Proinflammatory Cytokine mRNA Research Articles

Nobiletin derivative, 5-acetoxy-6,7,8,3’,4’-pentamethoxyflavone, inhibits neuroinflammation through the inhibition of TLR4/MyD88/MAPK signaling pathways and STAT3 in microglia

Objective Microglia in the central nervous system regulate neuroinflammation that leads to a wide range of neuropathological alterations. The present study investigated the anti-neuroinflammatory properties of nobiletin (Nob) derivative, 5-acetoxy-6,7,8,3′,4′-pentamethoxyflavone (5-Ac-Nob), in lipopolysaccharide (LPS)-activated BV2 microglia. Materials and Methods By using the MTT assay, Griess method, flow cytometry, and enzyme-linked immunosorbent assay (ELISA), we determined the cell viability, the levels of nitric oxide (NO), reactive oxygen species (ROS), and pro-inflammatory factors (interleukin 1 beta; IL-1β, interleukin 6; IL-6, tumor necrosis factor alpha; TNF-α and prostaglandin E2; PGE2) in LPS-stimulated BV2 microglia. Toll-like receptor 4 (TLR4)-mediated myeloid differentiation primary response gene 88 (MyD88)/nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK) signaling pathway and signal transducer and activator of transcription 3 (STAT3) were measured by western blotting. Analysis of NO generation and mRNA of pro-inflammatory cytokines was confirmed in the zebrafish model. Results 5-Ac-Nob reduced cell death, the levels of NO, ROS, inducible nitric oxide synthase (iNOS), cyclooxygenase 2 (COX-2), and pro-inflammatory factors in LPS-activated BV-2 microglial cells. TLR4-mediated MyD88/NF-κB and MAPK pathway (p38, ERK and JNK) after exposure to 5-Ac-Nob was also suppressed. Moreover, 5-Ac-Nob inhibited phosphorylated STAT3 proteins expression in LPS-induced BV-2 microglial cells. Furthermore, we confirmed that 5-Ac-Nob decreased LPS-induced NO generation and mRNA of pro-inflammatory cytokines in the zebrafish model. Conclusions Our findings suggest that 5-Ac-Nob represses neuroinflammatory responses by inhibiting TLR4-mediated signaling pathway and STAT3. As a result of these findings, 5-Ac-Nob has potential as an anti-inflammatory agent against microglia-mediated neuroinflammatory disorders.

Effects of the Dual FAAH/MAGL Inhibitor AKU-005 on Trigeminal Hyperalgesia in Male Rats.

The inhibition of endocannabinoid hydrolysis by enzymatic inhibitors may interfere with mechanisms underlying migraine-related pain. The dual FAAH/MAGL inhibitor AKU-005 shows potent inhibitory activity in vitro. Here, we assessed the effect of AKU-005 in a migraine animal model based on nitroglycerin (NTG) administration. Male rats were treated with AKU-005 (0.5 mg/kg, i.p.) or vehicle 3 h after receiving NTG (10 mg/kg, i.p.) or NTG vehicle. One hour later, rats were subjected to the open field test followed by the orofacial formalin test. At the end of the test, we collected serum samples for assessing calcitonin gene-related peptide (CGRP) levels as well as meninges, trigeminal ganglia, and brain areas to assess mRNA levels of CGRP and pro-inflammatory cytokines, and endocannabinoid and related lipid levels. AKU-005 reduced NTG-induced hyperalgesia during the orofacial formalin test but did not influence NTG-induced changes in the open field test. It significantly reduced serum levels of CGRP, CGRP, and pro-inflammatory cytokine mRNA levels in the meninges, trigeminal ganglia, and central areas. Surprisingly, AKU-005 caused no change in endocannabinoids and related lipids in the regions evaluated. The present findings suggest that AKU-005 may have anti-migraine effects by reducing CGRP synthesis and release and the associated inflammatory events. This effect, however, does not seem mediated via an interference with the endocannabinoid pathway.

Unrevealing the therapeutic potential of artesunate against emerging zoonotic Babesia microti infection in the murine model.

Babesiosis, a zoonotic blood protozoal disease, threatens humans and animals and is difficult to treat due to growing antimicrobial resistance. The study aimed to investigate the therapeutic efficacy of artesunate (AS), a well-known derivative of artemisinin, against Babesia microti (B. microti) using a murine infection model. Male BALB/c mice (6 weeks old; 15 per group) were chosen and randomly divided into 1) the control group, 2) the B. microti group, and 3) the B. microti + artesunate treatment groups. AS treatment at 2 mg/kg, 4 mg/kg, and 8 mg/kg of body weight significantly (p < 0.05) reduced the B. microti load in blood smears in a dose-dependent manner. Additionally, AS treatment mitigated the decrease in body weight and restored the normal state of the liver and spleen viscera index compared to the B. microti-infected group after 28 days. Hematological analysis revealed significant increases in RBC, WBC, and PLT counts post-AS treatment compared to the B. microti-infected group. Furthermore, AS administration resulted in significant reductions in total protein, bilirubin, ALT, AST, and ALP levels, along with reduced liver and spleen inflammation and lesions as observed through histopathological analysis. AS also elicited dose-dependent changes in mRNA and protein expression levels of apoptotic, proinflammatory, and anti-inflammatory cytokines in the liver compared to the control and B. microti-infected groups. Immunolabeling revealed decreased expression of apoptotic and inflammation-related proteins in AS-treated hepatic cytoplasm compared to the B. microti-infected group. AS also in dose-dependent manner decreased apoptotic protein and increased Bcl-2. Overall, these findings underscore the potential of AS as an anti-parasitic candidate in combating B. microti pathogenesis in an in vivo infection model, suggesting its promise for clinical trials as a treatment for babesiosis.

