LPS-induced Production Of Pro-inflammatory Cytokines Research Articles

Inulin alleviates inflammatory response and gut barrier dysfunction via modulating microbiota in lipopolysaccharide-challenged broilers

This study was conducted to explore the protective effects of inulin against lipopolysaccharide (LPS)-induced inflammatory response and intestinal barrier dysfunction in broilers. 108 broilers were allocated to 3 treatments: 1) non-challenged broilers (Control, CON); 2) LPS-challenged broilers (LPS); 3) LPS-challenged broilers fed the basal diet supplemented with 15 g/kg of inulin (Inulin+LPS). At 21 d of age, the LPS-challenged groups received an intraperitoneal injection of LPS, and the CON group received an equal volume of saline. After 4 h of LPS exposure, samples of blood, intestinal mucosa and cecal digesta were collected. The results showed that LPS challenge induced systemic inflammation and damaged intestinal barrier function, whereas inulin attenuated LPS-induced production of pro-inflammatory cytokines by inhibiting the activation of TLR4 and NF-κB p65, and enhanced intestinal barrier function. In addition, LPS stimulation caused cecal microbial dysbiosis as shown by increased abundance of pathogenic bacteria including Ruminococcus_torques_group, Escherichia-Shigella and Subdoligranulum, while supplementation of inulin increased abundance of beneficial bacteria Faecalibacterium and Anaeroplasma, and metabolite production including propionate and butyrate concentrations. In conclusion, dietary supplementation of inulin could partially alleviate LPS-induced inflammation and intestinal barrier injury by modulating intestinal microbiota, thereby minimizing growth retardation of broilers. Our results provide a basis for the rational utilization of inulin in alleviating immune stress in broiler production.

Origin recognition complex subunit 6 (ORC6) is a key mediator of LPS-induced NFκB activation and the pro-inflammatory response

Lipopolysaccharide (LPS)-activated pro-inflammatory responses play a critical role in sepsis, a life-threatening condition. This study investigates the role of origin recognition complex subunit 6 (ORC6) in LPS responses in macrophages and monocytes. Silencing ORC6 using targeted shRNA significantly reduced LPS-induced expression and production of IL-1β (interleukin-1 beta), TNF-α (tumor necrosis factor alpha), and IL-6 (interleukin-6) in THP-1 human macrophages, peripheral blood mononuclear cells (PBMCs), and bone marrow-derived macrophages (BMDMs). Additionally, ORC6 knockout (KO) via the CRISPR/Cas9 method in THP-1 macrophages inhibited LPS-induced pro-inflammatory responses, while ectopic overexpression of ORC6 enhanced LPS-induced expression and production of pro-inflammatory cytokines. ORC6 is crucial for the activation of the nuclear factor kappa B (NFκB) signaling cascade in macrophages and monocytes. LPS-induced NFκB activation was largely inhibited by ORC6 silencing or KO, but potentiated following ORC6 overexpression. Mechanistically, ORC6 associated with nuclear p65 after LPS stimulation, an interaction necessary for NFκB activation. Overexpression of ORC6 did not recover the reduced pro-inflammatory response to LPS in THP-1 macrophages with silenced p65. Furthermore, the NFκB inhibitor BMS-345,541 nearly eliminated the pro-inflammatory response enhanced by ORC6 overexpression in response to LPS. Further studies revealed that ORC6 depletion inhibited NFκB activation induced by double-stranded RNA (dsRNA) and high mobility group box 1 (HMGB1) in THP-1 macrophages. In vivo experiments demonstrated that macrophage-specific knockdown of ORC6 protected mice from LPS-induced septic shock and inhibited LPS-stimulated production of IL-1β, TNF-α, and IL-6 in mouse serum. ORC6 silencing also inhibited LPS-induced NFκB activation in ex vivo cultured PBMCs following macrophage-specific knockdown of ORC6. These findings highlight ORC6 as a pivotal mediator in LPS-induced NFκB activation and the pro-inflammatory response in sepsis, suggesting that targeting ORC6 could be a novel therapeutic strategy for managing sepsis and related inflammatory conditions.

