Effect Of Oxidative Stresss Induced Via Acrylonitrile On NF-κB Signaling Pathway In Male SD Rats Testis

Objective: To investigate the effect of oxidative stress induced via acrylonitrile(ACN) on NF-κB signaling pathway, as well as the regulation of apoptosis related gene after NF-κB activation, which may provide basis for the further reproductive toxicology mechanism.Methods: A total of 50 specific pathogen-free(SPF) adult male Sprague Dawley rats were divided into five groups randomly, each containing 10 Rats. Rats were fed in the Laboratory Animal Center in SPF Level of Gansu University of Chinese Medicine, which were provided water and chow ad libitum. After a week, they were treated with 0、5、25、50 mg/kg ACN or 50 mg/kg ACN+300 mg/kg N-acetylcysteine(NAC) respectively by gavaged for 13 weeks, The NAC gavaged in 30 min prior to gavage with ACN. Body weight was measured every four days. All rats were anesthetized with diethyl ether before collected blood from the heart and then killed by cervical dislocation. Then testis and epididymis were excised and weighted. Testis histology examination were executed. The activity of testicular superoxide dismutase(SOD), glutathione peroxidase(GSH-Px), glutathione(GSH) and contents of malondialdehyde(MDA) were determined by visible spectrophotometry. The activation of testicular nuclear factor κB(p65) and nuclear translocation was detected by immunofluorescence. The testicular mRNA level of Bax, Bcl-2 were determined by Real-Time PCR. The testicular protein level of p65, IKK, IκB, p-IκB, Bax, Bcl-2 were detected by Western Blot.Results: Our data showed that there was no significant difference in the weight of the body between treatment groups before and after treatment, the weight coefficient of testis significant decreased in medium and high concentration of ACN groups compared with control. Light microscopy showed that after treatment with ACN, the rats testis seminiferous tubules arranged sparse, low-intensive, smaller diameter, spermatogenic cells arranged in irregular, a small number of quantity, leydig sparse with few cells, the cell outline is not clear. The ratio of GSH/GSSG and the activity of GSH-Px significant decreased in low and high concentration of ACN group. The contents of MDA significant increased in medium and high concentration of ACN group. After pretreatment with NAC, the contents of MDA significant decreased in ACN+NAC group compared with high concentration of ACN group, the ratio of GSH/GSSG significant increased in ACN+NAC group compared with high concentration of ACN group. Immunofluorescence showed that the protein expression level of NF-κB(p65)increased and more p65 were translocated to the nucleus in high concentration of ACN group, which indicate that NF-κB was activated, the activation of NF-κB was significant inhibited after pretreatment with NAC. Western Blot showed that the protein expression level of IKK-α/β, p65, p-IκB significant increased and IκB significant decreased in high concentration of ACN group compared with control, while the protein expression level of IKK-α/β, p65, p-IκB significant decreased and IκB significant increased in ACN+NAC group compared with high concentration of ACN group. RT-PCR and Western Blot showed that the mRNA and protein expression level of Bax significant increased and Bcl-2 no significant changed in high concentration of ACN group compared with control, while the mRNA and protein expression level of Bax significant decreased in ACN+NAC group compared with high concentration of ACN group.Conclusion: ACN subchronic exposure may induce testicular pathological lesion, which may be related to oxidative stress. Oxidative stress activated the NF-κB signaling pathway, then upregulated the pro-apoptotic protein expression of Bax, this may be one of the mechanisms of testicular cells apoptosis in rats.