Molecular docking, dynamics, and preclinical experimental studies identify Morin hydrate as a potent PPAR-γ and NRF-2 transcription factor agonist that mitigates colon inflammation

Introduction: Peroxisome proliferator-activated receptor gamma (PPAR-γ) plays a vital role in regulating intestine inflammation. PPAR-γ is highly expressed in the adipose tissue and the colon. The nuclear erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein1 (Keap1) system protects cells from oxidative stress and inflammation. Therefore, PPAR-γ and Nrf2 ligands offer a drug-targeting system that can be exploited to treat inflammatory bowel diseases (IBDs), especially ulcerative colitis. IBD prevalence is on the rise globally. Current mainstream therapies for IBD offer protection but with many side effects; therefore, up to 30% of IBD patients turn to alternate therapy using bioactive phytochemicals. Morin hydrate, a naturally occurring bioflavonoid in many fruits, stems, and leaves of the Moraceae family plant, possesses potent anti-inflammatory properties. The docking studies reveal that it is a potent activator of the PPAR-γ and Nrf2 transcription factors. Therefore, in this study, we investigated the role of morin hydrate modulating colon epithelial PPAR-γ transcription in colon inflammation. Aim: Therefore, this study aims to investigate morin hydrate’s anti-inflammatory properties using both in vivo and in vitro models of colon inflammation. Methodology: C57BL/6J black mice were administered 2% dextran sodium sulfate (DSS) morin hydrate administered at concentrations of 25 &amp; 50mg/kg body for in vivo studies. The DAI, colon length, MPO, and histology changes were determined. Proinflammatory cytokines (IL-1β, IL-6, TNF-α, &amp; IL17A) were measured using ELISA and mRNA using real-time PCR. PPAR-γ and Nrf2 expression and phosphoNF-κB/NF-κB expression were estimated. HT-29 cells were cultured in DMEM media and were stimulated using TNF-α (1ng/ml). Different concentrations of morin hydrate were treated to HT-29 cells to determine a safe dose range for further in vitro experiments by estimating cell toxicity assay. CXCL-1, IL-8, and mRNA levels were determined using real-time PCR after morin hydrate co-treatment. Morin hydrate effect on PPAR-γ and Nrf2 promoter activity was determined using full-length promoter transfection studies. Results: Morin treatment significantly ( p&lt;0.01) decreased DAI, MPO level, and restored colon length dose-dependently in the treated group. Histological scoring for colonic inflammation was significantly ( p&lt;0.001) improved upon morin treatment. Morin treatment also inhibited the proinflammatory cytokines (IL-1β, IL-6, TNF-α, &amp; IL17A) significantly ( p&lt;0.01) both at protein and mRNA levels. Morin significantly inhibited COX-2 and iNOS both at protein and mRNA levels and also decreased tissue nitrite levels. Morin also activated PPAR-γ protein expression NRF2 nuclear translocation and inhibited NF-κB expression in DSS-administered mice. Morin significantly decreased antioxidant surrogate markers superoxide dismutase (SOD) and catalase activity. Morin significantly ( p&lt;0.01) decreased proinflammatory cytokine (CXCL-1 &amp; IL-8) mRNA expression when HT-29 cells were challenged with TNF-α. Morin also increased PPAR-γ and Nrf2 promoter activity. Conclusion: Morin hydrate is a potent PPAR-γ and Nrf2 transcription factor activator that mitigates colon inflammation through its anti-inflammatory and antioxidant activities. This is supported from UAE University grant, fund number 12R006 and 12R169. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Deciphering Microbiome, Transcriptome, and Metabolic Interactions in the Presence of Probiotic Lactobacillus acidophilus against Salmonella Typhimurium in a Murine Model.

Salmonella enterica serovar Typhimurium (S. Typhimurium), a foodborne pathogen that poses significant public health risks to humans and animals, presents a formidable challenge due to its antibiotic resistance. This study explores the potential of Lactobacillus acidophilus (L. acidophilus 1.3251) probiotics as an alternative strategy to combat antibiotic resistance associated with S. Typhimurium infection. In this investigation, twenty-four BALB/c mice were assigned to four groups: a non-infected, non-treated group (CNG); an infected, non-treated group (CPG); a group fed with L. acidophilus but not infected (LAG); and a group fed with L. acidophilus and challenged with Salmonella (LAST). The results revealed a reduction in Salmonella levels in the feces of mice, along with restored weight and improved overall health in the LAST compared to the CPG. The feeding of L. acidophilus was found to downregulate pro-inflammatory cytokine mRNA induced by Salmonella while upregulating anti-inflammatory cytokines. Additionally, it influenced the expression of mRNA transcript, encoding tight junction protein, oxidative stress-induced enzymes, and apoptosis-related mRNA expression. Furthermore, the LEfSe analysis demonstrated a significant shift in the abundance of critical commensal genera in the LAST, essential for maintaining gut homeostasis, metabolic reactions, anti-inflammatory responses, and butyrate production. Transcriptomic analysis revealed 2173 upregulated and 506 downregulated differentially expressed genes (DEGs) in the LAST vs. the CPG. Functional analysis of these DEGs highlighted their involvement in immunity, metabolism, and cellular development. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis indicated their role in tumor necrosis factor (TNF), mitogen-activated protein kinase (MAPK), chemokine, Forkhead box O (FOXO), and transforming growth factor (TGF-β) signaling pathway. Moreover, the fecal metabolomic analysis identified 929 differential metabolites, with enrichment observed in valine, leucine, isoleucine, taurine, glycine, and other metabolites. These findings suggest that supplementation with L. acidophilus promotes the growth of beneficial commensal genera while mitigating Salmonella-induced intestinal disruption by modulating immunity, gut homeostasis, gut barrier integrity, and metabolism.

