MicroRNA-183-5p protects human derived cell line SH-SY5Y cells from mepivacaine-induced injury

Abstract

With the gradual recognition of the side effects of local anesthetics, the nerve injury caused by local anesthetics has received growing attention. This research intended to delve into miR-183-5p changes in mepivacaine-mediated SH-SY5Y cell injury, as well as its modulatory mechanism on cell apoptosis. RT-qPCR was adopted for assaying miR-183-5p and PDCD4 mRNA expression. Our team respectively transfected miR-183-5p mimic and inhibitor to enhance or inhibit miR-183-5p function. We employed Western blot for detecting PDCD4 protein levels, as well as flow cytometry and Hoechst 33342/PI double staining for determining cell apoptosis rate. Additionally, our crew applied an ELISA kit for measuring TNF-α, IL-1β, IL-6, and IL-8 contents. The level of reactive oxygen species (ROS) production was examined by the Image-iT LIVE Green ROS detection Kit. As well as dual-luciferase reporter experiment for verifying the targeting link of miR-183-5p with PDCD4. In mepivacaine-induced cell apoptosis in SH-SY5Y cells, miR-183-5p expression was down-regulated. TNF-α, IL-1β, IL-6, and IL-8 contents were elevated. The rate of apoptosis increased visibly, cleaved caspase-3 and Bax levels waxed, whereas Bcl-2 level waned. MiR-183-5p could alleviate the damaging impact of mepivacaine. Dual-luciferase reporter experiments demonstrated that miR-183-5p directly targeted PDCD4. Collectively, we concluded that a high concentration of mepivacaine can cause SH-SY5Y cell damage, miR-183-5p functions crucially in mepivacaine-mediated cell damage. This study provides a theoretical basis for elucidating the mechanism of mepivacaine-induced nerve cell damage, and overexpressed miR-183-5p likely become a novel strategy to combat mepivacaine-induced nerve damage.Abbreviations:miRNA: Micro RNA; PDCD4: Programmed Cell Death 4; MDA: Malondialdehyde; SOD: Superoxide Dismutase; ROS: Reactive Oxygen Species; WT: Wild Type; Mut: Mutant; UTR: Untranslated Region; IL-6: Interleukin-6; IL-1β: Interleukin-1β; TNF-α: Tumor Necrosis Factor-α; IL-8: Interleukin-8; COX-2: Cyclooxygenase-2; iNOS: inducible NOS; MEP: Mepivacaine.

Keywords: Mepivacaine; PDCD4; apoptosis; miR-183-5p.

Graphical abstract

Figure 1.

Effect of mepivacaine on miR-183-5p and PDCD4 expression in SH-SY5Y cells. (a) Mepivacaine could decline miR-183-5p level in a concentration-dependent manner. (b) Mepivacaine could augment PDCD4 mRNA expression level in a concentration-dependent manner. (c) We detected miR-183-5p expression in cells to verify the transfection efficiency. (d) PDCD4 mRNA level was measured after cell transfection and mepivacaine (10 mM) treatment. (e) PDCD4 protein level was measured after cell transfection and mepivacaine (10 mM) treatment.*P< 0.05, **P< 0.01 vs. sham group; #P< 0.05, ##P< 0.01, ###P< 0.001 vs. mepivacaine group

Figure 2.

MiR-183-5p targeted PDCD4. (a) The forecasted target sequence of PDCD4 on the 3ʹ-UTR of miR-183-5p. Schematic diagrams showed the mutation in its binding site on the UTR. (b) Dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-183-5p and PDCD4 in SH-SY5Y cells. Luciferase activity was decreased by miR-183-5p mimics. WT: wild-type; Mut: mutant. *P< 0.05, **P< 0.01 (* miR-183-5p vs. miR-NC)

Figure 3.

Influence of miR-183-5p on apoptosis induced by mepivacaine. (a) Apoptosis was quantified by flow cytometry using propidium iodide exclusion. (b) Hoechst33342/PI double staining to monitor the apoptosis rate. (c) Western blot to further determine C-caspase-3, Bax, and Bcl-2 expression levels. MiR-183-5p mimics and miR-183-5p inhibitor changed the expression level of these proteins.*P< 0.05, **P< 0.01 vs. sham group; #P< 0.05, ##P< 0.01 vs. mepivacaine group

Figure 4.

MiR-183-5p attenuated ROS and oxidative damage in SH-SY5Y cells. (a~b) ELISA kit to assay the content of malondialdehyde (MDA) and superoxide dismutase(SOD). (c) Qualitative detection of the steady-state levels of intracellular reactive oxygen species (ROS) by fluorescence microscopy.**P< 0.01, ***P< 0.001 vs. sham group; #P< 0.05, ##P< 0.01 vs. mepivacaine group

Figure 5.

Impact of miR-183-5p on the release of inflammatory factors from SH-SY5Y cells. (a~d) Impact of miR-183-5p on the secretion of IL-6, TNF-α, IL-1β, and IL-8 by SH-SY5Y cells. (e) The protein expression of COX-2 and iNOS in the groups. β-actin was used as an invariant control for calculating protein fold changes. ***P< 0.001 vs. Sham group; #P< 0.05, ##P< 0.01, ###P< 0.001 vs. mepivacaine group