Abstract:AIM: To investigate whether catalpol protects against diabetic retinal vascular endothelial injury by targeting the methyltransferase-like 3 (METTL3)-m6A-thioredoxin-interacting protein (TXNIP) axis and inhibiting nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation. METHODS: A streptozotocin-induced diabetic mouse model (n=20 per group) was used to assess retinal function via electroretinogram (ERG) and vascular integrity via Evans Blue leakage. Human retinal vascular endothelial cells (HRVECs) were exposed to high glucose (HG, 30 mmol/L) with or without catalpol or the METTL3 inhibitor STM2457. NLRP3 inflammasome components (Western blot), oxidative stress (DCFH-DA probe), global m6A levels (Dot blot), and TXNIP expression were measured. The binding of catalpol to METTL3, NLRP3, TXNIP, and interleukin-1β (IL-1β) was analyzed via molecular docking and dynamics simulations. RESULTS: Catalpol treatment improved ERG amplitudes [a-wave, b-wave, oscillatory potentials (OPs)] and reduced vascular leakage in diabetic mice (P<0.05), while downregulating retinal vascular endothelial growth factor (VEGF), NLRP3, IL-1β, and IL-18 protein levels. In HG-stimulated HRVECs, catalpol inhibited the NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC)-caspase-1 inflammasome, reduced reactive oxygen species, and suppressed METTL3 expression and global m6A methylation (P<0.05). It also attenuated HG-induced TXNIP upregulation. METTL3 inhibition by STM2457 mimicked all protective effects of catalpol. Molecular simulations confirmed stable binding of catalpol to METTL3, NLRP3, TXNIP, and IL-1β. CONCLUSION: Catalpol alleviates diabetic retinal vascular endothelial injury by inhibiting the NLRP3 inflammasome. This effect is mediated, at least in part, through downregulating METTL3-dependent m6A RNA methylation of TXNIP.
