## 3 Results
### Effect of Extract on H\({}_{2}\)O\({}_{2}\)-Induced Oxidative Stress in Normal Fibroblast Cells  
The addition of hydrogen peroxide clearly changed the typical morphology of HFFF-2 fibroblasts (Figure 2A). The cells became more oblong in comparison to the controls. Cell numbers were considerably reduced and more dead (detached) cells appeared. Earlier incubation with FLE (100 \(\upmu\)g/mL) partially reduced this effect, but most cells did not have the typical HFFF-2 cell morphology (2b). The number of cells was higher and fewer dead cells were observed (Figure 2B). Application of the extract in cells without induction with hydrogen peroxide did not change the typical cell morphology (Figure 2D). The results of the LDH test showed that the addition of a crayfish extract at the concentration of 50-200 \(\upmu\)g/mL significantly reduced the cytotoxicity of hydrogen peroxide in HFFF-2 fibroblast cells (Table 3). The FLE concentration of 100 \(\upmu\)g/mL was found to be the most effective, which caused the cytotoxicity in cells induced with hydrogen peroxide  
Figure 1: Antioxidant activity of _F. limosus_ crayfish extract compared to gallic acid and \(\beta\)-carotene, expressed as % of DPPH inhibition. Different letters mean that the values for the bars differ statistically (\(p<0.05\)), \(N=6\). Figure 2: Morphology of cells treated with _F. limosus_ extract or PBS and/or H\({}_{2}\)O\({}_{2}\). (**A**) Cells treated only with 100 uM/mL hydrogen peroxide. (**B**) Cells treated with 100 ug/mL crayfish extract and 100 uM/mL hydrogen peroxide. (**C**) Control cells treated only with PBS. (**D**) Cells treated only with 100 ug/mL crayfish extract. Scale bar: 1 \(\upmu\)m.  
to be lower than in cells receiving PBS (not induced with H\({}_{2}\)O\({}_{2}\)). In the case of the highest dose of the extract (200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL), the cytotoxicity was low but significantly higher than for the 50 and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL dose. This indicates that the concentration of 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL is too high for HFFF-2 cells. Then, we planned to investigate selected mechanisms that may be responsible for this. First, the release of ROS was investigated in the same model (Figure 3a,b). The highest expression of ROS was observed in cells induced with hydrogen peroxide without the addition of an extract. Adding the extract in the amount of 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL abolished the effect of hydrogen peroxide. Concentrations of 50 and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, used without adding hydrogen peroxide, lowered ROS production below the values of cells treated with PBS only. At the extract concentration of 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, a trend towards higher ROS expression was observed than for the concentrations of 50 and 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, but this difference was not statistically significant. However, when combined with the LDH assay, where the 200 \(\mathrm{\SIUnitSymbolMicro g}\)/mL dose was less effective in reducing the cytotoxicity of H\({}_{2}\)O\({}_{2}\), this indicates that the dosage may be too high for HFFF-2 cells. Then, it was decided to check whether the decrease in cell death after applying the extract was also due to the increase in their proliferation. The results of the proliferation test are shown in Figure 4.  
Overall, the incubation of healthy cells with FLE increased their viability many times over. The strongest effect was demonstrated for the concentration of 100 \(\mathrm{\SIUnitSymbolMicro g}\)/mL, which increased the number of viable cells by 6.3 times compared to the control incubated with PBS. The addition of hydrogen peroxide alone reduced cell viability by 63%. In the oxidative stress model, the addition of _F. limosus_ extract increased the viability of cells above the control value (PBS only). This effect was proportional to the concentration of the extract.