TLR4 Expression Research Articles

Administration of a Recombinant Fusion Protein of IFN-γ and CD154 Inhibited the Infection of Chicks with Salmonella enterica

The cytokines IFN-γ and CD154 have been well established, and they play pivotal roles in immune protection against Salmonella in mice, but their effects and specific mechanisms in Salmonella-infected chickens are less understood. In this study, we conducted animal experiments to screen the highly immunoprotective chIFN-γ-chCD154 fusion protein compared with single protein chIFN-γ or chCD154 in white Leghorn chickens. The results showed that compared with separate pretreatments with chIFN-γ and chCD154, the fusion protein, chIFN-γ-chCD154, synergistically increased survival of infected chickens, reduced bacterial load in feces and organs, and attenuated pathological damage to the liver and cecum. Pretreatment with chIFN-γ-chCD154 also increased humoral immune responses, expression of the tight junction proteins zo-1, occludin, and claudin-1, and the relative abundance of Enterococcus_cecorum, Lactobacillus_helveticus, and Lactobacillus_agilis, which protect against intestinal inflammation. Compared with single protein pretreatment, chIFN-γ-chCD154 significantly upregulated STAT1, IRF1, and GBP1 in infected chickens while decreasing mRNA expression of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-1β. In summary, damage to the cecal epithelial barrier and the inflammation induced by S. typhimurium infection was alleviated by chIFN-γ-chCD154 pretreatment through a mechanism involving the TLR4/MyD88/NF-κB and IFN-γ/STAT/IRF1/GBP1 pathways.

Sinensetin attenuates LPS-induced acute pulmonary inflammation in mice and RAW264.7 cells by modulating NF-κB p65-mediated immune resistance and STAT3-mediated tissue resilience.

Acute pulmonary inflammation is a severe lower respiratory tract infection. Sinensetin (SIN), a polymethoxyflavone with strong anti-inflammatory properties, is known to ameliorate LPS-induced acute inflammatory lung injury, but its molecular mechanisms are not fully understood. This study aimed to provide insight into the pharmacological mechanisms of SIN in attenuating acute pulmonary inflammation. In LPS-induced inflammation assays in vivo and in vitro, SIN significantly reduced the mRNA levels of inflammatory genes including MCP-1, ICAM1, Ccl3, Ccl4, Ccl5, Ccl7, Cxcl9, Cxcl10, IL1α, IL1β, IL6, IL11, IL18, IL27, TNF-α, IFN-γ, TLR4, MyD88, F4/80, COX2, iNOS, NLRP3, ASC, JAK2, STAT3, STAT4, and Bcl2l1, as well as increased the mRNA levels of anti-inflammatory genes such as IL4, IL10, and IL12α. Besides, SIN markedly decreased the expression of CD68, TLR4, MyD88, phospho-IκBα (S32/S36), phospho-NF-κB p65 (S536), MCP-1, ICAM1, phospho-JAK2 (Tyr1008), phospho-STAT1 (S727), phospho-STAT3 (Y705), and phospho-STAT4 (Y693), inhibited NF-κB p65 translocation into the nucleus, thereby blocking in combination with STAT transcription factors to induce target gene expression. Further GC-MS/MS and LC-MS/MS metabolomic analysis revealed that SIN significantly increased the abundance of anti-inflammatory metabolites, such as L-alanine, L-carnitine, L-glutamic acid, Glycine, and L-cysteine. In conclusion, the results indicated that SIN attenuated LPS-induced acute pulmonary inflammation by modulating NF-κB p65-mediated immune resistance and STAT3-mediated tissue resilience. All these favorable findings presented critical insights into the remarkable abilities and health benefits of SIN in ameliorating inflammatory lung disease.

Prevention of fenitrothion induced hepatic toxicity by saponarin via modulating TLR4/MYD88, JAK1/STAT3 and NF-κB signaling pathways.

Fenitrothion (FEN) is an organophosphate insecticidal agent that is considered as major source of organs toxicity. Saponarin (SAP) is a naturally occurring novel flavone that exhibits a wide range of medicinal properties. The current trial was conducted to evaluate the ameliorative potential of SAP against FEN instigated liver toxicity in rats. Thirty-two male albino rats were apportioned into four groups including control, FEN (10 mg/kg), FEN (10 mg/kg) + SAP (80 mg/kg), and SAP (80 mg/kg) alone treated group. It was revealed that FEN administration upregulated the gene expression of TNF-α, TLR4, IL-1β, MYD88, IL-6, TRAF6, COX-2, NF-κB, JAK1 and STAT3 while reducing the gene expression of IκB. Moreover, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) were increased while the activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), heme-oxygenase-1 (HO-1) and glutathione reductase (GSR) were decreased after FEN exposure. Furthermore, FEN administration notably escalated the levels of hepatic enzymes including alanine transaminase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) whereas reduced the levels of total proteins and albumin. Besides, FEN intake upregulated the levels of Caspase-9, Bax and Caspase-3 while reducing the levels of Bcl-2. Hepatic histology was impaired after FEN intoxication. Nonetheless, SAP treatment remarkably protected the normal state of liver via regulating abovementioned irregularities. Our in-silico analysis confirmed that SAP hold that potential to interact with binding pocket of these proteins, highlighting its ability as a therapeutic compound to alleviate FEN-induced liver damage.

