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. 2023 Jul 12;15(7):455.
doi: 10.3390/toxins15070455.

Emodin, an Emerging Mycotoxin, Induces Endoplasmic Reticulum Stress-Related Hepatotoxicity through IRE1α-XBP1 Axis in HepG2 Cells

Affiliations

Emodin, an Emerging Mycotoxin, Induces Endoplasmic Reticulum Stress-Related Hepatotoxicity through IRE1α-XBP1 Axis in HepG2 Cells

Su Been Park et al. Toxins (Basel). .

Abstract

Emodin, an emerging mycotoxin, is known to be hepatotoxic, but its mechanism remains unclear. We hypothesized that emodin could induce endoplasmic reticulum (ER) stress through the inositol-requiring enzyme 1 alpha (IRE1α)-X-box-binding protein 1 (XBP1) pathway and apoptosis, which are closely correlated and contribute to hepatotoxicity. To test this hypothesis, a novel IRE1α inhibitor, STF-083010, was used. An MTT assay was used to evaluate metabolic activity, and quantitative PCR and western blotting were used to investigate the gene and protein expression of ER stress or apoptosis-related markers. Apoptosis was evaluated with flow cytometry. Results showed that emodin induced cytotoxicity in a dose-dependent manner in HepG2 cells and upregulated the expression of binding immunoglobulin protein (BiP), C/EBP homologous protein (CHOP), IRE1α, spliced XBP1, the B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 ratio, and cleaved caspase-3. Cotreatment with emodin and STF-083010 led to the downregulation of BiP and upregulation of CHOP, the Bax/Bcl-2 ratio, and cleaved caspase-3 compared with single treatment with emodin. Furthermore, the apoptosis rate was increased in a dose-dependent manner with emodin treatment. Thus, emodin induced ER stress in HepG2 cells by activating the IRE1α-XBP1 axis and induced apoptosis, indicating that emodin can cause hepatotoxicity.

