IL-6 mRNA Levels Research Articles

Penazaphilones J-L, Three New Hydrophilic Azaphilone Pigments from Penicillium sclerotiorum cib-411 and Their Anti-Inflammatory Activity.

Penazaphilones J-L (1-3), three new hydrophilic azaphilone pigments, as well as six known compounds, were discovered from the filamentous fungus Penicillium sclerotiorum cib-411. Compounds 1-3 were structurally elucidated by the detailed interpretation of their 1D and 2D NMR spectroscopic data. Compound 1 is an unprecedented hybrid of an azaphilone and a glycerophosphate choline. Compounds 2 and 3 each contain an intact amino acid moiety. The bioassay showed that compound 3 exhibited significant anti-inflammatory activity. Concretely, compound 3 significantly suppressed the NO production, the expression levels of COX-2, IL-6, IL-1β, and iNOS mRNA in LPS-stimulated RAW264.7 cells. Moreover, treatment of compound 3 prevented the translocation of NF-κB through inhibiting the phosphorylation of PI3K, PDK1, Akt, and GSK-3β. Thus, the inhibition of compound 3 against LPS-induced inflammation should rely on its inactivation on NF-κB.

Dietary supplementation with a complex of cinnamaldehyde, carvacrol, and thymol negatively affects the intestinal function in LPS-challenged piglets.

The effects of cinnamaldehyde, carvacrol and thymol complex (CCT) on the growth performance and intestinal function of piglets challenged with lipopolysaccharide (LPS) were determined. Colistin sulphate (CS) was as a positive control. Piglets (n = 24, 32 days of age) were allocated to four treatments: Control group (fed basal diet), LPS group (fed basal diet), CS+LPS group (fed basal diet + 50 mg/kg CS), and CCT+LPS group (fed basal diet + 50 mg/kg CCT). Results showed that diarrhea rates of piglets were significantly reduced by CCT and CS supplementation respectively. Further research showed that CS supplementation tended to improve the intestinal absorption function in LPS-challenged piglets. Moreover, CS supplementation significantly reduced the contents of cortisol in blood and malondialdehyde in the duodenum and the activities of inducible nitric oxide synthase in the duodenum and ileum and total nitric oxide synthase in the ileum in LPS-challenged piglets. CS supplementation significantly increased the activities of sucrase in the ileum and myeloperoxidase in the jejunum in LPS-challenged piglets. CS supplementation significantly alleviated the reduced mRNA levels of immune-related genes (IL-4, IL-6, IL-8, IL-10) in mesenteric lymph nodes and jejunum and mucosal growth-related genes (IGF-1, mTOR, ALP) in LPS-challenged piglets. These results suggested that CS supplementation improved the intestinal function in LPS-challenged piglets by improving intestinal oxidative stress, immune stress, and absorption and repair function. However, although CCT supplementation improved oxidative stress by reducing (p < 0.05) the content of malondialdehyde and the activity of nitric oxide synthase in the duodenum, CCT supplementation tended to aggravate the intestinal absorption dysfunction in LPS-challenged piglets. Furthermore, compared with the control and LPS groups, CCT supplementation remarkably elevated the content of prostaglandin in plasma and the mRNA levels of pro-inflammatory factor IL-6 in mesenteric lymph nodes and jejunum, and reduced the activity of maltase in the ileum in LPS-challenged piglets. These results suggested that CCT supplementation had a negative effect on intestinal function by altering intestinal immune stress response and reducing disaccharidase activity in LPS-challenged piglets. Compared to CS, CCT supplementation exhibited a negative effect on intestinal function, suggesting whether CCT can be as an effective feed additive still needs further study.

Cleistocalyx nervosum var. paniala Berry Seed Protects against TNF-α-Stimulated Neuroinflammation by Inducing HO-1 and Suppressing NF-κB Mechanism in BV-2 Microglial Cells

