IL-1β Gene Expression Research Articles

Abstract 4119913: Aging-Associate Peptide Medin Induces Proinflammatory Activation in Human Brain Vascular Smooth Muscle Cells

Background: Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. In vitro , medin has been shown to induce endothelial proinflammatory activation and in ex vivo human cerebral arterial tissue, medin induces both endothelial and smooth muscle dysfunction. The role of medin in vascular smooth muscle (VSMC) activation remains unknown. Aim: The aim of the study is to determine using gene transcription assay whether medin induces VSMC activation and phenotypic transformation. Methods: Human brain vascular smooth muscle cells (HBVSMCs, passages 6-10) were exposed to medin at doses observed in human tissues (0, 0.5, 1 and 5 υM) for 20 hours and reverse transcription polymerase chain reaction (rtPCR) was used to quantify gene expression of proinflammatory factors (interleukin (IL)-6, IL-8, IL-1b) and structural and enzyme proteins associated with VSMC phenotypic transformation (ACTA2, MYH11 and NOX4). In separate experiments, HBVSMCs were exposed to vehicle, medin 5 υM without or with small molecule NFκB inhibitor RO106-9920 (10 υM) or NLGM1 nanoliposome (70% phosphatidylcholine, 25% cholesterol, 5% monosialoganglioside, 100 υg/mL, agent shown to reverse medin-induced endothelial dysfunction and proinflammatory activation). Results: Medin induced a dose-dependent increase in gene expression of IL-6, IL-8 and IL-1b but did not alter gene expression of ACTA2, MYH11 and NOX4 (Fig. 1). Co-treatment of medin with RO106-9920 and NLGM1 showed a trend (not statistically significant) of reduced IL-6 and IL-8 (Fig. 2). Conclusions: Physiologic doses of medin induced pro-inflammatory activation of HBVSMCs but did not affect gene expression of structural proteins (ACTA2 and MYH11) and enzyme (NOX4) involved in VSMC phenotypic transformation. Although the mechanisms behind this effect remains to be explored, results suggest that medin may be an important mediator of aging-associated vascular inflammation.

Utilizing the apical-out enteroids in vitro model to investigate intestinal glucose transport, barrier function, oxidative stress, and inflammatory responses in broiler chickens

IntroductionConventional 2D intestinal epithelial cell lines have been widely used in investigating intestinal functions, yet with limitations in recapitulating the in vivo gut physiology of chickens. A recently established chicken enteroid model with apical-out nature and the presence of leukocyte components represents intestinal mucosal functions. The objectives of this study were to 1) evaluate basic gut nutrient transport and barrier functions in this model and 2) identify the model’s effectiveness in studying inflammation and oxidative stress responses.MethodsEnteroids were generated from individual villus units isolated from the small intestine of Cobb500 broiler embryos. Enteroid viability, morphology, and epithelial cell markers were monitored; barrier function was evaluated based on the permeability to fluorescein isothiocyanate–dextran (FD4) with or without EDTA and lipopolysaccharide (LPS) challenges; nutrient transport was evaluated by fluorescence-labeled glucose (2NBD-G) with or without transporter blockade; the oxidative status was indicated by reactive oxygen species (ROS). Inflammatory and oxidative challenges were induced by LPS and menadione treatment, respectively. Selected marker gene expressions, including tight junction proteins (CLDN-1, CLDN-2, ZO-1, and OCCL), epithelial cell markers (Lgr-5, LYZ, and MUC-2), cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, and INF-γ), and antioxidant enzymes (Nrf-2, catalase, and SOD), were determined by using RT-qPCR. Data were analyzed by one-way ANOVA among treatment groups.ResultsEnteroid cell activity was stable from day (d) 2 to d 6 and declined at d 7. Epithelial cell marker and cytokine expressions were stable from d 4 to d 6. FD4 permeability was increased after the EDTA treatment (P ≤ 0.05). Transporter-mediated 2NBD-G absorption was observed, which was reduced with glucose transporter blockade (P ≤ 0.05). Enteroids showed classic responses to LPS challenges, including upregulated gene expressions of IL-1β and IL-6, downregulated gene expressions of ZO-1 and OCCL, and increased FD4 permeability (P ≤ 0.05). Enteroids showed increased ROS generation (P ≤ 0.05) in response to oxidative stress.DiscussionIn conclusion, this apical-out enteroid model is a stable alternative in vitro model that exhibits intestinal barrier, nutrient transport, oxidation, and inflammation functions. With this enteroid model, we developed two challenge protocols for evaluating intestinal functions under oxidative stress and inflammation conditions.

