The protective effect of amitriptyline on experimental colitis through inhibiting TLR-4/MD-2 signaling pathway

Amitriptyline, a pleiotropic tricyclic antidepressant, possesses antioxidant and anti-inflammatory properties. Despite its diverse benefits, the specific effects of amitriptyline on inflammatory bowel disease (IBD) are not yet well defined. To explore this, we used a dextran sulfate sodium (DSS)-induced colitis model to examine the anti-inflammatory effects of amitriptyline and the underlying mechanisms by which it operates. Our research revealed that amitriptyline is effective in alleviating several pathological manifestations associated with colitis. This includes improving body weight retention, reducing disease activity index, lessening of colon length shortening, and repairing of colonic mucosal damage. Treatment with amitriptyline significantly protected mucosal injury by preserving the population of goblet cells and increasing the expression of tight junction proteins. Furthermore, we observed that amitriptyline effectively countered immune cell infiltration, specifically neutrophils and macrophages, while simultaneously lowering the levels of inflammatory cytokines such as tumor necrosis factor α, interleukin (IL)-1β, and IL-6. Additionally, RNA sequencing analysis pointed to the potential involvement of the Toll-like receptor (TLR) pathway in the anticolitic effects induced by amitriptyline. Subsequent Western blot analysis indicated that amitriptyline significantly inhibited the TLR-4-mediated nuclear factor (NF)-κB signaling pathway. To bolster our findings, in vitro studies demonstrated that amitriptyline downregulated the TLR-4/NF-κB/mitogen-activated protein kinase signaling cascades in mouse macrophages stimulated with lipopolysaccharide. Further molecular investigations revealed that amitriptyline was able to suppress the elevated expression of myeloid differentiation factor 2 that lipopolysaccharide stimulation typically induces. In summary, our findings suggest that amitriptyline effectively mitigates DSS-induced colitis in mice through the inhibition of TLR-4/myeloid differentiation 2 pathway signaling, indicating its potential repurposing for IBD treatment. SIGNIFICANCE STATEMENT: The potential of using amitriptyline in treating inflammatory bowel disease appears promising, leveraging its established safety and dosing profile as an antidepressant. The study results show that amitriptyline can alleviate pathological symptoms, inflammation, and intestinal mucosal damage in mice with colitis induced by DSS. The protective effect observed appears to be linked to the inhibition of TLR-4/myeloid differentiation 2 signaling pathway. By exploring novel applications for existing medications, we can optimize amitriptyline's efficacy and broaden its impact in both medical and commercial contexts.