| Colistin(also known as polymyxin E),is a kind of alkaline annular polypeptide antibiotic,and its sulfate has been mainly used in clinic,and it is one of the most effective drug for infections of multidrug resistant gram-negative bacteria.However,optimizing its clinical use is limited by nephrotoxicity and neurotoxicity.Our team has been committed to the research of colistin-induced neurotoxicity for many years,and obtained many experimental data about the neuroethology induces autophagy and apoptosis in nerve cells,and recent studies showed that colistin induces authophagy prior to apoptosis and that autophagy inhibits apoptosis.Simultaneously,p53 plays an important dual role in autophagy and apoptosis induced by colistin.The complex relationship of autophagy and apoptosis is a hot issue of research field today.Clearing the mechanism of p53 and JNK in nerve cells by colistin will enrich it's toxicology data,explore new attenuated measures and provide the information for clinical rational drug use.This study is carried out in PC12 cells,JNK signaling pathway was activated by JNK inhibitor/activator in colistin-treated PC12 cells,and MTT method was used to determine the dosages of JNK inhibitor(SP600125)and JNK activator(anisomycin).Cells were pretreated with SP600125(20 ?M)and anisomycin(4 ?M)for 1 h and followed by colistin(125 ?g/m L)for 12 h,and then autophagy and apoptosis were detected by real-time PCR,Western blot,electron microscopy and immunofluorescence microscopy.The aim of the present study was to investigate the relationship between JNK signaling pathway and colistin-induced autophagy in PC12 cells;Silencing of p53 by specific si RNA was examined by Western blot,and then anisomycin pretreated PC12-sip53 cells for 1 h following colistin treatment,and clarified the relationship among JNK,p53 and ROS in colistin treated PC12 cells.The results showed that:(1)Compared with control group,colistin induced activation of JNK and Bcl2 in PC12 cells and reached peak levels at 12 h,and Bax level significantly increased at 12 h in a time-dependent manner(p<0.01).(2)Compared with colistin group,SP600125 pretreatment induced the expression levels of autophagy related genes and protein significantly decreased(p<0.01),the number of autophagic vacuoles reduced by electron microscopy images;anisomycin led to autophagy related genes and protein significantly increased(p<0.01),different sizes and stages of autophagic vacuoles were visible by immunofluorescence staining with LC3 antibody and were evident by electron microscopy images and cell apoptosis rate slightly increased,nuclear pyknosis and migration.(3)Compared with colistin group,the activation of JNK by anisomycin induced the upregulation of autophagy related proteins in colistin-treated PC12 cells,and Bcl2 level was activated and reached peak at 6 h;the expression levels of apoptosis related protein significantly increased at 6 h,and Bax level significantly increased in a time-dependent manner.(4)Compared with colistin+sip53,anisomycin led to ROS production significantly increased,and p-JNK level significantly upregulated(p<0.01).PC12 cells were transfected with p53 si RNA or recombinant plasmid,and then western blot assay was used to identify the efficiency of p53 interference and subcellular localization of p53 protein was determined by immunofluorescence microscopy.Cells was treated by colistin up to 24 h,then the cells were divided into control group,colistin(12 h,24 h)group,colistin+NC(12 h,24 h)group,colistin+sip53(12 h,24 h)group,colsitin+pc DNA3.1(12 h,24 h)and colistin+pc DNA3.1+p53(12 h,24 h)group.Different groups were detected by the above experimental method and the aim of the present study was to investigate the changes of p53 protein level regulated colistin-induced autophagy in PC12 cells from morphology and biological changes.The results showed that:(1)Compared with colistin+NC or colistin+pc DNA3.1,Western blot and p53 immunofluorescence microscopic examination showed that silencing and overexpression of cytoplasmic p53 in PC12 cells were effective with specific si RNA and pc DNA3.1+p53 recombinant plasmid.(2)Compared with colistin+NC,silencing of p53 downregulated autophagy related genes and proteins at 12 h in colistin-treated PC12 cells(p<0.01),the number of autophagic vacuoles and fluorescence points were decreased;at 24 h time point had oppose results.(3)Compared with colistin+sip53,the index of autophagy flux had no changes in colsitin+sip53+EFA;the activation of apoptosis related proteins down-regulated in colistin+sip53+3-MA(p<0.01),nuclear pyknosis and migration were decreased.(4)Compared with colistin+pc DNA3.1,overexpression of p53 downregulated autophagy related genes and proteins in a time-dependent manner in colistin-treated PC12 cells(p<0.01),the number of autophagic vacuoles and fluorescence points were significantly decreased;the activation of apoptosis related proteins and gene significantly downregulated(p<0.01),nuclear pyknosis and migration were more obvious.Our study is the first to demonstrate that JNK was involved in colistin-induced autophagy,and mediated autophagy and apoptosis via JNK-Bcl2-Bax signaling pathway and ROS-JNK-p53 feedback loop in colistin-treated PC12 cells,and accelerated colistin-induced autophagy and apopotosis.The silencing of p53 inhibited autophagy in PC12 cells after colistin treatment for 12 h and then up-regulate defective autophagy at 24 h,and eventually promote apoptosis.Overexpression of p53 inhibited colistin-induced autophagy,and accelerated apoptosis in a time-dependent manner in PC12 cells. |
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