Evaluation of the inhibitory effects of emodin on Neospora caninum invasion and proliferation in vitro.

The impact of neosporosis on the livestock industry has worsening each year. Globally, annual losses were estimated to exceed $2.4 billion, with abortion rates reaching up to 47 % in affected herds. But no effective vaccines or drugs are currently available for prevention and treatment. This study aimed to investigate the inhibitory effects of emodin on Neospora caninum (N. caninum) infection and its potential mechanisms. The appropriates safe concentration of emodin was determined by the Cell Counting Kit-8 (CCK-8) assay. The anti-N. caninum effects of emodin in Vero cells, MDBK cells, macrophages, as well as the combined effects of emodin and niclosamide in Vero cells, were further verified using qPCR or Giemsa staining. Subsequently, the effects of direct incubation of tachyzoites with emodin on N. caninum invasion and proliferation were also examined. The activation of necroptosis by emodin was evaluated by measuring Lactate Dehydrogenase (LDH) levels in the supernatant and assessing cellular pMLKL levels. The CCK-8 results indicated that emodin exhibited low toxicity to Vero cells, MDBK cells, and macrophages. The qPCR results showed that emodin reduced the number of N. caninum in Vero cells, MDBK cells, and macrophages. Giemsa results showed that the number of tachyzoites within the parasitophorous vacuole was decreased accordingly. Notably, emodin directly affected tachyzoites, reducing its invasion capability by 55 %-63 % and its proliferation capacity by 62 %-88 %. Additionally, the combination of emodin with niclosamide significantly enhanced its antiparasitic effects compared with emodin alone. Western blot analysis showed that emodin significantly increased pMLKL levels and enhanced LDH release, assisting N. caninum in activating necroptosis. However, the inhibitory effects of emodin on N. caninum invasion and proliferation were not significantly altered in Mlkl-/- macrophages. In conclusion, emodin inhibited N. caninum infection may through a necroptosis-independent mechanism by directly targeting the parasite, highlighting its potential as a therapeutic candidate for neosporosis.