Increasing coil temperature of a third-generation e-cigarette device modulates C57BL/6 mouse lung immune cell composition and cytokine milieu independently of aerosol dose

ABSTRACT Higher coil temperature in e-cigarette devices increases the formation of aerosols and toxicants, such as carbonyls. At present, the health implications of vaping at higher temperatures, including exacerbation of pulmonary inflammation, are largely unknown when aerosol dose is considered. To isolate the pulmonary effects of coil temperature, C57BL/6 mice were exposed to e-cigarette aerosols generated at lower (190°C) or higher (250°C) temperature for 3 days, while maintaining a similar chamber aerosol concentration. Increasing coil temperature did not markedly alter aerosol mass-normalized emissions of select carbonyls formed from thermal degradation pathways including formaldehyde, acetaldehyde, propionaldehyde, and acetone under the tested environment. Total bronchoalveolar cells, primarily macrophages, were significantly decreased in mice exposed to aerosols generated with higher coil temperatures compared to lower temperature exposures. The gene expression of IFNβ, IL-1β, TNFα, and IL-10 in mouse lung tissue was significantly reduced following e-cigarette exposure under both conditions, compared to filtered air exposure. Higher temperature exposures further exacerbated downregulation of IFNβ and IL-1β. Data suggest that higher temperature vaping might modulate acute pulmonary immune responses, potentially inducing immune suppression, even when normalized for aerosol dose exposure. Coil temperature thus appears to be an important parameter that needs to be regulated to ensure harm reduction for e-cigarette users.

Experimental colitis is comorbid with social interaction deficits and anxiety-like behaviors in mice: mechanistic intuitions into neuroinflammation and Claudin 5 expression in the hippocampus.

Inflammatory bowel disease (IBD) is accompanied by psychiatric disorders, including Schizophrenic-like manifestations. Although incompletely illustrated, intestinal mucosal membrane damage and blood-brain barrier (BBB) penetrability may have significant roles in psychiatric symptoms of IBD. This study aimed to investigate role of the Claudin-5 (CLDN5) (a regulator of the permeability of BBB) and neuroinflammatory response in the comorbid behavioral disorders in experimental colitis in mice. Acetic acid was used to induce colitis in mice. 7 days after induction of colitis, behaviors including social interaction and locomotor activity as well as anxiety-like behaviors were evaluated. Then, the colon was extracted for gross and microscopic evaluations. The expression of CLDN5, TNF-α, IL1β and IL23 was measured by RT-PCR in the colon and hippocampus. Histopathologic evaluations demonstrated mucosal, submucosal, and crypt-related damages in the colon. The negative and positive number of social interactions significantly increased in the colitis group. A considerable increase in locomotor activities (horizontal and vertical components) shown in the colitis group. Mice in colitis group spent less time in the central zone in the open field apparatus. Gene expressions of TNF-α, IL1β, and IL23 increased and CLDN5 decreased in the colitis group. The barrier function of the intestine and brain would be impaired, partially at least, following colitis (as we observed decrease in CLDN5 gene expression). Furthermore, we found that beside inflammatory response in the colon, a neuro-immune response triggered in the hippocampus following colitis. These alterations probably, mediated comorbid behavioral disorders in acetic acid-induced colitis in mice.

The Effect of Variegated Grasshopper Meal (Zonocerous variegatus) on Growth, Nutrient Utilization and Gene Expression Study in Juvenile Clarias gariepinus (Burchell, 1822)

Over the years there has been research on how to improve fish yield. This study investigated the effects of variegated grasshopper meal (Zonocerous variegatus) on growth performance, nutrient utilization, and gene expressions in juvenile Clarias gariepinus (African catfish). The variegated grasshopper meal diets (VGDMs) were incorporated with the other feed ingredients at 25% (VGDM1), 50% (VGDM2), 75% (VGDM2), and 100% (VGDM3) diets while the remaining group served as control. Growth performance, nutrient utilization, and gene expression study were evaluated. The mRNA expression of Growth hormone (GH), Heat Shock Protein (HSP70), Melatonin (MEL 1C), tumour necrosis factor-alpha (TNF-α), and interleukin 1 beta (IL-1β) were investigated in the liver and muscle of C. gariepinus fed with varying doses of grasshopper meals (VGDMs) using reverse transcriptase–polymerase chain reaction. There were significant (p>0.05) variations in all parameters in all the treatments. There was a significant upregulation (p<0.05) in the gene expression of GH, HSP70, MEL 1C, and TNF-α in VGDM1 compared to control in both tissue groups. In addition, there was significant downregulation (p<0.05) in interleukin 1 (IL-1β) gene expression in liver VGDM2. This study suggests that dietary variations involving VGMD have the potential to boost growth and improve the immune responses of this fish species.

