iNOS In BV-2 Cells Research Articles

Rhein inhibits M1 polarization of BV2 microglia through MAPK/IκB signalling pathway and reduces neurotoxicity caused by neuroinflammation.

Rhein is an anthraquinone compound with anti-inflammatory pharmacological activity. It has been found to play a neuroprotective role in neurological diseases, but the neuroprotective mechanism of rhein remains unclear. SH-SY5Y cells serving as neuron-like cells and BV2 microglia were used. The toxicity of rhein on BV2 microglia and the viability of SH-SY5Y cells were measured by CCK-8 assay. The mRNA expression and secretion of pro-inflammatory cytokines were detected by qPCR and ELISA. Iba1, CD86 and pathway signalling protein in BV2 microglia were assessed by Western blot and immunofluorescence. Apoptosis of SH-SY5Y cells exposed to neuroinflammation was analysed through flow cytometry. Rhein inhibited MAPK/IκB signalling pathways. Further studies revealed that rhein inhibited the production of pro-inflammatory cytokines TNF-α, IL-6, IL-1β and iNOS in BV2 cells and also inhibited the expression of M1 polarization markers Iba1 and CD86 in BV2 cells. Furthermore, rhein reduced the apoptotic rate and restored cell viability of SH-SY5Y cells exposed to neuroinflammation. Our study demonstrated that rhein inhibited microglia M1 polarization via MAPK/IκB signalling pathway and protected nerve cells through suppressing neuroinflammation.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Network Pharmacology Analysis and Molecular Characterization of the Herbal Medicine Formulation Qi-Fu-Yin for the Inhibition of the Neuroinflammatory Biomarker iNOS in Microglial BV-2 Cells: Implication for the Treatment of Alzheimer's Disease.

Aberrant microglial activation drives neuroinflammation and neurodegeneration in Alzheimer's disease (AD). The present study is aimed at investigating whether the herbal formula Qi-Fu-Yin (QFY) could inhibit the inflammatory activation of cultured BV-2 microglia. A network pharmacology approach was employed to predict the active compounds of QFY, protein targets, and affected pathways. The representative pathways and molecular functions of the targets were analyzed by Gene Ontology (GO) and pathway enrichment. A total of 145 active compounds were selected from seven herbal ingredients of QFY. Targets (e.g., MAPT, APP, ACHE, iNOS, and COX-2) were predicted for the selected active compounds based on the relevance to AD and inflammation. As a validation, fractions were sequentially prepared by aqueous extraction, ethanolic precipitation, and HPLC separation, and assayed for downregulating two key proinflammatory biomarkers iNOS and COX-2 in lipopolysaccharide- (LPS-) challenged BV-2 cells by the Western blotting technique. Moreover, the compounds of QFY in 90% ethanol downregulated iNOS in BV-2 cells but showed no activity against COX-2 induction. Among the herbal ingredients of QFY, Angelicae Sinensis Radix and Ginseng Radix et Rhizoma contributed to the selective inhibition of iNOS induction. Furthermore, chemical analysis identified ginsenosides, especially Rg3, as antineuroinflammatory compounds. The herbal formula QFY may ameliorate neuroinflammation via downregulating iNOS in microglia.

PLGA Microspheres Loaded with β-Cyclodextrin Complexes of Epigallocatechin-3-Gallate for the Anti-Inflammatory Properties in Activated Microglial Cells.

