Inhibitory Effect Of Two Cold Atmospheric Plasma Activated Solution On Skin Basal Cell Carcinoma Cells And Analysis Of Differential Gene Expression

Objective Basal cell carcinoma of the skin,also known as basal cell epithelioma,is a common type of clinical skin tumor.This disease is more common in middle-aged and elderly people,and occurs in exposed areas.Its pathogenesis may be related to long-term sun exposure.Skin lesions of basal cell carcinoma of the skin are mostly single,more common are nodular ulcer types,and metastasis is rare.Treatment of basal cell carcinoma of the skin is usually based on the patient’s age,size and location of the lesion.At present,the main treatment methods include surgical treatment,laser treatment,photodynamic therapy,cryotherapy,and topical medication.Most patients can obtain satisfactory results.However,for some elderly and frail patients who cannot tolerate surgery and invasive treatment,a safer and more effective treatment with less pain is needed.Cold atmospheric plasma has been shown to inhibit proliferation and promote apoptosis in a variety of tumor cells cultured in vitro.The cold atmospheric plasma activating liquid is an active liquid that uses a cold atmospheric plasma to stimulate different solution media to produce a variety of active substances in the solution.On a large number of in vitro experiments,cold atmospheric plasma activating solution also has a good effect on tumor cell proliferation inhibition and promotion of apoptosis,and has less toxic and side effects on normal tissue cells.In this study,an air-source cold atmospheric plasma device was used to mediate the activation of complete cell culture medium DMEM and phosphate buffered saline PBS to prepare a cold atmospheric plasma activating solution,and to explore its effect on basal cell carcinoma cells cultured in vitro and related mechanisms.Methods This subject mainly involves two aspects of cell functional experiments and molecular biology experiments.An cold atmospheric plasma equipment was used to irradiate the DMEM and PBS solutions for 60s,120s,180s,240s,and 300s to prepare a cold atmospheric plasma activating solution.The changes in p H and active substance concentrations in the two plasma activating solutions were measured.Plasma activating solution was used to contact the TE354T cell line cultured in vitro,and the morphological changes of the cells were observed under a light microscope and a fluorescence 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.Skin keratinocytes Ha Ca T was used as a normal control group.MTT assay and detection of intracellular ROS were used to evaluate the selective effect of plasma activating fluid on basal cell cancer cells and keratinocytes.Apoptosis was detected by Annexin-V and PI staining using flow cytometry.Western blot was used to detect the expression of apoptotic proteins caspase9 and caspase3.RNA-seq was used to detect the expression of differential genes in cells after treatment with plasma activating solution.Results After treatment of the cells with a cold atmospheric plasma activating solution for several hours,shrinkage of the cell morphology and nuclear shrinkage were observed.The cell survival rate was measured by 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 basal cell carcinoma TE354T.At the same time,this effect was found to be selective by comparison with Ha Ca T,a keratinocyte.With the increase of the dose of plasma activating solution,the increase of ROS in basal cell cancer cells was detected,the increase of apoptosis rate was detected by Annexin-V/PI method,and the expression of apoptotic proteins caspase9and caspase3 was detected by Western blot.Through the detection of the p H in the solution,it was found that the p H of the activating solution gradually decreased with the extension of the irradiation time within the range of 60-300s,but the decline was still within the tolerance range of the cell biology,and the H₂O₂and NO₃-Rise.Significant differential expression of MAPK,TNF,and IL-17 signaling pathways was detected by RNA-seq.Conclusion The cold atmospheric plasma activating solution can selectively inhibit the proliferation and induce apoptosis of basal cell carcinoma cells in vitro.This effect of inducing apoptosis may be caused by changing intracellular reactive oxygen levels and through signal pathways such as MAPK,TNF,and IL-17.