Centipeda Minima Exerts Antioxidant And Neuroprotective Effects Via Activation Of ERK/Nrf2 Signaling

ObjectiveThe oxidative stress hypothesis is implicated in the pathogenesis of neurodegenerative diseases and other aging-related diseases,which are accompanied by extensive injury induced by high levels of oxidative stress in the central nervous system.However,there are no effective therapeutic drugs and synthetic antioxidants to stop the progression of diseases.We found that Centipeda minima had significant antioxidant activity.However,there is no report about the neuroprotective effect of Centipeda minima,and the potential mechanism of its antioxidant properties is unclear.The aim of this study was to investigate the antioxidant activities and potential mechanisms of the ethanol extract of Centipeda minima(ECM)and monomer compound 26(EBSC-26)from the perspective of antioxidant mediated neuroprotection in vivo and in vitro.and in vitro.MethodsFor in vi tro experiments,we selected neuroblastoma cells(SH-SY5Y)and highly differentiated mouse pheochromocytoma cells(PC12),and used tert-butyl hydrogen peroxide(tBHP)and glutamate to induce oxidative damage model.At the same time,we used reflux extraction method to extract ECM,and used high performance liquid chromatography to isolate and purify monomer compounds from ECM.cell viability was detected by MTT assay,DCFH-DA fluorescence probe assay was used to detect the production of reactive oxygen species(ROS)in cells.The content and activity of MDA,SOD and GSH were detected by related kits.JC-1 fluorescence probe assay was used to detect the mitochondrial membrane potential.and the expression of Bel-2 family members as well as MAPKs and Nrf2 pathways-related proteins was detected by Western blot.For in vivo experiments,40 6-month-old Kunming mice were selected to establish sub-acute aging modcl by vsubcutaneous injection of D-gal(120 mg/kg/d)and intragastric administration of AlC13(20 mg/kg/d).The experimental groups:Vehicle control group(containing 10%ethanol saline);D-gal(120 mg/kg)/AlCl3(20 mg/kg);D-gal/AlCl3+ECM(100 mg/kg);D-gal/AlCl3?ECM(200 mg/kg);D-gala/AlCl3+ECM(400 mg/kg);D-galactose/AlCl3+vitamin E(80 mg/kg).ECM or vitamin E were given for 30 days after 60 days treatment of D-gal/AlC13,and behavioral tests were started 90 days after intervention.Nissl staining and HE staining were used to observe the morphological changes of hippocampal neurons.The contents and activities of lipid peroxidation products and antioxidant proteins in hippocampus and cortex were detected by relevant kits.The expressions of PSD95 and SYN as well as MAPKs and Nrf2 pathways in hippocampus were detected by Western blot.The kits were used to detect the related functional indicators of liver and kidney in serum.ResultECM pretreatment can inhibit glutamate or tBHP-induced death of SH-SY5Y and PC12 cells.Meanwhile,ECM pretreatment can reduce ROS production and sustain mitochondrial membrane potential,reduce the level of malondialdehyde(MDA)in cells,and increase the content and activity of antioxidant molecules SOD and GSH.Western blot results showed that tBHP could increase p38 MAPK and JNK phosphorylation in SH-SY5Y and PC12 cells,which was blocked by ECM.In contrast,tBHP decreased ERK phosphorylation in SH-SY5Y and PC12 cells,while ECM maintained the phosphorylation level of ERK.Moreover,we found that ECM could induce translocation of nuclear factor-derived 2-like 2(Nrf2)and up-regulation of its downstream phase ? detoxification enzymes,including heme oxygenase-1(HO-1),superoxide dismutase-2(SOD2)and NAD(P)H:quinone oxidoreductase-1(NQO-1).In SH-SY5Y cells treated with Erk kinase inhibitor U0216,the up-regulation of ERK phosphorylation by ECM was attenuated,while the expression of Nrf2 and its downstream phase ? detoxifying enzymes were reduced.Further,ECM can maintain Bcl-2/Bax ratio and inhibit cell apoptosis.Four sesquiterpenoids,EBSC-26A-EBSC-26D,were isolated and purified from ECM by high performance liquid chromatography.The four compounds were identified as 6-O-angeloylprenolin,arnicolide D,arnicolide C and microhelenin C by spectral identification,.Among the isolated compounds,6-O-Angeloylprenolin and arnicolide D could significantly attenuate tBHP-induce cell damage in SH-SY5Y and PC12 cells,and exert a good effect at low doses.At the same time,6-O-Angeloylprenolin and arnicolide D could significantly reduce ROS production in SH-SY5Y cells,promote Nrf2 nuclear translocation,induce the upregulation of phase ? detoxification enzymes HO-1,NQO-1 and SOD2.In the sub-acute aging mouse model induced by D-galactose and AlCl3,ECM treatment group could significantly decrese the escape latency in Morris Water Maze test and increase the number of mice crossing the platform in space exploration test.Meanwhile,ECM can inhibit lipid peroxidation in hippocampus and cortex,increase the content and activity of antioxidant proteins GSH and SOD,maintain the hippocampal neuron morphology and synaptic structure,and sustain the expression levels of postsynaptic density protein 95(PSD95)and synaptophysin(SYN)in hippocampus.For in vivo experiments,ECM can also inhibit the activation of p38 MAPK and JNK,increase the phosphorylation level of ERK,activate Nrf2 signaling pathway,and restore the expression levels of phase? detoxification enzymes HO-1,NQO-1 and SOD2 in the hippocampus of mice.In toxicological study,there were no significant differences in serum ALT,AST,ALT/AST,BUN and Cr levels between ECM treatment groups and model group.ConclusionIn this study,we investigated the mechanism of ECM and its active ingredients from Centipeda minima in improving oxidative stress and neuroprotection.We found that ECM could activate ERK/Nrf2 signaling pathway to protect neurons and cells from oxidative stress-induced injusy.Among the four sesquiterpenes isolated,6-O-AngeloylPlenolin and Ariccolide D have significant antioxidant activities.As a source of antioxidants and neuroprotective compounds,Centipeda minima provide a new choice for the development of new drugs and treatment of neurodegenerative diseases.