LPS Induction Research Articles

The anti-inflammatory effects of photobiomodulation are mediated by cytokines: Evidence from a mouse model of inflammation.

There is an urgent need for therapeutic approaches that can prevent or limit neuroinflammatory processes and prevent neuronal degeneration. Photobiomodulation (PBM), the therapeutic use of specific wavelengths of light, is a safe approach shown to have anti-inflammatory effects. The current study was aimed at evaluating the effects of PBM on LPS-induced peripheral and central inflammation in mice to assess its potential as an anti-inflammatory treatment. Daily, 30-min treatment of mice with red/NIR light (RL) or RL with a 40 Hz gamma frequency flicker for 10 days prior to LPS challenge showed anti-inflammatory effects in the brain and systemically. PBM downregulated LPS induction of key proinflammatory cytokines associated with inflammasome activation, IL-1β and IL-18, and upregulated the anti-inflammatory cytokine, IL-10. RL provided robust anti-inflammatory effects, and the addition of gamma flicker potentiated these effects. Overall, these results demonstrate the potential of PBM as an anti-inflammatory treatment that acts through cytokine expression modulation.

Vinculin B inhibits NF-κB signaling pathway by targeting MyD88 in miiuy croaker, Miichthys miiuy

Myeloid differentiation factor 88 (MyD88) is the canonical adaptor for inflammatory signaling pathways downstream from members of the Toll-like receptor (TLR) and interleukin-1 (IL-1) receptor families, which activates the NF-κB signaling pathway and regulates immune and inflammatory responses. In this study, we found that Vinculin B (Vclb) is an inhibitor in the NF-κB signaling pathway, and its inhibitory effect was enhanced by LPS induction. Furthermore, Vclb inhibits NF-κB activation by targeting MyD88, thereby suppressing the production of inflammatory cytokines. Mechanistically, Vclb inhibits the NF-κB signaling pathway by targeting MyD88 ubiquitin-proteasome pathway. In summary, our study reveals that Vclb inhibits NF-κB signaling activation and mediates innate immunity in teleosts via the ubiquitin-proteasome pathway of MyD88.

Transcriptomic and Bioinformatic Analyses Identifying a Central Mif-Cop9-Nf-kB Signaling Network in Innate Immunity Response of Ciona robusta

The Ascidian C. robusta is a powerful model for studying innate immunity. LPS induction activates inflammatory-like reactions in the pharynx and the expression of several innate immune genes in granulocyte hemocytes such as cytokines, for instance, macrophage migration inhibitory factors (CrMifs). This leads to intracellular signaling involving the Nf-kB signaling cascade that triggers downstream pro-inflammatory gene expression. In mammals, the COP9 (Constitutive photomorphogenesis 9) signalosome (CSN) complex also results in the activation of the NF-kB pathway. It is a highly conserved complex in vertebrates, mainly engaged in proteasome degradation which is essential for maintaining processes such as cell cycle, DNA repair, and differentiation. In the present study, we used bioinformatics and in-silico analyses combined with an in-vivo LPS exposure strategy, next-generation sequencing (NGS), and qRT-PCR to elucidate molecules and the temporal dynamics of Mif cytokines, Csn signaling components, and the Nf-κB signaling pathway in C. robusta. A qRT-PCR analysis of immune genes selected from transcriptome data revealed a biphasic activation of the inflammatory response. A phylogenetic and STRING analysis indicated an evolutionarily conserved functional link between the Mif-Csn-Nf-kB axis in ascidian C. robusta during LPS-mediated inflammation response, finely regulated by non-coding molecules such as microRNAs (miRNAs).

Ozoile Reduces the LPS-Induced Inflammatory Response in Colonic Epithelial Cells and THP-1 Monocytes

