Lianweng formula restores intestinal barrier function in ulcerative colitis through the β-arrestin 1/NF-κB signaling axis.

Background: Ulcerative colitis (UC) is a chronic inflammatory disorder with a high incidence rate. UC has emerged as a significant global public health challenge mostly because of the limitations of current therapeutic strategies. The Lianweng formula (LWF) has exhibited promising efficacy in UC management; however, its precise mechanisms of action remain elusive, hindering its clinical translation and optimization.

Purpose: This study aimed to evaluate the effectiveness of LWF in treating UC and elucidate the underlying mechanisms.

Methods: This study systematically evaluated the therapeutic effects of LWF on the UC rat model using a multidimensional approach. First, disease progression was assessed using disease activity index (DAI) scoring, histopathological analysis, and proinflammatory cytokine quantification. Second, the restorative effects of LWF on colonic barrier integrity were investigated through western blotting, RT-qPCR, and immunofluorescence staining. Third, proteomic profiling was performed to identify the key signaling pathways mediating the therapeutic effects of LWF. Fourth, serum pharmacochemistry combined with high-throughput screening was applied to identify the bioactive components of LWF. Finally, in vitro mechanistic validation of target engagement and regulatory mechanisms was achieved through molecular dynamics simulations, a drug affinity responsive target stability (DARTS), cellular enthusiasm transfer assay (CETSA), dual-luciferase reporter assays, co-ip assay, small molecule pull-down experiments, and site-directed mutagenesis.

Results: LWF significantly alleviated pathological damage to the colon and suppressed the surge of proinflammatory cytokine in the UC rat model. Furthermore, LWF restored intestinal barrier integrity by increasing the goblet cell density and elevating the expressions of tight junction (TJ) proteins in colonic tissues. Proteomic profiling revealed that the β-arrestin 1 (Arrb1)/NF-κB axis is a central mechanism mediating the therapeutic effects of LWF. The results of high-throughput screening demonstrated that vanillin (VA), a blood-entry migratory component of LWF, might be a key compound involved in the therapeutic effects of LWF. The in vitro validation studies revealed that VA directly targeted the Ser302 residue of Arrb1, thereby inhibiting NF-κB transcriptional activation and nuclear translocation and ultimately preserving epithelial barrier function. These findings were rigorously validated through rescue experiments and site-directed mutagenesis assays, which confirmed the specificity of the VA-Arrb1 interaction and its functional relevance to barrier restoration.

Conclusion: LWF alleviated UC symptoms in a rat model by restoring intestinal barrier function through the modulation of the Arrb1/NF-κB signaling axis. Furthermore, VA was identified as the primary bioactive component of LWF, with its therapeutic efficacy mediated by the direct targeting of the Ser302 residue on Arrb1, resulting in suppressed NF-κB activation. This study establishes a novel paradigm that integrates serum pharmacochemistry and high-throughput screening technologies, which could serve as a viable solution to the longstanding challenge of identifying bioactive constituents in traditional Chinese medicine (TCM).