Silencing core circadian regulators CLOCK and BMAL1 inhibits autophagy in interstitial cells of Cajal in a gastroesophageal reflux disease model

Excessive autophagy of interstitial cells of Cajal (ICCs) in the esophagus and altered expression of circadian clock genes in the upper gastrointestinal tract are key pathological mechanisms in reflux esophagitis, a common form of gastroesophageal reflux disease (GERD). The circadian rhythm core regulators circadian locomotor output cycles kaput (CLOCK) and aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) participate in multiple diseases through autophagy modulation, but their roles in GERD remain unclear. This study aimed to investigate the roles of CLOCK and BMAL1 in ICCs autophagy within a GERD model. Primary esophageal ICCs were isolated and cultured. An in vitro GERD autophagy model was established by inducing ICCs autophagy with 25 μM deoxycholic acid. The cells were subsequently treated with CLOCK siRNA, BMAL1 siRNA, or combined CLOCK + BMAL1 siRNA. Cellular samples were collected, and ultrastructure and autophagy of ICCs were examined using transmission electron microscopy. The expression levels of autophagy-related proteins (LC3-II/I, Beclin-1) and CLOCK, BMAL1, Period Circadian Regulator 2 (PER2), Cryptochrome 2 (CRY2) were analyzed by Western blot and immunofluorescence assays. The corresponding mRNA levels were measured by Reverse Transcription Polymerase Chain Reaction (RT-PCR). Treatment with 25 μM deoxycholic acid significantly induced autophagy in ICCs and increased the expression levels of CLOCK, BMAL1, PER2, and CRY2. Silencing CLOCK or BMAL1 reduced the expression levels of PER2 and CRY2, decreased ICCs autophagy, and restored ICCs ultrastructure, with combined silencing producing a more pronounced effect. CLOCK and BMAL1 regulate excessive autophagy of ICCs in GERD by mediating the circadian-autophagy axis. Targeted silencing of both molecules effectively improves the function of ICCs, providing a potential therapeutic strategy for GERD.