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21 pages, 1259 KiB  
Article
Synthesis and Bioevaluation of New Stable Derivatives of Chrysin-8-C-Glucoside That Modulate the Antioxidant Keap1/Nrf2/HO-1 Pathway in Human Macrophages
by Javier Ávila-Román, Lirenny Quevedo-Tinoco, Antonio J. Oliveros-Ortiz, Sara García-Gil, Gabriela Rodríguez-García, Virginia Motilva, Mario A. Gómez-Hurtado and Elena Talero
Pharmaceuticals 2024, 17(10), 1388; https://doi.org/10.3390/ph17101388 - 17 Oct 2024
Abstract
Background/Objectives: The beneficial effects of the flavonoid chrysin can be reduced by its poor oral bioavailability. It has been shown that chrysin-8-C-glucoside (1) has a better absorption capability. The aim of this study was to evaluate the antioxidant and anti-inflammatory [...] Read more.
Background/Objectives: The beneficial effects of the flavonoid chrysin can be reduced by its poor oral bioavailability. It has been shown that chrysin-8-C-glucoside (1) has a better absorption capability. The aim of this study was to evaluate the antioxidant and anti-inflammatory activity of this glucoside, as well as the respective hexa-acetate derivative 1a and the hexa-ethyl carbonate derivative 1b since the inclusion of moieties in bioactive molecules may increase or modify their biological effects. Methods: THP-1 macrophages were used to determine the viability in the presence of chrysin derivatives, and non-cytotoxic concentrations were selected. Subsequently, lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) production and inflammatory mediators were examined. The involvement of chrysin derivatives with the Keap1 and Nrf2 antioxidant system was determined by docking and Western blotting studies. Results: Our data demonstrated, for the first time, that pretreatment with the three compounds caused a significant reduction in LPS-induced reactive oxygen species (ROS) production and pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β) levels, as well as in cyclooxygenase 2 (COX-2) expression. The mechanisms underlying these protective effects were related, at least in part, to the competitive molecular interactions of these phenolic compounds with Kelch-like ECH-associated protein 1 (Keap1)–nuclear factor erythroid 2-related factor 2 (Nrf2), which would allow the dissociation of Nrf2 and its translocation into the nucleus and the subsequent up-regulation of hemo-oxygenase 1 (HO-1) expression. Conclusions: Compared to the 8-C-glucoside parent chrysin, compound 1a exhibited the strongest antioxidant and anti-inflammatory activity. We hypothesized that the incorporation of an acetate group (1a) may reduce its polarity and, thus, increase membrane permeability, leading to better pharmacological activity. These findings support the potential use of these phenolic compounds as Nrf2 activators against oxidative-stress-related inflammatory diseases. Full article
(This article belongs to the Section Pharmacology)
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19 pages, 5830 KiB  
Article
Exploration of the Anti-Photoaging Mechanisms of Lactiplantibacillus plantarum TWK10 in a UVB-Induced Mouse Model
by Te-Hua Liu, Wan-Jyun Lin, Meng-Chun Cheng, Yi-Chen Cheng, Chia-Chia Lee, Jin-Seng Lin and Tsung-Yu Tsai
Appl. Sci. 2024, 14(20), 9497; https://doi.org/10.3390/app14209497 - 17 Oct 2024
Abstract
Functional foods have shown promise in mitigating skin aging. This study aimed to evaluate the effects of Lactiplantibacillus plantarum TWK10 (LPTWK10) and its spray-dried supernatant powder on ultraviolet B (UVB)-induced skin photoaging in female BALB/c nude mice. Over a 13-week period of UVB [...] Read more.
Functional foods have shown promise in mitigating skin aging. This study aimed to evaluate the effects of Lactiplantibacillus plantarum TWK10 (LPTWK10) and its spray-dried supernatant powder on ultraviolet B (UVB)-induced skin photoaging in female BALB/c nude mice. Over a 13-week period of UVB exposure and concurrent administration of high doses of LPTWK10 or its spray-dried fermentation supernatant, significant improvements were observed, skin wrinkles were notably reduced, transepidermal water loss rate decreased by 68.94–70.77%, and stratum corneum hydration increased by 76.97–112.24%. Furthermore, LPTWK10 was effective in reducing erythema and inflammation while enhancing skin lightness. Histological assessments revealed substantial reductions in epidermal hyperplasia and collagen degradation. Additionally, LPTWK10 was found to influence critical mechanisms associated with collagen metabolism and proinflammatory cytokine production. In summary, LPTWK10 attenuates photoaging through modulation of collagen metabolism and reduction in inflammatory responses, suggesting its potential as a functional ingredient for delaying photoaging. Full article
(This article belongs to the Section Food Science and Technology)
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Figure 1

Figure 1
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on dorsal wrinkles in UVB-induced nude mice. (<b>A</b>) NC (<b>B</b>) UVB (<b>C</b>) PC (<b>D</b>) LPLD (<b>E</b>) LPMD (<b>F</b>) LPHD (<b>G</b>) SPLD (<b>H</b>) SPMD (<b>I</b>) SPHD. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD, and LPHD: administration of LPTWK10 at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of TWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively. Red arrow: wrinkles.</p>
Full article ">Figure 2
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on (<b>A</b>) dorsal TEWL and (<b>B</b>) dorsal hydration of the stratum corneum in UVB-induced nude mice. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). Values with different uppercase letters were significantly different by Duncan’s multiple range test (<span class="html-italic">p</span> &lt; 0.05). TEWL: transepidermal water loss; UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of TWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively.</p>
Full article ">Figure 3
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on dorsal (<b>A</b>) erythema (<b>B</b>) a* value (<b>C</b>) melanin and (<b>D</b>) lightness in UVB-induced nude mice. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7−8). Values with different uppercase letters were significantly different in the same weeks by Duncan’s multiple range test (<span class="html-italic">p</span> &lt; 0.05). UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of LPTWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively.</p>
Full article ">Figure 4
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on dorsal (<b>A</b>) TNF-α, (<b>B</b>) IL-6, and (<b>C</b>) IL-1β levels in UVB-induced nude mice. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). Values with different uppercase letters were significantly different by Duncan’s multiple range test (<span class="html-italic">p</span> &lt; 0.05). TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; IL-1β: interleukin-1β; UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of LPTWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively.</p>
Full article ">Figure 5
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on dorsal (<b>A</b>) Type I procollagen, (<b>B</b>) Type III procollagen, (<b>C</b>) Type III procollagen/Type I procollagen, (<b>D</b>) MMP-1, (<b>E</b>) MMP-9 and (<b>F</b>) AP-1 levels in UVB-induced nude mice. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). Values with different uppercase letters were significantly different by Duncan’s multiple range test (<span class="html-italic">p</span> &lt; 0.05). UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of LPTWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively.</p>
Full article ">Figure 6
<p>Effects of the administration of LPTWK10 viable cells or LPTWK10 spray-dried supernatant powders on dorsal histopathological findings (<b>A</b>–<b>I</b>) H&amp;E stain, (<b>J</b>) epidermis thickness level and (<b>K</b>–<b>S</b>) MT stain in UVB-induced nude mice. (<b>A</b>,<b>K</b>) NC, (<b>B</b>,<b>L</b>) UVB, (<b>C</b>,<b>M</b>) PC, (<b>D</b>,<b>N</b>) LPLD, (<b>E</b>,<b>O</b>) LPMD, (<b>F</b>,<b>P</b>) LPHD, (<b>G</b>,<b>Q</b>) SPLD, (<b>H</b>,<b>R</b>) SPMD, and (<b>I</b>,<b>S</b>) SPHD. Original magnification, 400×; scale bar = 100 μm. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). H&amp;E: hematoxylin and eosin; UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of LPTWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively. Black arrow: epidermis thickness. Values with different uppercase letters were significantly different by Duncan’s multiple range test (<span class="html-italic">p</span> &lt; 0.05).</p>