Supplementation of Chlorogenic Acid Alleviates the Effects of H2O2-Induced Oxidative Stress on Laying Performance, Egg Quality, Antioxidant Capacity, Hepatic Inflammation, Mitochondrial Dysfunction, and Lipid Accumulation in Laying Hens

This research examined the impact of chlorogenic acid (CGA) on laying performance, antioxidant capacity, egg quality, hepatic inflammation, mitochondrial function, and lipid metabolism in hens subjected to hydrogen peroxide (H2O2)-induced oxidative stress (OS). Three hundred sixty healthy 43-wk-old Hy-Line brown hens were randomly assigned to six treatments: a basal diet + 0 (control and H2O2), 600 (600 mg/kg CGA and 600 mg/kg CGA + H2O2), and 800 (800 mg/kg CGA and 800 mg/kg CGA + H2O2) mg/kg CGA for 84 d. On the 64th and 78th days of the trial, hens in groups H2O2, 600 mg/kg CGA + H2O2, and 800 mg/kg CGA + H2O2 were injected intraperitoneally with 10% H2O2. The results demonstrated that 600 and 800 mg/kg CGA significantly improved the egg production rate (EPR) and egg quality and reduced lipid peroxidation compared to the control group. The 800 mg/kg CGA showed greater improvements in the EPR and average egg weight (AEW) compared to the 600 mg/kg dose. Conversely, H2O2 exposure significantly decreased the EPR, AEW, and egg quality and increased feed conversion rate and average daily feed intake. H2O2 exposure significantly decreased serum T-AOC and increased serum MDA levels while reducing hepatic T-SOD, GSH-Px, and CAT activities. Meanwhile, H2O2 exposure significantly elevated liver reactive oxygen species levels, pathological damage, and NF-κB, TNFα, and IL-1β gene expression. Additionally, H2O2 treatment disrupted hepatocyte mitochondrial structure and significantly increased the expression of VDAC1 protein, and IP3R, GRP75, MCU, Fis1, and MFF genes, while downregulating the expression of MFN2 protein and PGC1α gene. Oil Red O staining demonstrated that H2O2 induced significant lipid accumulation in hepatocytes. Concurrently, H2O2 significantly increased serum triglycerides, total cholesterol, and liver triglycerides levels while decreasing serum hepatic lipase activity. This was primarily attributed to the significant upregulation of liver SREBP1, FASN, and ACC genes and the downregulation of the liver CPT1 gene induced by H2O2. Furthermore, CGA pretreatment effectively prevented the degeneration in laying performance and egg quality, as well as OS, liver inflammation, pathological damage, and mitochondrial dysfunction induced by H2O2. CGA inhibited H2O2-induced hepatic lipid accumulation by upregulating fatty acid oxidation-related gene expression and downregulating fatty acid synthesis-related gene expression. These findings indicate that the dietary addition of 800 mg/kg of CGA is the optimum supplementation dose. CGA can enhance laying performance and egg quality while alleviating OS, hepatic inflammation, mitochondrial dysfunction, and lipid accumulation in H2O2-challenged laying hens.

Acute exposure to LPS induces cardiac dysfunction via the activation of the NLRP3 inflammasome

Systemic inflammation contributes to left ventricular (LV) dysfunction, however the role of the NLRP3 inflammasome in LV dysfunction in acute inflammatory conditions is unclear. This study investigated the role of the NLRP3 inflammasome in acute (24 h) cardiac structural and functional changes in vivo and in vitro in lipopolysaccharide (LPS)-induced inflammation. LPS-treated Sprague-Dawley (SD) rats showed increased LPS metabolite abundance in their LVs as measured by atmospheric pressure matrix-assisted laser desorption ionisation (AP-MALDI) mass spectrometry imaging (MSI). Echocardiography and histology showed that in LPS-exposed rats, LV internal diameter was decreased, with evidence of macrophage infiltration and oedema. However, there were no changes in LV wall thickness or collagen volume. Additionally, LPS-exposed rats exhibited impaired LV relaxation, potentially contributing to decreased stroke volume. While global systolic function was preserved, LPS exposure in SD rats resulted in impaired myocardial deformation assessed by speckle-tracking echocardiography. Exposure to LPS resulted in upregulation of the expression of components of the NLRP3 inflammasome in rodents. In vitro LPS exposure resulted in increased gene expression of NLRP3 and downstream cytokines IL-1β and IL-18, antioxidant SOD2, and elevated markers of pyroptosis (GSDMD) which were inhibited by treatment with a NLRP3 antagonist. However, LPS-induced increases in the gene expression of apoptosic markers (BAX/Bcl2) were not impacted by NLRP3 antagonism. These findings suggest that inflammation induced adverse cardiac structural and functional changes is, at least in part, mediated by the NLRP3 inflammasome in acute, high-grade inflammatory states. In addition, in vitro findings suggest that while the NLRP3 inflammasome mediates pyroptotic pathways, regulation of apoptosis that is independent of the inflammasome.

Transcriptome Analysis of Porphyromonas gingivalis Lipopolysaccharide-Induced Early Gene Expression in Human Gingival Keratinocytes.

