Effects And Related Mechanisms Of Plasma-Activated Solution On The Proliferation And Apoptosis Of Hemangioma Endothelial Cells

Objective Infantile hemangioma（IH） is a common benign tumor in the skin and soft tissue of infants.It is formed by the proliferation of embryonic vascular tissue and characterized by abnormal proliferation of vascular endothelial cells.IHs are usually found about 2 weeks after birth,and reach the peak of proliferation about 6 months.More than half of the IHs will gradually shrink after 3-5 years old,and some IHs will not completely disappear,leaving redundant fat tissue and other residues.Most IHs do not cause harm to the body,but only a few may have functional impact or severe destructive damage,and a few may even be life-threatening.At present,the commonly used clinical treatment methods include oral and local use of beta blockers,pulsed dye laser,local injection of anti proliferative drugs,surgical resection,etc.But the curative effect of these methods are not satisfactory.What’s more,there are serious adverse reactions.So it is necessary to find new safe and efficient treatment methods.Plasma is the fourth state of matter except solid,liquid and gas.It has been reported that cold atmosphere plasma（CAP） could inhibit proliferation and promote apoptosis of many kinds of tumor cells in vitro.Plasma-activated liquid（PAS） is a kind of liquid which can produce a variety of active substances by treating different solution media with low temperature plasma.In recent years,PAS has been widely reported to inhibit the proliferation of tumor cells in vitro,and it is selective,with relatively small side effects on human normal cells.In this study,phosphate buffer solution（PBS） was used as medium to activate PBS to form PAS through CAP.The effects of PAS on proliferation and apoptosis of hemangioma endothelial cells（HemECs） were observed,and the related mechanisms were further explored.Methods PBS was treated with CAP generator for 10s,20s,30s,40s and 50s respectively to form PAS.PBS with the treatment time of 0s was used as control group.The dose of PAS is defined as different treatment time under the same discharge parameters.The pH value of different doses of PAS was detected by electronic pH meter.The concentration of active medium in different doses of PAS was detected by nitrate ion,hydrogen peroxide and ozone detection kits.The proliferation of HemECs cells treated with different doses of PAS was detected by CCK-8 method.After Annexin-V and PI staining,flow cytometry was used to detect the apoptosis of HemECs treated with different doses of PAS.The intracellular reactive oxygen species（ROS） level of HemECs treated with different doses of PAS was labeled with DCFH-DA.Western blot was used to detect the protein expression levels of protein kinase B（AKT）,phosphorylated AKT（p-AKT）,phosphati DYlinositol-3 kinase（PI3K）and phosphorylated PI3K（p-PI3K） in HemECs cells treated with PAS at different doses.Results Compared with the control group,the pH value in PAS decreased gradually in a dose-dependent manner,while the concentrations of active media NO₃^-,H₂O₂ and O₃in the activation solution increased gradually in a dose-dependent manner.Compared with the control group,PAS significantly inhibited the proliferation of HemECs cells in a dose-dependent manner.When the dose of PAS was 50s,the survival rate of cells was 50.0%.Compared with the control group,PAS significantly promoted the apoptosis of HemECs in a dose-dependent manner.Compared with the control group,PAS increased the ROS levels in HEMECs in a dose-dependent manner.Compared with the control group,the intracellular AKT phosphorylation level of HemECs treated with PAS which was processed by CAP for 10s was not significantly changed.However,the phosphorylation level of AKT decreased rapidly when the CAP treatment time increased to 20-50s.However,the phosphorylation level of PI3K was not significantly changed.Conclusion PAS can significantly inhibit the proliferation and promote apoptosis of HemECs which may be related to the increase of ROS and the decrease of AKT phosphorylation level.