The Mechanism On Apoptosis Of Rat’s Leydig Cells Induced By Nickel Sulfate In Vitro

Objective To investigate the changes of ultrastructure, the protein expression of Bax and Bcl-2, and the oxidative stress levels of rat’s Leydig cells to explore the mechanism of apoptosis of Leydig cells induced by nickel sulfate in vitro.Methods (1) Healthy male Wistar rats were selected, and its testis were removed to isolate and purify the testicular Leydig cells by using collagenase digestion, the density gradient centrifugation with percoll separation medium and adherent culture to obtain the purification testicular Leydig cells in vitro.(2) The Leydig cells were collected to prepare the ultrathin sections after the cells of logarithmic growth phase treated by nickel sulfate (NiSO4) at doses of0,62.5,125,250,500and1000μmol/L at exposure time of6h, and then ultrastructure of Leydig cells were observed with transmission electron microscopy.(3) The Leydig cells of logarithmic growth phase administrated by NiSO4at concentration of250,500and1000μmol/L and the control group treated with culture solution. After exposure6,12and24h, the Leydig cells were collected to extract the total protein respectively, afterwards the protein expression levels of Bax and Bcl-2detected with Western blot technique.(4) The Leydig cells of logarithmic growth phase were treated by NiSO4at the same concentration and times, the cells were collected to prepare the cell lysis products through ultrasonic treatment and centrifugation, then the activities of catalase (CAT) and superoxide dismutase (SOD), the inhibiting capacity of hydroxyl free radical (·OH) and the content of malonyl dialdehyde (MDA) were measured with Microplate reader in spectrophotometry assay.Results (1) Transmission electron microscopy results showed that the ultrastructure changes of testicular Leydig cells included a few mild swelling of mitochondria and no significant change of endoplasmic reticulum in62.5μmol/L NiSO4group, mild swelling of mitochondria and no obvious change of endoplasmic reticulum expansion in125μmol/L NiSO4group, swelling of mitochondria and some expansion of endoplasmic reticulum in250μmol/L NiSO4group, mitochondria swelling obviously and vacuolization but degranulation of rough endoplasmic reticulum in500μmol/L NiSO4group, severe mitochodrial swelling and the significant expansion of endoplasmic reticulum in1000μmol/L NiSO4group.(2) Compared with control group, the level of protein expression of Bax upregulated, but the protein expression of Bcl-2decreased in NiSO4exposure concentration and exposure times (P<0.05).(3) The activities of CAT and SOD inhibited, the content of MDA and the inhibiting capacity of·OH decreased by nickel sulfate. Compared with control group at same exposure time in different concentration of nickel sulfate groups, the activity of CAT inhibited by nickel sulfate at dose of1000μmol/L and the inhibiting capacity of·OH decreased at dose of500μmol/L at the same exposure time of6h (P<0.01). The activity of CAT inhibited by NiSO4at dose of500μmol/L (P<0.01), and the activity of SOD and the inhibiting capacity of·OH decreased in NiSO4250,500and1000μmol/L groups at the same exposure time of12h (P<0.01). The activities of CAT, SOD and the inhibiting capacity of·OH decreased and the MDA content increased in all dosages of nickel sulfate at the same exposure time of24h (P<0.05or P<0.01). Compared with Oh group in same exposure concentration of nickel sulfate at different exposure time groups, the activity of CAT and MDA content decreased at exposure24h group, the activity of SOD and the inhibiting capacity of·OH decreased at exposure12h group in NiSO4250μmol/L (P<0.01). The inhibiting capacity of-OH decreased at exposure6,12and24h groups, and the activities of SOD and CAT decreased at exposure12and24h groups and the MDA content increased at exposure24h group in NiSO4500μmol/L respectively (P<0.01). The activity of CAT and the inhibiting capacity of OH decreased at exposure6,12and24h groups, and the activity of SOD decreased at exposure12and24h groups and the MDA content increased at exposure time24h group in NiSO41000μmol/L group (P<0.01).Conclusion The results showed that the abnormal changes of ultrastructure, protein expression of Bax and Bcl-2, and oxidative stress levels could be related to the apoptosis of rat’s Leydig cells induced by nickel sulfate.