Prevotella histicola ameliorates DSS-induced colitis by inhibiting IRE1α-JNK pathway of ER stress and NF-κB signaling

Inflammatory bowel disease (IBD) is a chronic and recurrent gastrointestinal inflammation regulated by intricate mechanisms. Recently, prebiotics is considered as promising nutritional strategy for the prevention and treatment of IBD. Prevotella histicola (P. histicola), an emerging probiotic, possesses apparently anti-inflammatory bioactivity. However, the role and underlying mechanism of P. histicola on IBD remain unclear. Hence, we probe into the effect of P. histicola on dextran sulfate sodium (DSS)-induced colitis and clarified the potential mechanism. Our results revealed that DSS-induced colonic inflammatory response and damaged epithelial barrier in mice were attenuated by oral administration of P. histicola. Moreover, supplementary P. histicola significantly enriched short-chain fatty acid (SCFA)-producing bacteria (Lactobacillus, and Bacillus) and reduced pathogenic bacteria (Erysipelotrichaceae, Clostridium, Bacteroides) in DSS-induced colitis. Notably, In DSS-treated mice, endoplasmic reticulum stress (ERS) was persistently activated in colonic tissue. Conversely, P. histicola gavage suppressed expansion of endoplasmic reticulum, downregulated PERK-ATF4-CHOP and IRE1α-JNK pathway. In vitro, the P. histicola supernatant eliminated LPS-induced higher production of pro-inflammatory cytokines regulated by NF-κB and impairment of epithelial barrier by inhibiting IRE1α-JNK signaling in Caco-2 cell. In summary, our study indicated that P. histicola mitigated DSS-induced chronic colitis via inhibiting IRE1α-JNK pathway and NF-κB signaling. These findings provide the new insights into the promotion of gut homeostasis and the application potential of P. histicola as a prebiotic for IBD in the future.

Induction of MTHFD2 in Macrophages Inhibits Reactive Oxygen Species-mediated NF-κB Activation and Protects against Inflammatory Responses.

The one-carbon metabolism enzyme methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) is critical for cancer cell proliferation and immune cell phenotypes, but whether it can contribute to macrophage inflammatory responses remains unclear. In this study, we show that MTHFD2 was upregulated by LPS in murine macrophages upon activation of the TLR4-MyD88-IKKα/β-NF-κB signaling pathway. MTHFD2 significantly attenuated LPS-induced macrophage proinflammatory cytokine production through its enzymatic activity. Notably, ablation of myeloid MTHFD2 rendered mice more sensitive to septic shock and CCl4-induced acute hepatitis. Mechanistically, MTHFD2 restrained IKKα/β-NF-κB activation and macrophage inflammatory phenotype by scavenging reactive oxygen species through the generation of NADPH. Our study reveals MTHFD2 as a "self-control" mechanism in macrophage-mediated inflammatory responses.

The Effects of Psilocybin on Lipopolysaccharide-Induced Inflammation in THP-1 Human Macrophages

Psilocybin, an innate compound produced by mushrooms belonging to the Psilocybe genus, is primarily known for its agonistic effects on the serotonin 5-HT2A receptor. This receptor’s functioning is involved in many neurological processes. In the context of this research, our primary aim was to comprehensively investigate the influence of psilocybin as a serotonin receptor agonist on the intricate cascade of events involved in THP-1 macrophages stimulated by lipopolysaccharide (LPS). THP-1 monocyte cells were subjected to differentiation into macrophages through a controlled incubation with phorbol 12-myristate 13-acetate (PMA). The next step involved the induction of an inflammatory response by exposing THP-1 macrophages to 500 ng/mL LPS for 4 h. Subsequently, we triggered the activation of the second phase of the NLRP3 inflammasome by introducing adenosine triphosphate (ATP) immediately following LPS stimulation. Our findings have revealed a dose-dependent inverse correlation between psilocybin exposure and the production of LPS-induced proinflammatory cytokines and proteins. Our work indicates that psilocybin likely mediates these responses by influencing key signaling pathways, including NF-κB, IL-6/TYK2/STAT3, and TYK2/STAT1.

Aquaporins (AQPs) as a marker in the physiology of inflammation and its interaction studies with garcinol.

