OBJECTIVE: Sepsis often triggers a systemic inflammatory response leading to multi-organ dysfunction, with complex and not fully understood pathogenesis. This study investigates the therapeutic effects of cimifugin on BV-2 cells under sepsis-induced stress conditions.
METHODS: We utilized a BV-2 microglial cell model treated with lipopolysaccharide (LPS) to mimic sepsis. Assessments included cellular vitality, inflammatory cytokine quantification (6 interleukin [6IL]-1β, interleukin 6 [IL-6], and tumor necrosis factor-α [TNF-α]) via enzyme-linked-immunosorbent serologic assay, and analysis of mRNA expression using real-time polymerase chain reaction. Oxidative stress and mitochondrial function were also evaluated to understand the cellular effects of cimifugin.
RESULTS: Cimifugin significantly attenuated LPS-induced inflammatory responses, oxidative stress, and mitochondrial dysfunction. It enhanced cell viability and modulated the secretion and gene expression of inflammatory cytokines IL-1β, IL-6, and TNF-α. Notably, cimifugin activated the deacetylase sirtuin 1-nuclear factor erythroid 2-related factor 2 pathway, contributing to its protective effects against mitochondrial damage.
CONCLUSIONS: Cimifugin demonstrates the potential of being an effective treatment for sepsis--induced neuroinflammation, warranting further investigation.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction activated by microglia. The potential pathological changes of SAE are complex, and the cellular pathophysiological characteristics remains unclear. This study aims to explore the ROS/TXNIP/NLRP3 pathway mediated lipopolysaccharide (LPS)-induced inflammatory response in microglia.
METHODS: BV-2 cells were pre-incubated with 10 μM N-acetyl-L-cysteine (NAC) for 2 h, which were then reacted with 1 μg/mL LPS for 24 h. Western blot assay examined the protein levels of IBA1, CD68, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. The contents of inflammatory factor were detected by ELISA assay. The co-immunoprecipitation assay examined the interaction between TXNIP and NLRP3.
RESULTS: LPS was confirmed to promote the positive expressions of IBA1 and CD68 in BV-2 cells. The further experiments indicated that LPS enhanced ROS production and NLRP3 inflammasome activation in BV-2 cells. Moreover, we also found that NAC partially reversed the facilitation of LPS on the levels of ROS, IL-1β, IL-18, TXNIP, NLRP3, ASC, and Cleaved Caspase-1 in BV-2 cells. NAC treatment also notably alleviated the interaction between TXNIP and NLRP3 in BV-2 cells.
CONCLUSIONS: ROS inhibition mediated NLRP3 signaling inactivation by decreasing TXNIP expression.

Carbendazim (CBZ) is a benzimidazole fungicide widely used worldwide in industrial, agricultural, and veterinary practices. Although, CBZ was found in all brain tissues causing serious neurotoxicity, its impact on brain immune cells remain scarcely understood. Our study investigated the in vitro effects of CBZ on activated microglial BV-2 cells. Lipopolysaccharide (LPS)-stimulated BV-2 cells were exposed to increasing concentrations of CBZ and cytokine release was measured by ELISA, and Cytometric Bead Array (CBA) assays. Mitochondrial superoxide anion (O2·-) generation was evaluated by Dihydroethidium (DHE) and nitric oxide (NO) was assessed by Griess reagent. Lipid peroxidation was evaluated by measuring the malonaldehyde (MDA) levels. The transmembrane mitochondrial potential (ΔΨm) was detected by cytometry analysis with dihexyloxacarbocyanine iodide (DiOC6(3)) assay. CBZ concentration-dependently increased IL-1β, IL-6, TNF-α and MCP-1 by LPS-activated BV-2 cells. CBZ significantly promoted oxidative stress by increasing NO, O2·- generation, and MDA levels. In contrast, CBZ significantly decreased ΔΨm. Pre-treatment of BV-2 cells with N-acetylcysteine (NAC) reversed all the above mentioned immunotoxic parameters, suggesting a potential protective role of NAC against CBZ-induced immunotoxicity via its antioxidant and anti-inflammatory effects on activated BV-2 cells. Therefore, microglial proinflammatory over-activation by CBZ may be a potential mechanism by which CBZ could induce neurotoxicity and neurodegenerative disorders.