Inappropriate activation of immune functions in intestinal epithelial cells can lead to inflammation that is characterized also by infiltration into intestinal tissue of monocytes/macrophages. Current therapies for intestinal inflammation include anti-inflammatory, immunosuppressive and biological drugs. Ozoile (stable ozonides) has been reported to exert anti-inflammatory effects. However, ozonated oil has been used mainly for topical applications and no data are available about its effects on intestinal cells or immune cells. In this study, we evaluated Ozoile effects on human HT-29 colonic cells and THP-1 monocytic cells stimulated with LPS to induce inflammation. HT-29 and THP-1 cells were treated with LPS in the presence/absence of Ozoile for 4 h. Biomarkers of inflammation, some members of tight junctions and the adhesion molecule ICAM were assessed by qRT-PCR. Protein expression was analyzed by Western blotting. The release of TNF-α and IL-1β was measured by ELISA. In HT-29, Ozoile inhibited LPS-induced expression of TNF-α, IL-1β, ZO-1, CLDN1, NOS2 and MMP-2 and increased the expression of Nrf2 and SOD2 antioxidant proteins. In THP-1 cells, the LPS induction of TNF-α, IL-1β and ICAM was counteracted by Ozoile treatment. Our in vitro results demonstrate the effectiveness of Ozoile in reducing the inflammatory response in intestinal and monocytic cells. Further in vivo studies are necessary to confirm its possible use for intestinal inflammatory conditions.

Evaluation of changes of apelin and apelin receptor (APJ) expression in cervix-uterus and placental axis in an LPS-induced preterm labor model.

Although preterm birth is among the preventable causes of maternal and infant death, its mechanism has not yet been clarified. When evaluated in terms of the results, the psycho-social burden of mother-infant losses and the costs of rehabilitation, care, and treatment for postpartum sequelae are high. When evaluated in terms of its causes, infection/inflammation has an important place. Therefore, it is essential to understand the role of pro- and anti-inflammatory proteins in the process. In our study, apelin and apelin receptor (APJ) expression in the cervix-uterus and placental axis were evaluated at tissue and protein levels in pregnant and non-pregnant control, sham, PBS, and LPS groups in the infection model in which LPS induction was performed by midline laparotomy, in CD-1 mice. The evaluation of this axis regarding apelin and apelin receptor in the preterm birth model is new in the literature. Apelin is expressed more intensely in uterine epithelial cells than in the cervix. In the placenta, expression is more intense in the junctional zone compared to other zones. Apelin protein levels decrease significantly in the cervix and placenta whereas it increases in the uterus. While no change was observed in the expression of the apelin receptor at the tissue and protein level in the cervix and uterus, it increased in both aspects in the placenta in the invasive procedure groups. We propose that the decrease in apelin protein due to LPS in the preterm delivery model may be related to the effort to compensate for the balance deteriorated in the pro-inflammatory direction with post-transitional modification at the tissue level. The tendency of apelin to increase with pregnancy has led to the conclusion that it is necessary for a healthy pregnancy. Although the apelin receptor does not change with inflammation, it is necessary to investigate the mechanisms associated with its stress and trauma-induced increase, since it increases in the invasive procedure group.

Transcription Factor Sox9 Exacerbates Kidney Injury through Inhibition of MicroRNA-96-5p and Activation of the Trib3/IL-6 Axis

Introduction: Our study investigated the possible mechanisms of the role of the transcription factor Sox9 in the development and progression of kidney injury through regulation of the miR-96-5p/Trib3/IL-6 axis. Methods: Bioinformatics analysis was performed to identify differentially expressed genes in kidney injury and normal tissues. An in vivo animal model of kidney injury and an in vitro cellular model of kidney injury were constructed using LPS induction in 8-week-old female C57BL/6 mice and human normal renal tubular epithelial cells HK-2 for studying the possible roles of Sox9, miR-96-5p, Trib3, and IL-6 in kidney injury. Results: Sox9 was highly expressed in both mouse and cellular models of kidney injury. Sox9 was significantly enriched in the promoter region of miR-96-5p and repressed miR-96-5p expression. Trib3 was highly expressed in both mouse and cellular models of kidney injury and promoted inflammatory responses and kidney injury. In addition, Trib3 promoted IL-6 expression, which was highly expressed in kidney injury, and promoted the inflammatory response and extent of injury in kidney tissue. In vivo and in vitro experiments confirmed that the knockdown of Sox9 improved the inflammatory response and fibrosis of mouse kidney tissues and HK-2 cells, while the ameliorative effect of silencing Sox9 was inhibited by overexpression of IL-6. Conclusion: Collectively, Sox9 up-regulates miR-96-5p-mediated Trib3 and activates the IL-6 signaling pathway to exacerbate the inflammatory response, ultimately promoting the development and progression of kidney injury.

