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. 2024 Sep 27;25(19):10456.
doi: 10.3390/ijms251910456.

Effects of Alkalinity Stress on Amino Acid Metabolism Profiles and Oxidative-Stress-Mediated Apoptosis/Ferroptosis in Hybrid Sturgeon (Huso dauricus ♀ × Acipenser schrenckii ♂) Livers

Cunhua Zhai  1   2 , Xiafei Liu  1   3 , Yutao Li  1 , Ruoyu Wang  1 , Weihua Lv  1 , Bo Ma  1   2 , Dingchen Cao  1 , Ying Zhang  1   3
Affiliations

Effects of Alkalinity Stress on Amino Acid Metabolism Profiles and Oxidative-Stress-Mediated Apoptosis/Ferroptosis in Hybrid Sturgeon (Huso dauricus ♀ × Acipenser schrenckii ♂) Livers

Cunhua Zhai et al. Int J Mol Sci. .

Abstract

Alkaline water is toxic to cultured aquatic animals that frequently live in pH-neutral freshwater. Overfishing and habitat destruction have contributed to the decline in the wild sturgeon population; consequently, the domestic hybrid sturgeon has become an increasingly important commercial species in China. Hybrid sturgeons are widely cultured in alkaline water, but little is known about the effects of alkalinity stress on hybrid sturgeon liver tissues. We exposed hybrid sturgeons to four alkaline concentrations (3.14 ± 0.02 mmol/L, 7.57 ± 0.08 mmol/L, 11.78 ± 0.24 mmol/L and 15.46 ± 0.48 mmol/L). Histopathology, biochemical index assessment, gene expression level detection and metabolomics analysis were used to investigate the negative effects on liver functions following exposure to NaHCO3. Livers exposed to alkaline stress exhibited severe tissue injury and clear apoptotic characteristics. With increased exposure concentrations, the hepatic superoxide dismutase, catalase, glutathione peroxidase and alkaline phosphatase activities significantly decreased in a dose-dependent manner. NaHCO3 exposure up-regulated the transcriptional levels of apoptosis/ferroptosis-related genes in livers. Similarly, the expression trends of interleukin-1β and heat shock protein genes also increased in high-alkalinity environments. However, the expression levels of complement protein 3 significantly decreased (p < 0.05). Hepatic untargeted metabolomics revealed the alteration conditions of various metabolites associated with the antioxidant response, the ferroptosis process and amino acid metabolism (such as beta-alanine metabolism; alanine, aspartate and glutamate metabolism; and glycine, serine and threonine metabolism). These data provided evidence that NaHCO3 impaired immune functions and the integrity of hybrid sturgeon liver tissues by mediating oxidative-stress-mediated apoptosis and ferroptosis. Our results shed light on the breeding welfare of domestic hybrid sturgeons and promote the economic development of fisheries in China.

Keywords: alkalinity stress; amino acid metabolism; ferroptosis; fishery breeding; sturgeon.

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Conflict of interest statement

None of the authors have any financial or personal relationships that could inappropriately influence or bias the content of the paper.

Figures

Figure 1
Figure 1
Histological observations (H&E staining, 200×) of sturgeon liver tissues exposed to different concentrations of NaHCO3 at 148 h: (a–d) liver tissues of C, T1, T2 and T3 groups, respectively. Note: HS, hepatic sinusoid; MV, muscular vein; HE, hepatocyte; BD, bile duct; hepatocytes hypertrophy, black arrows; HPV, hepatocellular vacuolation; PN, cellular peripheral nucleus; K, karyolysis; Pyknosis, red arrows.
Figure 2
Figure 2
Apoptosis rates of liver cells under different alkalinity concentrations were determined using the TdT-mediated dUTP Nick-End Labeling (TUNEL) method (×200). Green fluorescence represents apoptotic cells while blue represents the nucleus. Different lowercase letters above the bars represent significant differences (p < 0.05) between group C, group T1, group T2 and group T3 (mean ± SD, n = 3).
Figure 3
Figure 3
Effects of alkalinity stress on antioxidant parameters in the liver at high-alkaline exposure concentrations: (a–d) the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) and alkaline phosphatase (AKP) in sturgeon liver tissues, respectively. Different lowercase letters above the bars represent significant differences (p < 0.05) between group C, group T1, group T2 and group T3 (mean ± SD, n = 3). A one-way ANOVA test was used to identify the statistical significance.
Figure 4
Figure 4
(a,b) Effects of alkalinity stress on mRNA expression levels of apoptotic-related genes (Caspase3 and Bcl-2) in liver tissue. (c,d) Effects of alkalinity stress on mRNA expression levels of immune-related genes (C3 and IL-1β) in liver tissue. (e,f) mRNA expression levels of stress-related gene (HSP70 and HSP90). “*” above the bars represents significant differences (p < 0.05) between groups (mean ± SD, n = 9).
Figure 5
Figure 5
Metabolic analyses of the livers after alkalinity stress. The different metabolites between the NaHCO3 group (case) and the control group (con) were identified using PCA (A), PLS-DA (B), and OPLS-DA (C). The red triangles represent the control group. The blue square shapes represent the NaHCO3 group. (D) OPLS-DA permutation test. (E) S-Plot: the horizontal coordinate represents the effect of the metabolite on the NaHCO3 group and the control group, while the vertical coordinate represents the correlation between the sample and the metabolite. (F) The volcano plot of differential metabolites in livers. Red represents up-regulated, blue represents down-regulated and brown represents not significant. (G) Heatmap of differential metabolites in livers. (H) Scatter plot of significantly enriched KEGG pathways. The size of nodes indicates the gene number that mapped to the KEGG pathway; formula image represents the p-value decreasing from left to right. The Y-coordinate represents the pathway term. The X-coordinate represents the relative rich factors of each pathway.
Figure 6
Figure 6
Immunohistochemical staining for (a) apoptosis- and (b) ferroptosis-related proteins in sturgeon liver tissues (400×; scale: 100 μm). The nucleus is blue, and the positive expression area of the target protein is brown.
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