| Objective: This study was designed to investigate whether olfactory ensheathing cells conditioned medium（OECCM）could relieve the oxidative damage of neuroblastoma SH-SY5 Y cells induced by Aβ25-35 through inhibiting the mitochondrial apoptosis pathway.These data provide new insights into the roles of OECCM on oxidative stress-induced cell damage in Alzheimer’s disease（AD）model.Methods: SH-SY5 Y cells were induced by Aβ25-35 to establish in vitro model of AD,and the SH-SY5 Y cells were treated with OECCM.The cells were divided into three groups（control group,Aβ group,OECCM group）: cell viability was detected by Cell Count Kit 8（CCK8）assay,the apoptosis,intracellular reactive oxygen species and calcium imaging were detected by flow cytometry.The activities of malondialdehyde（MDA）and endogenous antioxidant enzymes were detected by biochemical detection kit.Fluorescence quantitative PCR and Western Blot were employed to detect the expression of endogenous antioxidant enzymes and mitochondrial apoptosis-related factors mRNA and protein.The expression of Bax and Bcl-2 were detected by immunofluorescence.Results:（1）The cell viability of Aβ group was lower than that in CTRL group（p<0.001）;the cell viability of OECCM group was increased when compared with Aβ group（p<0.001）.（2）Exposure to Aβ25-35 resulted in significantly higher level of ROS in SH-SY5 Y cells than that in normal controls（p=0.026）.The level of ROS in OECCM treated SH-SY5 Y cells were lower than those in Aβ group（p=0.046）.The level of MDA in Aβ group was higher than that in control group（p =0.018）.Compared with Aβ group,the level of MDA in OECCM group decreased（p = 0.02）.（3）The activity of antioxidant enzymes in Aβ group was significantly lower than that in normal group（T-SOD: p = 0.006,CAT: p = 0.03,GPx: p <0.001）,and the activity of antioxidant enzymes in OECCM group was significantly higher than that in Aβ group（T-SOD: p = 0.019,CAT: p = 0.019,GPx: p = 0.004）.The results of fluorescence quantitative PCR showed that the mRNA expression of antioxidant enzymes in Aβ group decreased in different degrees（SOD1: p <0.001,SOD2: p <0.001,CAT: p <0.001,GPx: p<0.001）,and the mRNA of antioxidant enzymes in OECCM group was significantly higher than that in Aβ group（SOD1: p =0.001,SOD2: p <0.001,CAT: p <0.001,GPx: p<0.001）.The results of Western Blot showed that antioxidant enzymes of SOD1 and SOD2 in Aβ group were significantly lower than those in normal group（SOD1,p <0.001;SOD2,p =0.046）and OECCM group（SOD1,p = 0.038;SOD2,p = 0.042）.（4）There is no significant difference of fluorescence intensity of Fluo-4 in SH-SY5 Y cells among the three groups（CTRL group vs Aβ group: p = 0.19;OECCM group vs Aβ group: p = 0.443;CTRL group vs OECCM group: p=0.769）.（5）The mRNA expression of Cytochrome C,Caspase-9,Caspase-3 and Bax in Aβ group was increased when compared with control group（Cytochrome C,p<0.001;Caspase-9,p<0.001;Caspase-3,p<0.001;Bax,p<0.001）and OECCM group（Cytochrome C,p<0.001;Caspase-9,p<0.001;Caspase-3,p<0.001;Bax,p<0.001）by fluorescence quantitative PCR.The mRNA expression of Bcl-2 in Aβ group was decreased when compared with control group（p<0.001）and OECCM group（p<0.001）.The protein expression of Cytochrome C,Caspase-9,Caspase-3 and Bax in Aβ group were increased when compared with OECCM group（Cytochrome C,p<0.001;Caspase-9,p<0.001;Caspase-3,p<0.001;Bax,p<0.001）and control group（Cytochrome C,p<0.001;Caspase-9,p<0.001;Caspase-3,p<0.001;Bax,p<0.001）.The protein expression of Bcl-2 in Aβ group was decreased when compared with control group（p=0.039）and OECCM group（p=0.004）.The fluorescence intensity of Bax in Aβ group was higer than that in control group（p<0.001）and OECCM group（p<0.001）.Compared with Aβ group,Bcl-2 fluorescence intensity increased in OECCM group（p<0.001）and control group（p<0.001）.Conclusion: Our data suggest that OECCM ameliorates Aβ25-35-induced oxidative damage in neuroblastoma SH-SY5 Y cells by regulate the activity of antioxidant enzymes inhibiting the mitochondrial intrinsic pathway. |
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