Atractylodin, β-eudesmol, and (+)-hinesol in Atractylodes chinensis rhizomes improve glomerular injuries in high immunoglobulin A mice

Background: The rhizome of Atractylodes chinensis (Asteraceae), a crude drug of Japanese Kampo medicines, has been administered to patients with edema, nephrotic syndrome, and gastrointestinal disorders. Essential oils, such as sesquiterpenoids (e.g., β-eudesmol and hinesol) and atractylodin, are rich in the rhizomes. Previously, we discovered that atractylodin, a polyacetylene compound, found in an ethyl acetate (EtOAc)-soluble fraction from a methanol extract of A. chinensis rhizomes, possessed marked anti-inflammatory activities. Oral administration of the EtOAc-soluble fraction reduced immunoglobulin A (IgA) deposition in the renal glomeruli of high immunoglobulin A (HIGA) mice, a model of human IgA nephropathy. An increased serum IgA forms immune complexes and causes the deposition on renal glomeruli, leading to inflammation by the complement-mediated pathway. Objective: The aim of this study is to identify the compounds that reduce IgA deposition in an EtOAc-soluble fraction of A. chinensis rhizomes. Methods: Metabolites in the serum of HIGA and control BALB/c mice were analyzed by gas chromatography–mass spectrometry. A standard diet including each compound was fed to HIGA and BALB/c mice for 20 weeks to evaluate the improvement of glomerular IgA deposition. Results: Metabolomic analysis of serum suggested that the HIGA mice exhibit a state near the early stage of chronic kidney disease, compared with the BALB/c mice. When mice were orally administered each hydrophobic compound of A. chinensis rhizomes, it was revealed that atractylodin, as well as β-eudesmol and (+)-hinesol, efficiently inhibited glomerular IgA deposition. Furthermore, the renal levels of complement component 3 (C3) and proinflammatory cytokine mRNAs were decreased, when the hydrophobic compounds were orally administered to HIGA mice. Conclusion: Atractylodin, β-eudesmol, and (+)-hinesol may inhibit glomerular IgA deposition, probably by attenuating complement-mediated injuries and suppressing proliferation of mesangial cells in the renal glomeruli. These compounds might improve the pathological findings of human IgA nephropathy. Keywords: Atractylodes chinensis; Crude drug; Immunoglobulin A nephropathy; Metabolomics; Complement

Anti-Inflammatory and Antioxidant Effects of Rosmarinic Acid Trimetallic (Cu0.5Zn0.5Fe2O4) Nanoparticles.

Newly, green metallic-nanoparticles (NPs) have received scientists' interest due to their wide variable medicinal applications owned to their economical synthesis and biologically compatible nature. In this study, we used rosmarinic acid (RosA) to prepare Cu0.5Zn0.5FeO4 NPs and later encapsulated them using PEG polymer. Characterization of NPs was done using the XRD method and SEM imaging. Further, we explored the encapsulated NPs for anti-inflammatory properties by downregulating the expression of pro-inflammatory cytokines mRNA in LPS-stimulated Raw 264.7 cells. Besides, employing DPPH, NO and ABTS radical scavenging assays to examine the antioxidant activity of the synthesized Cu0.5Zn0.5FeO4 NPs. Cu0.5Zn0.5FeO4 NPs revealed moderate antioxidant activity by scavenging DPPH and nitric oxide. We demonstrated that the NPs showed high potential anti-inflammatory activity by suppressing the mRNA and protein levels of pro-inflammatory cytokines in a dose-dependent manner, in LPS-induced Raw 264.7 cells. To our best knowledge, this is the first report where RosA was found to be a suitable phyto source for the green synthesis of Cu0.5Zn0.5FeO4 NPs and their in vitro anti-inflammatory and antioxidant effects. Taken together, our findings suggest that the RosA is a green resource for the eco-friendly synthesis of Cu0.5Zn0.5FeO4/PEG NPs, which further can be employed as a novel anti-inflammatory therapeutic agent.

