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.

E3 ubiquitin ligase BCA2 promotes breast cancer stemness by up-regulation of SOX9 by LPS.

Triple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer. Breast cancer stem cells (BCSCs) are believed to play a crucial role in the carcinogenesis, therapy resistance, and metastasis of TNBC. It is well known that inflammation promotes stemness. Several studies have identified breast cancer-associated gene 2 (BCA2) as a potential risk factor for breast cancer incidence and prognosis. However, whether and how BCA2 promotes BCSCs has not been elucidated. Here, we demonstrated that BCA2 specifically promotes lipopolysaccharide (LPS)-induced BCSCs through LPS induced SOX9 expression. BCA2 enhances the interaction between myeloid differentiation primary response protein 88 (MyD88) and Toll-like receptor 4 (TLR4) and inhibits the interaction of MyD88 with deubiquitinase OTUD4 in the LPS-mediated NF-κB signaling pathway. And SOX9, an NF-κB target gene, mediates BCA2's pro-stemness function in TNBC. Our findings provide new insights into the molecular mechanisms by which BCA2 promotes breast cancer and potential therapeutic targets for the treatment of breast cancer.

Baicalin nanodelivery system based on functionalized metal-organic framework for targeted therapy of osteoarthritis by modulating macrophage polarization.

Intra-articular drugs used to treat osteoarthritis (OA) often suffer from poor pharmacokinetics and stability. Nano-platforms as drug delivery systems for drug delivery are promising for OA therapy. In this study, we reported an M1 macrophage-targeted delivery system Bai@FA-UIO-66-NH2 based on folic acid (FA) -modified metal-organic framework (MOF) loaded with baicalin (Bai) as antioxidant agent for OA therapy. With outstanding biocompatibility and high drug loading efficiency, Bai@FA-UIO-66-NH2 could be specifically uptaken by LPS-induced macrophages to serve as a potent ROS scavenger, gradually releasing Bai at the subcellular level to reduce ROS production, modulate macrophage polarization to M2, leading to alleviation of synovial inflammation in OA joints. The synergistic effect of Bai@FA-UIO-66-NH2 on macrophage polarization and ROS scavenging significantly improved the therapeutic efficacy of OA, which may provide a new insight into the design of OA precision therapy.

Systemic LPS Administration Stimulates the Activation of Non-Neuronal Cells in an Experimental Model of Spinal Muscular Atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by deficiency of the survival motor neuron (SMN) protein. Although SMA is a genetic disease, environmental factors contribute to disease progression. Common pathogen components such as lipopolysaccharides (LPS) are considered significant contributors to inflammation and have been associated with muscle atrophy, which is considered a hallmark of SMA. In this study, we used the SMNΔ7 experimental mouse model of SMA to scrutinize the effect of systemic LPS administration, a strong pro-inflammatory stimulus, on disease outcome. Systemic LPS administration promoted a reduction in SMN expression levels in CNS, peripheral lymphoid organs, and skeletal muscles. Moreover, peripheral tissues were more vulnerable to LPS-induced damage compared to CNS tissues. Furthermore, systemic LPS administration resulted in a profound increase in microglia and astrocytes with reactive phenotypes in the CNS of SMNΔ7 mice. In conclusion, we hereby show for the first time that systemic LPS administration, although it may not precipitate alterations in terms of deficits of motor functions in a mouse model of SMA, it may, however, lead to a reduction in the SMN protein expression levels in the skeletal muscles and the CNS, thus promoting synapse damage and glial cells' reactive phenotype.

Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

Baicalin and baicalein from Scutellaria baicalensis Georgi alleviate aberrant neuronal suppression mediated by GABA from reactive astrocytes.

