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Conditioned medium obtained from human amniotic mesenchymal stem cells attenuates focal cerebral ischemia/ reperfusion injury in rats by targeting mTOR pathway
Journal of Chemical Neuroanatomy ( IF 2.7 ) Pub Date : 2019-12-01 , DOI: 10.1016/j.jchemneu.2019.101707
Donya Nazarinia 1 , Nahid Aboutaleb 1 , Raheleh Gholamzadeh 1 , Solmaz Nasseri Maleki 1 , Behnaz Mokhtari 1 , Mahin Nikougoftar 2
Affiliation  

Conditioned medium obtained from human amniotic mesenchymal stem cells (hAMSC-CM) was recently shown to have many antioxidant, antiapoptotic and proangiogenic growth factors. The present study was performed to investigate whether protective effects of hAMSC-CM against focal cerebral ischemia/ reperfusion (I/R) injury is associated with modulation of the mammalian target of rapamycin (mTOR) pathway. A rat model of middle cerebral artery occlusion (MCAO) was created and the animals were divided into three groups including sham, MCAO and MCAO + hAMSC-CM. Drug was administrated immediately after cerebral reperfusion (i.v). The expressions of mTOR, p-mTOR and LC3 were measured using Western blotting and real time-PCR, respectively. Apoptosis and neuronal loss were determined using TUNEL and Nissl staining, respectively. Infarct volume and the blood-brain barrier (BBB) damage were evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining and Evans Blue (EB) uptake, respectively. Compared with sham, significant infarct volume, apoptotic cell death, and neuronal loss were found in MCAO rats that reversed by hAMSC-CM (P < 0.05). Likewise, MCAO rats exhibited increased mRNA level of light-chain 3 (LC3) and the LC3II/LC3I ratio as well as decreased expression level of p-mTOR that reversed by hAMSC-CM (P < 0.05). There were no significant differences in the expression of total mTOR among the experimental groups. In summary, our results demonstrate that hAMSC-CM gives rise to neuroprotection following ischemic stroke by restoring mTOR activity and inhibiting autophagy.

中文翻译:

人羊膜间充质干细胞条件培养基靶向mTOR通路减轻大鼠局灶性脑缺血/再灌注损伤

从人类羊膜间充质干细胞 (hAMSC-CM) 获得的条件培养基最近被证明具有许多抗氧化、抗凋亡和促血管生成生长因子。本研究旨在调查 hAMSC-CM 对局灶性脑缺血/再灌注 (I/R) 损伤的保护作用是否与哺乳动物雷帕霉素靶标 (mTOR) 通路的调节有关。建立大脑中动脉闭塞(MCAO)大鼠模型,将动物分为假手术组、MCAO组和MCAO+hAMSC-CM三组。脑再灌注后立即给予药物(iv)。mTOR、p-mTOR 和 LC3 的表达分别使用蛋白质印迹和实时 PCR 测量。分别使用 TUNEL 和 Nissl 染色确定细胞凋亡和神经元丢失。分别使用 2,3,5-氯化三苯基四唑 (TTC) 染色和伊文思蓝 (EB) 摄取评估梗死体积和血脑屏障 (BBB) 损伤。与假手术相比,在被 hAMSC-CM 逆转的 MCAO 大鼠中发现了显着的梗死体积、凋亡细胞死亡和神经元丢失(P < 0.05)。同样,MCAO 大鼠表现出轻链 3 (LC3) 的 mRNA 水平和 LC3II/LC3I 比率增加,以及被 hAMSC-CM 逆转的 p-mTOR 表达水平降低(P < 0.05)。实验组间总mTOR的表达无显着差异。总之,我们的结果表明,hAMSC-CM 通过恢复 mTOR 活性和抑制自噬在缺血性中风后产生神经保护作用。分别为 5-三苯基氯化四唑 (TTC) 染色和伊文思蓝 (EB) 摄取。与假手术相比,在被 hAMSC-CM 逆转的 MCAO 大鼠中发现了显着的梗死体积、凋亡细胞死亡和神经元丢失(P < 0.05)。同样,MCAO 大鼠表现出轻链 3 (LC3) 的 mRNA 水平和 LC3II/LC3I 比率增加,以及被 hAMSC-CM 逆转的 p-mTOR 表达水平降低(P < 0.05)。实验组间总mTOR的表达无显着差异。总之,我们的结果表明,hAMSC-CM 通过恢复 mTOR 活性和抑制自噬在缺血性中风后产生神经保护作用。分别为 5-三苯基氯化四唑 (TTC) 染色和伊文思蓝 (EB) 摄取。与假手术相比,在被 hAMSC-CM 逆转的 MCAO 大鼠中发现了显着的梗死体积、凋亡细胞死亡和神经元丢失(P < 0.05)。同样,MCAO 大鼠表现出轻链 3 (LC3) 的 mRNA 水平和 LC3II/LC3I 比率增加,以及被 hAMSC-CM 逆转的 p-mTOR 表达水平降低(P < 0.05)。实验组间总mTOR的表达无显着差异。总之,我们的结果表明,hAMSC-CM 通过恢复 mTOR 活性和抑制自噬在缺血性中风后产生神经保护作用。在被 hAMSC-CM 逆转的 MCAO 大鼠中发现神经元丢失(P < 0.05)。同样,MCAO 大鼠表现出轻链 3 (LC3) 的 mRNA 水平和 LC3II/LC3I 比率增加,以及被 hAMSC-CM 逆转的 p-mTOR 表达水平降低(P < 0.05)。实验组间总mTOR的表达无显着差异。总之,我们的结果表明,hAMSC-CM 通过恢复 mTOR 活性和抑制自噬在缺血性中风后产生神经保护作用。在被 hAMSC-CM 逆转的 MCAO 大鼠中发现神经元丢失(P < 0.05)。同样,MCAO 大鼠表现出轻链 3 (LC3) 的 mRNA 水平和 LC3II/LC3I 比率增加,以及被 hAMSC-CM 逆转的 p-mTOR 表达水平降低(P < 0.05)。实验组间总mTOR的表达无显着差异。总之,我们的结果表明,hAMSC-CM 通过恢复 mTOR 活性和抑制自噬在缺血性中风后产生神经保护作用。
更新日期:2019-12-01
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