作为功能性脂肪酸,α-亚麻酸(ALA)是促进动物睾酮生物合成所必需的。本研究调查了ALA对公鸡原代睾丸间质细胞睾酮生物合成的影响及其信号通路的可能机制。
方法:用ALA(0、20、40或80μmol/L)或p38抑制剂(50μmol/L)预处理原代雄性睾丸间质细胞,c-JunNH2末端激酶(JNK)抑制剂(20μmol/L),或ALA治疗前的细胞外信号调节激酶(ERK)抑制剂(20μmol/L)。使用酶联免疫吸附测定(ELISA)检测条件培养基中的睾酮含量。使用实时荧光定量PCR(qRT-PCR)检测类固醇生成酶和JNK-SF-1信号通路因子的表达。
结果:补充ALA可显著增加培养基中睾酮的分泌(P<0.05),优化剂量为40μmol/L与对照组相比,类固醇生成急性调节蛋白(StAR),胆固醇侧链裂解酶(P450scc),在40μmol/LALA组中,3β-羟基类固醇脱氢酶(3β-HSD)mRNA表达明显升高(P<0.05);在40μmol/LALA组中,17-羟化酶/c17-20裂解酶(P450c17)和p38mRNA表达无明显差异;在40μmol/LALA组中,ERK和JNKmRNA表达明显上调(P<0.05)。在抑制剂组中,睾酮水平显著下调(P<0.05)。与40μmol/LALA组相比,StAR,P450scc,和P450c17mRNA表达显著降低(P<0.05),p38抑制剂组3β-HSDmRNA表达无变化;StAR,P450scc,3β-HSDmRNA表达水平明显下降(P<0.05),ERK抑制剂组P450c17mRNA表达无变化;StAR,P450scc,3β-HSD,JNK抑制剂组P450c17mRNA表达明显下降(P<0.05)。此外,当细胞与JNK和ERK抑制剂预孵育时,ALA诱导的类固醇生成因子1(SF-1)基因表达水平的增加被逆转.JNK抑制剂组明显低于对照组(P<0.05)。
结论:ALA可能通过激活JNK-SF-1信号通路上调StAR来促进睾酮生物合成,P450scc,3β-HSD,和P450c17在原代公鸡睾丸间质细胞中的表达。
As a functional fatty acid, α-linolenic acid (ALA) is essential in promoting animal testosterone biosynthesis. This study investigated the effects of ALA on testosterone biosynthesis and the possible mechanism underlying the signaling pathway in primary Leydig cells of the rooster.
METHODS: Primary rooster Leydig cells were treated with ALA (0, 20, 40, or 80 μmol/L) or pretreated with a p38 inhibitor (50 μmol/L), a c-Jun NH2-terminal kinase (JNK) inhibitor (20 μmol/L), or an extracellular signal-regulated kinase (ERK) inhibitor (20 μmol/L) before ALA treatment. Testosterone content in the conditioned culture medium was detected using an enzyme-linked immunosorbent assay (ELISA). The expression of steroidogenic enzymes and JNK-SF-1 signaling pathway factors was detected using real-time fluorescence quantitative PCR (qRT-PCR).
RESULTS: Supplementation with ALA significantly increased testosterone secretion within culture media (P < 0.05), and the optimized dose was 40 μmol/L. Compared with the control group, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA expression significantly increased (P < 0.05) in the 40 μmol/L ALA group; 17-hydroxylase/c17-20 lyase (P450c17) and p38 mRNA expressions were not significantly different in the 40 μmol/L ALA group; ERK and JNK mRNA expressions were significantly upregulated (P < 0.05) in 40 μmol/L ALA group. In the inhibitor group, testosterone levels were significantly downregulated (P < 0.05). Compared with the 40 μmol/L ALA group, StAR, P450scc, and P450c17 mRNA expressions were significantly decreased (P < 0.05), and 3β-HSD mRNA expression in the p38 inhibitor group did not change; StAR, P450scc, and 3β-HSD mRNA expressions were significantly decreased (P < 0.05), and P450c17 mRNA expression in ERK inhibitor group did not change; StAR, P450scc, 3β-HSD, and P450c17 mRNA expressions were significantly decreased (P < 0.05) in JNK inhibitor group. Additionally, the increased steroidogenic factor 1 (SF-1) gene expression levels induced by ALA were reversed when the cells were pre-incubated with JNK and ERK inhibitors. The levels in the JNK inhibitor group were significantly lower than those in the control group (P < 0.05).
CONCLUSIONS: ALA may promote testosterone biosynthesis by activating the JNK-SF-1 signaling pathway to upregulate StAR, P450scc, 3β-HSD, and P450c17 expression in primary rooster Leydig cells.