Ameliorative effects of Dictyota dichotoma on hepatotoxicity induced by gibberellic acid in albino rats

Abstract

Gibberellic acid (GA3) is a natural plant growth regulator that is crucial for plant structural and functional development. We examined the alleviating capacity of brown algae (Dictyota dichotoma) on biochemical and molecular degenerative processes caused by sub-chronic exposure to gibberellic acid resulting in hepatic cell apoptosis. Adult male albino rats were divided into five equal groups: the first group received distilled water, the second group was treated with GA3, the third group was administered D. dichotoma extract suspended in 1% carboxymethylcellulose (CMC), the fourth group was administered both GA3 and D. dichotoma simultaneously, and the fifth group received 1% CMC orally, 5 days per week for a total of 50 days. The results indicated that GA3 induced a significant increase in liver function parameters based on serum levels of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin, which indicate hepatotoxicity. A marked increase in malondialdehyde (MDA) levels and a marked decrease in reduced glutathione (GSH), glutathione-S-transferase (GST), and superoxide dismutase (SOD) were observed as a result of induction of lipid peroxidation and oxidative stress. Histopathology revealed severely degenerated hepatocytes including cytoplasmic vacuolations and many apoptotic cells with weak Bcl2 expression. Similarly, there was a significant up-regulation of gene and protein expression levels for the pro-apoptotic markers, Caspase-3 and Bax, and an increase in pro-inflammatory marker levels, tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) as well as C-reactive protein (CRP). The co-administration of D. dichotoma restored the disrupted biochemical, histopathological, molecular, and inflammatory changes resulting from GA3 toxicity. Our results confirm the antioxidant, anti-inflammatory, anti-apoptotic, and hepatoprotective potential of D. dichotoma.

Keywords: Apoptosis; Brown algae; Gibberellic acid; Hepatotoxicity; Plant growth regulators.

Fig. 1

a Western blot analysis of Caspase-3 and Bax expression. Lanes denoted as I, II, III, IV, and V represent the control, D. dichotoma extract, CMC, GA3, and GA3 + D. dichotoma treated groups, respectively. β-actin was used as a loading control. b Mean values of Caspase-3 and Bax protein expression levels, with standard deviation represented by error bars. Groups bearing different superscript letters differed significantly (p < 0.05)

Fig. 2

Effects of orally administered gibberellic acid (GA3) and/or brown algae D. dichotoma extract on Caspase-3 and Bax gene expression in the liver

Fig. 3

Effects of orally administered gibberellic acid (GA3) and/or brown algae D. dichotoma extract on TNF-α and IL-6 levels as measured by ELISA

Fig. 4

Effects of orally administered gibberellic acid (GA3) and/or brown algae D. dichotoma extract on CRP levels as measured by ELISA

Fig. 5

a–f Photomicrographs of liver tissues in all rat groups stained with hematoxylin and eosin (H&E stain). a, b Control group. a normal hepatic architecture, including hepatic lobule with plates of hepatocytes (H) radiating from the central vein (CV). The peripheral located portal area contained a branch of the hepatic artery (A), lymph vessel (V), and bile duct (B). Scale bar = 100 μm. b blood sinusoids (S) separate hepatic plates lined by endothelium (wrinkled arrow) and Von Kupffer cells (curved arrow). Hepatocytes with large polyhedral cells that contained acidophilic cytoplasm and vesicular rounded centrally located nuclei. Some cells were binucleated (thick arrow). Scale bar = 50 μm. c–e GA3-treated group. c severely degenerated hepatocytes with cytoplasmic vacuolations (arrows), dilatation and congestion of CV and blood sinusoids (S). Scale bar = 100 μm. d cytoplasm of hepatocytes occupied by one vacuole (arrows), and the nuclei are peripherally located (arrowhead); other cells show different signs of pyknosis (tailed arrows). Scale bar = 50 μm. e hepatocytes exhibiting ballooned structure with multiple intracytoplasmic vacuoles (arrows) and pyknotic nuclei (arrowhead). Notice congested blood sinusoids (S) and periportal lymphocytic infiltration (L). Scale bar = 50 μm. f GA3 + D. dichotoma-treated group shows hepatocytes with restored typical architecture with acidophilic cytoplasm and vesicular, centrally located nuclei and some cells appeared binucleated (thick arrow), whereas the CV are still congested. Scale bar = 50 μm

Fig. 6

a–i. Photomicrographs of PAS, bromophenol, and Bcl2 immunostained sections of the liver. (A, B, C) Photomicrographs of hepatic tissue in all rat groups stained by PAS (scale bar = 100 μm) for glycogen content. a Control group shows hepatocytes with standard cytoplasmic glycogen content indicated by a strong PAS-positive reaction (arrows). b GA3-treated group shows a marked reduction in the cytoplasmic glycogen content of hepatocytes indicated by a weak PAS reaction. c GA3 + D. dichotoma-treated group shows hepatocytes restored to normal glycogen content as manifested by a strong positive reaction to the PAS. d–f Photomicrographs of liver tissue in all rat groups stained by bromophenol stain (scale bar = 100 μm) for total protein content. d Control group shows normal total protein content of the hepatocytes as indicated by the dense blue coloration. e GA3-treated group shows a marked depletion of total cytoplasmic protein exhibited by a faint blue coloration. f GA3 + D. dichotoma-treated group shows marked improvement of total protein content as the hepatocytes have a restored deep blue coloration. g–i Photomicrographs of Bcl2 immunohistochemical staining (scale bar = 50 μm) of liver tissues from all rat groups. g Control group shows hepatocytes with a normal anti-apoptotic reaction exhibited by a strong positive reaction with brown coloration (arrows). h GA3-treated group shows an increase in hepatic apoptotic cells as indicated by a negative Bcl2 immunoreaction. i GA3 + D. dichotoma-treated group shows restoration of the anti-apoptotic reaction in hepatocytes (arrows) as most were strongly positive for Bcl2