The Apoptosis And Oxidative Stress In Methylglyoxal-induced SH-SY5Y Cells And The Protective Effects Of Ginsenoside Rbl

AimsThe aim of the present study is via simulating the injury of SH-SY5Y by methylglyoxal(MGO)to investigate the mechanism of oxidative stress,apoptosis,and PI3K/Akt pathway in MGO-induced neuron damage and the protective effects of ginsenoside Rb1(abbreviated as Rbl)at the cellular and molecular level.MethodsThe SH-SY5Y cell was incubated by MGO with a series of concentrations(2,1,0.75,0.5,0.25 and 0.125 mM)for 24 h,and the cell viability was measured by MTT to determine the optimal concentration of MGO.After that,the SH-SY5Y cell was pretreated with Rb1(1.5,0.75 and 0.375 μM)for 8 h,and then incubated with 0.5 mM MGO for 24 h.The cell viability and lactic dehydrogenase(LDH)were detected to verify the optimal protective concentration of Rb1.After the SH-SY5Y cell was treated with Rb1 and MGO,the levels of ROS,malonaldehyde(MDA),superoxide dismutase(SOD),catalase(CAT)and total glutathione were analyzed according to the manufactory’s instructions.Hoechst 33342/PI staining and flow cytometry with Annexin V/PI staining were performed to evaluate the apoptosis.The mitochondrial membrane potential was measured by the fluorescence probe JC-1,and the apoptosis-related proteins,including Bcl-2,Bax,cleaved Caspase-3 and cleaved Caspase-9 were measured by Western blot.The cell was pre-incubated with PI3K specific inhibitor LY294002(10 μM)for 1 h before Rb1 treatment to investigate the effects of Rbl on PI3K/Akt signal pathway.Results1.The cell viability of SH-SY5Y was inhibited by increasing concentrations of MGO.After incubation with 0.5 mM MGO for 24 h,the survival rate of SH-SY5Y was maintained at about 60%(p<0.01).Therefore,0.5 mM MGO incubation for 24 h was regarded as the optimal model condition.Rbl pretreatment could increase the cell viability of SH-SY5Y damaged by 0.5 mM MGO in a dose-dependent manner,and 1.5 μM Rb1 exerted the most significantly protective effects on SH-SY5Y cell(p<0.01).Rbl preadministration decreased LDH release in a dose-dependent way,and the effect of 1.5 μM Rbl was the best(p<0.01).2.MGO induced the remarkable increase in the generation of ROS(p<0.01),characterized by the enhanced fluorescence intensity of green.Pretreatment with Rbl could reduce the ROS level in SH-SY5Y cell(p<0.01).Furthermore,Rbl could reverse the decreased SOD,CAT and total glutathione,and increased MDA induced by MGO(p<0.01).3.MGO caused the breakage and shrinkage of cell nucleus,and the cell nucleus of SH-SY5Y in MGO group was characterized by bright blue fluorescence and increased red fluorescence by Hoechst 33342/PI staining.Rbl could improve the morphology of cell nucleus and decrease the red fluorescence intensity.Besides,Rbl(p<0.01)pretreatment could inhibit the elevated apoptosis rate in SH-SY5Y caused by MGO(p<0.01)detected by flow cytometry.4.The results from JC-1 staining showed that MGO induced the increase in green/red fluorescence ratio of SY-SY5Y cell(p<0.01).Rbl could inhibit the reduction of green/red fluorescence ratio(p<0.01).Compared with control group,MGO significantly reduced the protein ratio of Bcl-2/Bax(p<0.001),and increased the expression of cleaved Caspase-3 and cleaved Caspase-9 in SH-SY5Y(p<0.01).However,Rbl could increase the Bcl-2/bax ratio(p<0.01)and inhibit the levels of cleaved Caspase-3 and cleaved Caspase-9(p<0.01).5.The reduced expression of p-Akt caused by MGO(p<0.01)could also be reversed by Rbl(p<0.01).Moreover,PI3K specific inhibitor LY294002 could weaken the protective efficacy of Rbl in MGO-induced SH-SY5Y cell.LY294002 pretreatment significantly reversed the increased cell viability(p<0.01),decreased apoptosis(p<0.05)and enhanced p-Akt level(p<0.01)by Rb1 in MGO-induced SH-SY5Y cell.Conclusions1.MGO could cause oxidative stress,inhibit PI3K/Akt pathway,up-regulate the ratio of Bcl-2/Bax and the expression of Caspase family protein in the mitochondrial apoptosisi pathway,thus leading to the impairment in SH-SY5Y cells.2.Rbl could alleviate the oxidative stress induced by MGO,characterized by decreasing the production of ROS and MDA,raising the level of SOD,CAT total GSH.3.Rbl could distinctly attenuate the apoptosis induced by MGO through regulating the expression of Bcl-2 family and Caspse family proteins.4.Rb1 could avtivate the PI3K/Akt pathway by upregulating the expression of p-Akt,thus facilitating the survival and inhibiting the apoptosis of SH-SY5Y cells.