Role of NOX2 in bupivacaine-induced production of reactive oxygen species in nerve cells

Objective To evaluate the role of NOX2 in bupivacaine-induced production of reactive oxygen species (ROS) in nerve cells. Methods SH-SY5Y cells were seeded in culture plates and divided into 4 groups (n = 11 each) using a random number table: small interfering RNA (siRNA) negative control group (group NC), siRNA negative control plus bupivacaine group (group NC+ B), NOX2 siRNA group and NOX2 siRNA plus bupivacaine group (group NOX2 siRNA+ B). In NC and NOX2 siRNA groups, the cells were transfected with negative siRNA and NOX2 siRNA, respectively, and then incubated in the culture medium for 24 h. In NC+ B and NOX2 siRNA+ B groups, cells were transfected with negative siRNA and NOX2 siRNA, respectively, new plates were used, the cells were incubated for 3 h with bupivacaine at the final concentration of 1.5 mmol/L, the culture medium was then replaced, and the cells were incubated until 24 h. The level of intracellular ROS was measured using the fluorogenic probe dihydroethidium, the cell apoptosis was determined by TUNEL, and the expression of activated caspase-3 and caspase-9 was detected using Western blot.Apoptosis rate was calculated. Results Compared with group NC, the level of ROS and apoptosis rate were significantly increased, and the expression of activated caspase-3 and caspase-9 was up-regulated in group NC+ B (P 0.05), and no significant change was found in the level of ROS or apoptosis rate (P>0.05), and the expression of activated caspase-3 and caspase-9 was significantly up-regulated in group NOX2 siRNA (P<0.05). Compared with group NC+ B, the level of ROS and apoptosis rate were significantly decreased, and the expression of activated caspase-3 and caspase-9 was down-regulated in group NOX2 siRNA+ B (P<0.05). Conclusion NOX2 is involved in the pathophysiological mechanism of bupivacaine-induced burst production of ROS in nerve cells. Key words: NADPH oxidase; Reactive oxygen species; Bupivacaine; Neurotoxicity