Sustained inflammatory responses have been implicated in various neurodegenerative diseases (NDDs). Cleistocalyx nervosum var. paniala (CN), an indigenous berry, has been reported to exhibit several health-beneficial properties. However, investigation of CN seeds is still limited. The objective of this study was to evaluate the protective effects of ethanolic seed extract (CNSE) and mechanisms in BV-2 mouse microglial cells using an inflammatory stimulus, TNF-α. Using LC-MS, ferulic acid, aurentiacin, brassitin, ellagic acid, and alpinetin were found in CNSE. Firstly, we examined molecular docking to elucidate its bioactive components on inflammation-related mechanisms. The results revealed that alpinetin, aurentiacin, and ellagic acid inhibited the NF-κB activation and iNOS function, while alpinetin and aurentiacin only suppressed the COX-2 function. Our cell-based investigation exhibited that cells pretreated with CNSE (5, 10, and 25 μg/mL) reduced the number of spindle cells, which was highly observed in TNF-α treatment (10 ng/mL). CNSE also obstructed TNF-α, IL-1β, and IL-6 mRNA levels and repressed the TNF-α and IL-6 releases in a culture medium of BV-2 cells. Remarkably, CNSE decreased the phosphorylated forms of ERK, p38MAPK, p65, and IκB-α related to the inhibition of NF-κB binding activity. CNSE obviously induced HO-1 protein expression. Our findings suggest that CNSE offers good potential for preventing inflammatory-related NDDs.

Comparative efficacy of a novel Bacillus subtilis-based probiotic and pharmacological zinc oxide on growth performance and gut responses in nursery pigs

In this study, we assessed the efficacy of a novel Bacillus subtilis probiotic in improving growth performance and gut responses in comparison to pharmacological zinc oxide (ZnO) in nursery pigs. A total of 96 piglets were randomly assigned to four groups: Negative control (NC), Positive control (PC, 3000 mg Zn /kg feed), B.subtilis low dose (BS9-L, 2 × 107 CFU/pig) and B.subtilis high dose (BS9-H, 2 × 109 CFU/pig). Growth performance, diarrhea rate, gut mucosal gene expression and fecal microbial populations were evaluated. B.subtilis administration did not improve piglet bodyweight. BS9-L showed (P < 0.05) higher average daily gain (ADG) in Period 2 (D14-D28). BS9 groups had (P < 0.001) lower feed conversion ratio (FCR) in Period 2 (D14-D28) and overall. Like the ZnO-group, BS9 groups had lower (P < 0.01) diarrhea rate. A significant reduction (P < 0.05) in fecal E. coli, total coliforms, and an increase in lactic acid bacteria and Bacillus spp. in BS9 groups was observed. BS9 group had reduced (P < 0.05) mRNA levels of intestinal IL-8 and higher levels of MUC-1 and occludin and TJP-1 compared to negative control. These findings suggest that probiotic BS9, may promote growth performance, and ameliorate various indicators of intestinal health in piglets. Hence, it may serve as a prospective alternative to ZnO growth promoter in commercial swine production.

Maslinic acid attenuates UVB-induced oxidative damage in HFF-1 cells.

Oxidative damage is one of the major mechanisms of ultraviolet B (UVB)-induced damage to the skin. Maslinic acid (MA) is a natural compound of pentacyclic triterpene acids. It has been proved to have anti-inflammatory and antioxidant properties. This study aimed to explore the effects of MA on oxidative damage in human foreskin fibroblast cells (HFF-1) and the potential molecular mechanisms. A specific dose of UVB radiation was used to induce oxidative damage in HFF-1. Based on this, we performed measurements of cell proliferation, reactive oxygen species (ROS) levels, antioxidant enzyme activity, inflammation-related mediators, and NF-κB nuclear localization with or without the addition of MA. MA significantly promoted cell proliferation viability at 10 and 20 μM. The addition of MA 24 h before UVB irradiation was more effective at enhancing cell proliferation and also produced lower ROS levels compared to co-cultured fibroblasts and MA for 24 h after irradiation. However, there was no statistically significant difference between groups at concentrations of 10 and 20 μM. The pretreatment group with MA had elevated superoxide dismutase and catalase activities, decreased IL-6 generation, and lowered mRNA levels of IL-6, TNF-α and MMP3 in comparison with the UVB-irradiated group without additional MA. Meanwhile, the nuclear translocation of NF-κB and the degradation of IκB were inhibited by MA pretreatment. Taken together, these findings suggest that MA may alleviate UVB-induced oxidative damage in HFF-1 by inhibiting the nuclear translocation of NF-κB.

Effects of fatty acid-ethanol amine (FA-EA) derivatives on lipid accumulation and inflammation.