Aquaporins like AQP1, AQP3, and AQP4 are known to be involved in the pathophysiology of inflammation based on earlier reports. This study aimed to evaluate the involvement of Aquaporins as a potential target of inflammation. The study also investigates the efficacy of methanolic extract of Garcinia (GME) and its potent phytocompound (garcinol) against the Aquaporins involvedin inflammation. siRNA silencing of AQP3 was carried out in RAW264.7 cells followed by LPS stimulation (1µg/ml) and assessment of important markers of inflammation including NO, PGE2, TNF-α, IL-6, IL-1β, CCL20, iNOS and COX-2. To assess the anti-inflammatory potential of Garcinia extract and garcinol, cells were stimulated with 1µg/ml LPS in the absence and presence of increasing concentrations of GME and garcinol. During the experimental period, extract concentrations (115µg/ml and 230µg/ml for RAW264.7; 118µg/ml and 236µg/ml for THP-1) and garcinol concentrations (6µM and 12µM for RAW264.7; 3µM and 6µM for THP-1) were selected based on the IC50. The anti-inflammatory effects were assessed by measuring the levels of TNF-α, IL-1β, IL-6, and CCL20 in LPS-stimulated cells. The AQP expression was studied at transcriptional and translational levels using qPCR and Western blot analysis respectively. AQP3 knockdown significantly decreased the NO, PGE2, TNF-α, IL-1β levels along with iNOS andCOX-2 mRNA expression. LPS stimulation led to a significant increase in the mRNA and protein level expression AQP1, AQP3, and AQP4 in RAW264.7 cells; and AQP1 and AQP3 in THP-1 cells indicating their role as markers of inflammation. GME and garcinol effectively suppressed the LPS-induced proinflammatory cytokine production in both cell lines. The results indicate that AQP1, AQP3, and AQP4 could play a crucial role as markers of inflammation. Anti-inflammatory agents like Garcinia could potentially decrease the expression of such AQPs, thus inhibiting the inflammatory process.

Investigation of an aminothiazole-based scaffold as an anti-inflammatory agent: Potential application in the management of cytokine storm in SARS-CoV-19

Aminothiazole-based structures, including compounds like anakinra, nitazoxanide, amiphenazole, and tizoxanide, along with cytokine-blocking agents such as tocilizumab and siltuximab are currently under extensive investigation for their potential in managing severe COVID-19 by specifically targeting and inhibiting cytokines responsible for cytokine release syndrome (CRS). These targeted therapies have shown promising results in effectively mitigating the effects of severe COVID-19. Herein, we carried out a biological assessment of a novel aminothiazole-based derivative 5-(phenylthio)-4-(2-(trifluoromethyl) phenyl) thiazol-2-amine (X22) to gauge its potential as an anti-inflammatory agent. Utilizing in vitro assays, which included measuring nitric oxide (NO) inhibition and observing the production of LPS-induced pro-inflammatory cytokines (TNF-α and IL-6) in RAW 264.7 macrophages. Furthermore, the X22 was characterized by experimental FT-IR, NMR, and UV–visible spectral analysis. The optimized molecular geometry and vibrational wave numbers were calculated using the DFT and B3LYP/6–311++G (d,p) basis set. The vibrational assignments were calculated using VEDA's Potential Energy Distribution (PED) program. 1H and 13C NMR were calculated by the GIAO method. Additionally, compounds HOMO-LUMO and HF interactions were studied. Subsequently, the MEP map was traced to find the reactive sites of X22. In addition, Mulliken population analysis and thermodynamic properties with respect to various temperatures were also calculated. All the theoretical predictions were carried out by using the DFT-B3LYP method at the level of 6–311++G (d,p). Finally, the molecular docking interactions revealed that ligand (X22) binds to TNF-α and IL-6 via H-bonding with amino acids Tyr 151, Ser60, Gln61, and Arg179. These findings suggest that X22 could be a valuable therapeutic agent for mitigating the progression of severe SARS-CoV-2 infections.

Insulin Mediates Lipopolysaccharide-Induced Inflammatory Responses and Oxidative Stress in BV2 Microglia.

Insulin, the key hormone for glucose regulation, has garnered attention for its role as an immune modulator. Impaired insulin signaling in the central nervous system is linked to neuroinflammation and neurodegenerative diseases. Microglia, the resident macrophage-like immune cells in the brain, are key regulators of neuroinflammation. However, the mechanisms by which insulin influences microglial immune responses remain relatively unknown. This study aimed to assess the effects of post-treatment with insulin [30minutes after lipopolysaccharide (LPS) exposure] on LPS-induced inflammatory responses in BV2 microglial cells. Post-treatment with insulin potentiated LPS-induced production of nitric oxide and pro-inflammatory cytokines, such as TNF and IL-6, through activation of the Akt/NF-κB pathway. Insulin also enhanced the ability of BV2 cells to phagocytose bacteria particles and β-amyloid fibrils. Conversely, insulin inhibited activation of NADPH oxidase and reduced intracellular levels of reactive oxygen species in LPS-treated BV2 cells. Insulin enhances microglial immune competence when challenged by endotoxins but mitigates oxidative stress in these cells.

