Salivary gland protective and antiinflammatory effects of genistein in Sjögren's syndrome by inhibiting Xist/ACSL4-mediated ferroptosis following binding to estrogen receptor-alpha.

Background: Sjögren's syndrome (SS) is an autoimmune disease with limited effective treatment options. This study aimed to explore the underlying mechanism by which genistein-estrogen receptor alpha (ERα) complex targets X-inactive specific transcript (Xist) then leads to the inhibition of ferroptosis by regulating acyl-CoA synthetase long-chain family member 4 (ACSL4) expression in salivary gland epithelial cells (SGECs) to attenuate SS.

Methods: The effects of genistein treatment on the progression and underlying mechanism of SS were investigated using nondiabetic obese (NOD)/LtJ mice in vivo and Interferon-γ (IFNγ)-treated SGECs in vitro. Water intake and saliva flow rate were measured to evaluate the severity of xerostomia. Hematoxylin-eosin staining, real-time quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay were conducted to examine the pathological lesions. Western blotting and immunohistochemistry analysis were used to evaluate the protein expression. RNA sequencing and RNA fluorescence in situ hybridization were employed to verify the relationship between Xist and ACSL4. Surface plasmon resonance, molecular docking, and molecular dynamics were used to investigate the binding between genistein and ERα. Furthermore, a chromatin immunoprecipitation assay was used to analyze ERα-XIST promoter interactions. The levels of malondialdehyde, glutathione, Fe²⁺, and mitochondrial changes were measured to evaluate ferroptosis of SGECs.

Results: In NOD/LtJ mice, a ferroptosis phenotype was observed in salivary glands, characterized by downregulated Xist and upregulated X chromosome inactivation gene Acsl4. Genistein significantly alleviated SS symptoms, upregulated the Xist gene, and downregulated Acsl4 expression. Genistein upregulated Xist expression in the salivary gland of NOD/LtJ mice via the ERα signaling pathway. It downregulated Acsl4 and ferroptosis in the salivary glands of NOD/LtJ mice. IFNγ-treatment induced inflammation and ferroptosis in SGECs. Genistein binding to ERα upregulated XIST, and aquaporin 5 expression, downregulated ACSL4, and SS antigen B expression, and reversed ferroptosis in SGECs. Genistein mitigated inflammation and ferroptosis in SGECs by upregulated-XIST-mediated ACSL4 gene silencing.

Conclusions: Genistein binding to ERα targets Xist, leading to inhibiting ferroptosis by regulating ACSL4 expression in SGECs. This finding provides evidence for genistein as a treatment for SS and identifies Xist as a novel drug target for SS drug development, offering great promise for improving SS outcomes.