TNF‑α Inhibition Elicited by Mansoins A and B, Heterotrimeric Flavonoids Isolated from <i>Mansoa hirsuta</i>

Mansoins A (<b>1</b>) and B (<b>2</b>) isolated from the fruits of <i>Mansoa hirsuta</i> represent new glucosylated heterotrimeric flavonoids with a flavanone core linked to two 1,3-diarylpropane C₆–C₃–C₆ units. Their structures and absolute configurations were established by analysis of their NMR and electronic circular dichroism spectroscopic data. Compounds <b>1</b> and <b>2</b> inhibited TNF-α release by LPS-stimulated THP-1 cells with different potencies, with mansoin B (<b>2</b>) being active at lower concentrations than mansoin A (<b>1</b>) (IC₅₀ values 20.0 ± 1.4 and 48.1 ± 1.8 μM, respectively). These results indicate potential anti-inflammatory properties for this structural type of oligoflavonoids, especially for mansoin B (<b>2</b>)

TNF‑α Inhibition Elicited by Mansoins A and B, Heterotrimeric Flavonoids Isolated from <i>Mansoa hirsuta</i>

Mansoins A (<b>1</b>) and B (<b>2</b>) isolated from the fruits of <i>Mansoa hirsuta</i> represent new glucosylated heterotrimeric flavonoids with a flavanone core linked to two 1,3-diarylpropane C₆–C₃–C₆ units. Their structures and absolute configurations were established by analysis of their NMR and electronic circular dichroism spectroscopic data. Compounds <b>1</b> and <b>2</b> inhibited TNF-α release by LPS-stimulated THP-1 cells with different potencies, with mansoin B (<b>2</b>) being active at lower concentrations than mansoin A (<b>1</b>) (IC₅₀ values 20.0 ± 1.4 and 48.1 ± 1.8 μM, respectively). These results indicate potential anti-inflammatory properties for this structural type of oligoflavonoids, especially for mansoin B (<b>2</b>)

Mansoins C–F, Oligomeric Flavonoid Glucosides Isolated from <i>Mansoa hirsuta</i> Fruits with Potential Anti-inflammatory Activity

Continued investigation of the polyphenolic pool of the fruits of <i>Mansoa hirsuta</i> afforded four additional members of the new class of glucosylated oligomeric flavonoids comprising a flavanone core linked to 1,3-diarylpropane C₆–C₃–C₆ units. The structures and absolute configurations of mansoins C–F (<b>3</b>–<b>6</b>) were established by analysis of NMR and electronic circular dichroism data. Mansoin C (<b>3</b>) was identified as a diglucosylated heterodimer, whereas mansoins D (<b>4</b>), E (<b>5</b>), and F (<b>6</b>) were identified as triglucosylated heterotrimers, isomeric with mansoin A (<b>1</b>). Mansoin F (<b>6</b>) inhibited TNF-α release by lipopolysaccharide-stimulated THP-1 cells (IC₅₀ of 19.3 ± 1.3 μM) and, as with mansoin A (<b>1</b>), reduced the phosphorylation levels of p-65-NF-κB, when assayed at 50 μM. These results indicate that the potential anti-inflammatory properties of mansoin F (<b>6</b>) are probably due to inhibition of the NF-κB pathway and inhibition of TNF-α release

Lethality rate, hematological alterations and viral load upon DENV-2 infection.

<p>WT or CCR1^–/–, CCR2^–/– and CCR4^–/– (KO) mice were infected i.p. with 10 LD₅₀ of DENV-2 and then monitored for lethality until day 14. In panel A, percentages of survival (n = 10–12). Hematological analysis were done at day 6 p.i. for changes in platelets count (B) hematocrit (C) and lymphocytes (D) in the blood of non-infected and infected-WT and KO mice. In panel E, viral loads recovered from the liver of WT and KO mice at day 6 p.i. shown as the log of PFU/100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–12 mice). *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01 when compared to WT infected mice. NI: non-infected mice. NS: Not significant.</p

Histological changes in liver upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for tissue samples. Hematoxylin & Eosin stained liver sections from non-infected and DENV-2 infected WT, CCR1^–/–, CCR2^–/– and CCR4^–/– mice, showing different degrees of congestion, hemorrhage, hepatocyte degeneration and necrosis. Each slide presented in the panel is representative of at least 10 different fields (n = 5–6 mice). Magnification: 400X.</p

Liver inflammation and injury upon DENV-2 infection.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for blood and tissue samples. AST (A) and ALT (B) were dosed in serum of WT and KO mice as markers of hepatic injury. MPO activity, as an index of neutrophil accumulation, was evaluated in liver (C). Concentrations of cytokines IL-6 (D) and IFN-γ (E) were evaluated in liver homogenates by ELISA and are expressed as pg per 100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6). * P<0.05, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Cytokine and chemokine production in spleen upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for tissue samples. TNF-α (A), CCL2/JE (B), CCL3/MIP-1α (C), CCL5/RANTES (D) and CCL17/TARC (E) were evaluated in spleen homogenates by ELISA and are expressed as pg per 100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). * P<0.05, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Cytokine production in serum upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6 for blood samples. IL-6 (A) and IFN-γ (B) were evaluated in serum by ELISA and are expressed as pg/ml. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). ** P<0.01, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Chemokine production in liver upon DENV-2 infection in mice.

<p>WT or KO mice were infected i.p. with 10LD₅₀ of DENV-2 and then sacrificed at day 6 for tissue samples. CCL2/JE (A), CCL3/MIP-1α (B) and CCL5/RANTES (C) were evaluated in liver homogenates by ELISA and are expressed as pg per 100 mg of tissue. Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). *P<0.05, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01, ### P<0.001 when compared to WT infected mice. NI: non-infected mice.</p

Lymphocyte number and activation in CC chemokine receptors deficient mice upon DENV-2 infection.

<p>WT or KO mice were infected i.p. with 10 LD₅₀ of DENV-2 and then sacrificed at day 6. Splenic leukocytes were counted and then stained with specific antibodies. Flow cytometry, according to size and granularity, were performed as analysis. The numbers of specific cell populations are shown compared to total number of leukocytes in the spleen. Number of T lymphocytes CD3⁺CD4⁺ (A) and CD3⁺CD8⁺ (C) were evaluated in WT and KO mice. Activated T lymphocytes expressing CD69 were also evaluated for CD4⁺ (B) and CD8⁺ (D) populations. The number of CD3⁺DX5⁺ NKT cells (E) and their activation by CD69 expression as MFI, were also evaluated (F). Results are expressed as mean ± SEM and are representative of at least two experiments (n = 5–6 mice). *P<0.05, ** P<0.01, *** P<0.001 when compared to non-infected mice. # P<0.05, ## P<0.01 when compared to WT infected mice. NI: non-infected mice. MFI: Mean fluorescence intensity.</p