The neuroinflammatory process is considered one of the main characteristics of central nervous system diseases, where a pro-inflammatory response results in oxidative stress through the generation of reactive oxygen and nitrogen species (ROS and RNS). Olive (Olea europaea L.) pomace is a by-product of olive oil production that is rich in phenolic compounds (PCs), known for their antioxidant and anti-inflammatory properties. This work looked at the antioxidant and anti-neuroinflammatory effects of the bioavailable PC from olive pomace in cell-free models and microglia cells. The bioavailable PC of olive pomace was obtained through the process of in vitro gastrointestinal digestion of fractionated olive pomace (OPF, particles size < 2 mm) and micronized olive pomace (OPM, particles size < 20 µm). The profile of the PC that is present in the bioavailable fraction as well as its in vitro antioxidant capacity were determined. The anti-neuroinflammatory capacity of the bioavailable PC from olive pomace (0.03-3 mg L-1) was evaluated in BV-2 cells activated by lipopolysaccharide (LPS) for 24 h. The total bioavailable PC concentration and antioxidant activity against peroxyl radical were higher in the OPM than those observed in the OPF sample. The activation of BV-2 cells by LPS resulted in increased levels of ROS and nitric oxide (NO). The bioavailable PCs from both OPF and OPM, at their lowest concentrations, were able to reduce the ROS generation in activated BV-2 cells. In contrast, the highest PC concentration of OPF and OPM was able to reduce the NO levels in activated microglial cells. Our results demonstrate that bioavailable PCs from olive pomace can act as anti-neuroinflammatory agents in vitro, independent of particle size. Moreover, studies approaching ways to increase the bioavailability of PCs from olive pomace, as well as any possible toxic effects, are needed before a final statement on its nutritional use is made.

Animal models of cerebral ischemia have improved our understanding of the pathophysiology and mechanisms involved in stroke, as well as the investigation of potential therapies. The potential of zebrafish to model human diseases has become increasingly evident. The availability of these models allows for an increased understanding of the role of chemical exposure in human conditions and provides essential tools for mechanistic studies of disease. To evaluate the potential neuroprotective properties of minocycline against ischemia and reperfusion injury in zebrafish and compare them with other standardized models. In vitro studies with BV-2 cells were performed, and mammalian transient middle cerebral artery occlusion (tMCAO) was used as a comparative standard with the zebrafish stroke model. Animals were subjected to ischemia and reperfusion injury protocols and treated with minocycline. Infarction size, cytokine levels, oxidative stress, glutamate toxicity, and immunofluorescence for microglial activation, and behavioral test results were determined and compared. Administration of minocycline provided significant protection in the three stroke models in different parameters analyzed. Both experimental models complement each other in their particularities. The proposal also strengthens the findings in the literature in rodent models and allows the validation of alternative models so that they can be used in further research involving diseases with ischemia and reperfusion injury.

M1 microglia induce neuroinflammation-related neuronal death in animal models of spontaneous subarachnoid haemorrhage. Zileuton is a 5-lipoxygenase inhibitor that reduces the levels of downstream pro-inflammatory cytokines. This study aimed to investigate whether zileuton inhibits microglial activation and describe its underlying mechanisms. BV-2 cells were exposed to 1 mg/mL haemolysate for 30 min, followed by treatment with different concentrations (5, 10, 15, or 20 μM) of zileuton for 24 h. The cells were then assessed for viability, polarisation, and protein expression levels. Haemolysate increases the viability of BV-2 cells and induces M1 polarisation. Subsequent exposure to high concentrations of zileuton decreased the viability of BV-2 cells, shifted the polarisation to the M2 phenotype, suppressed the expression of 5-lipoxygenase, decreased tumour necrosis factor α levels, and increased interleukin-10 levels. Furthermore, high concentrations of zileuton suppressed the expression of myeloid differentiation primary response protein 88 and reduced the phosphorylated-nuclear factor-kappa B (NF-kB)/NF-kB ratio. Therefore, phenotype reversal from M1 to M2 is a possible mechanism by which zileuton attenuates haemolysate-induced neuroinflammation after spontaneous subarachnoid haemorrhage.

Clausena lenis Drake (C. lenis) is a folk medicinal herb to treat influenza, colds, bronchitis, and malaria. The 95% and 50% ethanol extract of C. lenis showed significant nitric oxide (NO) inhibition activity in BV-2 microglial cells stimulated by lipopolysaccharide (LPS). Bio-guided isolation of the active extract afforded five new compounds, including a chlorine-containing furoquinoline racemate, (±)-claulenine A (1), an amide alkaloid, claulenine B (2), a prenylated coumarin, claulenin A (3), a furocoumarin glucoside, clauleside A (4), and a multi-prenylated p-hydroxybenzaldehyde, claulenin B (5), along with 33 known ones. Their structures were determined via spectroscopic methods, and the absolute configurations of new compounds were assigned via the electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction analysis. Compounds 2, 23, 27, 28, 33, and 34 showed potent anti-neuroinflammatory effects on LPS-induced NO production in BV-2 microglial cells, with IC50 values in the range of 17.6-40.9 μM. The possible mechanism was deduced to interact with iNOS through molecular docking.

