核受体NR4A1是糖脂代谢和类固醇生成的关键因素,而脂滴是黄体细胞脂质代谢的关键动态细胞器。探讨NR4A1对山羊黄体脂滴代谢和孕酮(P4)合成的影响。分离来自中环黄体的黄体细胞,并用细胞孢子酮B处理(CSNB,激动剂)或NR4A1的siRNA。结果表明,低(1μM)和高(50μM)浓度的CSNB促进脂滴积累,而NR4A1敲除降低脂滴含量。CSNB增加,而siNR4A1降低总胆固醇含量;然而,CSNB和siNR4A1没有改变甘油三酯含量。CSNB在mRNA和蛋白质水平增加perilipin的表达,还增加了LDLR,SCARB1,SREBF,和HMGCRmRNA丰度。用siNR4A1治疗显示CSNB的相反结果,除了HMCGR和SREBF2。对于类固醇生成,1μMCSNB增加,但50μMCSNB抑制P4合成,NR4A1敲低也降低了P4水平。进一步的分析表明,1μMCSNB增加了StAR的蛋白质水平,HSD3B,P-HSL,当50μMCSNB降低StAR时,HSD3B,和CYP11A1蛋白水平。此外,50μMCSNB受损的活性线粒体,降低BCL2,并增加DRP1,Caspase3和裂解的Caspase3蛋白水平。siNR4A1始终下调P-HSL/HSL比率和类固醇生成蛋白水平。总之,NR4A1介导的脂滴参与调节山羊黄体细胞中孕酮的合成。
Nuclear receptor NR4A1 is a key factor in glycolipid metabolism and steroidogenesis, while lipid droplets serve as crucial dynamic organelles for lipid metabolism in luteal cells. To investigate the effects of NR4A1 on lipid droplet metabolism and progesterone (P4) synthesis in goat corpus luteum in vitro, luteal cells from the middle-cyclic corpus luteum were isolated and treated with Cytosporone B (CSNB, an agonist) or siRNA of NR4A1. Results showed that both low (1 μM) and high (50 μM) concentrations of CSNB promoted lipid droplet accumulation, while NR4A1 knockdown reduced lipid droplet content. CSNB increased while siNR4A1 decreased total cholesterol content; however, CSNB and siNR4A1 did not change triglyceride content. CSNB increased the expression of perilipins at mRNA and protein levels, also increased LDLR, SCARB1, SREBFs, and HMGCR mRNA abundance. Treatment with siNR4A1 revealed opposite results of CSNB, except for HMCGR and SREBF2. For steroidogenesis, 1 μM CSNB increased, but 50 μM CSNB inhibited P4 synthesis, NR4A1 knockdown also reduced the P4 level. Further analysis demonstrated that 1 μM CSNB increased the protein levels of StAR, HSD3B, and P-HSL, while 50 μM CSNB decreased StAR, HSD3B, and CYP11A1 protein levels. Moreover, 50 μM CSNB impaired active mitochondria, reduced the BCL2, and increased DRP1, Caspase 3, and cleaved-Caspase 3 protein levels. siNR4A1 consistently downregulated the P-HSL/HSL ratio and the steroidogenic protein levels. In conclusion, NR4A1-mediated lipid droplets are involved in the regulation of progesterone synthesis in goat luteal cells.