Protective effect of trans-nerolidol on vascular endothelial cell injury induced by lipopolysaccharides / Efectos protectores del trans-nerolidol en lesiones inducidas por lipopolisacáridos en células endoteliales vasculares

This article aimed to discuss the protection of trans - nerolidol on vascular endothelial cells (ECs) injured by lipopolysac charides. ECs were divided into four groups: normal, model, low and high dose trans - nerolidol treatment groups. The cell survival rate and the contents of NO in the cell culture supernatant were determined. The protein expression and transcript level of pe roxisome proliferator - activated receptor - γ (PPARγ), endothelial nitric oxide synthase (eNOS), and inducible nitric oxide synthase (iNOS) were determined by western blotting and RT - PCR respectively. Compared with the normal group, cell livability, protein e xpression and mRNA transcript level of PPARγ and eNOS decreased, NO contents, protein expression and mRNA transcript tlevel of iNOS increased in model group significantly. Compared with model group, all the changes recovered in different degree in treatmen t groups. Hence, it was concluded that trans - nerolidol can alleviate the ECs injuryby the regulation of iNOS/eNOS through activating PPARγ in a dose - dependent manner

Este artículo tiene como objetivo discutir la protección del trans - nerolidol en las células endoteliales vasculares (CE) dañadas por lipopolisacáridos. Las CE se di vidieron en cuatro grupos: normal, modelo, grupos de tratamiento con trans - nerolidol de baja y alta dosis. Se determinó la tasa de supervivencia de las células y los contenidos de óxido nítrico (NO) en el sobrenadante del cultivo celular. La expresión de p roteínas y el nivel de transcripción del receptor activado por proliferadores de peroxisomas - γ (PPARγ), el óxido nítrico sint et asa endotelial (eNOS) y el óxido nítrico sint et asa inducible (iNOS) se determinaron mediante western blot y RT - PCR, respectivamen te. En comparación con el grupo normal, la viabilidad celular, la expresión de proteínas y el nivel de transcripción de PPARγ y eNOS disminuyeron, los contenidos de NO, la expresión de proteínas y el nivel de transcripción de iNOS aumentaron significativam ente en el grupo modelo. En comparación con el grupo modelo, todos los cambios se recuperaron en diferentes grados en los grupos de tratamiento. Por lo tanto, se concluyó que el trans - nerolidol puede aliviar el daño en las CE regulando iNOS/eNOS a través d e la activación de PPARγ de manera dependiente de la dosis.

Cytokine production by bovine adipose tissue stromal vascular fraction cells upon Neospora caninum stimulation.

In bovines few studies addressed the contribution of adipose tissue to the host immune response to infection. Here we evaluated the in vitro response of bovine adipose tissue stromal vascular fraction (SVF) cells to the protozoan parasite Neospora caninum, using live and freeze-killed tachyzoites. Live N. caninum induced the production of IL-6, IL-1ß and IL-10 by SVF cells isolated from subcutaneous adipose tissue (SAT), while in mesenteric adipose tissue (MAT) SVF cell cultures only IL-1ß and IL-10 production was increased, showing slight distinct responses between adipose tissue depots. Whereas a clear IL-8 increase was detected in peripheral blood leucocytes (PBL) culture supernatants in response to live N. caninum, no such increase was observed in SAT or MAT SVF cell cultures. Nevertheless, in response to LPS, increased IL-8 levels were detected in all cell cultures. IL-10 levels were always increased in response to stimulation (live, freeze-killed N. caninum and LPS). Overall, our results show that bovine adipose tissue SVF cells produce cytokines in response to N. caninum and can therefore be putative contributors to the host immune response against this parasite.

The binding of PKCε and MEG2 to STAT3 regulates IL-6-mediated microglial hyperalgesia during inflammatory pain.

