When peripheral nerve injury, the cell body cannot provide nutritional support to the axons on the distal side of the broken end. At this time, Waller degeneration may occur (Conforti et al., 2014). In Wallerian degeneration, SCs are initially involved in the peripheral nerve regeneration process. After peripheral nerve injury, SCs began to proliferate. The number of SCs peaked 1 to 2 weeks after peripheral nerve injury, and then decreased. Together with SCs, macrophages phagocyte the myelin sheath of the deformed axons, and the SCs form Bungner bands, which promote axon growth. SCs can also secrete bioactive substances, such as nerve cell adhesion molecules and neurotrophic factors. It not only induces nerve reinnervation and myelination, but also stabilizes the network of peripheral glial cells, and promotes the myelination of new axons by means of temporary adhesion. The functions of SCs after peripheral nerve injury are diverse. They not only have the function of phagocytosis, but also provide scaffolds and neurotrophic factors for nerve regeneration, maintain the survival of neurons, guide the orderly extension of axons, promote the myelination of axons, and regulate the survival and apoptosis of the axons (Conforti et al., 2014). The function of SCs is so powerful that if the proliferation of SCs can be promoted when the nerves is damaged, it must speed up the repair of peripheral nerves. Although many studies have investigated the effect of CAP on cell proliferation (Kalghatgi et al., 2010, Liu et al., 2017, Duchesne et al., 2019, Sardella et al., 2020, Arndt et al., 2013, Lou et al., 2020, Kleineidam et al., 2019, Hasse et al., 2016), the relationship between CAP and cell behavior in the field of nerve regeneration, especially in the field of peripheral nerve regeneration, is still unclear. In this study, we choose SCs, which is an important cell that maintains the stability of the normal neural internal environment, as the research object. The effect of CAP on SCs proliferation was evaluated by cell morphology, cell viability, cell cycle and expression of related proteins in SCs.
The method proposed in this study was based on exposure of cells to the culture medium treated with CAP. In the culture medium treated by CAP, the effects of some less stable or less content substances, such as ONOO−、O3、O2−, are negligible compared with other stable substances produced. This is due to its preferred reaction with the composition of the medium at neutral pH (Tarabova et al., 2019). Rezaei et al. also showed that H2O2 and NO could be used as markers for all ROS and RNS species produced by CAP due to their stability and long effectiveness, although other ROS and RNS were present in the culture medium treated by CAP (Rezaei et al., 2019). In addition, the quantitative detection results of intracellular free radicals were difficult to obtain accurately with the current technology, and the concentration of free radicals could not be quantified (Kalyanaraman et al., 2017).Therefore, in this study, a common fluorescent probe was used to reflect the changing trend of free radicals, and the relative fluorescence intensity of each experimental group was analyzed with a fluorescence microplate reader to reflect the changing trend of intracellular ROS and RNS with the processing time. In fact, whether it was intracellular ROS and RNS or extracellular ROS and RNS, its amount was always positively correlated with processing time, which was consistent with the results of most researchers (Song et al., 2018, Liu et al., 2017, Xu et al., 2020, Zhang et al., 2020a).
ROS is a kind of oxygen-containing substance produced by aerobic cells in the process of aerobic metabolism, it has extremely high biological activity, and plays an important role in cell proliferation and apoptosis (Luo et al., 2019, Mo et al., 2019, Zhang et al., 2020b). A certain amount of intracellular ROS is necessary to maintain the normal physiological state of the cell (Forrester et al., 2018, Sies and Jones, 2020). However, high concentration of ROS can lead to oxidative stress and damage of DNA, protein and membrane in cells, leading to cell death (Sun et al., 2019, Nissanka and Moraes, 2018). As a special molecule, NO acts as a biological messenger in the body, transmitting information from nerves to cells, and Endogenous NO plays a role in regulating cellular function and messenger in health (Qi et al., 2020, Oláh et al., 2018, Xu et al., 2015). It is very meaningful to note that low concentrations of NO are beneficial to human health, but high concentrations are harmful (Islam, 2017, Zhang et al., 2017). The experimental results in this study confirm these views, the double effect of CAP on SCs was also proved by cell viability. The results showed that low-dose CAP could promote SCs proliferation, but high-dose CAP inhibite the growth of SCs, which was consistent with the effect of CAP treatment on many normal cells (Boehm et al., 2016, Xu et al., 2015, Hasse et al., 2016, Liu et al., 2017, Lou et al., 2020, Sardella et al., 2020, Kalghatgi et al., 2010, Kleineidam et al., 2019, Arndt et al., 2013). Besides, results proved that appropriate CAP treatment could promote the transformation of cells from G0/G1 phase to S + G2/M phase by cell cycle, thus promoting the proliferation of SCs.
SCs are the main glial cells of the peripheral nervous system to form the myelin sheath and realize the jump conduction of action potential (Castelnovo et al., 2017). They also secrete neurotrophic factors, which promote the survival of damaged neurons and the regeneration of axons, and participate in the formation of nerve fibers in the peripheral nervous system (Jessen and Mirsky, 2016, Feltri et al., 2016). For example, NRG-1/ErbB signaling is an important pathway for SCs development, and NRG-1/ErbB-mediated activation of the PI3K pathway converts phosphatidylinositol diphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3), then activating Akt (Park et al., 2008, Castelnovo et al., 2017). Other studies supported the importance of this pathway in SCs development (Ogata et al., 2006, Maurel and Salzer, 2000). Besides, the activity of PI3K is not only affected by ErbB signaling, but also affected by ROS (Chen et al., 2017, Yao et al., 2020), thus affecting the transformation of PIP2 to PIP3. In addition, some studies have shown that CAP-treated cells can increase ROS production and alter the expression of cyclinD1, thereby altering cell cycle progression (Liu et al., 2017, Zhang et al., 2020a). In this study, ROS produced by CAP activates PI3K, which converts PIP2 to PIP3, and activates Akt. Then, Akt activates cyclinD1 and mTOR respectively, which promotes cell cycle progression and cell proliferation at the same time. The specific process is shown in the Fig. 7. NAC preconditioning significantly inhibited the activation of PI3K/Akt/mTOR pathway, and also inhibited the change of cyclinD1 expression induced by CAP. The results showed that CAP can activate the PI3K/Akt/mTOR pathway at appropriate dose, and we speculate that the mechanism is closely related to CAP-induced ROS production.
In fact, many plasma sources with various design have been proposed for biomedical applications, but the relationship between source characteristics and application performance is not well-understood. This prevents researchers to directly compare different plasma sources to study the effect of plasma dose.. Therefore, in this study, we used the treatment time as the only variable to control the concentration of ROS and RNS produced by CAP. The effect of active substances produced by CAP on living cells is very complex, the properties and concentrations of active substances formed after CAP treatment may vary widely, which depends on the type of gas used in the experiment, the experimental apparatus (type of plasma reactor, e.g. DBD plasma or jet plasma), the voltage of the discharge apparatus and the type of plasma activated liquid (Przekora et al., 2019, Hasse et al., 2016, GÜMBEL et al., 2017). These parameters not only changed CAP-induced ROS, but also changed the effect of electromagnetic field radiation on cell proliferation in vitro. Therefore, maintaining other parameters unchanged, choosing time as the only variable can simply and effectively control the plasma dose problem. The experimental results also showed that the concentrations of ROS and RNS produced by CAP were obviously time-dependent when other parameters were maintained unchanged. Although some of the mechanisms discussed in this study could explain the regulation of CAP on SC proliferation, further studies are needed for clinical application of this technique.