The Effects Of NGF On Colistin-mediated Autophagy And Apoptosis In PC12 Cells

Colistin, a polymyxin antimicrobial agents is an effective means of combating multi-drug resistant Pseudomonas aeruginosa and Acinetobacter baumannii infections. However neurotoxicity and nephrotoxicity of colistin hindered its application in clinic. With the enhanced resistance of Gram-negative bacteria, colistin advantages as narrow-spectrum antibiotics gradually reflected. Reduceing side effects can improve the clinical application. Nerve growth factor is a neurotrophic factor. NGF can protect neurons, increase the activity of neurons. Autophagy and apoptosis are the normal course of physiological activity in cells. Autophagy is a genetically programmed process that degrades long lived cellular proteins and organelles. Various stress such as amino acid starvation or hormone stimulation induces atuophagy. In this study, NGF as a colistin protective agent was exposed with colistin together in the rat pheochromocytoma(PC12), to explore the protective effect of cell activity and autophagy in cell apoptosis in PC12 cells.In previous study, we investigated that PC 12 cells contacted with colistin(125,250 μg?mL-1) for 12 h reached a high level of autophagy and apoptotic cells also appeared, and the levels of caspase-3 expression reached peak when cells contacted with colistin for 24 h. In 250 μg?mL-1 colistin concentration group with the increase of autophagy degree, apoptosis was significantly increased and ultimately the concentration of colistin was detemined at 250 μg?mL-1. After PC12 cells were co-treated with colistin(250 μg?m L-1) and different concentrations of NGF(25, 50, 100 ng?m L-1) respectively for 12 and 24 h, autophagy was detected by monodansylcadaverine(MDC) labeled fluorescent particles, visualization of LC3 immunofluorescence microscopic examination labeled vacuoles. The autophagy-related proteins were detected by western blotting. Apoptosis was dectcted by visualization of Hoechst 33258 staining, mitochondrial membrane potential and cell viability were detcted by flow cytometry, apoptosis-related proteins by western blotting. The results showed that:(1) Colistin can cause autophagy and apoptosis in PC12 cells. 250 μg?m L-1 colistin can induce the highest rate of apoptosis in 24 h group and the highest rate of autophagy in 12 h group.(2) With the increase of the NGF concentration, the PC12 cell activity increased.(3) The expression levels of autophagy-related protein LC3 and Beclin1, and apoptosis rate decreased with the increase of the NGF concentration.(4) After coexposure different concentrations of NGF and 250 μg?mL-1 colistin for 12, 24 h, apoptosis-related protein Caspase-3 in the amount of protein in the cells also showed a downward trend.In summary, NGF can improve cell viability of PC12 cells; the high concentration of colistin induced autophagy has a negative effect on apoptosis; NGF was not only inhibit the colistin mediated apoptosis but also the autophagy in PC12 cells.