In Vitro Evaluation Reveals Effect and Mechanism of Artemether against Toxoplasma gondii

Due to the limited effectiveness of existing drugs for the treatment of toxoplasmosis, there is a dire need for the discovery of new therapeutic options. Artemether is an important drug for malaria and several studies have indicated that it also exhibits anti-T. gondii activity. However, its specific effect and mechanisms are still not clear. To elucidate its specific role and potential mechanism, we first evaluated its cytotoxicity and anti-Toxoplasma effect on human foreskin fibroblast cells, and then analyzed its inhibitory activity during T. gondii invasion and intracellular proliferation. Finally, we examined its effect on mitochondrial membrane potential and reactive oxygen species (ROS) in T. gondii. The CC50 value of artemether was found to be 866.4 μM, and IC50 was 9.035 μM. It exhibited anti-T. gondii activity and inhibited the growth of T. gondii in a dose-dependent manner. We also found that the inhibition occurred primarily in intracellular proliferation, achieved by reducing the mitochondrial membrane integrity of T. gondii and stimulating ROS production. These findings suggest that the mechanism of artemether against T. gondii is related to a change in the mitochondrial membrane and the increase in ROS production, which may provide a theoretical basis for optimizing artemether derivatives and further improving their anti-Toxoplasma efficacy.


Introduction
Toxoplasma gondii is an obligate intracellular parasite that infects a wide range of warmblooded mammals, including human beings [1]. About one in three people is infected by T. gondii in the world [2]. The infection is asymptomatic or mild for most immunocompetent individuals. For immunodeficiency patients, however, it may cause serious health problems such as encephalitis and retinochoroiditis [3].
The standard therapy for toxoplasmosis relies on a combination of sulfadiazine and pyrimethamine [4]. Both drugs inhibit T. gondii by inhibiting the parasite folate metabolism, and are only effective against tachyzoites [5]. Unfortunately, the side effects of these drugs are severe: about 40% of patients are forced to interrupt the treatment because they cannot tolerate it [6]. At the same time, prolonged treatment is necessary for immunocompromised patients [7]. Long-term treatment, side effects and resistance to drugs limit the use of these drugs. Therefore, it is necessary to find high-efficiency and low-toxic drugs in the treatment of toxoplasmosis.
Numerous studies have focused on screening new synthetic or semi-synthetic drugs for treating toxoplasmosis based on plant sources [8]. It is reported that only about 15% of plants have been explored. For instance, Aloe vera and Eucalyptus methanolic extracts not only have antimicrobial and antioxidant activities, but can also significantly inhibit the growth of T. gondii tachyzoites [9]. cells. To harvest tachyzoites, heavily infected cells were scraped and passed through a 27-gauge needle, 3-5 times. Cell debris was removed by a 3 µm membrane filter (Whatman, Maidstone, UK). Tachyzoites were counted using a hemocytometer for further experiments.

Drug Compounds
Artemether was purchased from Aladdin Reagent limited company, Shanghai, China. The powder was then dissolved in dimethyl sulfoxide (DMSO) with an initial concentration of 1 M. The solution was stored at −20 • C. The different concentrations of artemether were diluted with DMEM medium. The final concentration of DMSO in the samples used in the experiment was lower than 1% (v/v).

Cytotoxicity Assay
The cytotoxicity of artemether was determined in an HFF cell with a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, USA), according to the manufacturer's instructions. A total of 1 × 10 5 cells were seeded in 96-well plates and cultured at 37 • C, in an atmosphere containing 5% CO 2 , for 24 h. They were then treated with different concentrations of artemether (10,000 µM, 2000 µM, 1000 µM, 500 µM, 200 µM and 50 µM) in DMEM complete culture medium. After incubating for 24 h, adding 10 µL MTT solution to each well and incubating for 4 h, 200 µL of DMSO were added to dissolve the formazan crystals. The absorbance of the suspension was measured at 490 nm using an iMark TM Microplate Absorbance Reader (BioRad, Hercules, CA, USA). The 50% cytotoxic concentration (CC 50 ) of artemether was calculated using Graphpad Prism 8.0. The cytotoxicity experiment was performed in triplicate, using three separate plates.