KLF4 enhances transplantation-induced hematopoiesis by inhibiting TLRs and non-canonical NFκB signaling at a steady state.

The transcription factor Krüppel-like factor 4 (KLF4) acts as a transcriptional activator and repressor. KLF4 plays a role in various cellular processes, including the dedifferentiation of somatic cells into induced pluripotent stem cells. Although it has been shown to enhance self-renewal in embryonic and leukemia stem cells, its role in adult hematopoietic stem cells (HSC) remains underexplored. We demonstrate that conditional deletion of the Klf4 gene in hematopoietic cells led to an increased frequency of immunophenotypic hematopoietic stem cells (HSCs) in the bone marrow, along with a normal distribution of lymphoid and myeloid progenitor cells. Non-competitive bone marrow transplants showed normal engraftment and multi-lineage reconstitution, except for monocytes and T cells. However, the loss of KLF4 hindered hematological reconstitution in competitive serial bone marrow transplants, highlighting a critical role for KLF4 in stress-induced hematopoiesis. Transcriptome analysis revealed an upregulation of NFκB2 and toll-like receptors (e.g., TLR4) in Klf4-null HSCs during homeostasis. Flow cytometry and immunoblot analysis confirmed the increased cell surface expression of TLR4 and the activation of NFκB2 in HSCs under homeostatic conditions, whereas NFκB2 expression drops after radiation compared to steady-state levels. Our findings suggest that the constitutive activation of the TLR4-NFκB2 pathway inhibits the ability of HSCs to regenerate blood after transplantation in cytoablated bone marrow.

Protective effects of carnosic acid on growth performance, intestinal barrier, and cecal microbiota in yellow-feathered broilers under lipopolysaccharide challenge.

This research was performed to investigate protective effects of carnosic acid on growth performance, intestinal barrier, and cecal microbiota of lipopolysaccharide-challenged broilers. Three hundred 1-day-old yellow-feathered broilers (male) were allocated randomly into 5 treatments, with 6 replicates per treatment, and 10 birds per replicate cage. Birds in both the control group (CON) and the lipopolysaccharide-challenged group were provided with a basal diet, while others were fed a basal diet supplemented with 20, 40, and 60 mg/kg carnosic acid (CA20, CA40, CA60), respectively. At 17, 19, and 21 days of age, birds were injected intraperitoneally with lipopolysaccharide (500 μg/kg body weight), except those in CON, which were injected with saline. Compared with challenged birds, the CA20, CA40, and CA60 increased (P < 0.05) the final body weight, average daily gain, and average daily feed intake, and the CA40 and CA60 also decreased diarrhea rate. Compared with challenged birds, carnosic acid reduced (P < 0.05) plasmal levels of D-lactic acid and endotoxin, increased (P < 0.05) the villus height to crypt depth ratio, and the number of goblet cells in duodenum. The CA40 and CA60 elevated (P < 0.05) relative expression of cell junction proteins (Claudin-1/-2 and ZO-1/-2/-3) and MUC-2 in duodenum, while decreased (P < 0.05) relative expression of TLR2, TLR4, and the concentrations of IL-6, IL-10, TNF-α, TGF-β1 in duodenum. CA40 also increased (P < 0.05) the α-diversity of the cecal microbiota and boosted (P < 0.05) the relative abundance of beneficial phyla and genera, particularly Firmicutes, Anaerofilum, and Papilibacter. In conclusion, dietary supplementation with carnosic acid showed protective effects on the growth performance and intestinal health in challenged broilers by down-regulating the expression of TLRs (TLR2/4) and inhibiting the production of inflammatory cytokines, strengthening the tight junction in intestinal epithelial cells, and enhancing the diversity of microbiota and the relative abundance of beneficial bacteria. When supplemented to diet of broilers, 40 mg/kg carnosic acid was recommended.

