Effects And Mechanism Of MEK5/ERK5 Signaling Pathway On MN9D Cell Damage Induced By Manganese

Objective: To study the effects of manganese on dopaminergic neuron injury,apoptosis and function in MN9 D cells.And investigate the changes of MEK5/ERK5 signaling pathway and the mechanisms involved in the regulation under the action of manganese in MN9 D cells.Methods: Using in vitro cell culture methods,the cells were divided into 8 groups: blank control group(Mn2+: 0μmol/L,Group C),low dose group(Mn2+: 400μmol/L,Group L),medium dose group(Mn2+: 800μmol/L,Group M),high dose group(Mn2+: 1200μmol/L,Group H),inhibitor control group(BIX02189: 10μmol/L,Group BC),inhibitor & low dose group(BIX02189: 10μmol/L & Mn2+: 400μmol/L,Group BL),inhibitor & medium dose group(BIX02189: 10μmol/L & Mn2+: 800μmol/L,Group BM),inhibitor & high dose group(BIX02189: 10μmol/L & Mn2+: 1200μmol/L,Group BH),and each group was treated with Mn2+ for 24 hours.The cell morphology was observed by inverted microscope.Cell viability was detected by MTT colorimetric assay.Apoptotic bodies were observed by Hoechst 33258 fluorescent probe.Annexin V/PI double staining was used to detect the apoptotic rate by flow cytometry.Use the DCFH-DA probe to detect intracellular ROS levels,the content of DA in cell culture medium was detected by ELISA.The Western Blot and RT-q PCR were used to detect the expression of Bcl-2,Bax,Caspase-3,MEK5,p-MEK5,ERK5 and p-ERK5 proteins and genes.Results: 1.The MTT assay showed that the cell viability of the two time periods was statistically significant at the two concentrations of 24 hours and 48 hours(P<0.05).The cell viability of the other groups was significantly different from that of the control group of 72 hours(P<0.05).The survival rate of the other groups was lower than that of the blank control group(P<0.05).There was no significant difference in the apoptotic rate between Group BC and control group(P>0.05).With the dose of manganese increased gradually,MN9 D cell apoptosis rate also increased gradually,compared with Group C,the difference was statistically significant(P<0.05).Compared with Group BC,the apoptotic rateincreased gradually after the pretreatment with inhibitor,and the difference was statistically significant(P<0.05).And the rate of apoptosis after pretreatment with the inhibitor increased,the difference was statistically significant(P<0.05).2.Inverted microscope observation showed that cells in Group C and BC were normal.However,with the increase of manganese concentration,the cell synapses become shorter or disappear,and the cell float increases.The cells pretreated by the inhibitor pretreatment shrink and become more visible and produce large amounts of cell debris.3.The Hoechst 33258 fluorescent probe staining showed that the cells in the blank group and the inhibitor treatment group were weak blue.With the increase of the concentration of manganese,the nuclei showed dense cells.After pretreatment with the inhibitor,the cells were stained with dense cells,and the nuclei chromatin condensation and marginalization,nucleosome formation was obvious.4.In the bivariate flow scatter plot shows that with the manganese concentration increased,the apoptosis rate is gradually increased.Compared with Group C,the apoptotic rates of Group L,M and H were increased(P<0.05).5.DCFH-DA probe detection of intracellular ROS levels found that different concentrations of manganese on the cells,the cells in the ROS content were significantly increased.In addition to Group BC,the other groups were compared with Group C,ROS content were increased(P<0.05).6.The content of DA in the cell culture medium was detected by ELISA.The results showed that the contents of DA in the Group BM and BH were lower than those in the Group C(P<0.05).7.Western blot analysis showed that the expression of Bcl-2 in MN9 D cells was decreased compared with that in the control group(P<0.05).The expression of Bcl-2 protein in Group BL,BM and BH were decreased significantly(P<0.05).The expression of Bax protein increased with the increase of manganese concentration,and the expression of Bax protein increased with the increase of the concentration of Group BL,BM and BH(P<0.05).The protein expression of Bax protein was increased compared with Group L,M and H respectively(P<0.05).The ratio of Bcl-2 and Bax protein decreased with the increase of the dose of Mn,and the ratio of Group BL,BM and BH decreased after the pretreatment of the inhibitor(P<0.05).Compared with Group L,M and H,the difference was statistically significant(P<0.05).The expression of Caspase-3 protein was increased in the other groups except Group L(P<0.05).The expression of total protein of MEK5 and ERK5 was not statistically significant compared with the control group(P>0.05).The expression of p-MEK5 in MN9 D cells was significantly higher than that in control group(P<0.05).The expression of p-MEK5 in Group M and H was significantlyhigher than that in Group M and H(P<0.05).The ratio of p-MEK5 and MEK5 protein expression in Group M,H,BM and BH was significantly higher than that in control group(P<0.05).The expression of p-ERK5 protein in the Group BC,BL,BM and BH was significantly higher than that in the control group(P<0.05),and the expression of p-ERK5 protein was significantly higher than that of the control group,the difference was not statistically significant(P>0.05).The ratio of p-ERK5 and ERK5 protein expression in Group M and H increased,respectively,compared with the control group,the difference was statistically significant(P<0.05).8.The expression of Bcl-2 m RNA in MN9 D cells was decreased compared with that in the control group,and the expression of Bcl-2 m RNA in the Group BH was lower than that in the control group(P<0.05).Group M and BM compared with the Group C,Bax m RNA expression increased significantly difference was statistically significant(P<0.05).The ratio of Bcl-2 to Bax expression decreased with the dose of manganese(P<0.05).The expression of Caspase-3 m RNA in Group H and BM was higher than that in control group(P<0.05).The expression of MEK5 m RNA in each group was significantly higher than that in the control group(P<0.05),except for the increase of Group H and the decrease of Group BH.The expression of ERK5 m RNA in each group was significantly higher than that in control group(P<0.05),except Group M,H,BM and BH.Conclusion: Activation of the ERK5 pathway induced by Mn Cl2 plays an anti-apoptotic and anti-injury role in Mn-induced MN9 D cytotoxicity.