Keywords: ER stress; HepG2 cells; IRE1α−XBP1; apoptosis; emodin; hepatotoxicity; mycotoxin.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structures of (A) emodin and (B) STF-083010, as accessed from the Sigma-Aldrich (St. Louis, MO, USA) (https://www.sigmaaldrich.com/KR/ko/product/usp/1235059 (accessed on 12 April 2023)) and Selleckchem (Houston, TX, USA) (https://www.selleckchem.com/products/stf-083010.html (accessed on 12 April 2023)) websites, respectively.
Figure 2
Figure 2
Effect of emodin on the metabolic activity of HepG2 cells. HepG2 cells were treated with emodin for 24 h, and metabolic activity was measured with an MTT assay. The IC50 value was determined to be 20.93 μM. Data are shown as the mean ± standard deviation (SD) from three independent experiments. * p < 0.05, ** p < 0.01 (t-test) compared with the vehicle control group. The statistical power was ≥0.8, which means that a non-significant result is likely to be not really significant.
Figure 3
Figure 3
Morphological changes in HepG2 cells induced with emodin. Apoptotic nuclei appeared slightly smaller than nuclei in a normal state, and emodin treatment increased mitochondrial distribution. Hoechst 33342 and MitoTracker™ Deep Red were used to stain the nuclei and mitochondria, respectively.
Figure 4
Figure 4
Relative mRNA expression levels of (A) BiP, (B) IRE1α, (C) CHOP, and (D) sXBP1 after treatment with emodin (10, 20, and 30 μM) for 24 h. The relative mRNA expression levels (AD) increased after treatment with emodin. TM (2 μg/mL) served as the positive control. VCON, vehicle control; TM, tunicamycin. The data are shown as the mean ± SD from three independent experiments. * p < 0.05 and ** p < 0.01 (t-test) compared with the VCON group. The statistical power was ≥0.8, which means that a non-significant result is likely to be not really significant.
Figure 5
Figure 5
Relative protein expression levels of (B) BiP, (C) IRE1α, (D) CHOP, (E) Bax/Bcl-2 ratio, and (F) cleaved caspase-3 after treatment with emodin (10, 20, and 30 μM) for 24 h. (A) Relative protein expression levels of ER stress and apoptosis-related markers measured with a Western blot analysis. (BF) The relative protein expression levels were increased with emodin treatment. (G) The effects of emodin on the expression of sXBP1 cut with PstI. TM (2 μg/mL) served as the positive control. VCON, vehicle control; TM, tunicamycin. The data are shown as the mean ± SD from three independent experiments. * p < 0.05 and ** p < 0.01 (t-test) compared with the VCON group. The statistical power was ≥0.8, which means that a non-significant result is likely to be not really significant.
Figure 6
Figure 6
(A) Flow cytometry was used to measure the apoptosis rate using annexin V-FITC and PI double staining. (B) Treatment of HepG2 cells with emodin (10, 20, and 30 μM) increased the apoptosis rate in a dose-dependent manner. TM (2 μg/mL) served as the positive control. A total of 5000 events were collected per sample. VCON, vehicle control; TM, tunicamycin. The data are shown as the mean ± SD from three independent experiments. * p < 0.05 and ** p < 0.01 (t-test) compared with the VCON group. The statistical power was ≥0.8, which means that a non-significant result is likely to be not really significant.
Figure 7
Figure 7
Relative protein expression levels of (B) BiP, (C) CHOP, (D) Bax/Bcl-2 ratio, and (E) cleaved caspase-3 after cotreatment with EMO (30 μM) and STF-083010 (100 μM) for 24 h. (A) Relative protein expression levels of ER stress and apoptosis-related markers measured with a Western blot analysis. The relative protein expression levels of (B) BiP were decreased and (C) CHOP, (D) Bax/Bcl-2 ratio, and (E) cleaved caspase-3 were increased compared with treatment with EMO alone. (F) The effects of EMO and STF-083010 on the expression of sXBP1 cut with PstI. TM (2 μg/mL) served as the positive control. VCON, vehicle control; TM, tunicamycin; TM + STF, tunicamycin and STF-083010; EMO, emodin; EMO + STF, emodin and STF-083010. * p < 0.05 and ** p < 0.01 (t-test) compared with the VCON group. # p < 0.05 and ## p < 0.01 (t-test) compared with the single treatment of TM or EMO. The statistical power was ≥0.8 (except BiP; 0.6), which means that a non-significant result is likely to be not really significant.
Figure 8
Figure 8
Overview of the possible molecular mechanism of ER stress and apoptosis induced with emodin in HepG2 cells. Created with BioRender.com.

References

    1. Gajęcki M.T., Gajęcka M., Zielonka Ł. The presence of mycotoxins in feed and their influence on animal health. Toxins. 2020;12:663. doi: 10.3390/toxins12100663. - DOI - PMC - PubMed
    1. Wells J.M., Cole R.J., Kirksey J.W. Emodin, a toxic metabolite of Aspergillus wentii isolated from weevil-damaged chestnuts. Appl. Microbiol. 1975;30:26–28. doi: 10.1128/am.30.1.26-28.1975. - DOI - PMC - PubMed
    1. Gruber-Dorninger C., Novak B., Nagl V., Berthiller F. Emerging mycotoxins: Beyond traditionally determined food contaminants. J. Agric. Food Chem. 2017;65:7052–7070. doi: 10.1021/acs.jafc.6b03413. - DOI - PubMed
    1. Sulyok M., Krska R., Schuhmacher R. Application of an LC–MS/MS based multi-mycotoxin method for the semi-quantitative determination of mycotoxins occurring in different types of food infected by moulds. Food Chem. 2010;119:408–416. doi: 10.1016/j.foodchem.2009.07.042. - DOI
    1. Khoshal A.K., Novak B., Martin P.G.P., Jenkins T., Neves M., Schatzmayr G., Oswald I.P., Pinton P. Co-occurrence of DON and emerging mycotoxins in worldwide finished pig feed and their combined toxicity in intestinal cells. Toxins. 2019;11:727. doi: 10.3390/toxins11120727. - DOI - PMC - PubMed

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