TNF-α Production Research Articles

Baicalin promotes β-1,3-glucan exposure in Candida albicans and enhances macrophage response

Among the diverse fungal opportunistic pathogens, Candida albicans garners significant attention due to its wide range of infections and high frequency of occurrence. The emergence of resistance and the limited number of antifungals drives the need to develop novel antifungal drugs. Although the natural product baicalin has been shown to trigger apoptosis in C. albicans in previous experiments, its influence on cell wall (CW) structure along with immune recognition remains elusive. In this work, baicalin showed a significant killing effect against C. albicans SC5314. Moreover, CW destruction, characterized by β-1,3-glucan unmasking and chitin deposition, was observed as a consequence of the treatment with baicalin. The RNA sequencing analysis revealed that treatment with baicalin resulted in eight hundred forty-two differentially expressed genes (DEGs). Sixty-five genes, such as GSC1, ENG1, CHS3, GWT1, and MKC1, were associated with CW organization or biogenesis. Baicalin-pretreated C. albicans SC5314 was phagocytosed more efficiently by RAW264.7 macrophages, accompanied by increased TNF-α and IL-1β production. Accordingly, it is hypothesized that baicalin could stimulate β-1,3-glucan unmasking by governing CW-associated gene expression in C. albicans SC5314, which contributes to macrophage recognition and clearance.

Soy Protein and Safflower-Seed Oil Attenuate Inflammation and Immune Dysfunction in Rats with Hyperuricemia.

A plant-based diet is considered a promising approach for managing hyperuricemia (HUA). This study examined the effects of soy protein and plant-based oils on HUA-induced inflammation and immune dysfunction. Male Wistar rats, induced with HUA using oxonic acid and uric acid (UA), were fed casein or soy protein with palm or safflower oil (2 × 2 factorial design) for 8 weeks. HUA rats had lower serum albumin and T cell percentages in peripheral blood leukocytes (PBLs) and splenocytes, along with increased leukocyte counts and spleen weights, compared to healthy rats (p < 0.05). Soy protein improved HUA-induced reductions in albumin, while safflower-seed oil ameliorated reductions in albumin, plasma interleukin (IL)-4, and T-suppressor splenocytes, and mitigated elevated serum UA, plasma IL-6, and B leukocytes (two-way ANOVA, p < 0.05). In PBL, soy protein alleviated HUA-induced decreases in TNF-α, casein and palm oil increased IL-6, and casein further reduced IFN-γ production. Under Con A stimulation, casein and safflower-seed oil alleviated decreases in IL-6 and IL-10, respectively, while under LPS stimulation, casein further increased TNF-α production. In splenocytes, soy protein and safflower-seed oil reduced HUA-induced increases in TNF-α and increased IL-10, and safflower-seed oil increased IL-6 production. Under Con A stimulation, soy protein and safflower-seed oil reduced TNF-α and increased IL-10 production in splenocytes. The findings suggest that soy protein and safflower-seed oil may counteract HUA-related inflammation, alleviate monocyte activation, and enhance Th2 immune response in HUA. A plant-based diet rich in soy protein and safflower-seed oil may help manage HUA and associated inflammation and immune dysfunction.

Schisandrin B Inhibits LPS-Induced Endometritis Through Attenuating Ferroptosis via AMPK/PGC1α/Nrf2 Signalling Pathway.

Endometritis is one of the common reproductive diseases in human and animal. In recent years, a number of studies have found that Schisandra B (Sch B), as a natural Chinese medicine extract, has antioxidant, anti-inflammatory and other biological activities. Based on the above, in this study, mice were used to conduct an invivo experiment to investigate the effect and mechanism of Sch B on lipopolysaccharide (LPS)-induced endometritis. Haematoxylin and eosin (H&E) staining was used to detect the pathological changes of uterine tissue and western blot was used to detect the expression levels of signalling pathways and key genes for ferroptosis. The results showed that Sch B significantly inhibited the pathological injury of uterine tissue, myeloperoxidase (MPO) activity, the activation of NF-κB pathway and the production of TNF-α and IL-1β. Furthermore, Sch B effectively inhibited ferroptosis by inhibiting malondialdehyde (MDA) and iron production and promoting the expression of ferroptosis suppressor genes GPX4 and ferritin. In conclusion, Sch B inhibited LPS-induced endometritis through alleviating inflammatory response and ferroptosis via AMPK/PGC1α/Nrf2 signalling pathway.

