Protective Effects Of EGCG On Hydrogen Peroxide Induced Oxidative Damage In Astrocytes

PartⅠThe culture of primary astrocytes from spinal cord of mice in vitroObjective:To explore the dissociation,the purity and the culturing method of primary astrocytes from spinal cord of mice in vitro.Spinal astrocytes culturing in vitro serve as an available model to study ALS.Methods:Primary culture and passage of astrocytes in vitro.Under sterile environment,the spinal cord was obtained from ICR mice born in 1～3 days,dissected and prepared cell suspension by digestion with the use of trypsin and mechanical digestion,then transferred to culture flasks without any stratum,and cultured(37℃,5%CO2).The culture medium was DMEM/F12.The oligodentrocytes and microglial cells were reduced with orbital shaker when The cells were confluent by 80%～90% on days 9 through 12.The primary culture was passaged when it was confluent.Finally the secondary culture was observed and the astrocytes were identified by immunohistochemical staining.Results:By orbital shaker in constant temperature and passage,the purified astrocytes with typical shapes were obtained.Immunocytochemical staining of GFAP-positive cells can be up to 98%Conclusion:The study provided one method to culture astrocytes from spinal cord of mice successfully.Astrocytes from spinal cord of mice in vitro could be an ideal model to study some impaired or protective mechanisms of astrocytes. Astrocytes from spinal cord of mice could be identified by being marked with polyclonal antibody GFAP.PartⅡProtective effects of EGCG on hydrogen peroxide induced oxidative damage in astrocytesObjective:To study the protective effects and mechanism of EGCG on hydrogen peroxide (H2O2)induced oxidative damage in astrocytes.Methods:Based on the culture of primary astrocytes from spinal cord of mice,The third cultures were randomly divided into different groups:①control group:the DMEM culture medium;②EGCG group:the culture medium were added with five different concentration of EGCG,containing 1,5,10,20 and 50μmol/L.After 24 hour further culture,the astrocytes viability was analysed by MTT assay.Finallly,the selected drug concenratins were 5μmol/L and 10μmol/L.We divided astrocytes into 4 groups for further study:①normal control group:the DMEM culture medium;②H2O2 model group (25μmol/L);③5μmol/L EGCG pretreatment group(EGCG pretreated 24h+H2O2);④10μmol/L EGCG pretreatment group(EGCG pretreated 24h+H2O2),H2O2 was added to the medium for 1h further culture.Subsequently,the astrocytes and culture mediums of each group were collected. We measured the cell culture medium lactate dehydrogenase (LDH) content and the expression of protein NQO1 and GFAP by LDH assay and western blotting in different subgroups.10μmol/L EGCG was added to normal astrocytes.After 24 hour further culture,the expression of NQO1 protein was assayed by western blotting and compared with model group.Results:Treated by EGCG in the range of concentration from 1μmol/L to 50μmol/L,the cell viability of astrocytes were increased.MTT test showed that the difference of cell viability between EGCG group(1,5,10μmol/L)and control group was also significant (P<0.05).Morphological changes of cells were observed with invert microscope.The formation of the normal control group was normal;in H2O2 model group,astrocyte swelling,strong light refraction of cytoplasm was induced by H2O2 and stereognosis of cells died down;morphological changes of EGCG pretreatment group was not obvious.Compared with normal control group,H2O2 model group could result in an increase of LDH enzyme activity in culture medium.EGCG(5μmol/L,10μmol/L) pretreatment caused the level of LDH in culture medium decreased and the difference is significant(P<0.05).The expression of NQO1 and GFAP protein were mildly decreased in H2O2 group,compared with control group.The expression of NQO1 and GFAP protein were significant increased in 5μmol/L and 10μmol/L EGCG pretreatment group,compared with model group,the difference is significant(P<0.05,P<0.05).The protein of NQO1 were significant increased in 10μmol/L EGCG treated group,compared with control group(P<0.05).Conclusion:These data indicate that EGCG could induce functional expression of antioxidant enzyme NQO1 which locates downstream of Nrf2/ARE pathway. Upregulated expression of NQO1 contribute to antioxidative damage induced by hydrogen peroxide in a certain degree in astrocytes from spinal cord of mice.It is speculated that the activation of Nrf2/ARE induces the expression of a series of target gene,which coordinately strengthern the antioxidative ability and protect nerve cell.EGCG may be an effective agent for clinical neurodegenration.