy.

Introduction The pathogenesis of aging nephropathy is yet to be elucidated. Intrarenal Angiotensin-II (AngII) and activation of the NF-κB and NLRP3 inflammasome pathways exert a relevant pathogenic role in the progression of chronic kidney disease (CKD). We sought to investigate whether monotherapy with Losartan and combined treatment with Losartan and the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) would attenuate experimental aging nephropathy. Materials and Methods Forty adult Male Munich-Wistar rats were distributed among four groups: 12M (n=10), untreated 12-month-old rats; 15M (n=10), untreated 15-month-old rats; 15MLos (n=8), rats receiving Losartan (50 mg/kg/d) and 15MLos+PDTC (n=8), rats receiving Losartan and PDTC (60 mg/kg/d). All treatments were given by mouth from 12 to 15 months of age. Results Group 15M exhibited slightly decreased tail-cuff pressure, and marked increase in albuminuria, sclerotic glomeruli, cortical collagen-1 deposition and infiltration by myofibroblasts, AngII-positive cells and proinflammatory M1 macrophages, whereas the amount of anti-inflammatory M2 macrophages was reduced. In addition, the renal abundance of TLR4, nuclear p65 and IL-6 was increased, indicating activation of the NF-κB pathway, without evidence of simultaneous activation of the NLRP3 cascade. Losartan treatment decreased cortical collagen-1 deposition, myofibroblasts and AngII-positive cells, and partially restored renal M2, but had no significant effect on albuminuria, glomerulosclerosis or NF-κB activation. Combined Losartan+PDTC prevented all the observed abnormalities. Discussion/conclusion Simultaneous blockade of renal AngII and inhibition of the NF-κB pathway may represent a novel alternative to limit the decline of renal function with age.

Hydroethanolic Extract of Polygonum aviculare L. Mediates the Anti-Inflammatory Activity in RAW 264.7 Murine Macrophages Through Induction of Heme Oxygenase-1 and Inhibition of Inducible Nitric Oxide Synthase

Polygonum aviculare L. (PAL), commonly known as knotgrass, has been utilized as a traditional folk medicine across Asian, African, Latin American and Middle Eastern countries to treat various inflammatory diseases, including arthritis and airway inflammation. Numerous medicinal herbs exert anti-inflammatory and antioxidative effects that are mediated through the activation of nuclear factor-erythroid 2-related factor 2 (Nrf2) and the inhibition of nuclear factor kappa B (NF-κB). However, the underlying molecular mechanisms linking the antioxidative and anti-inflammatory effects remain poorly understood. Heme oxygenase-1 (HO-1) is an antioxidant enzyme that catalyzes heme degradation, ultimately leading to the production of carbon monoxide (CO). Elevated levels of CO have been correlated with the decreased level of inducible nitric oxide synthase (iNOS). In this study, we examined whether HO-1 plays a key role in the relationship between the antioxidative and anti-inflammatory properties of PAL. The anti-inflammatory and antioxidative activities of PAL in an in vitro system were evaluated by determining NF-κB activity, antioxidant response element (ARE) activity, pro-inflammatory cytokine and protein levels, as well as antioxidant protein levels. To examine whether HO-1 inhibition interfered with the anti-inflammatory effect of PAL, we measured nitrite, reactive oxygen species, iNOS, and HO-1 levels in RAW 264.7 murine macrophages pre-treated with Tin protoporphyrin (SnPP, an HO-1 inhibitor). Our results demonstrated that PAL increased ARE activity and the Nrf2-regulated HO-1 level, exerting antioxidative activities in RAW 264.7 macrophages. Additionally, PAL reduced cyclooxygenase-2 (COX-2) and iNOS protein levels by inactivating NF-κB in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Further investigation using the HO-1 inhibitor revealed that HO-1 inhibition promoted iNOS expression, subsequently elevating nitric oxide (NO) generation in LPS-activated RAW 264.7 macrophages treated with PAL compared to those in the macrophages without the HO-1 inhibitor. Overall, our findings suggest that HO-1 induction by PAL may exert anti-inflammatory effects through the reduction of the iNOS protein level. Hence, this study paves the way for further investigation to understand molecular mechanisms underlying the antioxidative and anti-inflammatory activities of medicinal herbs.

