宿主线粒体心磷脂的释放被认为是有助于梅毒中产生抗心磷脂抗体的主要因素。然而,在这种情况下,线粒体释放心磷脂的确切机制仍然难以捉摸。本研究旨在阐明梅毒线粒体心磷脂释放的机制。我们进行了心磷脂定量分析和免疫荧光分析,以检测人微血管内皮细胞(HMEC-1)中线粒体心磷脂的释放,有和没有梅毒螺旋体(Tp)感染。此外,我们探索了细胞凋亡,线粒体心磷脂释放的关键机制。然后通过RNA序列分析潜在的介质分子,随后使用由CRISPR-Cas9和途径特异性抑制剂介导的体外敲除技术进行验证。我们的发现证实,活Tp能够启动线粒体心磷脂的释放,而失活的Tp不表现出这种能力。此外,凋亡检测进一步支持线粒体心磷脂释放独立于凋亡发生的观点.RNA测序结果表明微管相关蛋白2(MAP2),轴突发生和树突发育基因,在用Tp处理的HMEC-1中上调,免疫荧光在梅毒性病变中进一步证实。值得注意的是,MAP2基因敲除抑制Tp诱导的HMEC-1线粒体心磷脂释放。机械上,Tp感染通过MEK-ERK-HES1通路调节MAP2表达,和MEK/ERK磷酸化抑制剂有效阻断Tp诱导的线粒体心磷脂释放。这项研究表明,活Tp的感染通过MEK-ERK-HES1途径增强了MAP2的表达,从而有助于我们了解抗心磷脂抗体在梅毒诊断中的作用。
The release of host mitochondrial
cardiolipin is believed to be the main factor that contributes to the production of anti-
cardiolipin antibodies in syphilis. However, the precise mechanism by which mitochondria release
cardiolipin in this context remains elusive. This study aimed to elucidate the mechanisms underlying mitochondrial
cardiolipin release in syphilis. We conducted a cardiolipin quantitative assay and immunofluorescence analysis to detect mitochondrial cardiolipin release in human microvascular endothelial cells (HMEC-1), with and without Treponema pallidum (Tp) infection. Furthermore, we explored apoptosis, a key mechanism for mitochondrial
cardiolipin release. The potential mediator molecules were then analyzed through RNA-sequence and subsequently validated using in vitro knockout techniques mediated by CRISPR-Cas9 and pathway-specific inhibitors. Our findings confirm that live-Tp is capable of initiating the release of mitochondrial cardiolipin, whereas inactivated-Tp does not exhibit this capability. Additionally, apoptosis detection further supports the notion that the release of mitochondrial cardiolipin occurs independently of apoptosis. The RNA-sequencing results indicated that microtubule-associated protein2 (MAP2), an axonogenesis and dendrite development gene, was up-regulated in HMEC-1 treated with Tp, which was further confirmed in syphilitic lesions by immunofluorescence. Notably, genetic knockout of MAP2 inhibited Tp-induced mitochondrial cardiolipin release in HMEC-1. Mechanically, Tp-infection regulated MAP2 expression via the MEK-ERK-HES1 pathway, and MEK/ERK phosphorylation inhibitors effectively block Tp-induced mitochondrial cardiolipin release. This study demonstrated that the infection of live-Tp enhanced the expression of MAP2 via the MEK-ERK-HES1 pathway, thereby contributing to our understanding of the role of anti-cardiolipin antibodies in the diagnosis of syphilis.