The role of FAM171A2-GRN-NF-κB pathway in TBBPA induced oxidative stress and inflammatory response in mouse-derived hippocampal neuronal HT22 cells

Tetrabromobisphenol A (TBBPA) is one of the brominated flame retardants (BFRs) widely used in industry, which has a broad toxic effect on organisms. However, there is still limited research on the neurotoxic mechanism of TBBPA. Using mouse hippocampal neurons (HT22) cells, the toxicity of TBBPA was evaluated, especially focusing on its alteration on the key molecules in FAM171A2-GRN-NF-κB signaling pathway. The results showed that TBBPA exposure could lead to an increase in the production of inflammation-related genes IL-6, iNOS, TGF-β1, COX2, and TNF-α in both HT22 cells and HT22-AD-model, intensifying the inflammatory response; it inhibits the mRNA expression of antioxidative enzymes genes Sod1, Cat, Gpx1, and Gsta1, resulting in reduced antioxidant enzyme activities of SOD, CAT, and GSH-Px/GPX. Mechanistically, TBBPA caused the upregulation of FAM171A2 expression level, alongside increased GRN, IκBα and p65 levels; whereas the expression of GRN, IκBα and p65 was decreased after FAM171A2 knockdown, demonstrating TBBPA-induced upregulation of FAM171A2 should be responsible for the increased GRN, IκBα and p65 expression. Therefore, for the first time, our data indicate that TBBPA-induced oxidative stress and inflammatory response is closely related to the FAM171A2-GRN-NF-κB pathway.

Cornus mas (Cornelian Cherry) Exerts Neuroprotective Effects on Cerebral Ischemia/Reperfusion Injury via Anti-Inflammatory and Antioxidant Properties

Background: Stroke is one of the most important causes of mortality and disability in the world. Over 80% of strokes are ischemic, resulting from blockages in cerebral arteries. Increasing evidence suggests that enriching the diet with nutritional antioxidants could decrease brain damage and enhance cognitive function. Cornelian cherry has anti-inflammatory and antioxidant properties and is introduced as a potential source of active ingredients, like anthocyanins, vitamin C, phenolic compounds, and minerals. Objective: Our research study aimed to evaluate the neuroprotective impact of Cornus mas L. (cornelian cherry) on a rat model of Cerebral Ischemia/Reperfusion Injury (CIRI). Methods: Middle cerebral artery blockage induced CIRI for 60 minutes. After inducing CIRI, intraperitoneal injections of Cornus mas Extract (CME) were administered for 14 days in a dosedependent manner (30, 60, and 120 mg/kg body weight). The effects of CME on learning and memory recovery were evaluated using a shuttle box behavioral test. Two weeks following CIRI, several factors of oxidative stress, such as nitrite (NO2-), Ferric ion Reducing Antioxidant Power (FRAP), and Malondialdehyde (MDA), were measured in the hippocampal tissues and serum. Anti-inflammatory genes, such as miR-125b, and the target genes (TNF-α and iNOS) were evaluated via real-time PCR assay. Additionally, neural damage in the CA1 and CA3 regions of the hippocampus was assessed using Hematoxylin and Eosin (H&amp;E) staining. Results: The avoidance time in the shuttle-box behavior test supported our finding that CME exhibited improved fear memory. Furthermore, it increased the CA1 and CA3 hippocampal post-stroke pyramidal cell layers. Levels of NO2- and MDA were decreased, and FRAP was considerably increased in both the hippocampus and serum by CME. Additionally, CME increased miR-125b expression, while modulating TNF-α and iNOS production in the hippocampal regions. Conclusion: Based on our findings, we can conclude CME to possess antioxidant and antiinflammatory properties, which confer neuroprotective potential against CIRI, thereby protecting neurons from ischemic death.

