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. 2021 Jan 22;10(2):158.
doi: 10.3390/antiox10020158.

Betulinic Acid Alleviates Spleen Oxidative Damage Induced by Acute Intraperitoneal Exposure to T-2 Toxin by Activating Nrf2 and Inhibiting MAPK Signaling Pathways

Li Kong  1 , Lijuan Zhu  1 , Xianglian Yi  1 , You Huang  1 , Haoqiang Zhao  1 , Yazhi Chen  1 , Zhihang Yuan  1 , Lixin Wen  1   2 , Jing Wu  1 , Jine Yi  1   2
Affiliations

Betulinic Acid Alleviates Spleen Oxidative Damage Induced by Acute Intraperitoneal Exposure to T-2 Toxin by Activating Nrf2 and Inhibiting MAPK Signaling Pathways

Li Kong et al. Antioxidants (Basel). .

Abstract

T-2 toxin, which is mainly produced by specific strains of Fusarium in nature, can induce immunotoxicity and oxidative stress, resulting in immune organ dysfunction and apoptosis. Betulinic acid (BA), a pentacyclic triterpenoids from nature plants, has been demonstrated to possess immunomodulating and antioxidative bioactivities. The purpose of the study was to explore the effect of BA on T-2 toxin-challenged spleen oxidative damage and further elucidate the underlying mechanism. We found that BA not only ameliorated the contents of serum total cholesterol (TC) and triglyceride (TG) but also restored the number of lymphocytes in T-2 toxin-induced mice. BA dose-dependently reduced the accumulation of reactive oxygen species (ROS), enhanced superoxide dismutase (SOD) activity, and decreased malondialdehyde (MDA) content, as well as increased the total antioxidant capacity (T-AOC) in the spleen of T-2-toxin-exposed mice. Moreover, BA reduced inflammatory cell infiltration in the spleen, improved the morphology of mitochondria and enriched the number of organelles in splenocytes, and dramatically attenuated T-2 toxin-triggered splenocyte apoptosis. Furthermore, administration of BA alleviated the protein phosphorylation of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinases (ERK); decreased the protein expression of kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein1 (Keap1); and increased the protein expression of nuclear factor erythroid 2 [NF-E2]-related factor (Nrf2) and heme oxygenase-1 (HO-1) in the spleen. These findings demonstrate that BA defends against spleen oxidative damage associated with T-2 toxin injection by decreasing ROS accumulation and activating the Nrf2 signaling pathway, as well as inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway.

