TRDMT1-dependent m5C modification of TLR4 mediates the protective effects of Shaoyao decoction in TNBS-induced Crohn's disease.
BACKGROUND AND AIM Crohn's disease (CD) is a chronic inflammatory disease of the digestive system with increasing morbidity worldwide. Shaoyao Decoction (SYD), a traditional Chinese medicine formula, has shown therapeutic potential in the treatment of CD. However, the underlying mechanisms remain unclear. This study aimed to elucidate the molecular mechanisms underlying the protective effects of SYD against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced CD-like colitis through the tRNA aspartic acid methyltransferase 1 (TRDMT1)/TLR4/NF-κB signaling pathway. EXPERIMENTAL PROCEDURE We used network pharmacology to identify potential targets and related pathways of SYD in the treatment of CD. The CD model was induced by TNBS. Immunofluorescence, Western blotting, and ELISA were employed to detect various cytokines and proteins associated with epithelial barrier, inflammation, apoptosis, and signaling pathway regulation. TLR4 mRNA expression and the 5-methylcytosine (m5C) modification level of TLR4 mRNA were assayed by qRT-PCR and MeRIP-qPCR. TRDMT1 silencing in Caco2 cells and TLR4 knockout (TLR4-/-) mice were used to verify the mechanism by which SYD attenuated CD symptoms. KEY RESULTS The TLR4/NF-κB pathway was identified as the core regulatory mechanism. Administration of SYD alleviated TNBS-induced CD-like colitis in mice, as evidenced by the restoration of intestinal mucosal barrier integrity, reduced inflammation, and decreased apoptosis. At the molecular level, SYD reduced the expression of TLR4 and MyD88, suppressed NF-κB phosphorylation and nuclear translocation, and increased TRDMT1 expression. SYD also enhanced the m5C modification level of TLR4 mRNA. Furthermore, mechanistic studies indicated that TRDMT1 and TLR4 were involved in the protective effects of SYD against CD-like colitis. CONCLUSIONS AND IMPLICATIONS SYD can alleviate intestinal inflammation, inhibit apoptosis, and protect the intestinal barrier in the CD model. This protective effect may be mediated through upregulation of TRDMT1, which enhances the m5C modification of TLR4 mRNA, thereby reducing TLR4 expression and suppressing TLR4/MyD88/NF-κB signaling.