关键词: 刚地弓形虫, 穿心莲内酯, 异戊烯基焦磷酸合成酶, 甲基赤藓醇磷酸途径

Abstract:

Objective To investigate the inhibitory effect of andrographolide (AG) on Toxoplasma gondii and unravel its potential therapeutic mechanisms. Methods Human foreskin fibroblasts (HFFs) were treated with 0, 5, 10, 20, 40, 80, 120, 160, or 320 μmol/L AG and 10 μl CCK-8 solutions. The absorbance (A₄₅₀ value) was measured in HFFs following treatment and cell proliferation curves were plotted. Screening for concentrations that were not significantly toxic to cells for subsequent studies. For invasion assays, HFFs were divided into dimethyl sulfoxide (DMSO) group, AG group, and pyrimethamine (PYR) group, which were incubated in SAG1 antibody, green fluorescence-labeled goat anti-rabbit IgG antibody (1:1 000), and red fluorescence-labeled goat anti-rabbit IgG antibody (1:1 000), respectively; and then, T. gondii invasion was observed under an optical microscope, and the invasive rate was calculated. For proliferation assays, HFFs were divided into DMSO and AG groups, treated with green fluorescence-labeled goat anti-mouse IgG antibody (1:1 000), and T. gondii proliferation was observed under an optical microscope. For plaque assays, HFFs were divided into DMSO and AG groups, which were treated with 100 μl DMSO and 40 μmol/L AG, respectively, and the size of plaques were observed under an optical microscope. Interacting proteins were screened using surface plasmon resonance imaging (SPRi), and the interaction of isopentenyl diphosphate isomerase (IspG) gene with AG was validated with drug affinity responsive target stability assays. The IspG protein expression was determined using Western blotting assay, and the relative mRNA levels of interacting protein-coding genes were quantified using real time quantitative reverse transcription PCR (qPCR) assay. All statistical analyses were performed using the software GraphPad Prism 8.0.2. Results The results of proliferation experiments showed that the relative viability of HFF cells in 0, 5, 10, 20, and 40 μmol/L AG was 100.00%, 107.45%, 100.66%, 109.21%, and 90.94%, respectively, and the cell viability was maintained at the same level without obvious toxicity, and the relative viabilities of HFFs were 57.83%, 34.16%, 48.25%, and 30.75% following treatment with AG at doses of 80, 120, 160 and 320 μmol/L, appearing a remarkable toxicity (F = 14.96, P < 0.01). Indirect immunofluorescence assay revealed lower invasion rates in AG [(8.06 ± 2.40) %] and PYR groups [(6.36 ± 1.79) %] than in the DMSO group [(42.49 ± 9.75) %] (F = 35.88, P < 0.01), and the number of T. gondii parasitophorous vacuoles was higher in the DMSO group [(5.78 ± 0.94) parasites/vacuole] than in the AG group [(1.40 ± 0.12) parasites/vacuole] (t = 7.98, P < 0.01). Plaque assays showed a higher plaque size in the AG group (0 μm²) than in the DMSO group [(3 210 ± 1 840) μm²] (t = 19.03, P < 0.01). SPRi identified that the AG-interacting T. gondii proteins with the highest mass spectrometry scores included ribosomal RNA processing protein (2.19), ATP synthase α subunit (4.01), IspG (4.01), and START domain protein (2.12), appearing high molecular relevance. Drug affinity responsive target stability assays showed higher IspG expression in HFFs treated with 10 μmol/L AG (0.25 ± 0.01) than in controls (0.12 ± 0.01) (F = 294.2, P < 0.01). qPCR assay quantified higher relative IspG mRNA expression (4.903 ± 1.546) in HFFs treated with 40 μmol/L AG than in those treated with DMSO (1.19 ± 0.20) (t = 4.123, P < 0.05), and Western blotting determined that the IspG protein expression appeared a tendency towards a concentration-dependent manner, with relative IspG protein expression of 0.57 ± 0.01, 0.52 ± 0.02, 0.24 ± 0.05, and 0.03 ± 0.01 in HFFs treated with 0 (DMSO), 10, 20, and 40 μmol/L AG, respectively (F = 313.4, P < 0.01). Conclusion AG inhibits T. gondii invasion and proliferation through targeting IspG, appearing a remarkable anti-T. gondii activity.

Key words: Toxoplasma gondii, Andrographolide, Isopentenyl diphosphate isomerase, Methylerythritol phosphate pathway