Kaurenoic Acid Possesses Leishmanicidal Activity by Triggering a NLRP12/IL-1β/cNOS/NO Pathway

Leishmania amazonensis (L. amazonensis) infection can cause severe local and diffuse injuries in humans, a condition clinically known as American cutaneous leishmaniasis (ACL). Currently, the therapeutic approach for ACL is based on Glucantime, which shows high toxicity and poor effectiveness. Therefore, ACL remains a neglected disease with limited options for treatment. Herein, the in vitro antiprotozoal effect and mechanisms of the diterpene kaurenoic acid [ent-kaur-16-en-19-oic acid] (KA) against L. amazonensis were investigated. KA exhibited a direct antileishmanial effect on L. amazonensis promastigotes. Importantly, KA also reduced the intracellular number of amastigote forms and percentage of infected peritoneal macrophages of BALB/c mice. Mechanistically, KA treatment reestablished the production of nitric oxide (NO) in a constitutive NO synthase- (cNOS-) dependent manner, subverting the NO-depleting escape mechanism of L. amazonensis. Furthermore, KA induced increased production of IL-1β and expression of the inflammasome-activating component NLRP12. These findings demonstrate the leishmanicidal capability of KA against L. amazonensis in macrophage culture by triggering a NLRP12/IL-1β/cNOS/NO mechanism

Kaurenoic Acid Possesses Leishmanicidal Activity by Triggering a NLRP12/IL-1 /cNOS/NO Pathway

Leishmania amazonensis (L. amazonensis) infection can cause severe local and diffuse injuries in humans, a condition clinically known as American cutaneous leishmaniasis (ACL). Currently, the therapeutic approach for ACL is based on Glucantime, which shows high toxicity and poor effectiveness. Therefore, ACL remains a neglected disease with limited options for treatment. Herein, the in vitro antiprotozoal effect and mechanisms of the diterpene kaurenoic acid [ent-kaur-16-en-19-oic acid] (KA) against L. amazonensis were investigated. KA exhibited a direct antileishmanial effect on L. amazonensis promastigotes. Importantly, KA also reduced the intracellular number of amastigote forms and percentage of infected peritoneal macrophages of BALB/c mice. Mechanistically, KA treatment reestablished the production of nitric oxide (NO) in a constitutive NO synthase-(cNOS-) dependent manner, subverting the NO-depleting escape mechanism of L. amazonensis. Furthermore, KA induced increased production of IL-1 and expression of the inflammasome-activating component NLRP12. These findings demonstrate the leishmanicidal capability of KA against L. amazonensis in macrophage culture by triggering a NLRP12/IL-1 /cNOS/NO mechanism

Antileishmanial Activity and Inducible Nitric Oxide Synthase Activation by RuNO Complex

Parasites of the genus Leishmania are capable of inhibiting effector functions of macrophages. These parasites have developed the adaptive ability to escape host defenses; for example, they inactivate the NF-κB complex and suppress iNOS expression in infected macrophages, which are responsible for the production of the major antileishmanial substance nitric oxide (NO), favoring then its replication and successful infection. Metal complexes with NO have been studied as potential compounds for the treatment of certain tropical diseases, such as ruthenium compounds, known to be exogenous NO donors. In the present work, the compound cis-[Ru(bpy)2SO3(NO)]PF6, or RuNO, showed leishmanicidal activity directly and indirectly on promastigote forms of Leishmania (Leishmania) amazonensis. In addition, treatment with RuNO increased NO production by reversing the depletion of NO caused by Leishmania. We also found increased expression of Akt, iNOS, and NF-κB in infected and treated macrophages. These results demonstrated that RuNO was able to kill the parasite by NO release and modulate the transcriptional capacity of the cell

Nitric Oxide and Brazilian Propolis Combined Accelerates Tissue Repair by Modulating Cell Migration, Cytokine Production and Collagen Deposition in Experimental Leishmaniasis

<div><p>The fact that drugs currently used in the treatment of <i>Leishmania</i> are highly toxic and associated with acquired resistance has promoted the search for new therapies for treating American tegumentary leishmaniasis (ATL). In this study, BALB/c mice were injected in the hind paw with <i>Leishmania (Leishmania) amazonensis</i> and subsequently treated with a combination of nitric oxide (NO) donor (cis-[Ru(bpy) ₂imN(NO)](PF₆)₃) (Ru-NO), given by intraperitoneal injection, and oral Brazilian propolis for 30 days. Ru-NO reached the center of the lesion and increased the NO level in the injured hind paw without lesion exacerbation. Histological and immunological parameters of chronic inflammation showed that this combined treatment increased the efficacy of macrophages, determined by the decrease in the number of parasitized cells, leading to reduced expression of proinflammatory and tissue damage markers. In addition, these drugs in combination fostered wound healing, enhanced the number of fibroblasts, pro-healing cytokines and induced collagen synthesis at the lesion site. Overall, our findings suggest that the combination of the NO donor Ru-NO and Brazilian propolis alleviates experimental ATL lesions, highlighting a new therapeutic option that can be considered for further <i>in vivo</i> investigations as a candidate for the treatment of cutaneous leishmaniasis.</p></div

Ru-NO plus propolis decreased protein nitration at the lesion site.