This study aimed to investigate the effect of fatty acid-ethanol amine (FA-EA) derivatives (L1-L10) on the mitigation of intracellular lipid accumulation and downregulation of pro-inflammatory cytokines in vitro. First, the series of FA-EA derivatives were synthesized and characterized. Then, their cytotoxic, intracellular lipid accumulation and inhibition of pro-inflammatory cytokines were evaluated. The oil red O staining experiment showed that the tested compounds L4, L6, L8, L9, and L10 could reduce intracellular lipid accumulation induced by palmitic acid (PA). Moreover, ω-3/ω-6 PUFA-EA derivatives showed inhibitory effect on the production of pro-inflammatory cytokines in lipopolysaccharide (LPS) -stimulated RAW 264.7 cells. ω-3/ω-6 PUFA-EA derivatives at a concentrations of 10μM could significantly decrease mRNA levels of IL-6, IL-1β, and TNF-α, inhibit NO production, and alleviate the protein expression of IL-1β in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. These data suggest that ω-3 PUFA-EA derivatives can be beneficial for further pharmaceutical development to treat chronic low-grade inflammation diseases such as obesity.

Hydroxytyrosol attenuates ethanol-induced liver injury by ameliorating steatosis, oxidative stress and hepatic inflammation by interfering STAT3/iNOS pathway

ABSTRACTObjective: Hydroxytyrosol (HT) is a polyphenol with a wide range of biological activities. Excessive drinking can lead to oxidative stress and inflammation in the liver, which usually develop into alcohol liver disease (ALD). At present, there is no specific drug to treat ALD. In this paper, the protection effect of HT on ALD and the underline mechanism were studied.Methods: HepG2 cells were exposed to ethanol in vitro and C57BL/6J mice were fed with a Lieber-DeCarli ethanol liquid diet in vivo.Results: triglyceride (TG) level in serum and the expression of fatty acid synthase (FASN) were reduced significantly by the treatment with HT The acetaldehyde dehydrogenase (ALDH) activity was increased, the serum level of malondialdehyde (MDA) was decreased, catalase (CAT) and glutathione (GSH) were increased, suggesting that HT may reduce its oxidative damage to the body by promoting alcohol metabolism. Furthermore, according to the mRNA levels of tnf-α, il-6 and il-1β, HT inhibited ethanol-induced inflammation significantly. The anti-inflammatory mechanism of HT may be related to suppress the STAT3/iNOS pathway.Dissussion: Our study showed that HT could ameliorate ethanol-induced hepatic steatosis, oxidative stress and inflammation and provide a new candidate for the prevention and treatment of ALD.

Activation of epithelial and inflammatory pathways in adolescent elite athletes exposed to intense exercise and air pollution

RationaleParticipation in high-intensity exercise in early life might act as stressor to the airway barrier.ObjectivesTo investigate the effect of intense exercise and associated exposure to air pollution on the airway...

Pathogenic NKT cells attenuate urogenital chlamydial clearance and enhance infertility.

Urogenital chlamydial infections continue to increase with over 127 million people affected annually, causing significant economic and public health pressures. While the role of traditional MHCI and II peptide presentation is well defined in chlamydial infections, the role of lipid antigens in immunity remains unclear. Natural killer (NK) T cells are important effector cells that recognize and respond to lipid antigens during infections. Chlamydial infection of antigen-presenting cells facilitates presentation of lipid on the MHCI-like protein, CD1d, which stimulates NKT cells to respond. During urogenital chlamydial infection, wild-type (WT) female mice had significantly greater chlamydial burden than CD1d-/- (NKT-deficient) mice, and had significantly greater incidence and severity of immunopathology in both primary and secondary infections. WT mice had similar vaginal lymphocytic infiltrate, but 59% more oviduct occlusion compared to CD1d-/- mice. Transcriptional array analysis of oviducts day 6 post-infection revealed WT mice had elevated levels of Ifnγ (6-fold), Tnfα (38-fold), Il6 (2.5-fold), Il1β (3-fold) and Il17a (6-fold) mRNA compared to CD1d-/- mice. In infected females, oviduct tissues had an elevated infiltration of CD4+ -invariant NKT (iNKT) cells, however, iNKT-deficient Jα18-/- mice had no significant differences in hydrosalpinx severity or incidence compared to WT controls. Lipid mass spectrometry of surface-cleaved CD1d in infected macrophages revealed an enhancement of presented lipids and cellular sequestration of sphingomyelin. Taken together, these data suggest an immunopathogenic role for non-invariant NKT cells in urogenital chlamydial infections, facilitated by lipid presentation via CD1d via infected antigen-presenting cells.