Early embryonic thermal programming and post-hatch flavonoid (Scutellaria baicalensis) supplementation enhanced immune response markers in broiler chickens

IntroductionGenetic selection in broiler chickens has led to increased muscle mass without comparable respiratory and cardiovascular system development, limiting the birds’ capacity to withstand high ambient temperatures and making them vulnerable to heat stress (HS). Early embryonic Thermal Manipulation (TM) has been suggested as an effective and sustainable way to mitigate the adverse effects of HS. This study investigated how these interventions influenced the immune status of broiler chickens exposed to HS.MethodsCobb 500 fertile eggs (n = 600) were incubated according to guidelines. On embryonic day (ED) 12, the eggs were split into two groups: (1) Control, kept at standard temperature until hatch day (ED 21) and (2) Thermal Manipulation (TM), exposed to 38.5°C with 55% humidity for 12 h daily from ED 12 to ED 18. After hatching, chicks were divided into (1) Control, (2) TM, (3) Control under Heat Stress (CHS), (4) TM under Heat Stress (TMHS), (5) Control with Heat Stress and Supplementation (CHSS), and (6) TM with Heat Stress and Supplementation (TMHSS). For the first 21 days, all chicks were raised under normal conditions. From day 22 to day 35, groups CHS, TMHS, CHSS, and TMHSS experienced chronic heat stress (32–33°C for 8 h daily), while the Control and TM groups remained in a thermoneutral environment (22–24°C).Results and discussionTM significantly increased (p &amp;lt; 0.05) AvBD11, IL4, and TLR21 expression in the spleen. TM and baicalein supplementation significantly decreased (p &amp;lt; 0.05) TLR15 expression. In the bursa, TM significantly increased (p &amp;lt; 0.05) IL4 expression. The combination of TM with baicalein significantly increased (p &amp;lt; 0.05) CD3 and decreased (p &amp;lt; 0.05) TLR1 expression. Interestingly, TM alone significantly decreased (p &amp;lt; 0.05) IFNg expression under HS condition. In the thymus, TM significantly decreased (p &amp;lt; 0.05) IL10 and TLR15, while incorporating baicalein with TM decreased (p &amp;lt; 0.05) AvBD6 expression.ConclusionTM improved the immune status of broiler chickens under normal conditions. When combined with baicalein, TM mitigated the negative effects of heat stress by boosting key immune-related gene expression in the spleen, bursa, and thymus.

Chrysanthemum extract mitigates high-fat diet-induced inflammation, intestinal barrier damage and gut microbiota disorder.

An effective intervention for obesity without side effects is needed. Chrysanthemum may be the preferred choice due to its influence in the improvement of glycolipid metabolism. This study assessed the efficacy of chrysanthemum and its flavonoids in mitigating high-fat diet (HFD) induced obesity, focusing on the integrity of the intestinal barrier, inflammation, and gut microbiota. Fifty male C57BL/6J mice were divided into 5 groups randomly: normal control (NC), HFD, HFD with chrysanthemum aqueous extract (CM), HFD with a low-dose flavonoid extract of chrysanthemum (FLL), and HFD with a high-dose flavonoid extract of chrysanthemum (FLH). The results showed that after 9 weeks of intervention with CM, FLL and FLH, the body weight and blood lipid levels of mice were reduced. The chrysanthemum treatment regimens down-regulated the gene expression and protein levels of TLR4, MyD88, TRAF6 and NF-κB, upregulated the gene expression levels of ZO-1 and occludin, and decreased the levels of LPS and diamine oxidase (DAO) in the serum. With CM, FLL and FLH, the levels of the inflammatory factors IL-1β, TNF-α, and IL-6 were decreased, and the abundance of pernicious bacteria Lachnoclostridium, Streptococcus and Enterococcus was decreased. Notably, the purified chrysanthemum flavonoid extract showed greater effects as compared to the CM. The study demonstrated that chrysanthemum extracts could achieve anti-obesity effects by strengthening the intestinal barrier function, relieving inflammation and modulating the gut microbial composition.

Minocycline Ameliorates Staphylococcus aureus-Induced Neuroinflammation and Anxiety-like Behaviors by Regulating the TLR2 and STAT3 Pathways in Microglia

