## 4 Discussion
### Non-Thermal Plasma Flux Induced Anti-Inflammatory Macrophages and Fibroblast Migration  
Non-thermal atmospheric pressure plasma might be beneficial in several health topics, including dentistry [46], hematology (coagulation) [47], microbiology (microbial eradication) [48], surgery (wound healing) [49] and oncology [50]. Interestingly, the effects of plasma flux on wound healing (in burns) and angiogenesis (in cancer) are dependent on the conditions and features of non-thermal plasma [51], which may cause varying intensities of bioactive molecules [52]. Indeed, plasma flux increased macrophage ROS, a mediator of several cell activities (apoptosis, proliferation and inflammation), inhibiting NF-kB [53; 54] in a variety of cells, including epithelial cells (H9C2) [55] and T cells (Jurkat cell) [56]. Here, plasma flux enhanced ROS and reduced NF-kB abundance in macrophages that might, at least in part, attenuate macrophage inflammatory responses. However, the reduced pro-inflammatory status of plasma-treated macrophages was not associated with the cell energy status, as the abundance of AMPK, a sensor of cell energy [39], was not different from control group.  
Figure 6: Characteristics of wounds in mouse models with burn injury with or without bacterial infection after treatment by plasma flux (Plasma) or argon gas alone (Untreat) as determined by cytokines from skin tissue (TNF-\(\alpha\), IL-6 and IL-10) (**A–C**), bacterial burdens (**D**) and collagen in the lesions with the representative [PERSON]’s trichrome stained histological pictures (**E**) (n = 5–7/ time-point or group) are demonstrated.  
Because (i) M2 polarized macrophages promote several processes of wound healing [25], (ii) excessive pro-inflammatory macrophages decelerate wound healing [57] and (iii) plasma flux could alter macrophage inflammatory responses [58], several genes are interesting to explore. Without LPS (an inflammatory activation), the macrophages in a neutral state did not produce inflammatory cytokines and plasma flux showed only a slight impact on macrophage as indicated by only the upregulation of _Fizz_, a biomarker of M2 macrophage polarization, but not other genes of M2 macrophages polarization. With LPS stimulation, plasma flux demonstrated a greater anti-inflammatory effect as indicated by reduced IL-6 production (at 24 h post-LPS), _IL-1\(\beta\)_ downregulation (6 h post-LPS) and _IL-10_ upregulation (3 and 6 h post-LPS). However, the expression of M2 associated genes was not different between the plasma flux treatment and the control group, implying that the plasma flux induced anti-inflammatory macrophages but did not profoundly activate M2 macrophage polarization. Nevertheless, the anti-inflammatory state could promote the wound healing process [25] and the wound healing promotion in our mouse models might partly have been due to the anti-inflammatory effect of plasma flux.  
Due to the influence of fibroblasts in wound healing processes (fibrin clot lysis and production of extra cellular matrix and collagen [45]), the impact of plasma flux on fibroblasts was tested. Indeed, plasma flux induced fibroblast migration without enhanced fibroblast proliferation. Despite the promotion of both proliferation and migration in human fibroblast-like cells by helium plasma flux [38], our argon-based plasma flux could not induce fibroblast proliferation, possibly due to the difference in plasma sources. It was mentioned that argon-based plasma demonstrates a lower electron temperature than helium-based plasma [59]. Nevertheless, the enhanced fibroblast migration is one of the factors that promotes wound healing [60]. Notably, the non-different proliferation between the plasma-treated group and the control imply a safety of plasma flux on fibroblasts.