Research On Protective Effects Of MgSO₄ On 6-hydroxydopamine-induced Neurotoxicity In Undifferentiated/Differentiated SH-SY5Y Cells

Objective:1.Compare the changes of active oxygen species、antioxidant capacity and protective effect of Mg SO4 on 6-hydroxydopamin induced neurotoxicity in undifferentiated and differentiated SH-SY5 Y cells.2.The expression of tyrosine hydroxylase(TH)in undifferentiated and differentiated SH-SY5 Y cells.Methods:1.Construct Parkinson’s disease cell model using the SH-SY5 Y treated with 6-OHDA.2.Cell viability was measured with different concentration of 6-OHDA in differentiated SH-SY5 Y cells by Cell Counting Kit-8(CCK-8).3.The effect of MgSO4 on cell viability in 6-OHDA treated undifferentiated and differentiated SH-SY5 Y cells was measured by CCK-8.4.Undifferentiated SH-SY5 Y cells were divided into three groups(control,6-OHDA,and Mg SO4).The 6-OHDA group was incubated with 50μM 6-OHDA for 24 h,Mg SO4 group was pretreated with Mg SO4(0.25,0.5,and 1m M)1h before 6-OHDA.The control was added equal volume of dissolvant.Undifferentiated SH-SY5 Y cells were treated with all-trans-retinoic acid(ATRA)for 72 h to differentiate SH-SY5 Y cells,and the differentiated cells were divided into three groups(control,6-OHDA,and Mg SO4).The concentration of6-OHDA in differentiated SH-SY5 Y cells was 200μM.5.The expression of TH in the cells was observed by immunocytochemistry.6.The total antioxidant capacity of cells were detected by 2,2-nitrogen-two(3-benzothiazole-6-sulfonic acid)-ammonium salt(ABTS).7.The intracellular ROS in the cells were detected using the fluorescent probe ’7’-twochloro-fluorescein diacetate(DCFH-DA).Results:1.The cell counting showed that differentiated SH-SY5 Y cells after incubated with 50 μM~200 μM 6-OHDA 24 h had less survival rate compared with the control(p<0.05).For example,the cell survival rate was decreased by 56.54% after treated with 200 μM(P<0.05).2.Mg SO4 could increase the survival rate in the undifferentiated and differentiated groups after treated with 6-OHDA(p<0.05).The damage concentration of 6-OHDA in the undifferentiated group was 50 μM,while the differentiated group was 200 μM and the optimal protective concentration of Mg SO4 in undifferentiated group was 0.5m M.Interestingly,the protective ability of Mg SO4 in differentiated group was increased as the increase of concentration of Mg SO4,which showed that the sensitivity of SH-SY5 Y cells to6-OHDA and MgSO4 was different in different differentiated form.3.The expression of TH in differentiated SH-SY5 Y cells was significantly higher than that in undifferentiated SH-SY5 Y cells.4.The antioxidant capacity and ROS detection results showed that the cells after treated with6-OHDA had low antioxidant capacity compared with control group(p<0.05),and produced a large number ROS.Mg SO4 can increase the total antioxidant capacity of damaged cells,and inhibit the formation of ROS.However,there was a significant difference between the undifferentiated and differentiated groups,suggesting that the protective effect of Mg SO4 may be related to the differentiation of SH-SY5 Y cells.Conclusions:MgSO4 can protect undifferentiated as well as differentiated SH-SY5 Y cells injured by6-OHDA.However,the protective effect may be related to the differentiation of SH-SY5 Y cells.