Copper Nanoclusters Triggers Apoptosis And Atrophy In C2C12 Muscle Cells

The copper nanoclusters(CuNCs)are increasingly used in nanomedicine due to their utilityin fluorescence and catalyst.And the nanotoxicology research of CuNCs is gaining attention.We report here the synthesis of CuNCs and their cytotoxic impact on mouse C2C12 cells,including the apoptosis in C2C12 myoblasts and the atrophy in C2C12 myotubes,and we preliminary explored the molecular mechanism of the nanotoxicity.A simple protein-directed synthesis of stable CuNCs was prepared,using BSA as the stabling agent.The morphology of CuNCs was analyzed by transmission electron microscope(TEM)and the fluorescence intensity was recorded on a fluorescence spectrometer.The prepared CuNCs exhibited good dispersion in aqueous solution and showed a color of lavenderblush.The TEM image of CuNCs exhibited a spherical shape.The luminescence of CuNCs revealing distinct excitation and emission maxima at 260 and 425 nm,respectively.This paper first investigated the nanotoxicity and mechanism of CuNCs on C2C12 myoblasts.The experimental results showed that:(1)To evaluate the possible toxicity of CuNCs on C2C12 myoblasts,cell viability and LDH release were analyzed after exposing C2C12 myoblasts to CuNCs with different dose and time.The results shown that CuNCs decreased the viability of C2C12 myoblasts and increased the LDH activity in a dose-dependent manner as detected by MTT and LDH release assay.These results indicated that CuNCs induced cytotoxicity in C2C12 myoblasts.(2)To determine whether exposure to CuNCs can result in apoptosis,cells were treated with CuNCs and apoptosis were measured by flow cytometry.These results shown that treatment with CuNCs resulted in a dose-dependent increase in both early and late apoptotic cells.These results indicate that the CuNCs induced apoptosis is mainly responsible for cell death of C2C12 myoblasts.(3)To further evaluate the possible mechanism of CuNCs induced apoptosis on C2C12 cells,the intracellular generation of ROS was measured..We found that the intracellular ROS levels were significantly increased after C2C12 cells exposure to CuNCs for 6 h.(4)CAT and GSH activity was measured..The CAT activity was significantly decreased,and the GSH levels were also reduced compared with control group in C2C12 cells exposed to CuNCs for 24 h.These data suggest that CuNCs induce ROS generation and cause oxidative damage in C2C12 myoblasts.(5)We speculate that CuNCs induce apoptosis through mitochondrial pathway.Indeed,treatment of C2C12 cells with CuNCs resulted in significant decrease in mitochondrial membrane potentia,western blotting analysis of Bcl-2 and Bax and the caspase-3/9 activity in C2C12 cells was performed.We found that the protein expression ratio of Bax/Bcl-2 and caspase-3/9 activity increased in C2C12 cells exposure to CuNCs for 24 h.These data suggest that CuNCs induce apoptosis through mitochondrial pathway in C2C12 myoblasts.Finally,we investigated the nanotoxicity and mechanism of CuNCs on C2C12 myotubes.CuNCs treatment resulted in atrophy of the C2C12 myotubes which was characterized by the decreased expression of the protein levels of myogenin,Caveolin 3,MyHC and pAkt and the increased expression of mRNA of ubiquitin-proteasome pathway-related genes(atrogin-1 and MuRF-1)and autophagy-lysosome pathway-related genes(beclin-1 and bnip-1).These datas sugges that CuNCs induce atrophy through the inhibit phosphorylation of Akt in C2C12 myotubes through inhibit the generation of muscle protein and promote muscle protein decomposition.In conclusion,our studies indicated that CuNCs induced the formation of reactive oxygen species(ROS),caused oxidative damageand actived the mitochondrial pathway,eventually lead to apoptosis in C2C12 myoblasts.Finally,CuNCs treatmentment resulted in atrophy of the C2C12 myotubes which was characterized by decreased expression of the protein levels of myogenin,Caveolin 3,MyHC and the increased expression of mRNA of ubiquitin-proteasome pathway-related genes(atrogin-1 and MuRF-1)and autophagy-lysosome pathway-related genes(beclin-1 and bnip-1),and maybe CuNCs induce atrophy through the inhibit phosphorylation of Akt in C2C12 myotubes.Taken together,these results suggest that exposure to CuNCs may be a possible risk factor to skeletal muscle system,and we should take the dose and time into consider during the application of it.