Although epigallocatechin-3-gallate (EG) is well-known as a potent antioxidant and free radical scavenger for neurodegenerative diseases, it still has disadvantages that reduce its treatment effectiveness due to low bioavailability, slow absorption, and water solubility. Therefore, the aim of this study is to improve the bioavailability of EG and increase the effectiveness of anti-inflammatory properties to microglial cells by using Poly(Lactide-co-Glycolide) (PLGA) microspheres as carriers. In this study, we used UV–Vis spectroscopy to show the formation of the complex of β-cyclodextrin (β-CD) and EG (CD-EG). The loading efficiency of EG in PLGA microspheres was optimized by the addition of β-CD. The highest loading efficiency of 16.34% was found among other formulations. The results of Fourier transform infrared spectroscopy indicated the loading of CD-EG in PLGA microspheres. The scanning electron microscopic images demonstrated the spherical PLGA particles with controlled particles size ranging from 1–14 µm. Moreover, the in vitro release of EG was conducted to explore the sustained release property of the PLGA formulations. In the in vitro model of mouse microglial cells (BV-2 cells) stimulated by lipopolysaccharide, the cytotoxicity test showed that for up to 1 mg/mL of PLGA microspheres no toxicity to BV-2 cells was found. PLGA microspheres can significantly suppress the nitric oxide production from BV-2 cells, indicating EG loaded in PLGA microspheres can suppress the inflammation of activated microglial cells. Furthermore, the intracellular iNOS in BV-2 cells was also found to be down regulated. In summary, we have successfully shown that the use of β-CD can increase the loading efficiency of EG in PLGA microspheres and provide neuroprotective effect on the activated microglial cells.

Inhibition of Lipopolysaccharide-Stimulated Neuro- Inflammatory Kuntze in BV-2 Microglial Cell Mediators by Tetragonia tetragonoides (Pall)

Purpose: To investigate the in vitro antioxidant and anti-neuroinflammatory effects of Tetragonia tetragonoides (Pall.) Kuntze extract (TKE) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells.Methods: To evaluate the effects of TKE, LPS-stimulated BV microglia were used and the expression and production of inflammatory mediators, namely, nitric oxide (NO), inducible NO synthase (iNOS) and tumor necrosis alpha (TNF-λ) were evaluated. Antioxidant activity of TKE was measured using 1, 1- diphenyl-2-picryl-hydrazyl (DPPH) assay. Cell viabilities were estimated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide (MTT) assay.Results: TKE significantly suppressed LPS-induced production of NO (p < 0.001 at 20, 40, 80 and 100 µg/ml) and expression of iNOS in BV-2 cells. TKE also suppressed LPS-induced increase in TNF-á level (p < 0.001at 100 µg/ml) in BV-2 cells. In addition, DPPH-generated free radicals were inhibited by TKE in a concentration-dependent manner.Conclusion: The results suggest that TKE can be explored as a potential therapeutic agent for regulating microglia-mediated neuroinflammatory responses observed in several neurodegenerative diseases.Keywords: Tetragonia tetragonoides, Anti-oxidant, Anti-inflammatory, Neurodegenerative diseases, Microglial cells, Lipopolysaccharide

Suppression of Lipopolysaccharide-Stimulated Neuroinflammatory Mediators by Chenopodiacea Mangrove, <i>Suaedea maritima</i> (L) Dumort, in BV-2 Microglial Cells

Purpose: To investigate the in-vitro antioxidant and anti-neuroinflammatory effects of Suaeda maritime (L) Dumort ethyl acetate (SM-EA) extract in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells.Methods: LPS-stimulated BV- microglia were used to study the expression and production of inflammatory mediators, viz, nitric oxide (NO), inducible NO synthase (iNOS, interleukin (IL)-6) and tumor necrosis alpha (TNF-α). Antioxidant activity was measured using 1, 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. Cell viability was estimated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl-tetrazolium bromide (MTT) assay.Results: SM-EA extract significantly suppressed LPS-induced production of NO (p < 0.001 at 80 and 100 ìg/ml) and expression of iNOS in BV-2 cells. SM-EA also suppressed LPS-induced increase in IL-6 and TNF-α levels (p < 0.001at 100 ìg/ml) in BV- cells. Further, DPPH-generated free radicals were inhibited by SM-EA extract in a concentration-dependent manner (p < 0.01 at 0.1 mg/ml and p < 0.001 at 1 mg/ml) with half maximal inhibitory concentration (IC50) of 0.42 mg/ml.Conclusion: The findings imply that SM-EA extract can be developed as a potential therapeutic agent in regulating microglia-mediated neuroinflammatory responses observed in several neurodegenerative diseases.Keywords: Suaedea maritime, Chenopodiaceae, Anti-oxidant, Anti-inflammatory, Microglial cells, Inducible nitric oxide synthase, Interleukin-6