## 3 Results
### Intracellular ROS Level  
To measure ROS levels, the H\({}_{2}\)DCFDA dye was selected. The increase in fluorescence of the H\({}_{2}\)DCF-DA probe in the cell is proportional to the amount of ROS, which may correlate with some types of cell death and/or metabolic processes. After 24 h, a 535.83%, 1442.81%, and 1492.25% increase in the ROS level was observed in the BJ cells treated with 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M concentrations of Les-4368, respectively, compared to the control (Figure 5A). Similarly, after the 48 h treatment of the cells with 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M of Les-4368, the ROS level increased by 458.74%, 1274.34%, and 1375.7%, respectively, compared to the control (Figure 5A).  
Figure 4: Effects of the increasing concentrations (1 nM–100 \(\upmu\)M) of Les-4368 (**A**,**C**) and Les-4370 (**B**,**D**) on the LDH release level in the BJ cell line (**A**,**B**) and the CACO-2 cell line (**C**,**D**) after treatment for 24 h and 48 h. Data are expressed as means (\(n=6\)) with standard deviation (SD); data marked with *, **, and *** are statistically different at \(p<0.05\), \(p<0.01\), and \(p<0.001\), respectively, compared to the control.  
The 24 h exposure of the BJ cells to 1 \(\upmu\)M, 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M of Les-4370 increased the ROS level by 48.61%, 202.79%, 319.97%, and 399.47%, compared to the control (Figure 5B). In turn, the 48 h treatment of the BJ cells to 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M of Les-4370 caused a 149.15%, 239.53%, and 301.18% increase in the ROS level, respectively, compared to the control (Figure 5B).  
After the 24 h exposure of the CACO-2 cells to Les-4368 at the concentrations of 1 \(\upmu\)M, 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M, the ROS level increased by 68.19%, 440.31%, 1074.63%, and 951.48%, respectively, compared to the control (Figure 5C). The 48 h exposure of the CACO-2 cells to Les-4368 at the concentrations of 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M resulted in a 358.41%, 978.88%, and 990.55% increase in the ROS level, respectively, compared to the control (Figure 5C).  
After the 24 h exposure of the CACO-2 cells to Les-4370, a 233.94%, 518.20%, and 431.86% increase in the ROS level was observed at the concentrations of 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M, respectively, compared to the control (Figure 5D). In turn, after the 48 h treatment of the 48 h  
the caspase-3 activity was increased by 29.34% and 191.01% in the 50 \(\upmu\)M and 100 \(\upmu\)M concentration variants, respectively, compared to the control (Figure 6A).  
In the BJ cell line, the 24 h treatment with 100 \(\upmu\)M of Les-4370 caused a 13.70% increase in the caspase-3 activity, compared to the control (Figure 6B). In turn, after the 48 h exposure of the BJ cells to Les-4370 in the concentration range of 10 \(\upmu\)M, 50 \(\upmu\)M, and 100 \(\upmu\)M the caspase-3 activity increased by 7.49%, 22.79%, and 18.41%, respectively, compared to the control (Figure 6B).  
After the 24 h exposure of the CACO-2 cells to Les-4368, a 23.10% and 51.12% increase in the caspase-3 activity was observed at the concentrations of 50 \(\upmu\)M and 100 \(\upmu\)M, respectively, compared to the control (Figure 6C). The activity of caspase-3 increased by 9.53% after the 48 h treatment of the CACO-2 cells with 100 \(\upmu\)M of Les-4368, compared to the control (Figure 6C).  
After the 24 h treatment of the CACO-2 cells with Les-4370, a 22.11% and 21.83% increase in the caspase-3 activity was observed at the 50 \(\upmu\)M and 100 \(\upmu\)M concentrations, respectively, compared to the control (Figure 6D). Similarly, the cells treated with the same concentrations of Les-4370 for 48 h were characterized by a 9.20% and 13.59% increase in the caspase-3 activity, respectively, compared to the control (Figure 6D).