## 4 Discussion
It is also known that plant substances may have various effects on the antioxidant activity of glutathione, e.g., galangin and isoramnetin antagonise the action of GSH [20], while methyl-ether derivatives of cyclitols (D-pinitol and L-quebrachtol) supported this action [21]. Next, the protective properties of FLE against fibroblasts, in which the addition of hydrogen peroxide caused oxidative stress, were examined. The extract did not change the morphology of healthy fibroblast cells, and when used before induction with H\({}_{2}\)O\({}_{2}\), the morphology was more similar to that of normal cells. _F. limosus_ extract reduced the cytotoxicity of the hydrogen peroxide used. At a concentration of 100 \(\upmu\)g/mL, the cytotoxicity of hydrogen peroxide was abolished entirely. Similar effects have been found for other aquaculture animals, e.g., protein isolated from mussels [22], oysters [23] and seahhorses [24]. Moreover, the water extract from the tissue of the pen shell (Atrina pectinate) reduced cell death and ROS secretion caused by oxidative stress, but the effect was weaker than that of FLE [25]. [PERSON] and [PERSON] (2011) showed that the aqueous extract of _Procambarus clarkii_ freshwater crayfish bodies decreased the markers of oxidative stress (MDA) and also increased the activity of antioxidant enzymes in the sera and livers of rats following CCl4 intoxication [26]. Similarly, in the reported study, it was shown that the addition of FLE reduced the release of ROS. Fibroblast cells incubated with FLE at 50 and 100 \(\upmu\)g/mL before induction with hydrogen peroxide secreted ROS at the same level as the control cells (PBS only). The level of ROS secretion in cells that had not received an extract before the administration of hydrogen peroxide increased more than threefold. It has also been shown that preincubation with FLE reduces the expression of caspase-3, a marker of early apoptosis, in H\({}_{2}\)O\({}_{2}\)-induced HFFF-2 cells. The activity in lowering the apoptosis of fibroblasts was demonstrated for the polyphenol-rich strawberry extract [27], as well as for silk lutein [28] and astaxanthin [29]. It should be noted, however, that the low increase in caspase-3 expression after the induction of oxidative stress indicates that this mechanism has little effect on HFFF-2 cell death following H\({}_{2}\)O\({}_{2}\) treatment. The FLE effect itself, although statistically significant, only slightly decreased the expression of caspase-3. The most promising mechanism of action of the FLE is to increase the proliferation. The presented study showed that the extract added to healthy fibroblast cells increased the number of viable cells in relation to cells treated only with PBS. The addition of hydrogen peroxide reduced the cell viability by over 60%. Adding the extract before the induction of oxidative stress completely eliminated the effects of death caused by hydrogen peroxide, and the protein fraction of the extract was likely responsible for this result. FLE has high lysine content, and this essential amino acid is one of the most important building blocks of many proteins in the body. It has long-known properties supporting tissue regeneration after injuries. In in vitro models, lysine enhanced the production of growth factors by fibroblasts. It also has the ability to induce strong inflammatory and immune responses (both humoral and cell-mediated), which could also have influenced the antioxidant effect of the FLE on HFFF-2 cells [30]. Moreover, proline is an amino acid proven to increase the regeneration of connective tissue after injuries. It is an essential component of collagen and also plays an important role in protein synthesis and structure and metabolism, as well as wound healing, antioxidant reactions and immune responses [31; 32]. Phenols present in the extract may also have been responsible for the increase in proliferation. It was shown that phenolic-rich plant extracts significantly increased the proliferation of normal fibroblasts [33; 34].