The regenerative capability of spinal cord neurons is limited to impossible. Thus, experimental approaches supporting reconstruction/regeneration are in process. This study focused on the evaluation of the protective potency of an extract from Gynostemma pentaphyllum (GP), a plant used in traditional medicine with anti-oxidative and neuroprotective activities, in vitro on organotypic spinal cord cultures, the motor-neuron-like NSC-34 cell line and the microglial cell line BV-2. Organotypic cultures were mechanically stressed by the slicing procedure and the effect of GP on motor neuron survival and neurite sprouting was tested by immunohistochemistry. NSC-34 cells were neuronal differentiated by using special medium. Afterwards, cell survival (propidium iodide/fluorescein diacetate labeling), proliferation (BrdU-incorporation), and neurite sprouting were evaluated. BV-2 cells were stimulated with LPS/interferon γ and subjected to migration assay and nanoparticle uptake. Cell survival, proliferation and the expression pattern of different microglial activation markers (cFOS, iNOS) as well as transcription factors (PPARγ, YB1) were analyzed. In organotypic cultures, high-dose GP supported survival of motor neurons and especially of the neuronal fiber network. Despite reduced neurodegeneration, however, there was a GP-mediated activation of astro- and microglia. In NSC-34 cells, high-dosed GP had degenerative and anti-proliferative effects, but only in normal medium. Moreover, GP supported the neuro-differentiation ability. In BV-2 cells, high-dosed GP was toxic. In lower dosages, GP affected cell survival and proliferation when combined with LPS/interferon γ. Nanoparticle uptake, migration ability, and the transcription factor PPARγ, however, GP affected directly. The data suggest positive effects of GP on injured spinal motor neurons. Moreover, GP activated microglial cells. The dual role of microglia (protective/detrimental) in neurodegenerative processes required further experiments to enhance the knowledge about GP effects. Therefore, a possible clinical use of GP in spinal cord injuries is still a long way off.

Alzheimer\'s disease (AD) is the most frequent type of dementia. Acteoside (ACT) is a compound isolated from Cistanche tubulosa, which possesses excellent neuroprotective properties. However, the underlying mechanism of ACT in regulating microglia polarization remains ill-defined. Therefore, a computational network model was established to identify the driving targets of ACT and predict its mechanism by integrating multiple available databases. The AlCl3-induced AD model in zebrafish larvae was successfully constituted to demonstrate the therapeutic efficacy of ACT. Subsequently, LPS-induced BV-2 cells uncovered the positive role of ACT in M1/M2 polarization. The NF-κB and AMPK pathways were further confirmed by transcriptomic analysis, metabolomics analysis, molecular biology techniques, and molecular docking. The research provided an infusive mechanism of ACT and revealed the connection between metabolism and microglia polarization from the perspective of mitochondrial function. More importantly, it provided a systematic and comprehensive approach for the discovery of drug targets, including the changes in genes, metabolites, and proteins.

Microglial cells are important resident innate immune components in the central nervous system that are often activated during neuroinflammation. Activated microglia can display one of two phenotypes, M1 or M2, which each play distinct roles in neuroinflammation. Rutin, a dietary flavonoid, exhibits protective effects against neuroinflammation. However, whether rutin is able to influence the M1/M2 polarization of microglia remains unclear. In this study, in vitro BV-2 cell models of neuroinflammation were established using 100 ng/mL lipopolysaccharide to investigate the effects of 1-hour rutin pretreatment on microglial polarization. The results revealed that rutin pretreatment reduced the expression of the proinflammatory cytokines tumor necrosis factor-α, interleukin-1β, and interleukin-6 and increased the secretion of interleukin-10. Rutin pretreatment also downregulated the expression of the M1 microglial markers CD86 and inducible nitric oxide synthase and upregulated the expression of the M2 microglial markers arginase 1 and CD206. Rutin pretreatment inhibited the expression of Toll-like receptor 4 and myeloid differentiation factor 88 and blocked the phosphorylation of I kappa B kinase and nuclear factor-kappa B. These results showed that rutin pretreatment may promote the phenotypic switch of microglia M1 to M2 by inhibiting the Toll-like receptor 4/nuclear factor-kappa B signaling pathway to alleviate lipopolysaccharide-induced neuroinflammation.