P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

BackgroundGuillain–Barré syndrome (GBS), a post-infectious, immune-mediated, acute demyelinating disease of the peripheral nerves and nerve roots, represents the most prevalent and severe acute paralyzing neuropathy. Purinergic P2X7 receptors (P2X7R) play a crucial role in central nervous system inflammation. However, little is known about their role in the immune-inflammatory response within the peripheral nervous system.MethodsInitially, we assessed the expression of purinergic P2X7R in the peripheral blood of patients with GBS using flow cytometry and qRT-PCR. Next, we explored the expression of P2 X7R in CD4+ T cells, CD8+ T cells, and macrophages within the sciatic nerves and spleens of rats using immunofluorescence labeling and flow cytometry. The P2X7R antagonist brilliant blue G (BBG) was employed to examine its therapeutic impact on rats with experimental autoimmune neuritis (EAN) induced by immunization with the P0180 − 199 peptide. We analyzed CD4+ T cell differentiation in splenic mononuclear cells using flow cytometry, assessed Th17 cell differentiation in the sciatic nerve through immunofluorescence staining, and examined the expression of pro-inflammatory cytokine mRNA using RT-PCR. Additionally, we performed protein blotting to assess the expression of P2X7R and NLRP3-related inflammatory proteins within the sciatic nerve. Lastly, we utilized flow cytometry and immunofluorescence labeling to examine the expression of NLRP3 on CD4+ T cells in rats with EAN.ResultsP2X7R expression was elevated not only in the peripheral blood of patients with GBS but also in rats with EAN. In rats with EAN, inhibiting P2X7R with BBG alleviated neurological symptoms, reduced demyelination, decreased inflammatory cell infiltration of the peripheral nerves, and improved nerve conduction. BBG also limited the production of pro-inflammatory molecules, down-regulated the expression of P2X7R and NLRP3, and suppressed the differentiation of Th1 and Th17 cells, thus protecting against EAN. These effects collectively contribute to modifying the inflammatory environment and enhancing outcomes in EAN rats.ConclusionsSuppression of P2X7R relieved EAN manifestation by regulating CD4+ T cell differentiation and NLRP3 inflammasome activation. This finding underscores the potential significance of P2X7R as a target for anti-inflammatory treatments, advancing research and management of GBS.

A Novel Lactobacillus brevis Fermented with a Vegetable Substrate (AL0035) Counteracts TNBS-Induced Colitis by Modulating the Gut Microbiota Composition and Intestinal Barrier.

Crohn's and ulcerative colitis are common conditions associated with inflammatory bowel disease as well as intestinal flora and epithelial barrier dysfunction. A novel fermented Lactobacillus brevis (AL0035) herein assayed in a trinitro benzene sulfonic acid (TNBS)-induced colitis mice model after oral administration significantly counteracted the body weight loss and improves the disease activity index and histological injury scores. AL0035 significantly decreased the mRNA and protein expression of different pro-inflammatory cytokines (TNFalpha, IL-1beta, IL-6, IL-12, IFN-gamma) and enhanced the expression of IL-10. In addition, the probiotic promoted the expression of tight junction proteins, such as ZO-1, keeping the intestinal mucosal barrier function to attenuate colitis symptoms in mice. Markers of inflammation cascade such as myeloperoxidase (MPO) and PPAR-gamma measured in the colon were also modified by AL0035 treatment. AL0035 was also able to reduce different lymphocyte markers' infiltration in the colon (GATA-3, T-Bet, NK1.1) and monocyte chemoattractant protein-1 (MCP-1/CCL2), a key chemokine involved in the migration and infiltration of monocytes/macrophages in the immunological surveillance of tissues and inflammation. In colonic microbiota profile analysis through 16S rRNA sequencing, AL0035 increased the microbial diversity depleted by TNBS administration and the relative abundance of the Lactobacillaceae and Lachnospiraceae families, whereas it decreased the abundance of Proteobacteria. Altogether, these data indicated that AL0035 could lower the severity of colitis induced by TNBS by regulating inflammatory cytokines, increasing the expression of tight junction proteins and modulating intestinal microbiota, thus preventing tissue damage induced by colitis.

Immunomodulatory responses of extracellular vesicles released by gram-positive fish pathogen Streptococcus parauberis

Bacterial extracellular vesicles (BEVs) are nanosized structures that play a role in intercellular communication and transport of bioactive molecules. Streptococcus parauberis is a Gram-positive pathogenic bacterium that causes “Streptococcosis” in fish. In this study, we isolated S. parauberis-derived extracellular vesicles (SpEVs), and then physicochemical and immunomodulatory properties were determined to elucidate their biological functions. Initially, the biogenesis of SpEVs was detected using field emission scanning electron microscopy, which revealed that secretory phase SpEVs attached to the outer surface of S. parauberis. SpEVs had an average particle diameter and zeta potential of 168.3 ± 6.5 nm and −17.96 ± 2.11 mV, respectively. Field emission transmission electron microscopy analysis confirmed the presence of round or oval-shaped SpEVs with clear membrane margins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed three sharp protein bands when SpEVs were stained with Coomassie blue. In vitro toxicity of SpEVs was assayed using the murine macrophage RAW 264.7 cells and we observed no significant (p < 0.05) viability reduction up to 50 μg/mL qRT-PCR results revealed that SpEVs-treated (5 and 10 μg/mL) RAW 264.7 cells significantly (p < 0.05) induced the mRNA of proinflammatory (Il1β, Il6, and Tnfα) and anti-inflammatory (Il10) cytokines in a concentration-dependent manner. In vivo immunomodulatory effects of SpEVs were investigated by injecting SpEVs (5 and 10 μg/fish) into adult zebrafish. Transcriptional analysis based on qRT-PCR indicates significant (p < 0.05) upregulation of proinflammatory (il1β, il6, and tnfα) and anti-inflammatory (il10) genes in a concentration-dependent manner in zebrafish kidney. Further, protein expression results in zebrafish spleen tissue confirmed the immunomodulatory activity of SpEVs. In conclusion, SpEVs display the characteristics of BEVs and immunomodulatory activities, suggesting their potential application as vaccine candidate.

