The Role And Mechanism Of NLRP3 Inflammasome In Methylmercury-induced Microglial Inflammation And Autophagy

Objective:By construct a vitro experimental model of methylmercury-exposed BV2 cells,the present study aimed to investigate the toxic effect of methylmercury（Me Hg）on BV2cells,clarify the relationship between methylmercury exposure and inflammatory reaction and autophagy in BV2 cells,explore the mechanism of NLRP3 inflammatory body in the neuroinflammatory reaction and autophagy triggered by methylmercury exposure,and provide novel research direction for the study on the mechanism of methylmercury neurotoxicity.Methods:The study used BV2 cells as the cell model and methylmercury as the test substance.Doses of exposure were set as 0μM,0.5μM,1.0μM,1.5μM and 2.0μM,with 3 h,6 h,12 h and 24 h as actuation durations.Cell survival rate was measured by MTT.Cell morphology was observed using inverted microscope.LDH method was used to detect the integrity of cell membrane.Intracellular ROS levels were observed with live cell imaging system.Flow cytometry was used to test intracellular ROS levels,mitochondrial transmembrane potential and intracellular Ca²⁺content.The activity of GSH-Px and SOD were measured by biochemistry method.Apoptosis was tested by Annexin-V/PI double staining detection method.ELISA was used to measure the level of inflammatory cytokines IL-1β,IL-18 and TNF-α.Autophagosomes were observed by confocal laser scanning microscope.The intensity of autophagy fluorescence was measured by flow cytometry.Western Blot was used to detect the expression of proteins related to intracellular inflammation and autophagy.SPSS 24.0 was used for statistical analysis,and the measurement data was represented by （?）s.For normal and homogeneous variance data,one-way ANOVA was used to compare the differences of each index among the different groups,and LSD（Least Significant Difference）method was used for pairwise comparison between the groups.The test level was set asα=0.05.Results:1.The effect of Me Hg on the viability and growth of BV2 cells.Results of MTT assay showed that,3 and 6 hours after the exposure to Me Hg,the survival rate of cell in 1.5μM and 2.0μM dose groups were signficantly lower（P<0.05）compare to other dose groups.12 and 24 hours after exposed to Me Hg,the survival rate of cell in 1.0μM,1.5μM and 2.0μM dose groups were significantly lower than cells in the control group（P<0.05）.By using an inverted microscopy,it was found that cells in the control group were in good growth condition,mostly round or with short tentacles.As the dose of Me Hg increased,the number of cell decreased,the morphology became irregular,tentacles were reclaimed,the body size increased,the protrusions became shorter and the intercellular space became fragmented.2.The effect of Me Hg on ROS in BV2 cells.After expose to Me Hg for 3 hours,the observational result showed that cells in the control group had lowest level of fluorescence,and the green fluorescence increased as cells exposed to higher concentrations of Me Hg,the effect was especially in 1.5μM and 2.0μM dose groups.The results of flow cytometry showed that the ROS levels in the 1.5μM and 2.0μM dose group were higher than the control group（P<0.05）.3.Effects of Me Hg on cell membrane integrity and mitochondrial membrane potential in BV2 cells.12 hours after the exposure to Me Hg,the LDH results showed that the difference in cell membrane integrity between the control group and each dose staining group were all statistically significant（P<0.05）.1 h after the exposure to Me Hg,the results of flow cytometry showed that the mitochondrial membrane potential decreased gradually as the concentration of Me Hg increased.The difference in mitochondrial membrane potential between the control group and dose groups（1.5μM and 2.0μM）were statistically significant（P<0.05）.4.The effect of Me Hg on intracellular Ca²⁺of BV2 cells.After expose to Me Hg for 12 hours,the intracellular Ca²⁺content gradually increased as the concentration of Me Hg increased.Ca²⁺contents in the 1.5μM and 2.0μM group were significantly higher than the control group and 0.5μM group（P<0.05）.The concent in the 2.0μM group was significantly higher than the 1.0μM group（P<0.05）.5.Effects of Me Hg on GSH-Px and SOD in BV2 cells.12 hours after the exposure to Me Hg,intracellular GSH-Px and SOD levels gradually decreased as the concentration of Me Hg increased.The difference between the control group and each dose group were all statistically significant（P<0.05）.6.The effect of Me Hg on apoptosis of BV2 cells.12 h after the exposure to Me Hg,both total and early apoptosis rates increased with the increase in Me Hg concentration.Compared to other dose groups,there was a statistically significant