Fatty Acids Derived from Royal Jelly Exert Anti-Inflammatory and Antibacterial Activities in the Treatment of Pseudomonas aeruginosa-Induced Acute Pneumonia.

Pseudomonas aeruginosa, an opportunistic pathogen, commonly causes hospital-acquired pneumonia. Royal jelly fatty acids (RJFAs), a mixture of various fatty acids extracted from royal jelly, exhibit antibacterial and anti-inflammatory properties in treating many infectious diseases. Nevertheless, the therapeutic mechanisms of RJFAs in treatment of acute P. aeruginosa pulmonary infection are still unclear. Herein, we initially extracted the fatty acids from royal jelly and characterized their chemical constituents using headspace gas chromatography-mass spectrometry. Furthermore, we examined the antibacterial effect of RJFAs in vitro and explored its therapeutic effect and molecular mechanisms in treating acute P. aeruginosa pulmonary infection invivo. The in vitro antibacterial studies revealed that RJFAs significantly inhibited P. aeruginosa growth. Moreover, the invivo studies showed that the RJFAs effectively mitigated the lung damage and inflammation induced by P. aeruginosa through impairing neutrophil infiltration, reducing the bacterial load in lung and diminishing the production of proinflammatory cytokines, including tumor necrosis factor (TNF-α), interleukin (IL-1β), IL-6, and macrophage inflammatory protein-2 (MIP-2). In addition, the mice treated with RJFAs exhibited reduced phosphorylation of extracellular signal-regulated kinase (ERK), p38, c-Jun N-terminal kinase (JNK), c-Jun, and nuclear factor-kappa B (NF-κB) p65 in the lung tissues in comparison with that of the mice without drug treatment. These findings demonstrated that RJFAs exhibited significant antibacterial and anti-inflammatory effects in treating the P. aeruginosa-induced acute pneumonia, and the anti-inflammatory effects were exerted through suppressing the mitogen-activated protein kinase/activator protein-1 (MAPK/AP-1) pathway and NF-κB activation, suggesting a promising therapeutic potential of RJFAs against acute bacterial pneumonia.

GLA-loaded Liposome Albumin Particles-mediated p53 Targets Cervical Cancer Cell Transplant Tumors

Background p53 is a phosphorylated glycoprotein whose expression is abnormally elevated in various human tumors. p53 also regulates a variety of tumor cells, but research on cervical cancer has not yet been reported. Purpose This study intends to investigate the role of targeting p53 in cervical cancer. Methods Electron microscopy was used to assess the size of protein particles, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) was used to detect the OD value of nanocomposites at different concentrations using a UV spectrophotometer, MTT and cell cloning were used to detect cell proliferation expression ability, and reverse transcriptase polymerase chain reaction (RT-PCR) to detect p53 expression. Immunofluorescence measured nuclear factor kappa B (NF-κB) expression in cells, and western blotting assessed the relative expression of p53 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) proteins. The detection mechanism was further explored through in vivo studies. Results Glutaraldehyde (GLA)-loaded liposomal albumin particles showed a positive zeta value, indicating that the CS coating process of GLA was successful. Our data show that the experimental group showed a concentration-dose-dependent growth relationship, indicating that GLA-loaded liposome albumin particles have better antitumor activity in cervical cancer cell lines. Compared with most GLA-loaded liposomal albumin particle groups, P53 expression was inhibited, and the expression of P53 was downregulated in GLA-loaded liposomal albumin particle cervical cancer. In vivo results showed that the measured tumor volume decreased after treatment with GLA-loaded liposomal albumin particles, and mice treated with GLA-loaded liposomal albumin particles had longer survival times. Induction of NF-κB in tumors may be a powerful strategy allowing GLA-loaded liposomal albumin particles to avoid apoptosis and possibly after cytotoxic chemotherapy. Conclusion This study demonstrates that GLA-loaded liposomal albumin particles may participate in the development and progression of human cervical cancer cells by targeted regulation of NF-κB activity.

