| DHEA (Dehydroepiandrosterone, DHEA) is the major steroid hormones produceed by the adrenal zona reticularis. One of the main characteristics is its aged-dependent pattern secretion, and this reduction has been shown to be associated with physical health. As a precursors of active steroid hormones, this steroid has generated major interest as an "anti-aging" hormone and is widely available as a dietary supplement H2O2 as a major form of active oxygen in the body, which can easily cause damage to cells through the cell membrane.In male,Leydig cells are the main target tuisse of synthesis and secretion testosterone. Studies had shown that exogenous DHEA has a protective affect that against H2O2-induced cell injury however the exact mechanism is not full understand. Thus, present study was carried to investigated the effect of exogenous DHEA on H2O2-induced primary rat Leydig cell antioxidant function, nuclear DNA damage and apoptosis-related factors which was based on establishment a reliable oxidative damage model in primary rat Leydig cells by H2O2-induced. According these results, we further study the repair effect of DHEA by dectected the apoptotic pathway associated factor mRNA and protein expression levels in the oxidative damage model of Leydig cells induced by H2O2, and further explore the role of PI3K/Akt signaling pathway in biology fuction. These resutls would provide the theoretical basis and experimental basis for further elucidate the anti-aging effects of DHEA.1 Establishment of a primary rat Leydig cells oxidative damage model induced by H2O2This study aimed to establish an oxidative damage model which was induced by hydrogen peroxide (H2O2) in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation method and the purity of Leydig cells was also determined by 3β-HSD staining method. The viability of Leydig cells was determined by MTT. The apoptosis, mitochondrial membrane potential, ROS and O2- content were determined by flow cytomertry methods. The content of ·OH, MDA and activity of SOD, GSH-Px, CAT, POD were detected by spectrophotometry. The results show that most of the cells present the fluorescence after immunofluorescence by 3β-HSD. The viability of primary rat Leydig cells had a significant decrease at 300-1000 μmol/L H2O2 concentration after 8 hours treated (P<0.01). The apoptosis ration was significantly increased after 300 μmol/L H2O2 treated when compared to control group (.P<0.05). Compared with control group, H2O2 treatment could enhance the content of eactive xygen pecies (ROS) and hydroxyl radical (·OH) in primary Leydig cells, while the mitochondrial membrane potential was significantly decreased (P<0.05). No differently change was observed on the activity of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) in primary rat Leydig cells after H2O2 treatment (P>0.05), while the content of malondialdehyde was markedly increased (P<0.01) when compared with control group. These results indicated that oxidative damage of Leydig cells can be induced by 300 μmol/L H2O2 after 8 hours.Thus, an oxidative damage model which was induced by H2O2 has been established.2 Protect effect of DHEA on the oxidative damage in Leydig cells induced by H2O2The aim of this study was investigated the protect effect of DHEA on the oxidative damage in primary Leydig cells.The oxidative damage of Primary rat Leydig cells were treated with 0 μmol/L,1 μmol/L,10 μmol/L,50 μmol/L,100 μmol/L DHEA for 24 h and then H2O2 containing a final concentration of 300 μmol/L of culture medium induced by primary rat Leydig cells 8 h, after the cells were collected and tested. Cell viability was measured by MTT assay, testosterone content was detected by radioimmunoassay, cell apoptosis, and mitochondrial membrane potential, ROS and O2- content were detected by flow cytometrythe content of ·OH, MDA and the activity of enzymes SOD, CAT, POD, GSH-Px were detected by colorimetry, single-cell gel electrophoresis technique to detect nuclear DNA damage, the mRNA expression levels of OGG1, Bcl-2, Bax, caspase-9, caspase-3, caspase-7were detected by Real-time PCR method. The results showed that DHEA treatment could significantly improve cell viability (P<0.05), and also restore between primary rat Leydig cells secrete testosterone biological function (P<0.01) when compared with the damage control group. DHEA treatment could significantly reduced intracellular ROS levels (P<0.05), MDA content (P<0.05) and ·OH content (P<0.01) than that of damage control group. The activity of POD was significantly increased in the DHEA-treated groups that damage control group (P<0.05),while it couldsignificantly decreased the activity of GSH-Px (P<0.05).No significantly difference were observed oth the O2- content and the activity of SOD,CAT in the DHEA-treated groups that damage control group (P>0.05). These results indicated that DHEA could against the decreased of cell viability and the content of testosterone