Treatment with sodium (S)-2-hydroxyglutarate prevents liver injury in an ischemia-reperfusion model in female Wistar rats

Abstract

Background: Ischemia-reperfusion (IR) injury is one of the leading causes of early graft dysfunction in liver transplantation. Techniques such as ischemic preconditioning protect the graft through the activation of the hypoxia-inducible factors (HIF), which are downregulated by the EGLN family of prolyl-4-hydroxylases, a potential biological target for the development of strategies based on pharmacological preconditioning. For that reason, this study aims to evaluate the effect of the EGLN inhibitor sodium (S)-2-hydroxyglutarate [(S)-2HG] on liver IR injury in Wistar rats.

Methods: Twenty-eight female Wistar rats were divided into the following groups: sham (SH, n = 7), non-toxicity (HGTox, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days), IR (n = 7, total liver ischemia: 20 minutes, reperfusion: 60 minutes), and (S)-2HG+IR (HGIR, n = 7, 25 mg/kg of (S)-2HG, twice per day for two days, total liver ischemia as the IR group). Serum ALT, AST, LDH, ALP, glucose, and total bilirubin were assessed. The concentrations of IL-1β, IL-6, TNF, malondialdehyde, superoxide dismutase, and glutathione peroxidase were measured in liver tissue, as well as the expression of Hmox1, Vegfa, and Pdk1, determined by RT-qPCR. Sections of liver tissue were evaluated histologically, assessing the severity of necrosis, sinusoidal congestion, and cytoplasmatic vacuolization.

Results: The administration of (S)-2HG did not cause any alteration in the assessed biochemical markers compared to SH. Preconditioning with (S)-2HG significantly ameliorated IR injury in the HGIR group, decreasing the serum activities of ALT, AST, and LDH, and the tissue concentrations of IL-1β and IL-6 compared to the IR group. IR injury decreased serum glucose compared to SH. There were no differences in the other biomarkers assessed. The treatment with (S)-2HG tended to decrease the severity of hepatocyte necrosis and sinusoidal congestion compared to the IR group. The administration of (S)-2HG did not affect the expression of Hmox1 but decreased the expression of both Vegfa and Pdk1 compared to the SH group, suggesting that the HIF-1 pathway is not involved in its mechanism of hepatoprotection. In conclusion, (S)-2HG showed a hepatoprotective effect, decreasing the levels of liver injury and inflammation biomarkers, without evidence of the involvement of the HIF-1 pathway. No hepatotoxic effect was observed at the tested dose.

Keywords: 2-hydroxyglutarate; Hmox1; Ischemia-reperfusion injury; Liver; Oxidative stress; Pdk1; Proinflammatory cytokines; Vegfa.

Figure 1. Effect of the administration of sodium (S)-2-hydroxyglutarate on the biochemical markers.

(A) Effect on the serum activity of ALP; (B) effect on the serum activity of ALT, *p < 0.0001 vs SH group, #p = 0.0002 vs IR group; (C) effect on the serum activity of AST, *p < 0.0001 vs SH group, #p = 0.0465 vs IR group; (D) effect on the serum concentration of glucose, *p = 0.0032 vs SH group, ‡p = 0.0012 vs SH group; (E) effect on the serum activity of LDH, *p < 0.0001 vs SH group, #p = 0.0109 vs IR group; (F) effect on the serum concentration of total bilirubin. One-way ANOVA test with Tukey post hoc test in (A-E) Kruskal–Wallis test with Dunn post hoc test in F. ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; LDH, lactate dehydrogenase. Values expressed as mean ± standard deviation.

Figure 2. Effect of the administration of sodium (S)-2-hydroxyglutarate on the proinflammatory cytokines.

(A) Effect on the tissue concentration of IL-1β, *p = 0.0228 vs SH group, #p = 0.0336 vs IR group; (B) effect on the tissue concentration of IL-6, #p = 0.0054 vs IR group, ‡p = 0.0002 vs SH group; (C) effect on the tissue concentration of TNF, ‡p = 0.0050 vs SH group. One-way ANOVA test with Tukey post hoc test. IL-1β, interleukin 1β; IL-6, interleukin 6; TNF, tumor necrosis factor. Values expressed as mean ± standard deviation.

Figure 3. Representative liver micrographs of the experimental groups.

Hematoxylin and eosin staining (original magnification: 400×). (A) SH group, (B) HGTox group. Tissue architecture was conserved in A and B, without significant cell necrosis or sinusoidal congestion. (C) IR group, (D) HGIR group. Severe cellular necrosis is observed in zone 3 of the liver acinus in C, while the severity of the damage was decreased in D.

Figure 4. Effect of the administration of sodium (S)-2-hydroxyglutarate on the expression of genes regulated by the HIF-1 pathway in liver tissue.

(A) Effect on the tissue expression of Hmox1; (B) effect on the tissue expression of Vegfa, †p = 0.0185 vs SH group; (C) effect on the tissue expression of Pdk1, †p = 0.0227 vs SH group, *p = 0.0204 vs SH group. One-way ANOVA test with Tukey post hoc test. Values expressed as mean ± standard deviation.