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Background Aluminum activates signal transducer and activator of transcription 3 (STAT3), causing microglial nucleotide-binding and oligomerization domain-like receptors protein 3 (NLRP3) inflammasome activation and inflammatory responses and producing neurotoxicity. Objective To explore the role of STAT3 regulated NLRP3 inflammasomes in the inflammatory response of mouse microglia cell line (BV2) cells induced by maltol aluminum [Al(mal)3]. Methods BV2 cells were assigned to five groups: one control group, three Al(mal)3 exposure groups (low, medium, and high doses at 40, 80, and 160 μmol·L−1 Al(mal)3 respectively), and one C188-9 (STAT3 antagonist) intervention group [10 μmol·L−1 C188-9 +160 μmol·L−1 Al(mal)3]. Cell viability was detected by CCK8. The expression of M1/M2 type markers, i.e. CD68/CD206, STAT3, p-STAT3, NLRP3, cleaved-casepase-1, and apoptosis-associated speck-like protein (ASC) in BV2 cells were detected by Western blotting, and proinflammatory cytokines interleukin (IL)-1β and IL-18, and anti-inflammatory cytokine IL-10 were determined by ELISA. Results The results of cell viability assay showed that cell viability gradually decreased with the increase of Al(mal)3 dose. Compared with the control group, the cell viability of the Al(mal)3 high-dose group was decreased by 18% (P<0.05); compared with the Al(mal)3 high-dose group, the cell viability of the C188-9 intervention group was significantly elevated by 14% (P<0.05). Compared with the control group, the expression levels of CD68 in the Al(mal)3 low-, medium-, and high-dose groups were elevated by 19%, 20%, and 21%, respectively (P<0.05); the expression level of CD206 in the Al(mal)3 high-dose group was decreased by 25% (P<0.05). Compared with the Al(mal)3 high-dose group, the expression level of CD68 in the C188-9 intervention group was reduced by 9% (P<0.05), whereas the expression level of CD206 was elevated by 22% (P<0.05). Compared with the control group, the p-STAT3 protein expression and the p-STAT3/STAT3 ratio in the Al(mal)3 high-dose group increased by 129% and 127%, respectively (P<0.05). Compared with the Al(mal)3 high-dose group, the p-STAT3 protein expression and the p-STAT3/STAT3 ratio in the C188-9 intervention group were decreased by 55% and 54%, respectively (P>0.05). Compared with the control group, the expression level of NLRP3 protein increased by 75% in the Al(mal)3 high-dose group (P<0.05), the expression levels of cleaved-casepase-1 protein increased by 28% and 35% in the Al(mal)3 medium- and high-dose groups (P<0.05), and the expression levels of ASC increased by 22%, 25%, and 53% in the Al(mal)3 low-, medium- and high-dose groups (P<0.05), respectively. Compared with the Al(mal)3 high-dose group, the expression levels of NLRP3, cleaved-casepase-1, and ASC proteins in the C188-9 intervention group decreased by 30%, 19%, and 32%, respectively (P<0.05). Compared with the control group, the levels of IL-1β in the Al(mal)3 medium- and high-dose groups increased by 18% and 21%, respectively (P<0.05), and the level of IL-18 in the Al(mal)3 high-dose group increased by 10% (P<0.05). Compared with the Al(mal)3 high-dose group, the IL-18 levels were reduced by 23% in the C188-9 intervention group (P<0.05). The content of anti-inflammatory factor IL-10 did not differ significantly between groups (P>0.05). Conclusion Aluminum can induce inflammatory responses in BV2 microglia and is predominantly pro-inflammatory, and the mechanism may involve STAT3 regulation of NLRP3 inflammasome secretion of inflammatory factors.
