Conclusions
Propofol protected hippocampal neurons from I/R injury through two independent signaling pathways, including the calcineurin/FKBP12.6-RyR/calcium overload pathway and the RhoA/Lats1/YAP/Bcl-2 pathway.
Methods
Hypoxia-reoxygenated (H/R) HT-22 cells were used to mimic I/R injury of the hippocampus in vitro. An MTT assay was used to determine cell viability. Cell apoptosis was detected by a TUNEL assay and a flow cytometry cell apoptosis assay. Expression levels of proteins were measured by Western blotting. Intracellular calcium was assessed by Fura-2/AM staining. Flow cytometry was used to determine the mitochondrial membrane potential (MMP). Coimmunoprecipitation was used to evaluate the stability of the FKBP-RyR complex. Calcineurin enzymatic activity was measured with a colorimetric method. YAP nuclear translocation was tested by immunofluorescence staining.
Results
H/R induced HT-22 cell viability depression, and apoptosis was reversed by propofol treatment. Propofol could alleviate H/R-induced intracellular calcium accumulation and MMP loss by inhibiting calcineurin activity and FKBP12.6-RyR disassociation in a concentration-dependent manner. In addition, YAP expression was crucial for propofol to protect HT-22 cell apoptosis from H/R injury. Propofol could activate YAP through dephosphorylation. Activated YAP stimulated the transcription of the Bcl2 gene, which promotes cellular survival. Our data also demonstrated that propofol activated YAP through the RhoA-Lats1 pathway without large G proteins or MST involvement. In addition, we showed that there was no interaction between calcineurin signaling and YAP activation in HT-22 cells. Conclusions: Propofol protected hippocampal neurons from I/R injury through two independent signaling pathways, including the calcineurin/FKBP12.6-RyR/calcium overload pathway and the RhoA/Lats1/YAP/Bcl-2 pathway.