Lumbricus Extract Prevents LPS-Induced Inflammatory Activation of BV2 Microglia and Glutamate-Induced Hippocampal HT22 Cell Death by Suppressing MAPK/NF-κB/NLRP3 Signaling and Oxidative Stress.

Microglia-induced inflammatory signaling and neuronal oxidative stress are mutually reinforcing processes central to the pathogenesis of neurodegenerative diseases. Recent studies have shown that extracts of dried Pheretima aspergillum (Lumbricus) can inhibit tissue fibrosis, mitochondrial damage, and asthma. However, the effects of Lumbricus extracts on neuroinflammation and neuronal damage have not been previously studied. Therefore, to evaluate the therapeutic potential of Lumbricus extract for neurodegenerative diseases, the current study assessed the extract's anti-inflammatory and antioxidant activities in BV2 microglial cultures stimulated with lipopolysaccharide (LPS) along with its neuroprotective efficacy in mouse hippocampal HT22 cell cultures treated with excess glutamate. Lumbricus extract dose-dependently inhibited the LPS-induced production of multiple proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-6, and IL-1β) and reversed the upregulation of proinflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2). Lumbricus also activated the antioxidative nuclear factor erythroid 2-relayed factor 2/heme oxygenase-1 pathway and inhibited LPS-induced activation of the nuclear factor-κB/mitogen-activated protein kinases/NOD-like receptor family pyrin domain containing 3 inflammatory pathway. In addition, Lumbricus extract suppressed the glutamate-induced necrotic and apoptotic death of HT22 cells, effects associated with upregulated expression of antiapoptotic proteins, downregulation of pro-apoptotic proteins, and reduced accumulation of reactive oxygen species. Chromatography revealed that the Lumbricus extract contained uracil, hypoxanthine, uridine, xanthine, adenosine, inosine, and guanosine. Its effects against microglial activation and excitotoxic neuronal death reported herein support the therapeutic potential of Lumbricus for neurodegenerative diseases.

Traditional Chinese medicine Pien-Tze-Huang ameliorates LPS-induced sepsis through bile acid-mediated activation of TGR5-STAT3-A20 signalling

Pien Tze Huang (PZH), a class-1 nationally protected traditional Chinese medicine (TCM), has been used to treat liver diseases such as hepatitis; however, the effect of PZH on the progression of sepsis is unknown. Here, we reported that PZH attenuated lipopolysaccharide (LPS)-induced sepsis in mice and reduced LPS-induced production of proinflammatory cytokines in macrophages by inhibiting the activation of mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signalling. Mechanistically, PZH stimulated signal transducer and activator of transcription 3 (STAT3) phosphorylation to induce the expression of A20, which could inhibit the activation of NF-κB and MAPK signalling. Knockdown of the bile acid (BA) receptor G protein-coupled bile acid receptor 1 (TGR5) in macrophages abolished the effects of PZH on STAT3 phosphorylation and A20 induction, as well as the LPS-induced inflammatory response, suggesting that BAs in PZH may mediate its anti-inflammatory effects by activating TGR5. Consistently, deprivation of BAs in PZH by cholestyramine resin reduced the effects of PZH on the expression of phosphorylated-STAT3 and A20, the activation of NF-κB and MAPK signalling, and the production of proinflammatory cytokines, whereas the addition of BAs to cholestyramine resin-treated PZH partially restored the inhibitory effects on the production of proinflammatory cytokines. Overall, our study identifies BAs as the effective components in PZH that activate TGR5-STAT3-A20 signalling to ameliorate LPS-induced sepsis.

Strontium Attenuates LPS-Induced Inflammation via TLR4/MyD88/NF-κB Pathway in Bovine Ruminal Epithelial Cells.

