• Parasites, Hosts and Diseases
• /
• v.53 no.4
• /
• pp.385-394
• /
• 2015

Leishmaniasis is a worldwide uncontrolled parasitic disease due to the lack of effective drug and vaccine. To speed up effective drug development, we need powerful methods to rapidly assess drug effectiveness against the intracellular form of Leishmania in high throughput assays. Reporter gene technology has proven to be an excellent tool for drug screening in vitro. The effects of reporter proteins on parasite infectivity should be identified both in vitro and in vivo. In this research, we initially compared the infectivity rate of recombinant Leishmania major expressing stably enhanced green fluorescent protein (EGFP) alone or EGFP-luciferase (EGFP-LUC) with the wild-type strain. Next, we evaluated the sensitivity of these parasites to amphotericin B (AmB) as a standard drug in 2 parasitic phases, promastigote and amastigote. This comparison was made by MTT and nitric oxide (NO) assay and by quantifying the specific signals derived from reporter genes like EGFP intensity and luciferase activity. To study the amastigote form, both B10R and THP-1 macrophage cell lines were infected in the stationary phase and were exposed to AmB at different time points. Our results clearly revealed that the 3 parasite lines had similar in vitro infectivity rates with comparable parasite-induced levels of NO following interferon-${\gamma}$/lipopolysaccharide induction. Based on our results we proposed the more reporter gene, the faster and more sensitive evaluation of the drug efficiency.

• The Korea Journal of Herbology
• /
• v.39 no.4
• /
• pp.37-45
• /
• 2024

Objectives : This study aimed to elucidate antioxidant activity of chrysin in polyinosinic-polycytidylic acid (poly-IC) and lipoteichoic acid-induced RAW 264.7 mouse macrophages. Methods : RAW 264.7 co-stimulated with poly-IC and lipoteichoic acid were incubated with chrysin at concentrations of 25 and 50 µM. Hydrogen peroxide production was measured with dihydrorhodamine 123 assay. Nitric Oxide (NO) production was evaluated by griess reagent assay. Results : For 16 h, 18 h, 20 h, 22 h, and 24 h incubation, chrysin at the concentration of 25 and 50 µM significantly suppressed hydrogen peroxide production in poly-IC and lipoteichoic acid-induced RAW 264.7. In details, production of hydrogen peroxide in 'poly-IC and lipoteichoic acid'-stimulated RAW 264.7 treated for 16 h with chrysin at concentrations of 25 and 50 µM was 83.84% and 79.3% of the control group treated with poly-IC and lipoteichoic acid only, respectively; the production of hydrogen peroxide for 18 h was 84.36% and 79.93%, respectively; production of hydrogen peroxide for 20 h was 85.68% and 80.22%, respectively; production of hydrogen peroxide for 22 h was 85.81% and 79.95%, respectively; production of hydrogen peroxide for 24 h was 86.01% and 80.18%, respectively. Additionally, chrysin at the concentration of 5, 10, 25, and 50 µM significantly inhibited NO production in THP-1 human monocytic cell line. Conclusions : Chrysin might have anti-oxidative activity related to its inhibition of hydrogen peroxide production in 'poly-IC and lipoteichoic acid'-stimulated RAW 264.7.