人牙髓干细胞条件培养基通过AMPK/PGC-1α途径改善氯化钴诱导的颏舌肌成肌细胞低氧损伤

目的： 研究氯化钴 (CoCl₂) 模拟的低氧对颏舌肌成肌细胞活性和氧化应激的影响,探讨人牙髓干细胞 （human dental pulp stem cells,hDPSCs）条件培养基 (conditioned medium,CM)保护作用的机制与AMPK/PGC-1α通路的关系。方法： 分离、培养及鉴定hDPSCs,通过超滤浓缩法提取条件培养基;分离、培养小鼠颏舌肌成肌细胞,分为对照组、CM组、CoCl₂组、CoCl₂+CM组。采用CCK-8法检测颏舌肌成肌细胞的活性,分别使用DCFH-DA和MitoSOX Red评估细胞内和线粒体活性氧 (reactive oxygen species,ROS) 水平,实时定量PCR分析NRF-1、NRF-2线粒体相关基因的表达,Western 印迹法检测PGC-1α、p-AMPK、总AMPK蛋白表达。采用SPSS 22.0软件包对数据进行统计学分析。结果： 颏舌肌成肌细胞经过200 μmol/L CoCl₂处理24 h后增殖显著下降（P<0.05）,细胞内和线粒体内ROS含量显著上升（P<0.05）;与CoCl₂组相比,加入hDPSCs-CM后,细胞活性显著增加（P<0.05）,胞内和线粒体内ROS含量显著降低 (P<0.05);hDPSCs-CM显著上调颏舌肌成肌细胞中pAMPK和PGC-1α蛋白的表达水平以及PGC-1α下游的线粒体效应物NRF-1、NRF-2的mRNA表达水平 (P<0.05)。结论： 人牙髓干细胞条件培养基能减轻CoCl₂诱导的颏舌肌成肌细胞低氧损伤,其机制可能与AMPK/PGC-1α通路的调节有关。

Human dental pulp stem cells conditioned medium protects genioglossus myoblast from cobalt chloride-induced hypoxia injury through AMPK/PGC-1α pathway

PURPOSE: To study the effect of hypoxia induced by cobalt chloride (CoCl₂) on viability and oxidative stress of genioglossus myoblast, and to explore the mechanism of the protective effect of conditioned medium (CM) on human dental pulp stem cells (hDPSCs). METHODS: The hDPSCs were isolated and cultured, and the conditioned medium was prepared by ultrafiltration concentration. Mouse genioglossus myoblasts were isolated and divided into control group, CM group, CoCl₂ group and CoCl₂+CM group. The cell viability of genioglossus myoblasts was detected by CCK-8. The intracellular and mitochondrial ROS levels were evaluated by DCFH-DA and MitoSOX, respectively. The expression level of mitochondria-related genes in NRF-1 and NRF-2 were analyzed by real-time PCR. The expression of PGC-1α, p-AMPK and total AMPK protein was detected by Western blot. Statistical analysis was performed using SPSS 22.0 software package. RESULTS: The proliferation of genioglossus myoblasts was significantly decreased after 200 μmol/L CoCl₂ treatment for 24 h (P<0.05), and the levels of reactive oxygen species (ROS) were significantly increased in intracellular and mitochondria (P<0.05). Compared with CoCl₂ group, the proliferation ability of hDPSCs-CM was dramatically raised (P<0.05), and the intracellular and mitochondrial ROS content was remarkably decreased(P<0.05). hDPSCs-CM up-regulated the protein expression levels of pAMPK and PGC-1α in genioglossus myoblasts and mitochondrial downstream effectors of PGC-1α, including mRNA expression levels of NRF-1, NRF-2 (P<0.05). CONCLUSIONS: Human dental pulp stem cells conditioned medium can alleviate hypoxia injury induced by CoCl₂ in genioglossus myoblasts, and its mechanism may be related to AMPK/PGC-1α signaling pathway.