Subacute ruminal acidosis (SARA) is a common nutritional metabolic disease in ruminants that causes significant economic losses to dairy farming. Strontium (Sr) is known to be involved in bone metabolism and exhibits potent anti-inflammatory effects. To evaluate the effect of Sr on inflammation in bovine ruminal epithelial cells, a model of LPS-induced inflammation was established in this study, and the cell viability of bovine ruminal epithelial cells was measured using CCK-8. The production of pro-inflammatory cytokines was measured by ELISA and real-time PCR, respectively. The related proteins of the TLR4/MyD88/NF-κB pathway were assayed through Western blotting, and the fluorescence of p-p65 and p-IκB were assayed by immunofluorescence. Molecular docking of Sr and TLR4/MyD88/NF-κB pathway-related proteins was performed using MIB2 ( http://bioinfo.cmu.edu.tw/MIB2/ ). Results showed that after treatment for 24 h, the cell viability was decreased at the high concentration of Sr (≥ 10 mmol/L). Sr significantly decreased the production of TNF-α, IL-1β, and IL-6, downregulated the related proteins expression of the TLR4/MyD88/NF-κB pathway, and reduced the fluorescence levels of p-p65 and p-IκB. The NF-κB pathway inhibitor PDTC and molecular docking further revealed that Sr reduced LPS-induced pro-inflammatory cytokines production via the TLR4/MyD88/NF-κB pathway. These results suggest that Sr reduces LPS-induced pro-inflammatory cytokines production via the TLR4/MyD88/NF-κB pathway, thereby exerting an anti-inflammatory effect in bovine ruminal epithelial cells, providing a basis for Sr in the treatment of bovine rumen acidosis disease.

6-Pentyl-α-Pyrone from Trichoderma gamsii Exert Antioxidant and Anti-Inflammatory Properties in Lipopolysaccharide-Stimulated Mouse Macrophages.

Filamentous fungi produce several beneficial secondary metabolites, including bioactive compounds, food additives, and biofuels. Trichoderma, which is a teleomorphic Hypocrea that falls under the taxonomic groups Ascomycota and Dikarya, is an extensively studied fungal genus. In an ongoing study that seeks to discover bioactive natural products, we investigated potential bioactive metabolites from the methanolic extract of cultured Trichoderma gamsii. Using liquid chromatography-mass spectrometry (LC-MS), one major compound was isolated and structurally identified as 6-pentyl-α-pyrone (6PP) based on nuclear magnetic resonance data and LC-MS analysis. To determine its antioxidant and anti-inflammatory activity, as well as the underlying mechanisms, we treated lipopolysaccharide (LPS)-stimulated Raw264.7 mouse macrophages with 6PP. We found that 6PP suppresses LPS-induced increase in the levels of nitric oxide, a mediator of oxidative stress and inflammation, and restores LPS-mediated depletion of total glutathione by stabilizing nuclear factor erythroid 2-related factor 2 (Nrf2), an antioxidative factor, and elevating heme oxygenase-1 levels. Furthermore, 6PP inhibited LPS-induced production of proinflammatory cytokines, which are, at least in part, regulated by heme oxygenase-1 (HO-1). 6PP suppressed proinflammatory responses by inhibiting the nuclear localization of nuclear factor kappa B (NF-κB), as well as by dephosphorylating the mitogen-activated protein kinases (MAPKs). These results indicate that 6PP can protect macrophages against oxidative stress and LPS-induced excessive inflammatory responses by activating the Nrf2/HO-1 pathway while inhibiting the proinflammatory, NF-κB, and MAPK pathways.

BRISC is required for optimal activation of NF-κB in Kupffer cells induced by LPS and contributes to acute liver injury

BRISC (BRCC3 isopeptidase complex) is a deubiquitinating enzyme that has been linked with inflammatory processes, but its role in liver diseases and the underlying mechanism are unknown. Here, we investigated the pathophysiological role of BRISC in acute liver failure using a mice model induced by D-galactosamine (D-GalN) plus lipopolysaccharide (LPS). We found that the expression of BRISC components was dramatically increased in kupffer cells (KCs) upon LPS treatment in vitro or by the injection of LPS in D-GalN-sensitized mice. D-GalN plus LPS-induced liver damage and mortality in global BRISC-null mice were markedly attenuated, which was accompanied by impaired hepatocyte death and hepatic inflammation response. Constantly, treatment with thiolutin, a potent BRISC inhibitor, remarkably alleviated D-GalN/LPS-induced liver injury in mice. By using bone marrow-reconstituted chimeric mice and cell-specific BRISC-deficient mice, we demonstrated that KCs are the key effector cells responsible for protection against D-GalN/LPS-induced liver injury in BRISC-deficient mice. Mechanistically, we found that hepatic and circulating levels of TNF-α, IL-6, MCP-1, and IL-1β, as well as TNF-α- and MCP-1-producing KCs, in BRISC-deleted mice were dramatically decreased as early as 1 h after D-GalN/LPS challenge, which occurred prior to the elevation of the liver injury markers. Moreover, LPS-induced proinflammatory cytokines production in KCs was significantly diminished by BRISC deficiency in vitro, which was accompanied by potently attenuated NF-κB activation. Restoration of NF-κB activation by two small molecular activators of NF-κB p65 effectively reversed the suppression of cytokines production in ABRO1-deficient KCs by LPS. In conclusion, BRISC is required for optimal activation of NF-κB-mediated proinflammatory cytokines production in LPS-treated KCs and contributes to acute liver injury. This study opens the possibility to develop new strategies for the inhibition of KCs-driven inflammation in liver diseases.

