Changes And Possible Mechanism Of MgSO₄ And Magnesium Transporters SLC41A1 On 6-hydroxydopamine-induced SH-SY5Y Cells And Parkinsonian Rats

Objective:To investigate the changes of MgSO₄ and Mg²⁺transporter SLC41A1 on6-OHDA-induced neurotoxicity in SH-SY5Y cells and parkinsonian rats,and to explore its role in PD pathogenesis and possible mechanisms.Methods1.Differentiated SH-SY5Y cells was induced by 10μmol/L all-trans retinoic acid（ATRA）for 72h,then the cells were randomly divided into control,200μM6-OHDA and MgSO₄ treatment groups（the final concentrations of 0.5mM,1mM,2mM,5mM,8mM and 10mM were added 1h before 6-OHDA treatment）.2.The effect of MgSO₄ on cell viability in 6-OHDA-treated SH-SY5Y cells was measured using a CCK-8（cell counting kit-8）kit.3.The protein expression of tyrosine hydroxylase（TH）and SLC41A1 in SH-SY5Y cells were detected by Western blot.4.The changes of total SOD activity of MgSO4 on 6-OHDA-damaged SH-SY5Y cells were detected using WST-8 method.5.A unilateral parkinsonian rat model was induced by injection of 6-OHDA into the right medial forebrain bundle.Some rats received MgSO₄（90 mg/kg/day,intraperitoneal injection）for 14 days starting from the day following the 6-OHDA injection.6.The extent of dopamine depletion was determined by assessing the density of tyrosine hydroxylase（TH）-immunoreactivity in the substantia nigra（SN）and ventral tegmental area（VTA）,and the apomorphine（APO）-induced rotational behavior.7.The protein expression of TH and SLC41A1 in striatum was evaluated by western blotting respectively.Results:1.Differentiated SH-SY5Y cells treated with 200μM 6-OHDA for 24 h significantly decreased cell viability,while if pre-incubated with 0.5mM⁸mM MgSO₄ for 1 h before adding 6-OHDA it partially prevented the cell damage（p<0.05）.However,when MgSO₄ concentration reached 10mM,the cell viability was significantly lower than that of the control group（p<0.05）.2.There was a significant decrease in protein expression of TH and SLC41A1 in 6-OHDA treated SH-SY5Y cells,and MgSO₄ is able to reverse its decline（p<0.05）.3.The SOD levels on 6-OHDA-induced SH-SY5Y cells were significantly lower than that of the control group（p<0.05）.The SOD levels of the cells were significantly higher than that of the injured group（p<0.05）when MgSO₄concentration were 5mM and 8mM.However,the SOD level was significantly reduced when the MgSO₄ concentration was 10mM（p<0.05）.4.APO-induced rotations increased significantly in the 6-OHDA lesioned rats compared with the controls,with MgSO₄ administration significantly improving the behavior.The densities of TH-immunoreactive neurons and processes in both the SN and VTA were significantly lower in the lesioned side than in the unlesioned side（p<0.001）.Administration of MgSO₄ for 14 days partly ameliorated the loss of TH-positive neurons and processes in the VTA（p<0.001）.5.The protein expressions of TH and SLC41A1 in the lesioned STR were lower in the 6-OHDA lesioned rats than in the controls at 14 days post-lesion induction（p<0.05）.MgSO₄ supplementation reversed the protein expressions of SLC41A1（p<0.01）.Conclusions:1.MgSO₄ may protect differentiated SH-SY5Y cells against 6-OHDA-induced cell injury,and that gene expression of SLC41A1 might be involved in Parkinson’s disease.2.The regulation of SLC41A1 expression is responsive to magnesium in a 6-OHDA-induced rat model,wherein 6-OHDA may alter magnesium transport in the brain.