Porphyromonas gingivalis lipopolysaccharide (PgLPS) is a significant virulence factor and a driver of early innate immune responses in epithelial cells. The presence of PgLPS in immediate proximity to gingival epithelium induces significant inflammatory responses. In primary human gingival keratinocytes (HGK), we utilized transcriptome analysis to elucidate the change in early gene expression induced by PgLPS. HGK cell cultures were treated with PgLPS (4 h), and RNA was extracted and prepared for RNA sequence (RNAseq) analysis. Differentially expressed genes (DEGs) were identified, and potential interactions between these genes were subsequently examined using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analytic approaches to identify significantly enriched pathways. Expression of genes associated with relevant pathways was evaluated using real-time quantitative reverse-transcription polymerase chain reaction (RT-qPCR). RNAseq analysis identified 25 DEGs, and GO and KEGG analytic approaches showed related genes expressed in two general pathways. First, pathways broadly related to urokinase and coagulation included the genes PLAU, PLAUR, and SerpinB2. In RT-qPCR analysis, these genes were induced by PgLPS over time (4-24 h), and these data were consistent with PgLPS induction of cell migration. Second, interleukin-1 (IL-1) receptor binding and cytokine-activity pathways were also enriched. Genes associated with these pathways included IL36G, IL1B, IL1RN, and CXCL14. RT-qPCR analysis confirmed PgLPS induction of genes associated with the IL-1family. When expression of IL1B and IL36G genes was examined in relation to their respective antagonists, only IL36G gene expression was increased. CXCL14 gene expression was reduced over time, and this was consistent with RNAseq analysis. Genes associated with significantly enriched GO and KEGG pathways are relevant to aspects of periodontal disease (PDD) pathogenesis. First, PgLPS induced expression of PLAU, PLAUR, and SerpinB2, and these changes were consistent with an increase in cell migration that was found. Second, both IL36G and IL1B gene expression was significantly induced, but only IL36G in relation to its selective antagonist (IL36RN) was increased. These data support that early upregulation of IL36G may serve as an alarmin that can drive early innate immune inflammatory responses in HGK. Further invivo testing of these findings is ongoing.

Anti-inflammatory oxazole-, nitro- and hexahydropyrrolo[2,1-b]oxazole-containing abietane diterpenoid alkaloids from Salvia miltiorrhiza

Nine undescibed abietane diterpenoid alkaloids (DAs), salviamines G‒H (1–2), isosalviamines G‒J (3–6), and miltiorramines A‒C (7–9) were isolated from the roots of Salvia miltiorrhiza. Their chemical structures including absolute configurations were elucidated by extensive spectroscopic analysis (including 1D and 2D NMR, and HRESIMS), combined with the calculated ECD method and single-crystal X-ray diffraction analysis. Among them, compounds 1–6 are unusual 20-nor- or 19,20-bisnor-abietane DAs with an oxazole ring. Compounds 7–8 are the first examples of DAs with a nitro group isolated from plant sources. Notably, compound 9 possesses a rare hexahydropyrrolo[2,1-b]oxazole unit that is fused in the ring B of the abietane skeleton. Bioactivity assay indicated that compound 3 showed significant anti-inflammatory activity by decreasing the gene expressions of IL-1β, IL-6, and TNF-α in LPS-induced RAW264.7 cells in a dose-dependent manner.

Responses of Intestinal Antioxidant Capacity, Morphology, Barrier Function, Immunity, and Microbial Diversity to Chlorogenic Acid in Late-Peak Laying Hens.

This study examined the influence of chlorogenic acid (CGA) on gut antioxidant status, morphology, barrier function, immunity, and cecal microbiota in late-peak laying hens. A total of 240 Hy-Line Brown hens, aged 43 weeks, were randomly assigned to four groups, the basal diet +0, 400, 600, and 800 mg/kg CGA, for 12 weeks. The results revealed that CGA significantly reduced ileal H2O2 and malondialdehyde levels; increased duodenal height, ileal villus height, and villus height-to-crypt depth ratio; while decreasing jejunal crypt depth. The 600 and 800 mg/kg CGA significantly upregulated the duodenal, jejunal, and ileal ZO-1 and occludin gene expression; increased IgG levels in serum and ileum; and upregulated ileal IgA gene expression. The 600 mg/kg CGA significantly upregulated CD3D and CD4 gene expression, while downregulating IL-1β gene expression in duodenum, jejunum, and ileum. Moreover, CGA changed the gut microbiota structure. The SCFA-producing bacteria unclassified_f__Peptostreptococcaceae, unclassified_f_Oscillospiraceae, Pseudoflavonifractor, Lachnospiraceae_FCS020_group, Oscillospira, Elusimicrobium, Eubacterium_ventriosum_group, Intestinimonas, and norank_f_Coriobacteriales_Incertae_Sedis were significantly enriched in the 400, 600, and/or 800 mg/kg CGA groups. The bacteria Lactobacillus, Bacillus, and Akkermansia were significantly enriched in the 600 mg/kg CGA group. Conclusively, dietary CGA (600-800 mg/kg) improved intestinal antioxidant status, morphology, barrier and immune function, and beneficial microbiota growth in late-peak laying hens.

The Addition of Hot Water Extract of Juncao-Substrate Ganoderma lucidum Residue to Diets Enhances Growth Performance, Immune Function, and Intestinal Health in Broilers.