Anti-T. gondii Activity of Artemether Evaluated by a Plaque Assay
A confluent monolayer of HFF cells was infected with 5 × 10 2 tachyzoites for 4 h in 6-well plates. Then, the extracellular parasites were removed, and a fresh medium containing different concentrations of artemether or 0.5% DMSO (vehicle control) was added to each well. Uninfected and untreated wells were used as controls. They were then incubated at 37 • C with a 5% CO 2 atmosphere for 7 days without any movement. HFF cells were washed with PBS, fixed with 4% paraformaldehyde and stained with 2% crystal violet for 30 min. Finally, the field of vision was randomly selected and photographed under the microscope. The plaque areas were analyzed.

Effects of Artemether on Intracellular T. gondii
Monolayer HFF cells were incubated in a 24-well plate, and RH-RFP tachyzoites were added at a multiplicity of infection (MOI) of 1 and allowed to settle at 37 • C with an atmosphere containing 5% CO 2 for 24 h. The medium containing extracellular parasites was removed and a fresh medium containing either artemether (31.25 µM, 15.63 µM, 7.81 µM, 3.91 µM and 1.95 µM), 0.3% DMSO (vehicle control) or pyrimethamine (positive control) was added, respectively. After incubating at 37 • C for 24 h, a fluorescence microscope was used to examine the growth of RH-RFP, and the growth rate was statistically analyzed using Image-Pro Express.

Invasion Assay
Invasion experiments were performed as described by Lim SSY et al. [33] In brief, HFF cells were cultured in a 6-well plate, and 3 mL DMEM with 2% FBS were added to each well. Then, 1 × 10 5 RH and 10 µM artemether were added simultaneously to the wells, respectively, incubating for 20 min, 40 min or 60 min. The supernatant was gently removed, cells were fixed with 2 mL 4% paraformaldehyde for 10 min, washed three times with PBS, blocked by 5% BSA in PBS (BSA/PBS) for 1 h, and washed three times with PBS. They were then incubated with mouse anti-T. gondii SAG1 at room temperature for 2 h, followed by Alexa Fluor 488 goat anti-mouse secondary antibodies for 2 h, then washed three times andpermeabilized with 0.1% Triton X-100/PBS for 1 h. Cells were stained with rabbit anti-T. gondii polyclonal antibodies followed by Alexa Fluor 594 goat anti-rabbit secondary antibodies. Nuclei were stained with DAPI for 1 h. Five visual fields were randomly selected for observation under the 40× objective of the fluorescence microscope and the parasites in each field were counted. Three repetitions were performed to increase the accuracy of the experiment. The difference between the tachyzoites of the two colors is termed the absolute invasion number of tachyzoites. The ratio of the invasion number to the total number of tachyzoites is termed the invasion rate of tachyzoites.

Intracellular Proliferation Assay
Freshly egressed tachyzoites were allowed to infect HFF monolayers for 2 h, then the medium was changed with containing artemether (30 µM, 15 µM, 7.35 µM, 4.15 µM and 1.37 µM), and 0.2% DMSO, respectively. Invaded parasites were co-cultured at 37 • C in a 5% CO 2 atmosphere for 24 h and 48 h. A total of 100 parasitophorous vacuoles (PV) were randomly selected and the number of parasites in each vacuole was counted.

Measurement of Mitochondrial Membrane Potential of Tachyzoites
A total of 1 × 10 7 fresh tachyzoites were incubated in DMEM containing different concentrations ofartemether (12.5 µM or 3.35 µM), or no drug (vehicle control) at 37 • C with a 5% CO 2 atmosphere for 60 min. Parasites were stained with JC-1 (Solarbio, Beijing, China) according to the manufacturer's protocol. The samples were analyzed by flow cytometry. Three independent biological experiments and three technical replicates were performed.