Postnatal budesonide improved lung function in preterm lambs exposed to antenatal steroids and chorioamnionitis.

A combination of budesonide and surfactant decreases the rates of BPD in infants and lung injury in preterm sheep. Whether this combination will show benefit in the setting of chorioamnionitis and antenatal steroids is not known. Ewes at 123 ± 1 day gestational age received intra-amniotic (IA) injections of 10 mg LPS before being randomized to receive either 0.25 mg/kg maternal betamethasone phosphate and acetate or saline by intramuscular (IM) injection at 48 and 24 h prior to delivery at 125 ± 1 day. Lambs (N = 6-9/group) underwent intentionally injurious ventilation for 15 min, then lambs received surfactant mixed with either: (1) saline; or (2) Budesonide 0.25 mg/kg and were ventilated for 4 h. Compared with LPS-exposed animals that received no IM steroid treatment, betamethasone exposed fetuses had improved hemodynamic stability, lung compliance, and ventilation efficiency. The addition of budesonide to surfactant further improved markers of injury and pro-inflammatory cytokine mRNA in both betamethasone IM or no IM lambs exposed to LPS IA. Antenatal betamethasone and IA LPS exposures decreased budesonide levels in the fetal lung and plasma. Antenatal betamethasone stabilizes physiologic parameters in LPS treated lambs. Budesonide mixed with surfactant further decreases injury and improves respiratory physiology in betamethasone treated animals. Antenatal betamethasone improved lung and systemic physiology in the setting of intra-amniotic LPS. The addition of budesonide to the surfactant further improved lung function. Budesonide levels in the plasma and lung were lower in lambs exposed to either LPS or LPS and Betamethasone animals, and these findings were not explained by increased esterification in the lungs. The combination of antenatal steroids and budesonide with surfactant had the lowest markers of pro-inflammatory cytokines in the lung of LPS exposed animals.

Comparative Analysis of Antibacterial and Wound Healing Activities of Chitosan and Povidone-Iodine-Based Hydrogels.

This study aimed to compare the antibacterial and wound healing efficacies of chitosan hydrogel with povidone-iodine (PI) hydrogel. The in vitro antibacterial activities of chitosan and PI hydrogels against Staphylococcus aureus and Escherichia coli were evaluated. Nine 6- to 8-week-old male Sprague-Dawley rats were divided into plain, PI, and chitosan hydrogel groups. Each rat received two 10-mm full-thickness dorsal wounds using an excisional splinting model. Each wound was treated with 0.2 mL of gel thrice over the course of 3 postoperative weeks. Weekly observations were conducted, and at the end of the third postoperative week, the rats were killed for histopathological and quantitative polymerase chain reaction evaluations. Data analysis included both 2- and 1-way analyses of variance. Chitosan hydrogel exhibited comparable in vitro antibacterial activity and a significantly enhanced in vivo wound closure rate compared with PI hydrogel. Three weeks after the surgery, the chitosan hydrogel group demonstrated marked differences in wound repair (P < 0.01). Histologically, increased collagen deposition was observed with chitosan hydrogel treatment. Immunohistochemistry for CD68 revealed a lower number of macrophages in the wounds treated with chitosan hydrogel. Quantitative polymerase chain reaction analysis indicated a superior collagen 1 to 3 ratio and reduced expression of proinflammatory cytokine mRNAs (interleukin 1b, interleukin 6, tumor necrosis factor α, and interferon γ) in the chitosan hydrogel group. Chitosan hydrogel demonstrates the potential to serve as an effective alternative to PI hydrogel, providing enhanced wound healing capabilities while maintaining comparable antimicrobial properties.

