陆军军医大学学报2026，Vol.48Issue(11)：1543-1554,12.DOI:10.16016/j.2097-0927.202601105
聚苯乙烯微塑料通过氧化应激促进Txnip表达及转位以激活ASK1-Caspase9/3信号通路诱导小鼠精母细胞凋亡
Polystyrene microplastics promote Txnip expression and translocation through oxidative stress to activate the ASK1-Caspase9/3 signaling pathway and induce apoptosis in mouse spermatocytes
摘要
Abstract
Objective Polystyrene microplastics(PS-MPs),as an emerging environmental pollutant,have attracted considerable attention regarding their toxic effects on the male reproductive system and underlying mechanisms.This study aims to investigate whether PS-MPs promote the expression and translocation of thioredoxin interacting protein(Txnip)by inducing oxidative stress,and thereby activate the apoptosis signal-regulating kinase 1(ASK1)-cysteinyl aspartate specific proteinase9/3(Caspase9/3)signaling pathway,ultimately mediating the apoptosis of mouse spermatocytes.Methods Mouse testicular spermatocyte line GC-2 was used as a model and treated with 5 μm PS-MPs(0,10,20,40 μg/mL).Cell morphology and viability were analyzed after 48 and 72 h of exposure,and cell morphology was observed by optical microscopy,and cell viability was detected by CCK-8 assay(n=3).The remaining mechanistic studies were based on PS-MPs exposure for 48 h.Flow cytometry was performed to determine the levels of reactive oxygen species(ROS)(n=7,9)and apoptosis rates(n=11,13).Transcriptome sequencing and GO function enrichment analysis were conducted to screen differentially expressed genes(DEGs)and signaling pathways.RT-qPCR and Western blotting were utilized to detect Txnip expression at mRNA and protein levels(n=3),and immunofluorescence assay was adopted to observe Txnip subcellular translocation.The protein levels of downstream apoptosis-related molecules,such as ASK1,p-ASK1,pro-cysteinyl aspartate specific proteinase 3(pro-Caspase3),cleaved-cysteinyl aspartate specific proteinase 3(Cleaved-Caspase3)and Caspase9 were detected by Western blotting.Small interfering RNA was used to knock down Txnip expression;RT-qPCR and Western blotting were employed to verify the knockdown efficiency,and flow cytometry and Western blotting were utilized to detect apoptosis rates(n=12,13)and the expression of downstream apoptosis-related molecules.CCK-8 assay was conducted to screen the appropriate intervention concentration of N-Acetylcysteine(NAC),and the expression of Txnip was detected by RT-qPCR and Western blotting(n=3).The apoptosis rate(n=7,8),Txnip translocation and expression of downstream apoptosis-related molecules were detected by flow cytometry and Western blotting.Results After 48 h of PS-MPs exposure,the number of cells was decreased and altered morphology was observed in all exposure groups.Compared with 0 μg/mL group,Except for the 10 μg/mL group,with no significant difference in cell viability,Compared with 0 μg/mL group,all other exposure groups demonstrated significantly decreased viability(P＜0.000 1).After 72 h of PS-MPs exposure,all exposure groups showed reduced cell numbers,altered morphology,and significantly decreased cell viability(P＜0.001).Compared with 0 μg/mL group,exposure to PS-MPs for 48 h significantly increased ROS levels(P＜0.001)and promoted cell apoptosis(P＜0.01).Transcriptome analysis showed that DEGs were significantly enriched in oxidative stress and apoptosis regulatory pathways,with Txnip showing the most significant expression.After 48 h of PS-MPs exposure,Compared with 0 μg/mL group,the mRNA and protein levels of Txnip were mildly increased in the 10 μg/mL group,while the levels in all other exposure groups were increased significantly(P＜0.05).Furthermore,knockdown of Txnip significantly inhibited cell apoptosis(P＜0.000 1).PS-MPs exposure induced the translocation of Txnip from the nucleus to mitochondria and accumulate in the cytoplasm,thereby promoting ASK1 phosphorylation,Caspase9 activation,and Caspase3 cleavage.Knockdown of Txnip significantly inhibited the activation of the ASK1-Caspase9/3 pathway.Co-exposure of PS-MPs with 1 mmol/L NAC for 48 h also inhibited Txnip expression(P＜0.01)and translocation,thereby blocking downstream pathway activation and ultimately reducing apoptosis(P＜0.000 1).Conclusion PS-MPs can upregulate Txnip expression and promote its translocation through oxidative stress,thereby activating the ASK1-Caspase9/3 signaling pathway and finally inducing apoptosis in mouse GC-2 cells.关键词
微塑料/硫氧还蛋白互作蛋白/氧化应激/细胞凋亡Key words
microplastics/thioredoxin interacting protein/oxidative stress/apoptosis分类
医药卫生引用本文复制引用
马爽,刘康乐,刘亚鑫,刘晋祎,姜晓,曹佳..聚苯乙烯微塑料通过氧化应激促进Txnip表达及转位以激活ASK1-Caspase9/3信号通路诱导小鼠精母细胞凋亡[J].陆军军医大学学报,2026,48(11):1543-1554,12.基金项目
国家自然科学基金面上项目(82473675) （82473675）
国家自然科学基金重点项目(82130097) Supported by the General Program of National Natural Science Foundation of China(82473675),and the Key Program of National Natural Science Foundation of China(82130097). （82130097）
