Empagliflozin mitigates lipopolysaccharide-induced tracheal injury in rats via downregulation of IL-6/JAK/STAT3 signaling pathway and stem cell preservation: histological and molecular study

Figures

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  Fig. 1. Tissue MDA content (nmol/gm) (a), CAT (b), and SOD (c) enzyme activities (nmol/gm) in the trachea of the study groups. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 6 rats/group)

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  Fig. 2. Serum level of IL-6 (a), IL-1β (b), and TNF-α (c) in the study groups. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 12 rats/group)

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  Fig. 3. H&E-stained tracheal sections from the study groups (a control group; b–e LPS group; f LPS + EMPA group). a Part of the tracheal wall shows mucosa (M), submucosa (SM), and hyaline cartilage (c). The mucosa is formed of pseudostratified columnar ciliated epithelium (arrows) with goblet cells (arrowheads) and underlying lamina propria (LP). Blood vessels (BV) are seen in the lamina propria and submucosa. b Disorganized tracheal epithelium with almost complete loss (arrows) in some areas. Most ciliated cells show disarranged or lost cilia (curved arrows). Dilated congested blood vessels (BV) are observed. c shows an area of mucosa lined with a single layer of cuboidal epithelial cells (arrows) with complete loss of cilia. d Part of the mucosa shows epithelial irregularity and hyperplasia (arrows). Some epithelial cells exhibit loss of their cilia (curved arrow). Dilated congested blood vessels (BV) are observed. e Lost epithelial cells (arrows) and heavy inflammatory cell infiltration (asterisks) in the lamina propria and submucosa are observed. f The tracheal mucosa (M) is relatively preserved and showed pseudostratified columnar ciliated epithelium (arrows) with goblet cells…

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  Fig. 4. a–d Masson’s trichrome-stained tracheal sections from the study groups (a control group; b, c LPS group; d LPS + EMPA group). a showing collagenous fibers (arrows) in the lamina propria and submucosa. b Area of denuded epithelium with excess amounts of collagenous fibers (arrows) in the underlying lamina propria and submucosa. c Area of hyperplastic epithelium showing extensive deposition of collagenous fibers (arrows) in the lamina propria, submucosa, and around congested blood vessels (BV). d showing moderate amount of collagenous fibers (arrows) in the lamina propria and submucosa (×400; scale bar = 25 μm). e Percentage area (%) of collagenous fibers in the trachea of study groups. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 6 rats/group)

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  Fig. 5. a–d E-cadherin immunostaining in the trachea of the studied groups (a control group; b, c LPS group; d LPS + EMPA group). a showing strong membranous E-cadherin immunostaining (arrows) in the epithelial cells. b, c showing moderate membranous E-cadherin immunostaining (arrows) in the epithelial lining with areas of focal loss of membrane staining (crossed arrows). d showing strong membranous E-cadherin immunostaining (arrows) in the epithelial cells (anti-E-cadherin ×400, scale bar = 25 μm). e Percentage area (%) of tracheal E-cadherin immunostaining of study groups. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 6 rats/group)

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  Fig. 6. a–d Vimentin immunostaining in the trachea of the study groups (a control group; b, c LPS group; d LPS + EMPA group). a showing mild cytoplasmic vimentin immune reaction in the connective tissue cells of lamina propria and submucosa (crossed arrows). Negative immunostaining is observed in the epithelial cells. b,c showing strong cytoplasmic vimentin immunostaining in the epithelial cells (arrows) and in the connective tissue cells of lamina propria and submucosa (crossed arrows). d showing moderate vimentin immunostaining in the connective tissue cells of lamina propria and submucosa (crossed arrows) with apparently negative immunostaining in the epithelial cells (anti-vimentin ×400; scale bar = 25 μm). e Percentage area (%) of tracheal vimentin immunostaining of study groups. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 6 rats/group)

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  Fig. 7. a–d Bax immunostaining in the trachea of the studied groups (a control group; b, c LPS group; d LPS + EMPA group). a showing apparently negative Bax immunostaining. b, c showing strong cytoplasmic Bax immunostaining in most cells of the epithelium (arrows), lamina propria, and submucosa (crossed arrows) and in submucosal glands (curved arrows). d showing mild cytoplasmic immunostaining in some epithelial (arrows), lamina propria, and submucosal cells (crossed arrows) (anti-Bax, ×400; scale bar = 25 μm). e Percentage area (%) of tracheal Bax immunostaining of study groups. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 6 rats/group)

