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. 2023 Jul 14;12(14):1852.
doi: 10.3390/cells12141852.

Zingiber officinale-Derived Extracellular Vesicles Attenuate Bleomycin-Induced Pulmonary Fibrosis Trough Antioxidant, Anti-Inflammatory and Protease Activity in a Mouse Model

Alma Aurora Ramírez-Hernández  1 , Edilburga Reyes-Jiménez  1 , Juan Manuel Velázquez-Enríquez  1 , Jovito Cesar Santos-Álvarez  1 , Adriana Soto-Guzmán  2 , Luis Castro-Sánchez  3 , Gabriela Tapia-Pastrana  4 , Honorio Torres-Aguilar  5 , Verónica Rocío Vásquez-Garzón  1   6 , Rafael Baltiérrez-Hoyos  1   6
Affiliations

Zingiber officinale-Derived Extracellular Vesicles Attenuate Bleomycin-Induced Pulmonary Fibrosis Trough Antioxidant, Anti-Inflammatory and Protease Activity in a Mouse Model

Alma Aurora Ramírez-Hernández et al. Cells. .

Abstract

Idiopathic pulmonary fibrosis (IPF) is the most frequent and severe idiopathic interstitial pneumonia. It is a chronic and progressive disease with a poor prognosis and is a major cause of morbidity and mortality. This disease has no cure; therefore, there is a clinical need to search for alternative treatments with greater efficacy. In this study, we aimed to evaluate the effect of extracellular vesicles (EVs) from Zingiber officinale (EVZO) in a murine model of bleomycin (BLM)-induced IPF administered through an osmotic minipump. EVZO had an average size of 373 nm and a spherical morphology, as identified by scanning electron microscopy. Label-free proteomic analysis of EVZOs was performed by liquid chromatography coupled to mass spectrometry, and 20 proteins were identified. In addition, we demonstrated the protease activity of EVZO by gelatin-degrading zymography assay and the superoxide dismutase (SOD) activity of EVZO by an enzymatic assay. In the BLM-induced IPF mouse model, nasal administration of 50 μg of EVZO induced recovery of alveolar space size and decreased cellular infiltrate, collagen deposition, and expression of α-SMA-positive cells. Additionally, EVZO inhibited inflammatory markers such as iNOS and COX-2, lipid peroxidation, and apoptotic cells. These results show that EVZO may represent a novel natural delivery mechanism to treat IPF.

Keywords: SOD; antioxidants; idiopathic pulmonary fibrosis; lung inflammation; planta extracellular vesicles; protease activity.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Scheme for obtaining EVs derived from ginger. Ginger extract was subjected to differential centrifugation (2000× g, 4500× g, and 15,000× g) to remove fibers and cell detritus, and the final supernatant was used to isolate EVs using the exoEasy Maxi kit. AF = aqueous phase; EVZO = extracellular vesicles of Zingiber officinale.
Figure 2
Figure 2
Experimental model. Administration of BLM by osmotic minipumps. Four experimental groups were established, and each group consisted of four CD1 mice, two control groups (saline and saline plus EVZO from day 15), and two BLM groups (BLM 100 U/kg and BLM plus 50 µg EVZO from day 15) every other day. All animals were euthanized on day 28.
Figure 3
Figure 3
Characterization of EVs derived from ginger. (A) The size of EVZO was measured using a Malvern Zetasizer NanoSZ. (B) The morphology of EVZO was identified with SEM. The scale bar indicates 100 nm. Results are the mean of two independent samples. SEM = scanning electron microscope; EVZO = extracellular vesicles Zingiber officinale; nm = nanometers.
Figure 4
Figure 4
GO analysis shows proteins involved in molecular functions and physiological processes. A GO term plot of molecular function (A) and biological processes (B) obtained from UniProt is shown.
Figure 5
Figure 5
Modulation of fibrosis with EVZO treatment. (A) Representative images were taken at 20× magnification of tissue sections were stained with hematoxylin-eosin (H&E), Masson’s trichrome, and Sirius red. (B) Quantification of the number (#) of cells, (C) measurement of the size of alveolar spaces, (D) score of fibrosis, (E) quantification of positive areas in Masson’s trichrome staining, and (F) quantification of positive areas of Sirius Red. Data are expressed as the mean ± SD (n = 4). Data are analyzed by one-way ANOVA followed by Tukey´s t-test for comparison between groups. Quantified with ImageJ 1.53t software and analyzed in GraphPad Prism. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. BLM: bleomycin, CT: control, EVZO: extracellular vesicles from Zingiber officinale.
Figure 6
Figure 6
Zymography on polyacrylamide gel polymerized with 1% gelatin. Fifty micrograms of EV and TE were loaded in duplicate for ginger and papaya, and arrows indicate positive bands with proteolytic activity. EVZO: extracellular vesicles from Zingiber officinale; TEZO: total extract of Zingiber officinale, EVPA: extracellular vesicles from papaya, TEPA: total extract of papaya, kDa: kilodaltons.
Figure 7
Figure 7
EVZO inhibits the expression of the myofibroblast marker α-SMA in cells. (A) Representative images were taken at 20× magnification of α-SMA expression and (B) quantification of positive areas. Data are expressed as the mean ± SD (n = 4). Quantification was performed with ImageJ 1.53t software and analyzed in GraphPad Prism. ** p < 0.01. BLM: bleomycin, CT: control, EVZO: extracellular vesicles from Zingiber officinale.
Figure 8
Figure 8
Modulation of inflammation and lipid peroxidation. (A) Representative images were taken at 20× magnification of iNOS, COX-2, and 4-HNE expression. (B) Quantification of positive areas of iNOS, (C) COX-2 and (D) 4-HNE expression. The results expressed are the mean ± SD (n = 4). Quantification was performed with ImageJ 1.53t software and analyzed in GraphPad Prism. ** p < 0.01, *** p < 0.001. BLM: bleomycin, CT: control, EVZO: extracellular vesicles Zingiber officinale, iNOS: inducible nitric oxide synthase, COX-2: cyclooxygenase-2, 4-HNE: 4-hydroxy-2-nonenal.
Figure 9
Figure 9
EVZO enzyme activity. SOD activity curve in standard and EVZO samples. ST: standard, EVZO: extracellular vesicles from Zingiber officinale.
Figure 10
Figure 10
Expression of caspase 3, an indicator of apoptosis. (A) Representative images were taken at 20× magnification of tissue sections labeled with Caspase 3. (B) Quantification of Caspase 3 positive areas. The results are expressed as the mean ± SD (n = 4). Quantification was performed with ImageJ 1.53t software and analyzed in GraphPad Prism. ** p < 0.01, **** p < 0.0001. BLM: bleomycin, CT: control, EVZO: extracellular vesicles from Zingiber officinale.
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Grants and funding

This research was funded by the Consejo Nacional de Ciencia y Tecnología CONAHCyT, Apoyo al Fortalecimiento y Desarrollo de la Infraestructura Científica y Tecnológica 2016 (No. 270189) to R.B.-H. and a grant from SEP, PFCE-2018 & 2019 to R.B.-H. and V.R.V.-G.