Aging and interaction of natriuretic factors on renal and vascular sodium pump

细胞色素450 CYP 11 A1（即类固醇生成的经典途径）对胆固醇的侧链切割与MBG的生物合成无关，而内源性哇巴因则是如此。先前对蟾蜍的研究（已知蟾蜍能产生蟾蜍二烯内酯）表明，给药标记胆固醇和胆烷酸，而不是胆固醇侧链裂解产物孕酮，会导致放射性标记掺入蟾蜍二烯内酯分子。由于胆汁酸的肝外合成最近已被描述，我们假设，在胎盘中，其中检测到高水平的MBG先兆子痫，蟾蜍二烯内酯可以从胆汁酸合成。在人滋养层JEG-3细胞中，我们研究了两个基因的转录后沉默的影响，编码甾醇27-水解酶（CYP 27 A1），一个起始酶胆固醇合成胆烷酸，和CYP 11 A1，一种酶，控制胆固醇侧裂解成双烯醇酮，生产MBG和孕酮。在细胞培养基中测量类固醇的水平。在JEG-3细胞中，与未转染和模拟转染的细胞相比，CYP 11 A1的沉默导致蛋白质量减少90%（蛋白质印迹），mRNA减少80%（qPCR），孕酮的产生减少77%，但不影响MBG的产生。 CYP 27 A1的沉默，伴随着CYP 27 A1蛋白的75%的减少，和mRNA水平的65%的减少，相反，不影响孕酮的生产，但抑制MBG的生产80%相比，在非转染和模拟转染的细胞。因此，在人滋养层细胞中，蟾蜍二烯类强心类固醇MBG是由胆固醇通过胆汁酸途径合成的，这代表了一种新的激素生物合成途径。

The side-chain cleavage of cholesterol by cytochrome 450 CYP11A1 (ie, classical pathway of steroidogenesis) is not implicated in the biosynthesis of MBG, as it is for endogenous ouabain. Previous studies in toads, known to produce bufadienolides, demonstrated that administration of labeled cholesterol and cholanic acid, but not of the product of cholesterol side-chain cleavage, progesterone, resulted in the incorporation of a radioactive label into bufadienolide molecules. Because the extra-hepatic synthesis of bile acids has recently been described, we hypothesized that in placenta, in which high levels of MBG are detected in preeclampsia, bufadienolides could be synthesized from bile acids. In human trophoblast JEG-3 cells, we examined the impact of post-transcriptional silencing of two genes, encoding sterol 27-hydrolase (CYP27A1), an initiating enzyme for synthesis of cholanic acid from cholesterol, and CYP11A1, an enzyme which controls side-cleavage of cholesterol into pregnenolone, on production of MBG and progesterone. Levels of steroids were measured in cell culture media. In JEG-3 cells, silencing of CYP11A1 resulted in 90% decrease of the protein amount (Western blot), in 80% decrease of mRNA (qPCR), and reduced production of progesterone by 77%, but did not affect production of MBG as compared to non-transfected and mock-transfected cells. Silencing of CYP27A1, accompanied by 75% reduction in CYP27A1 protein, and by 65% reduction in mRNA level, conversely, did not affect production of progesterone, but suppressed production of MBG by 80% vs. that in non-transfected and mock-transfected cells. Thus, in human trophoblast cells, bufadienolide cardiotonic steroid MBG is synthesized from cholesterol via bile acid pathway which represents a novel pathway of hormone biosynthesis.