Background: Anxiety disorders are the most common mental illnesses. S. aureus is a Gram-positive opportunistic pathogen most commonly associated with anxiety-like behaviors. Minocycline ameliorates Gram-negative bacterial LPS-induced anxiety-like behaviors by suppressing microglia activation. However, the effects of minocycline on anxiety-like behaviors caused by S. aureus infections have received little attention. In this study, we aimed to investigate the molecular mechanism and effect of minocycline on anxiety-like behaviors caused by S. aureus infection. Methods: BV2 and N9 microglial cells were treated in vitro. The effects of minocycline on lipoteichoic acid (LTA)-stimulated inflammatory responses, STAT3 activation, and GLS1 expression were assessed using Western blotting, and cytokine secretion was determined using an ELISA. A mouse model was used to evaluate the capacity of minocycline to ameliorate anxiety-like behaviors caused by S. aureus infection. Results: We found that ≥100 μmol/L of minocycline remarkably attenuated LTA-induced TLR2 signaling pathway activation and proinflammatory cytokine expression in microglial cells. Minocycline prevented LTA-stimulated STAT3 activation and GLS1 expression in vitro. LTA-induced TLR2, TNF-α, IL-6, and GLS1 expression was markedly reduced by the inhibition of STAT3 phosphorylation. Mice were pretreated with 50 mg/kg of minocycline, significantly attenuating microglial activation and neuroinflammation. Minocycline also effectively alleviated the anxiety-like behaviors induced by S. aureus infection. Conclusions: Our findings indicate that minocycline alleviates S. aureus infection-induced anxiety-like behaviors by suppressing microglia activation.

CD20 and CD19 promote proliferation driven by the IgM-TLR9-L265P MyD88 complex.

The cancer driver mutation L265P MyD88 is found in approximately 30 % of cases in the activated B cell-like subgroup of diffuse large B cell-like lymphoma (ABC DLBCL). L265P MyD88 forms a complex with TLR9 and IgM, referred to as the My-T-BCR complex, to drive proliferation. We here show that the B cell surface molecules CD19 and CD20 enhance proliferation mediated by the My-T-BCR complex. Using the IL-3-dependent Ba/F3 line transduced to express the IgM complex (IgM, CD79a, and CD79b) and TLR9, we observed proliferation in the presence of anti-IgM antibody and the TLR9 ligand CpG-B. TLR9 was constitutively associated with IgM and L252P MyD88. CD19 promoted proliferation with anti-IgM and CpG-B specifically in L252P MyD88-expressing Ba/F3 cells, while CD20 enhanced the proliferation in both wild-type- and L252P MyD88-expressing Ba/F3 cells. Additionally, CD20 uniquely enabled IgM-mediated proliferation in L252P MyD88-expressing Ba/F3 cells. Although CpG-B was not required for this proliferation, TLR9 expression remained indispensable. In the ABC DLBCL line TMD8, anti-IgM Ab mediated growth was impaired by the lack of CD20 and CD19 or of TLR9. Mechanistically, CD19 promoted IgM-dependent AKT phosphorylation, whereas CD20 increased expression of cell surface IgM, thereby enhancing the formation of the IgM-TLR9 complex. These findings suggest that CD19 and CD20 differentially contribute to the proliferation driven by the My-T-BCR complex.

MiR-125b-5p ameliorates ox-LDL-induced vascular endothelial cell dysfunction by negatively regulating TNFSF4/TLR4/NF-κB signaling

BackgroundOxidized low-density lipoprotein (ox-LDL)-induced endothelial cell dysfunction plays a crucial role in the progression of atherosclerosis (AS). Although miR-125b-5p is known to be involved in cardiovascular and cerebrovascular disorders, its function in ox-LDL-induced endothelial injury is still not well understood.MethodsAn in vitro AS cell model was established by exposing human umbilical vein endothelial cells (HUVECs) to 100 µg/mL ox-LDL for 24 h. A series of functional assays, including CCK-8 assay, flow cytometry, MDA and SOD kits, capillary-like network formation assay and ELISA assay were performed in vitro. TNFSF4/TLR4/NF-κB pathway-related protein expressions were measured by Western blot. Molecular mechanisms were elucidated through quantitative real-time PCR, western blot analysis, and luciferase reporter assays.ResultsOur investigation revealed that exposure to ox-LDL led to a downregulation in miR-125b-5p, while upregulating the expression of tumor necrosis factor (ligand) superfamily, member 4 (TNFSF4), TLR4, p-p65 and p-IkBa in HUVECs in a dose-dependent manner. We confirmed TNFSF4 as a direct target of miR-125b-5p. Ox-LDL exposure led to decreased cell viability and angiogenic capacity, along with increased apoptosis, inflammation, and oxidative stress in HUVECs. These effects were reversed by overexpressing miR-125b-5p or knocking down TNFSF4. Overexpression of TNFSF4 significantly reversed the effects brought about by miR-125b-5p in HUVECs exposed to ox-LDL. Moreover, miR-125b-5p inactivated the TLR4/NF-κB signaling pathway by negatively regulating TNFSF4.ConclusionsIn summary, our findings demonstrate that miR-125b-5p possessed an anti-inflammatory and anti-apoptosis against ox-LDL-induced HUVEC injury by regulating the TNFSF4/TLR4/NF-κB signaling, indicating that miR-125b-5p may have an important therapeutic function for AS.