Ang1 and Ang4 differentially affect colitis and carcinogenesis in an AOM-DSS mouse model.

Angiogenin-1 (Ang1) and angiogenin-4 (Ang4) are 14-kDa ribonucleases with potent angiogenic and antimicrobial properties. The role of Ang1 and Ang4 in chronic colitis and colitis-associated cancer has not been previously studied. Wild-type (WT) and angiogenin-1 knock-out (Ang1-KO) C57BL/6 mice were given azoxymethane, a colon carcinogen, 2 days in advance of three cycles of 3.5% dextran sodium sulfate (DSS). Disease activity index (DAI) was recorded, a colonoscopy was performed after each DSS treatment, and mice were euthanized (colitis, recovery, cancer) with tissue evaluated by histopathology. Ang1, Ang4, TNF-α, Il-1F062, IL-6, IL-10, IL-23, IL-33 mRNA levels were analyzed by RT-PCR. Ang1-KO mice exhibited more severe colitis compared to WT mice during both the acute (P<0.05) and recovery (P<0.05) phases of each DSS cycle. Consistent with these results, colonic TNF-α, IL1-β, IL-6, IL-10, and IL-33 mRNA levels were significantly upregulated in Ang1-KO mice (P<0.05). While Ang4 increased to similar levels in both WT and Ang1-KO mice during colitis and recovery phases, WT mice were distinguished by a significant upregulation of Ang1. Interestingly, despite the reduced colitis, WT mice developed significantly more tumors compared to Ang1-KO mice (P<0.05). 134 tumors formed in WT mice (4.6 tumors/mouse) while only 46 tumors formed (1.5 tumors/mice) in Ang1-KO mice, which were also characterized by a 34-fold decrease in Ang4 compared to WT mice and the complete absence of Ang1. In a mouse model of colitis-associated cancer, Ang1-KO mice develop more severe colitis, but fewer tumors compared to WT mice. Ang1 levels correlate with the severity of colitis and the development of colitis-associated cancer, while Ang4 was upregulated during both colitis and cancer. Ang1 and Ang4 play important regulatory roles in the response to chronic colitis and the development of colitis-associated cancer and may serve as novel therapeutic targets.

Network Pharmacology Combined with an Experimental Validation Study to Reveal the Effect and Mechanism of Eucommia ulmoides Leaf Polysaccharide against Immunomodulation

In the present study, the immuno-enhancing effect of Eucommia ulmoides leaf polysaccharide (ELP) was investigated in immunosuppressed mice induced by cyclophosphamide (CTX). To evaluate the immune enhancement mechanism of ELP, the immunoregulation effect of ELP was evaluated in vitro and in vivo. ELP is primarily composed of arabinose (26.61%), galacturonic acid (25.1%), galactose (19.35%), rhamnose (16.13%), and a small amount of glucose (12.9%). At 1000~5000 μg·mL-1, ELP could significantly enhance the proliferation and the phagocytosis of macrophages in vitro. Additionally, ELP could protect immune organs, reduce pathological damage, and reverse the decrease in the hematological indices. Moreover, ELP significantly increased the phagocytic index, enhanced the ear swelling response, augmented the production of inflammatory cytokines, and markedly up-regulated the expression of IL-1β, IL-6, and TNF-α mRNA levels. Furthermore, ELP improved phosphorylated p38, ERK1/2, and JNK levels, suggesting that MAPKs might be involved in immunomodulatory effects. The results provide a theoretical foundation for exploring the immune modulation function of ELP as a functional food.

Enhanced mucosal immune responses and reduced viral load in the respiratory tract of ferrets to intranasal lipid nanoparticle-based SARS-CoV-2 proteins and mRNA vaccines