Studies have suggested that microglial IL-6 modulates inflammatory pain; however, the exact mechanism of action remains unclear. We therefore hypothesized that PKCε and MEG2 competitively bind to STAT3 and contribute to IL-6-mediated microglial hyperalgesia during inflammatory pain. Freund's complete adjuvant (FCA) and lipopolysaccharide (LPS) were used to induce hyperalgesia model mice and microglial inflammation. Mechanical allodynia was evaluated using von Frey tests in vivo. The interaction among PKCε, MEG2, and STAT3 was determined using ELISA and immunoprecipitation assay in vitro. The PKCε, MEG2, t-STAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, GLUT3, and TREM2 were assessed by Western blot. IL-6 promoter activity and IL-6 concentration were examined using dual luciferase assays and ELISA. Overexpression of PKCε and MEG2 promoted and attenuated inflammatory pain, accompanied by an increase and decrease in IL-6 expression, respectively. PKCε displayed a stronger binding ability to STAT3 when competing with MEG2. STAT3Ser727 phosphorylation increased STAT3 interaction with both PKCε and MEG2. Moreover, LPS increased PKCε, MEG2, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and GLUT3 levels and decreased TREM2 during microglia inflammation. IL-6 promoter activity was enhanced or inhibited by PKCε or MEG2 in the presence of STAT3 and LPS stimulation, respectively. In microglia, overexpression of PKCε and/or MEG2 resulted in the elevation of tSTAT3, pSTAT3Tyr705, pSTAT3Ser727, IL-6, and TREM2, and the reduction of GLUT3. PKCε is more potent than MEG2 when competitively binding to STAT3, displaying dual modulatory effects of IL-6 production, thus regulating the GLUT3 and TREM2 in microglia during inflammatory pain sensation.

Anti-inflammatory and immunoregulatory effects of colistin sulphate on human PBMCs.

In previous studies, CST has been identified as having an immunostimulatory effect on Caenorhabditis elegans and macrophage of rats. Here, we further investigated its immunomodulatory effects on human peripheral blood mononuclear cells (PBMCs). LPS-stimulated PBMCs inflammatory model was established. Flow cytometry was applied to measure phagocytosis of PBMCs. Cytokine mRNA and protein expression levels of LPS-stimulated PBMCs with or without CST were measured by qRT-PCR and ELISA. The transcriptomic profile of CST-treated PBMCs was investigated by RNA-sequencing. Gene Ontology (GO) and Kyoto Encylopedia of Genes and Genomes (KEGG) were applied to find potential signalling pathways. PBMCs showed a significant increase in phagocytic activity at 6 h after being incubated with CST at the concentration of 10 µg/mL. In the presence of LPS, CST maintained and promoted the expression of TNF-α and chemokine CCL24. The content of pro-inflammatory cytokines, such as IL-1ß, IL-6 and IFN-γ, which were released from LPS-stimulated PBMCs, was reduced by CST at 6 h. Anti-inflammatory cytokines, such as IL-4, IL-13 and TGF-ß1, were significantly increased by CST at 24 h. A total of 277 differentially expressed immune-related genes (DEIRGs) were detected and cytokine-cytokine receptor interaction was highly enriched. CST presented obvious anti-inflammatory and immunoregulatory effects in LPS-induced PBMCs inflammatory model not only by improving the ability of PBMCs to clear pathogens but also by decreasing pro-inflammatory cytokines and increasing anti-inflammatory cytokines. And the mechanism may be related to cytokine-cytokine receptor interaction.

Disordered GPR43/NLRP3 expression in peripheral leukocytes of patients with atrial fibrillation is associated with intestinal short chain fatty acids levels.