Galacto-Oligosaccharides Alleviate LPS-Induced Immune Imbalance in Small Intestine through Regulating Gut Microbe Composition and Bile Acid Pool.

Recent evidence suggests that the protective effect of gut microbiota on intestinal inflammation can be achieved through a microbe-bile acids (BAs) mechanism. Galacto-oligosaccharides (GOS) are a kind of prebiotic that alter gut microbiota composition. To verify whether GOS has a protective effect on intestinal inflammation through a microbe-BAs mechanism, this research was performed in a lipopolysaccharide (LPS) porcine model with the presence or absence of GOS. GOS prevented LPS-induced production of pro-inflammatory cytokines, the decrease of bacterial bile salt hydrolase-containing bacteria abundance, and the decrease of chendoxycholic acid (CDCA) level in piglets. Additionally, CDCA decreased LPS-induced production of pro-inflammatory cytokines, induced the expression of the takeda G-protein receptor 5 (TGR5), and its downstream cyclic adenosine monophosphate (cAMP) production in lamina propria-derived CD11b+ cells. The cAMP inhibitor eliminated the protective effect of CDCA on lamina propria-derived CD11b+ cells. These results suggested that GOS reduced the production of pro-inflammatory cytokines and inhibited NF-κB activation via microbe-BA-dependent TGR5-cAMP signaling in LPS-challenged piglets.

Molecular binding mechanism and novel antidiabetic and anti-hypertensive bioactive peptides from fermented camel milk with anti-inflammatory activity in raw macrophages cell lines.

The investigation was to determine the effect of camel milk fermented with Limosilactobacillus fermentum KGL4 (MTCC 25515) on ACE-inhibiting, anti-inflammatory, and diabetes-preventing properties and also to release the novel peptides with antidiabetic and anti-hypertensive attributes with molecular interaction studies. Growth conditions were optimised on the basis of total peptide production by inoculating the culture in camel milk at different rates (1.5, 2.0, and 2.5%) along with different incubation periods (12, 24, 36, and 48h). However, after 48h of fermentation with a 2.5% rate of inoculum, the highest proteolytic activity was obtained. Reverse phase high-pressure liquid chromatography (RP-HPLC) was used to calculate the % Rpa from permeates of 3kDa and 10kDa fractions. Molecular weight distributions of fermented and unfermented camel milk protein fractions were compared using SDS-PAGE. Spots obtained from 2D gel electrophoresis were separated on the basis of pH and molecular weight. Spots obtained from 2D gel were digested with trypsin, and the digested samples were subjected to RP-LC/MS for the generation of peptide sequences. The inhibition of tumour necrosis factor alpha, interleukin-6, and interleukin-1 during fermentation was studied using RAW 264.7 macrophages. In the study, fermented camel milk with KGL4 (CMKGL4) inhibited LPS-induced nitric oxide (NO) production and pro-inflammatory cytokine production (TNF-α, IL-6, and IL-1β) by the murine macrophages. The results showed that the peptide structures (YLEELHRLNK and YLQELYPHSSLKVRPILK) exhibited considerable binding affinity against hPAM and hMGA during molecular interaction studies.

Inhibitory Effects of Ehretia tinifolia Extract on the Excessive Oxidative and Inflammatory Responses in Lipopolysaccharide-Stimulated Mouse Kupffer Cells.

