Abstract
Purpose: To determine the effect of quercetin on bupivacaine-induced neurotoxicity and to investigate the mechanisms involved. Methods: Cultured SH-SY5Y cells were divided into five treatment groups: control group with no drug, bupivacaine treatment group, quercetin group, bupivacaine--quercetin combination treatment group, and bupivacaine-mibefradil combination treatment group. Cell morphology in each group was examined by microscopy while cell viability was assessed by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2- tetrazolium bromide (MTT) assay after 24 h incubation. Cytosolic calcium ion concentration was determined by flow cytometry while Cav3.1 protein expression was evaluated by western blot. Results: Quercetin (50 μM) significantly ( p < 0.05) protected SH-SYS5 cells from bupivacaine-induced cell apoptosis and also significantly reduced intracellular calcium ion concentration ( p < 0.01) by approximately 40 %. Cav3.1 protein expression was normalized following quercetin treatment. Conclusion: These results show that quercetin reduces the neurotoxicity induced by bupivacaine, possibly through inhibition of T-type calcium channel. This finding implies a novel mechanism for neuroprotective effect of quercetin, and its potential for treating toxicity arising from the use of local anesthetic agents. Keywords: Quercetin, Bupivacaine, Local anaesthetic, Neuroprotection, Neurotoxicity, T-type calcium channel
Highlights
Local anesthetic agents are widely used in hospitals; they are associated with certain side effects, especially neurotoxicity
It has been shown that neurotoxicity caused by local anesthetics such as lidocaine and bupivacaine are related to changes in calcium homeostasis, resulting in intracellular calcium overload [1]
The sample cells were lysed and the total protein concentration in the supernatant was determined by the BCA protein assay kit (Fisher Scientific, Waltham, MA, USA). 20 μg of protein from each sample was separated by 12 % SDSPAGE before transferring to polyvinylidene difluoride (PVDF) membrane for western blotting
Summary
Local anesthetic agents are widely used in hospitals; they are associated with certain side effects, especially neurotoxicity. SH-SY5Y cells were cultured in DMEM/F12 medium with 10 % FBS, (with 150 μg/mL streptomycin and 150 units/mL penicillin) at 37 °C in a humidified CO2 incubator. The cells were seeded into 96-well plates (four thousand cells per well) with 200 μL culture media per well, and incubated overnight before drug treatment. Intracellular calcium ion concentration was analyzed using commercially available Fluo-8® AM ester probe in accordance with the product instruction manual. A testing solution (10 μM) was prepared with the cell culture media (without FBS) containing 0.02 % Pluronic® F-127. Each 200 μL of testing solution was added to the culture media in the plates with live cells (final probe concentration was kept at 5 μM). The dissociation constant (Kd), reflecting the affinity of the probe for calcium, was set at the theoretical value (389 nM) as provided in the manual
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