Human Bone Marrow Mesenchymal Stem Cells-Derived Exosomal miRNA-21-5p Inhibits Lidocaine-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells

Abstract

Background: Local anesthetic lidocaine is one of the most common pain therapies, but high concentration of lidocaine induced neurotoxicity and its mechanism is unclear. Exosomal microRNAs (miRNAs) is implicated in neuronal diseases, but its role in lidocaine induced neurotoxicity remains to be elucidated.

Methods: All the experiments were performed at Huzhou Key Laboratory of Molecular Medicine, Huzhou City, Jiangsu Province, China in 2022. Lidocaine was used to induce apoptosis of SH-SY5Y cells. Exosomes isolated from bone marrow mesenchymal stem cells (BMSC-exos) were used to co-treat SH-SY5Y cells with lidocaine. Cell apoptosis was measured using a flow cytometer. PKH-67 Dye was used for exosome uptake assay. miR-21-5p mimics/inhibitors, or negative controls were transfected with Lipo2000 to study its effect on lid-induced injury. Interactions between miR-21-5p and PDCD4 was analyzed by luciferase reporter assay.

Results: Administration of BMSC-exo protected SH-SY5Y cells against lidocaine induced apoptosis. Suppressing miR-21-5p dramatically enhanced PDCD4, but miR-21-5p overexpression sharply down-regulated PDCD4. Mechanism study showed that miR-21-5p bound to 3'-UTR of PDCD4 to inhibit it. Suppressing miR-21-5p reversed the effect of BMSC-exo on Lid-induced injury. Results also indicate that miR-21-5p regulated lidocaine-induced injury through targeting PDCD4.

Conclusion: BMSC-exos protected SH-SY5Y cells against lidocaine induced apoptosis through miR-21-5p by targeting PDCD4, which may develop new strategy in the management of lidocaine-induced neurotoxicity.

Keywords: Apoptosis; Exosomes; Lidocaine; Microrna; Neuronal toxicity; Programmed cell death protein 4 (PDCD4).

Fig. 1:

BMSC-exo protected against lidocaine-mediated apoptosis in a dose-dependent manner SH-SY5Y cells were provided with lidocaine (0, 1, 2, and 5mM) for 24 hours. (A) Apoptosis analysis. **P < 0.01, ***P < 0.001 vs. Vehicle; #P < 0.05 vs. 1 mM_Lid. (B) SH-SY5Y cells were treated with idocaine (2mM) and BMSC-exo (0, 50, 100, and 200 μg/ml) for 24 hours, and apoptosis was analyzed. *** P < 0.001 vs. Ctrl; ^# P < 0.05, ^## P < 0.01 vs. 2 mM_Lid+Vehicle; ⁺P < 0.05 vs. 2 mM_Lid+50 μg/ml BMSC_exo

Fig. 2:

Isolation and identification of exosomes from human bone mesenchymal stem cells (BMSC-exo). (A) Flow cytometry analysis of BMSC cell surface markers. (B-C) BMSC-exos were isolated using ultra-high-speed centrifugation and characterized by TEM (B) and immunoblotting analysis of exosomal markers (C). (D) Co-cultured of PKH-67-labeled BMSC-exos with SH-SY5Y cells showed that BMSC-exos could be endocytosed by SH-SY5Y cells. (E) miR-21-5p inhibitor or mimic was transfected into BMSCs and BMSC-exos were isolated using ultra-high-speed centrifugation. miR-21-5p expression was measured by Q-PCR. *P < 0.05, **P < 0.01 vs. NC

Fig. 3:

miR-21-5p negatively regulated PDCD4. WT or mutant 3′-UTR of PDCD4 and miR-21-5p inhibitor/mimic were transfected into SH-SY5Y cells. (A) Q-PCR measurement of miR-21-5p expression. (B-C) Q-PCR and immunoblot analysis of PDCD4. ** P < 0.01, *** P < 0.001 vs. NC. (D) Luciferase reporter assay of PDCD4 and miR-21-5p. **P < 0.01, ***P < 0.001 vs. WT+NC

Fig. 4:

Inhibition of miR-21-5p reversed the effect of BMSC-exo on lidocaine-induced injury. SH-SY5Y cells were treated with lidocaine (2mM) and exosomes isolated from BMSC-transfected with inhibitor-exo) or mimic-exo (100 μg/ml) for 24 hours. (A) Apoptosis analysis. (B) miR-21-5p levels. (C-D) PDCD4 levels. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Ctrl; ^# P < 0.05, ^## P < 0.01 vs. 2 mM_Lid+Ctrl; +P < 0.05, ++ P < 0.01 vs. 2 mM_Lid+NC-exo

Fig. 5:

miR-21-5p regulated lidocaine-induced injury probably through targeting PDCD4. PDCD4 plasmids were used to transfect SH-SY5Y to overexpress PDCD4. (A) Q-PCR and (B) immunoblot analysis of PDCD4. ***P < 0.001 vs. Vector. PDCD4-overexpression SH-SY5Y cells were treated with idocaine (2mM) and BMSC-exo (100μg/ml) for 24 hours. (C) Immunoblot analysis of PDCD4. (D) Flow cytometry analysis of apoptosis. **P < 0.01 vs. Control; ##P < 0.01 vs. 2 mM_Lid+Vehicle+Vector; +++P < 0.001 vs. 2 mM_Lid+exo+Vector