Brain derived neurotrophic factor alleviates neuronal injury induced by ropivacaine through threonine protein kinase signaling pathway

Abstract

Objective To investigate the protective effect of brain-derived neurotrophic factor(BDNF) on neuronal injury induced by ropivacaine (Rop) and its mechanism. Methods (1) Experiment one: 0, 1, 2, 3, 4 and 5 mmol/L Rop was used to stimulate SH-SY5Y cells for 48 h to induce neuronal injury; the morphological changes of the cells were observed under microscope; MTT assay was used to detect the cell activity; flow cytometry was used to detect the cell apoptosis, and immunohistochemistry was used to detect the expressions of protein kinase B (Akt) and proliferating cell nuclear antigen (PCNA). (2) Experiment two: SH-SY5Y cells were treated with 0, 1, 3, 5, 7 mmol/L Rop, respectively; the cell activity was measured 48 h after Rop treatment; the semi inhibitory concentration (IC50) of Rop was calculated by MTT assay; the SH-SY5Y cells were divided into control group (PBS for 48 h), Rop group (Rop at IC50 for 48 h), BDNF+Rop group (20 μg/L BDNF for 2 h, and Rop at IC50 for 48 h), Akt pathway activator SC79+Rop group (5 mg/L SC79 for 2 h, and Rop at IC50 for 48 h), and BDNF+Akt pathway inhibitor API-2+Rop group (20 μg/L BDNF+10 μmol/L API-2 for 2 h, Rop at IC50 for 48 h); the morphological changes of the cells were observed under microscope; MTT assay was used to detect the cell activity; flow cytometry was used to detect the cell apoptosis, and immunohistochemistry was used to detect the expressions of Akt and PCNA; the expressions of B lymphoma-2 (Bcl-2), Bcl-2-associated X (Bax) and cysteine protease-3 (Caspase-3) were detected by reverse transcription (RT)-PCR and Western blotting. Western blotting was used to detect the expressions of Akt and phosphatidylinositol 3-kinase (PI3K). Results (1) As compared with the 0 mmol/L Rop group, the 1, 2, 3, 4, and 5 mmol/L Rop group had significantly decreased cell activity, significantly increased apoptosis rate, and statistically smaller number of Akt and PCNA positive cells (P<0.05). (2) As compared with the control group, the Rop group had significantly decreased cell activity, statistically increased apoptosis rate, significantly smaller number of Akt and PCNA positive cells, significantly decreased Bcl-2 mRNA and protein expressions, significantly increased Bax and caspase-3 mRNA and protein expressions, and significantly decreased phosphorylated- (p-) Akt and p-PI3K protein expressions; as compared with the Rop group, the BDNF+Rop group and SC79+Rop group had significantly higher cell activity, significantly decreased apoptosis rate, significantly larger number of Akt and PCNA positive cells, significantly increased Bcl-2 mRNA and protein expressions, statistically decreased mRNA and protein expressions of Bax and Caspase-3, and significantly increased p-Akt and p-PI3K protein expressions (P<0.05); as compared with the BDNF+Rop group and SC79+Rop group, the BDNF+API-2+Rop group had significantly lower cell activity, significantly increased apoptosis rate, significantly smaller number of Akt and PCNA positive cells, significantly decreased Bcl-2 mRNA and protein expressions, statistically increased mRNA and protein expressions of Bax and Caspase-3, and significantly decreased p-Akt and p-PI3K protein expressions (P<0.05). Conclusion BDNF can alleviate ropivacaine-induced neuronal injury by activating Akt signaling pathway, consequently modulating the proliferation and apoptosis of neurons. Key words: Brain derived neurotrophic factor; Ropivacaine; Protein kinase B; Neuron