BACKGROUND: Atrial fibrillation (AF) is associated with circulating inflammation. Short-chain fatty acids (SCFAs) derived from gut microbiota (GM) regulate leukocyte function and inhibit the release of inflammatory cytokines, which are partly mediated by the G-protein-coupled receptor 43 (GPR43) signaling. This study aimed to investigate the expression of GPR43/NOD-like receptors family pyrin domain containing 3 (NLRP3) in leukocytes and the interaction with intestinal SCFAs levels in AF patients. METHODS: Expressions of GPR43 and NLRP3 mRNA in peripheral blood leukocytes from 23 AF patients and 25 non-AF controls were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Expressions of leukocyte GPR43 and NLRP3 protein were evaluated by western blot analysis. The levels of plasma IL-1ß were measured by enzyme-linked immunosorbent assay (ELISA). The fecal SCFAs levels based on GC/MS metabolome of corresponding 21 controls and 14 AF patients were acquired from our published dataset. To evaluate the expression of NLRP3 and GPR43 and the release of IL-1ß, human THP-1 cells were stimulated with or without SCFAs (acetate, propionate, and butyrate), lipopolysaccharide (LPS), and nigericin in vitro, respectively. RESULTS: Compared to the controls, the mRNA expression in peripheral leukocytes was significantly reduced in AF patients (P = 0.011) coupled with the increase in downstream leukocyte NLRP3 mRNA expression (P = 0.007) and plasma IL-1ß levels (P < 0.001), consistent with changes in GPR43 and NLRP3 protein expression. Furthermore, leukocyte GPR43 mRNA levels were positively correlated with fecal GM-derived acetic acid (P = 0.046) and negatively correlated with NLRP3 mRNA expression (P = 0.024). In contrast to the negative correlation between left atrial diameter (LAD) and GPR43 (P = 0.008), LAD was positively correlated with the leukocyte NLRP3 mRNA levels (P = 0.024). Subsequent mediation analysis showed that 68.88% of the total effect of intestinal acetic acid on AF might be mediated by leukocyte GPR43/NLRP3. The constructed GPR43-NLRP3 score might have a predictive potential for AF detection (AUC = 0.81, P < 0.001). Moreover, SCFAs treatment increased GPR43 expression and remarkably reduced LPS/nigericin-induced NLRP3 expression and IL-1ß release in human THP-1 cells in vitro. CONCLUSIONS: Disrupted interactions between GPR43 and NLRP3 expression in peripheral blood leukocytes, associated with reduced intestinal GM-derived SCFAs, especially acetic acid, may be involved in AF development and left atrial enlargement by enhancing circulating inflammation.

Resveratrol prevents the release of neutrophil extracellular traps (NETs) by controlling hydrogen peroxide levels and nuclear elastase migration.

Neutrophil extracellular traps (NETs) are defense mechanisms that trap and kill microorganisms and degrade cytokines. However, excessive production, dysregulation of suppression mechanisms, or inefficient removal of NETs can contribute to increased inflammatory response and the development of pathological conditions. Therefore, research has focused on identifying drugs that inhibit or delay the NET release process. Since reactive oxygen species (ROS) play a significant role in NET release, we aimed to investigate whether resveratrol (RSV), with a wide range of biological and pharmacological properties, could modulate NET release in response to different stimuli. Thus, human neutrophils were pretreated with RSV and subsequently stimulated with PMA, LPS, IL-8, or Leishmania. Our findings revealed that RSV reduced the release of NETs in response to all tested stimuli. RSV decreased hydrogen peroxide levels in PMA- and LPS-stimulated neutrophils, inhibited myeloperoxidase activity, and altered the localization of neutrophil elastase. RSV inhibition of NET generation was not mediated through A2A or A2B adenosine receptors or PKA. Based on the observed effectiveness of RSV in inhibiting NET release, our study suggests that this flavonoid holds potential as a candidate for treating NETs involving pathologies.

Flotillins affect LPS-induced TLR4 signaling by modulating the trafficking and abundance of CD14.

Lipopolysaccharide (LPS) induces a strong pro-inflammatory reaction of macrophages upon activation of Toll-like receptor 4 (TLR4) with the assistance of CD14 protein. Considering a key role of plasma membrane rafts in CD14 and TLR4 activity and the significant impact exerted on that activity by endocytosis and intracellular trafficking of the both LPS acceptors, it seemed likely that the pro-inflammatory reaction could be modulated by flotillins. Flotillin-1 and -2 are scaffolding proteins associated with the plasma membrane and also with endo-membranes, affecting both the plasma membrane dynamics and intracellular protein trafficking. To verify the above hypothesis, a set of shRNA was used to down-regulate flotillin-2 in Raw264 cells, which were found to also become deficient in flotillin-1. The flotillin deficiency inhibited strongly the TRIF-dependent endosomal signaling of LPS-activated TLR4, and to a lower extent also the MyD88-dependent one, without affecting the cellular level of TLR4. The flotillin depletion also inhibited the pro-inflammatory activity of TLR2/TLR1 and TLR2/TLR6 but not TLR3. In agreement with those effects, the depletion of flotillins down-regulated the CD14 mRNA level and the cellular content of CD14 protein, and also inhibited constitutive CD14 endocytosis thereby facilitating its shedding. Ultimately, the cell-surface level of CD14 was markedly diminished. Concomitantly, CD14 recycling was enhanced via EEA1-positive early endosomes and golgin-97-positive trans-Golgi network, likely to compensate for the depletion of the cell-surface CD14. We propose that the paucity of surface CD14 is the reason for the down-regulated signaling of TLR4 and the other TLRs depending on CD14 for ligand binding.

Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells inhibit cell apoptosis and alveolar bone loss in periodontitis.

OBJECTIVE: This study aimed to explore the effects of small extracellular vesicles derived from lipopolysaccharide-preconditioned dental follicle cells (L-D-sEV) on periodontal ligament cells from periodontitis affected teeth (p-PDLCs) in vitro and experimental periodontitis in mice. DESIGN: In vitro, the biological function of p-PDLCs and the underlying molecular mechanism were investigated by flow cytometry, Western blot, and quantitative real-time PCR (qRT-PCR) analysis. Eighteen-eight-week-old male C57BL/6 mice were randomly divided into three groups: control (Con), periodontitis (Peri), and L-D-sEV groups. Mice periodontitis model was induced by placing the 5-0 silk thread (around the maxillary second molar) and P.gingivalis (1 ×107 CFUs per mouse). In vivo, the alveolar bone loss, osteoclast activity, and macrophage polarization were measured by micro-computed tomography and histological analysis. RESULTS: In vitro, the RANKL/OPG ratio and phosphorylation of JNK and P38 protein levels of p-PDLCs were significantly decreased after L-D-sEV administration. Besides, flow cytometry and qRT-PCR analysis showed that L-D-sEV reduced apoptosis of p-PDLCs, down-regulated apoptosis-related genes Caspase-3 and BCL-2-Associated X expression, and up-regulated B-cell lymphoma-2 gene levels. In vivo, L-D-sEV administration significantly reduced alveolar bone loss, inhibited osteoclast activity, and induced M2 polarization. The histological analysis showed that iNOS/CD206, RANKL/OPG, p-JNK/JNK, and p-P38/P38 ratios were significantly lower in the L-D-sEV group than in the Peri group. CONCLUSIONS: L-D-sEV administration alleviated alveolar bone loss by mediating RANKL/OPG-related osteoclast activity and M2 macrophage polarization, alleviating p-PDLCs apoptosis and proliferation via the JNK and P38 pathways.

Threonine phosphorylation of STAT1 restricts interferon signaling and promotes innate inflammatory responses.

Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.

[Essential oil of Cinnamomum camphora mitigates depression-like behavior in mice by regulating Nrf2/HO-1 pathway and inhibiting inflammatory cytokines].

This study aims to investigate the essential oil(EOL) of Cinnamomum camphora regarding its anti-depression effect and mechanism in regulating inflammatory cytokines and the nuclear factor erythroid 2-related factor 2(Nrf2)/heme oxygenase-1(HO-1) pathway. A mouse model of depression was established by intraperitoneal injection of lipopolysaccharide(LPS). Open field, elevated plus maze, and forced swimming tests were carried out to examine mouse behaviors. Western blot and qRT-PCR were employed to determine the expression of proteins and genes in the Nrf2/HO-1 pathway in the hippocampus. The levels of tumor necrosis factor(TNF)-α, interleukin(IL)-6, and IL-1ß in the serum were measured by enzyme-linked immunosorbent assay(ELISA). The changes of apoptosis in mouse brain were detected by Tunel staining. Compared with the blank control group, the model group showed shortened distance travelled and time spent in the central zone and reduced number of entries in the central zone in the open field test. In the elevated plus maze test, the model group showed reduced open arm time(OT%) and open arm entries(OE%). In the force swimming test, the model group showed extended duration of immobility compared with the blank control group. Compared with the model group, the treatment with EOL significantly increased the distance travelled and time spent in the central zone and increased the number of entries in the central zone in the open field test. In addition, EOL significantly increased the OT% and OE% in the elevated plus maze and shor-tened the immobility duration in the forced swimming test. The model group showed lower expression levels of Nrf2 and HO-1 and hig-her levels of TNF-α, IL-6, and IL-1ß than the blank control group. Compared with the model group, the treatment with EOL up-regulated the expression levels of Nrf2 and HO-1 and lowered the levels of TNF-α, IL-6, and IL-1ß. The Tunel staining results showed that the apoptosis rate in the brain tissue of mice decreased significantly after the treatment with EOL. To sum up, EOL can mitigate the depression-like behaviors of mice by up-regulating the expression of Nrf2 and HO-1 and preventing hippocampal inflammatory damage. The findings provide empirical support for the application of EOL and aromatherapy in the treatment of depression.

