Anti-Toxoplasma gondii efficacy of beta, beta-dimethylacrylshikonin and isobutyrylshikonin in vitro and in vivo

Abstract

Background: Toxoplasma gondii is a widespread parasite that can infect almost all vertebrate species including humans, causing variable clinical symptoms from asymptomatic infection to serious diseases. Though extensive research has been done in recent decades, the prevention and control of T. gondii continue to present substantial challenges. Herbal medicines have long been a rich source of chemical entities and may provide new avenues for drug discovery against T. gondii. Thus, this study was performed to investigate the anti-T. gondii effect of two monomers, beta, beta-dimethylacrylshikonin (DMAS) and isobutyrylshikonin (IBS), extracted from the roots of a widely distributed and used medical plant.

Methods: The cytotoxicity of DMAS and IBS on Vero cells was evaluated using the MTT assay, and the toxicity in mice was assessed on the basis of the changes of body weight combined with the histopathologic examinations on spleen, liver, and kidney. The effects of DMAS and IBS on mice against T. gondii acute infection were evaluated by combining survival curves with splenic histopathologic examination. Ultrastructural change in T. gondii tachyzoites post co-incubation in vitro was observed by electron microscopy. ACT1-quantitative polymerase chain reaction (qPCR) was conducted to quantify T. gondii tachyzoites, including proliferation and the inhibitory efficacy of DMAS and IBS. Invasion and attachment, intracellular proliferation, and parasitophorous vacuole viability evaluations were conducted to assess the effects on the asexual life cycle of T. gondii. In addition, untargeted metabolomics analysis was performed to clarify the underlying mechanisms by which DMAS and IBS act against this parasite.

Results: Both DMAS and IBS, with higher half-maximal cytotoxic concentration (CC₅₀) values, exhibited concentration-dependent cytotoxicity in Vero cells and significantly inhibited the intracellular proliferation of T. gondii in vitro, showing lower half-maximal inhibitory concentration (IC₅₀) values and higher selectivity index (SI) values. DMAS showed a statistically more potent effect than IBS, but both were not significantly more potent than that of pyrimethamine (PM). The tachyzoites exhibited severe ultrastructural damage following treatment with DMAS or IBS. Metabolomics analysis indicated that this abnormal biological lesion was caused by the disruptions in purine and pyrimidine metabolism pathways in T. gondii, with mechanisms likely differing from that of PM. In vivo, a dose of 1.5 mg/kg of DMAS showed no significant toxicity in Kunming (KM) mice, with no significant pathological damage or weight loss. At this dosage, both DMAS and IBS significantly alleviated the splenic hyperemia and statistically prolonged the survival times of T. gondii-infected mice.

Conclusions: This study demonstrated that DMAS and IBS have an inhibitory effect on T. gondii infection in vitro and in vivo, probably associated with the disruption of nucleotide metabolism in the parasite. These results highlight that the two monomers, in particular DMAS, hold promise as a potential therapeutic medicine for toxoplasmosis.

Keywords: Toxoplasma gondii; Anti-infection; Beta, beta-dimethylacrylshikonin; Isobutyrylshikonin; Metabolomics.

Fig. 1

Cytotoxicity and potency analyses for IBS and DMAS compared with PM in vitro. Monomer cytotoxicity was demonstrated using percentages of cell viability compared with the blank control (zero gradient). Host Vero cells were treated with IBS (A), DMAS (B), PM (C), or DMSO (D) to calculate the CC₅₀ values of drugs. The solid red dots indicate the data from three independent experiments and the short black lines indicate the average values. Compared with zero gradient, the statistical difference was marked with * (down) or # (up). One * or # indicates P < 0.05, two indicates P < 0.01, and three indicates P < 0.001. E Monomer potency against T. gondii PRU tachyzoites at the drug concentration of 10 μg/mL. *P < 0.05. Inhibitory curves of IBS (F), DMAS (G), and PM (H) were drawn for calculating their IC₅₀ values against T. gondii infection

Fig. 2

Toxicity and potency assays for IBS and DMAS in comparison with PM in vivo. (A) Body weight of KM mice after drug administration. ns means no significance. (B) Survival curves and (C) survival days of KM mice infected with PRU tachyzoites. *P < 0.05, and ***P < 0.001. (D, E) Splenic histopathologic examination via H&E staining. (D) The monomer toxicity on spleen. (E) Splenic examination post-PRU acute infection and drug administration. Panels on the right are magnified versions of the boxed areas in images on the left

Fig. 3

Impact of IBS and DMAS on the asexual life cycle of Toxoplasma gondii PRU tachyzoites in vitro. Several kinds of curves with correlation index R² were fitted between the number of tachyzoites and OD₄₅₀ values. They are the (A) exponential function, (B) linear function, (C) logarithmic function, (D) power function, (E) quadratic function, (F) trinomial function, and (G) quadrinomial function, respectively. (H) The scatter diagram between the logarithm of the number of tachyzoites and OD₄₅₀ values. (I) Invasion and attachment analysis based on curve fitting. (J) Intracellular proliferation analysis and (K) egress experiments based on PV viability evaluation. The statistical differences are marked using *P < 0.05, **P < 0.01, ***P < 0.001. ns indicates no significance

Fig. 4

Ultrastructural alterations induced by IBS or DMAS compared with PM treatment in T. gondii PRU tachyzoites. Examination by (A) scanning electron microscopy and (B) transmission electron microscopy. Lower images are the magnified versions of the upper images, revealing significant ultrastructural damage on the surface and the intramembrane of T. gondii tachyzoites

Fig. 5

PCA ichnography, OPLS-DA score, volcano plots, heatmaps and two-way Venn diagrams post-monomer treatment in comparison with control (Ctrl). (A) PCA analyses between monomer-treated samples and control group in ESI+ and ESI−, respectively. (B) OPLS-DA score plot analyses between monomer-treated samples and control group. DMAS versus Ctrl (R²X = 0.583, R²Y = 1, and Q²Y = 0.976 in ESI+ ; R²X = 0.669, R²Y = 1, and Q²Y = 0.99 in ESI−), IBS versus Ctrl (R²X = 0.619, R²Y = 0.998, and Q²Y = 0.977 in ESI+ ; R²X = 0.678, R²Y = 1, and Q²Y = 0.988 in ESI−), and x- and y-axes indicate PC1 and PC2, respectively. (C) Volcano plots of all the metabolites marked with color points. x- and y-axes indicate log₂FC and −log₁₀(P-value in Student’s t-test), respectively. The point size indicates VIP values in the OPLS-DA model. The upregulated, downregulated, and unchanged metabolites were respectively colored with red, green, and black. (D) Heatmaps of the differential metabolites. (E) Venn diagrams showing the common and unique differential metabolites between DMAS versus Ctrl and IBS versus Ctrl, respectively. (F) Venn diagrams showing the common and unique different KEGG pathways between DMAS versus Ctrl and IBS versus Ctrl, respectively

Fig. 6

Pathway analysis of differential metabolites comparing DMAS and IBS with control. Differential metabolites involved in purine metabolism (ko00230); pyrimidine metabolism (ko00240); glycine, serine, and threonine metabolism (ko00260); cysteine and methionine metabolism (ko00270); tyrosine metabolism (ko00350); tryptophan metabolism (ko00380); and phenylalanine, tyrosine, and tryptophan biosynthesis (ko00400) are shown based on the KEGG database. SMT, S-methyl-5′-thioadenosine; PRPP, phosphoribosyl pyrophosphate; L-DOPA, levodopa; FAICAR, 5-formamidoimidazole-4-carboxamide ribotide