Study On The Mechanism Of Silica Induced Macrophage Pyroptosis By Caspase-3/GSDME Pathway

Objective:The macrophage pyroptosis model was constructed using an apoptosis-associated speck-like protein containing a CARD（ASC）-deficient mouse macrophage cell line（RAW264.7）to investigate whether silica（Si O₂）particles induce macrophage pyroptosis through the Caspase-3/Gasdermin E（GSDME）pathway when nucleotide-binding oligomerization domain-like receptor protein 3（NLRP3）inflammasome activation is blocked and to further understand the molecular mechanism of pyroptosis in the pathogenesis of pneumoconiosis.At the same time,the anti-pyroptosis effect of Prussian blue nanoparticles（PBNPs）on the classical pyroptosis pathway was used to explore whether PBNPs were involved in the regulation of macrophage pyroptosis induced by Si O₂ particles through the Caspase-3/GSDME pathway,so as to provide new ideas for the treatment of pneumoconiosis in the future.Method:RAW264.7 cells were divided into blank control group,lipopolysaccharide（LPS）group（500 ng/ml LPS）,LPS+Si O₂ group（500 ng/ml LPS+150μg/ml Si O₂）,LPS+Si O₂+Caspase-3 inhibitor（500 ng/ml LPS+150μg/ml Si O₂+100μM Ac-DEVD-CHO）and LPS+Si O₂+PBNPs group（500 ng/ml LPS+150μg/ml Si O₂+50/100/200 ppm PBNPs）.Cell viability was measured by CCK-8 assay;The membrane integrity of RAW264.7 was assessed by propidium iodide staining;The levels of LDH in supernatant were measured by ELISA;and protein expression levels of polyadenosine diphosphate ribose polymerase（PARP）,GSDMD,GSDME and related caspases were measured by Western blotting.Results:Compared with the blank control group,RAW264.7 cells treated with Si O₂ showed a decreasing trend in cell viability with the increase of Si O₂ concentration（P<0.01）,and the cell membrane integrity was destroyed,up-regulating the apoptosis-related activation of Caspase-9/8/3 and PARP,while GSDME was cleaved（P<0.01）.However,After pretreatment with specific inhibitor,the cell membrane destruction was reduced,and apoptosis and pyroptosis-related protein expression levels were also decreased（P<0.05）.In addition,PBNPs at a concentration of 200 ppm could effectively reduce the release of LDH（P<0.0001）and protect the integrity of cell membrane,but there was no effect on the expression levels of related proteins on the pyroptosis pathway（P>0.05）.Conclusion:In the presence of NLRP3/Caspase-1/GSDMD pathway blockade,silica can induce pyroptosis in RAW264.7 cells dependent on Caspase-3/GSDME pathway,which may be involved in the pathogenesis of pneumoconiosis.In addition,in this study,PBNPs couldn’t play an anti-pyroptotic role on Caspase-3/GSDME pathway at the protein expression level,but it had a certain protective effect on the cell membrane of RAW264.7 stained with silica dust.The role of PBNPs in the treatment of pneumoconiosis is still worthy of further study.