Measurement of Reactive Oxygen Species (ROS)
ROS was measured by ROS Assay Kit-Highly Sensitive DCFH-DA (Jiancheng, Nanjing, China) according to the manufacturer's instructions. Briefly, 1×10 7 purified tachyzoites were suspended in 0.1 M PBS and mixed with Highly Sensitive DCFH-DA Dye for 30 min, and then artemether (12.5 µM and 3.35 µM), PBS and H 2 O 2 were added, respectively, incubating for 2 h at 37 • C. Fluorescence acquisition was measured at 485 nm and 530 nm using a multifunctional microplate reader (BioTek Synergy 2, Genomics Co., Ltd., Waltham, MA, USA)

Statistical Analyses
All data were analyzed using Graphpad Prism 8.0. The differences between treatments and controls were assessed using analysis of variance (one-way and two-way ANOVA) and the results in comparisons between any two groups were considered as differences if p < 0.05. Flow cytometry assay was analyzed using CytExpert 2.3.

Cytotoxicity of Artemether
The cytotoxic potential of artemether on HFF cells was confirmed before anti-T. gondii activity study. According to the MTT assay result, the concentration of artemether that induced a 50% HFF cell mortality (CC 50 ) was 866.4 µM (Figure 1).
Cytotoxicity was evaluated using a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay. Cell viability was calculated by comparing the treatment group with the negative control group, and the base of log concentrations was used to analyze the CC 50 . All data are presented with error bars, the experiments were performed in triplicate and three technical replicates were included in each experiment.

Antiparasitic Activity of Artemether In Vitro
The antiparasitic effect of artemether was preliminarily evaluated by plaque assay. From the results, we found that the plaques were fewer and smaller in artemethertreated groups (25.25 µM or 7.5 µM), compared to DMSO-treated and untreated groups (Figure 2A,B). These results indicated that artemether could inhibit the parasite proliferation under the safe concentration. As seen in Figure 3, the IC 50 of artemether is 9.035 µM, this concentration is much lower than the safe concentration (866.4 µM). We could find that when the drug concentration reached 31.25 µM, the antiparasitic effect is similar to that of the pyrimethamine, within this concentration, the inhibition increased in a dose-dependent manner ( Figure 4A Cytotoxicity was evaluated using a CellTiter 96 ® AQueous One Solutio eration Assay. Cell viability was calculated by comparing the treatment gr negative control group, and the base of log concentrations was used to ana All data are presented with error bars, the experiments were performed in three technical replicates were included in each experiment.

Antiparasitic Activity of Artemether In Vitro
The antiparasitic effect of artemether was preliminarily evaluated by From the results, we found that the plaques were fewer and smaller in artem groups (25.25 μM or 7.5 μM), compared to DMSO-treated and untreated gr 2 A, B). These results indicated that artemether could inhibit the parasite under the safe concentration. As seen in Figure 3, the IC50 of artemether is 9 concentration is much lower than the safe concentration (866.4 μM). We co when the drug concentration reached 31.25 μM, the antiparasitic effect is sim the pyrimethamine, within this concentration, the inhibition increased in a d ent manner (Figure 4 A,B).  Cytotoxicity was evaluated using a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay. Cell viability was calculated by comparing the treatment group with the negative control group, and the base of log concentrations was used to analyze the CC50. All data are presented with error bars, the experiments were performed in triplicate and three technical replicates were included in each experiment.