Dengue virus induced autophagy is mediated by HMGB1 and promotes viral propagation

Dengue virus (DENV) exploits various cellular pathways including autophagy to assure enhanced virus propagation. The mechanisms of DENV mediated control of autophagy pathway are largely unknown. Our investigations have revealed a novel role for high-mobility group box1 protein (HMGB1) in regulation of cellular autophagy process in DENV-2 infected A549 cell line. While induction of autophagy by rapamycin treatment resulted in enhanced DENV-2 propagation, the blockade of autophagy flux with bafilomycin A1 suppressed viral replication. Furthermore, siRNA-mediated silencing of HMGB1 significantly abrogated dengue induced autophagy, while LPS induced HMGB1 expression counteracted these effects. Interestingly, silencing of HMGB1 showed reduction of BECN1 and stabilization of BCL-2 protein. On the contrary, LPS induction of HMGB1 resulted in enhanced BECN1 and reduction in BCL-2 levels. This study shows that the modulation of autophagy by DENV-2 is HMGB1/BECN1 dependent. In addition, glycyrrhizic acid (GA), a potent HMGB1 inhibitor suppressed autophagy as well as DENV-2 replication. Altogether, our data suggests that HMGB1 induces BECN1 dependent autophagy to promote DENV-2 replication.

Efek sitotoksisitas ekstrak etanol 70% curcuma xanthorrhiza roxb. Terhadap sel raw 264.7 yang diinduksi lipopolisakarida "lps" (Laporan Penelitian)

Background: Curcuma xanthorrhiza Roxb. has bioactive compounds that are beneficial as anti-inflammatory. To get these compounds need to be extracted. Ethanol 70% can increase the solubility of both polar and non-polar compounds. RAW 264.7 cells with LPS induction cause an inflammatory state due to the active pro-inflammatory cytokines. To suppress the development of inflammation, a cytotoxicity test can be carried out to see the toxic nature of the extract against inflamed cells. Goals: To determine the cytotoxicity effect of 70% ethanol extract of C. xanthorrhiza on LPS-induced RAW 264.7 cells. Methods: In vitro laboratory experimental research. Preparation of extracts of C. xanthorrhiza Roxb. carried out with 70% ethanol then made concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 g/mL. Cytotoxicity was tested using the MTS. Untreated culture medium was used as a negative control and a positive control of diclofenac sodium. Then it was incubated for 24 hours at 37oC. After 24 hours of incubation, MTS was added to the plate and incubated again for 3 hours. The results were read using spectrophotometry. The resulting absorption is equivalent to living cells. The research data were analyzed using one-way ANOVA and Dunnet T3 tests. Results: Extracts of C. xanthorrhiza with concentrations of 200 and 100 g/mL were weakly cytotoxic and at concentrations of 50, 25, 12.5, 6.25, and 3.125 g/mL showed non-toxic effects on LPS-induced RAW 264.7 cells. Based on the IC50, C. xanthorrhiza Roxb. extract had a strong cytotoxic effect on LPS-induced RAW 264.7 cells. Conclusion: C. xanthorrhiza extract was toxic to LPS-induced RAW 264 cells.

Metformin Improves Burn Wound Healing by Modulating Microenvironmental Fibroblasts and Macrophages.

Metformin, a biguanide, exerts different functions through various signaling pathways. In order to investigate the function and mechanism of metformin in burn wounds, we established burn rat models, subcutaneously injected metformin to treat the wounds, and observed the morphologies and the expression of collagen I, collagen III, fibronectin, and pro-inflammatory markers. In vitro experiments were performed to investigate the effects of metformin on the proliferation, migration, and collagen I synthesis of the mouse embryonic fibroblast (NIH 3T3) cell line and on the proliferation, apoptosis, and immune response of the mouse mononuclear macrophage (RAW 264.7) cell line. Finally, we studied the regulatory effects of metformin on a co-culture of RAW 264.7/NIH 3T3 cells. We found that 100 mM of metformin reduced dermal thickness, collagen I deposition, and mRNA expression of IL1β and CCL2 in rat burn wounds. In vitro experiments revealed that metformin inhibited the proliferation of NIH 3T3 and RAW 264.7 cells. Metformin attenuated NIH 3T3 cell migration via the AMPK/mTOR pathway and attenuated collagen I synthesis through the TGFβ1/Smad3 pathway. Metformin inhibited the apoptosis of RAW 264.7 cells induced by 10 μg/mL LPS. Metformin downregulated the mRNA expression of IL1β and CCL2 in RAW 264.7 cells under 1 μg/mL LPS induction by inhibiting NF-κB p65 phosphorylation. In a RAW 264.7/NIH 3T3 co-culture, metformin attenuated collagen I synthesis in NIH 3T3 cells by inhibiting RAW 264.7 paracrine secretion of TGF-β1. This provides new evidence related to the development of metformin for potentially improving burn wound healing.