BackgroundUnlike the injectable vaccines, intranasal lipid nanoparticle (NP)-based adjuvanted vaccine is promising to protect against local infection and viral transmission. Infection of ferrets with SARS-CoV-2 results in typical respiratory disease and pathology akin to in humans, suggesting that the ferret model may be ideal for intranasal vaccine studies.ResultsWe developed SARS-CoV-2 subunit vaccine containing both Spike receptor binding domain (S-RBD) and Nucleocapsid (N) proteins (NP-COVID-Proteins) or their mRNA (NP-COVID-mRNA) and NP-monosodium urate adjuvant. Both the candidate vaccines in intranasal vaccinated aged ferrets substantially reduced the replicating virus in the entire respiratory tract. Specifically, the NP-COVID-Proteins vaccine did relatively better in clearing the virus from the nasal passage early post challenge infection. The immune gene expression in NP-COVID-Proteins vaccinates indicated increased levels of mRNA of IFNα, MCP1 and IL-4 in lungs and nasal turbinates, and IFNγ and IL-2 in lungs; while proinflammatory mediators IL-1β and IL-8 mRNA levels in lungs were downregulated. In NP-COVID-Proteins vaccinated ferrets S-RBD and N protein specific IgG antibodies in the serum were substantially increased at both day post challenge (DPC) 7 and DPC 14, while the virus neutralizing antibody titers were relatively better induced by mRNA versus the proteins-based vaccine. In conclusion, intranasal NP-COVID-Proteins vaccine induced balanced Th1 and Th2 immune responses in the respiratory tract, while NP-COVID-mRNA vaccine primarily elicited antibody responses.ConclusionsIntranasal NP-COVID-Proteins vaccine may be an ideal candidate to elicit increased breadth of immunity against SARS-CoV-2 variants.

Dual red and near-infrared light-emitting diode irradiation ameliorates LPS-induced otitis media in a rat model.

Objective: Otitis media (OM) is an infectious and inflammatory disease of the middle ear (ME) that often recurs and requires long-term antibiotic treatment. Light emitting diode (LED)-based devices have shown therapeutic efficacy in reducing inflammation. This study aimed to investigate the anti-inflammatory effects of red and near-infrared (NIR) LED irradiation on lipopolysaccharide (LPS)-induced OM in rats, human middle ear epithelial cells (HMEECs), and murine macrophage cells (RAW 264.7). Methods: An animal model was established by LPS injection (2.0mg/mL) into the ME of rats via the tympanic membrane. A red/NIR LED system was used to irradiate the rats (655/842nm, intensity: 102mW/m2, time: 30min/day for 3 days and cells (653/842nm, intensity: 49.4mW/m2, time: 3h) after LPS exposure. Hematoxylin and eosin staining was performed to examine pathomorphological changes in the tympanic cavity of the ME of the rats. Enzyme-linked immunosorbent assay, immunoblotting, and RT-qPCR analyses were used to determine the mRNA and protein expression levels of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α). Mitogen-activated protein kinases (MAPKs) signaling was examined to elucidate the molecular mechanism underlying the reduction of LPS-induced pro-inflammatory cytokines following LED irradiation. Results: The ME mucosal thickness and inflammatory cell deposits were increased by LPS injection, which were reduced by LED irradiation. The protein expression levels of IL-1β, IL-6, and TNF-α were significantly reduced in the LED-irradiated OM group. LED irradiation strongly inhibited the production of LPS-stimulated IL-1β, IL-6, and TNF-α in HMEECs and RAW 264.7 cells without cytotoxicity in vitro. Furthermore, the phosphorylation of ERK, p38, and JNK was inhibited by LED irradiation. Conclusion: This study demonstrated that red/NIR LED irradiation effectively suppressed inflammation caused by OM. Moreover, red/NIR LED irradiation reduced pro-inflammatory cytokine production in HMEECs and RAW 264.7 cells through the blockade of MAPK signaling.

Dietary Bacillus licheniformis shapes the foregut microbiota, improving nutrient digestibility and intestinal health in broiler chickens.