CD209d/e promotes inflammation and lung injury during influenza virus infection.

Influenza virus infects millions each year, contributing greatly to human morbidity and mortality. Upon viral infection, pathogen-associated molecular patterns activate pattern recognition receptors on host cells, triggering an immune response. The CD209 protein family, homologs of DC-SIGN (dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin), is thought to modulate immune responses to viruses. The effects of the mouse functional DC-SIGN homolog CD209d/e on the lung immune responses during influenza viral infection are not known. Therefore, we generated mice that lack both CD209d and e isoforms to determine the role in influenza viral infection. We infected wild-type and CD209d/e gene-deficient (CD209d/e-/-) mice with influenza virus and measured the cellular response in bronchoalveolar lavage, the expression of proinflammatory cytokines, antiviral genes, toll-like receptors (TLRs) in the lung, and lung pathology. We found CD209d/e-/- mice had decreased viral burden, TLR3 and TLR9 expression, interferon response, macrophages in bronchoalveolar lavage, and parenchymal lung inflammation compared with control mice. We also found less influenza viral uptake in alveolar macrophages and bone marrow-derived macrophages isolated from CD209d/e-/- mice when compared with control mice. We further investigated the role CD209d/e by treating bone marrow-derived macrophages from control and CD209d/e-/- mice with TLR agonists. We found that lacking CD209d/e decreased the expression of TLR3, TLR9, RIG1, STAT1, and STAT2 compared with controls. Collectively these results show that CD209d/e plays an important role in viral sensing/uptake and inflammatory immune responses during influenza viral infection.

P0180 Bifidobacterium longum alleviates dextran sulfate sodium-induced colitis through the differential regulation of Toll-like receptor 5 on enteric glial cells

Abstract Background Inflammatory bowel disease (IBD) is a chronic immune-associated disease arising from gastrointestinal dysbiosis and subsequent immunological dysfunction. Numerous studies have reported the beneficial effects of probiotics, such as Bifidobacterium longum (BL), on IBD, while the related mechanism behind these effects have not been fully elucidated. Enteric nervous system abnormalities have increasingly been tied to the pathogenesis of IBD, with a particular focus on the role of enteric glial cells (EGC) in this context. In this study, we aim to explore the mechanism of probiotics in the treatment of IBD from the perspective of EGC. Methods On base of C57BL/6 mouse, TLR5 of EGC knockdown mouse model (TLR5 KD mouse) was constructed by adeno-associated virus (serum type 6) (AAV6) which carried the interference sequence of TLR5 gene (AAV6-TLR5-RNAi). Experimental colitis was induced by dextran sulfate sodium (DSS). Mice were treated with phosphate buffer solution (PBS) or BL. Disease activity index (DAI) and histological score were applied to assess the severity of colitis. The level of GDNF、IL-1β of colonic tissue were measured by ELISA. The expression of glial fibrillary acidic protein (GFAP)、toll-like receptor 5 (TLR5)、cleaved caspase-3 of colonic tissue were tested by immunofluorescence staining and quantitative PCR (qPCR). EGC cell line, EGC CRL2690, was exposed to probiotic (Bifidobacterium longum; BL) bacteria and pathogenic (Enterohemorrhagic Escherichia coli; EHEC). Cell apoptosis rate was assessed by flow cytometry. TLRs expression in EGC CRL2690 were evaluated at both baseline and after exposure to bacteria by qPCR and western blot analysis. GDNF and IL-1β release from EGC CRL2690, following exposure to bacteria, were measured in the presence or absence of specific TLR and pathway inhibitors. Results The results showed that BL ameliorated DSS-induced colitis accompanied by increased expression of GDNF, while pathogenic EHEC aggravated DSS-induced colitis combined with decreased expression of GDNF. Mechanistically, BL could up-regulate the secretion of GDNF via TLR5/p38 MAPK pathway in EGC, which was different from EHEC, which could up-regulate the secretion of IL-1β through TLR2/NF-κB pathway. Conclusion Our results suggested a new possible mechanism whereby probiotics ameliorate DSS-induced colitis in mice by regulating EGC via TLR5, namely, BL promotes GDNF secretion in EGC through activation of p38 MAPK signaling pathway by TLR5. Meanwhile, our results suggested that probiotic, pathogenic bacteria appeared to influence the development of intestinal inflammation by differentially modulating the secretion function of EGC via different TLRs.

