Bilirubin impacts microglial autophagy via the Akt-mTOR signaling pathway.

Abstract

Bilirubin encephalopathy is a severe complication of neonatal hyperbilirubinemia. With elevation of serum unconjugated bilirubin (UCB) levels, UCB crosses the blood-brain barrier and possibly leads to neurological dysfunction. Neuroinflammation is recognized as a prominent pathological feature in bilirubin encephalopathy. Recent studies have suggested that autophagy plays a crucial role in the inflammatory response. However, the potential effect of microglial autophagy in the pathogenesis of bilirubin encephalopathy remains uncertain. The invitro findings verified that in primary cultured microglia, UCB significantly reduced the ratio of LC3B-II to LC3B-I and downregulated the expression of ATG5, Beclin-1, and ATG7, while increasing the expression of p62/SQSTM1. The results showed that UCB could decrease the number of mCherry-EGFP-LC3 positive puncta, even when chloroquine (CQ) was applied to block the microglial autophagy flux. Mechanistically, UCB was found to upregulate the expression of TLR4 and increase the phosphorylation levels of Akt and mammalian target of rapamycin (mTOR). Promoting microglial autophagy by treatment with Rapamycin (RAPA), an mTOR inhibitor, decreased the levels of NOD-like receptor protein 3 (NLRP3) inflammasome components and IL-1β, rescued microglial overactivation, and improved neurological functions. These data indicated that UCB could impact microglial autophagy via the Akt-mTOR signaling pathway and synergistically promote neuroinflammatory responses. Enhancing autophagy might disrupt the assembly of NLRP3 inflammasome, attenuate UCB-induced neuroinflammation, and improve the prognosis of model rats with bilirubin encephalopathy. In conclusion, this study implies that regulating microglial autophagy might be a promising therapeutic strategy for bilirubin encephalopathy.