Bacillus licheniformis is considered a potential alternative to antibiotic growth promoters of animal growth and health. However, the effects of Bacillus licheniformis on the foregut and hindgut microbiota, and their relationships with nutrient digestion and health, in broiler chickens remain unclear. In this study, we aimed to identify the effects of Bacillus licheniformis BCG on intestinal digestion and absorption, tight junctions, inflammation, and the fore- and hind-gut microbiota. We randomly assigned 240 1-day-old male AA broilers into three treatment groups: CT (basal diet), BCG1 (basal diet + 1.0 × 108 CFU/kg B. licheniformis BCG), and BCG2 (basal diet + 1.0 × 109 CFU/kg B. licheniformis BCG). On day 42, the jejunal and ileal chyme and mucosa were subjected to analysis of digestive enzyme activity, nutrient transporters, tight junctions, and signaling molecules associated with inflammation. The ileal and cecal chyme were subjected to microbiota analysis. Compared with the CT group, the B. licheniformis BCG group showed significantly greater jejunal and ileal α-amylase, maltase, and sucrase activity; moreover, the α-amylase activity in the BCG2 group was higher than that in the BCG1 group (P < 0.05). The transcript abundance of FABP-1 and FATP-1 in the BCG2 group was significantly greater than that in the CT and BCG1 groups, and the GLUT-2 and LAT-1 relative mRNA levels were greater in the BCG2 group than the CT group (P < 0.05). Dietary B. licheniformis BCG resulted in significantly higher ileal occludin, and lower IL-8 and TLR-4 mRNA levels than observed in the CT group (P < 0.05). B. licheniformis BCG supplementation significantly decreased bacterial community richness and diversity in the ileum (P < 0.05). Dietary B. licheniformis BCG shaped the ileac microbiota by increasing the prevalence of f_Sphingomonadaceae, Sphingomonas, and Limosilactobacillus, and contributed to nutrient digestion and absorption; moreover, it enhanced the intestinal barrier by increasing the prevalence of f_Lactobacillaceae, Lactobacillus, and Limosilactobacillus. Dietary B. licheniformis BCG decreased microbial community diversity by diminishing Desulfovibrio, Alistipes, Campylobacter, Vibrio, Streptococcus, and Escherichia coli-Shigella levels, and down-regulating inflammatory associated molecule expression. Therefore, dietary B. licheniformis BCG contributed to digestion and absorption of nutrients, enhanced the intestinal physical barrier, and decreased intestinal inflammation in broilers by decreasing microbial diversity and optimizing the microbiota structure.

The homeostatic function of Regnase-2 restricts neuroinflammation.

The precise physiological functions and mechanisms regulating RNase Regnase-2 (Reg-2/ZC3H12B/MCPIP2) activity remain enigmatic. We found that Reg-2 actively modulates neuroinflammation in nontransformed cells, including primary astrocytes. Downregulation of Reg-2in these cells results in increased mRNA levels of proinflammatory cytokines IL-1β and IL-6. In primary astrocytes, Reg-2 also regulates the mRNA level of Regnase-1 (Reg-1/ZC3H12A/MCPIP1). Reg-2 is expressed at high levels in the healthy brain, but its expression is reduced during neuroinflammation as well as glioblastoma progression. This process is associated with the upregulation of Reg-1. Conversely, overexpression of Reg-2 is accompanied by the downregulation of Reg-1 in glioma cells in a nucleolytic NYN/PIN domain-dependent manner. Interestingly, low levels of Reg-2 and high levels of Reg-1 correlate with poor-glioblastoma patients' prognoses. While Reg-2 restricts the basal levels of proinflammatory cytokines in resting astrocytes, its expression is reduced in IL-1β-activated astrocytes. Following IL-1β exposure, Reg-2 is phosphorylated, ubiquitinated, and degraded by proteasomes. Simultaneously, the Reg-2 transcript is destabilized by tristetraprolin (TTP) and Reg-1 through the AREs elements and conservative stem-loop structure present in its 3'UTR. Thus, the peer-control loop, of Reg-1 and Reg-2 opposing each other, exists. The involvement of TTP in Reg-2 mRNA turnover is confirmed by the observation that high TTP levels correlate with the downregulation of the Reg-2 expression in high-grade human gliomas. Additionally, obtained results reveal the importance of Reg-2 in inhibiting human and mouse glioma cell proliferation. Our current studies identify Reg-2 as a critical regulator of homeostasis in the brain.

Lactate modulates microglial inflammatory responses after oxygen-glucose deprivation through HIF-1α-mediated inhibition of NF-κB