MITOCHONDRIAL DYSFUNCTION CAUSES CECAL PATCH (CP) INFLAMMATION IN ULCERATIVE COLITIS

Abstract BACKGROUND Up to 17% of distal ulcerative colitis (UC) patients have discontinuous (proximal) inflammation referred to as Cecal Patch (CP), associated with greater abdominal pain, bleeding and diarrhea. Despite these known associations, the pathogenesis of CP lesions is poorly understood. Recent data suggest that reduced mitochondrial (Mito) function predicts unfavorable clinical outcomes in active UC (Haberman et al; Nat Comm 2019). We posit that inflammation and tissue injury induced in UC-associated CP involves Mito dysfunction. METHOD UC patients with/without cecal disease and normal controls were biopsied. Tissue from CP lesions or normal caecum were collected in Allprotect® (AP) and formalin. Total RNA from AP-preserved biopsies were used for cDNA synthesis and RT-qPCR for Mito complexes, cytokines, anti-oxidant and calcineurin (CaN) signaling genes and tissue processed for IHC for Mito complexes. RESULT Transcriptomic analysis showed significant downregulation in genes associated with Mito biogenesis (PGC1α, TFAM, NRF2) and Mito complexes (Ndufa1, Ndufa4, Ndufb2, Ndufb6, mtCO1, mtCO2, Cox5B, Cox6A1, ATP5A1, ATP5B, ATP5E) in CP lesions compared to normal caecum from active patients. Major regulators of Mito biogenesis (PGC1α and TFAM) revealed ~39% and ~24% reductions of mRNA respectively, while NRF2 (anti-oxidant regulator) showed ~62% reduced mRNA in CP lesions compared to normal cecum from distal UC patients. Electron transport chain complex mRNAs showed 31%, 47%, and 48% reduction in complex I (Ndufa1, Ndufa4, Ndufb2, Ndufb6), complex IV (mtCO1, mtCO2, Cox5B, Cox6A1), and complex V (ATP5A1, ATP5B, ATP5E) levels, respectively in CP lesions compared to normal controls. Concomitantly, the IHC staining also showed significant reductions in Ndufb6, MTCO1 and Cox5A protein levels in CP lesions compared to normal cecum sections. The CP-related decreased markers of Mito biogenesis and function correlates with increased proinflammatory cytokine mRNA (IL-6: 2-fold; TGFβ1: 1.6-fold; IL-2: 2.6-fold) and decreased anti-inflammatory (IL10: 3.2-fold) gene expression in CP lesions. Further investigation demonstrated that repressed Mito function in CP lesions is associated with downregulation of anti-oxidant genes (SOD1, SOD2, Prdx3, Gpx4, Trx2) compared to normal cecum. Inflammation-induced Mito dysfunction in CP lesions also stimulates CaN signaling as demonstrated by significant increases in mRNA levels of CaN and downstream regulated genes (Cathepsin-L, Rel-A Glut4, CREB-BP and Igf-1r). CONCLUSION These data represent the first indication that Mito dysfunction occurs in CP associated with distal UC. Furthermore, we speculate that Mito dysfunction-induced CaN signaling promotes regional (CP) inflammatory responses. These important findings indicate that targeting Mito metabolism may be an important therapeutic goal of treating UC patients with CP.

Green synthesis of gold nanoparticles from the extract of Crocus sativus to study the effect of antidepressant in adolescence and to observe its aggressive and impulsive behavior in rat models

Depression is a unipolar condition caused by a brain monoamine deficit that affects emotion, thinking, and function. Depression treatments involve limitations such poor absorption, a delay in therapeutic efficacy, unwanted side effects, and high-dose toxicity. A simple, affordable, clean, non-toxic, and environmentally friendly nanoparticle manufacturing method was used to fix this. This research created C.sativus gold nanoparticles (CS-AuNPs) from Crocus sativus extract and auric chloride (HAuCl4). CS-AuNPs was spherical, 11–20 nm in size, and 550 nm absorbent after UV–vis, XRD, FTIR, SEM, and TEM analysis. Saffron action is increased using gold nanoparticles, a non-toxic drug delivery vehicle. Crocins, crocetin, and safranal are important antidepressants. Crocus sativus has been shown to effectively reduce proinflammatory cytokines mRNA expression (IL-1β, IL-6, and TNF-α) and inhibit NF-κB activation. With an IC50 of 256 μg/ml for DPPH, IC50 of 300 μg/ml for NBT assay and 90 % cell survival in cytotoxicity tests, this chemical is safe and effective as an antidepressant. The new study showed that C.sativus has therapeutic promise and that converting it from extract to nanoparticles will benefit depression treatment.

Utilizing molecular docking and cell validation to explore the potential mechanisms of lupenone attenuating the inflammatory response via NF‐κB pathway

Diabetic nephropathy (DN), a common microvascular complicating disease of diabetes. Lupenone, a pentacyclic triterpenoid, has anti-inflammatory effects and can prevent type 2 diabetes mellitus and treat renal damage, however, the effects and mechanisms of lupenone in DN remain unclear. Thereby,the MTT method was used to investigate the antiproliferative effect of lupenoneon the cell line rat glomerular mesangial cells (HBZY-1). Molecular docking was used to investigate the combination of lupenone and MCP-1, IL-1β, TNF-α, IKKβ, IκBα, and NF-κB p65 proteins. The expression of mRNA of the pro-inflammatory cytokines (MCP-1, IL-1β and TNF-α) and the NF-κB signalling pathway in HBZY-1 cells were assessed by RT-PCR. The protein expressions of pro-inflammatory cytokines and NF-κB pathway were got by Western blot. Result showed that lupenone inhibited the proliferative activity of HBZY-1 cells at non-cytotoxic concentrations. Molecular docking results showed that lupenone combined well with the target proteins. Moreover, lupenone could significantly reduced the mRNA and protein expressions for pro-inflammatory cytokines and IKKβ, p-p65 and p-IκBα. Lupenone may play an anti-inflammatory role in DN treatment by inhibiting the NF-κB signalling pathway. These results provided a new understanding of the pharmacological mechanisms of lupenone in treatment of DN.

Mesenchymal Stem Cells Combined With Electroacupuncture Treatment Regulate the Subpopulation of Macrophages and Astrocytes to Facilitate Axonal Regeneration in Transected Spinal Cord.