The purpose of this experiment was to investigate the effects of Hot Water Extract of Juncao-substrate Ganoderma lucidum Residue (HWE-JGLR) on the immune function and intestinal health of yellow-feather broilers. In an animal feeding experiment, 288 male yellow-feather broilers (1 day old) were randomly allocated to four treatment groups with six replicates of 12 birds each. The control (CON) group was fed a basal diet. HJ-1, HJ-2, and HJ-3 were fed a basal diet supplemented with 0.25%, 0.50%, and 1.00% HWE-JGLR, respectively. The feeding trial lasted for 63 d. The results showed increased ADFI (p = 0.033) and ADG (p = 0.045) of broilers in HJ-3, compared with the CON group. Moreover, higher contents of serum IL-4 and IL-10 and gene expression of IL-4 and IL-10 in jejunum mucosa and lower contents of serum IL-1β and gene expression of IL-1β in jejunum mucosa in HJ-3 were observed (p < 0.05). Additionally, the jejunal mucosal gene expression of Claudin-1 and ZO-1 in HJ-2 and HJ-3 was higher than that in the CON group (p < 0.05). As for the microbial community, compared with the CON group, the ACE index, Shannon index, and Shannoneven index of cecal microorganisms in HJ-2 and HJ-3 were elevated (p < 0.05). PCoA analysis showed that the cecal microbial structure of broilers in HJ-2 and HJ-3 was different from the CON group (p < 0.05). In contrast with the CON group, the broilers in HJ-2 and HJ-3 possessed more abundant Desulfobacterota at the phylum level and unclassified Lachnospiraceae, norank Clostridia vadinBB60 group and Blautia spp. at the genus level, while Turicibacter spp. and Romboutsia spp. were less (p < 0.05). In conclusion, dietary supplementation with HWE-JGLR can improve growth performance, enhance body immunity and intestinal development, and maintain the cecum microflora balance of yellow-feather broilers.

Aqueous Extracts of Rhus trilobata Inhibit the Lipopolysaccharide-Induced Inflammatory Response In Vitro and In Vivo.

Chronic noncommunicable diseases (NCDs) are responsible for approximately 74% of deaths globally. Medicinal plants have traditionally been used to treat NCDs, including diabetes, cancer, and rheumatic diseases, and are a source of anti-inflammatory compounds. This study aimed to evaluate the anti-inflammatory effects of Rhus trilobata (Rt) extracts and fractions in lipopolysaccharide (LPS)-induced inflammation models in vitro and in vivo. The aqueous extract (RtAE) and five fractions (F2 to F6) were obtained via C18 solid-phase separation and tested in murine LPS-induced J774.1 macrophages. Key inflammatory markers, such as IL-1β, IL-6, TNF-α, and COX-2 gene expression were measured using RT-qPCR, and PGE2 production was assessed via HPLC-DAD. The in vivo effects were tested in an LPS-induced paw edema model in Wistar rats. Results showed that RtAE at 15 μg/mL significantly decreased IL-1β and IL-6 gene expression in vitro. Fraction F6 further reduced IL-1β, TNF-α, and IL-6 gene expression, COX-2 expression, and PGE2 production. In vivo, F6 significantly reduced LPS-induced paw edema, inflammatory infiltration, and IL-1β and COX-2 protein expression. Chemical characterization of F6 by UPLC/MS-QTOF revealed at least eight compounds with anti-inflammatory activity. These findings support the anti-inflammatory potential of RtAE and F6, reinforcing the medicinal use of Rt.

Oral pre-treatment with Citronellol ameliorates Methotrexate-induced nephrotoxicity in Wistar rats via targeting oxidative stress and inflammation

Background Methotrexate (MTX) is a classical folic acid antagonist widely used in the treatment of malignant and non-malignant disorders. However, its clinical application is often restricted by concomitant adverse effects, including renal damage. Numerous studies have highlighted the role of oxidative stress and inflammation in mediating MTX-related nephrotoxicity. Therefore, the current study aimed to explore the possible renoprotective action of Citronellol (CT), a natural compound with prominent antioxidant and anti-inflammatory activities, against nephrotoxicity induced by MTX. Methods To fulfill our objective, 24 adult male Wistar rats were randomly allocated into four groups: control, MTX, 100 mg/kg CT plus MTX and 200 mg/kg CT plus MTX. At the end of the study, the experimental rats were anesthetized, blood samples were collected for biochemical assays, and the kidneys were surgically removed for biochemical and gene expression analyses, after which all rats were sacrificed by exsanguination. Results Compared to the MTX-treated group, our results revealed that pre-supplementation with 100 or 200 mg/kg CT remarkably ameliorated renal damage biomarkers, including serum urea, serum creatinine, and kidney injury molecule-1 (KIM-1). In addition, pre-treatment with 100 or 200 mg/kg CT enhanced the activity of the antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx), diminished renal malondialdehyde (MDA) contents, and attenuated inflammation by suppressing renal nuclear factor kappa B (NF-κB) signaling and diminishing tumor necrosis factor-alpha (TNF-α) gene expression. Moreover, pre-treatment with 200 mg/kg CT markedly reduced interleukin-1 beta (IL-1β) gene expression. Conclusion Our findings demonstrate, for the first time, that CT can serve as a new promising agent for mitigating nephrotoxicity induced by MTX through its antioxidant and anti-inflammatory properties.

