## 2 Results and Discussion
### ITC Reduces the Toxicity of V. cholera In Vitro and In Vivo
coli_ and _S. aureus_, where SFN treatment significantly reduced intracellular bacteria survival of THP-1 and PBMC-derived macrophages. Our results show significantly enhanced hemocytes infiltration to hemolymph 3 h after SFN treatment (Figure 6c) compared to PBS treated control larvae. We also observed the 27.7% enhancement of phagocytosis rate (Figure 6d,e) by hemocytes obtained from _G. mellonella_ injected with SFN. These results were assessed by fluorescence microscopy analysis of heat-killed FITC-labeled _V. cholerae_ bacteria. But yet, the detailed mechanism of this observation should be elucidated to define SFN-triggered cellular factors. [PERSON] et al. [64] proposed a combinatory mechanism where reduction of the inflammatory response through modulation of p38 and JNK signaling pathways accompanied antibacterial action of SFN in mice macrophages infected with _S. aureus_. A similar mode of action may operate in _G. mellonella_, especially as  
Figure 6: _G. mellonella_ larvae pretreatment with SFN improve their survival rate after _V. cholerae_ infection. (**a**) Kaplan–Meier survival analysis of _Galeria mellonella_ larvae infected with _Vibrio cholerae_ O395. Larvae (n = 15) were injected with (10 \(\mu\)L) PBS (blue circle) or _V. cholerae_ suspension in PBS at range of c.f.u/per dose: 10\({}^{2}\) (yellow circle) or 5 \(\times\) 10\({}^{2}\) (green deltoid) or 5 \(\times\) 10\({}^{3}\) (orange triangle) or 5 \(\times\) 10\({}^{4}\) (red triangle) or 10\({}^{6}\) c.f.u (gray squares). (**b**) Treatment efficacy assessed by survival analysis. Larvae (n = 15) were pre-treated (3 h before infection) with SFN (light green triangle) or AZT (yellow cross) or SFN + AZT (dark green deltoid). Infection groups (filled symbols) were then injected (t = 0 h) with 5 \(\times\) 10\({}^{2}\) c.f.u of bacterial suspension (_V. ch_), not treated control is represented by red squares. Not infected control groups are marked as empty symbols. Double injection with PBS was performed in the uninfected control group (blue circle). Larvae were injected and monitored as indicated on the timeline above graph. PT- pretreatment INF- infection. (**c**) Total hemocyte infiltration to hemolymph (3 h) in larvae injected with PBS (blue), SFN (light green), AZT (yellow), SFN + AZT (dark green) or not treated (white), individual. (**d**) Representative images of infected hemocytes are shown. The fluorescent signal of FITC-labeled bacteria is indicated by arrows. Scale bar upper left panel denotes 50 \(\mu\)m and applies to the rest of the micrographs. BF-bright light; FL-fluorescence (**e**) Quantification of the phagocytosis percentage. The significance of differences between the results and the control was tested using the Student’s _t_-test. Statistical significance is marked with asterisks: *** \(p<0.001\); *** \(p<0.01\). this organism possesses counterpart MAP kinases, and they are activated in response to infection with the entomopathogenic bacterium _Bacillus huringiensis_[65; 66] Finally, the dosages chosen for in vivo virulence assay were comparable to those used and reported as safe to treat human diseases. Therefore, based on its effects, as well as a low toxicity, SFN could be considered as a support to current therapies for the management of cholera as monotherapy and for the combination with other antibiotics. However, further studies are needed to elucidate the molecular mechanisms underlying the activity of ITCs and also to identify their new molecular targets.