AIMS: γ-aminobutyric acid (GABA) from reactive astrocytes is critical for the dysregulation of neuronal activity in various neuroinflammatory conditions. While Scutellaria baicalensis Georgi (S. baicalensis) is known for its efficacy in addressing neurological symptoms, its potential to reduce GABA synthesis in reactive astrocytes and the associated neuronal suppression remains unclear. This study focuses on the inhibitory action of monoamine oxidase B (MAO-B), the key enzyme for astrocytic GABA synthesis. METHODS: Using a lipopolysaccharide (LPS)-induced neuroinflammation mouse model, we conducted immunohistochemistry to assess the effect of S. baicalensis on astrocyte reactivity and its GABA synthesis. High-performance liquid chromatography was performed to reveal the major compounds of S. baicalensis, the effects of which on MAO-B inhibition, astrocyte reactivity, and tonic inhibition in hippocampal neurons were validated by MAO-B activity assay, qRT-PCR, and whole-cell patch-clamp. RESULTS: The ethanolic extract of S. baicalensis ameliorated astrocyte reactivity and reduced excessive astrocytic GABA content in the CA1 hippocampus. Baicalin and baicalein exhibited significant MAO-B inhibition potential. These two compounds downregulate the mRNA levels of genes associated with reactive astrogliosis or astrocytic GABA synthesis. Additionally, LPS-induced aberrant tonic inhibition was reversed by both S. baicalensis extract and its key compounds. CONCLUSIONS: In summary, baicalin and baicalein isolated from S. baicalensis reduce astrocyte reactivity and alleviate aberrant tonic inhibition of hippocampal neurons during neuroinflammation.

Inhibition of lipopolysaccharide-induced NF-κB maintains osteogenesis of dental pulp and periodontal ligament stem cells.

Dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) can differentiate into osteoblasts, indicating that both are potential candidates for bone tissue engineering. Osteogenesis is influenced by many environmental factors, one of which is lipopolysaccharide (LPS). LPS-induced NF-κB activity affects the osteogenic potencies of different types of MSCs differently. This study evaluated the effect of LPS-induced NF-κB activity and its inhibition in DPSCs and PDLSCs. DPSCs and PDLSCs were cultured in an osteogenic medium, pretreated with/without NF-κB inhibitor Bay 11-7082, and treated with/without LPS. Alizarin red staining was performed to assess bone nodule formation, which was observed under an inverted light microscope. NF-κB and alkaline phosphatase (ALP) activities were measured to examine the effect of Bay 11-7082 pretreatment and LPS supplementation on osteogenic differentiation of DPSCs and PDLSCs. LPS significantly induced NF-κB activity (p = 0.000) and reduced ALP activity (p = 0.000), which inhibited bone nodule formation in DPSCs and PDLSCs. Bay 11-7082 inhibited LPS-induced NF-κB activity, and partially maintained ALP activity and osteogenic potency of LPS-supplemented DPSCs and PDLSCs. Thus, inhibition of LPS-induced NF-κB activity can maintain the osteogenic potency of DPSCs and PDLSCs.

CD22 blockade aggravates EAE and its role in microglia polarization.

AIMS: Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease. Microglia are reportedly involved in the pathogenesis of MS. However, the key molecules that control the inflammatory activity of microglia in MS have not been identified. METHODS: Experimental autoimmune encephalomyelitis (EAE) mice were randomized into CD22 blockade and control groups. The expression levels of microglial CD22 were measured by flow cytometry, qRT-PCR, and immunofluorescence. The effects of CD22 blockade were examined via in vitro and in vivo studies. RESULTS: We detected increased expression of microglial CD22 in EAE mice. In addition, an in vitro study revealed that lipopolysaccharide upregulated the expression of CD22 in microglia and that CD22 blockade modulated microglial polarization. Moreover, an in vivo study demonstrated that CD22 blockade aggravated EAE in mice and promoted microglial M1 polarization. CONCLUSION: Collectively, our study indicates that CD22 may be protective against EAE and may play a critical role in the maintenance of immune homeostasis in EAE mice.

MicroRNA-671-5p regulates the inflammatory response of periodontal ligament stem cells via the DUSP8/p38 MAPK pathway.