P0170 The presence of creeping fat in a TNBS-induced colitis model is associated with greater acute inflammation and bacterial translocation

Abstract Background The hyperplasia of the mesenteric adipose tissue in Crohn’s disease, known as creeping fat, is associated with transmural lesions and the translocation of microbial antigens. In a rat model of TNBS-induced colitis, 50% of the animals presented adipose hyperplasia. Macroscopic evaluations showed that these animals had a more advanced progression of the disease, and colonoscopy could predict the presence of fat two days before euthanasia (L Clua et al, ECCO, 2024). Methods Colitis was induced using TNBS (n=10; half female), with the animals being sacrificed after five days. Tissue from the lesion along with mesenteric adipose tissue was obtained for histopathological staining and evaluation (haematoxylin-eosin, Masson’s trichrome, and Gram). Samples of intestinal mucosa and mesenteric fat were preserved in RNAlater to assess the expression of inflammatory markers by qRT-PCR. Results were statistically compared between animals with and without creeping fat, including sex. Results In histopathological analysis, using a validated scoring system, animals with creeping fat showed significantly greater severity of acute inflammation at the transmural level, with an increase in cryptitis and a more pronounced and extensive fibrosis pattern. Additionally, colonies of Gram-positive cocci were detected in all animals with creeping fat. In contrast, animals without fat hyperplasia exhibited appreciable adipogenesis in only two individuals, with an average of 105 adipocytes per field, compared to 185 in animals with creeping fat. qRT-PCR revealed higher expression of Il1b, Il6, Il10, Nod2, and Tlr2 in the intestinal mucosa and hyperplastic mesenteric fat of animals with creeping fat. A significant increase in Tnfa was also observed in hyperplastic adipose tissue, while the expression of Tgfbr1 was higher in the intestinal mucosa of animals without creeping fat. No differences were found between sexes. Conclusion The results indicated a more intense acute inflammatory and fibrotic component in animals with creeping fat, consistent with the macroscopic observations. The hyperplasia and inflammation observed in the mesenteric adipose tissue may be provoked by bacterial translocation, whereas animals without hyperplasia present a component influenced by TGF-β1 activity that could help counteract the inflammation. References L Clua, R Suau, M Guasch, D Monfort-Ferré, I Vañó, C Serena, E Domènech, R Bartolí, J Manyé, P022 Development of a novel experimental model for studying creeping fat in Crohn’s Disease, Journal of Crohn’s and Colitis, Volume 18, Issue Supplement_1, January 2024, Pages i275–i276, https://doi.org/10.1093/ecco-jcc/jjad212.0152

Alleviation of Acute Heat Stress in Broiler Chickens by Dietary Supplementation of Polyphenols from Shredded, Steam-Exploded Pine Particles

Reducing the detrimental effects of heat stress (HS) in poultry is essential to minimize production losses. The present study evaluates the effects of dietary polyphenols prepared from underutilized wood byproducts on the growth, gut health, and cecal microbiota in broilers subjected to acute heat stress (AHS). One hundred eight one-day-old Indian River broilers were fed with 0%, 0.5%, or 1% polyphenols from shredded, steam-exploded pine particles (PSPP) in their diet. On the 37th day, forty birds were equally distributed among four groups containing either a control diet at thermoneutral temperatures (NT0%) or AHS temperatures with 0% (AHS0%), 0.5% (AHS0.5%), and 1% (AHS1%) PSPP-supplemented diets. The temperature in the NT room was maintained at 21.0 °C, while, in the AHS room, it was increased to 31 °C. AHS negatively influenced performance parameters and increased rectal temperature (RT) in broilers. The AHS0% group showed a higher expression of NOX4, HSP-70, and HSP-90 genes, while the expression was lower in PSPP-supplemented birds. In the jejunum, mRNA expression of SOD was increased in all the birds under AHS compared to NT. The expression of the CLDN1 and ZO2 genes was increased in AHS0%, while that of the ZO1 and MUC2 genes was increased in PSPP-supplemented birds. HS tends to increase TLR2 and TLR4 gene expression in chickens. The significantly modified genera were Bariatricus, Sporobacter, Sporanaerobacter, and Natranaerovirga. Concludingly, AHS negatively influences the performance parameters, RT, stress, gut-health-related genes, and pathogenic penetration, but PSPP supplementation reduces its bad impact by overcoming the stress and gut-health-related genes, increasing favorable bacterial abundance and reducing pathogenic penetration in chickens.

PLGA co-loaded Salvia miltiorrhiza polysaccharide and Mn2+ as an adjuvant to induce potent immunity.

