| ObjectiveTo investigate the effect of Granylgeranylacetone(GGA)on the abnormal aggregation of TDP43 protein in paraquat-induced oxidative stress injury model of SH-SY5 Y cells and further study that the effect of GGA on the level of TDP43(35kDa)protein and autophagy in this cell model.Methods1 In order to observe the protective effect of GGA on paraquat-induced oxidative stress injury model of SH-SY5 Y cells(Experiment 1),SH-SY5 Y cells were established using 1mmol/L PQ to induce oxidative stress model.SH-SY5 Y cells were divided into group A(Control group),group B(100?mol/L GGA treatment group),group C(1 mmol/L PQ treatment group)D-F group(PQ-induced oxidative stress model were treated with concentrations of 20,50,and 100?mol/L GGA,respectively,24 hour).MTT and Annexin V/PI staining analysis were used to detect the survival rate and apoptosis rate of each group of SH-SY5 Y cells.2 To observe the effect of GGA on the protein level of TDP43(35kDa)(Experiment 2),the grouping method is the same as Experiment 1.Using Western blot to detect the expression level of TDP43(35kDa)protein.3.To observe the effect of GGA on autophagy in this cell model(Experiment 3),the grouping is the same as Experiment 1.Detection of the expression levels of autophagyrelated proteins LC3 B and P62 by Western blot.4.To observe whether the effect of GGA on TDP43(35kDa)protein level changes through autophagy(Experiment 4),SH-SY5 Y cells were divided into group A(control group),group B(1mmol/L PQ treatment group)C,D Group(the PQ-induced oxidative stress model was treated with GGA at a concentration of 100 and 100?mol/L for 24 hours,respectively,and group D was blocked with 5-mmol/L 3-MA to block autophagy).Western blot was used to detect TDP43(35kDa)and LC3 B protein Level.5.To observe the effect of GGA on TFEB(Experiment 5),SH-SY5 Y cells were divided into control group,group B(100?mol/L GGA treatment group),group C(PQinduced oxidative stress model),and group D(using the model was treated with a concentration of 100?mol/L GGA).After using immunochemistry,laser confocal microscopy to observe the entry of TFEB into the nucleus.Results1.Experiment(1)The survival rate of cells in the 1mmol/L PQ treatment group was significantly lower than that in the control group.As the therapeutic concentration of GGA increases(20,50,100?mol/L),the cell survival rate also increases P 0.01.The apoptosis rate of the 1mmol/L PQ treatment group was significantly higher than that of the control group,and the apoptosis rate gradually decreased with the increase of the therapeutic concentration of GGA P 0.01.The survival rate and apoptosis rate of SHSY5 Y cells in the 100?mol/L GGA-treated group were not different from the control group,indicating that GGA at this concentration was not toxic to SH-SY5 Y cells.GGA has a neuroprotective effect on PQ-induced SH-SY5 Y cells.2.Experiment(2)Western blot results showed that after treatment with 1mmol/L PQ,the protein level of TDP43(35kDa)increased significantly compared with the control groupP 0.01.After GGA treatment,the level of TDP43(35kDa)reduced and it was concentration-dependently.3.Experiment(3)the ratio of LC3?/? protein and P62 protein was used to reflect the intensity of autophagy.After GGA treatment,the ratio of LC3?/? protein increased and the expression of P62 protein decreased P 0.01,and GGA enhanced autophagy in PQinduced SH-SY5 Y cells.4.Experiment(4)Western blot results showed that after inhibiting autophagy with 3-MA,the clearance ability of GGA to TDP43(35kDa)protein level was weakened in PQinduced SH-SY5 Y cells P 0.01.This indicates that GGA clears TDP43(35kDa)protein by activating autophagy.5.Experiment(5)GGA can promote TFEB translocation into nucleus in PQ-induced SHSY5 Y cells P 0.05.ConclusionOur research indicates that GGA protect PQ-induced SH-SY5 Y cells from oxidative stress damage.GGA reduces TDP43(35kDa)protein level in this cell model by enhancing autophagy.The effect of GGA provides a reference for it to become a candidate for the treatment of amyotrophic lateral sclerosis. |
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