Ehretia tinifolia (E. tinifolia) L., an evergreen tree with substantial biological activity, including antioxidant and anti-inflammatory effects, has been used in many herbal and traditional medicines. To elucidate its antioxidant and anti-inflammatory activity and the underlying mechanisms, we applied a methanol extract of E. tinifolia (ETME) to lipopolysaccharide (LPS)-stimulated mouse immortalized Kupffer cells. ETME suppressed the LPS-induced increase in nitric oxide, a mediator for oxidative stress and inflammation, and restored LPS-mediated depletion of total glutathione level by stabilizing antioxidative nuclear factor erythroid 2-related factor 2 (Nrf2) and the subsequent increase in heme oxygenase-1 levels. Furthermore, ETME inhibited the LPS-induced production of pro-inflammatory cytokines, including tumor necrosis factor-α, interleukin (IL)-1β, and IL-6. The inhibitory effects of ETME on pro-inflammatory responses were regulated by ETME-mediated dephosphorylation of mitogen-activated protein kinases (MAPKs: p38, p44/p42, and stress-associated protein kinase/c-Jun N-terminal kinase) and inhibition of nuclear localization of nuclear factor kappa B (NF-κB). These results suggest that ETME is a possible candidate for protecting Kupffer cells from LPS-mediated oxidative stress and excessive inflammatory responses by activating antioxidant Nrf2/HO-1 and inhibiting pro-inflammatory NF-κB and MAPKs, respectively.

Lobeglitazone inhibits LPS-induced NLRP3 inflammasome activation and inflammation in the liver.

Liver inflammation is a common feature of chronic liver disease and is often associated with increased exposure of the liver to lipopolysaccharide (LPS). Kupffer cells (KCs) are macrophages in the liver and produce various cytokines. Activation of KCs through the NLRP3 inflammasome pathway leads to release of proinflammatory cytokines and induces hepatocyte injury and hepatic stellate cell (HSC) activation. Lobeglitazone is a peroxisome proliferator-activated receptor gamma ligand and a type of thiazolidinedione that elicits anti-inflammatory effects. However, there is no clear evidence that it has direct anti-inflammatory effects in the liver. This study showed that lobeglitazone reduces LPS-induced NLPR3 inflammasome activation and production of proinflammatory cytokines in primary KCs and hepatocytes. Cytokines secreted by activated KCs increased hepatocyte inflammation and HSC activation, and lobeglitazone inhibited these responses. In addition, lobeglitazone suppressed liver fibrosis by inhibiting LPS-induced transforming growth factor (TGF)-β secretion and TGF-β-induced CTGF expression. The inhibitory effect of lobeglitazone on inflammasome activation was associated with suppression of liver fibrosis. These results suggest that lobeglitazone may be a treatment option for inflammation and fibrosis in the liver.

Cocktail of three isoquinoline alkaloids derived from Glaucium grandiflorum Boiss. & A. Huet subsp. refractum (Nábelek) Mory inhibits the production of LPS-induced ROS, pro-inflammatory cytokines, and mediators through the down-regulation of p38 MAPK in BV-2 cells

Glaucium grandiflorum extracts have traditionally been used to treat brain-related disorders. G. grandiflorum extracts also exhibited inhibitory effects on cholinesterase enzymes, as well as antigenotoxic activity. However, no research has been done on the effect of G. grandiflorum alkaloid extracts on the anti-oxidative and anti-inflammatory mechanisms. In this study we aimed to evaluate the anti-oxidative and anti-inflammatory activities of the alkaloid extract obtained from G. grandiflorum as well as the mechanisms responsible for their neuroprotective effects in neuronal damage caused by LPS in BV2 cells. We used LC-MS/MS and 1H, 13C NMR analysis to determine the presence of major alkaloids (allocryptopine, tetrahydropalmatine, and tetrahydroberberine N-oxide (trans-cannadine-N-oxide) in the alkaloid extracts. We used flow cytometry to study the alkaloid extracts' effects on ROS production; we also employed qRT-PCR and Western Blot to analyze the effects of oxidative stress and inflammation-related genes and proteins. ROS production within the cell was inhibited by chloroform alkaloid extract (CAE). There occurred marked CAE-induced reductions in IL-1β, Cox-2, and iNOS mRNA expressions. We also observed marked reductions in IL-6 and TNF-α mRNA expressions with methanol alkaloid extract (MAE). CAE effectively suppressed IL-1β and iNOS protein levels, especially as in qRT-PCR studies, while MAE effectively reduced IL-6 and TNF-α protein levels. Additionally, MAE was found to be prominent in suppressing the levels of Cox-2 protein, unlike qRT-PCR studies. According to our study findings, oxidative stress brought about by inflammation was suppressed by alkaloid extracts from G. grandiflorum which can be attributed to their suppressor effects on the pro-inflammatory cytokines-mediators, and p38 MAPK. As a result, a drug active substance that suppresses oxidative stress and inflammation has been brought to the neuropharmacological field.

