双酚A通过上调Apoa1基因的表达抑制TM3细胞睾酮合成

doi:10.11843/j.issn.0366-6964.2024.08.024

摘要/Abstract

摘要：

旨在从脂质代谢的角度探讨载脂蛋白A1(apolipoprotein A1，Apoa1)是否介导了双酚A(bisphenol A，BPA)暴露所致小鼠睾丸间质细胞株(TM3)睾酮合成的降低。将TM3细胞随机分为不同浓度的BPA暴露剂量(0、5、10、20、40、60、80 μmol·L^-1)组，0 μmol·L^-1 BPA为对照组(CON)。给予相应剂量处理24 h后，运用CCK-8法检测TM3细胞活力，确定BPA最适染毒剂量；通过ELISA检测TM3细胞培养上清液睾酮(testosterone，T)含量；利用RT-qPCR检测TM3细胞脂质代谢相关基因Apoa1、Apoa2(apolipoprotein A2)、Apoc3(apolipoprotein C3)的mRNA表达水平；运用Western blot和免疫荧光方法检测APOA1蛋白表达水平；采用油红O染色观察细胞内脂滴累积情况。结果表明，20 μmol·L^-1 BPA处理24 h对TM3细胞活力无显著影响，40 μmol·L^-1 BPA处理24 h后，TM3细胞活力受到极显著抑制(P < 0.01)；此外，20 μmol·L^-1 BPA处理TM3细胞24 h后，培养上清液中睾酮含量极显著低于对照组(P < 0.01)，Apoa1基因的mRNA表达水平及蛋白表达量极显著升高(P < 0.001)，但Apoa2和Apoc3基因的mRNA表达水平无显著变化；与对照组相比，20 μmol·L^-1 BPA处理24 h，TM3细胞的脂滴累积量极显著降低(P < 0.000 1)。综上，BPA可通过上调Apoa1基因的表达水平，增强胆固醇逆向转运(reverse cholesterol transport, RCT)，引起TM3细胞内的脂滴含量减少，导致TM3细胞的睾酮合成分泌降低。

关键词: 双酚A(BPA), 载脂蛋白A1(Apoa1), 小鼠睾丸间质细胞(TM3), 睾酮(T)

Abstract:

The aim of the study was to investigate whether apolipoprotein A1 (apolipoprotein A1, Apoa1) mediates the reduction of testosterone synthesis in mouse Leydig cells line (TM3) induced by bisphenol A (BPA) exposure from the perspective of lipid metabolism. TM3 cells were randomly divided into 7 groups with different concentrations (0, 5, 10, 20, 40, 60, and 80 μmol·L^-1), and 0 μmol·L^-1 BPA was the control group (CON); After 24 h of treatment with the different concentrations, cell viability was detected using theby CCK-8 method to determine the optimal dose of BPA. Testosterone (testosterone, T) synthesiscontent in TM3 cell supernatant was detected by ELISA; mRNA expression levels of lipid metabolism-related genes Apoa1, Apoa2 (apolipoprotein A2) and Apoc3 (apolipoprotein C3) genes were measured in TM3 cells usingby RT-qPCR. APOA1 protein expression level was detected usingby Western blot and immunofluorescence method. Intracellular lipid droplet accumulation was observed usingby oil red O staining. The results showed that 20 μmol·L^-1 BPA treatment for 24 h had no significant effect on the viability of TM3 cells; However, an extremely significant inhibition of TM3 cell viability was observed followingin treatment with 40 μmol·L^-1 BPA for 24 h (P < 0.01); In addition, after 20 μmol·L^-1 BPA treatment on TM3 cells for 24 h, the testosterone content in the culture supernatant was extremely significantly lower than that in the CON group (P < 0.01), the mRNA expression level and protein expression of Apoa1 gene were extremely significantly elevated (P < 0.001), but there was no significant change in the mRNA expression level of Apoa2 andor Apoc3 genes; The accumulation of lipid droplets in TM3 cells was extremely significantly reduced by 20 μmol·L^-1 BPA treatment for 24 h compared with the CON group (P < 0.000 1). In conclusion, BPA can reduce the lipid droplets accumulation in TM3 cells by up-regulating Apoa1 expression levels, enhancing reverse cholesterol transport (Reverse cholesterol transport, RCT), leading to a decrease in testosterone synthesis in TM3 cells.

Key words: bisphenol A(BPA), apolipoprotein A1(Apoa1), mouse Leydig cells(TM3), testosterone(T)

中图分类号:

S814

赵彤, 杨文哲, 潘飞龙, 赵树臣, 刘克祥, 吕占军, 赵立佳. 双酚A通过上调Apoa1基因的表达抑制TM3细胞睾酮合成[J]. 畜牧兽医学报, 2024, 55(8): 3516-3525.

Tong ZHAO, Wenzhe YANG, Feilong PAN, Shuchen ZHAO, Kexiang LIU, Zhanjun LÜ, Lijia ZHAO. Bisphenol A Inhibits Testosterone Synthesis in TM3 Cells by Upregulating Apoa1 Gene Expression[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(8): 3516-3525.

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引物名称 Primer name	基因编号 Accession No.	引物序列(5′→3′) Primer sequence	产物长度/bp Product length
Apoa1	NM_009692.4	F: CAAAGACAGCGGCAGAGACT R: AGTTTTCCAGGAGATTCAGGTTCA	83
Apoa2	NM_013474.2	F: GAATCGCAGCACTGTTCCTA R: GTCTCTTAACCAAAGCTCCTTCC	107
Apoc3	NM_001289755.1	F: GAACAAGCCTCCAAGACGGT R: GTTGGTTGGTCCTCAGGGTT	173
Gapdh	NM_001289726	F: GCCTCCTCCAATTCAACCCT R: CCCAATACGGCCAAATCCGT	145

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