Preclinical evaluation of immunogenicity and protective efficacy of a recombinant chimeric protein vaccine against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a tropical disease that can be fatal if acute and untreated. Diagnosis is difficult, the treatment is toxic and prophylactic vaccines do not exist. Leishmania parasites express hundreds of proteins and several of them are relevant for the host's immune system. In this context, in the present study, 10 specific T-cell epitopes from 5 parasite proteins, which were identified by antibodies in VL patients’ sera, were selected and used to construct a gene codifying the new chimeric protein called rCHI. The rCHI vaccine was developed and thoroughly evaluated for its potential effectiveness against Leishmania infantum infection. We used monophosphoryl lipid A (MPLA) and polymeric micelles (Mic) as adjuvant and/or delivery system. The results demonstrated that both rCHI/MPLA and rCHI/Mic significantly stimulate an antileishmanial Th1-type cellular response, with higher production of IFN-γ, TNF-α, IL-12 and nitrite in vaccinated animals, and this response was sustained after challenge. In addition, these mice significantly reduced the parasitism in internal organs and increased the production of IgG2a isotype antibodies. In vivo and in vitro toxicity showed that rCHI is safe for the mammalians, and the recombinant protein also induced in vitro lymphoproliferative response and production of Th1-type cytokines by human cells, which were collected from healthy subjects and treated VL patients. These data suggest rCHI plus MPLA or micelles could be considered as a vaccine candidate against VL.

SMAC mimetics induce human macrophages to phagocytose live cancer cells.

Macrophages engulf apoptotic bodies and cellular debris as part of homeostasis, but they can also phagocytose live cells such as aged red blood cells. Pharmacologic reprogramming with the SMAC mimetic LCL161 in combination with T cell-derived cytokines can induce macrophages to phagocytose live cancer cells in mouse models. Here we extend these findings to encompass a wide range of monovalent and bivalent SMAC mimetic compounds, demonstrating that live cell phagocytosis is a class effect of these agents. We demonstrate robust phagocytosis of live pancreatic and breast cancer cells by primary human macrophages across a range of healthy donors. Unlike mouse macrophages where combination of SMAC mimetics with lymphotoxin enhanced phagocytosis, human macrophages were more efficiently polarized to phagocytose live cells by the combination of SMAC mimetics and IFNψ. We profiled phagocytic macrophages by transcriptional and proteomic methodologies, uncovering a positive feedback loop of autocrine TNFα production.

LDHB Mediates Histone Lactylation to Activate PD-L1 and Promote Ovarian Cancer Immune Escape

Background To investigate the effects of LDHB on lactylation of programmed cell death 1 ligand (PD-L1) and immune evasion of ovarian cancer. Methods Ovarian cancer cells were transfected with LDHB siRNA and cultured with primed T cells. Cell proliferation and viability were measured by cell counting kit 8 (CCK-8) and colony formation assay. The production of immune factors was detected by enzyme-linked immunosorbent assay (ELISA). The histone lactylation and activity of PD-L1 promoter were measured by chromatin immunoprecipitation (ChIP)-qPCR assay and luciferase reporter gene assay, respectively. Results Knockdown of LDHB notably inhibited the growth, glucose uptake, lactate production, and ATP production of ovarian cancer cells. Knockdown of LDHB enhanced the killing effects of T cells, led to increased production of immune activation factors IL-2, TNF-α, and IFN-γ, as well as elevated the levels of granzyme B and perforin. Mechanical study identified that LDHB regulated the H3K18 lactylation (H3K18la) modification on PD-L1 promoter region to promote its expression. Overexpression of PD-L1 abolished the immune activation effects that induced by siLDHB. Conclusion The LDHB modulated lactate production and the histone lactylation on PD-L1 promoter, which ultimately regulated its expression and participated in the immune evasion of ovarian cancer cells.

