Study On Intervention Effects Of Kinetin Against Glutamate Induced Oxidative Damage In HT22 Cells And Related Mechanism

Background:Alzheimer’s disease（AD）is a neurodegenerative disease that badly jeopardizes physical and psychological health,and living quality of the elderly.Increasing evidence has suggested that oxidative stress probably plays a crucial role in the initiation and development of AD.Kinetin is a methylfuran derivative of adenine,which is proved to be capable of exerting anti-oxidative stress and anti-aging effects on the levels of individuals,tissues and cells in animals.HT22 cells is an immortalized cell line deriving from hippocampal neurons of mice,which is a good cell model to study the oxidative stress in neurons because glutamate can induce oxidative stress and cell death in HT22 cells.This research investigated the protective effects of kinetin against gluatamate induced oxidative damage in HT22 cells,which has significance in screening drugs against AD.Purpose:Investigate the protective effects of kinetin against glutamate induced oxidative damage in HT22 cells and related mechanisms.Methods:Firstly,the anti-oxidative capabilities of kinetin itself in vitro were evaluated by DPPH scavenging assay,Fe³⁺reduction assay,Fe²⁺chelation assay,superoxide radical inhibition assay and hydroxyl radical inhibition assay,while the safe concentration range and optimal treatment time were selected by CCK8 cell viability assays.Then,the effects of kinetin against glutamate-induced cell viability decline and Lactate dehydrogenase（LDH）leakage were studied by CCK8 cell viability assays and LDH cytotoxicity assays;the effects of kinetin on intracellular reactive oxygen species（ROS）and Ca²⁺levels were investigated by flow cytometry;the effects of kinetin on intracellular GSH level,activities of anti-oxidative enzymes and cellular anti-oxidative capabilities were studied with related kits;the effects of kinetin against cell apoptosis were evaluated by flow cytometry;the effects of kinetin on mitochondrial function were evaluated by detecting intracellular ATP content（using an ATP kit）,mitochondrial membrane potential（MMP）（by flow cytometry）and apoptosis inducing factor（AIF）（by western blot）;kinetin’s effects against activation of apoptosis signal-regulating kinase 1（ASK-1）,c-Jun N-terminal kinase（JNK）and mitogen activated protein kinase p38（p38）were investigated by western blot（WB）.Finally,WB and quantitative real-time polymerase chain reaction（qRT-PCR）were used to study the effects of kinetin on Nrf2 nuclear translocation and expression of heme oxygenase-1（HO-1）,while siRNA silence and CCK8 cell vialiability assays were conducted to investigate the influence of Nrf2 and HO-1 on the cellular protective effects of kinetin.Results:a.Compared to positive control,kinetin itself hardly had anti-oxidative capabilities;b.The safe concentration range of kinetin was below 5mg/L,and its optimal treatment time was 8h;c.Kinetin can inhibit the decline of HT22 cell viability and the increase of LDH leakage;d.Kinetin can inhibit the elevation of intracellular ROS and Ca²⁺levels,increase activities of intracellular anti-oxidative enzymes and GSH level,and enhance cellular anti-oxidative capabilities;e.Compared to glutamate treatment group,kinetin can partly restore intracellular ATP level,inhibit depolarization of MMP and the nuclear translocation of AIF,and inhibit HT22 cell apoptosis induced by glutamate;f.Kinetin can inhibit glutamate-induced phosphorylation of ASK-1、JNK and p38;g.Kinetin can significantly promote the nuclear translocation of Nrf2 in a time dependent manner.It can also significantly promote the expression of downstream gene HO-1 both at transcription and translation levels,in time and dose dependent manners.In addition,targeted silence of Nrf2 using Nrf2 siRNA almost completely neutralized the cellular protective effects of kinetin,while targeted silence of HO-1 using HO-1 siRNA just partly neutralized the cellular protective effects of kinetin.Conclusion:Kinetin itself hardly has any anti-oxidative capabilities,but it can inhibit cytotoxicity,elevation of intracellular ROS and Ca²⁺levels,decline of intracellular anti-oxidative enzymes activities in HT22 cells induced by glutamate.Kinetin itself can also activate the cellular anti-oxidative enzyme system.Meanwhile,kinetin can inhibit mitochondrial dysfunction and cell apoptosis,and the activation of ASK-1,JNK and p38 in HT22 cells induced by glutamate.In addition,kinetin can activate Nrf2 signal pathway and enhance the expression of downstream anti-oxidative gene HO-1,and the cellular protective effects of kinetin depends on activation of Nrf2 and the expression of HO-1.These results indicate that kinetin is capable of neuroprotective effects,which may have application potentials in research and practice of AD’s precaution and treatment.