6681 Age and Sex Disparity in NASH and the Potential Role of Estradiol Treatment for NASH

Abstract Disclosure: M. Tripathi: None. S. Sakthivel: None. A. Suzuki: None. B.K. Singh: None. P.M. Yen: None. Introduction: NAFLD (nonalcoholic fatty liver disease) and its more severe form, NASH (nonalcoholic steatohepatitis) are sexually dimorphic conditions that exhibit higher prevalence in men than women during reproductive ages but have similar prevalence after menopause. Reproductive age women are protected from hepatic fibrosis relative to men, although they lose this protection after menopause. These sex differences in NASH have been attributed, at least partly, to different circulating estrogen levels. However, the underlying mechanism(s) is not fully understood. We thus conducted animal experiments to investigate: 1) sex differences in the histologic severity of NASH, necroinflammatory and fibrogenic activities, and other relevant pathways in young and old male and female mice and 2) the effects of estradiol administration on these parameters in old male and female mice to evaluate the therapeutic potential of estradiol in NASH. Methodology: Young/reproductive age (12-weeks old) and old/post-reproductive age female and male (48∼50-weeks weeks old) C57BL/6J mice were fed control chow diet (NCD) or high-fat methionine/choline-deficient (MCD) diet for six weeks to induce NASH. The old mice were administered 200nM 17β-estradiol (physiological) in drinking water. We analyzed serum and hepatic NASH parameters, ER stress, and lysosome-autophagy defects. Results and Conclusion: Among reproductive age mice with NASH, females developed less severe histologic inflammation and fibrosis and decreased gene expression of Il1b, Il6, Tgfb, Col1a1, Ccl2, and Ccl5, and lower hepatic collagen than males. Females with NASH also had less inhibition of hepatic autophagy and lysosomal protein expression than males with NASH. Among old mice, post-reproductive age females fed NCD or MCD exhibited more severe hepatic inflammation and fibrosis and had higher gene expression of inflammatory and fibrogenic markers with comparable autophagic inhibition and lysosomal defects as similar age males. Physiological doses of estradiol significantly reduced hepatic gene expression of inflammatory and fibrogenic markers, ER stress and lysosomal-autophagic defects in post-reproductive age females. In conclusion, similar to clinical NASH, we observed marked sex and age-related differences in the severity of NASH. Furthermore, physiological doses of estradiol treatment improved the hepatic histology, lysosome-autophagy, inflammation, and fibrosis in post-reproductive age female mice with NASH and suggests that estradiol supplementation may decrease or reverse NASH progression in post-menopausal women with NASH. Presentation: 6/2/2024

Proinflammatory factors inhibition and fish oil treatment: A promising therapy for neonatal seizures

Brain injury is one of the most important causes of infant mortality and chronic neurological disabilities. Hypoxia is an acute brain injury which led to various cognitive, behavioral, and memory disorders throughout life. Previous studies reported neuroprotective possibilities for fish oil (FO) in brain-injured situations. In this study, we evaluated the effect of the FO diet during the lactation period on seizure activity, behavioral performance, histomorphometry, and inflammatory changes in the brains of hypoxia rats. Male Wistar rats were randomly divided in to 4 groups: Sham (intact rats), hypoxia, FO and FO+hypoxia groups. Hypoxia was induced by keeping neonate rats at PND12 in a hypoxic chamber (7 % oxygen and 93 % nitrogen intensity) for 15 minutes. In the FO groups, rats received oral FO (1 ml/day) for 12 days during the lactation period. Seizure activity was assessed by measuring the number of tonic-clonic seizures and seizure thresholds. Novel object recognition tests (NORT), rotarod, and open field tests were used to measure behavioral performances. A Histological study was performed to evaluate histomorphometric changes in the hippocampus and cerebellum. The gene expression of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) was measured using RT-PCR. Findings showed that the number of tonic-clonic seizures, atrophy, and cell death in the hippocampus and cerebellum, the gene expression of TNF-α and IL-1β in the hippocampus, and behavioral disorders were significantly increased in the hypoxia rats compared to the sham group. Administration of FO in the hypoxia groups significantly decreased the gene expression of TNF-α and IL-1β, the number of tonic-clonic seizures, and neuronal cell death in the hippocampus and cerebellum compared to the hypoxia groups. Furthermore, it can improve behavioral tasks and cognitions.