Dual anti-inflammatory activities of COX-2/5-LOX driven by kratom alkaloid extracts in lipopolysaccharide-induced RAW 264.7 cells

Cyclooxygenase (COX) and lipoxygenase (LOX) enzymes play a pivotal role in producing pro-inflammatory eicosanoids, including prostaglandins (PGs) and leukotrienes (LTs), in the inflammation process. Mitragynine is a primary alkaloid contained in the kratom’s leaves and has been reported to show anti-inflammatory activity by suppressing COX-2 mRNA translation to lowering PGs synthesis. In this study, the Kratom’s alkaloid extract containing ~ 46% mitragynine was found to exhibit dual inhibition activity towards COX-2/5-LOX enzymes at concentrations below 25 ppm in the LPS-induced RAW 264.7 macrophage cells. At these levels, no cell toxicity was observed while the cells became death (e.g., 10–46% viability at 50–100 ppm) and only COX-2 inhibition activity was observed after exposed with more than 25 ppm of alkaloid extract. In contrast, the methanolic-crude extract of Kratom’s leaf containing ~ 5% mitragynine showed no inhibition toward COX-2/5-LOX enzymes and did not toxic onto the cells, even after treated at 100 ppm. The alkaloid extract suppressed several antiinflammation parameters, including ROS (64% reduction at 25 ppm), NO (30% reduction at 25 ppm), TNF-α (~ 50% reduction at 25 ppm), and IL-6 production (60% reduction at 6.25 ppm). In silico molecular studies indicated strong binding affinity of Kratom alkaloids to COX-2 and 5-LOX active sites, supporting the Kratom’s alkaloids to have great potential dual inhibition activity towards COX-2/5-LOX enzymes and to be developed as a safer NSAIDs with fewer side effects.

Methyl-esterification, degree of polymerization and ∆4,5-unsaturation of galacturonic acid oligosaccharides as determinants of immunomodulation

In recent years, immunomodulation by pectin and pectin-derived galacturonic acid oligosaccharides has been the subject of wide-spread scientific research due to the potential of different pectin structures as bioactive biomolecules. Yet, gaps remain in understanding the structure-dependent immunomodulation of galacturonic acid. This study describes in vitro immunomodulatory effects of well-characterized galacturonic acid oligosaccharides. Both methyl-esterified and non-methyl-esterified galacturonic acid oligosaccharides with a saturated non-reducing end (degree of polymerization 1–10) significantly induced cytokine production by THP-1 macrophages and directly activated TLR2 and TLR4 in transfected HEK-293 cells, even when accounting for minor endotoxin contamination. In contrast, both methyl-esterified and non-methyl-esterified galacturonic acid oligosaccharides with a Δ4,5-unsaturated non-reducing end (degree of polymerization 1–7) did not activate TLR2 and TLR4 and led to significantly reduced cytokine production (p < 0.05), suggesting Δ4,5-(un)saturation as a pivotal factor for immunomodulation by galacturonic acid oligosaccharides. Exposure to non-methyl-esterified saturated galacturonic acid oligosaccharides resulted in significantly lower TNF-α production, IL-1β production and TLR4 activation (p < 0.05) compared to methyl-esterified saturated galacturonic acid oligosaccharides, while IL-10 production and TLR2 activation remained unchanged. These findings establish galacturonic acid oligosaccharides as versatile immunomodulators with TLR2 and TLR4 binding capacity, fit for different immunomodulatory applications depending on their structural characteristics.

Inhibition of pro-inflammatory cytokines by homalolide A and homalomenol A isolated from rhizomes of Homalomena pendula.

