[双酚A体外改变大鼠支持细胞的葡萄糖代谢]

Huang Wei, Huang Hong-mei, Wang Hong, Zhao Ji-cun, Li Mian-zhou, Wang Hong-qiang, Wang Xin-sheng, Wang Pei-tao

Zhonghua Nan Ke Xue. 2015 Feb;21(2):119-23.

OBJECTIVE

To observe the influence of different concentrations of bisphenol A (BPA) on glucose metabolism and lactate dehydrogenase (LDH) expression in rat Sertoli cells in vitro and investigate the mechanisms of BPA inducing male infertility.

METHODS

Using two-step enzyme digestion, we isolated Sertoli cells from male Wistar rats and constructed a primary Sertoli cell system, followed by immunohistochemical FasL staining. We randomly divided the Sertoli cells into a control group to be cultured in the serum-free minimal essential medium (MEM) plus dimethyl sulfoxide (DMSO) and three experimental groups to be treated with 100 nmol/L, 10 μmol/L, and 1 mmol/L BPA, respectively, in the MEM plus DMSO. After 48 hours of treatment, we measured the proliferation of the cells by CCK-8 assay, determined the concentrations of metabolites by NMR spectroscopy, and detected the expression of LDH in the Sertoli cells by RT-PCR and Western blot.

RESULTS

The purity of the isolated Sertoli cells was (96.05 ± 1.28)% (n = 10). Compared with the control group, the 100 nmol/L, 10 μmol/L, and 1 mmol/L BPA groups showed no remarkable changes in the proliferation of Sertoli cells ([98 ± 8]%, [96 ± 3]%, and [95 ± 3]%, P >0.05), but the 10 μmol/L and 1 mmol/L of BPA groups exhibited significantly decreased concentrations of intracellular glucose ([3.89 ± 0.07] vs [3.36 ± 0.24] and [3.04 ± 0.21] pmol/cell, P <0.05) and lactate ([0.43 ± 0.06] vs [0.29 ± 0.05] and [0.20 ± 0.03] pmol/cell, P <0.05). The expression of LDH mRNA was decreased with the increased concentration of BPA, while that of LDH protein reduced only in the 1 mmol/L BPA group (P <0.05).

CONCLUSION

High-concentration BPA decreases the expression of LDH and alters glucose metabolism in Sertoli cells, and therefore may reduce the provision of lactate for germ cells and impair spermatogenesis.

目的

观察不同浓度双酚A（BPA）对体外培养的大鼠支持细胞葡萄糖代谢及乳酸脱氢酶（LDH）表达的影响，探讨BPA致男性不育的机制。

方法

采用两步酶消化法从雄性Wistar大鼠分离支持细胞，构建原代支持细胞体系，并行免疫组织化学FasL染色。将支持细胞随机分为对照组，用无血清的最低必需培养基（MEM）加二甲基亚砜（DMSO）培养，3个实验组分别用含100 nmol/L、10 μmol/L和1 mmol/L BPA的MEM加DMSO处理。处理48小时后，采用CCK-8法检测细胞增殖，用核磁共振波谱法测定代谢物浓度，通过RT-PCR和蛋白质印迹法检测支持细胞中LDH的表达。

结果

分离的支持细胞纯度为（96.05±1.28）%（n = 10）。与对照组相比，100 nmol/L、10 μmol/L和1 mmol/L BPA组支持细胞增殖无明显变化（分别为[98±8]%、[96±3]%和[95±3]%，P>0.05），但10 μmol/L和1 mmol/L BPA组细胞内葡萄糖浓度显著降低（分别为[3.89±0.07]对[3.36±0.24]和[3.04±0.21] pmol/细胞，P<0.05），乳酸浓度也显著降低（分别为[0.43±0.06]对[0.29±0.05]和[0.20±0.03] pmol/细胞，P<0.05）。LDH mRNA表达随BPA浓度升高而降低，而LDH蛋白表达仅在1 mmol/L BPA组降低（P<0.05）。