Potential Mechanisms Of Fas/FasL Regulated By TNF-α In The Cultured Immature Boar Sertoli Cells

Sertoli cells (SCs) play an important role in maintaining immune privilege function of testis,and form the blood-testosterone-barrier (BTB),highly express FasL and secrete immunosuppressive factor (TGF-beta, IL-1 and IGF-Ⅱ, et.). Tumor necrosis factor (TNF) belongs to the members of the family of TNFSFL, including TNF-a and TNF-β,of which. In testis, TNF-α is mainly secreted by SCs. Once binding to its receptors on SCs, TNF-α mediates the secretion of immunomodulatory factor, regulates spermatogenic function of SCs induced by FSH and the opening and closing of BTB. However, it is unclear how TNF-α regulates the expression of Fas/FasL in SCs.The objective of this study is to identify whether TNF-α could affect boar Sertoli cells apoptosis by increasing the expression of Fas/FasL and to explore the relevant mechanisms of TNF-αmediating the expression of Fas/FasL in the cultured immature boar Sertoli cells.Immature boar Sertoli cells were cultured in vitro and divided into the control group and treatment groups. Cell counting kit (CCK-8) was used to detect the effect on SCs viability of TNF-α.The effect on cell cycle progression and early apoptosis cells of TNF-α were assessed by flow cytometry (FCM). The expressions of Fas/FasL, MMP2/MMP9 and TIMP2/TIMP1 protein expression were examined by western blotting and their gene mRNA expression determined by quantitative (real-time) PCR. Finally, the expression of cytokines(IL-1β,IL-6,IGF,TGF-β) were measured by ELISA kits.The Results were as followed:(1)The effect of TNF-α on SCs viability showed a concentration and time dependence.The effect of TNF-α on SCs was most obvious at the concentration of 50 ng/mL, duration for 36h, cell proliferation activity is significantly inhibited and apoptosis of SCs was enhanced at this point (P<0.01).(2) TNF-α (50ng/mL) significantly enhanced the expression of Fas/FasL protein and mRNAlevel (P<0.01).(3)TNF-α(50ng/mL)significantly promoted the expression of MMP-2/MMP-9 protein and mRNA (P<0.05).(4)TNF-α(50ng/mL)significantly reduced the expression of TIMP-1 and TMP-2 mRNA(P<0.01).(5) TNF-α(50ng/mL)significantly increased the secration of IL-1β and IL-6, but decreased the secration of IGF-land TGF-β1(P<0.05).In conclusion, TNF-α reduces SCs prolifetation,but induces SCs apoptosis. The mechanism involved is that TNF-α increases the expression of Fas/FasL by promoting the secretion of inflammatory factors IL-1β and IL-6, enhancing the expression of MMP-2 and MMP-9, increasing the expression of Fas/FasL, but reducing the expression of TIMP-1 and TIMP-2 of SC. In addition, TNF-α also inhibits the secration of IGF-1 and TGF-β1. Therefore, TNF-α induces SCs apoptosis and injury.