N-乙酰基半胱氨酸对Aβ_(25-35)活化calpain活性的影响及可能机制

Possible effect of N-acetyl-L-cysteine on Aβ_(25-35)-induced increase of calpain activity

目的探讨N-乙酰基半胱氨酸(N-acetyl-L-cysteine,NAC)对β淀粉样蛋白毒性片段25-35(Aβ_(25-35))活化calpain活性的影响及可能机制。方法运用Aβ_(25-35)来诱导皮质神经元的损伤,实验分为空白对照组、Aβ_(25-35)组、NAC与Aβ_(25-35)共同作用组,采用荧光酶标法检测calpain活性、氧自由基(H2O2)和线粒体膜电位;用化学发光法测定神经元内ATP水平。结果 Aβ_(25-35)(20μmol·L_(-1))作用12h可明显升高calpain活性;与Aβ_(25-35)处理组相比,NAC(10 mmol·L_(-1))预先作用24h,然后再与Aβ_(25-35)共同作用12h,可明显降低calpain活性;与空白对照组相比,Aβ_(25-35)处理组中的H_2O_2明显升高、线粒体膜电位和ATP水平明显下降;而与Aβ_(25-35)处理组相比,NAC预处理组中的H_2O_2水平下降、线粒体膜电位和ATP水平升高,这些差异皆有统计学意义。结论这些研究结果提示,NAC可能通过线粒体的保护作用来降低Aβ_(25-35)诱导的异常活化的calpain活性。

Aim To explore the effect of N-acetyl-L-cysteine ( NAC ) on β amyloid peptide 25 - 35 ( Aβ25-35 )-induced the increase of calpain activity and its possible mechanism. Methods The activity of cal-pain was induced by 20μmol·L-1 Aβ 25-35 in primary cortical neuron. Neurons were incubated in the absent or present Aβ25-35 , or pre-incubated NAC ( 10 mmol ·L-1 ) , then co-incubated with Aβ25-35 . The meas-urement of calpain activity, H2 O2 level and mitochon-drial membrane potential was performed on a micro-plate fluorometer. The ATP level was detected using a luciferin/luciferase based ATP assay kit. Results In Aβ25-35 treated group, the activity of calpain and H2 O2 was obviously higher than that in control group. How-ever, in neurons pre-incubated in NAC and then co-in-cubated in Aβ25-35 , the calpain activity and H2 O2 level were significantly decreased compared with that in Aβ25-35 group. Upon Aβ25-35 exposure for 12 h, corti-cal neurons showed a significant decrease in mitochon-drial membrane potential and ATP level when com-pared to the control group. Pre-treatment with NAC showed an increase in mitochondrial membrane poten-tial and ATP level as compared to neurons treated with Aβ25-35 alone for 12h. Conclusion This result sug-gests that NAC can attenuate calpain activity induced by Aβ25-35 through protecting mitochondria.