Inflammation, a natural process of the innate immune system, involves elevated levels of various proinflammatory mediators, such as, nitric oxide (NO) and prostaglandin (PGE2), cytokines such as interleukin 6 (IL-6), interleukin 10 (IL-10) and tumor necrosis factor alpha (TNF-α), and enzymes including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). This study investigated the anti-inflammatory effects of homalolide A (1) and homalomenol A (2), two sesquiterpenoids isolated from the rhizome of Homalomena pendula, on lipopolysaccharide (LPS)- stimulated macrophage cells. The results demonstrated that both 1 and 2 dose-dependently inhibited the production of PGE2, TNF-α and IL-6 in RAW 264.7 macrophages. Furthermore, 2 also stimulated IL-10 production in RAW 264.7 cells. Consistent with these findings, these compounds suppressed the LPS-stimulated protein levels of iNOS and COX-2 in RAW 264.7 cells. These results suggested that 1 and 2 could be effective candidates for ameliorating inflammatory-associated complications.

Potential role of the antimicrobial peptide Tachyplesin III in regulating nontypeable Haemophilus influenzae-induced inflammation in airway epithelial cells.

The respiratory bacterium nontypeable (non-encapsulated) Haemophilus influenzae (NTHi) is a key pathogen driving exacerbations in chronic obstructive pulmonary disease (COPD), and is associated with an excessive airway inflammation. Increasing issues with tolerance and unwanted side effects of existing pharmaceuticals present an urgent need for new, effective and minimally toxic therapeutic options. This study aimed to investigate the potential role of Tachyplesin III, an antimicrobial peptide derived from the hemolysates of Southeast Asian horseshoe crabs, in regulating NTHi-induced airway inflammation. The results revealed that Tachyplesin III effectively inhibited the production of IL-1β in NTHi-stimulated human lung epithelial cells (A549), without causing cytotoxic effects. Additionally, Tachyplesin III significantly reduced TNF-α, PGE2 and NO production in NTHi-stimulated A549 cells. Moreover, this peptide inhibited the nuclear translocation of the NF-κB p65 subunit in NTHi-stimulated lung epithelial cells. It also reduced transcriptional activation of NF-κB target genes, as shown by lower mRNA levels of IL-1β, TNF-α, COX-2 and iNOS, which correlated with corresponding decreases in their protein expression. Tachyplesin III peptide also inhibited pro-IL-1β and NLRP3 protein expression and prevented NTHi-induced caspase-1 cleavage and IL-1β maturation. Together, our findings demonstrate that Tachyplesin III effectively reduced NTHi-mediated inflammation via the NF-κB/NLRP3 inflammasome signaling pathway, highlighting its important anti-inflammatory activity. Complementing these findings, in silico analysis revealed key pharmacokinetic and toxicological attributes, establishing a foundational understanding of Tachyplesin III as a promising therapeutic agent for managing NTHi-associated inflammation.

Fetal microchimeric cells influence maternal lung health following term and preterm births

Fetal microchimerism, the presence of fetal cells in maternal tissues, has garnered interest for its potential role in maternal physiology. In this study, we aimed to explore the impact of fetal microchimeric cells on maternal lung health following term and preterm delivery, particularly in the context of infection-induced preterm birth and subsequent allergic challenges. We characterized the immune cells in maternal lungs using a transgenic mouse model (mT+ Ve, Td Tomato) and high dimensional mass cytometry (CyTOF) techniques. We evaluated their influence on lung function and inflammation. Our findings revealed distinct differences in the immune cell composition of maternal lungs between term and preterm deliveries. Mice delivered preterm significantly increased in fetal-specific cells, such as activated macrophages and Tbet + Ve memory B-cells, compared to term-delivered mice. Conversely, term deliveries showed elevated levels of CD4 cells. Furthermore, preterm-delivered dams demonstrated heightened airway hyperresponsiveness, pro-inflammatory cytokine expression, cellular infiltration, and lung mucous production compared to term-delivered dams. Co-culture experiments demonstrated that microchimeric cells from preterm births stimulated the production of inflammatory cytokines IL-6 and TNF-α in lung epithelial cells. These findings shed light on the complex immune dynamics postpartum and their role in lung complications after preterm birth. Understanding these mechanisms could provide insights for targeted interventions to improve maternal lung health in at-risk populations.

