Dexmedetomidine protects PC12 cells from lidocaine-induced cytotoxicity via downregulation of Stathmin 1

Abstract

Background: Understanding of lidocaine-induced neurotoxicity is not complete, resulting in the unsuccessful treatment in some clinical settings. Dexmedetomidine (DEX) has been shown to alleviate lidocaine-induced neurotoxicity in our previous cell model. However, the rationale for DEX combined with lidocaine to reduce lidocaine-induced neurotoxicity in the clinical setting remains to be further clarified in the detailed molecular mechanism. Methods: In this study, we established a cellular injury model by lidocaine preconditioning. Cell Counting Kit-8 (CCK-8) and 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay kit were used to analyze cell proliferation. Cell apoptosis was measured by flow cytometry and Hoechst 33342 staining. Cell cycle progression was detected by flow cytometry. The protein expression levels were detected by Western blotting and immunofluorescence staining. Results: Our results showed that DEX dose-dependently restored impaired proliferation of PC12 cells induced by lidocaine，as reflected by the increased cell viability and EdU positive cells, which were consistent with the decreased expression of tumor suppressor protein p21 and increased expression of cell cycle-related cyclin D1 and CDK1. In addition, DEX dose-dependently reduced apoptotic PC12 cells induced by lidocaine，as reflected by the decreased expression of apoptosis-related Bax, caspase-3 and caspase-9 and increased expression of anti-apoptotic Bcl-2 compared to the cells only treated with lidocaine. Mechanistically, with gain-or-loss-of-function of STMN1, we showed that DEX-mediated neuroprotection by lidocaine-induced damage is associated with downregulation of STMN1 which might be an upstream molecule involved in regulation of mitochondria death pathway. Conclusion: Our results reveal that DEX is likely to be an effective adjunct to alleviate chronic neurotoxicity induced by lidocaine.

Keywords: STMN1; apoptosis; dexmedetomidine; lidocaine; neurotoxicity; proliferation.

Figure 1

DEX restores proliferation and cell cycle progression impaired by lidocaine in PC12 cells. (A) The effect of DEX on cell proliferation. (B) The effect of DEX on cell viability in lidocaine-treated cells. Cell viability at the indicated time points was detected by CCK8 assay. (C) The effect of DEX on cell proliferation was detected by EdU staining assay. The statistical analysis was displayed on the right. (D) The effect of DEX on lidocaine-inhibited cell cycle progression was determined by flow cytometry analysis. (E) The statistical analysis of cell cycle distribution influenced by DEX. (F) The expressions of cell cycle related proteins in lidocaine or lidocaine/DEX combination treated cells were analyzed by Western blotting. The experiments were performed in triplicate. The experiments were performed in triplicate and each value represents mean ± SD. Values with the same letter within groups indicate no significant difference with P≥0.05.

Figure 2

DEX inhibits lidocaine-induced apoptosis in PC12 cells. (A) The effect of DEX on lidocaine-induced apoptosis in PC12 cells was analyzed by flow cytometry (up). The effect of DEX on lidocaine-induced apoptosis in PC12 cells was detected by Hoechst 33,342 staining (down). (B) The statistical analysis of apoptosis rates influenced by DEX. (C) The expression of Bax, Bcl-2, caspase-3 and caspase-9 in lidocaine or lidocaine/DEX combination treated cells was analyzed by Western blotting. The experiments were performed in triplicate and each value represents mean ± SD. Values with the same letter within groups indicate no significant difference with P≥0.05.

Figure 3

DEX depresses the expression of STMN1 induced by lidocaine in PC12 cells. (A)The expression of STMN1 in lidocaine or lidocaine/DEX combination-treated cells was detected by immunofluorescence staining. (B) The expression of STMN1 in lidocaine or lidocaine/DEX combination-treated cells was analyzed by Western blotting. The experiments were performed in triplicate.

Figure 4

DEX restores cell proliferation impaired by lidocaine through downregulating STMN1. (A) The expression of STMN1 in lidocaine-treated or lidocaine/DEX combination-treated cells transfected with STMN1-shRNA or pcDNA3.0-STMN1 vector. The expression of STMN1 was detected by immunofluorescence staining. (B) The effect of STMN1 knockdown and overexpression on cell proliferation in lidocaine or lidocaine/DEX combination treated cells was detected by EdU-staining assay. (C) The statistical analysis of EdU positive cells proportion. (D) The effect of STMN1 knockdown and overexpression on cell viability in lidocaine or lidocaine/DEX combination treated cells was detected by CCK8 assay. NC means co-transfection of scramble shRNA and pcDNA3.0 vector. The experiments were performed in triplicate and each value represents mean ± SD. Values with the same letter within groups indicate no significant difference with P≥0.05.

Figure 5

DEX restores cell cycle progression inhibited by lidocaine through downregulating STMN1. (A) The effect of STMN1 knockdown and overexpression on cell cycle progression in lidocaine-treated or lidocaine/DEX combination-treated PC12 cells was determined by flow cytometry analysis. (B) The statistical analysis of cell cycle distribution with assay of the gain- or loss-of-function of STMN1 in lidocaine-treated or lidocaine/DEX combination-treated PC12 cells. (C) The expression of cell cycle related proteins was analyzed by Western blotting. The experiments were performed in triplicate.

Figure 6

DEX protects PC12 cells from lidocaine-induced apoptosis through downregulating STMN1. (A) The effect of STMN1 knockdown and overexpression on cell cycle progression in lidocaine-treated or lidocaine/DEX combination-treated cells was analyzed by flow cytometry (up). The effect of STMN1 knockdown and overexpression on cell cycle progression in lidocaine-treated or lidocaine/DEX combination-treated cells was detected by Hoechst 33,342 staining(down). (B) The statistical analysis of apoptosis rates with assay of the gain or loss-of-function of STMN1 in lidocaine-treated or lidocaine/DEX combination-treated PC12 cells. (C) The expression of Bax, Bcl-2, caspase-3 and caspase-9 was analyzed by Western blotting. The experiments were performed in triplicate.

Figure 7

The possible mechanism by which DEX alleviates neurotoxicity provoked by lidocaine in PC12 cell.