Genetic Arg-304-His substitution in GRK5 protects against sepsis progression by alleviating NF-κB-mediated inflammation

Previous studies have demonstrated that G protein-coupled receptor kinase 5 (GRK5) exerts a pivotal regulatory effect on the inflammation associated with sepsis. The present study aimed to investigate the clinical association of GRK5 genetic variants with sepsis and to further explore the underlying genetic mechanisms involved in regulating sepsis-induced inflammatory responses and the pathogenesis of sepsis. This case-control study enrolled 1081 septic patients and 1147 matched controls for genotyping of GRK5 rs2230349 and rs2230345 polymorphisms. The effect of these genetic variants on GRK5-mediated inflammatory responses was analyzed in peripheral blood mononuclear cells (PBMCs) and THP-1 macrophages. A clinically relevant polymicrobial sepsis model was established by subjecting wild-type (WT) and GRK5-knockout mice to cecal ligation and puncture (CLP) to evaluate the role of GRK5 in sepsis. We identified significant differences in the genotype/allele distribution of rs2230349 G>A, but not rs2230345, between the sepsis subtype and septic shock subgroups (GA+AA vs. GG genotype, OR=0.698, 95% CI=0.547-0.893, P=0.004; A vs. G allele, OR=0.753, 95% CI=0.620-0.919, P=0.005) and between the survivor and nonsurvivor subgroups (GA+AA vs. GG genotype, OR=0.702, 95% CI=0.531-0.929, P=0.015; A vs. G allele, OR=0.753, 95% CI=0.298-0.949, P=0.017). PBMCs carrying the sepsis-associated protective A allele produced significantly lower levels of TNF-α and IL-1β upon LPS stimulation. The results from the in vitro experiment showed that the Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 significantly decreased the LPS-induced production of several proinflammatory cytokines, such as TNF-α, IL-6, IL-1β and MCP-1, via the IκB-α/NF-κB signaling pathway in THP-1 macrophages. Furthermore, GRK5-knockout mice exhibited a significant decrease in IκB-α phosphorylation/degradation, the p-p65/p65 ratio, the p-p50/p50 ratio, p65 nuclear translocation and downstream cytokine (TNF-α, IL-6, IL-1β and VCAM-1) production compared to WT mice after CLP surgery. A significant improvement in 7-day survival rate in GRK5-KO septic mice was observed in the presence of antibiotics. The Arg-304-His substitution caused by the rs2230349 G-to-A mutation in GRK5 might disrupt GRK5 function and alleviate IKB-α/NF-κB-mediated inflammatory responses, which ultimately conferred a genetic protective effect against susceptibility to sepsis progression and mortality. These results may, to some extent, explain the heterogeneity of the clinical prognoses of septic patients and provide novel opportunities for individualized approaches for sepsis treatment.

RCsHscB Derived from Clonorchis sinensis: A Carcinogenic Liver Fluke Ameliorates LPS-Induced Acute Hepatic Injury by Repression of Inflammation.

Sepsis-associated acute liver injury caused by spillovers of bacteria and endotoxins (lipopolysaccharide, LPS) into the liver remains a public health issue due to the lack of specific therapeutic approaches. Previous studies showed that the recombinant protein HscB (rCsHscB) of Clonorchis sinensis, a carcinogenic liver fluke, had an anti-inflammatory effect and could alleviate inflammatory diseases such as enteritis; however, whether it can prevent sepsis-associated acute liver injury induced by LPS is still unknown. In our current study, the therapeutic effects and the potential mechanisms of rCsHscB on LPS-induced acute liver injury were investigated both in vivo and in vitro. The data showed that rCsHscB prevented LPS-induced liver damage, as demonstrated by histopathological observation and hepatic damage markers (the activities of serum ALT and AST) in a murine model of sepsis-associated acute liver injury. rCsHscB also significantly reversed the high levels of serum IL-6 and MCP-1 induced by LPS. In addition, rCsHscB attenuated the production of LPS-induced proinflammatory cytokines, including IL-6 and TNF-α, in a macrophage cell line-RAW264.7, through possible mediation by the MAPK signaling pathway in vitro. In conclusion, the present study demonstrates that rCsHscB derived from a fluke C. sinensis protects against sepsis-associated acute liver injury induced by LPS, which may be attributed to the inhibition of the MAPK signaling pathway. Our present study provides a potential therapeutic strategy for sepsis-associated acute liver injury.