High glucose and bupivacaine-induced cytotoxicity is mediated by enhanced apoptosis and impaired autophagy via the PERK-ATF4-CHOP and IRE1-TRAF2 signaling pathways

Abstract

Bupivacaine has previously been reported to induce neurotoxicity, which is further enhanced by high glucose levels. In the present study, the underlying molecular mechanisms via which bupivacaine induces cytotoxicity under high glucose conditions were investigated in cultured human SH-SY5Y cells. In order to identify the optimal concentrations of glucose and bupivacaine that induced cytotoxicity, SH-SY5Y cells were treated with 30-100 mM glucose and 0.5-1.0 mM bupivacaine. Based on the dose response experiments, 50 mM glucose and 0.5 mM bupivacaine was used in the present study. The effects that 3-MA (autophagy inhibitor) and rapamycin (RA PA; autophagy inducer) exerted on cell apoptosis, autophagy and the expression of protein kinase R- like endoplasmic reticulum kinase (PER K)-activating transcription factor 4 (ATF4)-C /EBP-homologous protein (CHOP) and inositol-requiring enzyme 1 (IRE 1)-tumor necrosis factor receptor associated factor 2 (TRA F2) signaling proteins were measured in high glucose and bupivacaine-treated cells. Cell viability was measured using a Cell Counting Kit-8 assay, cell apoptosis was assessed using flow cytometry, and protein expression was determined using western blot analyses. Compared with the control group, high glucose and bupivacaine significantly increased ATF4, CHOP and caspase-12 expression, increased apoptosis, and decreased p-IRE 1, TRA F2, LC 3-II /LC 3-I and Beclin1 expression. Promoting autophagy with RA PA partly reversed the high glucose and bupivacaine-induced changes in p-PER K, CHOP, TRA F2, Beclin1, caspase-12 and apoptosis, while inhibiting autophagy with 3-MA further enhanced the changes in ATF4, CHOP, p-IRE 1, TRA F2 and apoptosis. High glucose and bupivacaine induced cytotoxicity in SH-SY5Y cells, at least in part, through enhancing cell apoptosis and inhibiting autophagy via the PER K-A TF4-C HOP and IRE 1-TRA F2 signaling pathways.