Full article ">Figure 7
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on dorsal immunohistochemistry stain (claudin-1) in UVB-induced nude mice. (<b>A</b>) NC, (<b>B</b>) UVB, (<b>C</b>) PC, (<b>D</b>) LPLD, (<b>E</b>) LPMD, (<b>F</b>) LPHD, (<b>G</b>) SPLD, (<b>H</b>) SPMD, and (<b>I</b>) SPHD. Original magnification, 100×; scale bar = 200 μm. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). MT: Masson’s trichrome; UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD, and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of LPTWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively. Black arrow: claudin-1 protein in the epidermis layer.</p>
Full article ">Figure 8
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on dorsal immunohistochemistry stain (laminin) in UVB-induced nude mice. (<b>A</b>) NC, (<b>B</b>) UVB, (<b>C</b>) PC, (<b>D</b>) LPLD, (<b>E</b>) LPMD, (<b>F</b>) LPHD, (<b>G</b>) SPLD, (<b>H</b>) SPMD, and (<b>I</b>) SPHD. Original magnification, 100×; scale bar = 200 μm. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). MT: Masson’s trichrome; UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD, and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup> CFU/kg bw/day, 2 × 10<sup>9</sup> CFU/kg bw/day and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of LPTWK10 spray-dried supernatant powders at 0.30 g/kg bw/day, 0.60 g/kg bw/day, and 1.20 g/kg bw/day, respectively. Black arrow: laminin protein in the basement membrane.</p>
Full article ">Figure 9
<p>Effects of the administration of LPTWK10 or LPTWK10 spray-dried supernatant powders on dorsal immunohistochemistry stain (type IV collagen) in UVB-induced nude mice. (<b>A</b>) NC, (<b>B</b>) UVB, (<b>C</b>) PC, (<b>D</b>) LPLD, (<b>E</b>) LPMD, (<b>F</b>) LPHD, (<b>G</b>) SPLD, (<b>H</b>) SPMD, and (<b>I</b>) SPHD. Original magnification, 100×; scale bar = 200 μm. Data are presented as mean ± SD (<span class="html-italic">n</span> = 7 or 8). MT: Masson’s trichrome; UVB: ultraviolet B; NC: normal control; PC: positive control; LPLD, LPMD and LPHD: administration of LPTWK10 viable cells at 2 × 10<sup>8</sup>, 2 × 10<sup>9</sup> and 2 × 10<sup>10</sup> CFU/kg bw/day, respectively; SPLD, SPMD and SPHD: administration of LPTWK10 spray-dried supernatant powders at 0.30, 0.60 and 1.20 g/kg bw/day, respectively. Black arrow: type IV collagen protein in the basement membrane.</p>
Full article ">
20 pages, 6780 KiB  
Article
AIBP Protects Müller Glial Cells Against Oxidative Stress-Induced Mitochondrial Dysfunction and Reduces Retinal Neuroinflammation
by Seunghwan Choi, Soo-Ho Choi, Tonking Bastola, Keun-Young Kim, Sungsik Park, Robert N. Weinreb, Yury I. Miller and Won-Kyu Ju
Antioxidants 2024, 13(10), 1252; https://doi.org/10.3390/antiox13101252 - 17 Oct 2024
Abstract
Glaucoma, an optic neuropathy with the loss of retinal ganglion cells (RGCs), is a leading cause of irreversible vision loss. Oxidative stress and mitochondrial dysfunction have a significant role in triggering glia-driven neuroinflammation and subsequent glaucomatous RGC degeneration in the context of glaucoma. [...] Read more.
Glaucoma, an optic neuropathy with the loss of retinal ganglion cells (RGCs), is a leading cause of irreversible vision loss. Oxidative stress and mitochondrial dysfunction have a significant role in triggering glia-driven neuroinflammation and subsequent glaucomatous RGC degeneration in the context of glaucoma. It has previously been shown that apolipoprotein A-I binding protein (APOA1BP or AIBP) has an anti-inflammatory function. Moreover, Apoa1bp−/− mice are characterized by retinal neuroinflammation and RGC loss. In this study, we found that AIBP deficiency exacerbated the oxidative stress-induced disruption of mitochondrial dynamics and function in the retina, leading to a further decline in visual function. Mechanistically, AIBP deficiency-induced oxidative stress triggered a reduction in glycogen synthase kinase 3β and dynamin-related protein 1 phosphorylation, optic atrophy type 1 and mitofusin 1 and 2 expression, and oxidative phosphorylation, as well as the activation of mitogen-activated protein kinase (MAPK) in Müller glia dysfunction, leading to cell death and inflammatory responses. In vivo, the administration of recombinant AIBP (rAIBP) effectively protected the structural and functional integrity of retinal mitochondria under oxidative stress conditions and prevented vision loss. In vitro, incubation with rAIBP safeguarded the structural integrity and bioenergetic performance of mitochondria and concurrently suppressed MAPK activation, apoptotic cell death, and inflammatory response in Müller glia. These findings support the possibility that AIBP promotes RGC survival and restores visual function in glaucomatous mice by ameliorating glia-driven mitochondrial dysfunction and neuroinflammation. Full article
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Figure 1

Figure 1
<p>AIBP deficiency exacerbates visual dysfunction induced by oxidative stress. (<b>a</b>) Representative graphs of total recordings of pERG analysis among groups. (<b>b</b>) Quantification analysis of pERG test among groups. <span class="html-italic">N</span> = 8 mice. (<b>c</b>) Quantification analysis of optomotor response among groups. <span class="html-italic">N</span> = 8 mice. (<b>d</b>) Quantification analysis of pVEP tests among groups. <span class="html-italic">N</span> = 8 mice. (<b>e</b>) TLR4 and IL-1β immunohistochemistry in retina. Representative images show TLR4- and IL-1β-positive Müller glial cells in retina. Note that quantification analysis showed significant increase in IL-1β immunoreactive intensity under oxidative stress with AIBP deficiency compared with oxidative stress alone. <span class="html-italic">N</span> = 10 sections from middle area of retina from 3 mice. Images were taken with 20X magnification. Scale bar: 20 μm. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001. pERG, pattern electroretinogram; PQ, paraquat; pVEP, pattern visual evoked potential; WT, wild-type.</p>
Full article ">Figure 2
<p>AIBP deficiency intensifies impairment of retinal mitochondrial dynamics, OXPHOS activity, and mitochondrial biogenesis induced by oxidative stress. (<b>a</b>) Total DRP1, phospho-DRP S616, and phospho-DRP1 S637 expression in retina. <span class="html-italic">N</span> = 3 mice. (<b>b</b>) OPA1, MFN1, and MFN2 expression in retina. <span class="html-italic">N</span> = 3 to 6 retinas from 3 mice. (<b>c</b>) AIBP, PGC-1α, and TFAM expression in retina. <span class="html-italic">N</span> = 3 mice. (<b>d</b>) OXPHOS complex expression in retina. <span class="html-italic">N</span> = 3 retinas from mice. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001. PQ, paraquat; WT, wild-type.</p>
Full article ">Figure 3
<p>Oxidative stress worsens structural and functional impairment of mitochondria in Müller glia cells lacking AIBP. (<b>a</b>) Oligomycin A, FCCP and rotenone were sequentially added at indicated time point. Basal respiration indicates starting basal OCR and value which was set to 100%. Maximum respiration represents ratio between FCCP uncoupled OCR and basal OCR. (<b>b</b>) Quantitative analyses of basal, maximal, and ATP-linked respiration and spare respiratory capacity in rMC-1 cells. <span class="html-italic">N</span> = 8 replicated wells. (<b>c</b>) Quantitative analysis of MMP and mitochondrial ROS. <span class="html-italic">N</span> = 3 independent experiments in rMC-1 cells. (<b>d</b>) AIBP, total DRP1, phospho-DRP S616, and phospho-DRP1 S637 expression in rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>e</b>) Oligomycin A, FCCP and rotenone were sequentially added at indicated time point. Basal respiration indicates starting basal OCR and value which was set to 100%. Maximum respiration represents ratio between FCCP uncoupled OCR and basal OCR. (<b>f</b>) Quantitative analyses of basal, maximal, and ATP-linked respiration and spare respiratory capacity in rMC-1 cells. <span class="html-italic">N</span> = 8 replicated wells. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001. PQ, paraquat; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate.</p>
Full article ">Figure 4