Keywords: MAPK signaling pathway; Nrf2 signaling pathway; T-2 toxin; betulinic acid; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structure of betulinic acid (BA).
Figure 2
Figure 2
Effects of BA on serum biochemical indicators and blood indicators in T-2 toxin-treated mice. Blood samples were collected, and then the levels of total cholesterol (TC) (A) and triglyceride (TG) (B) were assayed using the oxidase method. An automatic blood analyzer was used to detect the levels of red blood cells (RBCs) (C), white blood cells (WBCs) (D), lymphocytes (LYMs) (E) and platelets (PLTs) (F). Data were presented as the mean ± standard error of the mean (SEM) (10 mice per group). * p < 0.05 and ** p < 0.01 vs. the control group. # p < 0.05 and ## p < 0.01 vs. the T-2 toxin group.
Figure 3
Figure 3
BA protects against T-2 toxin-triggered spleen oxidative stress. The reactive oxygen species (ROS) level in the spleen was determined by a fluorescence microscope using dihydroethidium (DHE). Notes: control group (A), T-2 toxin group (B), 0.25 mg/kg BA + T-2 toxin group (C), 0.5 mg/kg BA + T-2 toxin group (D), and 1 mg/kg BA + T-2 toxin group (E). Scale bar, 50 μm. Quantitative analysis of ROS level (F). The levels of malondialdehyde (MDA) (G), glutathione (GSH) (H), superoxide dismutase (SOD) (I), and total antioxidant capacity (T-AOC) in the spleen, (J) Data were presented as the mean ± SEM (10 mice per group). ** p < 0.01 vs. the control group. # p < 0.05 and ## p < 0.01 vs. the T-2 toxin group.
Figure 4
Figure 4
Effects of BA on spleen injury in T-2 toxin-exposed mice. Morphological changes in the spleen were examined using hematoxylin-eosin (H&E) staining. Notes: control group (A), T-2 toxin group (B), 0.25 mg/kg BA + T-2 toxin group (C), 0.5 mg/kg BA + T-2 toxin group (D), and 1 mg/kg BA + T-2 toxin group (E). Red arrow: macrophages. Yellow arrow: LYMs. Scale bar, 50 μm. The ultrastructure of the spleen was observed using transmission electron microscopy (TEM). Notes: control group (F), T-2 toxin group (G), and 0.5 mg/kg BA + T-2 toxin group (H). Red arrow: mitochondria. Black arrow: nucleus. Green arrow: endoplasmic reticulum. Blue arrow: Golgi apparatus. Scale bar, 2 μm. Splenocyte apoptosis was detected by terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay (I). Scale bar, 50 μm. Percentage of apoptotic cells (J). Data were presented as the mean ± SEM (10 mice per group). ** p < 0.01 vs. the control group. ## p < 0.01 vs. the T-2 toxin group.
Figure 4
Figure 4
Effects of BA on spleen injury in T-2 toxin-exposed mice. Morphological changes in the spleen were examined using hematoxylin-eosin (H&E) staining. Notes: control group (A), T-2 toxin group (B), 0.25 mg/kg BA + T-2 toxin group (C), 0.5 mg/kg BA + T-2 toxin group (D), and 1 mg/kg BA + T-2 toxin group (E). Red arrow: macrophages. Yellow arrow: LYMs. Scale bar, 50 μm. The ultrastructure of the spleen was observed using transmission electron microscopy (TEM). Notes: control group (F), T-2 toxin group (G), and 0.5 mg/kg BA + T-2 toxin group (H). Red arrow: mitochondria. Black arrow: nucleus. Green arrow: endoplasmic reticulum. Blue arrow: Golgi apparatus. Scale bar, 2 μm. Splenocyte apoptosis was detected by terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling (TUNEL) assay (I). Scale bar, 50 μm. Percentage of apoptotic cells (J). Data were presented as the mean ± SEM (10 mice per group). ** p < 0.01 vs. the control group. ## p < 0.01 vs. the T-2 toxin group.
Figure 5
Figure 5
Effects of BA on the relative proteins expression of the mitogen-activated protein kinase (MAPK) signaling pathway in the spleen of T-2 toxin-intoxicated mice. The protein expression and phosphorylation of ERK, JNK, and p38 were evaluated by Western blot (A). Protein bands for each region were quantified, and the intensity was normalized to the intensity of the β-actin band present on the same blot. The values were expressed as the ratios of p-p38/p38 (B), p-JNK/c-Jun N-terminal kinase (JNK) (C), and p-ERK/extracellular signal-regulated kinases (ERK) (D). Data were presented as the mean ± SEM (3 mice per group). * p < 0.05 and ** p < 0.01 vs. the control group. # p < 0.05 and ## p < 0.01 vs. the T-2 toxin group.
Figure 6
Figure 6
Effects of BA on the relative protein expression of the Nrf2 signaling pathway in the spleen of T-2 toxin-intoxicated mice. The protein expressions of nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2), kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1) were evaluated by Western blot (A). Quantitative analysis of Nrf2 (B), Keap1 (C), and HO-1 (D). Data were presented as the mean ± SEM (3 mice per group). ** p < 0.01 vs. the control group. # p < 0.05 and ## p < 0.01 vs. the T-2 toxin group.
Figure 6
Figure 6
Effects of BA on the relative protein expression of the Nrf2 signaling pathway in the spleen of T-2 toxin-intoxicated mice. The protein expressions of nuclear factor erythroid 2 [NF-E2]-related factor 2 (Nrf2), kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1 (Keap1), and heme oxygenase-1 (HO-1) were evaluated by Western blot (A). Quantitative analysis of Nrf2 (B), Keap1 (C), and HO-1 (D). Data were presented as the mean ± SEM (3 mice per group). ** p < 0.01 vs. the control group. # p < 0.05 and ## p < 0.01 vs. the T-2 toxin group.
Figure 7
Figure 7
The main mechanism of BA in protecting splenocytes against oxidative damage caused by T-2 toxin.
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