Short term high‐fat diet induced liver ILC1 differentiation associated with the TLR9 activation

The health impact of dietary fat is a significant nutritional concern. However, the effects of high-fat diet on immune system particularly the liver regional immune function remains still unclear. Liver ILC1 has been recently identified as playing crucial roles in anti-viral defense, liver regeneration, and protection against acute liver injury. Here, in a mouse model, we uncovered that short term high-fat diet for 2 weeks obviously increased the frequency and number of ILC1 in liver. The production of TNF-α and expressions of TRAIL, CXCR3 and CXCR6 were also increased. Furthermore, EASY-RNAseq and ATAC-seq of liver ILC1 clarified the transcriptome characteristics and chromatin accessibility in response to short term high-fat diet, which were involved with lymphocyte differentiation. Mechanistically, we demonstrated that accumulation of liver ILC1 induced by short term high-fat diet was dependent on a TLR9-mediated differentiation through TLR9 inhibitor. Taken together, these findings shed light on the effect and underlying mechanism of short term high-fat diet on liver ILC1 differentiation and provide nutritional strategies and theoretical basis for the liver regional immune function regulation.

Intraperitoneal Administration of S100A8 Ameliorates Experimental Acute Colitis in Rats.

S100A8 is a protein that is abundant in neutrophils and macrophages (MΦ), but its role in inflammation remains unclear. This study aimed to assess the immunological role(s) of S100A8 in acute intestinal inflammation in rats and its role in MΦ. Rat recombinant S100A8 (rr-S100A8, 1.0 mg/kg) was intraperitoneally administered daily to rats with 3% dextran sulfate sodium (DSS) (DSS + A8 group)-induced experimental acute colitis. The histological severity score (6.50 ± 0.51, p = 0.038) in the DSS + A8 group rats remained lower than that (9.75 ± 1.48) of the rats without S100A8 (DSS group) administration. The tumor necrosis factor-alpha (TNF-α) production in the colon tissues of the rats in the DSS + A8 group (4.76 ± 0.90 pg/mL/g, p = 0.042) was significantly suppressed, compared with that of the DSS group (10.45 ± 2.04 pg/mL/g). To stimulate rat peritoneal MΦ, rr-S100A8, the anti-rat S100A8 antibody, and a lipopolysaccharide (LPS) were used in the in vitro experiments. In the MΦ stimulated with rr-S100A8 for 2 h, the mRNA level of intracellular S100A8 (47.41 ± 24.44, p = 0.002) increased in an autocrine manner, whereas that of S100A9 (0.24 ± 0.43, p = 0.782) was not significant. The TNF-α mRNA level in the MΦ treated with LPS and the anti-rat S100A8 antibody significantly increased (102.26 ± 18.60, p = 0.001) compared to that with LPS alone (16.9 ± 8.56). These results indicate that S100A8 can serve as an anti-inflammatory protein in acute inflammation by negatively regulating S100A9 and TNF-α production through inflammatory signaling pathways in MΦ.

Loss of O6-methylguanine DNA methyltransferase (MGMT) in macrophages alters responses to TLR3 stimulation and enhances DNA double-strand breaks and mitophagy

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA damage repair enzyme. The roles of this enzyme in immune cells remain unclear. In this study, we explored the roles of MGMT in bone marrow-derived murine macrophages (BMMs) via the use of MGMT knockout (KO) mice. Loss of MGMT altered the response to TLR3 agonists (poly (I:C)), such as dampening the production of TNFα and IL-6. Increased DNA double-strand breaks (DSBs) were observed in MGMT-KO macrophages but did not result in increased cell death. MGMT localized to both nuclei and mitochondria at increasing levels during poly (I:C) stimulation. MGMT deficiency increased the production of mitochondrial reactive oxygen species (mtROS), which was correlated with increased mitophagy. The underlying mechanism involves mediation through activation of the AMPKα pathway. Taken together, our findings reveal the roles of MGMT in macrophages in regulating the response to TLR3, which links DSBs to mtROS and mitophagy via the AMPKα pathway. These roles may have consequences for the inflammatory response and chronic inflammation.