<p>Oxidative stress exacerbates MAPK activation and apoptotic cell death and inflammatory response in Müller glia cells lacking AIBP. (<b>a</b>) p38, phospho-p38 (pp38), ERK1/2, phospho-ERK1/2 (pERK1/2) expression in rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>b</b>) caspase-1, cleaved caspase-1, caspase-3, and cleaved caspase-3 expression in rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>c</b>,<b>d</b>) Representative images show cleaved caspase-3-positive rMC-1 cells in the retina. Note that quantification analysis showed a significant increase in cleaved caspase-3 immunoreactive intensity in rMC-1 cells under oxidative stress with AIBP knockdown compared with control rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>e</b>) Quantitative real-time PCR analysis of <span class="html-italic">Nlrp3</span>, <span class="html-italic">Il-1β</span>, <span class="html-italic">Il-6</span>, and <span class="html-italic">Tnfα</span> mRNA expression in rMC-1 cells. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. PQ, paraquat.</p>
Full article ">Figure 5
<p>Administration of rAIBP prevents visual dysfunction, restores mitochondrial dynamics, and enhances OXPHOS activity in retina. (<b>a</b>) Representative graphs of total recordings of pERG analysis among groups. (<b>b</b>) Quantification analysis of pERG test among groups. <span class="html-italic">N</span> = 6 mice. (<b>c</b>) Quantification analysis of optomotor response among groups. <span class="html-italic">N</span> = 6 mice. (<b>d</b>) Quantification analysis of pVEP tests among groups. <span class="html-italic">N</span> = 6 mice. (<b>e</b>) OPA1, total DRP1, phospho-DRP S616, and phospho-DRP1 S637 expression in retina. <span class="html-italic">N</span> = 3 mice. (<b>f</b>) OXPHOS complex expression in retina. <span class="html-italic">N</span> = 3 mice. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001. BSA, bovine serum albumin; PQ, paraquat; pERG, pattern electroretinogram; pVEP, pattern visual evoked potential.</p>
Full article ">Figure 6
<p>Administration of rAIBP reduced TLR4-associated lipid rafts in Müller glia exposed to oxidative stress. (<b>a</b>) Representative images of TLR4 (green)-LR (red) immunoreactivity (red). Scale bar: 10 μm. (<b>b</b>) Quantitative fluorescent intensity of TLR4-LR immunoreactivity in rMC-1 cells. a. <span class="html-italic">N</span> = 3 independent experiments. Scale bar: 10 μm. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001. LR, lipid raft; PQ, paraquat; BSA, bovine serum albumin.</p>
Full article ">Figure 7
<p>Administration of rAIBP preserves mitochondrial function and dynamics in Müller glia exposed to oxidative stress. (<b>a</b>) Quantitative analysis of MMP and mitochondrial ROS in rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>b</b>) Total DRP1, phospho-DRP S616, and phospho-DRP1 S637 expression in rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>c</b>) Oligomycin A, FCCP and rotenone were sequentially added at indicated time point. Basal respiration indicates starting basal OCR and value which was set to 100%. Maximum respiration represents ratio between FCCP uncoupled OCR and basal OCR. (<b>d</b>) Quantitative analyses of basal, maximal, and ATP-linked respiration and spare respiratory capacity in rMC-1 cells. <span class="html-italic">N</span> = 4 replicated wells. (<b>e</b>) Glucose, oligomycin A and 2DG were sequentially added at indicated time point. (<b>f</b>) Quantitative analyses of glycolysis, glycolytic capacity, and glycolytic reserve in rMC-1 cells. <span class="html-italic">N</span> = 5 replicated wells. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; *** <span class="html-italic">p</span> &lt; 0.001; **** <span class="html-italic">p</span> &lt; 0.0001. BSA, bovine serum albumin; PQ, paraquat; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; OCR, oxygen consumption rate; ECAR, extracellular acidification rate.</p>
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<p>Administration of rAIBP inhibits MAPK activation, apoptotic cell death, and inflammatory response in Müller glia exposed to oxidative stress. (<b>a</b>) p38, phospho-p38 9 (pp38), ERK1/2, phospho-ERK1/2 (pERK1/2) expression in rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>b</b>) caspase-1, cleaved caspase-1, caspase-3, and cleaved caspase-3 expression in rMC-1 cells. <span class="html-italic">N</span> = 3 independent experiments. (<b>c</b>) Representative images show cleaved caspase-3-positive rMC-1 cells. (<b>d</b>) Note that quantification analysis showed significant decrease in cleaved caspase-3 immunoreactive intensity in rMC-1 cells with rAIBP treatment compared with BSA-treated cells under oxidative stress. <span class="html-italic">N</span> = 3 independent experiments. (<b>e</b>) Quantitative real-time PCR analysis of <span class="html-italic">Nlrp3</span>, <span class="html-italic">Il-1β</span>, <span class="html-italic">Il-6</span>, and <span class="html-italic">Tnfα</span> mRNA expression in rMC-1 cells. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. * <span class="html-italic">p</span> &lt; 0.05; ** <span class="html-italic">p</span> &lt; 0.01; **** <span class="html-italic">p</span> &lt; 0.0001. PQ, paraquat; BSA, bovine serum albumin.</p>
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16 pages, 2122 KiB  
Article
Assessing a “Least-Concern” Red List Tree Species from Madagascar Used in Traditional Medicine: Morella spathulata (Myricaceae) Phyto-Compounds and Anti-Inflammatory Properties
by Annachiara Fioccardi, Dario Donno, Zoarilala Rinah Razafindrakoto, Nantenaina Tombozara, Sylvia Henintsoa, Elyna Mahitasoa, Valeria Torti, Marcellin Solofoniaina, Lorenzo Rosso, Giovanni Gamba, Charles Andrianjara, David Ramanitrahasimbola and Gabriele Loris Beccaro
Plants 2024, 13(20), 2899; https://doi.org/10.3390/plants13202899 - 17 Oct 2024
Viewed by 182
Abstract
Morella spathulata (Myricaceae family) is a common plant from Madagascar and is present on the IUCN Red List of threatened species classified at the ’least concern’ level, used by the local population to treat numerous illnesses and pain. Despite its frequent use, comprehensive [...] Read more.
Morella spathulata (Myricaceae family) is a common plant from Madagascar and is present on the IUCN Red List of threatened species classified at the ’least concern’ level, used by the local population to treat numerous illnesses and pain. Despite its frequent use, comprehensive phytochemical and pharmacological research on the species is limited. This study evaluated the antioxidant, analgesic, and anti-inflammatory properties, as well as the toxicity of methanol extracts from the leaves (MS_L) and bark (MS_B) of M. spathulata. The research involved the analysis of nutritional traits such as sugars, organic acids, vitamin C, polyphenolic content (TPC) and the main phytochemicals by HPLC analysis. Antioxidant capacity was assessed through DPPH and FRAP assays. Analgesic and anti-inflammatory activities were evaluated using acetic acid-induced writhing and carrageenan-induced paw oedema tests in mice. The results showed a high content of phenolic and bioactive components in the leaf and bark extracts, associated with antioxidant, analgesic and anti-inflammatory properties. The interaction of key compounds such as ferulic acid and ellagic acid with proteins involved in pH regulation and immune modulation provides clues to the mechanisms underlying the therapeutic effects. However, conservation efforts are crucial due to habitat loss and illegal logging, and further studies are needed to fully explore the plant’s therapeutic potential. Full article
(This article belongs to the Special Issue Phytochemistry and Pharmacological Properties of Medicinal Plants)
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<p>Percentage contribution of phenolic compounds and vitamins in the analyzed samples. MS_L: <span class="html-italic">M. spathulata</span> leaf extract; MS_B: <span class="html-italic">M. spathulata</span> bark extract. Mean values are shown (N = 3).</p>
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17 pages, 774 KiB  
Review
Perceived Pain in People Living with Amyotrophic Lateral Sclerosis—A Scoping Review
by Debora Rosa, Laura Ingrande, Ilaria Marcomini, Andrea Poliani, Giulia Villa, Martina Sodano and Duilio Fiorenzo Manara
Nurs. Rep. 2024, 14(4), 3023-3039; https://doi.org/10.3390/nursrep14040220 - 17 Oct 2024
Viewed by 217
Abstract
(1) Background: Pain is a common symptom in patients with Amyotrophic Lateral Sclerosis (ALS). There are no evidence-based pharmacological treatments for pain in ALS; recommendations are based on guidelines for chronic non-oncological pain and clinical experience. The aim is to map the literature [...] Read more.