BACKGROUND: MicroRNAs are differentially expressed in periodontitis tissues. They are involved in cellular responses to inflammation and can be used as markers for diagnosing periodontitis. Microarray analysis showed that the expression level of microRNA-671-5p in periodontal tissues of patients with periodontitis was increased. In this study, we investigated the mechanism of action of microRNA-671-5p in human periodontal ligament stem cells (hPDLSCs) under inflammatory conditions. METHODS AND RESULTS: HPDLSCs were treated with lipopolysaccharide (LPS) to establish an inflammation model. The cell survival rate was determined using the cell counting kit-8 (CCK8). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blot analyses were used to detect the expression of microRNA-671-5p and dual-specificity phosphatase (DUSP) 8 proteins, respectively, Interleukin (IL)-6, IL-1ß, and tumor necrosis factor (TNF)-α were detected using qRT-PCR and Enzyme-linked immunosorbent assay (ELISA). A dual-luciferase reporter system was employed to determine the relationship between micoRNA-671-5p and DUSP8 expression. Activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway was confirmed using western blot analysis. Following the treatment of hPDLSCs with LPS, the expression levels of microRNA-671-5p in hPDLSCs were increased, cell viability decreased, and the expression of inflammatory factors displayed an increasing trend. MicroRNA-671-5p targets and binds to DUSP8. Silencing microRNA-671-5p or overexpressing DUSP8 can improve cell survival rate and reduce inflammatory responses. When DUSP8 was overexpressed, the expression of p-p38 was reduced. CONCLUSIONS: microRNA-671-5p targets DUSP8/p38 MAPK pathway to regulate LPS-induced proliferation and inflammation in hPDLSCs.

GC-MS based antioxidants characterization in Saussurea heteromalla (D. Don) Hand-Mazz by inhibition of nitric oxide generation in macrophages.

For centuries, medicinal plants have served as the cornerstone for traditional health care systems and same practice is still prevalent today. In the Himalayan region, Saussurea heteromalla holds a significant place in traditional medicine and is used to address various health issues. Despite its historical use, little exploration has focused on its potential for scavenging free radicals and reducing inflammation. Hence, our current study aims to investigate the free radical scavenging capabilities of S. heteromalla extracts. The n-hexane extract of entire plant revealed promising activity. This extract underwent extensive extraction on a larger scale. Subsequent purification, employing column chromatography, HPLC-DAD techniques, led to the identification of active compounds, confirmed via GC-MS and the NIST database as 1-O-butyl 2-O-octyl benzene-1,2-dicarboxylate and 2,4-ditert-butylphenol. Assessing the free radical scavenging properties involved utilizing RAW-264.7 macrophages activated by lipopolysaccharides. Notably, the compound 2,4-di-tert-butylphenol exhibited remarkable scavenging abilities, demonstrating over 80% inhibition of Nitric oxide. This study stands as the inaugural report on the isolation of these compounds from S. heteromalla.

LPS-induced monocarboxylate transporter-1 inhibition facilitates lactate accumulation triggering epithelial-mesenchymal transformation and pulmonary fibrosis.

The epithelial-mesenchymal transformation (EMT) process of alveolar epithelial cells is recognized as involved in the development of pulmonary fibrosis. Recent evidence has shown that lipopolysaccharide (LPS)-induced aerobic glycolysis of lung tissue and elevated lactate concentration are associated with the pathogenesis of sepsis-associated pulmonary fibrosis. However, it is uncertain whether LPS promotes the development of sepsis-associated pulmonary fibrosis by promoting lactate accumulation in lung tissue, thereby initiating EMT process. We hypothesized that monocarboxylate transporter-1 (MCT1), as the main protein for lactate transport, may be crucial in the pathogenic process of sepsis-associated pulmonary fibrosis. We found that high concentrations of lactate induced EMT while moderate concentrations did not. Besides, we demonstrated that MCT1 inhibition enhanced EMT process in MLE-12 cells, while MCT1 upregulation could reverse lactate-induced EMT. LPS could promote EMT in MLE-12 cells through MCT1 inhibition and lactate accumulation, while this could be alleviated by upregulating the expression of MCT1. In addition, the overexpression of MCT1 prevented LPS-induced EMT and pulmonary fibrosis in vivo. Altogether, this study revealed that LPS could inhibit the expression of MCT1 in mouse alveolar epithelial cells and cause lactate transport disorder, which leads to lactate accumulation, and ultimately promotes the process of EMT and lung fibrosis.