Anti-aging potential of Cephalotaxus harringtonia extracts: the role of harringtonine and homoharringtonine in skin protection

Photoaging damages the skin layers. The tumor necrosis factor-alpha (TNF-α) plays a crucial role in the central mechanism of photoaging. TNF-α production leads to direct damage to skin cells and facilitates the degradation of vital extracellular matrix (ECM) proteins. TNF-α stimulates matrix metalloproteinase-1 (MMP-1) activation This accelerates the loss of skin elasticity and wrinkle formation. Thus, preventing photoaging and delaying the skin aging process are important research objectives, and the development of new anti-aging substances that target the TNF-α and MMP-1 pathways is promising. In this context, the efficacies of four extracts derived from two types of Cephalotaxus harringtonia (CH) buds (CH-10Y-buds, CH-200Y-buds) and leaves (CH-10Y-leaves, CH-200Y-leaves) were investigated, exhibiting a significant reduction in reactive oxygen species (ROS). Among the four extracts, CH-10Y-buds was the most effective in reducing ROS and exhibited the highest amounts of harringtonine and homoharringtonine. The activities of harringtonine, homoharringtonine, and ginkgetin were evaluated; harringtonine exhibited a high efficacy in inhibiting TNF-α-induced inflammatory responses and MMP-1 activation, thereby reducing collagen degradation. These findings suggest that CH-10Y-buds and their components herringtonin are promising candidates for preventing damage caused by photoaging. Our results can facilitate the development of new methods for maintaining skin health and inhibiting the skin aging process. Further research is necessary to comprehensively evaluate the potential efficacy of these candidate substances and investigate their applicability to actual skin. Such studies will aid in the development of more effective anti-aging strategies in the future.

Anti-Inflammatory Effects and Metabolomic Analysis of Ilex Rotunda Extracted by Supercritical Fluid Extraction.

Ilex rotunda is a famous medicinal plant with many ethnopharmacological uses. It is traditionally employed for treating inflammation and cardiovascular diseases. In this study, we established green technology to extract the leaves and twigs of I. rotunda. The obtained extracts and their fractions were evaluated for their anti-inflammatory potential. In cytokine assays, the extract, n-hexane (H), methylene chloride (MC), and EtOAc (E) fractions of the twigs of I. rotunda significantly inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α production in RAW264.7 macrophages. Furthermore, the extract, H, and MC fractions of the leaves of I. rotunda modulated cytokine expression by downregulating LPS-induced NO, IL-6, and TNF-α production in RAW264.7 macrophages. Western blotting analysis revealed that the extracts and fractions of the leaves and twigs of I. rotunda inhibited inflammatory cytokines by inactivating nuclear factor kappa B (NFκB) action by reducing the phosphorylation of transcript factor (p65) and nuclear factor-kappa B inhibitor alpha (IκBα) degradation, or by inactivating mitogen-activated protein kinase (MAPK) through the p38 or ERK signaling pathways via the active ingredients of the leaves and twigs of I. rotunda. Ultra-high-resolution liquid chromatography-Orbitrap mass analysis (UHPLC-ESI-Orbitrap-MS/MS)-based molecular networking, in cooperation with social open platform-guided isolation and dereplication, led to the identification of metabolites in this plant. Our findings indicate that the leaves and twigs of I. rotunda could be promising candidates for developing therapeutic strategies to treat anti-inflammatory diseases.

