Inhibition of chloride intracellular channel protein 1 (CLIC1) ameliorates liver fibrosis phenotype by activating the Ca2+-dependent Nrf2 pathway

Persistent damage to liver cells leads to liver fibrosis, which is characterized by the accumulation of scar tissue in the liver, ultimately leading to cirrhosis and serious complications. Because it is difficult to reverse cirrhosis once it has progressed, the primary focus has been on preventing the progression of liver fibrosis. However, studies on therapeutic agents for liver fibrosis are still lacking. Here, we investigated that the natural dipeptide cyclic histidine-proline (CHP, also known as diketopiperazine) shows promising potential as a therapeutic agent in models of liver injury by inhibiting the progression of fibrosis through activation of the Nrf2 pathway. To elucidate the underlying biological mechanism of CHP, we used the Cellular Thermal Shift Assay (CETSA)-LC–MS/MS, a label-free compound-based target identification platform. Chloride intracellular channel protein 1 (CLIC1) was identified as a target whose thermal stability is increased by CHP treatment. We analyzed the direct interaction of CHP with CLIC1 which revealed a potential interaction between CHP and the E228 residue of CLIC1. Biological validation experiments showed that knockdown of CLIC1 mimicked the antioxidant effect of CHP. Further investigation using a mouse model of CCl4-induced liver fibrosis in wild-type and CLIC1 KO mice revealed the critical involvement of CLIC1 in mediating the effects of CHP. Taken together, our results provide evidence that CHP exerts its anti-fibrotic effects through specific binding to CLIC1. These insights into the mechanism of action of CHP may pave the way for the development of novel therapeutic strategies for fibrosis-related diseases. [Display omitted] •Label-free CETSA-LC-MS/MS is a powerful tool for target identification of an unmodified chemical.•CLIC1 was newly identified as the target protein of Cyclic(His-Pro).•In silico docking assay reveals the hotspots of Cyclic(His-Pro) and CLIC1 interactions.•Cyclic(His-Pro) regulates the Nrf2 pathway through CLIC1 and contributes to the alleviation of fibrosis.