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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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OBJECTIVE: To establish a PC12 cell line with stable expression of human apolipoprotein E( ApoE4) gene by transfection with a lentiviral vector carrying human ApoE4 gene and to investigate the effect of maltol aluminum on the viability of transfected PC12 cells. METHODS: The lentiviral vector carrying human ApoE4 gene was transfected into PC12 cells. PC12 cells with overexpression of ApoE4 gene and negative control vector were obtained after puromycin screening.The mRNA relative expression of R-Apo E and( or) H-Apo E-FLAG of cells in PC12,PC12-NC and PC12-ApoE4 groups were detected by real-time fluorescent quantitative polymerase chain reaction,and the effect of cell construction was identified. PC12-ApoE4 cells and PC12 cells were exposed to maltol aluminum solution at concentrations of 0. 00,100. 00,200. 00 and 400. 00 μmol/L respectively for 24 hours,and cell viability was detected by Cell Counting Kit-8( CCK-8)assay. RESULTS: PC12-ApoE4 and PC12-NC cells under the fluorescence microscope showed fluorescence expression,suggesting that transfection was successful. The expression of PC12 cells showed no fluorescence. The relative expression of H-Apo E-FLAG gene mRNA( the median amount) of PC12-ApoE4 cells was 148. 74,which was higher than the R-Apo E gene in PC12 cells( 1. 00) and PC12-NC cells( 1. 01)( P < 0. 01). After exposure to maltol aluminum,the cell survival rates in terms of the main effect and interaction effect of dose and cell type were statistically significant( P < 0. 01),among them,the cell viabilities were decreased in the concentration range of 0. 00-400. 00 μmol/L with the dose of maltol aluminum exposure increased,showing dose-effect relationship( P < 0. 01). CONCLUSION: The cell line stably expressed human ApoE4 gene was constructed successfully. There was interaction between the effects of maltol aluminum and ApoE4 gene on the survival rate of PC12 cells,and ApoE4 gene could enhance the cytotoxicity of maltol aluminum on PC12 cells.