PiR-mmu-1019957 Promotes 1,2-dichloroethane-induced Testicular Pyroptosis Via Upregulating IRF7 In Vitro And In Vivo

Background1,2-dichloroethane(1,2-DCE)is a ubiquitous environmental pollutant with male reproductive toxicity.We have previously shown that 1,2-DCE mediates reproductive toxicity in male NIH mice through the CREM/CREB pathway.PiRNAs are a class of small non-coding RNAs that are specifically expressed in animal germ cells and play an important role in reproductive damage caused by exogenous chemicals.However,the role of piRNAs in 1,2-DCE-induced male reproductive toxicity remains unclear.ObjectivesThis study aims to investigate the toxic phenotype of mouse spermatocytes(GC-2 spd)exposed to 1,2-DCE in vitro.We explored whether the specific molecular events of 1,2-DCE-induced male reproductive damage in mice are related to piRNAs,so as to provide a potential biomarker and therapeutic target for 1,2-DCE poisoning.Methods(1)1,2-DCE exposure in vitro and in vivo:The concentration of 1,2-DCE at the cellular level was determined by the CCK8 assay and set up as control(0 mM),low dose(7.5 mM),medium dose(15 mM)and high dose(30 mM)groups.NIH male mice were randomly divided into 4 groups and exposed to corresponding concentrations of 1,2-DCE(0,100,350,700 mg/m3)for 28 days by dynamic inhalation dosing.(2)1,2-DCE reproductive toxicity phenotype study:cellular level using CCK8 assay,cell death assay,electron microscopy morphology,qRT-PCR and western blot at the molecular level,lactate dehydrogenase(LDH)release assay,flow cytometry to confirm the toxicity phenotype and validation by western blot at the animal level.(3)Exploration of the mechanism of reproductive toxicity:Based on the sequencing results of piRNAs from mouse testis tissues,differential piRNAs were screened and validated by qRT-PCR in vivo.The key piRNAs were identified at the molecular level of GC-2 spd treated with differential piRNAs overexpression,combined with the mRNA sequencing of GC-2 spd cells treated with 1,2-DCE and overexpression of the key piRNAs,and the molecular regulatory interactions,we explored the molecular regulatory mechanism of 1,2-DCE inducing GC-2 spd cell death through piRNA.Results(1)1,2-DCE induces GC-2 spd and mouse testicular tissue pyroptosis:The decrease in GC-2 spd cells activity induced by 1,2-DCE exposure in vitro was rescued by the pyroptosis inhibitors VX-765 and Disulfriam,and pan-Caspase inhibitor Z-VAD-FMK.1,2-DCE caused upregulation of mRNA and protein levels of pyroptosis-related molecules(NLPR3,GSDMD,CASP-1(CASP-1),IL-1β,ASC,IL-18).Pores appeared on the cell membrane surface,accompanied by lactate dehydrogenase(LDH)leakage and an increased proportion of double positive cells for pyroptosis in a 1,2-DCE dose-dependent manner.Exposure to 1,2-DCE in vivo caused an increase in the levels of pyroptosis-related proteins(NLRP3,GSDMD,N-GSDMD,CASP-1,Cleaved-CASP-1,IL-1β,Cleaved-IL-1β,ASC,IL-18)in mouse testis tissue.(2)1,2-DCE induces GC-2 spd cells pyroptosis by upregulating piR-mmu-1019957:1,2-DCE upregulates piR-mmu-1019957 both in vitro and in vivo,and the high expression of piR-mmu-1019957 promotes pyroptosis-related molecules(NLPR3,GSDMD,CASP-1,IL-1β,ASC),piR-mmu-1019957 inhibitor had the opposite effect.Inhibitior of piR-mmu-1019957 restrained the 1,2-DCE-induced increases of levels of pyroptosis-related molecules in mRNA and protein,the proportion of double positive cells for pyroptosis and LDH leakage.(3)PiR-mmu-1019957 promotes 1,2-DCE-induced pyroptosis in GC-2 spd cells by up-regulating IRF7:The RIG-Ⅰ-like cell signaling pathway was activated in the GC-2 spd cells treated with 1,2-DCE(30 mM)and piR-mmu-1019957 overexpression group,among which Irf7 expression was the strongest in both groups(P<0.05).The mRNA and protein levels of IRF7 were up-regulated by 1,2-DCE in vitro and in vivo,and the level of IRF7 was positively regulated by piR-mmu-1019957.Overexpression of Irf7 increased the levels of pyroptosis-related proteins(NLRP3,GSDMD,N-GSDMD,CASP-1,Cleaved-CASP-1,IL-1β,Cleaved-IL-1β,ASC,IL-18)and LDH release,while Irf7 knockdown had the opposite effect.Knockdown of Irf7 inhibited the 1,2-DCE-induced increases of pyroptosis-related protein levels,the proportion of double positive cells for pyroptosis and the level of LDH released.Overexpression of piR-mmu-1019957 and knockdown of Irf7,pyroptosis-related proteins,the proportion of double positive cells for pyroptosis and the level of LDH released had no significant differences compared with the control group.Conclusions(1)1,2-DCE induce pyroptosis of GC-2 spd cells and mouse testis tissue in vitro and in vivo.(2)1,2-DCE could upregulate the level of piR-mmu-1019957 in vitro and in vivo,and the high expression of piR-mmu-1019957 could induce pyroptosis in GC-2 spd cells.(3)1,2-DCE exposure promotes IRF7-mediated activation of RIG-Ⅰ-like cell signaling pathway through piR-mmu-1019957 to induce pyroptosis in GC-2 spd cells and cause reproductive toxicity.