Propofol alleviates OGD/R-induced cerebral ischemia-reperfusion injury-related neuronal injury by regulating NEDD4L-mediated ubiquitination of HTR2B.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is an important pathological process in ischemic brain injury. Previous studies have shown that propofol can alleviate CIRI-related neuronal injury; however, the underlying molecular mechanisms remain incompletely understood. METHODS: An in vitro CIRI-related injury model was established by exposing SK-N-SH cells to oxygen-glucose deprivation/reoxygenation (OGD/R), followed by treatment with different concentrations of propofol. Cell viability, apoptosis, inflammatory cytokine levels, oxidative stress, and mitochondrial membrane potential (MMP) were assessed using the Cell Counting Kit-8 (CCK-8) assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, enzyme-linked immunosorbent assay (ELISA), specific assay kits, and JC-1 staining, respectively. Potential targets of propofol were predicted using the SwissTargetPrediction database. The messenger RNA (mRNA) and protein expression levels of related genes were detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot. UbiBrowser was used to predict ubiquitin ligases related to 5-hydroxytryptamine receptor 2B (HTR2B). Ubiquitination assay, MG132 treatment, and cycloheximide (CHX) chase assay were performed to verify the regulatory effect of neural precursor cell expressed developmentally down-regulated 4-like (NEDD4L) on HTR2B ubiquitination and protein stability. Finally, a middle cerebral artery occlusion (MCAO) model was established for in vivo validation. RESULTS: Propofol significantly alleviated OGD/R-induced SK-N-SH cell injury, inflammatory responses, oxidative stress, and mitochondrial dysfunction (P < 0.05). HTR2B knockdown attenuated these OGD/R-induced injury phenotypes (P < 0.05). HTR2B overexpression weakened the protective effects of propofol against OGD/R-induced SK-N-SH cell injury (P < 0.05). NEDD4L promoted K48-linked ubiquitination of HTR2B and reduced HTR2B protein stability (P < 0.05). HTR2B overexpression reversed NEDD4L overexpression-mediated protection in OGD/R-treated SK-N-SH cells (P < 0.05). NEDD4L knockdown aggravated OGD/R-induced cell injury and weakened propofol-mediated protection, whereas NEDD4L overexpression further enhanced the protective effects of propofol (P < 0.05). In vivo, propofol reduced infarct volume, improved neurological deficits, alleviated brain histopathological injury, and regulated the NEDD4L/HTR2B axis and inflammatory cytokine levels in MCAO mice (P < 0.05). CONCLUSION: Propofol alleviated CIRI-related neuronal injury at least partly by promoting NEDD4L-mediated ubiquitination of HTR2B, providing new experimental evidence for its neuroprotective effects.