Harmine inhibits oxidative phosphorylation, thus regulating the polarization of macrophages mediated by extracellular adenosine in endometriosis

Abstract

Study question: Can harmine mitigate endometriosis by modulating oxidative phosphorylation (OXPHOS) suppression and macrophage polarization?

Summary answer: Harmine induces apoptosis in endometriotic lesions by inhibiting OXPHOS while also modulating macrophage polarization through the reduction of extracellular adenosine (eADO), a metabolic byproduct of OXPHOS.

What is known already: Endometriosis is associated with mitochondrial dysfunction while ectopic endometrial stromal cells (EESCs) exhibit heightened susceptibility to oxidative stress. Macrophages accumulate in the peritoneal cavity and peritoneal fluid, constituting a dominant immune cell population within endometriotic lesions.

Study design, size, duration: A total of 25 patients diagnosed with ovarian endometriosis and 25 healthy donors undergoing surgical treatment at a university-affiliated hospital within 6 months were included. Primary normal endometrial stromal cells (NESCs) and EESCs were isolated, cultured, and subjected to pharmacological drug intervention, small interfering RNA (siRNA) transfection, OXPHOS analysis, and transcriptomic profiling. Macrophage cell line RAW264.7 was used to study macrophage polarization. The in vivo study involved 24 C57BL/6 female mice to establish an endometriosis model.

Participants/materials, setting, methods: The impact of harmine on cellular viability in vitro was assessed using Cell Counting Kit-8 (CCK-8) clonogenic assays, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, and flow cytometry. OXPHOS inhibition was evaluated through mitochondrial ultrastructural changes observed via transmission electron microscopy and fluorescence staining. Oxidative stress was quantified using JC-1 mitochondrial membrane potential assays and reactive oxygen species (ROS) detection kits. Seahorse oxygen consumption rate assay was conducted to measure OXPHOS activity. Immunohistochemistry was employed to examine key protein expression in endometrial tissues. ATP and eADO levels were quantified using an ATP Assay Kit and Adenosine Assay Kit, respectively. We used RNA sequencing (RNA-seq) to identify enriched biological pathways. Flow cytometry was used to characterize macrophage populations in peritoneal fluid, and CIBERSORTx software was applied to generate immune cell expression profiles. Samples with P-values <0.05 were selected for analysis, with macrophage subtype (Mφ2/Mφ1) alterations statistically evaluated using the Wilcoxon test.

Main results and the role of chance: EESCs exhibited greater susceptibility to harmine treatment compared to NESCs (P < 0.05). RNA-seq analysis revealed that harmine primarily influences pathways related to cell proliferation, oxidative stress, and HIF-1 signaling. Differentially expressed genes were enriched in complexes regulating mitochondrial OXPHOS. Harmine intervention significantly increased mitochondrial fission in EESCs compared to NESCs and markedly suppressed OXPHOS activity in EESCs. Treatment with harmine decreased total ROS levels in cultured EESCs (P < 0.05 vs NESCs). The eADO/CD73-mediated adenosine pathway was upregulated in endometriotic lesions, accompanied by a predominance of Mφ2 macrophages in the microenvironment (Wilcoxon test, P < 0.05). In the murine model, harmine treatment decreased eADO production and shifted macrophage polarization toward a pro-inflammatory Mφ1 phenotype, contributing to endometriotic lesion regression.

Large scale data: N/A.

Limitations, reasons for caution: The study's reliance on primary cells was constrained by their limited passage potential and inherent heterogeneity, precluding co-culture investigations. Further mechanistic elucidation requires cell line-based studies. More subtypes should be included in the research, not just models of ovarian endometriosis. Eutopic endometrial cells should be included in the experiment if conditions permit. Additionally, primate models with menstrual cycles, such as rhesus monkeys, should be considered for future in vivo validation.

Wider implications of the findings: These findings indicate that harmine may serve as a promising alkaloid-based therapeutic for endometriosis by modulating energy metabolism and immune responses.

Study funding/competing interest(s): This research was supported by the National Natural Science Foundation of China (NSFC No. 82201814), the Guangdong Basic and Applied Basic Research Foundation (2021A1515110444), and the Foundation of Shenzhen Hospital of Southern Medical University (PY2022YM03). The authors declare no conflicts of interest.

Trial registration number: N/A.

Keywords: OXPHOS; adenosine pathway; endometriosis; harmine; macrophage.