Catalpol inhibits Hedgehog signaling pathway to suppress proliferation and promote lipid accumulation in rat meibomian gland epithelial cells.

Meibomian gland dysfunction (MGD) is an ocular surface disease lacking optimal treatment strategy. The Hedgehog pathway is involved in regulating MGEC proliferation and differentiation. Catalpol (CAT) is the main active ingredient in Rehmannia glutinosa with therapeutic potential. Exploring the effects and biological mechanisms of CAT on meibomian gland epithelial cells (MGECs). Primarily cultured rat MGECs were co-cultured with 3T3 cells for 7 days. MGECs were exposed to 2.5, 5, and 10 mmol/L CAT, 10 μg/mL Azithromycin (AZM), and 0.6 μmol/L Smoothened receptor agonist (SAG) for 48 h. Colony formation assays, Cell counting kit-8, Ki67 immunofluorescence, Nile red and Oil red O staining, and HSD LipidTOX Green kits were used to assess cell proliferation and lipid accumulation. Real-time quantitative PCR and Western blot analysis were used to measure gene expressions related to Hedgehog- and peroxisome proliferator-activated receptor (PPAR)-γ. This study successfully isolated primarily rat MGECs (expressed P63 and K14). AZM and 5, and 10 mmol/L CAT inhibited colony number, cell viability, and Ki67 mean fluorescence intensity (MFI), while they enhanced MFI of Nile red and LipidTOX Green, as well as increasing the ratio of Oil red O staining area. Additionally, transcription and translation levels of the Hedgehog pathway were significantly suppressed, meanwhile, PPAR-γ and SREBP-1 expression were increased. Interestingly, SAG reversed the effects of 10 mmol/L CAT on MGECs. CAT suppresses MGEC proliferation and promotes lipid accumulation by inhibiting the Hedgehog signaling pathway. This study offers a potential therapeutic strategy for MGD. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-025-00769-9.