The Mechanism Of Cold Atmospheric-pressure Plasma Induced Apoptosis In HepG2Hepatoma Cells

Cold atmospheric pressure plasma with its many advantages, including high efficiency, safety and harmless operation, has a wide range of applications, such as food sterilization, disinfection of medical equipment and material handling. In recent years, people pay more attention on the emerging field of the combination of plasma technology and biomedicine. Plasma medicine has become one of the most important development direction of the plasma. It has been reported that people have obtained good results in the inactivation of bacteria, promotion blood coagulation clotting and inhibition of tumor cell proliferation. Therefore, we have confidence to expect that the atmospheric pressure plasma could play a huge role in the area of clinical treatment in the future. The present research is to clarify the mechanism of plasma-induced apoptosis in HepG2cells by the basic experimental techniques of biochemistry and molecular biology, cytology and enzymology, so as to provide the basic theory of plasma in medicine, especially cancer treatment. The major results are as follows:1. MTT assay was used to detect to the anti-proliferation effect of plasma treatment on HepG2cells. Hoechst33342and AnnexinV/PI staining method were used to detect the apoptosis in HepG2cells. And the detection of gene and protein expression related to cell apoptosis at the transcriptional and translational levels by RT-PCR and Western-blot analysis. The results showed that HepG2cell proliferation had obviously been suppressed while treated by cold atmospheric pressure plasma. After plasma treatment, cells had changes in cell morphology, including cells shrinking, chromatin condensing and apoptotic cells beginning to appear. And these phenomena had a significant dose-dependent manner. With the increase of the processing time, the cell viability decreased and the rate of apoptosis increased. Then the mitochondrial apoptotic pathway was activated. Furthermore, the plasma treatment also suppressed the expression of Bcl-2 and increased that of Bax in both mRNA and protein levels.The expression of the p21Cdk inhibitor, as well as that of tumor suppressor p53,is enhanced. Caspase cascade was activated, eventually leading to apoptosis of HepG2cells.2. DCFH probe was used to detect the changes of the intracellular reactive oxygen species(ROS), and further to test the intracellular antioxidant capacity, mainly including the test of the content changes of superoxide dismutase(SOD), catalase(CAT) and glutathione. The experimental results showed that the plasma could effectively control the intracellular concentrations of ROS, while the superoxide dismutase activity and catalase activity declined. Excessive reactive oxygen caused intracellular oxidative stress in HepG2cells. NAC could significantly improve cell viability. The results illustrated that the plasma induce HepG2cell apoptosis through oxidative stress.3. Plasma treatment could induce apoptosis in HepG2cells. However, after pretreatment with NAC for30min, apoptosis rate of HepG2cells could be significantly decreased. NAC could antagonize the occurrence of oxidative stress-mediated apoptosis in HepG2cells by preventing the generation of oxygen free radicals. The results showed that the plasma could induce apoptosis in HepG2cells through oxidative stress.