Shaoyao decoction inhibits neutrophil migration and restores epithelial integrity in ulcerative colitis model mice:...

Ulcerative colitis (UC) is a chronic inflammatory disease that seriously endangers human health. Shaoyao Decoction (SYD) has been widely used in clinical practice to treat UC; however, its active components and molecular mechanisms remain unclear.

To elucidate the pharmacological mechanisms by which SYD and its active constituents ameliorate UC using single-cell RNA sequencing (scRNA-seq).

We first assessed the therapeutic efficacy of SYD in a dextran sulfate sodium (DSS)-induced UC mouse model. We then applied scRNA-seq to identify the potential cell populations and molecular pathways involved in SYD-mediated protection. Guided by these findings, we verified the inferred mechanisms in vivo and screened potential active compounds from SYD. We further examined their inhibitory effects on formyl-methionyl-leucyl-phenylalanine (fMLP)-induced neutrophil migration and their protective effects on DSS-induced Caco-2 cell injury in vitro. Finally, we validated the underlying mechanisms using inhibitors, agonists, gene silencing, and overexpression approaches in vitro.

SYD markedly alleviated weight loss, reduced disease activity index (DAI) scores, and mitigated colon shortening and histopathological damage in mice with UC. The scRNA-seq analysis revealed alterations in neutrophils and intestinal epithelial cells. Functional pathway analysis indicated that neutrophil migration, epithelial tight junction regulation, and apoptosis were key processes modulated by SYD. In vivo, SYD decreased neutrophil infiltration and downregulated the expression of RAC1, RAC2, S100A8, and S100A9. Concurrently, SYD upregulated the tight junction proteins TJP1 and OCLN and suppressed epithelial apoptosis. In vitro, Ferulic acid emerged as the potential compound responsible for suppressing neutrophil migration. RAC1 inhibition and Ferulic acid treatment produced comparable suppressive effects on neutrophil migration, whereas RAC1 activation effectively antagonized the inhibitory effect of Ferulic acid. Chrysin-7-O-glucuronide was identified as the potential component enhancing tight junction integrity and suppressing apoptosis in intestinal epithelial cells; silencing KLF4 eliminated these protective effects of Chrysin-7-O-glucuronide. Conversely, KLF4 overexpression exerted protective effects comparable to those of Chrysin-7-O-glucuronide treatment.

SYD exerts potent therapeutic effects against UC. Mechanistically, Ferulic acid in SYD suppresses RAC1-mediated neutrophil migration, while Chrysin-7-O-glucuronide enhances tight junction integrity and suppresses apoptosis in intestinal epithelial cells through a KLF4-dependent mechanism.