Abietane-Type Diterpenoids from the Arils of Torreya grandis.

A chemical investigation of the arils of Torreya grandis led to the isolation of seven abietane-type diterpenoids (compounds 1-7) including three previously undescribed compounds, one unreported natural product, and three known analogs. The structures of these compounds were determined by means of spectroscopy, single-crystal X-ray diffraction, and ECD spectra. An antibacterial activity assay showed that compounds 5 and 6 had significant inhibitory effects on methicillin-resistant Staphylococcus aureus, with MIC values of 100 µM. Moreover, compounds 1, 3, 4, and 7 exhibited anti-neuroinflammatory activity in LPS-stimulated BV-2 microglia cells, with the IC50 values ranging from 38.4 to 67.9 µM.

2-Substituted-4,7-dihydro-4-ethylpyrazolo[1,5-a]pyrimidin-7-ones alleviate LPS-induced inflammation by modulating cell metabolism via CD73 upon macrophage polarization.

Macrophage polarization towards the M1 phenotype under bacterial product-related exposure (LPS) requires a rapid change in gene expression patterns and cytokine production along with a metabolic rewiring. Metabolic pathways and redox reactions are such tightly connected, giving rise to an area of research referred to as immunometabolism. A role in this context has been paid to the master redox-sensitive regulator Nuclear factor erythroid 2-related factor 2 (Nrf2) and to the 5'-ectonucleotidase CD73, a marker related to macrophage metabolism rearrangement under pro-inflammatory conditions. In this light, a cell model of LPS-stimulated macrophages has been established and nine 4,7-dihydro-4-ethylpyrazolo[l,5-a]pyrimidin-7-ones with a potential anti-inflammatory effect have been administered. Our data highlight that two selected compounds (namely, 5 and 8) inhibit the LPS-induced Nrf2 nuclear translocation and ameliorate the activity rate of the antioxidant enzyme catalase. Additionally, the pyridine-containing compound (8) promotes the shift from the pro-inflammatory immunophenotype M1 to the pro-resolving M2 one, by downregulating CD80 and iNOS and by enhancing CD163 and TGFß1 expression. Most importantly, CD73 is modulated by these compounds as well as the lactate production. Our data demonstrate that pyrazolo[l,5-a]pyrimidine derivatives are effective as anti-inflammatory compounds. Furthermore, these pyrazolo[l,5-a]pyrimidines exert their action via CD73-related signaling and modulation of cell metabolism of activated macrophages.

Inhibition of PI3K p110δ rebalanced Th17/Treg and reduced macrophages pyroptosis in LPS-induced sepsis.

Sepsis is a systemic inflammatory response syndrome caused by trauma or infection, which can lead to multiple organ dysfunction. In severe cases, sepsis can also progress to septic shock and even death. Effective treatments for sepsis are still under development. This study aimed to determine if targeting the PI3K/Akt signaling with CAL-101, a PI3K p110δ inhibitor, could alleviate lipopolysaccharide (LPS)-induced sepsis and contribute to immune tolerance. Our findings indicated that CAL-101 treatment improved survival rates and alleviated the progression of LPS-induced sepsis. Compared to antibiotics, CAL-101 not only restored the Th17/regulatory T cells (Treg) balance but also enhanced Treg cell function. Additionally, CAL-101 promoted type 2 macrophage (M2) polarization, inhibited TNF-α secretion, and increased IL-10 secretion. Moreover, CAL-101 treatment reduced pyroptosis in peritoneal macrophages by inhibiting caspase-1/gasdermin D (GSDMD) activation. This study provides a mechanistic basis for future clinical exploration of targeted therapeutics and immunomodulatory strategies in the treatment of sepsis.

