13 research outputs found
Antiviral Effect of Methylated Flavonol Isorhamnetin against Influenza
<div><p>Influenza is an infectious respiratory disease with frequent seasonal epidemics that causes a high rate of mortality and morbidity in humans, poultry, and animals. Influenza is a serious economic concern due to the costly countermeasures it necessitates. In this study, we compared the antiviral activities of several flavonols and other flavonoids with similar, but distinct, hydroxyl or methyl substitution patterns at the 3, 3′, and 4′ positions of the 15-carbon flavonoid skeleton, and found that the strongest antiviral effect was induced by isorhamnetin. Similar to quercetin and kaempferol, isorhamnetin possesses a hydroxyl group on the C ring, but it has a 3′-methyl group on the B ring that is absent in quercetin and kaempferol. Co-treatment and pre-treatment with isorhamnetin produced a strong antiviral effect against the influenza virus A/PR/08/34(H1N1). However, isorhamnetin showed the most potent antiviral potency when administered after viral exposure (post-treatment method) <i>in vitro</i>. Isorhamnetin treatment reduced virus-induced ROS generation and blocked cytoplasmic lysosome acidification and the lipidation of microtubule associated protein1 light chain 3-B (LC3B). Oral administration of isorhamnetin in mice infected with the influenza A virus significantly decreased lung virus titer by 2 folds, increased the survival rate which ranged from 70–80%, and decreased body weight loss by 25%. In addition, isorhamnetin decreased the virus titer <i>in ovo</i> using embryonated chicken eggs. The structure-activity relationship (SAR) of isorhamnetin could explain its strong anti-influenza virus potency; the methyl group located on the B ring of isorhamnetin may contribute to its strong antiviral potency against influenza virus in comparison with other flavonoids.</p></div
<i>In vivo</i> antiviral activity of isorhamnetin against influenza virus infection.
<p>(A)The rate of loss in body weight of six-week-old female mice(C57BL/6)after influenza A/PR/8/34 (H1N1) virus infection and isorhamnetin treatment, and Tamiflu was used as a positive anti-influenza material; *P < 0.05. (B) The survival rate in six-week-old female mice (C57BL/6) after influenza A/PR/8/34 (H1N1) virus infection and flavonoids treatment. *P < 0.05.</p
Effect of isorhamnetin on the inhibition of the influenza virus-induced ROS generation and ERK phosphorylation.
<p>(A) After influenza virus infection and isorhamnetin treatment, ROS generation was measured using ROS fluorescent probe, H<sub>2</sub>DCFDA. Scale bar = 200 μm. (B) Western blot analysis for phosphorylation of ERK. After influenza A virus infection and isorhamnetin treatment, we checked the phosphorylation level of ERK in a time-dependent manner (6 hr, 9 hr, and 12 hr) after virus infection (post-treatment).</p
Effect of isorhamnetin treatment on the virus-induced autophagy.
<p>(A) Fluorescent microscope data for AVOs staining. MDCK cells were infected with the virus and then treated with isorhamnetin and finally stained with vital stains specific for AVOs (AO and MDC staining). (B) Western blot analysis for detection of LC3B protein lipidation. After infection with influenza A virus and treatment with isorhamnetin, we checked the lipidation of LC3B in a time-dependent manner (6 hr, 9 hr, and 12 hr). (C)RT-PCR data showing the expression level of autophagy genes. After virus infection and isorhamnetin treatment, we checked the expression level of autophagy related genes (Atg-5, Atg-7, and LC3B) using RT-PCR. Loading control = GAPDH.</p
Measurement of antiviral activity of isorhamnetin by NA assay and RT-PCR.
<p>(A) NAI assay was assessed by mixing of 50 μL of the flavonoids at the indicated concentrations with 50 μL of the virus, and then adding 100μL of the substrate solution (4-MU-NANA; (2′-(4-methylumbelliferyl)-α-d-<i>N</i>-acetylneuraminicacid, sodium salt hydrate; Sigma-Aldrich) that dissolved in the enzyme buffer [33mM 2-(<i>N</i>-morpholino) ethanesulfonic acid (MES), pH 6.5, and 4 mM CaCl2].This reaction mixture was protected from light and incubated at 37°C for 2hr under shaking condition. The optical density was measured for calculating the fluorescence intensity of 4-methylumbelliferone using fluorescence spectrophotometer at excitation of 365 nm and emission at 460 nm. *P < 0.05, **P <0.01, ***P <0.001. (B) RT-PCR was carried out to detect the expression level of HA and NA genes. MDCK cells were seeded in a 6-well plate infected with influenza A/PR/8/34 (H1N1) viruses for 2 hr, followed by virus removal and flavonoids treatment in a dose dependent manner (10 μM, 50 μM, and 100 μM) for 48 hr. After incubation, RT-PCR was performed using specific primers for influenza virus HA and NA.</p
Schematic diagram represents the detailed mechanism of isorhamnetin in its antiviral activity against influenza A virus.
