Study On The Mechanism Of Orchitis Induced By DBP Exposure During Gestation In Progeny Of Mouse

The plasticizer dibutyl phthalate(DBP)is a kind of environmental chemical substance as environmental endocrine disruptor widely existing in the environment,which can cause male reproductive function problems,such as testicular hypoplasia and spermatogenesis disorder.Testitis is closely related to male reproductive dysfunction.However,whether exposure to DBP in pregnant mice will lead to testitis in offspring is unclear.In this study,we investigated the molecular mechanism of inflammatory response of Sertoli cells(SCs)in testis after DBP exposure.1.Purpose1.1 To observe the effect of DBP exposure on testis tissue structure of offspring mice;1.2 To explore the molecular mechanism of SCs inflammation induced by DBP exposure in pregnant mice.2.Method2.1 To establish the animal model;2.2 At the tissue level,H&E staining was used to observe the testicular tissue;2.3 q RT-PCR,immunofluorescence and immunohistochemistry were used to detect or observe the level of testis inflammation;2.4 At the cellular level,q RT-PCR,ELISA,Western blotting and immune-flurescence were used to detect or observe the inflammatory levels of SCs exposed to different concentrations of MBP(Monobutyl phthalate,metabolite of DBP),NF-?B and other inflammatory pathways as well as their upstream and downstream changes;2.5 Immunofluroscence,Western blotting,ELISA and q RT-PCR were used to detect the inhibitory effect of INF39,a non-cytotoxic NLRP3 specific inhibitor,on MBP induced inflammation;2.6 At the animal and histological level,Western blotting,immunofluroscence,q RT-PCR and immunohistochemistry were used to detect the activation of Pellino2(Peli2)dependent NLRP3 inflammatory bodies and downstream proteins.3.Results3.1 H&E staining showed that the testis tissue structure of the offspring mice was destroyed,the number of seminiferous tubules and spermatogenic cells decreased,and the arrangement of spermatogenic cells was disordered or dropped.3.2 The m RNA expression of TNF-?,IL-6,MCP-1 and CXCL-10 was up-regulated in the testis of the offspring mice.The immunofluorescence co-location of TNF-? and IL-6 showed that the distribution of TNF-? and CXCL-10 were basically coincident.The expression of TNF-? and IL-6increased with the increase of DBP concentration.The number of macrophages in the stroma increased with the increase of DBP concentration in the concentration group,and it was observed that macrophages entered the seminiferous tubules due to the destruction of blood-testis-barrier.3.3 The expression of TNF-?,IL-6,MCP-1 and CXCL-10 was continuously high after MBP was exposed to TM4 cells.The expression of TNF-? and IL-6 was continuously high after MBP was exposed to primary SCs.3.4 One hour after MBP exposure to TM4 cells,Western blotting showed that NF-?B signaling pathway had been activated.With the extension of exposure time,the expression of NLRP3,CD36,caspase-1,IL-1?,Peli2 protein increased significantly.The non-cytotoxic NLRP3 specific inhibitor INF39 had a significant inhibitory effect on MBP induced inflammation.3.5 NF-?B/NLRP3 signaling pathway was activated in DBP induced offspring,and Peli2,a protein dependent on NLRP3,was increased in DBP induced offspring.4.Conclusion4.1 The disorder of testis tissue,the abnormal development of seminiferous tubules,the abnormal development of sperm in the high concentration group,or the absence of sperm production in the offspring mice suggested testis damage.4.2 Inflammatory cell aggregation appeared in testis of offspring mice after DBP exposure,indicating inflammation.4.3 NF-?B signaling pathway was activated within 1 hour after exposure of SCs to MBP,and it was transmitted to the downstream,resulting in the activation of NLRP3 inflammatory bodies,which was involved in the production of testitis induced by DBP.