Quercetin Ameliorates Lipopolysaccharide-Induced Duodenal Inflammation through Modulating Autophagy, Programmed Cell Death and Intestinal Mucosal Barrier Function in Chicken Embryos.

Diarrhea has been a global health problem for centuries, and the treatment has become increasingly difficult duo to the antibiotics overuse and resistance. Quercetin is a common flavonoid of extracts of vegetables, fruits, and traditional Chinese herbs, however, the mechanism of quercetin alleviating LPS-induced duodenal inflammation remains elusive. Specific pathogen-free chicken embryos (n = 120) were allocated to groups including control, PBS with or without alcohol, LPS (125 ng/egg) with or without quercetin (10, 20, or 40 nmol/egg, respectively), and quercetin groups (10, 20, or 40 nmol/egg). Fifteen day-old embryonated eggs were inoculated with abovementioned solutions via the allantoic cavity. At embryonic day 19, the duodena of the embryos were collected for histopathological examination, RNA extraction and real-time polymerase chain reaction, immunohistochemical investigations, and Western blotting. The results demonstrated quercetin enhanced the inflammatory cell infiltration in the Peyer's patch of the intestinal mucosa after LPS induction. The LPS-induced expressions of these inflammation-related factors (TLR4, IL-1β, MMP3, MMP9, NFKB1, IFNγ, IL-8, IL-6) were completely blocked by quercetin. Quercetin also decreased the protein expression of TLR4, IL-1β, MMP3, and MMP9 after LPS induction. Quercetin could down-regulate autophagy gene expression (ATG5, LC3-1, LC3-2, and LKB1), and decreased the protein expression of ATG5, and LC3-1/LC3-2 after LPS induction. Quercetin treatment prevented LPS-induced increases of the gene expressions of programmed cell death factors (TNFα, Fas, CASP1, CASP3, CASP12, Drp1, and RIPK1); meanwhile, quercetin decreased the protein expression of CASP1 and CASP3 after LPS challenge. LPS reduced the gene expression of mucin 2, but upregulated the mRNA and protein expression of claudin 1, occludin, and ZO-1, and this was balanced by quercetin. This evidence suggests that quercetin can alleviate duodenal inflammation induced by LPS through modulating autophagy, programmed cell death, intestinal barrier function.

Effect and Mechanism Analysis of Pig FUT8 Gene on Resistance to Escherichia coli F18 Infection.

Post-weaning diarrhea caused by enterotoxigenic Escherichia coli F18 (E. coli F18) causes significant economic losses for pig producers. Fucosyltransferase 8 (FUT8) is a glycosyltransferase that catalyzes core fucosylation; however, its role in mediating the resistance to E. coli F18 infection in pigs remains unknown. In this study, we systematically verified the relationship between FUT8 expression and E. coli resistance. The results showed that FUT8 was expressed in all detected tissues of Meishan piglets and that its expression was significantly increased in the duodenum and jejunum of E. coli F18-sensitive individuals when compared to E. coli F18-resistant individuals. FUT8 expression increased after exposure to E. coli F18 (p < 0.05) and decreased significantly after LPS induction for 6 h (p < 0.01). Then, the IPEC-J2 stable cell line with FUT8 interference was constructed, and FUT8 knockdown decreased the adhesion of E. coli F18ac to IPEC-J2 cells (p < 0.05). Moreover, we performed a comparative transcriptome study of IPEC-J2 cells after FUT8 knockdown via RNA-seq. In addition, further expression verification demonstrated the significant effect of FUT8 on the glycosphingolipid biosynthesis and Toll-like signaling pathways. Moreover, the core promoter of FUT8, which was located at −1213 bp to −673 bp, was identified via luciferase assay. Interestingly, we found a 1 bp C base insertion mutation at the −774 bp region, which could clearly inhibit the transcriptional binding activity of C/EBPα to an FUT8 promoter. Therefore, it is speculated that FUT8 acts in a critical role in the process of E. coli infection; furthermore, the low expression of FUT8 is conducive to the enhancement of E. coli resistance in piglets. Our findings revealed the mechanism of pig FUT8 in regulating E. coli resistance, which provided a theoretical basis for the screening of E. coli resistance in Chinese local pig breeds.

