Protective Effect Of MgSO₄ On 6-hydroxydopamine-induced Neurotoxicity In SH-SY5Y Cells

Objective:1. To study whether there is a protective effect of Mg SO4 on 6-hydroxydopamine induced neurotoxicity in SH-SY5 Y cells. 2. To investigate the effect of Mg SO4 on tyrosine hydroxylase(TH) expression in SH-SY5 Y cells treated with 6-OHDA. 3. To investigate the change of m RNA and protein expression of magnesium transporters SLC41A1, NIPA1, Mag T1 and CNNM2 on 6-OHDA-induced neurotoxicity in SH-SY5 Y cells. 4. To investigate the impact of Mg SO4 in cell anti-oxidative capacity and the production of reactive oxygen species(ROS) on 6-OHDA injured SH-SY5 Y.Methods:1. Using Cell Counting Kit-8(CCK-8) to measure the cell viability under different concentration of Mg SO4 and 6-OHDA, and investigate the effect of Mg SO4 on cell viability in 6-OHDA treated SH-SY5 Y cells. 2. The cultured SH-SY5 Y cells were divided into 5 groups as follows: the control group; the 50μM 6-OHDA group; the Mg SO4 groups(the final concentrations were 0.25 m M, 0.5m M and 1m M, Mg SO4 were added 1h before 6-OHDA). 3. Detect the expression of TH under above treatment by Western Blot and immunofluorescence. 4. Measure the m RNA and protein expression of magnesium transporters SLC41A1, NIPA1, Mag T1 and CNNM2 under above treatment by q PCR and Western Blot.5. Using ABTS to measure the effect of Mg SO4 to the total anti-oxidative capacity of SH-SY5 Y cells treated with 6-OHDA. 6. Using DCFH-DA to detect the intracellular ROS level of SH-SY5 Y cells under above treatment.Results:1. CCK-8 test data illustrates that SH-SY5 Y cells preincubated with 25μM~50μM 6-OHDA 24 h could decrease the cell viability(p<0.05,versus control group), while add 0.125 m M~1m M Mg SO4 1h before 6-OHDA could prevent the cell damage of 6-OHDA(p<0.05,versus 6-OHDA group). 2. Western blot data shows that SH-SY5 Y treated with 50μM 6-OHDA lead 52.23% loss of TH versus control group(p<0.05). While Mg SO4 can prevent SH-SY5 Y from 6-OHDA induced TH loss. 3. q PCR and Western Blot test indicate that there is significantly decrease of cellular m RNA and proteins expression level of SLC41A1, NIPA1, Mag T1 and CNNM2 in 6-OHDA treated SH-SY5 Y. Mg SO4 can reverse that decreasing. 4. Mg SO4 attenuate 6-OHDA-induced reducing of cell total anti-oxidative capacity and increasing in intracellular ROS level.Conclusions:MgSO4 may protect SH-SY5 Y cells against 6-OHDA-induced cell injury. And the magnesium transporters SLC41A1, NIPA1, Mag T1 and CNNM2 may attend the developing of PD.