Abstract:

Abstract:Objective
To investigate the time order of lysosomal and mitochondrial cell death  in human HeLa cells after oxidative stress injury and its significance,and to clarify the role of lysosomal membrane permeabilization in the process of mitochondrial apoptosis induced by oxidative stress.Methods The  HeLa cells line was used for the study.Oxidative stress model of cervical cancer HeLa cells was established by vitamin K3 (VK3).The cells were divided into control,V3 1 h,VK3 3 h,VK3 6 h and VK 12 h groups.The vitalities of HeLa cells in various groups were measured by MTT assay.The levels of reactive oxygen species(ROS) were determined by DCFH-DA.The lysosomal membrane permeabilization was observed by AO staining,and the mitochondrail membrane permeabilization in HeLa cells was detected by Rhodamine 123  staining.The expression of apoptosis-related protein cleaved caspase-3 was determined by Western blotting method.Results Compared with control group,the survival rates of HeLa cells in VK3 6 h and VK3 12 h groups were decreased (P<0.05).Compared with control group,the cleaved caspase-3 expression  levels  in VK 36 h and VK3  12 h groups were increased detected by Western blotting method(P<0.05).The DCFH-DA results showed  that after VK3 treatment for  1 h,3 h, and 6 h,the levels of green fluoresence  were increased in HeLa cells.The AO staining results showed that  VK3 treatment for 3 and 6 h caused a decline in the red staining of lysosomes and increase in green staining of  cytoplasm.The Rhodamine 123 results revealed that VK3 treatment for 6 h induced a drop in Rhodamine 123 red staining of cytoplasm.Conclusion Oxidative stress damage can induce lysosomal and mitochondrial cell death in HeLa cells,and lysosomal cell death eventuates ahead of mitochondrial,which prompting the lysosomal membrane permeabilization plays an important role in mitochondrial apoptosis pathway induced by oxidative stresss..
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Key words: oxidative stress, lysosome, mitochondria, apoptosis