Anti-Inflammatory Effects of Apostichopus japonicus Extract in Porphyromonas gingivalis-Stimulated RAW 264.7 Cells

Apostichopus japonicus has been used both as a food and in traditional medicine. However, its anti-inflammatory effects in periodontal diseases have not been studied. We examined the anti-inflammatory properties of Apostichopus japonicus extract in RAW 264.7 cells stimulated by Porphyromonas gingivalis. The cytotoxicity of Apostichopus japonicus extract was evaluated using the MTS assay. Its effect on NO production was then measured using the NO assay. The mRNA expression of inducible nitric oxide synthase (iNOS) and the pro-inflammatory cytokines IL-1β and IL-6 were assessed using quantitative real-time PCR (qRT-PCR). Western blotting was performed to investigate the expression of regulatory proteins involved in the NF-κB and MAPK signaling pathways. Apostichopus japonicus extract significantly inhibited NO production without cytotoxicity in RAW 264.7 cells. Following Porphyromonas gingivalis stimulation, treatment with the extract decreased iNOS mRNA expression and protein levels, which are responsible for NO production. The extract also suppressed the mRNA expression of pro-inflammatory cytokines. Additionally, Apostichopus japonicus extract inhibited NF-κB activation by regulating signaling molecules such as IKK and IκBα, while also preventing the phosphorylation of MAPK, including ERK, p38, and JNK, showing anti-inflammatory potential. Therefore, it may be a promising natural candidate for the development of new preventive and therapeutic strategies for periodontitis.

The Annexin A1 Protein Mimetic Peptide Ac2-26 prevents cellular senescence of CHON-001 chondrocytes against tumor necrosis factor-α via the Nrf2/NF-κB pathway.

Osteoarthritis (OA) is a degenerative joint disorder characterized by progressive cartilage degradation. Excessive oxidative stress (OS), inflammatory responses, extracellular matrix breakdown, and cellular senescence of chondrocytes play crucial roles in the pathological development of OA. Currently, curing OA remains a significant challenge. In this study, we aimed to elucidate the protective effects of Annexin A1 protein Mimetic Peptide (Ac2-26) against tumor necrosis factor-α (TNF-α)-induced damage in CHON-001 chondrocytes by assessing cellular senescence, OS, and the expression levels of matrix metalloproteinase-13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4. Our results show that Ac2-26 mitigated the reduction of telomerase activity and the exacerbation of cellular senescence induced by TNF-α in CHON-001 chondrocytes. Treatment with TNF-α led to decreased expression of the human telomerase reverse transcriptase gene and increased expression of the telomeric repeat-binding factor 2 gene, which were reversed by Ac2-26 treatment. The TNF-α-induced increases in the gene and protein expressions of p53 and p16 were restored by Ac2-26 in a dose-dependent manner. Additionally, we found that TNF-α caused elevations in the mRNA and protein levels of MMP-13 and ADAMTS-4, which were reduced by Ac2-26 in a dose-dependent fashion. Furthermore, TNF-α triggered the activation of nuclear factor κ-B (NF-κB) by increasing the levels of phosphorylated NF-κB p65 and the luciferase activity of NF-κB. Notably, Ac2-26 alleviated OS by reducing mitochondrial reactive oxygen species levels and promoting the activation of NF-E2-related factor 2 (Nrf2) in TNF-α-challenged CHON-001 chondrocytes. Silencing Nrf2 abolished the Ac2-26-induced activation of NF-κB and cellular senescence in CHON-001 chondrocytes. Collectively, these findings offer new insights into the potential therapeutic use of Ac2-26 for treating OA.

Complex IIa formation and ABC transporters determine sensitivity of OSCC to Smac mimetics