Ginsenoside Rg1 Alleviates Sepsis-Induced Acute Lung Injury by Reducing FBXO3 Stability in an m6A-Dependent Manner to Activate PGC-1α/Nrf2 Signaling Pathway.

BACKGROUND: Sepsis-induced acute lung injury (ALI) is one of the serious life-threatening complications of sepsis and is pathologically associated with mitochondrial dysfunction. Ginsenoside Rg1 has good therapeutic effects on ALI. Herein, the pharmacological effects of Rg1 in sepsis-induced ALI were investigated. METHODS: Sepsis-induced ALI models were established by CLP operation and LPS treatment. HE staining was adopted to analyze lung pathological changes. The expression and secretion of cytokines were measured by RT-qPCR and ELISA. Cell viability and apoptosis were assessed by MTT assay, flow cytometry and TUNEL staining. ROS level and mitochondrial membrane potential (MMP) were analyzed using DHE probe and JC-1 staining, respectively. FBXO3 m6A level was assessed using MeRIP assay. The interactions between FBXO3, YTHDF1, and PGC-1α were analyzed by Co-IP or RIP. RESULTS: Rg1 administration ameliorated LPS-induced epithelial cell inflammation, apoptosis, and mitochondrial dysfunction in a dose-dependent manner. Mechanically, Rg1 reduced PGC-1α ubiquitination modification level by inhibiting FBXO3 expression m6A-YTHDF1 dependently. As expected, Rg1's mitigative effect on LPS-induced inflammation, apoptosis and mitochondrial dysfunction in lung epithelial cells was abolished by FBXO3 overexpression. Moreover, FBXO3 upregulation eliminated the restoring effect of Rg1 on CLP-induced lung injury in rats. CONCLUSION: Rg1 activated PGC-1α/Nrf2 signaling pathway by reducing FBXO3 stability in an m6A-YTHDF1-dependent manner to improve mitochondrial function in lung epithelial cells during sepsis-induced ALI progression.

A Robust In Vitro Culture Model and Generation of Memory Monocytes.

Innate monocytes can be trained or reprogrammed to adopt distinct memory states, such as low-grade inflammation and immune exhaustion, bearing fundamental relevance to the pathogenesis of both acute diseases such as sepsis as well as chronic diseases such as atherosclerosis. Therefore, it is critically important to develop a regimen for generating memory monocytes in vitro in order to better define key monocyte memory states with diverse potentials for proliferation, differentiation, and activation, as well as underlying mechanisms. Here, we describe an efficient in vitro system to propagate a large number of highly purified murine memory monocytes through sustaining bone marrow-derived monocytes with macrophage colony-stimulating factor (M-CSF, 10 ng/mL)-containing medium, together with other polarization agents such as lipopolysaccharide (LPS) for a 5-day period. This method can yield high-purity monocytes, capable of exhibiting dynamic memory behaviors upon training with various polarizing agents.

Forskolin-mediated cAMP activation upregulates TNF-α expression despite NF-κB downregulation in LPS-treated Schwann cells.