Rocaglamide regulates iron homeostasis by suppressing hepcidin expression.

The anemia of inflammation (AI) is characterized by the presence of inflammation and abnormal elevation of hepcidin. Accumulating evidence has proved that Rocaglamide (RocA) was involved in inflammation regulation. Nevertheless, the role of RocA in AI, especially in iron metabolism, has not been investigated, and its underlying mechanism remains elusive. Here, we demonstrated that RocA dramatically suppressed the elevation of hepcidin and ferritin in LPS-treated mice cell line RAW264.7 and peritoneal macrophages. In vivo study showed that RocA can restrain the depletion of serum iron (SI) and transferrin (Tf) saturation caused by LPS. Further investigation showed that RocA suppressed the upregulation of hepcidin mRNA and downregulation of Fpn1 protein expression in the spleen and liver of LPS-treated mice. Mechanistically, this effect was attributed to RocA's ability to inhibit the IL-6/STAT3 pathway, resulting in the suppression of hepcidin mRNA and subsequent increase in Fpn1 and TfR1 expression in LPS-treated macrophages. Moreover, RocA inhibited the elevation of the cellular labile iron pool (LIP) and reactive oxygen species (ROS) induced by LPS in RAW264.7 cells. These findings reveal a pivotal mechanism underlying the roles of RocA in modulating iron homeostasis and also provide a candidate natural product on alleviating AI.

Low-grade systemic inflammation stimulates microglial turnover and accelerates the onset of Alzheimer's-like pathology.

Several in vivo studies have shown that systemic inflammation, mimicked by LPS, triggers an inflammatory response in the CNS, driven by microglia, characterized by an increase in inflammatory cytokines and associated sickness behavior. However, most studies induce relatively high systemic inflammation, not directly compared with the more common low-grade inflammatory events experienced in humans during the life course. Using mice, we investigated the effects of low-grade systemic inflammation during an otherwise healthy early life, and how this may precondition the onset and severity of Alzheimer's disease (AD)-like pathology. Our results indicate that low-grade systemic inflammation induces sub-threshold brain inflammation and promotes microglial proliferation driven by the CSF1R pathway, contrary to the effects caused by high systemic inflammation. In addition, repeated systemic challenges with low-grade LPS induce disease-associated microglia. Finally, using an inducible model of AD-like pathology (Line 102 mice), we observed that preconditioning with repeated doses of low-grade systemic inflammation, prior to APP induction, promotes a detrimental effect later in life, leading to an increase in Aß accumulation and disease-associated microglia. These results support the notion that episodic low-grade systemic inflammation has the potential to influence the onset and severity of age-related neurological disorders, such as AD.

Electrospraying and electrospinning of tannic acid-loaded zein: Characterization and antioxidant effects in astrocyte culture exposed to E. coli lipopolysaccharide.

Tannic acid (TA) exhibits low bioavailability in the gastrointestinal tract, limiting its benefits due to small amounts reaching the CNS. Thus, the objective of this study was to develop zein capsules and fibers by electrospraying/electrospinning for encapsulation of TA. Polymeric solutions were evaluated by electrical conductivity, density, and viscosity. In zein capsules, up to 2 % TA was added, and in fibers, up to 1 % TA was added. Zein capsule and fiber with TA were evaluated by morphology, size distribution, encapsulation efficiency, thermal and thermogravimetric properties, and functional groups. Zein capsule with 1.5 % TA was evaluated in astrocyte culture for cytotoxicity and antioxidant activity. TA zein capsules and fibers exhibited high encapsulation efficiency and homogeneous morphology. TA encapsulated in zein presented higher thermal stability than free TA. TA zein capsule did not present toxicity and elicited antioxidant action in lipopolysaccharide-induced astrocyte culture. Capsules and fibers were successfully produced by electrospraying/electrospinning techniques.

