The activation of cGAS-STING pathway promotes the epithelial-mesenchymal transition and inflammation in intrauterine adhesion

Intrauterine adhesion (IUA) is characterized by endometrial fibrosis due to basal layer injury, leading to hypomenorrhea, recurrent abortions, and infertility, with significant clinical implications. Maintaining homeostasis in the endometrial epithelium is crucial for its normal physiological functions. Epithelial-mesenchymal transition (EMT) in endometrial epithelial cells is a key mechanism driving endometrial fibrosis in IUA. Accumulating evidences substantiate the pivotal role of the cGAS-STING pathway in inflammatory fibrotic diseases. In this study, we identified endometrial epithelium-specific activation of the cGAS-STING pathway as a driver of IUA through mtDNA leakage-induced EMT, a previously unreported mechanism in reproductive fibrosis. We quantitatively demonstrated cGAS-STING pathway activation in endometrial tissues from 20 IUA patients compared to 20 normal controls. Pharmacological inhibition of STING with C-176 (10 μM) significantly reduced TGF-β1-induced EMT markers: N-cadherin (0.37 ± 0.08-fold, p = 0.0044) and α-SMA (1.34 ± 0.13-fold, p = 0.0047), while restoring E-cadherin (0.27 ± 0.05-fold, p = 0.0016). Conversely, STING activation by 2',3''-cGAMP (10 μM) exacerbated EMT, increasing N-cadherin (0.25 ± 0.01-fold, p = 0.0377) and α-SMA (1.79 ± 0.28-fold, p = 0.0007). TGF-β1 triggered mitochondrial DNA (mtDNA) leakage into the cytoplasm, as evidenced by TOMM20/dsDNA co-localization (76 % reduction with VDAC1 inhibitor VBIT-4, p = 0.0077), activating cGAS-STING signaling, reflected by upregulated cGAS (1.15 ± 0.24-fold, p = 0.0093), STING (4.37 ± 0.46-fold, p = 0.0007), and p-IRF3 (0.4 ± 0.04, p = 0.0007) in an IUA cell model. Administration of C-176 reduced endometrial fibrosis by 73 % (p < 0.0001), suppressing collagen I (75 %, p < 0.0001), α-SMA (74 %, p = 0.0015), and N-cadherin (83 %, p < 0.0001), while increasing E-cadherin expression (2.44 ± 0.44-fold, p = 0.0007). It also reduced pro-inflammatory cytokines IL-1β (mRNA: 6.08 ± 0.69-fold, p = 0.0047; protein: 0.08 ng/g, p = 0.0019) and IL-6 (mRNA: 5.90 ± 0.05-fold, p < 0.0001; protein: 0.46 ± 0.50 pg/g, p = 0.0215). In contrast, 2',3''-cGAMP increased fibrosis by 50 % (p = 0.0001) and amplified IL-1β (mRNA: 67.69 ± 0.45-fold, p < 0.0001; protein: 0.20 ± 0.02 ng/g, p < 0.0001) and IL-6 (mRNA: 7.52 ± 0.12-fold, p < 0.0001; protein: 0.95 ± 0.16 pg/g, p < 0.0001).These findings suggest that the mtDNA-cGAS-STING axis plays a critical role in IUA development, highlighting potential therapeutic targets for intervention.