Safety Study Of Subcutaneous Action In Rats With Plasma-activated Solution

Objective: Studies have illustrated that Cold-temperature plasma(CAP)is an effective method to treat many skin diseases,and performs a significant function in promoting wound healing,cancer treatment,sterilization and other aspects.Cold atmosphere Plasma-activated solutions(PAS)is an indirect method of plasma treatment that produces similar biological utility as direct treatment,facilitates storage and transportation,so it is convenient for the treatment of larger skin tumors and deep skin infections.The safety of PAS interaction with living mammals cannot be accurately assessed because of the lack of data available on toxicological studies of PAS.In this study,Plasma-Activated Solution was prepared by air-source DBD cold temperature plasma device mediated to activate the phosphate buffer PBS,and then hypodermic injection was performed in rats to explore the safety of plasma-activated solution in rats in the course of subcutaneous injection,and provide theoretical support for further clinical treatment.Methods: Cold atmospheric plasma activating solutions were prepared by an cold atmospheric plasma equipment with ordinary air as the gas source to irradiate the PBS for 1 minute,3 minutes,and 5 minutes.PBS was considered as the control group,the changes in p H and active substance concentrations in the control group and the treatment groups were detected.After 10 hypodermic injections on the back of rats,abdominal aorta blood was drawn for routine blood routine and biochemical tests of liver and kidney function to evaluate the status and function of the hematopoietic system,liver and kidney.Histopathological examination of the lung,heart,liver and kidney were used to evaluate structural changes of the main organs.Experiments were used to evaluate local injection site safety : 1.Histopathological examination and Masson trichrome staining were performed to detect the changes in the skin tissue in subtle structural.2.SOD viability and MDA concentration were measured to assess the oxidative stress status.3.The changes in γ-H2 AX and Ki67 in the skin tissue cells were measured to detect the DNA damage conditions and the proliferation of skin histiocytes.4.The changes in TNF-α concentration were measured by Enzyme Linked Immunosorbent Assay to detect the inflammatory response of cells.5.The expression of differential genes in cells after treatment with plasma activating solution was detected by RNA-seq.6.Western blot was used to detect the expression of growth factor TGF-β 、VEGFA.Results: The changes in p H in cold atmospheric plasma activating solutions of PAS1 minute,PAS3 minutes,and PAS5 minutes gradually decreased with the extension of the irradiation time.The changes in active substance concentrations in the solution were increase gradually(P<0.05).There were no manifest abnormalities in the blood routine and liver and kidney function biochemical tests,and there was no statistically significant difference compared to the control group(P>0.05).Histopathological examination of the lungs,heart,liver,and kidneys showed no abnormal tissue structure changes.The injection site of skin was measured: 1.Visual inspection of the rat’s skin did not reveal any damage,including redness,rash,and skin breakdown.Moreover,no significant changes in Histopathological examination and Masson trichrome staining were observed under microscope.The epidermis,dermis and subcutaneous tissue structure of the skin were complete,and the collagen fibers in the dermis layer were arranged in an orderly manner,and there is no collagen hyperplasia.2.The levels of SOD and MDA recorded in the experimental group did not differ significantly from those recorded in the control group(P>0.05).3.The Immunohistochemistry analysis showed that there were no abnormal expression of γ-H2 AX and Ki-67 proliferation marker.4.Enzyme Linked Immunosorbent Assay analysis showed that compared the amount of TNF-α in each group and found no clear difference(P>0.05).5.RNA-seq was used to detect the increase in the gene expression of transforming growth factor receptor signaling pathways and angiogenesis in biological processes;6.The expression of TGF-β and VEGFA was detected by Western blot,compared with the control group,the expression of each treatment group was significantly increased(P<0.05).However,there were no significant difference in experimental groups.Conclusion: This study proved that multiple subcutaneous injection of cryogenic plasma activation fluid has no toxic side effects on biological individuals;The cold atmospheric plasma activating solution promoted the conversion of growth factor receptor signaling pathway,angiogenic biological process gene expression,and the protein expression of TGF-β and VEGFA,indicating that cold atmospheric plasma activating solution had a positive effect on promoting tissue regeneration.These results indicate that multiple subcutaneous injections of PAS seem to be safe,but this finding needs to be further confirmed by future clinical studies.