Lopachev A V, Lopacheva O M, Abaimov D A, Koroleva O V, Vladychenskaya E A, Erukhimovich A A, Fedorova T N
Research Center of Neurology, Moscow, 125367, Russia.
Biochemistry (Mosc). 2016 May;81(5):511-20. doi: 10.1134/S0006297916050084.
Dipeptide carnosine (β-alanyl-L-histidine) is a natural antioxidant, but its protective effect under oxidative stress induced by neurotoxins is studied insufficiently. In this work, we show the neuroprotective effect of carnosine in primary cultures of rat cerebellar cells under oxidative stress induced by 1 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH), which directly generates free radicals both in the medium and in the cells, and 20 nM rotenone, which increases the amount of intracellular reactive oxygen species (ROS). In both models, adding 2 mM carnosine to the incubation medium decreased cell death calculated using fluorescence microscopy and enhanced cell viability estimated by the MTT assay. The antioxidant effect of carnosine inside cultured cells was demonstrated using the fluorescence probe dichlorofluorescein. Carnosine reduced by half the increase in the number of ROS in neurons induced by 20 nM rotenone. Using iron-induced chemiluminescence, we showed that preincubation of primary neuronal cultures with 2 mM carnosine prevents the decrease in endogenous antioxidant potential of cells induced by 1 mM AAPH and 20 nM rotenone. Using liquid chromatography-mass spectrometry, we showed that a 10-min incubation of neuronal cultures with 2 mM carnosine leads to a 14.5-fold increase in carnosine content in cell lysates. Thus, carnosine is able to penetrate neurons and exerts an antioxidant effect. Western blot analysis revealed the presence of the peptide transporter PEPT2 in rat cerebellar cells, which suggests the possibility of carnosine transport into the cells. At the same time, Western blot analysis showed no carnosine-induced changes in the level of apoptosis regulating proteins of the Bcl-2 family and in the phosphorylation of MAP kinases, which suggests that carnosine could have minimal or no side effects on proliferation and apoptosis control systems in normal cells.
二肽肌肽（β-丙氨酰-L-组氨酸）是一种天然抗氧化剂，但其在神经毒素诱导的氧化应激下的保护作用研究尚不充分。在本研究中，我们展示了肌肽在大鼠小脑细胞原代培养物中，在1 mM 2,2'-偶氮二异丁脒二盐酸盐（AAPH）和20 nM鱼藤酮诱导的氧化应激下的神经保护作用。AAPH可在培养基和细胞中直接产生自由基，鱼藤酮则会增加细胞内活性氧（ROS）的含量。在这两种模型中，向孵育培养基中添加2 mM肌肽可降低通过荧光显微镜计算的细胞死亡率，并提高通过MTT法评估的细胞活力。使用荧光探针二氯荧光素证明了肌肽在培养细胞内的抗氧化作用。肌肽将20 nM鱼藤酮诱导的神经元中ROS数量增加减少了一半。通过铁诱导化学发光，我们发现用2 mM肌肽预孵育原代神经元培养物可防止1 mM AAPH和20 nM鱼藤酮诱导的细胞内源性抗氧化潜力的降低。使用液相色谱-质谱联用技术，我们发现用2 mM肌肽孵育神经元培养物10分钟会导致细胞裂解物中肌肽含量增加14.5倍。因此，肌肽能够穿透神经元并发挥抗氧化作用。蛋白质免疫印迹分析显示大鼠小脑细胞中存在肽转运体PEPT2，这表明肌肽有可能转运进入细胞。同时，蛋白质免疫印迹分析表明肌肽对Bcl-2家族凋亡调节蛋白水平和MAP激酶磷酸化没有诱导变化，这表明肌肽对正常细胞的增殖和凋亡控制系统可能具有最小或无副作用。