The role and mechanism of NLRP3 inflammasome-mediated astrocyte activation in dehydrocorydaline against CUMS-induced depression.

Background: Depression is a common and potentially life-threatening mental illness, and currently, there is a lack of effective treatment. It has been reported that dehydrocorydaline (DHC) can inhibit monoamine transporter uptake in depressed CUMS mice, but more possible mechanisms of action remain to be further studied. Methods: C57BL/6 mice were exposed to chronic unpredictable mild stress (CUMS) for five consecutive weeks. The mice were administrated with dehydrocorydaline or fluoxetine (FLU) for four consecutive weeks. Behavioral tests including sucrose preference test (SPT), tail suspension test (TST), and forced swimming test (FST) were applied. In parallel, hematoxylin and eosin (H&E) staining and Nissl staining were used to explore the effect of DHC on pathological changes in the hippocampus. The concentrations of depression-related factors (5-HT and DA) and inflammatory factors (TNF-α, IL-6, and IL-1β) in the hippocampus and serum were assessed by ELISA assay. NLRP3 inflammasome pathway-related proteins (NLRP3, IL-18, IL-1 IL-1α, and caspase-1) were detected by western blot. The activation of microglia and astrocytes was subjected to immunofluorescent staining. Additionally, microglia were treated with DHC (100mg/L) for 24h following incubation with 100ng/ml LPS for 12h. ov-NC or ov-NLRP3 plasmid was transfected into microglia 6h before LPS induction for exploring the effect of NLRP3 overexpression on DHC-inhibited microglia activation. Then, conditioned media of microglia were collected from each group, followed by intervention of astrocytes for 24h to explore the effect of NLRP3 overexpression of microglia on astrocyte activation. Results: In vivo administration of DHC was found to ameliorate depressive-like behaviors and attenuate neuron damage of CUMS mice. DHC increased neurotransmitter concentration, reduced the proinflammatory factor levels, attenuated NLRP3 inflammasome activation, and decreased A1 astrocyte and microglia activation in the hippocampus of CUMS mice. Furthermore, in vivo results showed that activated microglia induced activation of A1 astrocytes but not A2 astrocytes. Conclusion: Taken together, we provided evidence that DHC exhibited antidepressive effects on CUMS mice possibly via NLRP3 inflammasome-mediated astrocyte activation.

Leptospiral lipopolysaccharide mediated Hog1 phosphorylation in Saccharomyces cerevisiae directs activation of autophagy

Cells have developed a variety of mechanisms to counteract stress to give a specific and adaptive response. Yeast Hog1 is a homolog to mammalian p38, which is a mitogen-activated protein kinase. In this work, we analyze the Hog1 signaling during the induction of leptospiral LPS (100 ng/mL) and the hyperosmotic element NaCl (0.8 M). After the addition of stress elements, the stress-activated protein kinase was phosphorylated within 30 min of exposure and led to the expression of various genes responsible for cell survival. We found that leptospiral lipopolysaccharide mediated Hog1 phosphorylation leads to activation of autophagy-related genes phosphorylation; thereby cells encounter and digest the metabolic waste or organelles for their energy during starvation. And, the wild-type cells accumulate lipid droplets and trigger vacuole calcium release, to maintain cell survival. Loss of Hog1 leads to shrinkage in the cell wall, condensation of the cytoplasmic part, and high-level ROS production. This led to the Hog1 mutant cell death under LPS treatment or stress condition. The phosphorylation of stress-activated kinase during exposure to leptospiral LPS provides insight and knowledge about the organization of cellular metabolic products and cell survival during stress conditions and identifies the pathogenic mechanisms of leptospirosis.