Although Schwann cells have been found to play a key role in inflammation and repair following nerve injury, the exact pathway is still unknown. To explore the mechanism by which Schwann cells exert their effects in the neuron microenvironment, we investigated two main inflammatory pathways: the NF-κB and cAMP pathways, and their downstream signaling molecules. In this study, lipopolysaccharide (LPS), a bacterial endotoxin, was used to activate the NF-κB pathway, and forskolin, a plant extract, was used to activate the cAMP pathway. The rat RT4-D6P2T Schwann cell line was treated with 0.1, 1, or 10 µg/mL of LPS, with or without 2 µM of forskolin, for 1, 3, 12, and 24 hours to determine the effects of elevated cAMP levels on LPS-treated cell viability. To investigate the effects of elevated cAMP levels on the expression of downstream signaling effector proteins, specifically NF-κB, TNF-α, AKAP95, and cyclin D3, as well as TNF-α secretion, RT4-D6P2T cells were incubated in the various treatment combinations for a 3-hour time period. Overall, results from the CellTiter-Glo viability assay revealed that forskolin increased viability in cells treated with smaller doses of LPS for 1 and 24 hours. For all time points, 10 µg/mL of LPS noticeably reduced viability regardless of forskolin treatment. Results from the Western blot analysis revealed that, at 10 µg/mL of LPS, forskolin upregulated the expression of TNF-α despite a downregulation of NF-κB, which was also accompanied by a decrease in TNF-α secretion. These results provide evidence that cAMP might regulate TNF-α expression through alternate pathways. Furthermore, although cAMP activation altered AKAP95 and cyclin D3 expression at different doses of LPS, there does not appear to be an association between the expression of AKAP95 or cyclin D3 and the expression of TNF-α. Exploring the possible interactions between cAMP, NF-κB, and other key inflammatory signaling pathways might reveal a potential therapeutic target for the treatment of nerve injury and inflammation.

SS-31 inhibits the inflammatory response by increasing ATG5 and promoting autophagy in lipopolysaccharide-stimulated HepG2 cells.

SS-31 is a mitochondria-targeting short peptide. Recent studies have indicated its hepatoprotective effects. In our study, we investigated the impact of SS-31 on LPS-induced autophagy in HepG2 cells. The results obtained from a dual-fluorescence autophagy detection system revealed that SS-31 promotes the formation of autolysosomes and autophagosomes, thereby facilitating autophagic flux to a certain degree. Additionally, both ELISA and qPCR analyses provided further evidence that SS-31 safeguards HepG2 cells against inflammatory responses triggered by LPS through ATG5-dependent autophagy. In summary, our study demonstrates that SS-31 inhibits LPS-stimulated inflammation in HepG2 cells by upregulating ATG5-dependent autophagy.

S100A9 exacerbates sepsis-induced acute lung injury via the IL17-NFκB-caspase-3 signaling pathway.

BACKGROUND: Sepsis-induced acute lung injury (ALI) is associated with considerable morbidity and mortality in critically ill patients. S100A9, a key endothelial injury factor, is markedly upregulated in sepsis-induced ALI; however, its specific mechanism of action has not been fully elucidated. METHODS: The Gene Expression Omnibus database transcriptome data for sepsis-induced ALI were used to screen for key differentially expressed genes (DEGs). Using bioinformatics analysis methods such as Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and protein-protein interaction network analyses, the pathogenesis of sepsis-induced ALI was revealed. Intratracheal infusion of lipopolysaccharide (LPS, 10 mg/kg) induced ALI in wild-type (WT) and S100A9 knockout mice. Multiomics analyses (transcriptomics and proteomics) were performed to investigate the potential mechanisms by which S100A9 exacerbates acute lung damage. Hematoxylin-eosin, Giemsa, and TUNEL staining were used to evaluate lung injury and cell apoptosis. LPS (10 µg/mL)-induced murine lung epithelial MLE-12 cells were utilized to mimic ALI and were modulated by S100A9 lentiviral transfection. The impact of S100A9 on cell apoptosis and inflammatory responses were identified using flow cytometry and PCR. The expression of interleukin (IL)-17-nuclear factor kappa B (NFκB)-caspase-3 signaling components was identified using western blotting. RESULTS: Six common DEGs (S100A9, S100A8, IFITM6, SAA3, CD177, and MMP9) were identified in the six datasets related to ALI in sepsis. Compared to WT sepsis mice, S100A9 knockout significantly alleviated LPS-induced ALI in mice, with reduced lung structural damage and inflammatory exudation, decreased exfoliated cell and protein content in the lung lavage fluid, and reduced apoptosis and necrosis of pulmonary epithelial cells. Transcriptomic analysis revealed that knocking out S100A9 significantly affected 123 DEGs, which were enriched in immune responses, defense responses against bacteria or lipopolysaccharides, cytokine-cytokine receptor interactions, and the IL-17 signaling pathway. Proteomic analysis revealed that S100A9 knockout alleviated muscle contraction dysfunction and structural remodeling in sepsis-induced ALI. Multiomics analysis revealed that S100A9 may be closely related to interferon-induced proteins with tetratricopeptide repeats and oligoadenylate synthase-like proteins. LPS decreased MLE12 cell activity, accompanied by high expression of S100A9. The expression of IL-17RA, pNFκB, and cleaved-caspase-3 were increased by S100A9 overexpression and reduced by S100A9 knockdown in LPS-stimulated MLE12 cells. S100A9 knockdown decreases transcription of apoptosis-related markers Bax, Bcl and caspase-3, alleviating LPS-induced apoptosis. CONCLUSIONS: S100A9 as a key biomarker of sepsis-induced acute lung injury, and exacerbates lung damage and epithelial cell apoptosis induced by LPS via the IL-17-NFκB-caspase-3 signaling pathway.