(1) Background: Pain is a common symptom in patients with Amyotrophic Lateral Sclerosis (ALS). There are no evidence-based pharmacological treatments for pain in ALS; recommendations are based on guidelines for chronic non-oncological pain and clinical experience. The aim is to map the literature on how people with ALS experience pain, and how this affects their daily activities and social relationships. (2) Methods: This scoping review included studies concerning patients with spinal/bulbar ALS aged ≥ 18 years who experience pain, focusing on perception, characteristics, treatment, and impact on quality of life. Temporal and linguistic criteria were applied when searching the MEDLINE, CINAHL, and SCOPUS databases. (3) Results: The management of pain in these patients is complex and involves the use of anti-inflammatory drugs, analgesics, and opioids. Pain is associated with other conditions such as depression and anxiety, which contribute to a deterioration in the quality of life. Moreover, pain may also negatively influence patient compliance with prescribed treatment regimens and the quality of care they perceive themselves to be receiving. (4) Conclusions: It is of the most importance to identify effective ways to assess and treat this issue, with health care professionals taking an active role in this process. Full article
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<p>PRISMA flow diagram.</p>
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14 pages, 2623 KiB  
Article
Fortification of Orange and Apple Juices with Ferulic Acid: Implications for Food Safety and Quality
by Maribel Abadias, Gloria Bobo, Marina Anguera, Jordi Ortiz-Solà and Ingrid Aguiló-Aguayo
Foods 2024, 13(20), 3288; https://doi.org/10.3390/foods13203288 - 16 Oct 2024
Viewed by 324
Abstract
In recent years, the awareness of healthier lifestyles among consumers has driven to an increased interest in more natural, nutritious, and low-processed foods. Ferulic acid, one of the most abundant phenolic acids in plants, has demonstrated a wide spectrum of antimicrobial activities and [...] Read more.
In recent years, the awareness of healthier lifestyles among consumers has driven to an increased interest in more natural, nutritious, and low-processed foods. Ferulic acid, one of the most abundant phenolic acids in plants, has demonstrated a wide spectrum of antimicrobial activities and a range of biomedical effects, including antioxidant, antiallergic, hepatoprotective, anticarcinogenic, anti-inflammatory, and antithrombotic, among others. The objective of this work was to study the antilisterial effect of ferulic acid (FA, 1500 mg/L) on fresh, ready-to-eat orange (FOJ) and apple (FAJ) juices and evaluate its effect on product quality. The results showed that FA reduced the Listeria monocytogenes population after 9 days of storage at 4 °C, but no effect on the indigenous microbiota was observed. The titratable acidity and color significantly changed. The antioxidant capacity and total phenolic content significantly increased with the addition of FA, being at least two times greater in fortified juices. FAJ and FOJ containing FA were scored lower (6.8 and 5.7 on a 9-point hedonic scale, respectively) than their respective controls. Overall, our results demonstrated that FA treatment could be a useful strategy to maintain the safety of fresh apple and orange juices and increase the antioxidant activity and phenolic content. The potential industrial applications and health benefits of the fortification of fruit juices with FA should be further explored. Full article
(This article belongs to the Section Food Quality and Safety)
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<p><span class="html-italic">L. monocytogenes</span> population in fresh orange juice (<b>A</b>) and fresh apple juice (<b>B</b>) with (FOJ-FA, FAJ-FA) or without (FOJ-CT, FAJ-CT) ferulic acid at 1500 mg/L and stored at 4 °C for 9 days. The values are the means of three samples. The vertical bars indicate the standard error of the mean; if they are not visible, they are smaller than the symbol size. DL: detection limit (0.70 log cfu/mL).</p>
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<p>Populations of total aerobic mesophilic (TAM) or psychrotrophic (TPA) microorganisms and molds and yeasts (M&amp;Y) in (<b>A</b>) fresh orange juice (FOJ) and (<b>B</b>) fresh apple juice without ferulic acid (CT) or with 1500 mg/L ferulic acid (FA) stored at 4 °C. The values are the means of at least three values, and the vertical bars indicate the standard error of the mean. For each storage time and microorganism group, * indicates significant differences due to the presence of FA. For each microorganism group and treatment, different letters indicate significant differences among storage times, according to a Duncan test (<span class="html-italic">p</span> &lt; 0.05).</p>
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<p>Total color difference (ΔE*) between fresh orange juice (FOJ) and fresh apple juice (FAJ) due to the addition of ferulic acid. The samples were stored at 5 °C for 10 days. The vertical bars indicate the standard deviation of the mean.</p>
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<p>Images of apple (<b>A</b>,<b>C</b>) and orange (<b>B</b>,<b>D</b>) juices without FA (<b>A</b>,<b>B</b>) or with FA (<b>C</b>,<b>D</b>).</p>
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<p>Antioxidant capacity (ascorbic acid equivalents, mg/100 mL juice) measured by the DPPH· method in fresh orange juice (FOJ, gray lines) and fresh apple juice (FAJ, black lines) with (dotted lines) or without (solid lines) the addition of ferulic acid (FA). The samples were stored at 5 °C for 10 days. The vertical bars indicate the standard deviation of the mean, and where not visible, they are smaller than the symbol size. Within each fruit juice and evaluation time, different capital letters indicate significant differences according to a <span class="html-italic">t</span>-test. For each treatment, different lower-case letters indicate significant differences according to the storage time.</p>
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<p>Total phenolic content (ferulic acid equivalents, mg/100 mL juice) of fresh orange juice (FOJ, gray lines) and fresh apple juice (FAJ, black lines) with (dotted lines) or without (solid lines) the addition of ferulic acid (FA). The samples were stored at 5 °C for 10 days. The vertical bars indicate the standard deviation of the mean and, where not visible, they are smaller than the symbol size. Within each fruit juice and evaluation time, different capital letters indicate significant differences according to a <span class="html-italic">t</span>-test. For each treatment, different lower-case letters indicate significant differences according to the storage time.</p>
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<p>Global acceptability of fresh apple and orange juices with or without the addition of 1500 mg FA/L. Scores represent the mean value of a 1 to 9 hedonic scale. For each fruit, different letters indicate significant differences according to a <span class="html-italic">t</span>-test (<span class="html-italic">p</span> &lt; 0.05).</p>
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17 pages, 5472 KiB  
Article
The Nephroprotective Effect of Punica granatum Peel Extract on LPS-Induced Acute Kidney Injury
by Sena Sahin Aktura, Kazim Sahin, Levent Tumkaya, Tolga Mercantepe, Atilla Topcu, Esra Pinarbas and Zihni Acar Yazici
Life 2024, 14(10), 1316; https://doi.org/10.3390/life14101316 - 16 Oct 2024
Viewed by 285
Abstract
Sepsis is an exaggerated immune response resulting from systemic inflammation, which can damage tissues and organs. Acute kidney injury has been detected in at least one-third of patients with sepsis. Sepsis-associated acute kidney injury increases the risk of a secondary infection. Rapid diagnosis [...] Read more.
