Cold Atmospheric Plasma-activated Ringer’s Solution Inhibits The Proliferation Of Osteosarcoma Cells Through The Mitochondrial Apoptosis Pathway

Objective: Osteosarcoma is one of the most common malignant primary bone tissue tumors.And often occurs in the dry bone of children and adolescents.In the early stages of the initial diagnosis,some patients developed metastases.Osteosarcoma is highly invasive and highly metastatic and can cause lung metastases and progressive bone destruction.At present,the clinical treatment of osteosarcoma is mainly surgical resection,combined with chemotherapy or radiotherapy.Although the therapeutic effect of osteosarcoma has been appropriately improved in the past few decades,the overall 5-year survival rate of osteosarcoma patients is still relatively low.Intraoperative contamination of radical surgery,as well as micro-lesions and micro-residual tumors in the tissue surrounding the tumor that cannot be removed entirely,are the leading causes of local recurrence in patients with osteosarcoma.Therefore,new treatment methods are urgently needed to control the contamination and residue of tumors during the operation,reduce the recurrence rate,and improve the survival rate of patients.Low temperature plasma has been proven in many studies to have the effect of inhibiting the proliferation of a variety of in vitro cultured tumor cells and can promote tumor cell apoptosis.Besides,a low-temperature plasma activating medium such as a plasma activating liquid is an active liquid that uses low-temperature plasma to stimulate different types of solution substances,so that a variety of biologically active materials are produced in the solution.These activating fluids have the advantages of convenient transportation and more excellent permeability.Also,in large numbers of in vitro experimental surfaces,low-temperature plasma activating fluids have functional proliferation inhibition and apoptosis promotion effects on tumor cells and are toxic to normal tissue cells.Fewer side effects.The research of this subject is to use the air-source low-temperature plasma device to mediate and activate Ringer’s solution to prepare low-temperature plasma activating solution,and use this active liquid to study the low-temperature plasma-activated Ringer’s solution on osteosarcoma cells MG63 and U2 OS.And explore its possible mechanism of action.Methods: An air-source Cold atmospheric plasma equipment was used to irradiate the Ringer’s solution for the 60 s,120s,180 s,240s,and 300 s to prepare a low-temperature plasma activating solution.A plasma activating solution was used to contact the osteosarcoma MG63 and U2 OS cell lines cultured in vitro,and the cell morphological changes were observed under an optical microscope.MTT cell viability method was used to detect the cell survival rate of the two plasma activating solutions between different groups and at different time points.Human osteoblast h FOB 1.19 was used as a normal control group,and the MTT method was used to evaluate the selective effect of plasma-activated Ringer’s solution on osteosarcoma and human osteoblasts.Apoptosis was detected by Annexin-V and PI staining using flow cytometry.Western blot was used to identify the expression of apoptotic proteins caspase3,cleaved caspase3,cytochrome c,and PARP.Changes in mitochondrial membrane potential were detected by JC-1 staining and verified by detecting intracellular reactive oxygen species(ROS)levels.Results: Cell morphological shrinkage and nuclear shrinkage were observed several hours after the cells were treated with cryogenic plasma-activated Ringer’s solution.The cell survival rate was measured by the MTT method.It was found that the Cold atmospheric plasma activating solution had a dose-dependent and time-dependent effect on the proliferation inhibition of osteosarcoma MG63 and U2 OS.At the same time,by comparing with human osteoblast h FOB1.19,this effect was found to be selective.Annexin-V / PI method detected the increase of apoptosis rate and Western blot method to identify the changes of apoptotic proteins caspase3,cleaved caspase3,and PARP.JC-1 recognized the mitochondrial membrane potential of the cells and found that the mitochondrial membrane potential decreased,and it was found that cytochrome c was released from the mitochondria into the cytoplasm.And we found that with the increase of the dose of plasma-activated Ringer’s solution,the growth of ROS in osteosarcoma cells was detected.Still,with the rise in the treatment time,the ROS content in the cells decreased relatively.Conclusion: Cold atmospheric plasma-activated Ringer’s solution can selectively inhibit osteosarcoma cell proliferation and induce apoptosis in vitro.This effect of inducing apoptosis may be through changing the level of intracellular reactive oxygen species,further causing a decrease in mitochondrial membrane potential levels and the release of cytochrome c,thereby promoting a cascade of apoptosis.