which was induced by H2O2 in primary rat Leydig cells. The protective effect of DHEA on the oxidative damage by H2O2-induced in primary rat Leydig cells were via enhanced the POD activity, reduced the ·OH content and decreased the MDA content which was produced by ·OH attack cell membrane, and ultimately reduce ROS levels in the Leydig cells.Compared with the damage control group, DHEA-treatment could significantly reduce the rate of apoptosis (P<0.05) and significantly reduce the nuclear DNA Olive tail moment (P<0.01). DHEA treatment could significantly increased OGG1 mRNA expression levels (P<0.05)than that of damage control group., while it could could significantly reduce the expression levels of Bax, caspase-9, caspase-3 and caspase-7 (P<0.05) in primary rat Leydig cells. These results suggest that DHEA has a protective effect on DNA damage which was induced by H2O2 in primary rat Leydig cell, and this action was mainly by enhancing the expression levels of OGG1. Also, our results indicated that DHEA has a protective effect on the cell apoptosis which was induced by H2O2 in primary rat Leydig cell, and it mainly via reduceing the expression levels of Bax, caspase-9, caspase-3, caspase-7 and the ratio of Bax/Bcl-2.3 Repair effect of DHEA on the oxidative damage in Leydig cells induced by H2O2The aim of this study was investigated the repair effect of DHEA on the oxidative damage in primary Leydig cells.The oxidative damage of Primary rat Leydig cells were treated with H2O2 containing a final concentration of 300 umol/L for 8 h and then 0 μmol/L, 1 μmol/L,10 μmol/L,50μmol/L,100 μmol/L DHEA of culture medium induced by primary rat Leydig cells 24 h, after the cells were collected and tested. Cell viability was measured by MTT assay, testosterone content was detected by radioimmunoassay, cell apoptosis, and mitochondrial membrane potential, ROS and O2- content were detected by flow cytometrythe content of ·OH, MDA and the activity of enzymes SOD, CAT, POD, GSH-Px were detected by colorimetry, single-cell gel electrophoresis technique to detect nuclear DNA damage, the mRNA expression levels of OGG1, PI3K, Akt, Bcl-2, Bax, caspase-9, caspase-3, caspase-7 were detected by Real-time PCR method. The protein levels of PI3K, Akt, Bax and caspase-3 were detected by Western blot.The results showed that DHEA treatment could significantly improve cell viability (P<0.01), and also restore between primary rat Leydig cells secrete testosterone biological function (P<0.01) when compared with the damage control group. DHEA treatment could significantly reduced intracellular ROS levels (P<0.05), MDA content (P<0.05) and ·OH content (P<0.01) than that of damage control group. The activity of SOD, CAT, POD were significantly increased in the DHEA-treated groups that damage control group (P<0.05),while it could significantly decreased the activity of GSH-Px (P<0.05). No significantly difference were observed oth the O2- content in the DHEA-treated groups that damage control group (P>0.05). These results indicated that DHEA could against the decreased of cell viability and the content of testosterone which was induced by H2O2 in primary rat Leydig cells. The repair effect of DHEA on the oxidative damage by H2O2-induced in primary rat Leydig cells were via enhanced the SOD, CAT, POD activity, reduced the ·OH content and decreased the MDA content which was produced by ·OH attack cell membrane, and ultimately reduce ROS levels in the Leydig cells.Compared with the damage control group, DHEA-treatment could significantly reduce the rate of apoptosis (P<0.05) and significantly reduce the nuclear DNA Olive tail moment (P<0.01). DHEA treatment could significantly increased the mitochondrial membrane potential. DHEA treatment could significantly increased OGG1, PI3K, Akt and Bcl-2 mRNA expression levels (P<0.05) than that of damage control group, while it could could significantly reduce the expression levels of DNMT1, Bax, caspase-9, caspase-3 and caspase-7 (P<0.05) in primary rat Leydig cells. The protein level of PI3K and p-Akt were significantly increased when compared to the control group (P<0.05), the protein level of Bax and caspase-3 were decreased when compared to the control group (P<0.05), but there was no significant change in protein levels of Akt (P>0.05). After adding the inhibitor LY294002 of PI3K, the effect of DHEA on PI3K, p-Akt, Bax, caspase-3 could be completely reversed. These results suggest that DHEA has a repair effect on DNA damage which was induced by H2O2 in primary rat Leydig cell, and this action was mainly by enhancing the expression levels of OGG1. Also, our results indicated that DHEA has a repair effect on the cell apoptosis which was induced by H2O2 in primary rat Leydig cell, and it mainly via increased the protein expression levels of PI3K and p-Akt, and reduced the protein expression levels of Bax and caspase-3. |
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