P53 inhibitor pifithrin-α inhibits ropivacaine-induced neuronal apoptosis via the mitochondrial apoptosis pathway

The neurotoxicity of local anesthetics (LAs) has attracted more and more attention, However, they lack preventive and therapeutic measures. Many studies have shown that apoptosis plays an important role in the process of LA-induced neurotoxicity. As an important signaling molecule to activate apoptosis, p53 has been proved to be involved in the neurotoxicity induced by LAs, but the mechanism is unclear. In this study, we explored the effect of pifithrin-alpha (PFT-alpha), a p53 inhibitor, on apoptosis by ropivacaine (Rop) in vivo and in vitro. Cell viability and apoptosis detected by CCK-8 and a JC-1 apoptosis detection kit, the changes of spinal cord structure observed after hematoxylin and eosin staining, apoptosis of the spinal cord measured by terminal deoxynucleotidyl transferase dUTP nick end labeling staining, behavioral assessment of the nerve Injury evaluated by the detection of sciatic nerve conduction velocity (SNCV) andmechanical withdrawal threshold (MWT), the expression of p53 and many apoptosis-related genes included Bax, Bcl-2, and caspase-3 detected by quantitative real-time polymerase chain reaction, Western blot analysis, immunofluorescence, and immunohistochemistry. Results showed that PC12 cell viability decreased because of Rop, but the pretreatment of PFT-alpha could protect it. And PFT-alpha reduced the injuries in the spinal cord by Rop included vacuoles or edema. The results of immunofluorescence and immunohistochemistry testing showed that PFT-alpha inhibited the p53 protein upregulated by Rop. Apoptosis rate and many proapoptotic genes include p53, Bax, caspase-3 messenger RNA, and proteins were increased by Rop, but PFT-alpha could decrease it. In conclusion, PFT-alpha inhibited cell apoptosis and spinal cord injuries induced by Rop.