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

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

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((reac)tive 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:颇舌肌成肌细胞经过200μmol/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。

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,pAMPK 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 200μmol/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-1αin 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-1αsignaling pathway.