Extracellular Vesicles of Mesenchymal Stem Cells Are More Effectively Accessed through Polyethylene Glycol-Based Precipitation than by Ultracentrifugation.

Extracellular vesicles (EVs) have been identified as cell-cell communication agents, and EVs derived from mesenchymal stem cells (MSCs) exhibit therapeutic effects similar to those of the cells of origin. Precipitation methods have been used extensively for EV harvests, such as UC- (ultracentrifugation-) or PEG- (polyethylene glycol-) based methods, and the difference in EVs derived from MSCs by UC and PEG is not fully understood. We harvested EVs from amniotic fluid MSCs (AF-MSCs) by UC- or PEG-based precipitation methods and conducted a comparison study of those EVs derived by the two methods: output, RNA, and protein expression of EVs and EV biological reaction in a THP-1-cell model of LPS induction, which was considered an infection model. There was no difference in morphology, size, or specific marker-positive ratio of PEG-EVs and UC-EVs, but PEG obtained more EV particles, protein, and RNA than the UC method. In our THP-1 model of LPS induction, MSC-EVs did not lead to a change in protein expression but inhibited the LPS-induced increase in cytokine secretion. UC-EVs were more effective for TNF-α inhibition, and PEG-EVs were more effective for IL10 inhibition. Thus, our findings provide evidence that PEG-based precipitation is a more efficient mesenchymal stem cell-extracellular vesicle-derived method than UC.

In vitro two-step granuloma formation model for testing innate immune response to implants and coatings

The extensive innate immune response to implanted biomaterials contributes significantly to their sub-par performance and failure. Granuloma formation is one of such reactions which results in multi-cell type clusters in line with the immune reaction to implanted materials. However, currently no in vitro model of granuloma formation exists that takes into account the arrival of multiple cell types (immune cells and connective tissue cells) to the implant insertion site. In this study, we developed a two-step model based on stimulated macrophage seeding followed by fibroblast introduction after a physiologically relevant time period for mimicking initial steps of immune reaction to biomaterials and inducing granuloma like behavior. Both LPS and TNF-α induction resulted in granuloma like formations which persisted longer than the control conditions. Introduction of human fibroblasts resulted in the colonization of the surfaces where the cell numbers and the collagen secretion were dependent on the microenvironment. In order to demonstrate the capacity of our model system to monitor the reaction to a given coating, a validated antimicrobial coating (Polyarginine (PAR)/Hyaluronic acid (HA)) was used as a testing bed. The coating prevented the adhesion of macrophages while allowing the adhesion of the fibroblast at the time of their arrival. Similar to its antimicrobial activity, macrophage metabolic activity and M2 differentiation in the presence of PAR was dependent to its chain length. The incorporation of fibroblasts resulted in decreased TNF-α and increased IL-1RA secretion especially in stimulation conditions. The pro- and anti-inflammatory cytokine secretions were low for PAR/HA coatings in line with the decreased number of macrophage presence. In the presence of complex PBMC population, the coating resulted in slightly less cellular attachment, without any significant cytokine secretion; the absence of inflammatory reaction was also demonstrated in vivo in a mouse model. The described in vitro granuloma testing system can control the macrophage reaction as a function of stimulation. It can also be used for testing new biomaterials for the potential innate immune responses and also for validation of implant coatings beyond their primary function from the immune response point of view.

Cross‐linking by tissue transglutaminase‐2 alters fibrinogen‐directed macrophage proinflammatory activity