Metabolic adaption drives microglial inflammatory responses, and lactate shapes immunological and inflammatory states. However, whether lactate was involved in the regulation of microglial inflammatory responses after cerebral ischemia remains unclear. In this study, the expression of iNOS, arginase-1, phosphorylated NF-κB p65 and IκB-α, and HIF-1α in BV2 cells after oxygen-glucose deprivation (OGD) were detected by western blotting and immunofluorescence. The mRNA levels of microglial responsive markers and inflammatory factors were assessed by real-time-qPCR. The effect of lactate-treated BV2 cells on the survival of primary neurons was observed using transwell co-culture. The results showed that the protein levels of iNOS and arginase-1, the ratio of mRNA levels of iNOS/CD206, CD86/Ym1, IL-6/IL-10, TNF-α/IL-10 and the mRNA levels of IL-6 and TNF-α, as well as the protein levels of phosphorylated NF-κB p65 and IκB-α, were increased after OGD. Lactate treatment inhibited the OGD-induced increase in the protein levels of iNOS, phosphorylated NF-κB p65 and IκB-α, as well as iNOS/CD206, CD86/Ym1, IL-6/IL-10, TNF-α/IL-10, IL-6 and TNF-α mRNA levels in BV2 cells, while promoted arginase-1 protein expression as well as IL-10 and TGF-β mRNA level. Interestingly, lactate activated HIF-1α and the HIF-1α inhibitor YC-1 reversed the effect of lactate on levels of microglial responsive markers and phosphorylated NF-κB p65 and IκB-α in BV2 cells. Moreover, knockdown of HIF-1α by lentivirus-delivered shRNA also reversed the effect of lactate on phosphorylated NF-κB p65 and IκB-α in BV2 cells after OGD. Finally, and importantly, lactate-treated BV2 microglia increased the viability and decreased the apoptosis of neurons after OGD. These findings revealed that lactate inhibited NF-κB pathway and skewed BV2 microglia toward the protective response through activation of HIF-1α after OGD, thereby improving neuronal survival.

Tea Polyphenols Reduce Inflammation of Orbital Fibroblasts in Graves’ Ophthalmopathy via the NF-κB/NLRP3 Pathway

ObjectiveThis study aimed to explore the effects of tea polyphenols (TP) on inflammation of orbital fibroblasts in Graves’ ophthalmopathy (GO) and to provide new ideas for GO treatment.MethodsPrimary orbital fibroblasts were extracted from orbital adipose/connective tissues of patients with and without GO. Real-time quantitative PCR (RT-qPCR) was used to detect the expression of interleukin (IL)-6, IL-1β, and monocyte chemotactic protein (MCP)-1 in non-GO and GO orbital fibroblasts. The CCK-8 assay was used to determine the appropriate concentration of TP for subsequent experiments. RT-qPCR and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the effects of TP on lipopolysaccharide (LPS)-induced production of inflammatory cytokines. Nuclear factor-κB (NF-κB) expression was measured using Western blotting analysis. NOD-like receptor 3 (NLRP3) expression was detected using both Western blotting analysis and immunofluorescence staining.ResultsThe mRNA levels of IL-6, IL-1β, and MCP-1 in GO orbital fibroblasts were significantly higher than those in non-GO cells. TP treatment significantly inhibited LPS-induced production of inflammatory factors, including IL-6, IL-1β, and MCP-1. TP also inhibited the expression levels of NF-κB and NLRP3. Inflammation in the GO orbital fibroblasts was higher than that in non-GO cells. TP inhibited the production of inflammatory cytokines in GO orbital fibroblasts in vitro through the NF-κB/NLRP3 pathway.ConclusionThese findings suggest that TP may have a potential role in GO treatment.

Gambogic acid affects high glucose-induced apoptosis and inflammation of retinal endothelial cells through the NOX4/NLRP3 pathway.

This study aimed to investigate the effect and mechanism of gambogic acid (GA) on the apoptosis and inflammation of human retinal endothelial cells (HRECs) under high glucose conditions. HRECs were cultured in a high glucose medium to simulate retinal endothelial cell injury induced by diabetic retinopathy. Flow cytometry was used to analyze the apoptosis level of HRECs. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Western blotting was applied to detect the intracellular apoptosis-related proteins and expression levels of NADPH oxidase 4 (NOX4), nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), and interleukin (IL)-1β. Enzyme linked immunosorbent assay (ELISA) was utilized to detect the expression of IL-6, IL-8, IL-10, and tumor necrosis factor-α (TNF-α) in the cell supernatants. The messenger RNA (mRNA) levels of IL-6, IL-8, IL-10, and TNF-α were detected by reverse transcription-polymerase chain reaction (RT-qPCR). We observed that high glucose induced the apoptosis and inflammation of HRECs. In addition, the high glucose environment promoted NOX/NLRP3 pathway activation. The activity of HRECs was not significantly affected by the presence of 20 μM or less of GA, and 15 μM of GA could restore the diminished activity of HRECs induced by high glucose. The apoptosis of HRECs cultured under high glucose conditions was significantly inhibited (P<0.05), the levels of IL-6, IL-8, and TNF-α in the cell supernatant were significantly decreased (P<0.05), and the levels of IL-10 were significantly increased (P<0.05). Meanwhile, the relative mRNA expression levels of IL-6, IL-8, and TNF-α in HRECs were significantly decreased (P<0.05), while those of IL-10 were significantly increased (P<0.05). The activity of the high glucose-induced NOX4/NLRP3 pathway in HRECs was significantly inhibited after treatment with 15 μM of GA (P<0.05). Following activation of the NOX4/NLRP3 pathway in HRECs, the apoptosis level was significantly increased (P<0.05), and the inflammatory response was aggravated (P<0.05). Inhibiting the activity of the intracellular NOX4/NLRP3 pathway markedly inhibited cell apoptosis and the inflammatory response (P<0.05). GA can inhibit the apoptosis and inflammation of HRECs under high glucose conditions by inhibiting the activity of the NOX4/NLRP3 pathway. This has a significant inhibitory effect on diabetic retinopathy, which is worthy of further study.

