Bupivacaine at clinically relevant concentrations induces toxicity in human intervertebral disc cells via the induction of autophagy in vitro

Abstract

It has been reported that bupivacaine, the most widely used local anesthetic to relieve discogenic back pain in clinical settings, is cytotoxic to intervertebral disc (IVD) cells invitro; however, the precise mechanisms of cytotoxicity induced by bupivacaine remain unclear. Autophagy is an intracellular lysosomal degradation process that is important for cellular survival. The present study investigated the role of autophagy in the survival of IVD cells subjected to bupivacaine treatment. Human nucleus pulposus (NP) cells isolated from IVD cells were exposed to various concentrations of bupivacaine for 2, 6 and 12h, and analyzed for cellular viability using MTT assay and western blotting. Additionally, autophagosome formation and autophagy‑associated biomarkers were evaluated by electron microscopy and western blotting to determine the autophagic activity and signaling alterations in NP cells under bupivacaine treatment. Furthermore, autophagic activity was inhibited invitro using 3‑methyladenine to further analyze the association between autophagy and apoptosis in bupivacaine‑treated NP cells. Bupivacaine exhibited time‑ and dose‑dependent cytotoxic effects on human IVD cells at clinically relevant concentrations. Bupivacaine increased autophagic activity by promoting autophagosome formation, and LC3‑II and Beclin‑1 production. Additionally, bupivacaine inhibited protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/S6 kinase (S6K) signaling, which is a negative regulator of autophagic activity. Of note, pharmacological inhibition of autophagy alleviated bupivacaine‑induced cytotoxicity of IVD cells. The findings indicated that application of clinically relevant concentrations of bupivacaine upregulated autophagic activity via inhibition of Akt/mTOR/S6K signaling. In addition, the inhibition of autophagic activation served as a protective mechanism against bupivacaine‑induced cytotoxicity. Collectively, these findings may provide novel insight into the mechanisms underlying cytotoxicity induced by bupivacaine when controlling spine‑associated pain.