Cystatin C alleviates unconjugated bilirubin-induced neurotoxicity by promoting bilirubin clearance from neurocytes via exosomes, dependent on hepatocyte UGT1A1 activity.

There is an urgent need to identify effective drugs for the treatment of nerve injury caused by unconjugated bilirubin (UCB). Our previous research found that cystatin C (CST3) alleviates UCB-induced neurotoxicity by promoting autophagy in nerve cells, but that autophagy inhibitors did not completely inhibit the effects of CST3. This study investigated whether CST3 could alleviate the neurotoxicity of UCB by promoting the secretion and transport of exosomes containing UCB to the liver for metabolism. It demonstrated that hyperbilirubinemia mice treated with CST3 had a higher number of serum exosomes than those in hyperbilirubinemia mice treated with phosphate-buffered saline. CST3-mediated protection against UCB-induced damage was abolished when autophagy and extracellular vesicle inhibitors were used in combination. The number of exosomes in the CST3 overexpression group was higher than that in the control group. Molecular docking experiments showed that UCB and CST3 had high docking score (-8.2). These results suggest that UCB may be excreted from cells by exosomes, and CST3 may promote this process by binding to UCB and entering the exosomes. We demonstrated that the effect of CST3 relied on liver cells with normal UDP-glucuronyl transferase1A1 (UGT1A1) activity in a coculture system of HT22 and L02 cells. CST3 levels were lower in exosomes secreted by L02 cells than in those secreted by human umbilical vein endothelial cells (HUVECs), whereas CST3 levels were higher in the culture supernatants of L02 cells than in the culture supernatants of HUVECs. This suggests that UCB exosomes in L02 cells may be released and internalized by CST3 and that UCB is then processed by UGT1A1 to conjugate UCB, thus reducing its toxicity. These results suggest that CST3 might alleviate UCB-induced neurotoxicity by promoting the clearance of UCB from cells via exosomes and that these effects are dependent on UGT1A1 activity in liver cells.