[Le miR-224-5p régulé sert de biomarqueur pour l'insuffisance hépatique aiguë pédiatrique et régule l'inflammation en modulant ZBTB20]. / Upregulated miR-224-5p served as a biomarker for pediatric acute liver failure and regulate inflammation by modulating ZBTB20.

Pediatric acute liver failure (PALF) is a severe liver dysfunction with complex pathological mechanisms and rapid development. MiRNAs have been identified as promising biomarkers for human disease screening and monitoring. This study focused on evaluating the clinical significance of miR-224-5p in PALF and revealing its potential molecular mechanism in regulating liver cell injury. This study enrolled 103 children with PALF and 55 healthy children without liver diseases. Serum miR-224-5p levels were compared between the two groups, and their clinical significance was estimated by analyzing the correlation with clinicopathological features and outcomes of PALF children. In vitro, a normal liver cell was treated with lipopolysaccharide (LPS), and cell growth and inflammation were assessed by CCK8 and ELISA assay. Upregulated miR-224-5p in PALF showed significance in screening PALF children from healthy children with the sensitivity and specificity of 78.64% and 84.47%, respectively. Increasing serum miR-224-5p in PALF children was closely associated with increasing prothrombin time, alanine transaminase, international normalized ratio, total bilirubin, ammonia, and aspartic transaminase and decreasing albumin of PALF children. MiR-224-5p was also identified as a risk factor for adverse outcomes in children with PALF. In LPS-treated liver cells, miR-224-5p could negatively regulate ZBTB20, and silencing miR-224-5p could alleviate the inhibited cell growth and promoted inflammation by LPS, which was reversed by ZBTB20 knockdown. Increasing miR-224-5p distinguished PALF children, predict severe disease development and risk of adverse prognosis. miR-224-5p also reguled LPS-induced liver cell injury via negatively regulating ZBTB20.

L-AP Alleviates Liver Injury in Septic Mice by Inhibiting Macrophage Activation via Suppressing NF-κB and NLRP3 Inflammasome/Caspase-1 Signal Pathways.

