## 4 Discussion
This suggests that this approach may represent the optimal timing/window of response (the interval between irradiation and exposure to damage) for effectively modulating reactive species levels. This aligns with results reported in a recent clinical trial evaluating the response time of PBMT-sMF in muscle recovery [41]. ROS and NO are important cellular signaling molecules and may be directly related to the effects generated by PBMT. Regarding NO, no increase was observed after exposureto PBMT, as proposed in the literature [42]. Nonetheless, it is noteworthy that NO is a dual molecule synthesized by nitric oxide synthases (NOS), and at elevated levels, it can mediate inflammatory responses [43] and nitrosative stress [44]. NO can promote the enzymatic inhibition of complex I and III of the electron transport chain, favoring the formation of superoxide (O\({}_{2}\)\({}^{-}\)), which results in the formation of peroxnitrite (ONOO\({}^{-}\)), leading to macromolecular damage and cell death [45]. NO plays a crucial role in cellular signaling during physiological processes, such as resistance to fatigue and adaptation to prolonged activity (resistance training). However, when NO signaling is dysregulated, these processes can become pathophysiological, causing disruption of the calcium channel involved in muscle contraction, resulting in fatigue and injury [46]. Therefore, this parameter should be maintained within homeostatic levels, and PBMT was able to normalize the NO increase caused by hydrogen peroxide. It is believed that the primary photoreceptor for the energy provided by PBMT is the enzyme known as cytochrome c oxidase (CCO), part of COX [47]. It has been proposed that the photosensitization of metal sites in the structure of this enzyme leads to an increase in proton pumping by the mitochondrial respiratory chain, thereby enhancing ATP biosynthesis [48; 49]. A previous study reported that the association of PBMT with electromagnetic fields would synergistically accelerate electron transfer [23], leading to the measurement of the effects of combined therapies on CCO activity. A significant increase was observed at all doses compared to the control, with the highest peak of activity at the 60 J. Supporting findings, [PERSON] et al. [50] demonstrated a significant increase in ATP synthesis and mitochondrial membrane potential in intact myotubes 3 to 6 h after irradiation. Furthermore, it was observed that the increase in COX activity occurred after applying PBMT in fibroblast mitochondria, in a dose-dependent manner [51]. This response was corroborated by the present study. Myoblasts exposed to H\({}_{2}\)O\({}_{2}\) exhibit mitochondrial dysfunction, which can lead to the loss of membrane potential [52], as observed in the present study. Similarly, studies have demonstrated that acute exposure of C2C12 cells to H\({}_{2}\)O\({}_{2}\)--300 uM (1 to 5 h) [53] and 250 uM (6 h) [54]--induced a slow fragmentation of the mitochondrial network due to decreased membrane potential and maximum respiratory frequency in a dose/time-dependent manner. On the other hand, in healthy cells, an increase in membrane potential may be associated with the stimulation of COX activity, resulting in a brief/modest increase in ROS generation [55]. The induction of increased COX activity without an increase in membrane potential by H\({}_{2}\)O\({}_{2}\) may be explained, at least in part, as a compensatory mechanism by the other complexes of the electron transport chain (CI-CIII). However, this hypothesis needs to be tested in future studies. In an environment with pre-existing oxidative stress or mitochondrial dysfunction, membrane potential may be compromised or depolarized. In contrast, in normal cells, PBMT could induce a non-deleterious increase in membrane potential and subsequent normalization of ROS production, as previously shown [33; 56]. In the present study, \(\Delta\)Ym was positively modulated by PBMT-sMF at 9 and 60 J compared to H\({}_{2}\)O\({}_{2}\).