Immunoprotective effect of recombinant Lactobacillus plantarum expressing largemouth bass virus MCP on largemouth bass

Largemouth bass virus (LMBV) is an infectious pathogen that causes high mortality rates in largemouth bass, and outbreaks of this virus can significantly harm the aquaculture industry. Currently, no vaccine has been developed that can effectively prevent the transmission of LMBV. In this study, we constructed a recombinant Lactobacillus plantarum (L. plantarum) strain capable of expressing the MCP gene of LMBV and displaying this protein on its surface; then, we evaluated the immunoprotective effect of this recombinant bacterium on largemouth bass. Western blotting, immunofluorescence, and flow cytometry confirmed that MCP was successfully expressed and anchored on the surfaces of NC8 cells. Immunization of largemouth bass with NC8-pSIP409-pgsA'-MCP via the oral feeding route induced CD4, CD8, IL-1β, and IL-6 gene expression. In addition, NC8-pSIP409-pgsA'-MCP at different CFUs increased the survival of largemouth bass after LMBV infection; in particular, NC8-pSIP409-pgsA'-MCP (109 CFU) resulted in approximately 30% survival. NC8-pSIP409-pgsA'-MCP immunization alleviated the pathological changes in the liver and spleen, exerting a more advantageous protective effect. These data suggest that the recombinant L. plantarum strain NC8-pSIP409-pgsA'-MCP can increase the resistance of largemouth bass to LMBV infection and that this strain is a promising candidate oral vaccine for the prevention of LMBV infection.

Role for NLRP3 inflammasome-mediated, Caspase1-dependent response in glaucomatous trabecular meshwork cell death and regulation of aqueous humor outflow

PurposeAcute ocular hypertension (AOH) is the defining feature of acute glaucoma. The mechanical stress and excessive production of reactive oxygen species (ROS) during episodes can directly or indirectly damage the trabecular meshwork (TM). Despite its significance, a clear understanding of its pathogenesis and an effective therapeutic target remain lacking in acute glaucoma. In the present study, we explored the potential molecular mechanisms underlying TM cell death following oxidative damage and AOH. The use of NAC/VX-765 as a potential pharmaceutical intervention for reducing intraocular pressure (IOP) was discussed. MethodsThe levels of NLRP3 and caspase-1 were compared between normal and glaucomatous TM samples. An in vitro oxidative damage model and an AOH rat model were used to investigate the potential molecular mechanism behind TM cell death. The ROS scavenger N-acetyl-L-cysteine (NAC) and caspase-1 inhibitor VX-765 were used to counteract TM damage. ResultsElevated levels of NLRP3 and caspase-1 were observed in patients with acute glaucoma. H2O2 exposure decreased the viability of human trabecular meshwork (HTM) cells and increased intracellular ROS levels. Both Gene and protein expressions of NLRP3, caspase-1, GSDMD-N, and IL-1β were notably upregulated in H2O2-induced HTM cells and the rodent AOH model. Both NAC and VX-765 demonstrated protective effects against TM injury by inhibiting pyroptosis. The IOP-lowering effects of NAC and VX-765 persisted for 7 days. ConclusionsOur findings indicate that the classical pyroptosis pathway, NLRP3/caspase-1/IL-1β, plays a key role in acute glaucomatous TM injury. Targeting pyroptosis provides novel therapeutic avenues for treating AOH-induced irreversible TM injury. This provides not only a promising therapeutic target for glaucoma but also introduces a new approach to intervention.

PSI-8 Inflammatory cytokine concentrations and gene expression in mature and aging sedentary horses

Abstract Chronic inflammation associated with aging, termed “inflamm-aging” may negatively impact overall animal health. However, the mechanisms of inflamm-aging and, therefore, effective interventions, remain unknown. Nuclear factor erythroid 2-related factor 2 (Nrf2; NFE2L2) is a transcription factor that controls antioxidant gene expression and could be a target for promoting anti-inflammatory pathways. To test the hypothesis that supplementation of nonenzymatic derived antioxidants (Protandim Nrf2 Synergizer, LifeVantage Corporation) would activate Nrf2 and mitigate inflammation, 40 mature, sedentary horses [32 mares, 8 geldings; 15.7 ± 4.9 yr of age, body weight (BW) = 519 ± 46 kg] were stratified by age, sex, and BW and randomly assigned to one of four dietary treatment groups for 56 d: 1) 0 mg (CON); 2) 675 mg (Pro1); 3) 2,025 mg (Pro3); or 4) 4,050 mg (Pro6) Protandim Nrf2 Synergizer per day (n = 10 per group). Blood samples were collected at d 0 (September, 27°C, 61.6% humidity), d 28 (October, 21.6°C, 55.8% humidity), and d 56 (November, 15.1°C, 73.8% humidity) of supplementation prior to the morning feeding. Plasma cytokine concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-6, IL-8, and IL-10 were quantified using custom Meso Scale Discovery U-Plex kits and whole blood gene expression of IL-1β, NFE2L2, and heme oxygenase 1 (HMOX1) were determined using qRT-PCR. Data were analyzed using PROC MIXED in SAS v9.4 with sex, time, treatment, age (under or over 15 yr), and time × treatment as fixed effects and time as a repeated effect with horse(treatment) as the subject. Day 0 was included as a covariate when treatment groups differed at d 0. Relationships between age and cytokine concentrations or mRNA expression were determined using PROC CORR. Plasma IL-4 (r = 0.4054) concentrations as well as whole blood HMOX1 (r = 0.3437) and NFE2L2 (r = 0.3382) expression on d 0 were positively correlated with age (P ≤ 0.04). Regardless of treatment or age, IL-1β and NFE2L2 expression and IL-4 and IL-8 concentrations decreased from d 0 to 28 (P ≤ 0.03) and remained depressed at d 56 (P ≤ 0.02) while IL-10 was not different from d 0 to 28 but decreased from d 0 to 56 (P = 0.02). TNFα concentrations were not different between treatments or over time. These data corroborate increased inflammatory markers with age and suggest a change in inflammatory status over time potentially due to seasonal environmental factors.

