Shasha Liu, Ling Li, Jing Li, Siyu Li, Jiaqi Chen, Qian Sun, Yan Zhang, Ziyu Hua
In summary, UCB-induced microglial lipophagy is a key mechanism to link UCB to pathogenic LDs accumulation and neuroinflammation in BE. Pharmacological restoration of lipophagic flux through AMPK-mTOR activation mitigates these effects, reduces neuroinflammation, and attenuates neuronal synaptic injury. Therefore, modulating microglial lipophagy might be a promising therapeutic strategy for BE.
BACKGROUND: Hyperbilirubinemia arises from disrupted bilirubin homeostasis, which can lead to bilirubin encephalopathy (BE). Unconjugated bilirubin (UCB)-induced neuroinflammation plays a pivotal role in the pathogenesis and progression of BE.
PURPOSE: To delineate the role and molecular mechanisms of lipophagy in UCB-evoked neuroinflammation.
METHODS: Western blot, qPCR, immunofluorescence, ELISA, transmission electron microscopy, mCherry-EGFP-LC3 tandem fluorescent reporter, BODIPY staining, Nissl staining, and behavioral tests were employed to evaluate differences in lipophagy, LDs accumulation, neuroinflammation, and neurological function.
RESULTS: UCB significantly increased PLIN2 expression and BODIPY mean fluorescence intensity, indicating excessive LDs accumulation in microglia. This was accompanied by impaired autophagic flux, as evidenced by a reduced LC3B-II/LC3B-I ratio, elevated p62/SQSTM1, and decreased mCherry-EGFP-LC3 puncta. Furthermore, the loss of colocalization between LC3 and BODIPY confirmed impaired lipophagy. The AMPK activator AICAR restored AMPK phosphorylation, suppressed mTOR activity, and reinstated autophagic flux, thereby reducing LDs accumulation. Concurrently, AICAR reduced NLRP3 and ASC levels and decreased the release of IL-6, IL-1β, and TNF-α. Moreover, compared with UCB-MCM, UCB+AICAR-MCM reduced neuronal NLRP3 and ASC levels and restored synaptic proteins synaptophysin (SYN) and HOMER1. Furthermore, these findings were corroborated in vivo, as AICAR mitigated UCB-induced neuroinflammation, attenuated neuronal synaptic injury, and improved neurological function in BE model rats.
CONCLUSION: In summary, UCB-induced microglial lipophagy is a key mechanism to link UCB to pathogenic LDs accumulation and neuroinflammation in BE. Pharmacological restoration of lipophagic flux through AMPK-mTOR activation mitigates these effects, reduces neuroinflammation, and attenuates neuronal synaptic injury. Therefore, modulating microglial lipophagy might be a promising therapeutic strategy for BE.