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To investigate the mechanism by which Schisandra Chinensis mediates the phenotypic transformation of microglia via microRNA-124 (miR-124)-based regulation of the Toll-like receptor 4 (TLR4) pathway, a model was established using lipopolysaccharide (LPS) stimulation of BV2 cells. Cells were treated with different doses of Schisandra Chinensis extract (SCE). MiR-124 inhibitors and negative control sequences (NC inhibitor) were transfected into LPS-induced BV2 cells and treated with SCE. The MTT assay was used for cell activity detection; an NO kit was used to measure NO release; ELISA kits were used to measure the levels of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α). Microglia markers, including ionized calcium binding adapter molecule-1 (IBA-1) and arginase-1 (Arg-1), and the nuclear translocation of nuclear factor-kappa B (NF-κB) were evaluated by immunofluorescent staining. NF-κB p65, IBA-1, Arg-1, TLR4, myeloid differentiation primary factor 88 (MyD88), inhibitor of nuclear factor-kappa B kinases-α (IKK-α), IL-10, TNF-α were detected by immunoblot. SCE at concentrations ranging from 31.25 to 250 μg·mL-1 had no significant effect on cell activity. SCE treatment significantly inhibited NO release induced by LPS (P < 0.001, P < 0.01), increased the level of IL-10 (P < 0.05), and decreased the level of TNF-α (P < 0.001). In addition, SCE significantly reduced the expression of TNF-α, IBA-1, TLR4, and MyD88 (P < 0.01, P < 0.001) and elevated the expression of IL-10, Arg-1, NF-κB P65 and IKK-α (P < 0.001, P < 0.01, P < 0.05). SCE treatment could also promote the expression of miR-124 (P < 0.01). However, transfection with the miR-124 inhibitor increased TNF-α (P < 0.001), decreased the level of IL-10 (P < 0.05), increased the mRNA level and the protein expression of TNF-α and IBA-1 (P < 0.05, P < 0.01, P < 0.001), and decreased the mRNA level and protein expression of IL-10 and Arg-1 (P < 0.001, P < 0.01). In addition, the inhibition of TLR4 and MyD88 was attenuated. In conclusion, SCE appears to inhibit the activation of TLR4 signaling pathway by upregulating miR-124 so as to inhibit microglia M1 polarization and promote microglia M2 polarization.
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Background Programmed necrosis is closely related to the occurrence and development of neurodegenerative diseases, but whether lead causes programmed cell necrosis has not been reported. Objective This experiment is designed to probe into the function of programmed necrosis and the effect of its inhibitor on lead-induced microglia (BV2 cell) injury. Methods The BV2 cells at logarithmic growth phase were treated with 0, 1, 5, 10, 25, 50, 100, and 200 μmol·L−1 lead acetate for 12, 24, 36, and 48 h, respectively, and methylthiazolyldiphenyl-tetrazolium bromide (MTT) was used to determine cell viability. After treatment with 0, 25, 50, and 100 μmol·L−1 lead acetate for 24 h, enzyme-linked immunosorbent assay, Western blotting, and flow cytometry were used to determine the expressions of tumor necrosis factor-α (TNF-α), receptor-interacting protein kinase 3 (RIPK3), receptor-interacting protein kinase 1 (RIPK1), and mixed lineage kinase domain-like protein (MLKL) in the cells, and the effect of RIPK1 inhibitor Nec-1 pretreatment on lead-induced BV2 cell injury . Results The BV2 cell viability decreased with higher lead concentration (r12 h=−0.995, r24 h=−0.984, r36 h=−0.983, r48 h=−0.981, all P<0.01) and time extension (only for 5 μmol·L−1 lead acetate, r=−0.994, P<0.01). Compared with the control group, the BV2 cell viability decreased at the same exposure time when the concentration of lead was above 10 μmol·L−1 (P<0.01). Compared with the control group, the expressions of RIPK1 and MLKL were increased in the 25, 50, and 100 μmol·L−1 lead groups (P<0.05 or 0.01), accompanied by an increase in the contents of inflammatory cytokine TNF-α, especially in the 100 μmol·L−1 lead group, the increment was the highest (P<0.01). The expression levels of p-RIPK1 and p-MLKL in BV2 cells were both increased when the concentration of lead acetate was above 50 μmol·L−1 (P<0.01). In addition, pretreatment with Nec-1 increased the cell viability rate and decreased the necrosis and late apoptosis rate of BV2 cells exposed to lead compared with corresponding lead exposure groups (P<0.05). Conclusions Lead can reduce BV2 cell viability, increase necrosis rate, and up-regulate the expressions of RIPK1, RIPK3, amd MLKL, and the phosphorylation levels of RIPK1 and MLKL. The RIPK1 inhibitor Nec-1 has an intervention effect on lead-induced damage in BV2 cells, indicating that programmed necrosis may play a role in lead neurotoxicity.
