A Synergistic Effect of Reactive Oxygen and Reactive Nitrogen Species in Plasma Activated Liquid Media Triggers Astrocyte Wound Healing

Abstract

Astrocyte proliferation and migration toward injured Central Nervous System (CNS) areas are key features of astrogliosis and glial scar formation. Even though it is known that intracellular and environmental Reactive Oxygen and Nitrogen Species (RONS) affect astrocyte behaviour in physiological and pathophysiological conditions, their effects on the migration and growth of astrocytes are still unclear. Plasma-technologies are emerging in medicine as a tool to generate RONS for treating cells directly or through Plasma Activated Liquid Media (PALM). In this paper, we show for the first time how the use of PALM can modulate both astrocyte growth and migration as a function of active species produced by plasma in liquids. Our results show that PALM, generated by means of cold atmospheric pressure plasmas fed with N2, air or O2, can modulate astrocyte behaviour depending on the content of hydrogen peroxide and nitrite in the liquid. In particular, H2O2 enriched PALM induced a negative effect on cell growth associated with the mild wound healing improvement of primary astrocytes, in a scratch assay. Nitrite enriched PALM induced a selective effect on the wound healing without affecting cell growth. PALM containing a more balanced level of H2O2 and NO2− were able to affect cell growth, as well as significantly ameliorate wound healing. None of the PALM investigated induced upregulation of the gliotic inflammatory marker glial fibrillary acidic protein (GFAP), or of the astrocyte markers Aquaporin-4 (AQP4) and Connexin-43 (Cx-43) analysed by Western blot. Finally, immunofluorescence analysis revealed the presence of NO2- able to induce elongated protrusions at the front end of wounded astrocytes in the direction of cell migration. With our study we believe to have shown that PALM offer a novel tool to modulate astrocyte behaviour and that they are promising candidates for controlling astrogliosis in the case of CNS injuries.

Plasma Treated Water Solutions in Cancer Treatments: The Contrasting Role of RNS

“…The presence of NO 2 − anions in PT- culture media, in fact, could act as a source of nitrosative stress as well, due to the biological conversion, internal to cells, in the more reactive species of ● NO and ONOO − [ 6 ]. Indeed, nitrite ions are considered the largest intravascular and tissue source of ● NO in the human body [ 64 ].…”

“…As a consequence, the response of Saos2 cells to PT-DMEM (++)H 2 O 2 containing about 300 µM of H 2 O 2 was a strong decrease in cell density, as expected, despite the absence of nitrites. Moreover, it is well known that high doses of H 2 O 2 can trigger many apoptotic pathways in cancer cells, most of them involving the passage of exogenous H 2 O 2 through aquaporin (AQP) channels and the direct intracellular damage via Fenton reaction, as discussed by us [ 6 ], and by Yusupov et al [ 69 ]. It has also been suggested by Yan et al [ 70 ] that the increased concentration of AQP channels on tumor cells could explain the anti-tumor effects of PTWS.…”

“…N. G7513, Sigma–Aldrich), penicillin and streptomycin solution (20 units mL −1 /20 mg mL −1 , T4049, Sigma–Aldrich) was used for plasma treatments and, when untreated, as a reference in all biological experiments; for Electron Paramagnetic Resonance (EPR) analyses also serum free DMEM was used to assess the ability of components of DMEM to scavenge ● NO; water and water solutions were also used for the EPR experiments. The energy dose delivered during the discharges was calculated with the voltage–charge (i.e., the time integral of the current) Lissajous method described in our previous works [ 5 , 6 ]. Data are summarized in Table 1 .…”

“…Since it has been established that the efficacy of conventional anti-cancer therapies, chemo- and radiotherapy, actually relies on a release of ROS in cancer cells, PTWS have been widely tested as novel anti-cancer ROS-generating systems, and proved to cause the selective damaging of cancer cells in vitro [ 3 , 4 , 5 , 6 ], but also a significant inhibition of tumors growth in vivo [ 7 , 8 ]. The mechanism of PTWS-induced cancer inhibition, however, still needs to be fully elucidated [ 9 , 10 , 11 ].…”

Adjusted multiple gases in the plasma flow induce differential antitumor potentials of plasma‐activated solutions

“…[ 33,34 ] Moreover, recent investigation has demonstrated that plasma‐activated liquid media (PALM) produced with nitrogen, air, or oxygen induced different biological activities in astrocyte, depending on the amount of H 2 O 2 and NO 2 − in the liquid. [ 35 ] The study by Azzariti et al [ 36 ] clearly indicated that PALM generated with air or oxygen in a closed plasma exposure system induced an antiproliferative activity and immunogenic cell death by oxygen feed gas condition rather than air, indicating that ROS might be more effective than RNS in cancer treatment. Although a lot of studies are focused on the characteristics of RONS production and stability in the various plasma systems, [ 37,38 ] it is not yet known how to control them in an effective and efficient manner that will enhance or optimize their biological effects for the purposeful use.…”