Antiparasitic Activity of Artemether In Vitro
The antiparasitic effect of artemether was preliminarily evaluated by plaque assay. From the results, we found that the plaques were fewer and smaller in artemether-treated groups (25.25 μM or 7.5 μM), compared to DMSO-treated and untreated groups ( Figure  2 A, B). These results indicated that artemether could inhibit the parasite proliferation under the safe concentration. As seen in Figure 3, the IC50 of artemether is 9.035 μM, this concentration is much lower than the safe concentration (866.4 μM). We could find that when the drug concentration reached 31.25 μM, the antiparasitic effect is similar to that of the pyrimethamine, within this concentration, the inhibition increased in a dose-dependent manner (Figure 4 A,B).  The relative areas of plaque in each group were calculated. All the above experiments were repeated three times and 3 technical replicates were included in each experiment. Red arrows (T. gondii plaques) *** p < 0.001, **** p < 0.0001. RH-RFP was cultured in monolayers of HFF cells and treated with different concentrations of artemether for 24 h. The growth rate of RH-RFP tachyzoites was statistically counted using Image-Pro Express and plotted by Graphpad Prism 8.0, and the base of log concentrations were used to analyze the IC50. The data are presented as the mean ± SD. Three independent experiments were performed, and three technical replicates were included in each experiment.   RH-RFP was cultured in monolayers of HFF cells and treated with different concentrations of artemether for 24 h. The growth rate of RH-RFP tachyzoites was statistically counted using Image-Pro Express and plotted by Graphpad Prism 8.0, and the base of log concentrations were used to analyze the IC50. The data are presented as the mean ± SD. Three independent experiments were performed, and three technical replicates were included in each experiment.  RH-RFP was cultured in monolayers of HFF cells and treated with different concentrations of artemether for 24 h. The growth rate of RH-RFP tachyzoites was statistically counted using Image-Pro Express and plotted by Graphpad Prism 8.0, and the base of log concentrations were used to analyze the IC 50 . The data are presented as the mean ± SD. Three independent experiments were performed, and three technical replicates were included in each experiment.

Effect of Artemether on the T. gondii Invasion
The invasion ability of T. gondii is an important prerequisite for its proliferation in different hosts. To test whether the artemether inhibited T. gondii proliferation in HFF by inhibiting invasion, the related assay was performed. From the results, we could find that tachyzoites were treated with 10 µM artemether for 20, 40 and 60 min, the invasion rates were 20.07%, 36.64% and 48.58%, respectively. In the untreated group, the invasion rates were 31.77%, 53.53% and 66.92% ( Figure 5). Although the invasion rate was slightly inhibited by artemether (p < 0.05), the differences were not significant, which indicated that the artemether-inhibited T. gondii proliferation was not related to the invasion.

Effect of Artemether on the T. gondii Invasion
The invasion ability of T. gondii is an important prerequisite for its proliferation in different hosts. To test whether the artemether inhibited T. gondii proliferation in HFF by inhibiting invasion, the related assay was performed. From the results, we could find that tachyzoites were treated with 10 μM artemether for 20, 40 and 60 min, the invasion rates were 20.07%, 36.64% and 48.58%, respectively. In the untreated group, the invasion rates were 31.77%, 53.53% and 66.92% ( Figure 5). Although the invasion rate was slightly inhibited by artemether (p < 0.05), the differences were not significant, which indicated that the artemether-inhibited T. gondii proliferation was not related to the invasion. Figure 5. Effect of artemether on the invasion of T. gondii was evaluated by two immunofluorescent dyes. T. gondii invasion rate was evaluated using two immunofluorescent dyes. RH tachyzoites were treated with artemether for 20, 40 and 60 min, respectively. The ratio of the invasion number to the total number of tachyzoites is termed the invasion rate. Three independent experiments were performed and three technical replicates were included in each experiment. * p < 0.05.

Inhibition of Artemether on T. gondii Intracellular Proliferation
The inhibition of artemether on T. gondii intracellular proliferation was evaluated by replication assays on HFF cells. One hundred PV were randomly selected to count the tachyzoite number in each group. As shown in Figure 6 A, we could find that nearly 80% of PVs contained one or two tachyzoites in the 30 μM artemether group, which indicated that the proliferation rate was significantly inhibited by artemether. Compared to the control group, artemether-treated groups showed a significantly reduced proliferation rate in a dose-dependent manner treated by artemether for 48 h (Figure 6 A). Similar results were found when tachyzoites were treated for 24 h (Figure 6 B). Figure 5. Effect of artemether on the invasion of T. gondii was evaluated by two immunofluorescent dyes. T. gondii invasion rate was evaluated using two immunofluorescent dyes. RH tachyzoites were treated with artemether for 20, 40 and 60 min, respectively. The ratio of the invasion number to the total number of tachyzoites is termed the invasion rate. Three independent experiments were performed and three technical replicates were included in each experiment. * p < 0.05.