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Objective: To study the chemical constituents of 95% ethanol aqueous extract of Chuanxiong Rhizoma and the bioactivities of inhibition on nitric oxide (NO) production in lipopolysaccharides (LPS)-activated murine macrophage RAW264. 7 and mouse microglia BV2 cell lines. Methods: The compounds were separated and purified by silica gel column and high performance liquid chromatographies, and their structures were determined by spectroscopic data analysis. Using LPS-activated RAW264. 7 and BV2 cell line models in vitro, all of the isolated compounds were evaluated for the inhibition against NO production. Results: Three butylphthalide derivatives were obtained and identified as Z-3', 8', 3'a, 7'a-tetrahydro-6, 3', 7, 7'a-diligustilide-8'-one (1), Z, Z'-3.3'a, 7. 7'a-diligustilide (2), and chuanliguspirolide (3), respectively. For the inhibition of NO production in the LPS-activated two cell lines, the half inhibitory concentration (IC50) values of compounds 1-3 and indomethacin as a positive control drug in RAW 264. 7 cell line model were (31.60 ± 2.62), (21.20 ± 0.61), (30.12 ± 2.90), and (54.62 ± 7.53) μmol/L, respectively, while IC50 values of compounds 1-3 and curcumin as a positive control drug in BV2 cell line model were (21.99 ± 4.40), (15.43 ± 1.34), (12.20 ± 3.40), and (10.58 ± 1. 41) μmol/L, respectively. Conclusion: Compound 3 named as chuanliguspirolide is a new one. The results of bioactivity assays indicated that compounds 1-3 are potential anti-inflammatory agents.
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The effects of catechin on inflammatory response of BV-2 cells were investigated using the lipopolysaccharide (LPS) model. BV-2 cells were incubated with LPS (1 mg·L-1) for 12 h in the microglia inflammatory model in vitro. After catechin and LPS co-incubation for 12 h, MTT, ELISA and Western blot were used to detect cell viability, cytokines, cell migration and protein expression. In addition, transwell assay was conducted to investigate the effect of catechin on cell chemokaxis. Catechin did not show any cytotoxicity effect on BV-2 cells, but reversed the change in cell morphology and inhibited the release of TNF-α and IL-1β, cell chemotaxis and phosphorylation of NF-κB/p65. In conclusion, Catechin could inhibit the LPS-induced inflammatory response in BV-2 cells.
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Introduction: Neurodegeneration resulting from pathogen invasion or tissue damage has been associated with activation of microglia, and exacerbated by the release of neurotoxic mediators such as pro-inflammatory cytokines, chemokines and reactive oxygen species. Activation of microglia stimulated by lipopolysaccharide is mediated in part by GSK-3 signaling molecule. Induced IL-10 expression via GSK-3 inhibition is noteworthy since IL-10 has been remarkably shown to suppress inflammation. Objectives: We aimed to inactivate microglia through inhibition of GSK-3 signaling and to determine its effects on the production of pro- and anti-inflammatory mediators. Methods: LPS-stimulated BV-2 cells were treated with a GSK-3 inhibitor (LiCl, NP12, SB216763 or CHIR99021). Inhibition of GSK-3 was determined by the phosphorylation status of GSK-3β. The effects of GSK-3 inhibition on microglial inflammatory response were investigated by examining various mediators and CD200R marker. Production of nitric oxide (NO), glutamate and pro- and anti-inflammatory cytokines were measured using flow cytometry, Griess assay, glutamate assay and Cytometric Bead Array (CBA) respectively. Results: GSK-3β signaling in LPS-stimulated microglia was blocked by GSK-3 inhibitor through increased phosphorylation at Serine 9 residue. GSK-3 inhibitors had also led to reducing in microglia activity via increased expression of CD200R. Inhibition of GSK-3 also diminished inflammatory mediators such as nitric oxide (NO), glutamate, pro-inflammatory cytokines (TNF-α and IL-6) and chemokine, MCP-1. Reduction of pro-inflammatory mediators by GSK-3 inhibitor was coincided with increased IL-10 production. Conclusions: Suppression of microglia-mediated inflammatory response was facilitated by GSK-3 inhibition with associated increased in IL-10 production.
