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. 2023 Aug 29;21(9):477.
doi: 10.3390/md21090477.

The Preventive Mechanism of Anserine on Tert-Butyl Hydroperoxide-Induced Liver Injury in L-02 Cells via Regulating the Keap1-Nrf2 and JNK-Caspase-3 Signaling Pathways

Ming Chen  1 , Jing Luo  1 , Hongwu Ji  1   2   3   4   5 , Wenkui Song  1 , Di Zhang  1 , Weiming Su  1   2 , Shucheng Liu  1   2   3   4   5   6
Affiliations

The Preventive Mechanism of Anserine on Tert-Butyl Hydroperoxide-Induced Liver Injury in L-02 Cells via Regulating the Keap1-Nrf2 and JNK-Caspase-3 Signaling Pathways

Ming Chen et al. Mar Drugs. .

Abstract

Anserine is a naturally occurring histidine dipeptide with significant antioxidant activities. This study aimed to investigate the preventive mechanism of anserine on tert-butyl hydroperoxide (TBHP)-induced liver damage in a normal human liver cell line (L-02 cells). The L-02 cells were pretreated with anserine (10, 20, and 40 mmol/L) and then induced with 400 μmol/L of TBHP for 4 h. The results showed that the survival rates of L-02 cells and the contents of GSH were significantly increased with the pretreatment of anserine; the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the extracellular fluid were sharply decreased; and the formation of reactive oxygen species (ROS), nuclear fragmentation, and apoptosis were significantly inhibited. In addition, anserine could bind to the Kelch domain of Kelch-like ECH-associated protein 1 (Keap1) with a binding force of -7.2 kcal/mol; the protein expressions of nuclear factor-erythroid 2-related factor-2 (Nrf2), quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), and Bcl-2 were upregulated by anserine in TBHP-induced L-02 cells, with the downregulation of p-JNK and caspase-3. In conclusion, anserine might alleviated liver injury in L-02 cells via regulating related proteins in the Keap1-Nrf2 and JNK-Caspase-3 signaling pathways.

Keywords: JNK-Caspase-3; Keap1-Nrf2; L-02 cell; anrerine; apoptosis; liver injury; oxidative stress.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of anserine.
Figure 2
Figure 2
The effect of anserine on the survival rate of L-02 cells (A) and the survival rate of TBHP-induced L-02 cells (B). Data are displayed as the mean ± SD (n = 3). ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the model group.
Figure 3
Figure 3
Effect of anserine on ALT, AST, ROS, GSH, and the nucleus. (A) ALT, (B) AST, (C) ROS, and (D) GSH. Data are displayed as mean ± SD (n = 3). ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the model group.
Figure 4
Figure 4
DAPI staining and fluorescence observation of L-02 cells.
Figure 5
Figure 5
Molecular docking between anserine and Keap1 and the expressions of Nrf2, HO-1, and NQO1 in L-02 cells. (A) The proposed docking mode of anserine binding with the Keap1 Kelch domain. (B) The binding modes of anserine with Keap1 Kelch domain showing interacting amino acids and H-bonds. (C–F) The expressions of Nrf2, HO-1, and NQO1 in L-02 cells. Data are displayed as mean ± SD (n = 6). ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the model group.
Figure 6
Figure 6
Delaying effect of anserine on the apoptosis of L-02 Cells. (A) Observations using a flow cytometer. (B) Number of apoptotic cells.
Figure 7
Figure 7
The expressions of JNK, p-JNK, BCL-2, Bax, and Cas-3 in L-02 cells. (A) Western blot bands of JNK, p-JNK, BCL-2, Bax, and Cas-3. (B–D) Quantitative analysis of p-JNK/JNK, Bcl-2/Bax, and Cas-3. Data are displayed as mean ± SD (n = 3). ## p < 0.01 compared with the control group; * p < 0.05, ** p < 0.01 compared with the model group.
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