Inhibition of Artemether on T. gondii Intracellular Proliferation
The inhibition of artemether on T. gondii intracellular proliferation was evaluated by replication assays on HFF cells. One hundred PV were randomly selected to count the tachyzoite number in each group. As shown in Figure 6A, we could find that nearly 80% of PVs contained one or two tachyzoites in the 30 µM artemether group, which indicated that the proliferation rate was significantly inhibited by artemether. Compared to the control group, artemether-treated groups showed a significantly reduced proliferation rate in a dose-dependent manner treated by artemether for 48 h ( Figure 6A). Similar results were found when tachyzoites were treated for 24 h ( Figure 6B).

Artemether Impaired Mitochondrial Membrane Potential of T. gondii
To investigate the potential anti-Toxoplasma mechanism of the artemether, tachyzoites were treated with different concentrations of artemether. From the results, we could find that 61.91% and 31.23% of mitochondrial membranes were damaged after treatment by 12.5 µM and 3.35 µM artemether, respectively ( Figure 7A). According to the mean fluorescence intensity, the mitochondrial membrane potential in the 12.5 µM artemether group was significantly lower than the control group (p < 0.01) ( Figure 7B). This result suggested that artemether may exert antiparasitic effects by disrupting the mitochondrial membrane of T. gondii.

Artemether Impaired Mitochondrial Membrane Potential of T. gondii
To investigate the potential anti-Toxoplasma mechanism of the artemether, tachyzoites were treated with different concentrations of artemether. From the results, we could find that 61.91% and 31.23% of mitochondrial membranes were damaged after treatment by 12.5 μM and 3.35 μM artemether, respectively ( Figure 7A). According to the mean fluorescence intensity, the mitochondrial membrane potential in the 12.5 μM artemether group was significantly lower than the control group (p < 0.01) (Figure 7 B). This result suggested that artemether may exert antiparasitic effects by disrupting the mitochondrial membrane of T. gondii.

Artemether Increased ROS Production of T. gondii
Since the mitochondrial membrane potential of T. gondii was changed, we wanted to further analyze the changes in ROS in extracellular tachyzoites. As shown in Figure 8, we found that artemether could increase ROS activity of extracellular tachyzoites, especially in the 12.5 µM artemether-treated group, ROS activity was significantly higher compared to that in the control group (p < 0.001) (Figure 8). From this result, we speculated that the antiparasitic activity of artemether is associated with the induction of ROS accumulation in Toxoplasma.

Artemether Increased ROS Production of T. gondii
Since the mitochondrial membrane potential of T. gondii was changed, we wanted to further analyze the changes in ROS in extracellular tachyzoites. As shown in Figure 8, we found that artemether could increase ROS activity of extracellular tachyzoites, especially in the 12.5 μM artemether-treated group, ROS activity was significantly higher compared to that in the control group (p < 0.001) (Figure 8). From this result, we speculated that the antiparasitic activity of artemether is associated with the induction of ROS accumulation in Toxoplasma.

Discussion
Toxoplasmosis is one of the most challenging protozoan diseases in public health. In the absence of a safe and effective vaccine or drug to eradicate toxoplasmosis, the disease remains one of the great global challenges [34]. Hence, the development of new therapeutic drugs is urgent in the treatment of toxoplasmosis. Chinese medicine from natural plants is widely used to control different parasites due to their safety and effectiveness. In recent years, many researchers focused on screening anti-Toxoplasma drugs from natural plant extracts, such as plant essential oils and other products [35,36]. Numerous studies The results were analyzed using one-way ANOVA. ** p < 0.01, *** p < 0.001.

