The Protective Mechanism Study Of Baicalein Against Hydrogen Peroxide-induced Neurotoxicity In HT22 Cells

Introduction:Neurological diseases such as cerebrovascular diseases and neurodegenerative diseases have become major health problems in China and the world.Oxidative stress and endoplasmic reticulum stress are the main molecular pathological mechanisms of nerve cells damage.Baicalein is one of the important flavonoids,which is mainly isolated from the root of Scutellaria baicalensis.It can effectively alleviate neurons injury via various pharmacological mechanisms,including anti-oxidant stress,anti-inflammation,anti-excitotoxicity,anti-apoptosis,stimulating neurogenesis and promoting the expression of neuroprotective factors.Aims:We established an experimental model of H₂O₂-induced HT22 cells injury in vitro,and explored the protective effect and the changes of proteins expression after baicalein pretreatment in HT22 cells.We furthered to explore the targets and potential molecular mechanisms of baicalein against H₂O₂-induced neurotoxicity in HT22 cells.Mmethods:HT22 cells were exposed to different concentrations of H₂O₂（0μM,150μM,200μM,250μM,300μM,350μM,400μM）for 24h and the half-inhibitory concentration（IC50）was calculated.HT22 cells pretreated with different concentrations of baicalein（0μM,1.563μM,3.125μM,6.25μM,12.5μM,25μM）for 2h,then damaged by H₂O₂ for 24h.Then the inverted phase contrast microscope were used to observe the morphological changes of cells.And the cell vitality of HT22 cells in each group was detected by CCK-8.The content of lactate dehydrogenase（LDH）in cell culture medium was detected by LDH assay kit,and the cells apoptosis was detected by Hoechst 33342 staining.The changes of proteins expression after baicalein pretreatment were analyzed by 2D-PAGE,and the differentially expressed proteins were identified by MALDI-TOF-MS mass spectrometry.The expression of Prdx2,ATF4,Bcl-2 and Bax in HT22 cells was analyzed by Western blot.Results:The results were shown that cell viability decreased significantly with the increase of H₂O₂ concentrations,and IC50 was 228.5μM（95%CI:215.3 to 242.5μM R²=0.9509）.The morphology of cells injury changed significantly after treatment with H₂O₂（250μM）,while baicalein could significantly alleviate the cells injury.When HT22 cells were treated alone with various doses of baicalein,there was no changes in cell viability（p?>?0.05）.Pretreatment with different concentrations of baicalein in HT22cells had a protective effect on H₂O₂-induced cells injury in a dose-dependent manner.Among them,1.563μM group（p?<?0.05）,3.125μM group（p?<?0.01）,6.25μM group（p?<?0.05）and 12.5μM group（p?<?0.05）had significant difference compared with H₂O₂group,while 25μM group had no statistical difference（p?>?0.05）,of which 3.125μM group had the most significant protective effect.Baicalein pretreatment could significantly reduce the increase of LDH induced by H₂O₂（p?<?0.01）.The cells apoptosis was significantly increased in H₂O₂ group by Hoechst 33342 staining,while it was reversed after baicalein pretreatment.Seven differentially expressed proteins were detected and identified by 2D-PAGE and mass spectrometry after baicalein pretreatment.Three proteins,including Peroxiredoxin 2,Tropomyosin alpha-1 chain and Peptidyl-prolyl cis-trans isomerase A,were up-regulated,while four proteins,including Fascin,S-formylglutathione hydrolase,Eukaryotic translation initiation factor-3 subunit I and Hemoglobin subunit epsilon-Y2,were down-regulated.The expression of Prdx2 in baicalein pretreated group was significantly up-regulated,which was consistent with the result of two-dimensional electrophoresis.The expression of Bcl-2 protein was significantly decreased in H₂O₂ group,while the expression of ATF4and Bax proteins was significantly up-regulated compared with the control group.However,compared with the H₂O₂ group,the expression of Bcl-2 was significantly up-regulated,the expression of ATF4 and Bax proteins was down-regulated in baicalein pretreated group.Conclusion:This study found that baicalein can attenuate the injury of HT22 cells induced by H₂O₂ in a concentration-dependent manner and play a neuroprotective effect.The protective mechanism of baicalein on HT22 cells may be through up-regulating the expression of Prdx2 protein,inhibiting oxidative stress,and then regulating ATF4-mediated Bcl-2/Bax apoptosis pathway,thus protecting HT22 cells from H₂O₂-induced neurotoxicity by attenuating nerve cell apoptosis.