Ciprofol Promotes SIRT1-Mediated Raf Regulation of ERK1/2 Hyperphosphorylation.

Epilepsy (EP) is characterized by sudden abnormal discharges of neurons in the brain. Its complex pathological mechanisms limit effective treatment strategies. In recent years, Ciprofol, as a novel 2,6-disubstituted phenol derivative, has attracted extensive attention due to its unique molecular structure and diverse biological functions. This study aims to investigate the mechanism by which Ciprofol exerts anti-temporal lobe epilepsy (TLE) effects: specifically, Ciprofol upregulates SIRT1 (a NAD+-dependent deacetylase) expression to trigger its deacetylase activity, thereby downregulating Raf (an upstream factor of MAPK/ERK pathway) and further modulating ERK phosphorylation in the MAPK/ERK signaling pathway. Behavioral analysis showed that Ciprofol significantly reduced the severity of acute seizures in kainic acid (KA)- and pentylenetetrazol (PTZ)-induced acute TLE mouse models. Further molecular biology experiments, using techniques such as electrophysiological recordings, Western blotting, metabolomics, transcriptome sequencing, and qPCR, revealed that Ciprofol decreased the amplitude of induced excitatory postsynaptic currents (EPSCs) in the hippocampus and significantly downregulated the expression levels of postsynaptic NMDA2B and NMDA2A receptor proteins; transcriptome sequencing indicated prominent enrichment of MAPK/ERK pathway-related genes; LC-MS (metabolomics) showed GABA release remained unchanged while glutamate (Glu) levels were significantly reduced. LC-MS measurements demonstrated that GABA release remained unchanged following Ciprofol treatment, while glutamate (Glu) levels were significantly reduced. In addition, bubble plot analysis indicated that the MAPK/ERK signaling pathway was prominently involved in Ciprofol’s anti-epileptic effects.Further pharmacological interventions confirmed the critical role of the MAPK/ERK signaling pathway. Administration of the ERK1/2 inhibitor Ulixertinib and experiments using ERK1/2-specific knockout mice significantly enhanced the anti-epileptic effects of Ciprofol. In contrast, treatment with the broad-spectrum ERK1/2 activator Okadaic Acid markedly weakened these effects. These results further validated the key role of ERK1/2 in the MAPK/ERK signaling pathway in mediating the anti-temporal lobe epilepsy effects of Ciprofol. Moreover, this study identified that SIRT1 regulates the expression of the upstream factor Raf through its deacetylation activity, thereby influencing the phosphorylation state of ERK1/2 in the MAPK/ERK signaling pathway. Therefore, for the first time, this study elucidated that Ciprofol exerts anti-TLE effects by upregulating SIRT1 to mediate Raf deacetylation and inhibit ERK1/2 hyperphosphorylation in the MAPK/ERK pathway—directly linking Ciprofol, SIRT1, and anti-epileptic action. Ciprofol exerts its antiepileptic effects on temporal lobe epilepsy by mediating the downregulation of Raf expression through SIRT1, thereby modulating the excessive phosphorylation of ERK1/2 in the MAPK/ERK signaling pathway. This, in turn, reduces neuronal eEPSCs and the release of excitatory neurotransmitters.