Sepsis is an exaggerated immune response resulting from systemic inflammation, which can damage tissues and organs. Acute kidney injury has been detected in at least one-third of patients with sepsis. Sepsis-associated acute kidney injury increases the risk of a secondary infection. Rapid diagnosis and appropriate initiation of antibiotics can significantly reduce mortality and morbidity. However, microorganisms are known to develop resistance to antibiotics. Estimations indicate that the annual casualties caused by microbial resistance will surpass cancer fatalities by 2050. The prevalence of bacterial infections and their growing antibiotic resistance has brought immediate attention to the search for novel treatments. Plant-derived supplements contain numerous bioactive components with therapeutic potential against a variety of conditions, including infections. Punica granatum peel is rich in phenolic compounds. The purpose of this study was to determine the anti-inflammatory and anti-bacterial properties of P. granatum peel extract (PGPE) on lipopolysaccharide (LPS)-induced acute kidney injury. Experimental groups were Control, LPS (10 mg/kg LPS, intraperitoneally), PGPE100, and PGPE300 (100 and 300 mg/mL PGPE via oral gavage, respectively, for 7 days). According to biochemical results, serum blood urea nitrogen (BUN), creatinine (Cr) and C-reactive protein (CRP), kidney tissue thiobarbituric acid reactive substances (TBARS), and reduced glutathione (GSH) levels significantly decreased in the PGPE groups compared to the LPS group. Histopathological and immunohistochemical findings revealed that toll-like receptor 4 (TLR4) level and nuclear factor kappa B (NF-κB) expression increased in the LPS group compared to the Control group. In addition, the anti-Gram-negative activity showed a dose-dependent effect on Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa with the agar well diffusion method and the minimal inhibitory concentration (MIC). The MIC value was remarkable, especially on A. baumannii. We conclude that PGPE has the potential to generate desirable anti-bacterial and anti-inflammatory effects on LPS-induced acute kidney injury in rats. Full article
(This article belongs to the Special Issue Bioactive Natural Compounds: Therapeutic Insights and Applications)
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<p>Serum blood urea nitrogen; BUN (<b>A</b>), creatinine; Cr (<b>B</b>), and C-reactive protein; CRP (<b>C</b>) Levels.</p>
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<p>Representative light microscopy image of kidney tissue sections. Sections stained with H&amp;E. Distal tubules (d), Proximal tubules (p), Renal corpuscle (RC). The brush border (spiral arrow). (<b>A</b>) (×20), (<b>B</b>) (×40), Sections from the Control group exhibiting normal renal tubules (p, d) and renal corpuscle structure (RC). The brush border (spiral arrow) is particularly evident in the proximal (p) tubules, and the distal (d) tubules are observed to be normally structured. [TNS: 0 (0–0.5)]. (<b>C</b>) (×20), (<b>D</b>) (×40)<b>:</b> Vacuolization in the renal epithelial cells and loss of brush border (arrowhead) structures was observed in the proximal epithelial cells in the LPS group. In addition, debris accumulation is observed in the renal tubules [TNS: 7.5 (5–8)]. (<b>E</b>) (×20), (<b>F</b>) (×40) Sections of PGPE100 treatment group exhibit a decrement in the loss of epithelial cells in the kidney tubules [TNS: 1.5 (0.5–2)]. (<b>G</b>) (×20), (<b>H</b>) (×40) In addition to exhibiting a decrement in the loss of epithelial cells in the kidney tubules, widespread tubule epithelial cells with typical epithelial appearance are observed [TNS: 1 (0.5–1)].</p>
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<p>Representative light microscopy image of the effect of PGPE treatment on inflammatory (TLR4) changes after LPS-induced kidney injury. Typical tubule epithelium (arrow). (<b>A</b>) (×40)<b>,</b> Control group renal tubule epithelium (arrow) was observed to have a normal structure in the renal cortex [TRL4 positivity score: 0 (0–0.5)]. (<b>B</b>) (×40), LPS group sections demonstrated many apoptotic renal epithelial cells (spiral arrow) showed intensive TRL-4 positivity [TRL4 positivity score: 1.5 (1–2)]. (<b>C</b>) (×40)<b>,</b> PGPE100 group sections showed decreased apoptotic cells (spiral arrow) in renal tubule epithelium that showed TRL-4 positivity [TRL4 positivity score: 0.5 (0–0.5)]. (<b>D</b>) (×40)<b>,</b> A decreased TLR4 positive cell in the apoptotic (spiral arrow) renal tubule epithelium and typical epithelial cells (arrow) were observed widely in the PGPE300 group sections [TRL4 positivity score: 0.5 (0–0.5)].</p>
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<p>Representative light microscopy image of the effect of PGPE treatment on inflammatory (NF-κB/p65) changes after LPS-induced kidney injury. Typical tubule epithelium (arrow). (<b>A</b>) (×40), Control group kidney sections exhibit renal tubule epithelium (arrow) with a normal structure in the renal cortex [NF-κB/p65 positivity score: 0 (0–0)]. (<b>B</b>) (×40), LPS group sections demonstrated many apoptotic renal epithelial cells (tailed arrow) showed intensive NF-κB/p65 positivity [NF-κB/p65 positivity score: 3 (2–3)]. (<b>C</b>) (×40), PGPE100 group sections showed decreased NF-κB/p65 positive cells (tailed arrow) in the kidney cortex [NF-κB/p65 positivity score: 0 (0–1)]. (<b>D</b>) (×40), PGPE300 group sections showed decreased immune-positive cells in the renal tubule epithelial cells, and widespread typical epithelial cells (arrow) were observed NF-κB/p65 positivity score: 0 (0–0.5)].</p>
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<p>Inhibition zones of PGPE on <span class="html-italic">A. baumannii</span>, <span class="html-italic">E. coli</span>, and <span class="html-italic">P. aeruginosa</span>, respectively.</p>
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21 pages, 868 KiB  
Review
Citrus Seed Waste and Circular Bioeconomy: Insights on Nutritional Profile, Health Benefits, and Application as Food Ingredient
by S. Seyyedi-Mansour, M. Carpena, P. Donn, P. Barciela, A. Perez-Vazquez, J. Echave, A. G. Pereira and M. A. Prieto
Appl. Sci. 2024, 14(20), 9463; https://doi.org/10.3390/app14209463 - 16 Oct 2024
Viewed by 322
Abstract
Citrus fruits are widely grown, processed, and distributed in more than 140 countries, with annual global production exceeding 124.3 million metric tons. This substantial consumption generates significant organic waste, accounting for approximately 50–60% of the total fruit mass, primarily in the form of [...] Read more.
Citrus fruits are widely grown, processed, and distributed in more than 140 countries, with annual global production exceeding 124.3 million metric tons. This substantial consumption generates significant organic waste, accounting for approximately 50–60% of the total fruit mass, primarily in the form of peel, pulp, and seeds. Often discarded or reused as animal feed, these wastes contribute to significant environmental pollution and economic losses. Therefore, the valorization of these by-products represents an important opportunity to mitigate these challenges and improve the sustainability of the Citrus-related industry. This review highlights Citrus seed waste concerning its invaluable bioactive compounds, including fatty acids, phenolic compounds, limonoids, dietary fibers, vitamins, and carotenoids. Chemical compositions of Citrus seed biowaste differ depending on a variety of factors, such as Citrus variety, fruit maturity, environmental conditions, waste storage conditions, and extraction methods. The extraction and purification of phytochemicals from Citrus seed biowaste are one of the major procedures for valorizing waste. The two types of effective extraction methods are traditional (conventional extraction) and innovative (green extraction). Furthermore, Citrus seeds have been demonstrated to exhibit several biological activities and health-promoting properties including antioxidative, anti-inflammatory, and anti-cancer activities. Therefore, these wastes are safe and beneficial compounds used in the production of functional foods, nutraceuticals, pharmaceuticals, and cosmetics. A conclusion can be reached by emphasizing the abundance of bioactive compounds in Citrus seed wastes, which makes them an excellent opportunity for increased environmental and economic utilization. Full article
(This article belongs to the Special Issue Novel Food Technologies and Applications)
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<p>Examples of <span class="html-italic">Citrus</span> seeds waste applications in food and non-food industry. Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>.</p>
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12 pages, 1620 KiB  
Review
Omega-3 and Sports: Focus on Inflammation
by Roberto Cannataro, Diana Marisol Abrego-Guandique, Natascia Straface and Erika Cione
Life 2024, 14(10), 1315; https://doi.org/10.3390/life14101315 - 16 Oct 2024
Viewed by 250
Abstract
Inflammation is expected in sports, especially when practiced at a high level. The human body is pushed toward its limit, and this is perceived as a “stressogenic agent”. Athletes, especially elite ones, desire it because their bodies can react with super-compensation, i.e., improve [...] Read more.
Inflammation is expected in sports, especially when practiced at a high level. The human body is pushed toward its limit, and this is perceived as a “stressogenic agent”. Athletes, especially elite ones, desire it because their bodies can react with super-compensation, i.e., improve muscle mass, strength, speed, resistance, and, therefore, athletic performance. Thus, the inflammatory stimuli should be there during training but also counteracted to have the body placed in the optimal conditions for reacting with super-compensation. In this sense, omega-3 fatty acids have been shown to have anti-inflammatory biochemical activity. In this review, we will present the biochemical mechanisms of action of omega-3 fatty acids through their mediators, specialized pro-resolving mediators, which have anti-inflammatory activity. A focus will be on studies on omega-3 fatty acid supplementation in sports, and we will provide indications for possible practical applications and future studies, which are undoubtedly necessary to clarify the omega-3 fatty acids used in sports practice. Full article
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<p>Biochemistry of omega-3. Utilization of ALA and synthesis of EPA and DHA. Created with BioRender (<a href="https://www.biorender.com/" target="_blank">https://www.biorender.com/</a>).</p>
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<p>Precursors AA giving the lipoxin A/A (in red), DHA giving D Resolving D1-6 (in blue), Protectins (in light green) and Maresin (in dark green) and EPA giving E Resolving E1-3 (in blue). The fatty acids are converted via biosynthetic enzymes into SPMs, in turn activate their specific receptors to stimulate specific anti-inflammatory actions. Created with BioRender.</p>
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32 pages, 5551 KiB  
Review
Unveiling the Interplay—Vitamin D and ACE-2 Molecular Interactions in Mitigating Complications and Deaths from SARS-CoV-2
by Sunil J. Wimalawansa
Biology 2024, 13(10), 831; https://doi.org/10.3390/biology13100831 - 16 Oct 2024
Viewed by 316
Abstract
The interaction of the SARS-CoV-2 spike protein with membrane-bound angiotensin-converting enzyme-2 (ACE-2) receptors in epithelial cells facilitates viral entry into human cells. Despite this, ACE-2 exerts significant protective effects against coronaviruses by neutralizing viruses in circulation and mitigating inflammation. While SARS-CoV-2 reduces ACE-2 [...] Read more.
