Genistein-Supplemented Diet Decreases Malaria Liver Infection in Mice and Constitutes a Potential Prophylactic Strategy (original) (raw)
Figure 2
Genistein suppresses the early development of parasites within hepatocytes.
(A) Hepa1-6 cells were incubated with genistein or DMSO and inoculated with 4×104 P. berghei ANKA sporozoites. The two images show EEFs observed 24 hours after infection of control (left) or genistein-treated cells (right) at a 1000× magnification. Bar = 2.5 μm. (B) Distribution of EEFs according to size. Hepa1-6 cells were treated with genistein or DMSO (control) immediately prior to inoculation with 4×104 P. berghei ANKA sporozoites and the size of 100 EEFs from different coverslips was determined 24 hours later. The figure shows the frequency of EEFs with sizes between 0 and 40 μm2. ○ control; • 25 μM genistein. P = 5.9×10−25 (TTest relative to the control group). The results are representative of 3 independent experiments. (C) Incubation of cultured HepG2 cells with 25 μM genistein or DMSO (control) at various times after addition of 4×104 P. berghei ANKA sporozoites. Infection was determined 24 hours after sporozoite addition by counting the total number of EEFs per coverslip. The results are expressed as the percentage of EEFs relative to the average number of EEFs in the control samples, taken as 100%. (D) Incubation of cultured HepG2 cells with 25 μM genistein or DMSO (control) at the time of addition of 4×104 P. berghei ANKA sporozoites and washed at various time-points. Infection was determined 24 hours after sporozoite addition by counting the total number of EEFs per coverslip. The results are expressed as the percentage of EEFs relative to the average number of EEFs in the control samples, taken as 100%. ★ p<0.05, ★★ p<0.01 ★★★ p<0.001 (TTest relative to control group). Bar plots show one representative of 3 independent experiments, error bars represent s.d. of mean number of EEFs in each condition (n = 3). (E) Flow cytometry analysis of the invasion rate in genistein-treated and control Huh7 cells at 2 hours after addition of 3×104 PbGFP sporozoites. Bar plots show one representative of 3 independent experiments, error bars represent s.d. of mean percentage of GFP positive cells in each condition (n = 3). (F) Flow cytometry analysis of genistein-treated and control Huh7 cells at 6, 30 and 44 hours after addition of 3×104 PbGFP sporozoites, and of primaquine-treated and control cells at 44 hours after adition of 3×104 PbGFP sporozoites. Red line represents Genistein treated cells and black line represents control cells. The graphs show one representative dataset of triplicate samples. (G) Quantification of the GFP intensity of PbGFP-infected genistein-treated (red bars) and non-treated (black bars) cells at the same time points as in E. ★★ p<0.01 (TTest relative to control group). Bar plots show one representative of 3 independent experiments, error bars represent s.d. of mean GFP intensity in each condition (n = 3).