Developing a novel and potent adjuvant with excellent biocompatibility for immune response augmentation is crucial for enhancing vaccine efficacy. Here, we prepared a stable PLGA nanoparticle by encapsulating MnCl2/Salvia miltiorrhiza polysaccharide (MS-PLGA) and employed it as an adjuvant in the model antigen OVA (MS-PLGA-OVA) to elicit potent immunity. The biological experiments indicated that the MS-PLGA-OVA could effectively recruit APCs to the injection site and provoke long-term antibodies. Compared with the conventional Alum adjuvanted group, the MS-PLGA-OVA increased the IgG2a antibody titers and CD8+T cells maturation, triggering cytotoxic T lymphocyte response and inducing the activation of memory T cells. Importantly, the MS-PLGA could up-regulate the expression of TLRs and cGAS-STING pathway-related genes, thus increasing the DCs maturation, as well as the secretion of interleukin and IFN-β. Collectively, the MS-PLGA system may provide a novel and efficient adjuvant platform for various prophylactic vaccines and insights for the development of the next-generation nano adjuvant.

Afobazole alleviates streptozotocin-induced diabetic nephropathy in rats via hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic properties: Role of the S1R/Nrf2 antioxidant axis.

Sigma-1 receptor (S1R) activation was recently identified as a promising target for preventing diabetic nephropathy (DN) by mitigating hypoxia, oxidative stress, and inflammation. This study aimed to investigate the potential reno-protective effect of the S1R agonist afobazole against streptozotocin (STZ)-induced DN in rats compared to metformin. Rats were split into six groups: the normal control group; the diabetic control group received STZ (55mg/kg i.p.); the other four groups received STZ and were treated with different doses of either afobazole (10, 15, and 20mg/kg) or metformin (200mg/kg). Metabolic parameters and renal function were assessed. Expression levels of oxidative stress markers and inflammatory cytokines were measured using ELISA, apoptosis-related proteins were evaluated using immunohistochemistry, and gene expression of S1R, Nrf2, NF-κB, and TLR-4 was determined. Histopathological analysis was performed on kidney tissues. Both afobazole and metformin exerted hypoglycemic effects, alleviating renal injury, reducing blood urea nitrogen (BUN) and serum creatinine, and restoring oxidant/antioxidant balance in diabetic rats. Both treatments boosted renal S1R and Nrf2 levels, suppressed inflammatory proteins and cytokines, and reduced apoptotic features. The study revealed that afobazole provided nephroprotection in STZ-induced DN through a hypoglycemic, antioxidant, anti-inflammatory, and anti-apoptotic potential mediated by activating the S1R/Nrf2 antioxidant axis. The 15mg/kg dose elicited the most pronounced nephroprotective effects, outperforming other treatment groups.

Sustained Release of HIF-2α Inhibitors Using Biodegradable Porous Silicon Carriers for Enhanced Immunogenic Cell Death of Malignant Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine skin cancer with limited treatment options, often associated with Merkel cell polyomavirus (MCPyV) and marked by hypoxic tumor microenvironments that promote resistance to therapies. Belzutifan, an FDA-approved hypoxia-inducible factor-2α (HIF-2α) inhibitor, has shown promise in inhibiting tumor growth; however, its clinical efficacy is hindered by its low solubility, rapid clearance, and limited bioavailability. In this study, we present a strategy using porous silicon (pSi) microparticles and nanoparticles as carriers for the sustained delivery of benzoate to MCC cells. The pSi carriers were engineered to securely encapsulate and gradually release belzutifan, overcoming the limitations of free drug administration. Microparticles provided sustained extracellular release, while nanoparticles enabled efficient intracellular delivery, enhancing HIF-2α inhibition. Moreover, the use of biodegradable silicon particles enables long-term consistent release of belzutifan over 10 days in vitro with a single dose administration in the tumor microenvironment, while free belzutifan is rapidly deactivated within 1 day postadministration. In vitro studies demonstrated significant immunogenic cell death (ICD) in MCC cells, marked by the cytosolic localization of HMGB1 and elevated expression of pro-inflammatory cytokines as well as strong upregulation of TLR9. Particularly, the increased TLR9 expression in both MCC cell lines with pSi carrier treatment reinforces immune activation through toll-like receptor signaling, enhancing both innate and adaptive immune responses within the tumor microenvironment. These findings indicate that pSi carriers not only enhance belzutifan's stability and release profile but also amplify antitumor immune responses within the tumor microenvironment. Our results suggest that belzutifan-loaded pSi carriers offer a potent and targeted therapeutic strategy for MCC, potentially addressing key challenges in cancer immunotherapy by combining HIF-2α inhibition with robust immune activation. This platform highlights the universal utility of pSi-based delivery systems to advance MCC treatment with implications for broader cancer therapies.