Wogonoside alleviates microglia-mediated neuroinflammation via TLR4/MyD88/NF-κB signaling axis after spinal cord injury.

Wogonoside (WG) is a natural flavonoid extracted from Scutellariae Radix, recognized for its established anti-inflammatory properties. However, the role of WG in the context of neuroinflammation after spinal cord injury (SCI) remains inadequately elucidated. This study employed in silico, in vitro, and in vivo methodologies to investigate the impact of WG on microglia-mediated neuroinflammation after SCI. In the in silico experiment, we identified 15 potential target genes of WG associated with SCI. These genes were linked to the regulation of inflammatory response and immune defense. Molecular docking maps revealed toll-like receptor 4 as a molecular target for WG, demonstrating binding through a hydrogen bond (Lys263, Ser120). In lipopolysaccharide-stimulated BV2 cells and SCI mice, WG significantly attenuated microglial activation and facilitated a phenotype shift from M1 to M2. This was evidenced by the reversal of the increased expressions of Iba1, GFAP, and iNOS, as well as the decreased expression of Arg1. WG also suppressed the production of pro-inflammatory mediators (NO, TNF-α, IL-6, IL-1α, IL-1ß, C1q). WG exerted these effects by suppressing the TLR4/MyD88/NF-κB signaling axis in microglia. Furthermore, by reducing levels of TNF-α, IL-1α, and C1q in supernatant of LPS-induced microglia, WG indirectly induced astrocytes change to A2 phenotype, evidenced by transcriptome sequencing result of primary mouse astrocytes. All these events above collectively created a favorable microenvironment, contributing to a significant alleviation of weight loss and neuronal damage at the lesion site of SCI mice. Our findings substantiate the efficacy of WG in mitigating neuroinflammation after SCI, thereby warranting further exploration.

Extracellular mixed histones are neurotoxic and modulate select neuroimmune responses of glial cells.

Although histone proteins are widely known for their intranuclear functions where they organize DNA, all five histone types can also be released into the extracellular space from damaged cells. Extracellular histones can interact with pattern recognition receptors of peripheral immune cells, including toll-like receptor 4 (TLR4), causing pro-inflammatory activation, which indicates they may act as damage-associated molecular patterns (DAMPs) in peripheral tissues. Very limited information is available about functions of extracellular histones in the central nervous system (CNS). To address this knowledge gap, we applied mixed histones (MH) to cultured cells modeling neurons, microglia, and astrocytes. Microglia are the professional CNS immunocytes, while astrocytes are the main support cells for neurons. Both these cell types are critical for neuroimmune responses and their dysregulated activity contributes to neurodegenerative diseases. We measured effects of extracellular MH on cell viability and select neuroimmune functions of microglia and astrocytes. MH were toxic to cultured primary murine neurons and also reduced viability of NSC-34 murine and SH-SY5Y human neuron-like cells in TLR4-dependent manner. MH did not affect the viability of resting or immune-stimulated BV-2 murine microglia or U118 MG human astrocytic cells. When applied to BV-2 cells, MH enhanced secretion of the potential neurotoxin glutamate, but did not modulate the release of nitric oxide (NO), tumor necrosis factor-α (TNF), C-X-C motif chemokine ligand 10 (CXCL10), or the overall cytotoxicity of lipopolysaccharide (LPS)- and/or interferon (IFN)-γ-stimulated BV-2 microglial cells towards NSC-34 neuron-like cells. We demonstrated, for the first time, that MH downregulated phagocytic activity of LPS-stimulated BV-2 microglia. However, MH also exhibited protective effect by ameliorating the cytotoxicity of LPS-stimulated U118 MG astrocytic cells towards SH-SY5Y neuron-like cells. Our data demonstrate extracellular MH could both damage neurons and alter neuroimmune functions of glial cells. These actions of MH could be targeted for treatment of neurodegenerative diseases.

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.