BackgroundThe blood coagulation factor fibrin(ogen) can modulate inflammation by altering leukocyte activity. Analyses of fibrin(ogen)‐mediated proinflammatory activity have largely focused on leukocyte integrin binding activity revealed by conversion of fibrinogen to a stabilized fibrin polymer by blood coagulation enzymes. In addition to coagulation enzymes, fibrinogen is a substrate for tissue transglutaminase‐2 (TG2), a widely expressed enzyme that produces unique fibrinogen Aα‐γ chain cross‐linked products. ObjectivesWe tested the hypothesis that TG2 dependent cross‐linking alters the proinflammatory activity of surface‐adhered fibrinogen. MethodsMouse bone marrow‐derived macrophages (BMDMs) were cultured on tissue culture plates coated with fibrinogen or TG2‐cross‐linked fibrinogen (10 µg/ml) and then stimulated with lipopolysaccharide (LPS, 1 ng/ml) or vehicle for various times. ResultsIn the absence of LPS stimulation, TG2‐cross‐linked fibrin(ogen) enhanced inflammatory gene induction (e.g., Tnfα) compared with unmodified fibrinogen. LPS stimulation induced mitogen‐activated protein kinase phosphorylation, IκBα degradation, and expression of proinflammatory cytokines (e.g., tumor necrosis factor α) within 60 min. This initial cellular activation was unaffected by unmodified or TG2‐cross‐linked fibrinogen. In contrast, LPS induction of interleukin‐10 mRNA and protein and STAT3 phosphorylation was selectively attenuated by TG2‐cross‐linked fibrinogen, which was associated with enhanced proinflammatory cytokine secretion by LPS‐stimulated BMDMs at later time points (6 and 24 h). ConclusionsThe results indicate that atypical cross‐linking by TG2 imparts unique proinflammatory activity to surface‐adhered fibrinogen. The results suggest a novel coagulation‐independent mechanism controlling fibrinogen‐directed macrophage activation.

Pirfenidone ameliorates early pulmonary fibrosis in LPS-induced acute respiratory distress syndrome by inhibiting endothelial-to-mesenchymal transition via the Hedgehog signaling pathway

Pulmonary vascular endothelial dysfunction is a key pathogenic mechanism in acute respiratory distress syndrome (ARDS), resulting in fibrosis in lung tissues, including in the context of COVID-19. Pirfenidone (PFD) has become a novel therapeutic agent for treating idiopathic pulmonary fibrosis (IPF) and can improve lung function, inhibit fibrosis and inhibit inflammation. Recently, endothelial-to-mesenchymal transition (EndMT) was shown to play a crucial role in various respiratory diseases. However, the role of PFD in the course of EndMT in LPS-induced ARDS remains poorly understood. The purpose of this study was to explore the anti-EndMT effects of PFD on pulmonary fibrosis after LPS-induced ARDS. First, we determined that PFD significantly reduced LPS-induced ARDS, as shown by significant pathological alterations, and alleviated the oxidative stress and inflammatory response in vitro and in vivo. Furthermore, PFD decreased pulmonary fibrosis in LPS-induced ARDS by inhibiting EndMT and reduced the expression levels of Hedgehog (HH) pathway target genes, such as Gli1 and α-SMA, after LPS induction. In summary, this study confirmed that inhibiting the HH pathway by PFD could decrease pulmonary fibrosis by downregulating EndMT in LPS-induced ARDS. In conclusion, we demonstrate that PFD is a promising agent to attenuate pulmonary fibrosis following ARDS in the future.

Local nebulization of 1\u03b1,25(OH)2D3 attenuates LPS-induced acute lung inflammation

BackgroundEvidence supports a critical role of vitamin D status on exacerbation in chronic obstructive pulmonary disease, indicating the need to avoid vitamin D deficiency in these patients. However, oral vitamin D supplementation is limited by the potential risk for hypercalcemia. In this study, we investigated if local delivery of vitamin D to the lungs improves vitamin D-mediated anti-inflammatory action in response to acute inflammation without inducing hypercalcemia.MethodsWe studied vitamin D sufficient (VDS) or deficient (VDD) mice in whom 1α,25(OH)2D3 (0.2 μg/kg) or a vehicle followed by lipopolysaccharide (LPS 25 µg) were delivered to the lung as a micro-spray.ResultsLocal 1α,25(OH)2D3 reduced LPS-induced inflammatory cells in bronchoalveolar lavage (BAL) in VDS (absolute number of cells: − 57% and neutrophils − 51% p < 0.01) and tended to diminish LPS-increased CXCL5 BAL levels in VDS (− 40%, p = 0.05) while it had no effect on CXCL1 and CXCL2 in BAL and mRNA in lung of VDS and VDD. It also significantly attenuated the increased IL-13 in BAL and lung, especially in VDD mice (− 41 and − 75%, respectively). mRNA expression of Claudin-18 in lung was significantly lower in VDS mice with local 1α,25(OH)2D3 while Claudin-3, -5 and -8 mRNA levels remained unchanged. Finally, in VDD mice only, LPS reduced lung mRNA expression of adhesion junction Zona-occludens-1, in addition to increasing uric acid and total protein in BAL, which both were prevented by local 1α,25(OH)2D3.ConclusionUnder normal levels of vitamin D, local 1α,25(OH)2D3 nebulization into the lung efficiently reduced LPS induction of inflammatory cells in BAL and slightly attenuated LPS-increase in CXCL5. In case of severe vitamin D deficiency, although local 1α,25(OH)2D3 nebulization failed to significantly minimize cellular inflammation in BAL at this dose, it prevented epithelial barrier leakage and damage in lung. Additional research is needed to determine the potential long-term beneficial effects of local 1α,25(OH)2D3 nebulization on lung inflammation.