Herein, we investigated whether mesenchymal stem cells (MSCs) transplantation combined with electroacupuncture (EA) treatment could decrease the proportion of proinflammatory microglia/macrophages and neurotoxic A1 reactive astrocytes and inhibit glial scar formation to enhance axonal regeneration after spinal cord injury (SCI). Adult rats were divided into 5 groups after complete transection of the spinal cord at the T10 level: a control group, a nonacupoint EA (NA-EA) group, an EA group, an MSC group, and an MSCs+EA group. Immunofluorescence labeling, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blots were performed. The results showed that MSCs+EA treatment reduced the proportion of proinflammatory M1 subtype microglia/macrophages, but increased the differentiation of anti-inflammatory M2 phenotype cells, thereby suppressing the mRNA and protein expression of proinflammatory cytokines (tumor necrosis factor-α and IL-1β) and increasing the expression of an anti-inflammatory cytokine (interleukin [IL]-10) on days 7 and 14 after SCI. The changes in expression correlated with the attenuated neurotoxic A1 reactive astrocytes and glial scar, which in turn facilitated the axonal regeneration of the injured spinal cord. In vitro, the proinflammatory cytokines increased the level of proliferation of astrocytes and increased the expression levels of C3, glial fibrillary acidic protein, and chondroitin sulfate proteoglycan. These effects were blocked by administering inhibitors of ErbB1 and signal transducer and activator of transcription 3 (STAT3) (AG1478 and AG490) and IL-10. These findings showed that MSCs+EA treatment synergistically regulated the microglia/macrophage subpopulation to reduce inflammation, the formation of neurotoxic A1 astrocytes, and glial scars. This was achieved by downregulating the ErbB1-STAT3 signal pathway, thereby providing a favorable microenvironment conducive to axonal regeneration after SCI.

Reparative effect of different dietary additives on soybean meal-induced intestinal injury in yellow drum (Nibea albiflora).

Soybean meal (SBM) is an acceptable replacement for unsustainable marine fish meal (FM) in aquaculture. However, we previously reported that high dietary SBM supplementation causes intestinal inflammatory injury in yellow drum (Nibea albiflora). Accordingly, a 4-week SBM-induced enteritis (SBMIE) in yellow drum trial was conducted first, followed by a 4-week additive-supplemented reparative experiment to evaluate the reparative effect of five additives on SBMIE in yellow drum. The control diet comprised 50% FM protein substituted with SBM. The additive-supplemented diet was added with 0.02% curcumin (SBMC), 0.05% berberine (SBM-BBR), 0.5% tea polyphenols (SBM-TPS), 1% taurine (SBM-TAU), or 0.8% glutamine (SBM-GLU) based on the control diet, respectively. The weight gain (WG), specific growth rate (SGR), feed efficiency ratio (FER), and survival rate (SR) of fish fed the additive-supplemented diets were significantly higher than those of fish fed the SBM diet. The WG, SGR, and FER of fish fed the SBMC, SBM-GLU and SBM-TAU diets were significantly higher than those of fish fed other diets. Moreover, fish fed the additive-supplemented diets SBMC and SBM-GLU, exhibited significantly increased intestinal villus height (IVH), intestinal muscular thickness (IMRT), and intestinal mucosal thickness (IMLT) and significantly decreased crypt depth (CD) in comparison with those fed the SBM diets. The relative expression of intestinal tight junction factors (ocln, zo1), cytoskeletal factors (f-actin, arp2/3), and anti-inflammatory cytokines (il10, tgfb) mRNA was remarkably elevated in fish fed additive-supplemented diets than those of fish fed the SBM diet. Whereas, the relative expression of intestinal myosin light chain kinase (mlck) and pro-inflammatory cytokines (il1, il6, tnfa) mRNA was markedly lower in fish fed the additive-supplemented diets. The highest relative expression of intestinal ocln, f-actin, and arp2/3 and the lowest relative expression of intestinal mlck were found in fish fed the SBMC diet. Hence, all five dietary additives effectively repaired the intestinal injury induced by SBM, with curcumin exhibiting the strongest repair effect for SBMIE in yellow drum.

Regnase-1 overexpression as a therapeutic approach of Marfan syndrome

Rupture or dissection of thoracic aortic aneurysms is still the leading cause of death for patients diagnosed with Marfan syndrome. Inflammation and matrix digestion regulated by matrix metalloproteases (MMPs) play a major role in the pathological remodeling of the aortic media. Regnase-1 is an endoribonuclease shown to cleave the mRNA of proinflammatory cytokines, such as interleukin-6. Considering the major anti-inflammatory effects of regnase-1, here, we aimed to determine whether adeno-associated virus (AAV)-mediated vascular overexpression of the protein could provide protection from the development and progression of aortic aneurysms in Marfan syndrome. The overexpression of regnase-1 resulted in a marked decrease in inflammatory parameters and elastin degradation in aortic smooth muscle cells invitro. Intravenous injection of a vascular-targeted AAV vector resulted in the efficient transduction of the aortic wall and overexpression of regnase-1 in a murine model of Marfan syndrome, associated with lower circulating levels of proinflammatory cytokines and decreased MMP expression and activity. Regnase-1 overexpression strongly improved elastin architecture in the media and reduced aortic diameter at distinct locations. Therefore, AAV-mediated regnase-1 overexpression may represent a novel gene therapy approach for inhibiting aortic aneurysms in Marfan syndrome.

Gastric coinfection with thiopeptide-positive Cutibacterium acnes decreases FOXM1 and pro-inflammatory biomarker expression in a murine model of Helicobacter pylori-induced gastric cancer.