difference in the apoptosis rate between the 1.5μM and 2.0μM group（P<0.05）.7.The effect of Me Hg on inflammatory response of BV2 cells.After expose to Me Hg for 12 hours,Western Blot showed that the NLRP3 expression was up-regulated in response to the increase in Me Hg concentration.The NLRP3 expression in the 1.5μM,2.0μM and LPS group were significantly higher than the control group（P<0.05）.ASC expression was also up-regulated.The expression in the 1.0μM,1.5μM,2.0μM and LPS group were significantly higher than the control group（P<0.05）.Moreover,the Caspase-1 20 k D expression was up-regulated with statistically significant differences were found between the control group and all dose groups（P<0.05）.Results of ELISA showed that cell supernatant IL-1βlevels elevated,and the level was significantly higher in the 1.0μM,1.5μM,2.0μM and LPS groups than the control group（P<0.05）.Cell supernatant IL-18 levels also elevated.Statistically significant differences were found between the control group and all dose groups（P<0.05）.In addition,the cellular supernatant TNF-αlevel was elevated.The level in the 1.0μM,1.5μM,2.0μM and LPS group were all significantly higher than the control group（P<0.05）.8.The effect of Me Hg on autophagy of BV2 cells.12 houre after the exposure to Me Hg,the observational results from a confocal laser scanning microscope revealed that autophagic vesicles were almost undetectable in lower Me Hg concentration groups,and the number of autophagic vesicles increased in the 1.5μM and 2.0μM dose group.The results of flow cytometry showed that the autophagic fluorescence intensity of cells in the 1.0μM,1.5μM and 2.0μM group were significantly higher than the control group（P<0.05）.Western Blot showed that,as the concentration of Me Hg increased,the expression of Beclin-l was significantly higher in the 1.0μM,1.5μM and 2.0μM group compare to the control group（P<0.05）.LC3Ⅱ/LC3Ⅰexpression was up-regulated,and the expression in the 2.0μM group was significantly higher than the control group（P<0.05）.On the other hand,SQSTM1/p62 expression was down-regulated,with statistically significant differences（P<0.05）between the other dose groups and the control group.9.The effect of NLRP3 inflammasome in the inflammatory response and autophagy of BV2 cells triggered by methylmercury.After using the NLRP3inflammasome inhibitor,MTT results showed that cell survival rate in Me Hg group was significantly lower than the control group（P<0.05）,the cell survival rate of INF4E+Me Hg group was lower than the control group（P<0.05）,but significantly higher than the Me Hg group（P<0.05）.The results of flow cytometry showed that the autophagy fluorescence intensity in the Me Hg group was significantly higher than the control group（P<0.05）,and the autophagy fluorescence intensity in the INF4E+Me Hg group was significantly lower than the Me Hg group（P<0.05）.Western Blot results showed that,compare to the Me Hg group,NLRP3,Caspase-1 activated fragment and LC3Ⅱ/LC3Ⅰexpression were down-regulated in the INF4E+Me Hg group（P>0.05）.ASC and Beclin-l expression was down-regulated（P<0.05）,SQSTM1/p62 expression was up-regulated（P<0.05）.Results of the ELISA showed that,after INF4E inhibited NLRP3 inflammatory vesicles,the level of IL-1β,IL-18 and TNF-αinflammatory factors released by Me Hg-induced BV2 cells were significantly lower than those in the Me Hg group（P<0.05）.After using the autophagy inhibitor,MTT results showed that cell survival rate in Me Hg group was significantly lower than the control group（P<0.05）,and the cell survival rate of 3-MA+Me Hg group was significantly lower than the control group and the Me Hg group（P<0.05）.Western Blot results showed that,compare to the Me Hg group,NLRP3,Caspase-1 activation fragment and ASC expression were all up-regulated in the 3-MA+Me Hg group with statistically significant differences between two groups（P<0.05）.Beclin-l and LC3Ⅱ/LC3Ⅰexpression were down-regulated and SQSTM1/p62 expression was up-regulated（P>0.05）.ELISA results showed that the level of IL-1β,IL-18 and TNF-αinflammatory factors released by Me Hg-induced BV2 cells were significantly higher than those in the Me Hg group after 3-MA inhibited autophagy in BV2 cells（P<0.05）.Conclusions:1.Me Hg reduced the survival rate of BV2 cells and caused changes in cell morphology.2.Me Hg could damage the integrity of BV2 cells membrane,reduce mitochondrial membrane potential,distrupt calcium homeostasis and cause oxidative damages.3.Me Hg could trigger inflammatory reaction in BV2 cells by activate NLRP3inflammasomes.4.Me Hg could cause autophagy by trigger inflammatory reactions in BV2 cells.5.Me Hg could affect the incidence rate of autophagy through NLRP3inflammasomes,and NLRP3 inflammasome has a positive effect on autophagy.