The Mechanism Investigation of (-)-Epigaiiocatechin-3-galiate(EGCG) Against the BV2Cell Damage Induced by Hydrogen Peroxide(H2O2)
|School||North Sichuan Medical College|
|Keywords||EGCG H2O2 BV2cell Apoptosis Signaling transduction pathway|
Objective: EGCG is a kind of polyphenol compound with the highestpharmacological activity extracted from green tea. In recent years, growingevidence has shown that EGCG might be a potent substance against nervoussystem damage. This study aims to investigate the protective mechanism of（）-Epigallocatechin-3-gallate（EGCG） against the BV2cell damage inducedby hydrogen peroxide（H2O2）, and hopefully to provide theoretical basis forits use in the treatment of neurodegenerative diseases and ischemic stroke.Method：1. The optimal concentration of H2O2, which can induceoxidative stress injury as well as apoptosis, was Screened by CCK-8assay，and then this concentration of H2O2（200umol/L） was used to make theoxidative stress injury mode of BV2cells, and the best protectiveconcentrations of EGCG were subsequently screened by CCK-8assay（10μmol/L和20μmol/L）.2.The cells in the period of Exponential growth were randomly dividedinto four groups:①blank control group,②H2O2damage group,③EGCG10μmol/L and④EGCG20μmol/L protection group. The protective effectof EGCG on BV2cell damage, which was induced by hydrogenperoxide（H2O2）, was investigated by immunofluorescence （Hoechst33258staining）.3.The expressions of Bcl-2、Bax、ERK1/2and p-ERK1/2proteins inevery group were analyzed qualitatively by immunocytochemistry and semi-quantitatively by Western blotting, respectively. The expressions ofBcl-2and bax gene in all groups were investigated by RT-PCR.Results:1. The BV2cell proliferation rate declined variably with theconcentration of H2O2increasing. After incubated with the concentration of200umol/L H2O2for24hours, The BV2cell viability decreased to88.2±3.5%, which reached the significant difference（P＜0.05）compared withthe blank control group. This concentration of200umol/L H2O2was acted asthe optimal concentration to induce BV2cells apoptosis. After EGCG wasadded on, the BV2cell viability increased to different extent. But only inEGCG10μmol／L and20μmol／L group, the increase of the cell viabilityreached significant level compared with the non-EGCG H2O2group（P＜0.05）, namely the concentrations of10μmol／L and20μmol／L EGCGhaving optimal protection against H2O2damage.2. Hoechst33258staining showed that a lot of densely stained nucleiappeared in the H2O2group. These nuclei took on strong fluorescence andwhite color, which implied apoptotic cells. After EGCG was added on, thenumber of apoptotic cells decreased, which declined to24±2.1%and16±4.3%in the EGCG10μmol／L and20μmol／L group, respectively. Comparedwith H2O2damaged group （57±6.4%）, this decrease in apoptotic cells inboth EGCG treated groups reached the significant level （P<0.05）.3. Immunocytochemistry showed that the BV2cells in all groupsexpressed Bcl-2、Bax、ERK1/2and p-ERK1/2. Western blotting analysisdisplayed that the expressions of Bcl-2and bax was significantly reduced andincreased respectively in the H2O2group when compared with that in theblank control group （both P<0.05）. However, the expression of Bcl-2,compared with the blank control group and H2O2group （P<0.05）, and the expression of Bax, compared with H2O2group （P<0.05）, were significantlyincreased and decreased respectively after10μmol／L and20μmol／LEGCG treatment for24hours. The expression of ERK1/2reduced graduallyin the order of control group, H2O2group,10μmol／L EGCG group and20μmol／L EGCG group. Conversely the expression of p-ERK1/2increasedgradually in the same order. The increase of p-ERK1/2expression and thedecrease of ERK1/2expression in the10μmol／L EGCG group and20μmol／L EGCG group were significant when compared with counterpart in thecontrol group and H2O2group（both P<0.05）.RT-PCR disclosed that theexpression of GAPDH as a reference gene was stable in all groups. The expression of Bcl-2gene rose significantly in the H2O2group compared with that inthe control group（P＜0.05）. In addition, the expression of Bcl-2geneincreased more significantly in10μmol／L EGCG group. Otherwise itsexpression decreased remarkably in20μmol／L EGCG group. But both ofthem had significant difference compared with control group and H2O2group（both P＜0.05） Besides, the expression of Bax gene increased significantly inthe H2O2group compared with that in the control group（P＜0.05）.However,its expression declined remarkably after adding10μmol／L or20μmol／LEGCG on to the culture medium and co-incubating for24hours（p＜0.05compared with H2O2group）.Conclusion: EGCG may be able to inhibit BV2cell apoptosis inducedby H2O2. This effect might be related to the activation of ERK1/2signalingtransduction pathway, the rise in p-ERK1/2expression, and the regulation ofsome genes’ transcription. These changes directly or indirectly results in theupregulation of Bcl-2and the downregulation of Bax in gene and protein level which are related to anti-apoptosis and pro-apoptosis, respectively.