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

与内源性哇巴因不同，细胞色素450Cyp11a1(即类固醇合成的经典途径)对胆固醇的侧链断裂与MBG的生物合成无关。此前在蟾蜍身上进行的研究表明，服用标记的胆固醇和胆酸，而不是胆固醇侧链裂解的产物黄体酮，会导致放射性标记加入蟾酥二烯内酯分子中。由于胆汁酸的肝外合成是最近被描述的，我们假设在胎盘中，在先兆子痫中检测到高水平的MBG，可以从胆汁酸合成蟾酥二烯内酯。在人滋养层细胞JEG-3中，我们检测了两个基因转录后沉默的影响，这两个基因编码类固醇27-水解酶(CYP27A1)和Cyp11a1，这两个基因编码的是从胆固醇合成胆汁酸的起始酶，Cyp11a1是控制胆固醇侧切成孕烯醇酮的酶，对MBG和孕酮的产生有影响。测定细胞培养上清液中的类固醇水平。在JEG-3细胞中，与未转染组和模型转染组相比，Cyp11a1沉默导致蛋白质含量减少90%(Western印迹)，mRNA减少80%(QPCR)，孕酮产生减少77%，但不影响MBG的产生。与未转染组和模型转染组相比，沉默细胞色素P27A1蛋白表达降低75%，基因表达水平降低65%，不影响孕酮的产生，但抑制MBG的产生达80%。因此，在人滋养层细胞中，蟾酥二烯内酯强心类固醇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.