目的:子宫内膜异位症异位基质细胞(EESCs)中的骨桥蛋白(OPN)如何参与子宫内膜异位症的发病机制并实现体外无创检测?
结论:靶向OPN通过抑制RhoA/活性氧(ROS)轴来调节子宫内膜异位症的坏死和炎症状态,从而减轻子宫内膜异位症并能够在体外进行经血的非侵入性检测。
背景:子宫内膜异位症是一种慢性炎症性疾病。最近的研究表明,OPN通过调节细胞死亡和炎症在疾病进展中发挥重要作用。
方法:该研究包括20例诊断为子宫内膜异位症的患者(经腹腔镜检查和组织学证实)和10例无子宫内膜异位症的对照。从子宫内膜样品中分离出子宫内膜间质细胞,同时从月经血中分离出子宫内膜细胞(MESCs)。然后在体外培养这些细胞并用于随后的实验。
方法:使用炎症因子测序评估EESCs中OPN的表达,免疫组化染色(IHC),实时定量PCR(qRT-PCR)分析,和蛋白质印迹(WB)。使用EdU检查OPN的生物学行为及其对炎症因子的影响。伤口愈合,Transwell,和ELISA测定。通过qRT-PCR检测EESCs的坏死及其对炎症因子的影响,WB,和钙黄绿素-AM/PI荧光测定。EESC中线粒体应激的检查涉及线粒体膜电位(ΔkWm)测定的使用,ROS检测,和钙黄绿素-AM加载/氯化钴淬火。qRT-PCR,WB,等实验验证OPN通过RhoA/ROS轴对EESCs凋亡和炎症因子水平的调控作用。使用AAV9病毒证实了OPN的敲除及其对子宫内膜异位症病灶大小的抑制作用,IHC,qRT-PCR,WB,和其他实验。此外,使用转录组测序检测MESCs中的OPN表达,RT-PCR,WB,和其他实验。
结果:体外实验证明了EESCs中OPN的显著上调,OPN的敲除能有效抑制细胞凋亡和炎症因子的释放。OPN通过介导RhoA依赖性ROS产生和阻断混合谱系激酶结构域样蛋白在细胞膜上的磷酸化来抑制坏死和炎症因子释放。在体内,靶向OPN可以抑制子宫内膜异位症病灶的生长。临床上,子宫内膜异位症患者的经血中OPN也明显上调。
方法:不适用。
结论:由于获取手术标本的局限性,我们的研究主要涉及在月经周期的增生期和分泌期收集女性子宫内膜异位症组织。我们在用于我们研究的样品中观察到OPN的显著过表达。然而,月经间期子宫内膜异位症组织中OPN的表达尚不清楚。
结论:我们的研究结果强调了OPN/RhoA/ROS轴在调节坏死性凋亡和炎症因子释放中的关键作用。OPN敲低在体内发挥治疗作用,体外检测经血中OPN的高表达。总之,靶向OPN为子宫内膜异位症的治疗和检测提供了可能。
背景:本研究得到国家自然科学基金(82071626)的资助,浙江省公益科技应用研究项目(LGF21H040010),和温州医科大学附属第二医院临床研究项目(1010293)。作者没有利益冲突。
OBJECTIVE: How does osteopontin (OPN) in endometriosis ectopic stromal cells (EESCs) participate in the pathogenesis of endometriosis and achieve non-invasive detection in vitro?
CONCLUSIONS: Targeted OPN regulates endometriosis\'s necroptosis and inflammatory state by inhibiting the RhoA/reactive oxygen species (ROS) axis, thereby alleviating endometriosis and enabling non-invasive detection of menstrual blood in vitro.
BACKGROUND: Endometriosis is a chronic inflammatory disease. Recent studies have shown that OPN plays an important role in disease progression by regulating cell death and inflammation.
METHODS: The study included 20 patients diagnosed with endometriosis (confirmed by laparoscopy and histology) and 10 controls without endometriosis. Endometriotic stromal cells were isolated from endometrial samples, while menstrual blood endometrial cells (MESCs) were isolated from menstrual blood. These cells were then cultured in vitro and utilized in subsequent experiments.
METHODS: OPN expression in EESCs was assessed using inflammatory factor sequencing, immunohistochemical staining (IHC), quantitative real-time PCR (qRT-PCR) analysis, and Western blotting (WB). The biological behavior of OPN and its effects on inflammatory factors were examined using EdU, wound-healing, Transwell, and ELISA assays. Necroptosis in EESCs and its impact on inflammatory factors were detected through qRT-PCR, WB, and Calcein-AM/PI fluorescence assays. The examination of mitochondrial stress in EESCs involved the use of the Mitochondrial Membrane Potential (ΔΨm) Assay, ROS detection, and Calcein-AM Loading/cobalt chloride Quenching. qRT-PCR, WB, and other experiments were conducted to verify the regulation of necroptosis and inflammatory factor levels in EESCs by OPN through the RhoA/ROS axis. Knockdown of OPN and its inhibitory effect on endometriosis lesion size were confirmed using AAV9 virus, IHC, qRT-PCR, WB, and other experiments. Additionally, OPN expression in MESCs was detected using transcriptome sequencing, RT-PCR, WB, and other experiments.
RESULTS: In vitro assays demonstrated a significant upregulation of OPN in EESCs, and the knockdown of OPN effectively inhibited necroptosis and the release of inflammatory factors. OPN inhibited necroptosis and inflammatory factor release by mediating RhoA-dependent ROS production and blocking mixed lineage kinase domain-like protein phosphorylation at the cell membrane. In vivo, targeting of OPN can inhibit the growth of endometriosis lesions. Clinically, OPN was also significantly upregulated in the menstrual blood of patients with endometriosis.
METHODS: N/A.
CONCLUSIONS: Due to limitations in obtaining surgical specimens, our study primarily involved collecting endometriosis tissues from women during the proliferative and secretory phases of the menstrual cycle. We observed a significant overexpression of OPN in the samples used for our investigation. However, the expression of OPN in endometriosis tissues during the intermenstrual phase remains unknown.
CONCLUSIONS: Our findings highlight the pivotal role of the OPN/RhoA/ROS axis in the regulation of necroptosis and the release of inflammatory factors. OPN knockdown exerts a therapeutic effect in vivo, and the high expression detection of OPN in menstrual blood in vitro. In summary, targeting OPN provides possibilities for the treatment and detection of endometriosis.
BACKGROUND: This study was supported by the National Natural Science Foundation of China (82071626), the Zhejiang Province Public Welfare Technology Application Research Project (LGF21H040010), and the Clinical Research project of the Second Affiliated Hospital of Wenzhou Medical University (1010293). The authors have no conflicts of interest.