Potential of nobiletin against Alzheimer's disease through inhibiting neuroinflammation.

This study aimed to explore the mechanism of Nobiletin attenuating Alzheimer's disease (AD) by inhibiting neuroinflammation. The expression of inflammatory cytokines and HMGB-1 in serum of AD patients were examined. Microglia (MGs) were treated with different doses of Nobiletin before LPS and Nigericin induction. Cell viability and apoptosis were determined by CCK-8 and TUNEL assays, respectively. APP/PS1 mice were gavaged with Nobiletin, and Morris water maze (MWM) was established to record swimming speed, escape latency, the number of platform crossings, and time spent in the platform quadrant. MGs activation in brain tissues was evaluated by immunofluorescence. The expression of pyroptosis-related proteins, inflammatory cytokines, and HMGB-1 was determined in the hippocampus and MGs. The levels of inflammatory cytokines and HMGB-1 were high in serum of AD patients. Treatment with different concentrations of Nobiletin prominently enhanced cell viability and reduced apoptosis and the expression of inflammatory cytokine and pyroptosis-related proteins in LPS + Nigericin-induced MGs. Gavage of different doses of Nobiletin into APP/PS1 mice shortened the escape latency in mice, diminished MGs activation in brain tissues, and remarkably elevated the number of platform crossings and the time spent in the platform quadrant without obvious change in swimming speed, suggesting that Nobiletin improved the spatial learning and memory abilities in APP/PS1 mice. The expression of pyroptosis-related proteins, HMGB-1, and inflammatory cytokines was decreased dramatically by Nobiletin in the hippocampus of APP/PS1 mice. Nobiletin can inhibit neuroinflammation by inhibiting HMGB-1, pyroptosis-related proteins, and inflammatory cytokines, thus mitigating AD.

Protective effect and mechanism of loganin and morroniside on acute lung injury and pulmonary fibrosis

BackgroundLoganin and morroniside are two iridoid glycosides with anti-inflammatory, antioxidant and anti-tumor effects. Whether they have effect on acute lung injury and pulmonary fibrosis are still unknown. PurposeTo explore the potential effects of loganin and morroniside against acute lung cancer and pulmonary fibrosis, and the underlying molecular mechanism. Study design and methodsCell and animal models of acute lung injury were established by the induction of LPS. After intervention with loganin and morroniside, the pathological symptom of lung tissue was assessed, pro-inflammatory factors in cells and lung tissues were detected, NF- κB/STAT3 signaling pathway related proteins were detected by western blotting. Mice pulmonary fibrosis model was induced by bleomycin, pathological symptom was assessed by HE and Masson staining. Fibrosis related indicators were detected by qPCR or western blot. CD4+/CD8+ was detected by flow cytometry. ResultsLoganin and morroniside relieved the pathological symptom of lung tissue in acute lung injury, pro-inflammatory factors such as IL-6, IL-1β, TNF-α mRNA were inhibited. Expression of p-p65 and STAT3 in lung tissues were also downregulated. In addition, loganin and morroniside downregulated the expression of collagen fiber, hydroxyproline and TGF-β1, collagen I and α-SMA mRNA in lung tissues of pulmonary fibrosis model. This study proved that loganin and morroniside have protective effect on acute lung injury and pulmonary fibrosis, and may provide theoretical basis for the development of new clinical drugs.