Taxifolin ameliorates cigarette smoke-induced chronic obstructive pulmonary disease via inhibiting inflammation and apoptosis.

Chronic obstructive pulmonary disease (COPD) is a significant cause of morbidity and mortality worldwide and is characterized by chronic airway inflammation and lung parenchymal cell apoptosis. Cigarette smoke is the major risk factor for the occurrence and development of COPD. Taxifolin (TAX) showed promising pharmacological effects in the management of inflammation, oxidative stress, and apoptosis. In the present study, our results demonstrated that TAX significantly alleviated cigarette smoke-induced inflammation and apoptosis both in vivo and in vitro. TAX notably lowered the elevated total cell count in mouse BALF compared with that in the COPD group. The cigarette smoke-induced emphysematous changes were remarkably reversed by TAX. In addition, treatment with TAX suppressed the elevated mRNA and protein levels of IL-1β, IL-6 and TNF-α in COPD mouse lung tissue and cigarette smoke extract (CSE)-treated human bronchial epithelial cells (HBECs). Additionally, TAX significantly decreased the ratios of p-iκB to iκB and p-p65 to p65 compared with the COPD group and CSE-treated HBECs. Moreover, the results of the TUNEL assay and flow cytometry also demonstrated the anti-apoptotic effect of TAX in mouse lung tissue and HBECs. Furthermore, the elevated Bax and CCP3 levels and decreased Bcl-2 levels induced by cigarette smoke were significantly reversed by TAX treatment in vivo and in vitro. Our results highlight the ameliorating effects of TAX against cigarette smoke-induced inflammation and apoptosis in the pathogenesis of COPD.

AdipoRon Effect on Expression of Lipid Metabolism Genes in Cultured Human Primary Macrophages

Atherosclerosis is characterized by excessive uptake of cholesterol-rich low-density lipoprotein (LDL) by vascular wall macrophages. The macrophages are transformed into foam cells, lipids accumulate in the intima of arteries, atherosclerotic plaques arise, and cardiovascular diseases develop. Adiponectin is an adipose tissue adipokine and possess anti-atherogenic and anti-inflammatory activities, which are mediated by adiponectin binding to its receptors AdipoR1 and AdipoR2. To exert its anti-atherogenic effect, adiponectin may regulate the reverse cholesterol transport and prevent foam cells formation. The small-molecule adiponectin receptor agonist AdipoRon was assumed to modulate expression of reverse cholesterol transport and inflammation genes in human macrophages. Several AdipoRon concentrations (0, 5, 10, and 20 μM) were tested for effect on expression of the lipid metabolism genes ABCA1, ABCG1, APOA1, NR1H3 (LXRα), NR1H2 (LXRβ), PPARG, and ACAT1 and the inflammation genes IL6, TNFA, and TLR4 in cultured human primary macrophages and the THP-1 macrophage cell line. Cell viability was measured using the MTS assay. ABCA1, ABCG1, APOA1, NR1H3, NR1H2, PPARG, ACAT1, IL6, TNFA, and TLR4 mRNA levels in human primary macrophages were assessed by real-time PCR. The PPARG and ABCA1 relative mRNA levels were found to increase in human primary macrophages treated with 5 or 10 μM AdipoRon for 24 h. A higher AdipoRon concentration (20 μM) was cytotoxic to macrophages, especially THP-1 cells. The effect of AdipoRon on human macrophages and potential adiponectin receptor agonists are of interest to study in view of the need to develop new approaches to atherosclerosis prevention and treatment.