The role of low-carbohydrate, high-fat diet in modulating autophagy and endoplasmic reticulum stress in aortic endothelial dysfunction of metabolic syndrome animal model

Figures

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  Figure 1. A Photomicrograph of sections in the aorta stained with H&E. (A). In the control group, the wall of the aorta consists of very thin tunica intima lined by endothelial cells (arrows), tunica media which is formed of wavy elastic fibers (crossed arrows), and smooth muscle fibers (curved arrows) with large oval nuclei and tunica adventitia (AV) which is composed of slightly dense connective tissue. (B). In the DEX-treated group, there are giant multinucleated cells (G), foam cells (F), and plump nuclei (P) of some smooth muscle fibers of the tunica media. (C). In the DEX + LCHF treated group, the aorta is improved apart from round nuclei (arrowheads) of some tunica media smooth muscle fibers. (D). In the DEX + HCLF treated group, some endothelial cells are lost (arrow), and others are shed into the lumen (arrowheads) of the aorta. In the tunica media, there is the disorientation of the smooth muscle cells with the presence of giant multinucleated cells (G), foam cells (F), and apoptotic cells (A) with dark pyknotic nuclei. Inflammatory cells (I) are seen in the tunica adventitia (AV).

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  Figure 2. A photomicrograph of sections of the aorta immunohistochemically stained with Endothelin and PCNA. (A). The control group shows negative (arrowheads) to weak (curved arrow) endothelin expression in the cytoplasm of the endothelial cells and (B). Negative PCNA expression (arrowhead) in the nuclei of the smooth muscle fibers of the tunica media. (C) DEX group showing strong dark brown (arrows) expression of Endothelin (C) and PCNA (D). DEX + LCHF group shows weak (curved arrows) to moderate (crossed arrows) expression of Endothelin (E) and PCNA (F). DEX+ HCLF group showing moderate (crossed arrows) to strong dark brown (arrows) expression of Endothelin (G) and PCNA (H). (I,J) represent the mean area % of Endothelin and PCNA expression. Data were expressed as mean ± SEM. Statistically significant if *p ≤ 0.05: **p < 0.01: ***p < 0.001: ****p < 0.0001.

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  Figure 3. LCHF and HCLF impact on the gene expression of endothelial dysfunction markers (A) Endothelin gene expression; (B) PCNA gene expression; and (C) Nrf-2α gene expression. (D) eNOS gene expression. Y axis of gene expression (Fold expression relative to control). Data were expressed as mean ± SEM. DEX, DEX + LCHF, and DEX + HCLF groups. Statistically significant if *p ≤ 0.05: **p < 0.01: ***p < 0.001: ****p < 0.0001.

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  Figure 4. LCHF and HCLF impact on oxidative stress markers in aortic tissue homogenate. (A) Tissue Malondialdehyde (MDA); (B) total Antioxidant Capacity (TAC); (C) Oxidative stress Index (D) Tissue 8-OH-dG. (E) Tissue Nitric Oxide (NO). (F) Tissue Nrf-2 α/ARE. Data were expressed as mean ± SEM. DEX, DEX + LCHF, and DEX + HCLF groups. Statistically significant if *p ≤ 0.05: **p < 0.01: ***p < 0.001: ****p < 0.0001.

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  Figure 5. LCHF and HCLF diets impact on Autophagy markers. (A) p62 gene expression; (B) LC3 gene expression; and (C) BECN-1 gene expression. Y axis of gene expression (Fold expression relative to control). Data were expressed as mean ± SEM. DEX, DEX + LCHF, and DEX + HCLF groups. Statistically significant if *p ≤ 0.05: **p < 0.01: ***p < 0.001: ****p < 0.0001.

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  Figure 6. LCHF and HCLF diets impact on ER stress markers. (A) PINK1 gene expression; (B) CHOP gene expression; and (C) BNIP3 gene expression. Y axis of gene expression (Fold expression relative to control). Data were expressed as mean ± SEM. DEX, DEX + LCHF, and DEX + HCLF groups. Statistically significant if *p ≤ 0.05: **p < 0.01: ***p < 0.001: ****p < 0.0001.

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  Figure 7. Electron micrographs of sections in the aorta. (A). In the control group, the endothelial cell of the tunica intima appears with an euchromatic nucleus (N), and its cytoplasm contains mitochondria (M), rough endoplasmic reticulum (R), and a few lysosomes (L). The subendothelial connective tissue layer shows collagen fibers (*). The internal elastic lamina (EL) separates the endothelial cell from the tunica media’s smooth muscle (SM) fibers. (B). In the DEX-treated group, the endothelial cell has a shrunken heterochromatic nucleus (n), and its cytoplasm shows numerous lysosomes (L), autophagosome (AP), dilated rough endoplasmic reticulum (R) with fewer attached ribosomes and mitochondria (M). (C). In the DEX + LCHF treated group, the endothelial cell appears nearly similar to the control group with a euchromatic nucleus (N), and its cytoplasm contains mitochondria (M), rough endoplasmic reticulum (R) and few lysosomes (L). (D). In the DEX + HCLF treated group, the endothelial cell has a shrunken nucleus (n), appears partially separated from the subendothelial connective tissue layer (*) and its cytoplasm shows vaccules (V) autophagosome (AP) and dilated rough endoplasmic reticulum…

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  Figure 8. Pearson’s correlations of all measured and calculated data in all studied rats. Blue indicates a strong positive correlation, and white suggests a strong negative correlation.