Effects Of Dhea On Biological Characteristics And Its Biotransformation In TM-3Cell

Steroid hormones have an important role in animal growth and development. Cholesterol could convert to androgens, estrogen and progesterone by steroidogenic enzymes in peripheral target tissues. Dehydroepiandrosterone (DHEA) as the most important intermediate metabolites of steroid hormones has numerous biological effects, for example anti-aging, regulate lipid metabolism, enhance immunity and promote animal health, and the biological functions of DHEA major attributed to its intermediate/final metabolites. Leydig cell is the main cell to synthesis and secret testosterone in vivo. Some researches indicated serum androgen significantly increased by exogenous DHEA treatment, however, the effect of DHEA on the biological characteristics and its biotransformation in Leydig cells is not yet reported. So, a study was carried out for an investigation of the influences of DHEA on cell cycle and ultrastructure and functions in TM-3cell, and biotransformation of DHEA and dynamic change, relationships and variation of its intermediate and final metabolites using LC-MS, and its effect on steroidogenic enzymes in TM-3cell. The purpose of this study is to enrich the theory of steroids metabolism in vivo and provide theoretical basis for animal health and human-related disease prevention.1Influences of DHEA on Cell cycle in TM-3cellIn this study main explore the effect of DHEA on cell cycle and mRNA transcriptional level of cycle factors in TM-3cell. The cell was treated with medium included the final concentration Oμmol/L,1μmol/L,50μmol/L and100μmol/L DHEA (dissolved in DMSO). Cells were collected after6h,12h,24h and48h administrated with DHEA respectively. The effect of DHEA on growth of TM-3cell was observated using inverted phase contrast microscope; cell cycle was measured by flow cytometer; the mRNA transcriptional level of cyclin dependent kinase2(CDK2) genes and cyclin A, cyclin B genes in TM-3cell were determined after treatment for24h using the real time RT-PCR; determined activity of the glucise-6-phosphate dehydrogenase (G6PD) by assay kit. The data shown that, compared with control group, a significantly growth inhibition in TM-3cell after administration50μ.mol/L and100μmol/L DHEA for24h; treatment with50μmol/L and100μmol/L DHEA, the cell ratio of S and G2/M phase increased and G1phase decreased after treatment for12h, however, significantly difference occurred only at48h; as compared with the control group (P<0.01), the mRNA level of cyclin A and cyclin B significantly decreased in100μmol/L DHEA group (P<0.05); CDK2mRNA transcriptional level had no significant difference however the tendency of decrease was found; Meanwhile, the activity of G6PD significantly reduced (P<0.05) after50μmol/L and100μmol/L DHEA administration. The above consequences manifested that, DHEA could inhibit TM-3cell growth through significantly reduced the activity of G6PD and arrested TM-3cell cycle in S phase and G2/M phase attributed to down-regulated mRNA expression level of cyclinA, cyclinB and CDK2.2Effects of DHEA on Ultrastructure and Functions of Mitochondria in TM-3cellIn this study main explore the effect of DHEA on ultrastructure and function of mitochondria in TM-3cell. The cell was incubated with medium included the final concentration Oμmol/L,1μmol/L,50μmol/L and100μmol/L DHEA (dissolved in DMSO) for48h. The shape and quantity of mitochondria were investigated treatment with DHEA by TEM, and determined the impacts of DHEA on mitochondrial membrane potentials (ΔΨm) using flow cytometer, and measured activity of SDH by assay kit. The result showed that no significant differences in both shape and quantity of mitochondria in TM-3cell treated with1μmol/L,50μmol/L and100μmol/L DHEA (P>0.05); compared to the control group, TM-3cell treated with DHEA resulted in a lower ΔΨm and a significantly difference at100μmol/L DHEA group (P<0.05), the activity of SDH had no significant difference in1μmol/L and50μmol/L DHEA administration, and a significantly increased at100μmol/L DHEA group (P<0.05). These results indicated that the administration of exogenous DHEA have no influence on shape and quantity of mitochondria; however,100μmol/L DHEA could declined ΔΨm and increased mitochondrial membrane permeability, meanwhile, elevation of cellular oxidative metabolism to meet cellular energy need through increased activity of SDH.3Biotransformation of DHEA and its effects on expression of steroidogenic enzymes in TM-3cellThe intent of this study was to investigate the biotransformation of DHEA and its effect of steroidogenic enzymes expression. The final concentration of100μmol/L DHEA medium were incubated TM-3cells, and an equal volume of DMSO (0.1%) was administered to control group, cell and medium were gathered after0,3,12,24and48h treatment with DHEA. The concentration of DHEA, testosterone, estradiol, estrone, progesterone, cortisol, corticosterone and17a-hydroxyprogesterone in cell lysate were measured by LC-MS. The expression of steroidogenic enzymes were determined using the real time RT-PCR and western blot. The present study demonstrated that, after0,3,12,24and48h treatment with100μmol/L DHEA, the concentration of DHEA from34.12ng/mL (0h) to17.90ng/mL (48h) in TM-3cell lysate, the concentration of testosterone increased from0.26ng/mL to5.41ng/mL with DHEA treatment extending at3h-48h, and significant negative (P<0.05) between DHEA and testosterone. The concentration of estradiol increased from15.26ng/mL (0h) to167.16ng/mL (48h), and significant negative (P<0.05) between DHEA and estradiol. The concentration of estrone and cortisol decreased with DHEA treatment, and significant positive correlation compared with DHEA(r=0.958, P<0.01; r=0.752, P<0.01). Although progesterone not detected within0-3h after DHEA treatment, but concentration of progesterone significantly increased in cell lysates in12-48h. The corticosterone and17a-hydroxyprogesterone not detected in cell lysates. Compared with control group, the concentration of total cholesterol significantly decreased after treatment with DHEA for12h-48h (P<0.05). The expression analysis of steroidogenic enzymes shown that, after100μmol/L DHEA administration, compared with control group, mRNA expression level of StAR significant decreased at3h (P<0.05), then gradually elevated and significant increased (P<0.01) at24h and48h. The mRNA level of P450scc significant increased compared with control group at3h (P<0.05), then decreased and returned to normal levels. The mRNA transcriptional level of CPY17a significant increased at3h (P<0.05), then decreased at12h, however, the expression of CPY17a significant increased compared with control group in24h-48h again. The protein expression of3β-HSD showed significant increased within12-48h after DHEA administration, compared with respective control group, the expression of3β-HSD had a notable increased in24-48h. The protein level of17β-HSD merely had a significantly elevated at48h compared with respective control group (P<0.05), and there no difference of protein level of17β-HSD in treated group (P>0.05). The protein expression of aromatase in TM-3cell was decreased after DHEA administrated, compared with respective control group, protein expression of aromatase significant after decreased in24-48h (P<0.05). These results indicated that the significant growth-decline correlation between DHEA and its intermediates and final metabolites, the concentration of most of its metabolic intermediates decreased with estradiol and testosterone increased, and the expression of steroidogenic enzymes shown a timing-manner, moreover, consistent with the biotransformation of DHEA in TM-3cell.