Methyl 3-Bromo-4,5-dihydroxybenzoate Attenuates Inflammatory Bowel Disease by Regulating TLR/NF-κB Pathways.

Inflammatory bowel disease (IBD) is characterized by uncontrolled, chronic relapsing inflammation in the gastrointestinal tract and has become a global healthcare problem. Here, we aimed to illustrate the anti-inflammatory activity and the underlying mechanism of methyl 3-bromo-4,5-dihydroxybenzoate (MBD), a compound derived from marine organisms, especially in IBD, using a zebrafish model. The results indicated that MBD could inhibit the inflammatory responses induced by CuSO4, tail amputation and LPS in zebrafish. Furthermore, MBD notably inhibited the intestinal migration of immune cells, enhanced the integrity of the gut mucosal barrier and improved intestinal peristalsis function in a zebrafish IBD model induced by trinitro-benzene-sulfonic acid (TNBS). In addition, MBD could inhibit ROS elevation induced by TNBS. Network pharmacology analysis, molecular docking, transcriptomics sequencing and RT-PCR were conducted to investigate the potential mechanism. The results showed that MBD could regulate the TLR/NF-κB pathways by inhibiting the mRNA expression of TNF-α, NF-κB, IL-1, IL-1β, IL6, AP1, IFNγ, IKKβ, MyD88, STAT3, TRAF1, TRAF6, NLRP3, NOD2, TLR3 and TLR4, and promoting the mRNA expression of IL4, IκBα and Bcl-2. In conclusion, these findings indicate that MBD could be a potential candidate for the treatment of IBD.

Pir-hsa-216911 inhibit pyroptosis in hepatocellular carcinoma by suppressing TLR4 initiated GSDMD activation

Hepatocellular carcinoma (HCC) is a global health concern, ranking as the fourth leading cause of cancer-related deaths worldwide. However, the role of piwi-interacting RNAs (piRNAs) in HCC processes has not been extensively explored. Through small RNA sequencing, our study identified a specific piRNA, pir-hsa-216911, which is highly expressed in HCC cells. This overexpression of pir-hsa-216911 promotes HCC cell invasion and inhibits cell death, particularly pyroptosis. Knocking out pir-hsa-216911 led to increased cell pyroptosis activity, resulting in the activation of caspase-1 and GSDMD. Further analysis revealed that pir-hsa-216911 targets and suppresses TLR4, a key gene associated with pyroptosis in HCC. In the Huh7 cell line, pir-hsa-216911 knockout confirmed its role in suppressing the TLR4/NFκB/NLRP3 pathway by silencing TLR4. Knocking out pir-hsa-216911 significantly inhibited the formation of Huh7 xenograft tumor. In HCC patients, pir-hsa-216911 was highly expressed in HCC tumor samples with steatosis, suppressing TLR4 expression and inhibiting GSDMD activation. This study introduces pir-hsa-216911 as a new high-expressing piRNA in HCC, which inhibits pyroptosis by silencing TLR4 to suppress GSDMD activation. These findings have significant implications for HCC molecular subtyping and as a potential target for cancer therapy.

Citronellal Alleviates Insulin Resistance in High-Fat Diet/Streptozocin Model: Role of Asprosin/Olfactory Receptor Axis.

Ectopic olfactory receptors are expressed in nonolfactory tissues and perform diverse roles including regulation of glucose homeostasis. We explored the effect of citronellal treatment on olfactory receptor 4M1 subtype (OR4M1) signaling in insulin resistance and Type II diabetes in rats. We aimed to validate the anti-diabetic effect of citronellal through Asprosin/OR4M1 modulation. Exploring new antidiabetics and pharmacological targets is important to improve quality of life and limit complications. The model was established in Sprague-Dawley rats by a high-fat diet for 4weeks followed by a single low-dose streptozotocin (STZ) (35 mg/kg/ip). One week after STZ injection, oral citronellal (100mg/kg) was administered for 4weeks. Citronellal lowered serum glucose and triglycerides and ameliorated OGTT and HOMA-IR results. Docking results revealed that citronellal blocked the Asprosin binding site at OR4M1. The hepatic expression of OR4M1 and Asprosin was reduced. Citronellal lowered cAMP levels causing attenuated levels of protein kinase A and downstream gluconeogenic enzymes: glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. Citronellal also inhibited the expression of hepatic TLR-4 and inhibited JNK phosphorylation. Citronellal attenuated hepatic levels of NF-κB, p-NF-κB, and downstream proteins MCP-1 and TNF-α. These results suggest that citronellal alleviates insulin resistance by mitigating Asprosin/OR4M1 and Asprosin/TLR4/JNK signaling.