<p>Expression of <b>A)</b> iNOS and <b>B)</b> Nitrotyrosine determined by immunohistochemical assays of paw sections of infected mice at 12 weeks post-infection. Infected BALB/c mice were treated with Ru-NO (0.385 μmol.kg-1 day-1,i.p.), propolis (5 mg.kg-1 day-1, p.o.) or Glucantime (33 μmol.kg-1 day-1, i.p.) for 4 weeks post-lesion appearance. The dotted line (—) represents the uninfected control. The result was expressed as the mean ± SEM of five animals per group. Significant difference relative to infected control * P<0.05, unpaired t-test.</p

Effect of Ru-NO and propolis treatment on lesion development and parasite load of BALB/c mice infected with <i>L</i>. <i>amazonensis</i>.

<p>Animals were inoculated in the right hind paw with 1 x 10⁵ promastigote forms. <b>A)</b> After eight weeks of infection, mice were treated with saline (i.p.) (■), Glucantime (33 μmol.kg^-1 day^-1, i.p.) (□), propolis (5 mg.kg^-1 day^-1, p.o.) (▼),Ru-NO (0.385 μmol.kg^-1 day^-1, i.p.) (◆) or Ru-NO plus propolis (0.385μmol.kg^-1 day^-1, i.p. + 5 mg.kg^-1 day^-1, p.o.) (◯) for 30 days, and the lesion was measured once a week. <b>B)</b> At the end of treatment, the number of <i>Leishmania</i> kDNA was determined by real-time quantitative PCR. The results represent the mean ± SEM of lesion size for each group (n = 5). Significant difference relative to the infected control * <i>P</i><0.05 and ** <i>P</i><0.01, unpaired t-test.</p

Ru-NO by intraperitoneal injection is able to reach the center of the lesion.

<p>Animals were inoculated with 1 x 10⁵<i>L</i>. <i>amazonensis</i> promastigote forms in the right hind paw, and after lesion appearance, they were treated intraperitoneally with Ru-NO (0.385 μmol.kg^-1 day^-1). <b>A)</b> EDS analysis of paw sections was performed in infected control and <b>B)</b> RuNO treated group, to identify the elements present at the lesion site.</p

Effect of Ru-NO + propolis on healing process at the lesion site.

<p>The paw sections were analyzed for <b>A)</b> number of fibroblasts (H&E), <b>B)</b> TGF-β1 by Western blotting. <b>C)</b> Photomicrographs of paw sections stained with picrosirius technique <b>D)</b> and the quantification of collagen deposition. The paw sections were analyzed at a final magnification of 200x. The data represent the mean±SEM of five animals per group. The dotted line (—) represents the uninfected control. Significant difference relative to infected control ** <i>P</i><0.01, ***<i>P</i><0.0001, unpaired t-test. Scale bars = 50 μm.</p

Effect of Ru-NO plus propolis on inflammatory process at lesion site.

<p>BALB/c mice were infected with 1x10⁵ promastigotes of <i>L</i>. <i>amazonensis</i> in the right hind paw and treated with Ru-NO (0.385 μmol.kg^-1 day^-1,i.p.), propolis (5 mg.kg^-1 day^-1, p.o.) or Glucantime (33 μmol.kg^-1 day^-1, i.p.) for 4 weeks post-lesion appearance. The paw sections were analyzed for number of <b>A)</b> macrophages (H&E), <b>B)</b> lymphocytes (H&E), <b>C)</b> CD4⁺ T (IHC), and <b>D)</b> CD8⁺ T (IHC) at a final magnification of 200x. The paw supernatants were assayed for expression of <b>E)</b> NF-κB(p65), <b>F)</b> TNF-α, <b>G)</b> IL-10 and <b>H)</b> STAT3 by Western blotting. The dotted line (—) represents the uninfected control. Bars represent the mean ± SEM (n = 5). Significant difference relative to the infected control * <i>P</i><0.05, ** <i>P</i><0.01 and ***<i>P</i> < 0.0001, unpaired t-test.</p