The interaction of the SARS-CoV-2 spike protein with membrane-bound angiotensin-converting enzyme-2 (ACE-2) receptors in epithelial cells facilitates viral entry into human cells. Despite this, ACE-2 exerts significant protective effects against coronaviruses by neutralizing viruses in circulation and mitigating inflammation. While SARS-CoV-2 reduces ACE-2 expression, vitamin D increases it, counteracting the virus’s harmful effects. Vitamin D’s beneficial actions are mediated through complex molecular mechanisms involving innate and adaptive immune systems. Meanwhile, vitamin D status [25(OH)D concentration] is inversely correlated with severity, complications, and mortality rates from COVID-19. This study explores mechanisms through which vitamin D inhibits SARS-CoV-2 replication, including the suppression of transcription enzymes, reduced inflammation and oxidative stress, and increased expression of neutralizing antibodies and antimicrobial peptides. Both hypovitaminosis D and SARS-CoV-2 elevate renin levels, the rate-limiting step in the renin-angiotensin-aldosterone system (RAS); it increases ACE-1 but reduces ACE-2 expression. This imbalance leads to elevated levels of the pro-inflammatory, pro-coagulatory, and vasoconstricting peptide angiotensin-II (Ang-II), leading to widespread inflammation. It also causes increased membrane permeability, allowing fluid and viruses to infiltrate soft tissues, lungs, and the vascular system. In contrast, sufficient vitamin D levels suppress renin expression, reducing RAS activity, lowering ACE-1, and increasing ACE-2 levels. ACE-2 cleaves Ang-II to generate Ang(1–7), a vasodilatory, anti-inflammatory, and anti-thrombotic peptide that mitigates oxidative stress and counteracts the harmful effects of SARS-CoV-2. Excess ACE-2 molecules spill into the bloodstream as soluble receptors, neutralizing and facilitating the destruction of the virus. These combined mechanisms reduce viral replication, load, and spread. Hence, vitamin D facilitates rapid recovery and minimizes transmission to others. Overall, vitamin D enhances the immune response and counteracts the pathological effects of SARS-CoV-2. Additionally, data suggests that widely used anti-hypertensive agents—angiotensin receptor blockers and ACE inhibitors—may lessen the adverse impacts of SARS-CoV-2, although they are less potent than vitamin D. Full article
(This article belongs to the Special Issue SARS-CoV-2 and Immunology)
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<p>Infections and immune-related broader functions of vitamin D (calcitriol, 1,25(OH)<sub>2</sub>D). The figure illustrates muti-system-wide functions of vitamin D related through the modulation of innate and adaptive immune systems, resulting in lowering complications from infections and chronic disease burdens [⇧ = increased; ⇩ = reduced; RAS: renin-angiotensin-system; CVS: cardiovascular system] (after Wimalawansa, Nutrients, 2022) [<a href="#B51-biology-13-00831" class="html-bibr">51</a>].</p>
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<p>Pathological and physiological responses of the renin-angiotensin system. Peach and green boxes illustrate the renin-angiotensin system’s regulatory and counter-regulatory physiologic pathways. When excess angiotensin-II (Ang-II) is synthesized, as in the case of hypovitaminosis D and SARS-CoV-2 infection, this leads to the over-activation of the AT1 receptors (AT1-R) with pathological manifestations, as indicated in the peach colored boxes [⇧ = increased; ⇩ = reduced; ARDS = acute respiratory distress syndrome; RAS, renin-angiotensin system; ACE, angiotensin-converting enzyme; ACE-2, angiotensin-converting enzyme 2; Ang 1–7, angiotensin 1–7; Ang-I, angiotensin-I; Ang-II, angiotensin-II; AT1R, type 1 angiotensin-II receptor; MasR, MAS proto-oncogene receptor. PHT, pulmonary hypertension].</p>
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<p>This diagram outlines the status of the renin-angiotensin axis (RAS) axis: (<b>A</b>) physiological status, (<b>B</b>) pathological/activated status in the presence of vitamin D deficiency, and (<b>C</b>) following SARS-CoV-2 infection. RAS axis homeostasis is disrupted by hypovitaminosis D. SARS-CoV-2 or other coronal viral infections markedly activate the RAS, leading to pathologically elevated levels of angiotensin -II and the suppression of ACE-2. This hyperactivation of the RAS leads to increased complications and mortality (⇧ = increased; ⇩ = reduced; ACE: angiotensin-converting enzyme; ARB: angiotensin receptor blockers; AT1R: type 1 angiotensin-II receptor; ARDS: acute respiratory distress syndrome).</p>
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<p>Vit D strengthens innate and adaptive immune systems. This summary outlines the correlation between vitamin D, angiotensin-converting enzyme-2 (ACE-2), angiotensin-converting enzyme inhibitors (ACEi), and angiotensin II receptor blockers (ARBs) concerning severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and their impact on COVID-19 morbidity and mortality ([↑ = increased; ↓ = reduced; RAS: renin-angiotensin-system; CVS: cardiovascular system; ACE: angiotensin-converting enzyme; ARB: angiotensin receptor blockers; AT1R: type 1 angiotensin-II receptor; ARDS: acute respiratory distress syndrome; HTN: hypertension).</p>
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Article
Dual-Function Femtosecond Laser: β-TCP Structuring and AgNP Synthesis via Photoreduction with Azorean Green Tea for Enhanced Osteointegration and Antibacterial Properties
by Marco Oliveira, Liliya Angelova, Liliana Grenho, Maria Helena Fernandes and Albena Daskalova
Materials 2024, 17(20), 5057; https://doi.org/10.3390/ma17205057 - 16 Oct 2024
Viewed by 307
Abstract
β-Tricalcium phosphate (β-TCP) is a well-established biomaterial for bone regeneration, highly regarded for its biocompatibility and osteoconductivity. However, its clinical efficacy is often compromised by susceptibility to bacterial infections. In this study, we address this limitation by integrating femtosecond (fs)-laser processing with the [...] Read more.