Sujets)
MicroglieRésumé
Betulinic acid (BA), a natural pentacyclic triterpene found in many medicinal plants is known to have various biological activity including tumor suppression and anti-inflammatory effects. In this study, the cell-death induction effect of BA was investigated in BV-2 microglia cells. BA was cytotoxic to BV-2 cells with IC50 of approximately 2.0 μM. Treatment of BA resulted in a dose-dependent chromosomal DNA degradation, suggesting that these cells underwent apoptosis. Flow cytometric analysis further confirmed that BA-treated BV-2 cells showed hypodiploid DNA content. BA treatment triggered apoptosis by decreasing Bcl-2 levels, activation of capase-3 protease and cleavage of PARP. In addition, BA treatment induced the accumulation of p62 and the increase in conversion of LC3-I to LC3-II, which are important autophagic flux monitoring markers. The increase in LC3-II indicates that BA treatment induced autophagosome formation, however, accumulation of p62 represents that the downstream autophagy pathway is blocked. It is demonstrated that BA induced cell death of BV-2 cells by inducing apoptosis and inhibiting autophagic flux. These data may provide important new information towards understanding the mechanisms by which BA induce cell death in microglia BV-2 cells.
Sujets)
Apoptose , Autophagie , Mort cellulaire , ADN , Concentration inhibitrice 50 , Microglie , Plantes médicinalesRésumé
Objective To investigate the effects of Annexin-A1 ( Anxa1 ) gene silencing induced by siRNA on the growth and migration of microglial BV-2 cells and its possible mechanisms.Methods A synthesized siRNA duplex targeting Anxa1 gene was transfected into BV-2 cells.The efficiency of siRNA-in-duced Anxa1 gene silencing was evaluated on both mRNA and protein levels by using reverse-transcription PCR and Western blot assay.MTT assay was performed to measure the proliferation of BV-2 cells with si-lenced expression of Anxa1 gene.Flow cytometry with Annexin V-FITC/PI double staining was used to de-tect the apoptosis rate of BV-2 cells.Transwell chambers were used to analyze the effects of siRNA-induced Anxa1 gene silencing on the migration of BV-2 cells.Western blot assay was performed to detect the expres-sion of signaling proteins related to cell cycle and migration.Results Compared with the siRNA negative control ( siRNA-NC) group, the inhibitory rates of siRNA-induced Anxa1 gene silencing on the proliferation of BV-2 cells were significantly increased at the time points of 24 h, 48 h and 72 h after intervention [(16.9 ±2.1)%, (23.1±3.6)%and (42.4±1.7)%vs (1.35±0.5)%, (2.06±0.7)% and (8.65±0.9)%, P<0.05 ].The apoptosis rate of BV-2 cells transfected with Anxa1 siRNA was (18.4±2.1)%, which was significantly elevated as compared with that of the siRNA-NC group (5.2±0.3)%and control group (4.3±0.2)%.Cell migration of the Anxa1 siRNA transfected BV-2 cells was inhibited remarkably at 48 h as com-pared with that of the siRNA-NC group (28.7±5.2 vs 173.4±11.4, P<0.01).Moreover, the suppressed expression of Cyclin D1 protein and activation of p38 and JNK signaling pathways were induced by silenced expression of Anxa1 gene in BV-2 cells.Conclusion The growth and migration of BV-2 cells were signifi-cantly inhibited by silencing the expression of Anxa1 gene with siRNA, the possible mechanisms might be associated with the suppressed expression of Cyclin D1protein and the activation of p38 and JNK signaling pathways.