Exposure of RAW264.7 macrophages to exhaust emissions (gases and PAH) and non-exhaust emissions (tire particles) induces additive or synergistic TNF-α production depending on the tire particle size

Road traffic is a major contributor to air pollution and consequently negatively affects human health. Car pollution originates both from exhaust emissions (EE) and non-exhaust emissions (NEE, such as tire and brake wear particles, erosion of road surfaces and resuspension of road dust). While the toxicity of EE and NEE has been characterized separately, their combined effects are poorly documented. However, we are constantly exposed to a mixture of pollutants and their interactions should not be neglected as they may significantly impact their toxicological profile resulting in additive, synergistic or antagonistic effects. To fill this gap, we investigated in vitro the combined toxicity of exhaust gases and benzo[a]pyrene (representative of EE) and tire particles (representative of NEE). Macrophages from the RAW264.7 cell line were exposed for 24 h to tire particles (TP) of variable size (6–113 µm), alone or in combination with exhaust gases (CO2, CO, NO, NO2) and benzo[a]pyrene (B[a]P) as an archetype of polycyclic aromatic hydrocarbon (PAH). The cell response was assessed in terms of cytotoxicity, proinflammatory response and oxidative stress. TP, gases and B[a]P, alone or in combination triggered neither cytotoxicity nor oxidative stress. On the contrary, a proinflammatory response was elicited with two different profiles depending on the size of the TP: TNF-α production was either slightly (with the finest TP) or strongly (with coarse TP) increased in the presence of gases and B[a]P, suggesting that the effects of TP, gases and B[a]P were either additive or synergistic, depending on TP size.

IP-10 acts early in CV-A16 infection to induce BBB destruction and promote virus entry into the CNS by increasing TNF-α expression.

The mechanisms underlying pathological changes in the central nervous system (CNS) following Coxsackievirus A16 (CV-A16) infection have not yet been elucidated. IFN-γ-inducible protein-10 (IP-10) is often used as a predictive factor to monitor early virus infection. It has also been reported that IP-10 plays a pivotal role in neuroinflammation. In this study, we aimed to explore the role of IP-10 in the neuropathogenesis of CV-A16 infection. We observed that the level of IP-10, as well as the TLR3-TRIF-TRAF3-TBK1-NF-κB and RIG-I/MDA5-MAVS-TRAFS-TBK1-NF-κB pathways, which are the upstream of IP-10, were significantly elevated during the course of CV-A16 infection. This increase was accompanied by an increase in a series of inflammatory cytokines at different time-points during CV-A16 infection. To determine whether IP-10 influences BBB integrity, we examined junctional complexes. Our results revealed that the expression levels of Claudin5, Occludin, ZO-1 and VE-Cadherin were notably decreased in CV-A16-infected HUVECs, but these indicators were restored in CV-A16-infected HUVECs with Eldelumab treatment. Nevertheless, IP-10 is only a chemokine that primarily traffics CXCR3-positive immune cells to inflammatory sites or promotes the production of inflammatory cytokines. Therefore, the interactions between IP-10 and inflammatory cytokines were evaluated. Our data revealed that IP-10 mediated the production of TNF-α, which was also observed to change the junctional complexes. Moreover, in a suckling mouse model, IP-10 and TNF-α treatments exacerbated clinical symptoms, mortality and pathological changes in the brain of CV-A16-infected mice, but Anti-IP-10 and Anti-TNF-α treatments alleviated these changes. Our data also revealed that IP-10 may be detected early in CV-A16 infection, whereas TNF-α was detected late in CV-A16 infection, and the production of TNF-α was also found to be positively correlated with IP-10. In addition, IP-10 and TNF-α were observed to reduce junctional complexes and enhance virus entry into the CNS. Taken together, this study provides the first evidence that CV-A16 activates the IP-10/TNF-α regulatory axis to cause BBB damage and accelerate the formation of neuroinflammation in infected hosts, which not only provides a new understanding of the neuropathogenesis caused by CV-A16, but also offers a promising target for the development of CV-A16 antiviral drugs.