Study On The Molecular Mechanism Of Colistin Induced Cell Apoptosis And Autophagy And Targeted Modulation

Polymyxins(polymyxin B and colistin)is one of the most important antibiotics for the therapy of the infections caused by multiple resistant(MDR)Gram-negative in clinic.Neurotoxicity and nephrotoxicity remain the poorly characterized adverse effect associated with polymyxin therapy and are also the mainly dose-limiting factors.In the present study,we established the mouse and in vitro cell model to investigate the molecular mechanism of apoptosis and autophagy induced by colistin,and further explored the effect of targeted modulation involved apoptosis and autophagy signal pathway on the colistin nephrotoxicity and neurotoxicity.The main findings are as follows:(1)Colistin induces nephrotoxicity and neurotoxicity,which involved oxidative stress,mitochondrial function,in cascade to actiating mitochondria,death receptors and endoplasmic reticulum apoptotic pathway in a dose-dependent by using the mouse and in vitro HEK293 and N2a cell models.Colistin treatment can also activate NF-?B,MAPKs,p53,Nrf2/ARE and Akt signaling pathways.Furthermore,it found that the activation of JNK and Erk plays the protective role;meanwhile,the activation of p53 and the inhibition of Akt signal pathway promotes cell death caused by colistin treatment by in vitro cell model.(2)Colistin exposure markedly up-regulated the expression of Beclinl and the ratio of LC3?/? in the mouse kidney tissues and N2a cells.Colistin treatmen also increased the formation of the autophagosome-lysosomes and autophagosome,and the punctum formation using GFP-LC3 transfection.These results indicated that colistin could activate cell autophagy.Furthermore,in the N2a cell model,it was shown that autophagy inhibition by chloroquine(CQ)aggravates colistin-induced apoptosis,which was dependent the ROS-mediated oxidative stress damage and casapse activation,suggesting that autophagy play a protective role in colistin induced apoptosis.Rapamycin,could specifically inhibit the mTOR/p70s6k signaling pathway,followed to up-regulate Akt/CREB,Nrf2/ARE signaling pathways and ULK1-dependent cell autophagy as well as inhibit p53 signaling pathway and p53-Nrf2 protein co-localization in cytoplasm,which contributed to improve colistin-induced oxidative stress damage,mitochondrial dysfunction and caspase activation-mediated apoptosis.Furthermore,the protective role of autophagy was certified by using mouse primary neuronal cell and animal model.(3)Natural active substances curcumin and baicalein could up-regulate anti-oxidative stress,anti-inflammatory ability,activate Nrf2/HO-1 pathways and ULK1-dependent cell autophagy and down-regulate p53 protein expression and colistin receptor protein megalin,followed to attenuate colistin idncued-oxidative stress and caspase-dependent apoptosis,NF-?B mediated inflammatory response,finally reducing colistin induced neurotoxicity and nephrotoxicity.(4)In mouse and N2a cell model,antibiotics substance minocycline could scavenge cellular ROS,abolish oxidative stress,caspase activation,and apoptosis,finally improving the neurotoxicity and nephrotoxicity caused by colistin.The present finding has the important clinical value.In conclusion,colistin neurotoxicity and nephrotoxicity involved exogenous and endogenous apoptotic pathways,ULK1-mediated cell autophagy and NF-?B-mediated inflammatory response.We also demonstreated that activation of Akt/CREB,Nrf2/HO-1 pathways and autophagy could protect against colistin caused neurotoxicity and nephrotoxicity by regulating oxidative stress,mitochondrial dysfunction,inflammatory response and apoptosis.Our study highlights an important insight into the development of new generation of polymyxin lipopeptide antibiotics,combination therapy,and attenuated strategy in clinic.