Small molecule inhibitors of apoptosis proteins (IAPs) antagonists, known as Smac mimetics (SMs), activate non-canonical NF-κB and sensitize cancer cells to TNF-induced cell death. SMs are currently in phase III clinical trials for head and neck squamous cell carcinoma (HNSCC) after promising phase II trials. To explore the utility of SMs in oral squamous cell carcinoma (OSCC), we tested nine human OSCC cell lines and correlated SM sensitivity with both IAP mutation and expression levels. cIAP1 protein expression was shown to be higher in OSCC and a predictor of poor prognosis. However, our in vitro and in vivo testing demonstrated differential sensitivity to SMs, which did not correlate with cIAP1 and cIAP2 expression in these OSCC cell lines. Exogenous TNF failed to effectively increase the sensitivity of SM-resistant OSCC cells to SM-induced cell death. SM resistance was associated with a deficiency in Complex IIa formation, but activation of non-canonical NF-κB was not a determinant of SM efficacy. Finally, metabolic analysis revealed that the ABC transporter pathway was activated in SM-resistant OSSC cells, and SMs combined with ABC transporter inhibitors improved cell death sensitivity to overcome SM resistance. These studies highlight the therapeutic potential of SMs in OSCC and support patient stratification to improve efficacy with the addition of adjuvant therapy.

Neuronal PLPP/CIN exaggerates the immune response of hippocampal microglia to LPS challenge dependent on PAK1-NF-κB-COX-2 signaling pathway

Recently, we have reported that pyridoxal-5′-phosphate phosphatase/chronophin (PLPP/CIN) selectively dephosphorylates neurofibromin 2 (NF2, also known as merlin) at serine (S) 10 site. Since NF2 inhibits p21-activated kinase 1 (PAK1)-mediated nuclear factor-κB (NF-κB) activation, in the present study, we investigated the role of PLPP/CIN-mediated NF2 S10 dephosphorylation in lipopolysaccharide (LPS)-induced neuroinflammation and explored its related signaling pathways in the mouse hippocampus. PLPP/CIN overexpression increased NF2 S10 dephosphorylation and PAK1 S204 autophosphorylation under physiological condition, which were reversed by PLPP/CIN deletion. Following LPS injection, PLPP/CIN overexpression exacerbated microglial activation, although microglial PLPP/CIN expression was undetectable. In addition, PLPP/CIN overexpression enhanced PAK1 and NF-κB phosphorylations, and upregulated cyclooxygenase-2 (COX-2) and prostaglandin E synthase 2 (PTGES2) expressions in CA1 neurons. PLPP/CIN overexpression also augmented microglial interleukin-1β induction. PLPP/CIN ablation and 1,1′-dithiodi-2-naphthtol (IPA-3, a PAK1 inhibitor) pretreatment ameliorated these LPS-induced neuroinflammatory responses. These findings indicate that PLPP/CIN-mediated NF2 S10 dephosphorylation may facilitate PAK1-NF-κB-COX-2-PTGES2 signaling pathway in CA1 neurons, which would subsequently exaggerate immune response of microglia following LPS treatment. Therefore, our findings suggest that this PLPP/CIN-mediated neuron-microglia interaction may play an important role in the pathogenesis of inflammation-related neurological diseases.

Antimicrobial Peptide CATH-2 Attenuates Avian Pathogenic E. coli-Induced Inflammatory Response via NF-κB/NLRP3/MAPK Pathway and Lysosomal Dysfunction in Macrophages

Cathelicidins have anti-inflammatory activity and chicken cathelicidin-2 (CATH-2) has shown to modulate immune response, but the underlying mechanism of its anti-inflammation is still unclear. Therefore, in this study, we investigated the anti-inflammatory activity of CATH-2 on murine peritoneal macrophages during avian pathogenic E. coli (APEC) infection. The results showed that CATH-2 priming significantly reduced the production of IL-1β, IL-6, IL-1α, and IL-12. In addition, CATH-2 significantly attenuated APEC-induced caspase-1 activation and the formation of an adaptor (ASC) of NLRP3 inflammasome, indicating that CATH-2 inhibits APEC-induced NLRP3 inflammasome activation. Furthermore, CATH-2 remarkably inhibited NF-κB and MAPK signaling pathways activation. Moreover, CATH-2 significantly inhibited mRNA expression of cathepsin B and inhibited lysosomal acidification, demonstrating that CATH-2 disrupts lysosomal function. In addition, promoting lysosomal acidification using ML-SA1 hampered the anti-inflammatory effect of CATH-2 on APEC-infected cells. In conclusion, our study reveals that CATH-2 inhibits APEC-induced inflammation via the NF-κB/NLRP3/MAPK pathway through the dysfunction of lysosome.