■发现四氢生物蝶呤(BH4)及其氧化衍生物二氢生物蝶呤(BH2)在体位不耐受(MEOI)的ME/CFS患者中强烈升高。
■然而,在ME+OI受试者中生物蝶呤生物发生的分子机制知之甚少。这里,我们报告说,非氧化性磷酸戊糖途径(PPP)的激活在ME+OI受试者中生物蝶呤(BH4和BH2)的生物发生中起关键作用。
■基于微阵列的基因筛选,然后进行基于实时PCR的验证,ELISA测定,最后是葡萄糖-6-磷酸脱氢酶(G6PDH)的酶动力学研究,转醛缩酶(TALDO1),和转酮醇酶(TK)酶表明,厌氧PPP的增加对生物蝶呤的调节至关重要。为了进一步调查,我们设计了一种新的细胞培养策略,通过在85%N2/10%CO2/5%O2的低氧条件下用5-磷酸核糖(R5P)处理人小胶质细胞来诱导非氧化性PPP,然后通过ELISA分析生物蝶呤代谢。免疫印迹,和双重免疫细胞化学分析。此外,taldo1基因的siRNA敲除强烈抑制磷酸核糖焦磷酸(PRPP)的生物利用度,减少嘌呤生物合成酶的表达,减毒GTP环化水解酶1(GTPCH1),并抑制了随后在R5P处理和缺氧诱导的C20人小胶质细胞中BH4的产生及其向BH2的代谢转化。这些结果证实,通过嘌呤生物合成途径上调BH4和BH2确实需要非氧化性PPP的活化。为了测试ME+OI血浆衍生的生物蝶呤的功能作用,外源性添加人小胶质细胞中具有高BH4上调的诱导型一氧化氮合酶(iNOS)和一氧化氮(NO)的ME+OI血浆的血浆样本,表明非氧化性PPP诱导的生物蝶呤可刺激ME+OI患者的炎症反应.
■放在一起,我们目前的研究强调,非氧化性PPP的诱导调节生物蝶呤的生物发生,有助于ME/CFS发病机制.
四氢生物蝶呤(BH4)代谢在健康个体中受到严格调节。最近,我们的研究表明,在体位不耐受的ME/CFS患者的血浆样本中,BH4水平上调.在研究分子机制的同时,我们目前的研究表明,戊糖磷酸途径(PPP)的诱导是关键的上调表达的BH4。引入了一种新型的基于缺氧的细胞培养模型来研究人小胶质细胞中的PPP。随后,全面的RNA阵列研究,不同的免疫测定,酶活性的生化分析证实,小胶质细胞中非氧化性PPP的诱导增强了PPP调节基因和酶的表达,诱导酶活性,活性嘌呤生物合成,最终上调了生物蝶呤的生物合成。
UNASSIGNED: Tetrahydrobiopterin (BH4) and its oxidized derivative dihydrobiopterin (BH2) were found to be strongly elevated in ME/CFS patients with orthostatic intolerance (ME + OI).
UNASSIGNED: However, the molecular mechanism of biopterin biogenesis is poorly understood in ME + OI subjects. Here, we report that the activation of the non-oxidative pentose phosphate pathway (PPP) plays a critical role in the biogenesis of biopterins (BH4 and BH2) in ME + OI subjects.
UNASSIGNED: Microarray-based gene screening followed by real-time PCR-based validation, ELISA assay, and finally enzyme kinetic studies of glucose-6-phosphate dehydrogenase (G6PDH), transaldolase (TALDO1), and transketolase (TK) enzymes revealed that the augmentation of anaerobic PPP is critical in the regulations of biopterins. To further investigate, we devised a novel cell culture strategy to induce non-oxidative PPP by treating human microglial cells with ribose-5-phosphate (R5P) under a hypoxic condition of 85%N2/10%CO2/5%O2 followed by the analysis of biopterin metabolism via ELISA, immunoblot, and dual immunocytochemical analyses. Moreover, the siRNA knocking down of the taldo1 gene strongly inhibited the bioavailability of phosphoribosyl pyrophosphate (PRPP), reduced the expressions of purine biosynthetic enzymes, attenuated GTP cyclohydrolase 1 (GTPCH1), and suppressed subsequent production of BH4 and its metabolic conversion to BH2 in R5P-treated and hypoxia-induced C20 human microglia cells. These results confirmed that the activation of non-oxidative PPP is indeed required for the upregulation of both BH4 and BH2 via the purine biosynthetic pathway. To test the functional role of ME + OI plasma-derived biopterins, exogenously added plasma samples of ME + OI plasma with high BH4 upregulated inducible nitric oxide synthase (iNOS) and nitric oxide (NO) in human microglial cells indicating that the non-oxidative PPP-induced-biopterins could stimulate inflammatory response in ME + OI patients.
UNASSIGNED: Taken together, our current research highlights that the induction of non-oxidative PPP regulates the biogenesis of biopterins contributing to ME/CFS pathogenesis.
Tetrahydrobiopterin (BH4) metabolism is tightly regulated in a healthy individual. Recently, our research showed that BH4 level is upregulated in the plasma samples of ME/CFS patients with orthostatic intolerance. While investigating the molecular mechanism, our current study identified that the pentose phosphate pathway (PPP) induction is critical for the upregulated expression of BH4. A novel hypoxia-based cell culture model is introduced to study PPP in human microglial cells. Subsequently, a comprehensive RNA array study, different immunoassay, and biochemical analyses of enzyme activities confirmed that the induction of non-oxidative PPP in microglial cells enhanced expressions of PPP-regulatory genes and enzymes, induced enzyme activities, activated purine biosynthesis, and finally upregulated biopterin biogenesis.