Spinal Cathepsin S promotes visceral hypersensitivity via FKN/CX3CR1/p38 MAPK signaling pathways

BACKGROUND: Irritable bowel syndrome (IBS) is one of the typical representatives of chronic functional visceral pain that lacks effective treatment. Recently, attention has been given to the role of microglia in IBS, particularly the activation of spinal microglia and the subsequent release of Cathepsin S (Cat S), a proteolytic enzyme. However, the specific role of spinal Cat S in IBS remains to be elucidated. The purpose of this study is to investigate the mechanisms underlying the regulation of visceral hypersensitivity in IBS-like rats by Cat S. METHODS: An IBS-like rat model was developed, and visceral sensitivity was tested via the electromyographic (EMG) response to colorectal distention (CRD) and pain threshold. Western blot and immunofluorescence were used to examine the expressions of proteins. The effects of inhibitors or neutralizing antibodies on visceral pain and the downstream molecular expressions were detected. The open-field test was performed to evaluate locomotor activity and anxiety-like behaviors in rats. RESULTS: We discovered that spinal Cat S was upregulated and colocalized with microglia in IBS-like rats. Treatment with LY3000328, a selective inhibitor of Cat S, dose-dependently down-regulated EMG amplitude and Fractalkine (FKN) expression, indicating that Cat S regulated visceral hypersensitivity via activating FKN in IBS-like rats. Furthermore, the expressions of FKN, CX3CR1, and p-p38 MAPK were elevated in IBS-like rats whereas inhibition of these molecules could alleviate visceral pain. Moreover, pharmacological inhibitor experiments suggested the activation of CX3CR1 by FKN facilitated p38 MAPK phosphorylation, which in turn promoted Cat S expression in IBS-like rats. CONCLUSIONS: Neonatal adverse stimulation might enhance the expression of spinal microglial Cat S, thereby activating the FKN/CX3CR1/p38 MAPK pathway and lead to visceral hypersensitivity in IBS-like rats. As a selective inhibitor of Cat S, LY3000328 could become a potential therapeutic option for IBS.