β-Tricalcium phosphate (β-TCP) is a well-established biomaterial for bone regeneration, highly regarded for its biocompatibility and osteoconductivity. However, its clinical efficacy is often compromised by susceptibility to bacterial infections. In this study, we address this limitation by integrating femtosecond (fs)-laser processing with the concurrent synthesis of silver nanoparticles (AgNPs) mediated by Azorean green tea leaf extract (GTLE), which is known for its rich antioxidant and anti-inflammatory properties. The fs laser was employed to modify the surface of β-TCP scaffolds by varying scanning velocities, fluences, and patterns. The resulting patterns, formed at lower scanning velocities, display organized nanostructures, along with enhanced roughness and wettability, as characterized by Scanning Electron Microscopy (SEM), optical profilometry, and contact angle measurements. Concurrently, the femtosecond laser facilitated the photoreduction of silver ions in the presence of GTLE, enabling the efficient synthesis of small, spherical AgNPs, as confirmed by UV–vis spectroscopy, Transmission Electron Microscopy (TEM), and Fourier Transform Infrared Spectroscopy (FTIR). The resulting AgNP-embedded β-TCP scaffolds exhibited a significantly improved cell viability and elongation of human bone marrow mesenchymal stem cells (hBM-MSCs), alongside significant antibacterial activity against Staphylococcus aureus (S. aureus). This study underscores the transformative potential of combining femtosecond laser surface modification with GTLE-mediated AgNP synthesis, presenting a novel and effective strategy for enhancing the performance of β-TCP scaffolds in bone-tissue engineering. Full article
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<p>Representative image of four β-TCP samples prepared for fs-laser treatment.</p>
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<p>Schematic diagram illustrating the laser setup utilized for the surface treatment of β-TCP samples and the subsequent synthesis of AgNPs, each carried out as distinct steps.</p>
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<p>SEM micrographs illustrating the morphological changes in β-TCP samples induced by fs-laser treatment at various fluences (6.1 and 4.1 J/cm<sup>2</sup>), scanning velocities (1, 3.44, 5, 10, and 15 mm/s), and patterns (linear and crossed). Subfigures: (<b>A</b>) V = 1 mm/s, F = 4.1 J/cm<sup>2</sup>; (<b>B</b>) V = 3.44 mm/s, F = 4.1 J/cm<sup>2</sup>; (<b>C</b>) V = 5 mm/s, F = 4.1 J/cm<sup>2</sup>; (<b>D</b>) V = 1 mm/s, F = 6.1 J/cm<sup>2</sup>; (<b>E</b>) V = 3.44 mm/s, F = 6.1 J/cm<sup>2</sup>; (<b>F</b>) V = 5 mm/s, F = 6.1 J/cm<sup>2</sup>; (<b>G</b>) V = 10 mm/s, F = 4.1 J/cm<sup>2</sup>; (<b>H</b>) V = 15 mm/s, F = 4.1 J/cm<sup>2</sup>; (<b>I</b>) V = 1 mm/s (Crossed), F = 4.1 J/cm<sup>2</sup>; (<b>J</b>) V = 10 mm/s, F = 6.1 J/cm<sup>2</sup>; (<b>L</b>) V = 15 mm/s, F = 6.1 J/cm<sup>2</sup>. All micrographs were acquired with an acceleration of 20 kV and a magnification of 5000×.</p>
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<p>3D optical profilometry images illustrating the groove depth variations in β-TCP samples induced by fs-laser treatment at various fluences (6.1 and 4.1 J/cm<sup>2</sup>), scanning velocities (1, 3.44, 5, 10, and 15 mm/s), and patterns (linear and crossed), acquired with a magnification of 20×.</p>
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<p>Multivariable bubble plot (<b>A</b>) and biplot of PCA analysis (<b>B</b>) illustrating the effects of fluence, scanning velocity, and patterns on the surface roughness parameter Sa.</p>
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<p>(<b>A</b>) Graph illustrating the variation in contact angle over time for non-laser-treated samples, indicating changes in wettability. (<b>B</b>) Representative image of a water droplet on a non-laser-treated sample, demonstrating its wettability. (<b>C</b>) Comparison image of a water droplet on an fs-laser-treated sample, highlighting the increased wettability of the treated surface.</p>
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<p>UV–vis spectra showing the SPR peaks of synthesized AgNPs at laser fluences of 8.1 J/cm<sup>2</sup> (<b>A</b>) and 16.3 J/cm<sup>2</sup> (<b>B</b>). Graphs depicting the pseudo-first-order kinetics for the reduction of Ag<sup>+</sup> ions at fluences of 8.1 J/cm<sup>2</sup> (<b>C</b>) and 16.3 J/cm<sup>2</sup> (<b>D</b>).</p>
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<p>FTIR spectra showing the characteristic vibrational bands of the GTLE and the synthesized AgNPs.</p>
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<p>TEM micrographs illustrating the morphology of AgNPs synthesized at fluences of 8.1 J/cm<sup>2</sup> (<b>A</b>) and 16.3 J/cm<sup>2</sup> (<b>B</b>); corresponding histograms showing the size distribution of AgNPs synthesized at these fluences, 8.1 J/cm<sup>2</sup> (<b>C</b>) and 16.3 J/cm<sup>2</sup> (<b>D</b>).</p>
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<p>Metabolic activity of hBM-MSCs cultured over the fs-laser-treated β-TCP scaffolds for periods up to 12 days. Results are presented relative to the untreated samples (control, set up at 1.0, dotted line). Statistically different from control: * <span class="html-italic">p</span> &lt; 0.05, ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>SEM images of human hBM-MSCs cultured over the fs-laser-treated β-TCP scaffolds for 12 days. Low (<b>A</b>–<b>C</b>) and high (<b>D</b>–<b>F</b>) magnification images (1000× and 5000×, respectively). Red arrows: examples of mineralized deposits.</p>
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<p>Antibacterial activity of the fs-laser-treated β-TCP scaffolds against sessile (<b>A</b>) and planktonic (<b>B</b>) growth of <span class="html-italic">S. aureus</span>. Results are presented relative to the untreated samples (control, set up at 1.0, dotted line). Statistically different from control: ** <span class="html-italic">p</span> &lt; 0.01 and *** <span class="html-italic">p</span> &lt; 0.001.</p>
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<p>Representative SEM micrographs showing <span class="html-italic">S. aureus</span> cells (yellow) adhered to the non-laser-treated region of the β-TCP surface (<b>A</b>) and to the bottom of the laser-treated region (<b>B</b>).</p>
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20 pages, 526 KiB  
Review
The Role of Resveratrol in Cancer Management: From Monotherapy to Combination Regimens
by Eduarda Ribeiro and Nuno Vale
Targets 2024, 2(4), 307-326; https://doi.org/10.3390/targets2040018 - 16 Oct 2024
Viewed by 288
Abstract
Resveratrol, a naturally occurring polyphenol found in grapes, berries, and peanuts, has garnered significant attention for its potential anti-cancer properties. This review provides a comprehensive analysis of its role in cancer therapy, both as a standalone treatment and in combination with other therapeutic [...] Read more.
Resveratrol, a naturally occurring polyphenol found in grapes, berries, and peanuts, has garnered significant attention for its potential anti-cancer properties. This review provides a comprehensive analysis of its role in cancer therapy, both as a standalone treatment and in combination with other therapeutic approaches. This review explores the molecular mechanisms underlying resveratrol’s anti-cancer effects, including its antioxidant activity, modulation of cellular signaling pathways, antiproliferative properties, anti-inflammatory effects, and epigenetic influences. This review also examines in vitro and in vivo studies that highlight resveratrol’s efficacy against various cancer types. Furthermore, the synergistic effects of resveratrol when used in conjunction with conventional treatments like chemotherapy and radiotherapy, as well as targeted therapies and immunotherapies, are discussed. Despite promising preclinical results, this review addresses the challenges and limitations faced in translating these findings into clinical practice, including issues of bioavailability and toxicity. Finally, it outlines future research directions and the potential for resveratrol to enhance existing cancer treatment regimens. This review aims to provide a thorough understanding of resveratrol’s therapeutic potential and to identify areas for further investigation in the quest for effective cancer treatments. Full article
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<p>Chemical structure of resveratrol (3,5,4′-trihydroxylstilbene).</p>
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16 pages, 1324 KiB  
Article
Association between Serum Fatty Acids Profile and MetScore in Women with Severe Obesity
by Emilly Santos Oliveira, Fabiana Martins Kattah, Glaucia Carielo Lima, Maria Aderuza Horst, Nayra Figueiredo, Gislene Batista Lima, Renata Guimarães Moreira Whitton, Gabriel Inacio de Morais Honorato de Souza, Lila Missae Oyama, Erika Aparecida Silveira and Flávia Campos Corgosinho
Nutrients 2024, 16(20), 3508; https://doi.org/10.3390/nu16203508 - 16 Oct 2024
Viewed by 241
Abstract
Background: Metabolic syndrome (MetS) is a set of conditions associated with an increased cardiovascular risk. Several serum fatty acids (FAs) seem to play an essential role in the development of cardiometabolic diseases and mortality. Thus, it is imperative to explore the impact of [...] Read more.