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  Fig. 8. a–d CK5/6 immunostaining in trachea of the study groups (a control group; b, c LPS group; d LPS + EMPA group). a showing strong positive cytoplasmic CK5/6 immunostaining mainly confined to the basal stem cells (arrows). Weak reaction appears in few suprabasal cells (crossed arrows). b Apparent loss of CK5/6 expression in the basal stem cells (zigzag arrows) is seen. c Positive CK5/6 immunoreactivity is observed in some epithelial areas in both basal (arrows) and suprabasal (crossed arrows) cells. d Restoration of CK5/6 expression predominantly in the basal stem cells (arrows) with limited suprabasal (crossed arrows) reactivity (anti-CK5/6 × 400; scale bar = 25 μm). e Percentage area (%) of tracheal CK5/6 immunostaining of study groups. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 6 rats/group)

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  Fig. 9. Electron micrographs of the tracheal mucosa of control group. a, b The tracheal pseudostratified columnar ciliated epithelium reveals the columnar ciliated cells (C), the more electron-dense goblet cells (G), and basal cells (B). The columnar ciliated cells contain rounded to oval euchromatic nuclei (N) with small patches of peripheral heterochromatin. Regularly arranged cilia (arrows) are present on their apical surface. The goblet cells exhibit basally located heterochromatic nuclei (N), and their apical cytoplasm contains large electron-lucent mucin granules (asterisks). The basal cells appear small, triangular, and show heterochromatic nuclei (N). c The apical region of the columnar ciliated cells shows cilia (arrows) with numerous basal bodies (arrow heads) anchoring the cilia to the cytoplasm. The cytoplasm contains mitochondria (M), rough endoplasmic reticulum (rER). d A ciliated cell shows part of a euchromatic nucleus (N), its cytoplasm contains mitochondria with well-defined cristae (M), rough endoplasmic reticulum (rER), and free ribosomes (R). e The apical region of a goblet cell shows large mucin granules of variable electron density (asterisks). The cytoplasm shows…

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  Fig. 10. Electron micrographs of the tracheal mucosa of the LPS group. a, b The ciliated cells (C) show shrunken, irregular, heterochromatic nuclei (N) surrounded by wide perinuclear space (curved arrow), with loss of the apical cilia (arrows). A ciliated cell with a fragmented nucleus (FN) is also observed. Goblet cells (G) contain irregular nuclei (N), wide perinuclear spaces (curved arrows), and display prominent secretory activity; some of them contain numerous, large mucin granules (asterisks), while others show apparent mucin extrusion (crossed arrows). The basal cell shows nucleus (N) with wide perinuclear space (curved arrows). c Ciliated cells (C) exhibit vacuoles (V), rarefied cytoplasm (stars), containing residual electron-lucent nuclei (N) and mitochondria with lost cristae (M). d Part of a ciliated cell contains an electron-dense nucleus (N); its cytoplasm shows degenerated swollen mitochondria (M) with cristolysis, dilated rough endoplasmic reticulum (rER), and dilated Golgi saccules (Go). e Goblet cells (G) show dilated rough endoplasmic reticulum (rER) cisternae, dilated Golgi saccules (Go), and mucin granules (asterisks) with variable electron density. Their nuclei (N)…

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  Fig. 11. Electron micrographs of the tracheal mucosa of the LPS + EMPA group. a The ciliated cells (C) show oval euchromatic nuclei (N), intact mitochondria (M), and regularly arranged cilia (arrows). The goblet cells (G) contain heterochromatic nuclei (N), and the apical cytoplasm contains some mucin granules (asterisks) with variable electron density. The basal cells (B) contain heterochromatic nuclei (N). b, c (c is a higher magnification of b): The ciliated (C) cells contain mitochondria with well-defined cristae (M), dilated rough endoplasmic reticulum (rER), and few vacuoles (V). Another ciliated cell (C1) shows some degenerative changes; its cytoplasm contains degenerated mitochondria (M), dilated rough endoplasmic reticulum (rER). Wide perinuclear space is also noticed (curved arrow). The goblet cells (G) show preserved structure with heterochromatic nucleus (N) and mucin granules (asterisks). d shows a basal cell with heterochromatic nucleus (N), mitochondria (M), rough endoplasmic reticulum (rER), and free ribosomes (R). (TEM, a ×1500; scale bar = 0.5 µm, b ×2500; scale bar = 2 µm, c ×6000; scale bar = 1 µm, d ×8000; scale bar = 1 µm) (n = 6 rats)

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  Fig. 12. Relative expression of IL-6 (a), JAK2 (b), and STAT3 (c) genes in the different study groups by RT-qPCR. One-way ANOVA test is used. Data are expressed as mean ± SD. P < 0.05 is significant; a indicates significance against control group a, b significance against control group b, c significance against EMPA group, d significance against LPS group. (n = 6 rats/group)