In vitro antileishmanial activity of fisetin flavonoid via inhibition of glutathione biosynthesis and arginase activity in Leishmania infantum

Abstract

With the increasing emergence of drug resistant Leishmania sp. in recent years, combination therapy has been considered as a useful way to treat and control of Leishmaniasis. The present study was designed to evaluate the antileishmanial effects of the fisetin alone and combination of fisetin plus Meglumine antimoniate (Fi-MA) against Leishmania infantum. The IC50 values for fisetin were obtained 0.283 and 0.102 μM against promastigotes and amastigote forms, respectively. Meglumine antimoniate (MA, Glucantime) as control drug also revealed IC50 values of 0.247 and 0.105 μM for promastigotes and amastigotes of L. infantum, respectively. In order to determine the mode of action of fisetin and Meglumine antimoniate (MA, Glucantime), the activities of arginase (ARG), catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) were measured. Moreover, intracellular glutathione (GSH) and nitric oxide (NO) levels in L. infantum-infected macrophages and L. infantum promastigotes which were treated with IC50 concentrations of fisetin, MA and Fi-MA were investigated. Our results showed that MA decreased CAT and SOD activity and increased NO levels in L. infantum-infected macrophages. In promastigotes, MA inhibited parasite SOD activity and reduced parasite NO production. The decreased levels of most of the antioxidant enzymes, accompanying by the raised level of NO in treated macrophages with MA, were observed to regain their normal profiles due to Fi-MA treatment. Furthermore, fisetin could prevent the growth of promastigotes by inhibition of ARG activity and reduction of GSH levels and NO production. In conclusion, these findings showed that fisetin improves MA side effects.

Keywords: Leishmania infantum; antioxidant enzymes; arginase; fisetin; glutathione; nitric oxide.

Figure 1.

Effects of different concentrations of fisetin and meglumine antimoniate (MA) on the multiplication index (MI) of amastigotes and viability of Leishmania infantum promastigotes in comparison with corresponding control groups.

Figure 2.

Effects of inhibitory concentration (IC50) of fisetin (F), Meglumine antimoniate (MA) and fisetin in combination with MA (Fi-MA) on GSH and No levels in Leishmania infantum-infected macrophages and promastigotes are shown in Figure 2(a) and (b), respectively.

Figure 3.

Effects of inhibitory concentration (IC50) of fisetin, Meglumine antimoniate (MA) and fisetin plus MA on catalase (CAT), glutathione peroxidase (GPx), arginase (ARG) and superoxide dismutase (SOD) activity levels in Leishmania infantum-infected macrophages and ARG and SOD activity levels in promastigotes are shown in Figure 3(a) and (b), respectively.

Figure 4.

The activities of the enzymes arginase (ARG) and inducible nitric oxide synthase (iNOS) are explained in parasite and macrophage. L-Arginine is a precursor for the synthesis of urea and L-ornithine by the ARG enzyme and for the synthesis of nitric oxide (NO) and L-citrulline by the enzyme iNOS. L-ornithine is metabolized by the enzymes ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) to produce polyamines (including putrescine, spermidine and spermine) and L-proline, respectively. Superoxide anions (O₂^–) can combine with nitric oxide (NO) to generate peroxynitrites (ONOO^–) or with water (H₂O) to produce hydrogen peroxide (H₂O₂). The dismutation of superoxide to oxygen (O₂) and H₂O₂ catalyzes by superoxide dismutase (SOD). Both the H₂O₂ from O₂^– disproportionation and the ONOO^– may be reduced at the expense of glutathione (GSH) in reactions that are catalyzed by glutathione peroxidase (GPx) and catalase (CAT). GSH is formed from glutamate (Glu), cysteine (Cys) and glycine (Gly) in two ATP-dependant steps catalyzed by γ-glutamylcysteine synthetase (γ-GCS) and glutathione synthetase (GluS). The generated spermidine by spermidine synthase (SpS) accompanying with two molecules of GSH produce glutathionylspermidine (GSP) as a precursor of trypanothione (T (SH)₂). GSP is then converted into T (SH)₂ by trypanothione synthase (TryS). Dithiol trypanothione (T (S)₂) is reduced to T (SH)₂ by trypanothione reductase (TryR) with the consumption of NADPH. Tryparedoxin-dependent peroxidases (TXNPx) and peroxiredoxin (PRX) are peroxidases in trypanosomatids that obtain their reducing equivalents from NADPH via the T (SH)₂/TXN couple and detoxify ONOO^–, H₂O₂ and radiation-induced radicals produced by host defense mechanisms. CAT-2B and AAP3 are transporters of L-arginine in Macrophage and promastigote membrane, respectively. PV: parasitophorous vacuole.