Ageing results in an exacerbated inflammatory response to LPS by resident lung cells

BackgroundAgeing is associated with an increased risk of lung infection and chronic inflammatory lung disease. Innate immune responses are the first line of defence in the respiratory tract, however, age-related changes to innate immunity in the lung are not fully described. Both resident haematopoietic cells, such as alveolar macrophages, and non-haematopoeitic cells, such as epithelial and endothelial cells can contribute to inflammatory and immune responses in the lung. In this study we aimed to determine the impact of ageing on early innate responses of resident cells in the lung.ResultsAged and young mice were inoculated intranasally with lipopolysaccharide (LPS). After 4 h, aged mice recruited higher numbers of neutrophils to the airways and lung. This exacerbated inflammatory response was associated with higher concentrations of chemokines CXCL1, CXCL2 and CCL2 in the airways. Next, precision cut lung slices (PCLS) were stimulated ex vivo with LPS for 16 h. Gene expression of Cxcl2, Tnf and Il1b were all higher in PCLS from aged than young mice and higher levels of secretion of CXCL2 and TNF were detected. To determine which lung cells were altered by age, LPS was intranasally administered to aged and young mice and individual populations of cells isolated by FACS. RT-PCR on sorted cell populations demonstrated higher expression of inflammatory cytokines Cxcl2, Ccl2 and Tnf in epithelial cells and alveolar macrophages and higher expression of Cxcl2 by endothelial cells of aged mice compared to young. These differences in expression of pro-inflammatory cytokines did not correspond to higher levels of Tlr4 expression.ConclusionsAgeing leads to a heightened neutrophilic inflammatory response in the lung after LPS exposure, and higher expression and production of pro-inflammatory cytokines by resident lung cells, including alveolar macrophages, epithelial cells and endothelial cells. The responses of multiple resident lung cell populations are altered by aging and contribute to the exacerbated inflammation in the lung following LPS challenge. This has implications for our understanding of respiratory infections and inflammation in older people.

Protective Effects of Tea Tree Oil on Inflammatory Injury of Porcine Intestinal Epithelial Cells Induced by Lipopolysaccharide In Vitro.

Tea tree oil (TTO) improves the intestinal mucosal immunity of weaning piglets, but its underlying mechanism is not clear. We hypothesized that TTO may alleviate inflammatory injury by regulating the function of intestinal epithelial cells. Ileum epithelial cells (IPI-2I) were chosen and an inflammatory injury cell model was generated. The cell viability, cytokine secretion, and gene expression of TLR4 and NF-κB were measured to further evaluate the effects of TTO on the inflammatory injury in immune-stressed cells. The results showed that lipopolysaccharide (LPS; content: ≥30 μg/mL; time: 3 h, 6 h, or 9 h) decreased cell viability (p < 0.01), and 50 μg/mL LPS stimulated for 6 h resulted in an increased secretion of proinflammatory cytokines and a dramatically decreased secretion of anti-inflammatory cytokines (p < 0.05) in IPI-2I cells. Concentrations of 0-0.05% of TTO improved cell viability, while the 0.03% TTO treatment resulted in the highest cell viability and alleviated LPS-induced cell death (p < 0.01). In addition, 0.03% TTO alleviated the LPS-induced increase in the gene expression of IL-1β, TNFα, and IFNγ, as well as the decrease in the expression of IL-10 in IPI-2I cells (p < 0.05). LPS also upregulated the gene expression of TLR4 and NF-κB (p < 0.05); while TTO supplementation alleviated this effect (p < 0.05), 0.03% and 0.05% TTO supplementation had greater effects (p < 0.05). In conclusion, 50 μg/mL LPS stimulated for 6 h can be used to establish an immune-stressed cell model in IPI-2I cell lines, and 0.03% TTO treatment for 6 h alleviated inflammatory injury in the intestinal epithelial cells of pigs.