Exploring The Protective Effect Of Trazalin On Colistin-induced Nerve Injury Based On The Mitochondrial Apoptosis Pathway Mediated By The Nrf2/HO-1 Pathwa

Background: Polymyxin E(Colistin)is the last line of defense in the clinical treatment of multidrug resistant and pan-drug resistant gram-negative infection,but its neurotoxicity limits its wide application.Therefore,it is of great significance for clinical application of colistin to explore the dose-toxicity relationship and toxic mechanism of colistin and to find effective measures to prevent and treat the neurotoxicity of colistin.Objective: Neural damage model in mice was established by intraventricular injection of colistin and in vitro neurotoxicity model was established by treating N2 a cells with colistin.To investigate the molecular mechanism of colistin induced neurotoxicity and the effect of natural compound Piceatannol 3’-O-glucopyranoside(PG)on the neurotoxicity induced by colistin.Methods: In vivo experiments: The mice model of neurotoxicity was established by intraventricular injection of colistin.The damage effect of colistin on hippocampal neurons and the protective effect of caudal vein injection PG combined with were observed and analyzed by HE staining and Nissl staining of brain tissue sections.After drug intervention,the Change of oxidative stress related indexes Superoxide dismutase(SOD),Glutathione(GSH),Catalase(CAT)and Malondialdehyde(MDA)in mice brain were detected by microplate reader.Finally,Western Blot assay was used to detect the mechanism of PG improving the neurotoxicity induced by colistin.In vitro experiments: CCK8 was used to detect the effect of colistin alone and combined with PG on the activity of N2 a cells.Flow cytometry was used to detect the effect of drug intervention on the apoptosis level of N2 a cells.The effects of drug intervention on oxidative stress levels of SOD,GSH,CAT and MDA in N2 a cells were determined by microplate reader.Immunofluorescence was used to detect the effect of drug intervention on ROS content in N2 a cells.The effects of drug intervention on mitochondrial morphology,mitochondrial membrane potential,and protein expression of Nrf2 and p Nrf2 in N2 a cells were observed and analyzed by laser scanning confocal microscope.The effects of drug intervention on Nrf2/HO-1 pathway,apoptosis and autophagy related protein expression in N2 a cells were detected by Western Blot.The Nrf2 knockdown N2 a cell model was constructed by lectin virus transfection.The apoptosis level was detected by flow cytometry,the oxidative stress level was detected by enzyme label,and the expression levels of Nrf2/HO-1 pathway,apoptosis and autophagy related proteins were detected by Western blot.Results: In vivo animal experiments showed that Colistin induced hippocampal neuronal cell damage and brain oxidative stress in a dose-dependent manner after 5 consecutive days of intraverebral injection of Colistin 8 and 16U/g/d,decreased Nrf2 and HO-1 protein levels,and increased Cyt c and Caspase-3 protein levels.PG intervention can relieve the inhibition of Colistin on Nrf2/HO-1 pathway related proteins,increase the content of antioxidant factors in tissues,reduce the level of apoptosis,and achieve the therapeutic effect of improving the nerve injury caused by Colistin.In vitro cell experiments showed that Colistin single inhibited N2 a cell viability in a time-and dose-dependent manner,and significantly induced apoptosis,oxidative stress and mitochondrial function impairment in N2 a cells at 400μM,while reducing Nrf2,p Nrf2(s40)and HO-1 protein levels.The protein levels of Cyt c,Caspase-3 and Caspase-9 and the ratio of Bax/Bcl-2 and LC3BⅡ/LC3BⅠ were increased,while the protein level of p62 and the ratio of p ULK1(s757)/ULK1 were decreased.PG could improve Colistin induced apoptosis,oxidative stress,mitochondrial function injury and autophagy in a dose-dependent manner.PG intervention did not improve Colistin-induced oxidative stress,apoptosis,and up-regulation of autophagy in N2 a cell lines with Nrf2 knockdown.Conclusion: PG regulates Nrf2/HO-1 pathway and increases the activity of antioxidant enzymes in the body,thus alleviating oxidative stress induced by Colistin,reducing mitochondrial damage,reducing apoptosis level and autophagy level,and maintaining neuronal cell homeostasis,which has a protective effect on the neurotoxicity induced by Colistin in mice.