## 4 Discussion
Indeed, the addition of long-lived hydrogen peroxide at concentrations generated by LTP treatment (50-100 \(\upmu\)M) did not significantly affect the mitochondrial potential of BMSC (Figure 3K,L), nor the cell survival and proliferation (data not shown), which corresponds to data from the literature on the effect of such concentrations of hydrogen peroxide on cell cultures [41]. At the same time, the further effect on BMSC might be related to examples of long-lived ROS (e.g., H\({}_{2}\)O\({}_{2}\), which is able to spread across the whole Petri dish via diffusion). Lipid peroxidation and oxidative stress are generally considered as common triggers that initiate the process of cell death [34]. One could assume that mitochondria play a key role in this process. Indeed, it can be seen that the acute action of ROS during the first days after LTP exposure initiates mitochondrial dysfunction, presumably through mitophagy, as evidenced by a decrease in the membrane potential of mitochondria which, most likely, leads to an increase in the expression of PINK1/Parkin and the triggering of the signaling pathway, leading to the formation of autophagosomes and subsequent delivery to lysosomes for complete destruction [42]. This is accompanied by the suppression of mitochondrial biogenesis (decreased expression of PGC-1a), as well as the processes of fusion and fission of the organelles (decreased expression of OPA1, MFN1,MFN2 and DRP1). Eventually, the BMSC show signs of massive degradation of damaged organelles (increased FIS1 expression and decreased mtDNA/nDNA ratio). Destructive changes are also found at the cellular level. Indeed, as can be seen from the microscopy data (Figures 4 and 5), the BMSC located in the zone of direct action of LTP showed morphological changes specific to apoptosis, manifested in the formation of apoptotic bodies (Figure 4B). The Hoechst 33342/PI dual-staining assay also showed typical apoptotic morphology for the BMSC upon LTP treatment (Figure 5B). The results of real-time PCR analysis confirmed the data obtained and indicated a significant increase in the expression of the protein factors of apoptosis-caspase 3 and JNK during the first hour after LTP exposure (Figure 6). Caspase-3 is a cysteine-aspartic acid executioner protease that is best known for its enzymatic function at the end of the intrinsic apoptotic cascade [43]. Caspase 3 is known to be activated in an apoptotic cell both externally (via death ligands) and intrinsically, primarily due to the release of the cytochrome \(c\) from mitochondria, induced by mitochondrial dysfunction and the opening of mitochondrial permeability transition (MPT) pores [34; 35; 44]. In turn, the activation of JNK leads to its translocation into the mitochondrial membrane, where it blocks the anti-apoptotic protein Bcl-2, which contributes to the sequential activation of proapoptotic members of the Bcl-2 family, which are critical for the release of the cytochrome \(c\) from mitochondria and the induction of apoptosis [34]. Both of these cascades are directly initiated by ROS under the condition of oxidative stress [34]. Hence, one could assume that the initiation of cell death by apoptosis due to the overproduction of ROS and mitochondrial degradation underlies the acute action of LTP, which was observed during the first hour after plasma exposure to the cell culture. It can be seen that the intensity of oxidative stress due to the LTP-induced hyperproduction of ROS showed a tendency to decrease after reaching its maximum in the first hour after LTP exposure. This was manifested both in the decreased concentration of hydrogen peroxide in the medium and in the lowered concentration of lipid peroxidation products (DC and MDA) in the cell culture. One could assume that this was a consequence of the activation of intracellular antioxidant defense systems that normalize the ROS concentration in the cell [45].