Liver injury and progressive liver failure are severe life-threatening complications in sepsis, further worsening the disease and leading to death. Macrophages and their mediated inflammatory cytokine storm are critical regulators in the occurrence and progression of liver injury in sepsis, for which effective treatments are still lacking. l-Ascorbic acid 6-palmitate (L-AP), a food additive, can inhibit neuroinflammation by modulating the phenotype of the microglia, but its pharmacological action in septic liver damage has not been fully explored. We aimed to investigate L-AP's antisepticemia action and the possible pharmacological mechanisms in attenuating septic liver damage by modulating macrophage function. We observed that L-AP treatment significantly increased survival in cecal ligation and puncture-induced WT mice and attenuated hepatic inflammatory injury, including the histopathology of the liver tissues, hepatocyte apoptosis, and the liver enzyme levels in plasma, which were comparable to NLRP3-deficiency in septic mice. L-AP supplementation significantly attenuated the excessive inflammatory response in hepatic tissues of septic mice in vivo and in cultured macrophages challenged by both LPS and ATP in vitro, by reducing the levels of NLRP3, pro-IL-1ß, and pro-IL-18 mRNA expression, as well as the levels of proteins for p-I-κB-α, p-NF-κB-p65, NLRP3, cleaved-caspase-1, IL-1ß, and IL-18. Additionally, it impaired the inflammasome ASC spot activation and reduced the inflammatory factor contents, including IL-1ß and IL-18 in plasma/cultured superannuants. It also prevented the infiltration/migration of macrophages and their M1-like inflammatory polarization while improving their M2-like polarization. Overall, our findings revealed that L-AP protected against sepsis by reducing macrophage activation and inflammatory cytokine production by suppressing their activation in NF-κB and NLRP3 inflammasome signal pathways in septic liver.

Octreotide attenuates intestinal barrier damage by maintaining basal autophagy in Caco2 cells.

The intestinal mucosal barrier is of great importance for maintaining the stability of the internal environment, which is closely related to the occurrence and development of intestinal inflammation. Octreotide (OCT) has potential applicable clinical value for treating intestinal injury according to previous studies, but the underlying molecular mechanisms have remained elusive. This article is based on a cell model of inflammation induced by lipopolysaccharide (LPS), aiming to explore the effects of OCT in protecting intestinal mucosal barrier function. A Cell Counting Kit­8 assay was used to determine cell viability and evaluate the effectiveness of OCT. Gene silencing technology was used to reveal the mediated effect of somatostatin receptor 2 (SSTR2). The changes in intestinal permeability were detected through trans­epithelial electrical resistance and fluorescein isothiocyanate­dextran 4 experiments, and the alterations in tight junction proteins were detected using immunoblotting and reverse transcription fluorescence­quantitative PCR technology. Autophagosomes were observed by electron microscopy and the dynamic changes of the autophagy process were characterized by light chain (LC)3­II/LC3­I conversion and autophagic flow. The results indicated that SSTR2­dependent OCT can prevent the decrease in cell activity. After LPS treatment, the permeability of monolayer cells decreased and intercellular tight junctions were disrupted, resulting in a decrease in tight junction protein zona occludens 1 in cells. The level of autophagy­related protein LC3 was altered to varying degrees at different times. These abnormal changes gradually returned to normal levels after the combined application of LPS and SSTR2­dependent OCT, confirming the role of OCT in protecting intestinal barrier function. These experimental results suggest that OCT maintains basal autophagy and cell activity mediated by SSTR2 in intestinal epithelial cells, thereby preventing the intestinal barrier dysfunction in inflammation injury.

Insights from bioinformatics analysis reveal that lipopolysaccharide induces activation of chemokine-related signaling pathways in human nasal epithelial cells.

Lipopolysaccharide (LPS) is known to elicit a robust immune response. This study aimed to investigate the impact of LPS on the transcriptome of human nasal epithelial cells (HNEpC). HNEpC were cultured and stimulated with LPS (1 µg/mL) or an equivalent amount of normal culture medium. Subsequently, total RNA was extracted, purified, and sequenced using next-generation RNA sequencing technology. Differentially expressed genes (DEGs) were identified and subjected to functional enrichment analysis. A protein-protein interaction (PPI) network of DEGs was constructed, followed by Ingenuity Pathway Analysis (IPA) to identify molecular pathways influenced by LPS exposure on HNEpC. Validation of key genes was performed using quantitative real-time PCR (qRT-PCR). A total of 97 DEGs, comprising 48 up-regulated genes and 49 down-regulated genes, were identified. Results from functional enrichment analysis, PPI, and IPA indicated that DEGs were predominantly enriched in chemokine-related signaling pathways. Subsequent qRT-PCR validation demonstrated significant upregulation of key genes in these pathways in LPS-treated HNEpC compared to control cells. In conclusion, LPS intervention profoundly altered the transcriptome of HNEpC, potentially exacerbating inflammatory responses through the activation of chemokine-related signaling pathways.