Withaferin-A induced iNOS expression to restore the protective Th-1 response in Miltefosine treated experimental Visceral Leishmaniasis

• Withferin A + Miltefosine treatment significantly reduced parasite burden in mice. • Withferin A significantly induced iNOS expression leads to increase NO production. • Combination therapy heightened IFN-γ/IL-10 ratio by decreasing IL-10 cytokine. • Withaferin A displays a potent natural additive along with Miltefosine treatment. • Combination therapy confirms the safety of the treatment with no harmful effects. Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis. It is caused by Leishmania donovani , a macrophage-specific intracellular parasite that survives due to immunosuppression. The chemotherapeutic treatments available against VL have limitations such as exorbitant cost and the emergence of resistance. Current study reveals the therapeutic potential of Withaferin-A (WA), a natural steroidal lactone, in combined with Miltefosine (Milt) as an alternative approach to treat VL. VL model of L. donovani was established in BALB/c mice and subjected to combination therapy. Parasite load was determined by the serial dilution method and microscopy in spleen and estimation of parasite k-DNA in liver. iNOS expression were determined by Western blot. Nitric oxide and cytokine levels were determined by ELISA and flow cytometry. Biochemical parameters of blood, liver and kidney functions were analysed to determine the drug toxicity. Combination therapy showed a significant reduction in parasite load in the spleen and liver of mice. WA treatment induced iNOS expression, resulting in an increase in NO production. WA treatment significantly reduced IL-10 cytokine and improved IFN-γ/IL-10 ratio, resulting in efficient parasite clearance in visceral organs of mice. Analysis of blood biochemistry revealed that there are no detrimental effects on liver or kidney function upon combining WA treatment along with Milt. WA plus Milt treatment has shown a promising additive effect by correcting the host immune response to clear the intracellular parasite more efficiently than Milt alone. In conclusion, WA along with Milt treatment could be a novel strategy to overcome the emergence of Milt-resistant parasites.