Antiparasitic activity of artemether in vitro
The anti-T. gondii effect of artemether was preliminarily evaluated by plaque assay. From the results we found that the plaques were less and smaller in artemether treated groups (3.125 mM or 0.78 mM), comparing to DMSO-treated and untreated groups (Fig. 2A, B). These results indicated that artemether could inhibit the parasite proliferation under the safe concentration. As seen in Fig. 3, the IC50 of artemether is 0.905 mM and the inhibition increased in a dose-dependent manner, especially, the inhibition was slightly better than pyrimethamine at 3.125 mM (Fig. 4A, B).
Effect of artemether on the invasion of T. gondii
To test whether the artemether inhibited parasite invasion, invasion assay was carried on. From the results, we could find when parasites were treated by 1mM artemether for 20 min, 40 min and 60, the invasion rates were 20.07%, 36.64%, 48.58%, respectively. In untreated group, the invasion rates were 31.77%, 53.53%, 66.92% at the same time points (Fig. 5). Although, the invasion rates were slightly inhibited by artemether, the differences were not significant.
Inhibition of artemether on intracellular proliferation of T. gondii
The inhibition of artemether on intracellular T. gondii proliferation was evaluated on HFF cells. As shown in Fig. 6A, comparing with the control group, most of parasitophorous vacuoles (PVs) contained 4, 2, 1 tachyzoites in artemether treated groups. Especially, intracellular growth was significantly inhibited at artemether group at 3 mM, from the results we could find that most PVs contained 1 or 2 tachyzoites treated by artemether for 24 h. The similar situation was also found, when cells and parasites were treated with different concentrations artemether for 48 h (Fig. 6B).
Artemether impaired mitochondrial membrane potential of T. gondii
To investigate the potential mechanism of the artemether anti-Toxoplasma, tachyzoites were treated by different concentration artemether. From the results we could find that 61.91% and 31.23% mitochondrial membrane were damaged after treated by 0.5 mM and 0.05 mM artemether, respectively (Fig. 7A). According to the mean fluorescence intensity, T. gondii mitochondrial membrane potential was significantly lower to the control group in 0.5 mM artemether group (P < 0.01) (Fig. 7B). This result indicated that artemether played anti-parasite role by destroying T. gondii mitochondrial membrane.
ROS produced with artemether anti- T. gondii effect
Due to the change of T. gondii mitochondrial membrane potential, the ROS production in extracellular T. gondii tachyzoites was further analyzed. From the results we found that artemether could increase ROS activity, especially in 0.5 mM artemether group, the difference was significant compare to the control group (P < 0.001) (Fig. 8). We speculated that the anti-parasitic activity of artemether related to inducing accumulation of ROS in Toxoplasma.