Exploratory experiment designed to discover new patterns targeting CLOCK, BMAL1, PER2, CRY2 in primary esophageal interstitial cells of Cajal. Primary outcome: ICC autophagy levels and cellular ultrastructure
This study investigated the roles of circadian clock genes CLOCK and BMAL1 in regulating excessive autophagy of interstitial cells of Cajal (ICCs) in gastroesophageal reflux disease (GERD). Primary esophageal ICCs were isolated and cultured, then treated with deoxycholic acid to establish an in vitro GERD autophagy model. The cells were subsequently treated with siRNA targeting CLOCK, BMAL1, or both genes in combination. The study examined cellular ultrastructure using transmission electron microscopy and measured autophagy-related proteins (LC3-II/I, Beclin-1) and circadian clock proteins (CLOCK, BMAL1, PER2, CRY2) using Western blot, immunofluorescence, and RT-PCR. The research aimed to understand how circadian rhythm regulators modulate autophagy in GERD pathogenesis and identify potential therapeutic targets.
ICC isolation and culture, deoxycholic acid treatment (25 ΞM), siRNA transfection (CLOCK, BMAL1, or combined), transmission electron microscopy, Western blot analysis, immunofluorescence assays, RT-PCR
Deoxycholic acid increased autophagy and circadian gene expression; CLOCK/BMAL1 silencing reduced autophagy, decreased PER2/CRY2 expression, and restored ICC ultrastructure
Reduced autophagy markers (LC3-II/I, Beclin-1), improved cellular ultrastructure, decreased circadian gene expression
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