Effects of N-acetylcysteine on endoplasmic reticulum stress and oxidative stress in cerebral micro-vascular endothelial cells / 中华行为医学与脑科学杂志

ABSTRACT

Objective:To investigate the effect of N-acetylcysteine (NAC) on endoplasmic reticulum stress (ERS) and oxidative stress(OS) induced by tunicamycin (Tm) and its mechanism.Methods:Mouse derived brain microvascular endothelial cells cultured in vitro were divided into control group (normal cell culture), TM group (cells were intervened with 5 μg/mL Tm for 24 h), NAC + TM group (cells were pretreated with 1 mmol/L NAC for 1 h, then were intervened with 5 μg/mL Tm for 24 h) and NAC group (cells were intervened with 1 mmol/L NAC for 24 h) according to different intervention methods.CCK-8 and FITC-Annexin V/PI were used to detect the survival rate and apoptosis rate of cells.Western blot was used to detect the expression of GRP78、CHOP、p-eNOS and caspase-12 protein. Laser confocal microcopy was used to detect the expression of ROS, and colorimetry was used to detect the activity of MDA and SOD.Results:There were significant differences in apoptosis rate and survival rate among the four groups ( F=62.57, 35.00, both P<0.05). The apoptosis rate of TM group ((25.49±1.55)%) was higher than that of Control group ((13.76±1.48)%)( P<0.01), while the apoptosis rate of NAC+ TM group ((17.65±1.00)%) was lower than that of TM group ( P<0.01). The survival rate of TM group ((66.33±5.69)%) was lower than that of Control group ((100.00±2.12)%)( P<0.01), while the survival rate of NAC+ TM group ((85.67±4.04)%) was higher than that of TM group ( P<0.01). Western blot showed that there were significant differences in the expression levels of GRP78、CHOP and p-eNOS among the four groups ( F=32.39, 68.66, 13.12, all P<0.01). The expression levels of GRP78 and CHOP protein in TM group were higher than those of Control group (both P<0.05), while the expression level of p-eNOS was lower than that of Control group ( P<0.01). The expression levels of GRP78 and CHOP protein in NAC+ TM group were lower than those of TM group (both P<0.05), while the expression level of p-eNOS was higher than that of TM group ( P<0.01). There was no significant difference in the expression level of caspase-12 protein among the four groups ( F=0.33, P>0.05). Laser confocal showed that there was significant difference in the average fluorescence intensity of ROS among the four groups ( F=77.66, P<0.01). The average fluorescence intensity of ROS in TM group (32.67±1.53) was higher than that in Control group (12.67±2.08) and NAC+ TM group (18.33±1.53) (both P<0.01). Colorimetry showed that there were significant differences in the activity of SOD and the concentration of MDA among the four groups ( F=40.53, 34.99, both P<0.01). The results of colorimetry showed that the activity of SOD in TM group((41.60±1.53)U/mg) was lower than that in Control group((65.39±4.60)U/mg) and NAC+ TM group((58.72±1.64)U/mg)(both P<0.01). The concentration of MDA in TM((2.27±0.11)μmol/mg) group was higher than that in Control group((1.39±0.13)μmol/mg) and NAC+ TM group((1.44±0.11)μmol/mg) (both P<0.01). Conclusion:NAC can reduce Tm-induced apoptosis of cerebral micro-vascular endothelial cells, which may be related to its inhibition of ERS/ OS-related pathways.