Piezo1 mediates hypoxia-induced endometriosis fibrosis via the mtDNA -dependent cGAS-STING pathway

BACKGROUND: Endometriosis (EMs) is a prevalent gynecological disorder characterized by the ectopic growth of functional endometrial tissue, and its fibrotic pathology represents a primary contributor to chronic pain and infertility in affected patients. Hypoxia, a hallmark of the endometriotic microenvironment, is a well-established driver of fibrogenesis. However, the precise molecular mechanisms translating hypoxic signaling into pro-fibrotic responses remain elusive. The mechanosensitive ion channel Piezo1 is recognized as a contributor to fibrosis in various diseases; whereas its specific role in endometriosis is poorly understood. We demonstrate that hypoxia-inducible factor 1-alpha (HIF-1α) and Piezo1 are co-upregulated alongside fibrosis in EMs patient lesions. Functionally, both hypoxia and Piezo1 agonism promote fibrogenesis in HESCs. Mechanistically, hypoxia acts through Piezo1 to induce Ca²⁺-dependent mitochondrial damage and mitochondrial DNA (mtDNA) leakage, which in turn activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling and subsequently drives fibrosis. Therefore, we identifying the axis as a promising therapeutic target for future intervention. METHODS: This study combines clinical tissue analysis with in vitro cellular experiments. Normal endometrial tissue, eutopic and ectopic endometrial samples from patients with endometriosis were collected. Immunohistochemistry (IHC), immunofluorescence (IF), western blotting, and quantitative reverse transcription PCR (qRT-PCR) were used to analyse the expression levels of HIF-1α, Piezo1, cGAS, STING, and the fibrosis markers α-smooth muscle actin (α-SMA) and type I collagen (Collagen I). In vitro experiments, we utilized HESCs to establish a hypoxic culture model. IF, western blotting, and qRT-PCR were employed to assess the expression of HIF-1α, Piezo1, and fibrosis-related markers across experimental conditions. Combined flow cytometric analysis and EdU assays, 24 h was selected as the optimal intervention duration for subsequent experiments. To clarify the role of Piezo1, its activity was modulated via lentiviral knockdown or agonist (Yoda1) activation. Cytosolic and mitochondrial Ca²⁺ fluxes were tracked with fluorescent probes; mitochondrial morphology and function were evaluated by immunofluorescence and JC-1 staining. Cytosolic mtDNA leakage was quantified by immunofluorescence and qRT-PCR. RESULTS: We report that Piezo1 and HIF-1α are co-upregulated in ectopic endometrial lesions, correlating with fibrotic severity. The expression of HIF-1α and Piezo1 was time-dependently induced by hypoxia in HESCs. Mechanistically linking hypoxia to fibrosis, we found that hypoxia-induced Piezo1 upregulation drives mitochondrial damage and mtDNA release into the cytosol, which activates the cGAS-STING pathway and ultimately upregulates the fibrosis markers α-SMA and collagen I. Consequently, genetic or pharmacological inhibition of Piezo1, as well as blockade of cGAS with RU.521 or STING with C-176, effectively suppressed cGAS-STING pathway activation and attenuated fibrosis in HESCs. CONCLUSIONS: Piezo1 exerts a crucial regulatory function in the process of hypoxia-driven fibrogenesis in endometriosis. This study reveals the central role of the hypoxia/Piezo1/mtDNA/cGAS-STING signaling axis in EMs fibrosis and provides a novel therapeutic strategy for targeting this pathway to delay EMs progression.