Discussion
Toxoplasmosis is one of the most challenging protozoan diseases in public health. In the absence of a safe and effective vaccine or drug to eradicate toxoplasmosis, the disease remains one of the great global challenges [34]. Hence, the development of new therapeutic drugs is urgent in the treatment of toxoplasmosis. Chinese medicine from natural plants is widely used to control different parasites due to their safety and effectiveness. In recent years, many researchers focused on screening anti-Toxoplasma drugs from natural plant extracts, such as plant essential oils and other products [35,36]. Numerous studies have shown that artemisinin and its derivatives not only effectively treat malaria, but also inhibit the proliferation of cancer cells and promote their apoptosis and ferroptosis [37][38][39][40].
In this study, we systematically studied the effect of artemether on T. gondii inhibition in HFF cells and explored its potential inhibition mechanism. We found that the IC 50 of artemether was 9.035 µM (Figure 3), while other studies had different results, such as one study indicating the EC 50 of artemether was 0.286 µM and another showing the IC 50 was14.64 µM [20,41]. This difference might be caused by the drugs coming from different companies, different solvents and host cells. The anti-Toxoplasma effect of artemether was further assessed using the growth assay, and inhibition showed a dose-dependent manner ( Figure 4). It is well known that the lysis cycle of T. gondii includes the processes of adhesion and invasion of host cells, the exponential growth of 2 n in intracellular proliferation, and the escape to find a new host after the disintegration of the host cells. To study the inhibited mechanism, an invasion assay was first carried on. We found that artemether did not significantly inhibit the T. gondii invasion, although the invasion rate was slightly influenced by it. Intracellular proliferation was then analyzed and we happily found that PVs contained fewer tachyzoites in the artemether-treated group, compared to the untreated group, which indicated artemether inhibited the T. gondii intracellular proliferation. Shaw et al. proved that once T. gondii enters the host cell, it will stop moving and proliferate in the way of the daughter cell budding [42]. Parts of the host's mitochondria and endoplasmic reticulum are recruited to form PV, allowing T. gondii to proliferate. The specific molecular mechanism of artemether inhibiting T. gondii intracellular replication is unclear and needs further investigation.
The mitochondrial membrane potential is often detected with a JC-1 fluorescent probe to analyze mitochondrial configuration and function [43]. The ratio of monomers to aggregates is often used to measure the proportion of mitochondrial depolarization. Mitochondria are "little power stations", they are important organelles that provide energy to host cells. The mitochondrial electron transport chain (ETC) is an essential pathway for providing energy and participating in the oxidation-reduction process of T. gondii [44]. Mitochondrial disorders lead to disruptions in metabolism, which in turn release ROS [45]. More evidence indicates that intracellular ROS production is mainly due to changes in mitochondria, in which either NADH or FADH 2 react with O 2 arising from more ROS at complexes I and III of the electron transport chain (ETC) [46]. The production of intracellular ROS leads to disturbances in mitochondrial DNA, lipids and protein synthesis [47].
In this study, we explored the relationship between the mechanism of artemether against T. gondii and mitochondrial membrane potential, and then explored its association with ROS release. Flow analysis revealed that the mitochondria membrane potential of T. gondii was reduced and ROS was increased when treated with artemether. These results suggested that Toxoplasma was inhibited due to oxidative stress treated by artemether.
Cytotoxicity of artemisinin and its derivatives to cancer cells are thought to involve the generation of oxidative stress, following cleavage of the endoperoxide bridge. Heme synthesis can modulate artemisinin cytotoxicity towards cancer cells [48]. Additionally, artemisinin and artesunate were found to exert anti-malarial effects by destroying the basic chemical of an endoperoxide bridge that generates carbon-centered free radicals which increase oxidative stress and arbitrarily modify molecular structure such as lipid, protein and DNA damage via alkylation [49]. Taking the anti-plasmodium principle as a reference, whether artemether acts against T. gondii by breaking the intramolecular peroxide bridges to activate divalent iron or heme iron needs further study.

Conclusions
In conclusion, artemether inhibited T. gondii intracellular proliferation in a dosedependent manner in vitro. The results speculated that the mechanism of artemether against T. gondii was related to the change in mitochondrial membrane and the increase in ROS production, which provided a theoretical basis for optimizing artemether derivatives and further improving their anti-Toxoplasma efficacy, and the results were expected to bring new hope for the treatment of toxoplasmosis.