<p>Isorhamnetin possesses potent direct or indirect anti-influenza activity via direct suppression of virus adsorption onto host cells (HI) and NA activity (NAI) or indirect inhibition of the expression of influenza A surface proteins (HA and NA), virus-induced ROS generation and ERK phosphorylation, and the autophagic changes (AVOs formation and lipidation of LC3B) after influenza A virus infection.</p
The <i>in vivo</i> anti-influenza virus activity of isorhamnetin.
<p>Anti-influenza virus activity of the isorhamnetin against influenza A/PR/8/34 (H1N1)<i>in vivo</i> by determining the virus titer in embryonated chicken egg and in mice lung tissues that represented in EID<sup>50</sup>(50% embryo infectious dose).</p><p>*P < 0.05,</p><p>**P <0.01.</p><p>The <i>in vivo</i> anti-influenza virus activity of isorhamnetin.</p
The thermo-stability of the antioxidant component of H-PE was assessed.
<p>(A) H-PEs were stored for 60 min at 55°C to 60°C, and their cell viability-rescuing effect was analyzed in H<sub>2</sub>O<sub>2</sub>-treated HaCaT cells. The graph shows the effect of heated H-PE on the cell viability of HaCaT cells. HaCaT cells were exposed to 1 mM H<sub>2</sub>O<sub>2</sub> and cultured with H-PE heated at 55°C to 60°C (n = 3, *p<0.05). (B) HaCaT cells were treated with 1 mM H<sub>2</sub>O<sub>2</sub> and cultured with H-PE heated at 60°C for 0 to 60 min. The cell viability was determined using the MTT assay. The data are presented as the percentage of control (non-treated cells) and the mean ± SD (n = 3, *p<0.05).</p
Male- and female-derived somatic and germ cell-specific toxicity of silver nanoparticles in mouse
<p>Silver nanoparticles (AgNPs) are widely used as an antibiotic agent in textiles, wound dressings, medical devices, and appliances such as refrigerators and washing machines. The increasing use of AgNPs has raised concerns about their potential risks to human health. Therefore, this study was aimed to determine the impact of AgNPs in germ cell specific complications in mice. The administration of AgNPs results in toxicity in mice; however, a more detailed understanding of the effects of AgNPs on germ cells remains poorly understood. Here, we demonstrate the effects of AgNPs (20 nm in diameter) in a mouse Sertoli and granulosa cells <i>in vitro</i>, and in male and female mice <i>in vivo</i>. Soluble silver ion (Ag<sup>+</sup>)-treated cells were used as a positive control. We found that excessive AgNP-treated cells exhibited cytotoxicity, the formation of autophagosomes and autolysosomes in Sertoli cells. Furthermore, an increase in mitochondrial-mediated apoptosis by cytochrome <i>c</i> release from mitochondria due to translocation of Bax to mitochondria was observed. In <i>in vivo</i> studies, the expression of pro-inflammatory cytokines, including tumor necrosis factor α, interferon-γ, −6, −1β, and monocyte chemoattractant protein-1 were significantly increased (<i>p</i> < 0.05). Histopathological analysis of AgNP-treated mice shows that a significant loss of male and female germ cells. Taken together, these data suggest that AgNPs with an average size of 20 nm have negative impact on the reproduction.</p
The proliferation and viability of human keratinocytes were analyzed after treatment with different concentrations of homogenized placental extract (H-PE) or acid-hydrolyzed placental extract (A-PE).
<p>Human keratinocytes (HaCaT) were incubated for 24 hr in the presence of different concentrations of (A) H-PE or A-PE; (B) H-PE or A-PE under 0.5 mM H<sub>2</sub>O<sub>2</sub>; and (C) H-PE or A-PE under 1 mM H<sub>2</sub>O<sub>2</sub>. Cell viability in the presence of H-PE or A-PE under oxidative stress was measured by the MTT assay. The data are presented as the percentage of control (non-treated cells) and the mean ± SD of at least three independent experiments. *p<0.05.</p