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

》》文章原文链接：The activation of cGAS-STING pathway promotes the epithelial-mesenchymal transition and inflammation in intrauterine adhesion

》》Journal：International immunopharmacology

》》相关产品： 2',3'-cGAMP sodium (SJ-MN0890)、C-176 (SJ-MX0351) 和 VBIT-4 (SJ-MX0612)

》》产品引用描述：

》》Abstract：

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.

》》部分实验数据展示：

Fig. 2.  STING regulators modulate the inflammatory cytokine secretion and EMT in HEECs.

C. qRT-PCR was performed to quantify the relative mRNA levels of IL-1B and IL-6 in HEECs: Compared to the TGF-β1 treatment group, C-176 (10 μM) addition significantly reduced IL-1B mRNA level by 0.67 ± 0.02- fold (p = 0.0101) and IL-6 mRNA level by 0.82 ± 0.02-fold (p < 0.0001); while 2′,3′-cGAMP (10 μM) addition significantly increased IL-1B mRNA level by 2.31 ± 0.21-fold (p < 0.0001) and IL-6 mRNA level by 0.29 ± 0.05-fold (p = 0.0079). D. ELISA was conducted to detected the protein concentrations of IL-1B and IL-6 in HEECs: Compared to the TGF-β1 treatment group, C-176 (10 μM) addition significantly reduced IL-1B protein level by 7.73 ± 0.76 pg/ml (p = 0.0001) and IL-6 protein level by 3.90 ± 0.39 pg/ml (p = 0.0023); while 2′,3′-cGAMP (10 μM) addition significantly increased IL-1B protein level by 19.89 ± 0.76 pg/ml (p < 0.0001) and IL-6 protein level by 5.96 ± 0.97 pg/ml (p < 0.0001). 

Fig. 4.  VDAC oligomerization inhibitor VBIT-4 attenuates mtDNA cytoplasmic release and EMT in HEECs.

A. Immunofluorescent double-labeling of DNA (red) and mitochondrial (TOMM20, green) was observed by confocal microscopy: Compared to the TGF-β1 treatment group (20 ng/ml for 48 h), the addition of VBIT-4 (pretreated with 10 μM VBIT-4 for 2 h before incubation with TGF-β1 for 48 h) significantly reduced the release of mitochondrial DNA into the cytoplasm by 76 % (p = 0.0077). 63×, Oil mirror, Scale bar: 5um. B. Compared to the TGF-β1 treatment group(20 ng/ml for 48 h), the addition of VBIT-4 (pretreated with 10 μM VBIT-4 for 2 h before incubation with TGF-β1 for 48 h) significantly alleviated EMT detected by Western blot: with a 0.61 ± 0.01-fold increase in E-cadherin (p < 0.0001), a 0.90 ± 0.09-fold decrease in N-cadherin (p = 0.0038), and a 4.08 ± 0.16-fold decrease in α-SMA (p = 0.0018). (n = 3 technical replicates for each group) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)