H. pylori infects half of the world population and is the leading cause of gastric cancer. We previously demonstrated that gastric cancer risk is associated with gastric microbiota. Specifically, gastric urease-positive Staphylococcus epidermidis and Streptococcus salivarius had contrasting effects on H. pylori-associated gastric pathology and immune responses in germ-free INS-GAS mice. As gastritis progresses to gastric cancer, the oncogenic transcription factor Foxm1 becomes increasingly expressed. In this study, we evaluated the gastric commensal C. acnes, certain strains of which produce thiopeptides that directly inhibit FOXM1. Thiopeptide-positive C. acnes was isolated from Nicaraguan patient gastric biopsies and inoculated into germ-free INS-GAS mice with H. pylori. We, therefore, asked whether coinfection with C. acnes expressing thiopeptide and H. pylori would decrease gastric Foxm1 expression and pro-inflammatory cytokine mRNA and protein levels. Our study supports the growing literature that specific non-H. pylori gastric bacteria affect inflammatory and cancer biomarkers in H. pylori pathogenesis.

8-Oxoguanine DNA Glycosylase 1 Inhibition Suppresses Inflammatory Responses in Sickle Cell Disease

Background: The continuous production of reactive oxygen species (ROS) and oxidative stress contribute to ongoing inflammation and the pathology of sickle cell disease (SCD). ROS are known to mediate DNA damage through oxidation of nucleotide bases with guanine being the primary target. Although guanine base lesions vary according to the nature of the oxidants, 7,8-dihydro-8-oxoguanine (8-oxoG) is the most frequent oxidation product of DNA, which is repaired by 8-Oxoguanine-DNA glycosylase-1 (OGG1) via the DNA base excision repair pathway (OGG1-BER). The release of the 8-oxoG base from the genome by OGG1-BER is a prerequisite for increasing expression of pro-inflammatory cytokines and immune response. OGG1 inhibition has been shown to attenuate proinflammatory responses via the mitochondrial DNA-cGAS-STING axis in mice (Qin et al, 2020), a pathway also involved in the activation of neutrophils in SCD (Tumburu et al, 2021). Here, we evaluate the effect of OGG1 inhibition on expression of pro-inflammatory cytokines and immune response in SCD. Methods: Blood samples were obtained from ethnic-matched healthy controls (HbAA) and patients with SCD (HbSS) enrolled under protocols NCT03049475 and NCT00047996. CD14+ monocytes were isolated from PBMC of HbAA and HbSS subjects using EasySep™ Human Monocyte Isolation Kit (STEMCELL Technologies). 8-oxoG was measured by staining CD14+ monocytes with anti-8-oxoG antibody; OGG1 and pro-inflammatory cytokine levels were evaluated by qRT-PCR and western blotting. After differentiation, macrophages derived from CD14+ monocytes were treated with or without 10 µM SU0268 (OGG1 inhibitor) for 1 hour and then incubated with plasma from healthy controls or HbSS patients in steady or crisis status, followed by qRT-PCR quantitation of IL-1ꞵ, IL-6, TNF-α, and OGG1 mRNA levels. These measurements were repeated after OGG1 knockdown using OGG1 siRNA in macrophages derived from CD14+ monocytes. We repeated the study on peritoneal macrophages (PMs) from Townes HbAA and HbSS mice that were collected 4 days after a 3% Brewer's thioglycolate medium intraperitoneal injection. The level of 8-oxoG was measured by staining PMs with anti-8-oxoG antibody. The mRNA and protein level of OGG1 and pro-inflammatory cytokines were evaluated by qRT-PCR and western blotting. PMs from HbAA mice were treated with 10 µM SU0268 (OGG1 inhibitor) or vehicle for 1 hour, before incubation with plasma from HbAA or HbSS mice, the mRNA levels of IL-1ꞵ, IL-6, TNF-α, and OGG1 were measured by qRT-PCR. Results: CD14+ monocytes isolated from human HbSS PBMC showed increased levels of 8-oxoG nuclear staining when compared with human HbAA CD14+ monocytes, consistent with an increased oxidative burden in SCD patients (Fig 1A). The mRNA and protein levels of OGG1 were also increased in HbSS monocytes, suggestive of a potential therapeutic target of OGG1. Consistent with previous studies, mRNA levels of pro-inflammatory cytokines IL-1ꞵ, IL-6, and TNF-α were increased in SS compared with AA monocytes. To investigate the role of OGG1 in regulating cytokine expression, we treated the macrophages derived from CD14+ monocytes with SU0268, a selective OGG1 inhibitor (Tahara et al, 2018). Macrophages treated with plasma from HbSS patients (steady and crisis status) showed increased expression of OGG1, IL-1ꞵ, IL-6, and TNF-α compared with cells treated with normal plasma, and the increase was largely blocked by SU0268 pretreatment (Fig1B). Further, siRNA knockdown of OGG1 decreased the expression of IL-1ꞵ and TNF-α, confirming the role of OGG1 in regulating pro-inflammatory cytokine expression. We then evaluated the effect of OGG1 in regulating cytokines expression in cells form SCD mice. The levels of 8-oxoG and OGG1, as well as cytokines (IL-1ꞵ, IL-6, and TNF-α), were increased in PMs obtained from HbSS mice compared with macrophages obtained from HbAA mice. Similar to the results obtained from human macrophages, murine cells treated with plasma from SS mice showed increased expression of IL-1ꞵ, IL-6, TNF-α and OGG1 compared with cells treated with plasma from HbAA mice, and pretreatment with SU0268 largely blocked their increased expression. Conclusion: OGG1 has a regulatory role in the expression of pro-inflammatory cytokines in SCD. OGG1 inhibition could be a potential therapeutic approach to control sickle cell inflammation.