Cytotoxicity activity
The MTT assay revealed that different concentrations of both lumefantrine and sulfadiazine had no cytotoxicity compared with the negative control cells (Fig. 1a and 1b). Thus, different concentrations of lumefantrine (high 94.5 nmol/L, medium 17.7188 nmol/L, and low 2.9531 nmol/L) and sulfadiazine (10 µg/mL) were used to carry out further experiments against T. gondii in vitro.
Anti-invasion activity
Vero cell counts using the MTT assay showed that pre-treatment with 94.5 nmol/L, 17.7188 nmol/L and 2.9531 nmol/L lumefantrine reduced tachyzoites invasion by 7.12%, 7.08%, and 6.72% for 18 h, respectively (Fig. 2A). Sulfadiazine caused a 4.15% reduction after 18 h treatment (Fig. 2A). The invasion ability of pre-treated tachyzoites with different concentrations of lumefantrine and sulfadiazine were significantly reduced compared with the untreated group at 18 h (Toxoplasma group) (P ≤ 0.01).
Anti-Proliferation activity
Further evaluation of the ability of lumefantrine and sulfadiazine to inhibit the intracellular tachyzoite replication within Vero cells was examined using the MTT assay at 24 h and 48 h post-treatment (Fig. 2B). Post-treatment with 94.5 nmol/L, 17.7188 nmol/L or 2.9531 nmol/L lumefantrine reduced the proliferation of tachyzoites by 27.31%, 22.79%, and 20.85% at 24 h and 47.18%, 42.34% and 42.48% at 48 h, respectively (Fig. 2B). A 21.12% reduction at 24 h and 41.2% reduction at 48 h for tachyzoite post-treatment with sulfadiazine were observed (Fig. 2B). This was an indication that lumefantrine could significantly inhibit tachyzoite proliferation compared with the Toxoplasma group (P ≤ 0.01).
Flow cytometry
The anti-proliferation activity of lumefantrine was further examined using flow cytometry. Samples were stained with annexin V-FITC and Propidium Iodide after treatment with lumefantrine and sulfadiazine for 24 h. Different quadrants represent different states of the cells (Q1:Necrotic and damaged cells. Q2: Late apoptotic cells. Q3: Living cells. Q4: Early apoptotic cells). The more living cells in Q3 quadrant, reflects the good effect of lumefantrine on anti-parasite proliferation (Fig. 3a). These results indicated that different concentrations of lumefantrine could inhibit the proliferation of T. gondii by flow cytometry in Fig. 3a and 3b.
Survival rate of acutely infected mice treated with lumefantrine
Mice were observed daily and survival time was recorded at 11 days post-treatment. Compared with the Toxoplasma group, mice started to die at 6 days post-treatment. However, mice treated with 94.5 nmol/L, 17.7188 nmol/L, and 2.9531 nmol/L lumefantrine started to die at day 7, 8, and 9 post-treatemt, respectively. The positive group (sulfadiazine group) started to die at day 7 post-treatment. After 11 days, 80%, 66.7% and 53.3% of mice treated with 94.5 nmol/L, 17.7188 nmol/L, 2.9531 nmol/L lumefantrine, respectively had survived, while only 46.7% living mice treated with 10 µg/mL sulfadiazine had survived (Fig. 4).
Parasite load in mice tissues
To further evaluate the parasite load in the mice after lumefantrine treatment, liver, heart, spleen, and lung samples from infected mice were determined by qPCR, and the results are shown in Fig. 5. Treatment with different concentrations of lumefantrine significantly (**p ≤ 0.01 and *p ≤ 0.05) reduced the parasite load in the liver, heart, spleen and lung tissues compared with the Toxoplasma group (PBS group). The parasite load in different tissues except the liver was also reduced in the positive control group (sulfadiazine group).
Regulation of cytokine levels by lumefantrine in mice infected by T. gondii
In order to determine whether lumefantrine treatment enhances Th1 or Th2 cytokine response, IFN-γ, IL-4, and IL-10 levels in the serum of mice were determined in Fig. 6. Significantly higher levels of IFN-γ were observed in mice treated with a high concentration lumefantrine compared to the control groups (p ≤ 0.01). In addition, IL-4 and IL-10 were significantly produced in mice treated with a low concentration lumefantrine compared to the control groups (p ≤ 0.01).