Background: Metabolic syndrome (MetS) is a set of conditions associated with an increased cardiovascular risk. Several serum fatty acids (FAs) seem to play an essential role in the development of cardiometabolic diseases and mortality. Thus, it is imperative to explore the impact of FAs on MetS parameters, using an early MetS screening tool such as MetScore, which is readily available in clinical practice. Aim: The aim of this study was to assess the potential correlation between serum FAs and cardiovascular risk using a MetScore. Methods: This cross-sectional study involved 41 women with severe obesity. The MetScore was calculated, and participants were categorized into high- and low-cardiovascular-risk groups based on the median MetScore value. Gas chromatography was used to quantify serum FAs. Generalized Linear Models were used to compare group means. The association was assessed through simple logistic regression, and an adjusted logistic regression was conducted to validate the association between Metscore and serum FAs. Results: The high-cardiovascular-risk group exhibited elevated values of HOMA-IR, palmitic, oleic, cis-vaccenic, and monounsaturated fatty acids, as well as the SCD-18C, indicating a heightened cardiovascular risk. Conversely, HDL-c, QUICK, gamma-linolenic, and eicosatetraenoic fatty acids showed lower values compared to the low-risk group. Conclusions: Women with severe obesity and high cardiovascular risk have lower values of some omega-3 and omega-6 FAs, considered cardioprotective and anti-inflammatory, and have higher lipogenic activity and FAs, correlated with high cardiovascular risk. These findings emphasize the need to address lipid metabolism in this population as a therapeutic target to reduce cardiovascular risk. Future research should explore clinical interventions that modulate fatty acid metabolism to mitigate cardiometabolic complications. Full article
(This article belongs to the Section Nutrition in Women)
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<p>Graphical illustration of the study design: stages of participant recruitment and data collection.</p>
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<p>Illustration of linoleic acid bioconversion pathway, eicosanoid synthesis, and cardiovascular risk. Green arrows: anti-inflammatory pathways; Red arrow: inflammatory pathways.</p>
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<p>The mechanism by which PUFA [n3] decreases triacylglycerol biosynthesis.</p>
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21 pages, 1748 KiB  
Review
The Role of Dietary Ingredients and Herbs in the Prevention of Non-Communicable Chronic Liver Disease
by Monika Maćków, Tomasz Dziubyna, Tatiana Jamer, Dmytro Slivinskyi, Tomasz Pytrus, Katarzyna Neubauer, Małgorzata Zwolińska-Wcisło, Andrzej Stawarski, Ewa Piotrowska and Dorian Nowacki
Nutrients 2024, 16(20), 3505; https://doi.org/10.3390/nu16203505 - 16 Oct 2024
Viewed by 307
Abstract
Background: Liver diseases are among the most commonly diagnosed conditions, with the main risk factors being inappropriate lifestyles, including poor diet, excessive alcohol consumption, low physical activity and smoking, including electronic cigarettes. Non-communicable chronic liver diseases also often develop as a result of [...] Read more.
Background: Liver diseases are among the most commonly diagnosed conditions, with the main risk factors being inappropriate lifestyles, including poor diet, excessive alcohol consumption, low physical activity and smoking, including electronic cigarettes. Non-communicable chronic liver diseases also often develop as a result of accompanying overweight and obesity, as well as type 2 diabetes. Methods: The literature on risk factors for non-communicable chronic liver diseases, which show a high strong influence on their occurrence, was analysed. Results: Measures to prevent non-communicable chronic liver disease include the selection of suitable food ingredients that have proven protective effects on the liver. Such ingredients include dietary fibre, probiotics, herbs, various types of polyphenols and fatty acids (omega-3). Conclusions: Because of their liver-protective effects, nutritionists recommend consuming vegetables, fruits, herbs and spices that provide valuable ingredients with anti-inflammatory and anti-cancer effects. These components should be provided with food and, in the case of probiotics, supplementation appears to be important. As a preventive measure, a diet rich in these nutrients is therefore recommended, as well as one that prevents overweight and other diseases that can result in liver disease. Full article
(This article belongs to the Special Issue Nutrition in the Liver Damage)
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<p>Negative and positive factors influencing the development of non-communicable chronic liver diseases.</p>
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<p>Schematic representation of key herbal components in liver disease prevention. Signs: ↑ increasees, ↓ decreases.</p>
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17 pages, 3785 KiB  
Article
Dual Functionality of Papaya Leaf Extracts: Anti-Coronavirus Activity and Anti-Inflammation Mechanism
by Yujia Cao, Kah-Man Lai, Kuo-Chang Fu, Chien-Liang Kuo, Yee-Joo Tan, Liangli (Lucy) Yu and Dejian Huang
Foods 2024, 13(20), 3274; https://doi.org/10.3390/foods13203274 (registering DOI) - 16 Oct 2024
Viewed by 440
Abstract
Papaya leaves have been used as food and traditional herbs for the treatment of cancer, diabetes, asthma, and virus infections, but the active principle has not been understood. We hypothesized that the anti-inflammatory activity could be the predominant underlying principle. To test this, [...] Read more.
Papaya leaves have been used as food and traditional herbs for the treatment of cancer, diabetes, asthma, and virus infections, but the active principle has not been understood. We hypothesized that the anti-inflammatory activity could be the predominant underlying principle. To test this, we extracted papaya leaf juice with different organic solvents and found that the ethyl acetate (EA) fraction showed the most outstanding anti-inflammatory activity by suppressing the production of nitric oxide (NO, IC50 = 24.94 ± 2.4 μg/mL) and the expression of pro-inflammatory enzymes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX-2), and cytokines including interleukins (IL-1β and IL-6), and a tumor necrosis factor (TNF-α) in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Transcriptomic analysis and Western blot results revealed its anti-inflammatory mechanisms were through the MAPK signaling pathway by inhibiting the phosphorylation of ERK1/2, JNKs, and p38 and the prevention of the cell surface expression of TLR4. Furthermore, we discovered that the EA fraction could inhibit the replication of alpha-coronavirus (HCoV-229E) and beta-coronavirus (HCoV-OC43 and SARS-CoV-2) and might be able to prevent cytokine storms caused by the coronavirus infection. From HPLC-QTOF-MS data, we found that the predominant phytochemicals that existed in the EA fraction were quercetin and kaempferol glycosides and carpaine. Counter-intuitively, further fractionation resulted in a loss of activity, suggesting that the synergistic effect of different components in the EA fraction contribute to the overall potent activity. Taken together, our results provide preliminary evidence for papaya leaf as a potential anti-inflammatory and anti-coronavirus agent, warranting further study for its use for human health promotion. Full article
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<p>The inhibitory effects of papaya leaf juice extract (PLJE) on NO production in LPS-induced RAW 264.7 cells (<b>A</b>). Cytotoxicity of PLJE (<b>B</b>). Data points and bar represent arithmetic means ± SD. ns, not significant. * <span class="html-italic">p</span> &lt; 0.05, **** <span class="html-italic">p</span> &lt; 0.0001 compared to DMSO or control group.</p>
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<p>Suppressive effects of fractions extracted from PLJE on NO production in LPS-induced RAW 264.7 cells (<b>A</b>). Cell viability of RAW 264.7 cells treated with five fractions (<b>B</b>). IC<sub>50</sub> of EA fraction on NO production in LPS-induced RAW 264.7 cells (<b>C</b>). Data points and bar represent arithmetic means ± SD. ns, not significant. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 compared to DMSO group, <sup>#</sup> <span class="html-italic">p</span> &lt; 0.05, <sup>##</sup> <span class="html-italic">p</span> &lt; 0.01, and <sup>####</sup> <span class="html-italic">p</span> &lt; 0.0001 compared within group.</p>
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<p>The inhibitory effects of EA fraction on inflammation-related protein expression in LPS-stimulated RAW 264.7 models (<b>A</b>). The expression levels of iNOS (<b>B</b>), COX-2 (<b>C</b>), and TLR4 (<b>D</b>) were determined by Western blot. The mRNA expression levels of IL-1β (<b>E</b>), IL-6 (<b>F</b>), and TNF-α (<b>G</b>) were tested by qRT-PCR. Data points and bar represent arithmetic means ± SD. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 compared to DMSO group.</p>
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<p>GO (<b>A</b>) and KEGG (<b>B</b>) enrichment scatter plots of EA (25 μg/mL) vs. DMSO group. GeneRatio is the ratio of the number of DEGs annotated to the GO or KEGG term to the total number of DEGs. The size of the dot represents the number of genes annotated to the terms.</p>
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<p>Effects of EA fraction on MAPK pathway in LPS-induced RAW 264.7 cell (<b>A</b>). Suppressive effects of EA fraction on the LPS-induced phosphorylation ratio of ERK1/2 (<b>B</b>), JNK (<b>C</b>), and p38 (<b>D</b>). Data points and bar represent arithmetic means ± SD. ** <span class="html-italic">p</span> &lt; 0.01, *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 compared to DMSO group.</p>
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<p>Schematic diagram of potential contribution of papaya leaves in LPS-induced signaling pathways.</p>
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<p>Plaque reduction neutralization tests (PRNT) of EA fraction and nirmatrelvir against infectious HCoVs-OC43 (<b>A</b>), HCoV-229E (<b>B</b>), and SARS-CoV-2 (<b>C</b>). Data points and bar represent arithmetic shown which are the mean ± SD of at least two independent tests performed. ns, not significant. *** <span class="html-italic">p</span> &lt; 0.001, and **** <span class="html-italic">p</span> &lt; 0.0001 compared between indicated groups.</p>
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