Long-Term Consumption of Food-Derived Chlorogenic Acid Protects Mice against Acetaminophen-Induced Hepatotoxicity via Promoting PINK1-Dependent Mitophagy and Inhibiting Apoptosis

Abstract

Hepatotoxicity brought on by acetaminophen (APAP) is significantly impacted by mitochondrial dysfunction. Mitophagy, particularly PINK1-mediated mitophagy, maintains the stability of cell function by eliminating damaged mitochondria. One of the most prevalent dietary polyphenols, chlorogenic acid (CGA), has been shown to have hepatoprotective properties. It is yet unknown, nevertheless, whether its defense against hepatocyte apoptosis involves triggering PINK1-mediated mitophagy. In vitro and in vivo models of APAP-induced hepatotoxicity were established to observe CGA's effect and mechanism in preventing hepatotoxicity in the present study. Serum aminotransferase levels, mouse liver histology, and the survival rate of HepG2 cells and mice were also assessed. The outcomes showed that CGA could reduce the activities of serum enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), and lactate dehydrogenase (LDH), and alleviate liver injury in mice. It could also significantly increase the cell viability of HepG2 cells and the 24-h survival rate of mice. TUNEL labeling and Western blotting were used to identify the hepatocyte apoptosis level. According to data, CGA could significantly reduce liver cell apoptosis in vivo. Additionally, Tom20 and LC3II colocalization in mitochondria may be facilitated by CGA. CGA considerably increased the levels of genes and proteins associated with mitophagy (PINK1, Parkin, LC3II/LC3I), while considerably decreasing the levels of p62 and Tom20, suggesting that it might activate PINK1/Parkin-mediated mitophagy in APAP-induced liver damage. Additionally, the protection of CGA was reduced when PINK1 was knocked down by siPINK1 in HepG2 cells, and it did not upregulate mitophagy-related proteins (PINK1, Parkin, LC3II/LC3I). In conclusion, our findings revealed that long-term consumption of food-derived CGA could prevent APAP hepatotoxicity via increasing PINK1-dependent mitophagy and inhibiting hepatocyte apoptosis.

Keywords: APAP-induced liver injury; PINK1/Parkin pathway; apoptosis; chlorogenic acid; mitophagy.

Figure 1

Schematic diagram of APAP-induced hepatotoxicity. NOPQI is metabolized in combination with cysteine and mercaptoacetic acid when GSH is abundant. With the depletion of GSH, the accumulated NAPQI binds to some large proteins to induce mitochondrial damage, oxidative stress, an inflammatory response, and apoptosis.

Figure 2

A schematic diagram of the treatment schedule.

Figure 3

CGA alleviated hepatotoxicity in APAP-induced mice. (A) 24 h survival rate of APAP-induced liver injury mice (log-rank test, n = 20). (B–D) Serum levels of ALT, AST, and LDH (n = 5). (E) Microscopic pictures of the H&E-stained hepatic sections (n = 5, scale bar = 20 μm); the arrows indicate leukocyte infiltration and the dotted line represents the necrotic area. (F) Histological necrosis scores (n = 5). (G) Inflammation scores (n = 5). Data are displayed as mean ± S.E.M. ^# p < 0.05 vs. Ctrl group; * p < 0.05 vs. APAP group.

Figure 4

CGA suppressed liver cell apoptosis in APAP hepatotoxicity mice. (A) Representative microscopic images of liver cell apoptosis (TUNEL assay, 200 (×), scale bar = 50 μm); the arrows indicate apoptotic cells. (B) TUNEL-positive stained cells (n = 4). (C) Representative Western blotting images of Bcl-2 and Bax in liver tissue. GAPDH was used as an internal standard. (D–F) Quantitative analysis of Bcl-2, Bax, and the ratio of Bcl-2/Bax in the liver in different groups by Bio-Rad Quantity One v4.62 software. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 vs. Ctrl group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs. APAP group.

Figure 5

CGA triggered PINK1-dependent mitophagy in APAP hepatotoxicity mice. (A) Representative immunofluorescence images of LC3II and Tom20 (n = 4, scale bar = 40 μm). (B) Quantitative analysis of Tom20 mean fluorescence intensity by Image J. (C) The expression of PINK1 mRNA, Parkin mRNA, LC3II mRNA, and p62 mRNA (n = 3). (D) Representative Western blotting images of PINK1, Parkin, LC3I, LC3II, and p62 in liver. GAPDH was used as an internal standard. (E) Quantitative analysis of PINK1, Parkin, LC3II/LC3I, and p62 in liver in different groups by Bio-Rad Quantity One (n = 3). ^# p < 0.05, ^## p < 0.01 vs. Ctrl group; * p < 0.05, ** p < 0.01 vs. APAP group.

Figure 6

siPINK1 reversed the protective effect of CGA in APAP-induced HepG2 cells. (A) Cell viability: HepG2 cells were treated with different concentrations of CGA for 24 h, and cell viability was measured by MTT assay. (B,C) The expression of PINK1 mRNA and protein in siPINK1 HepG2 cells. (D) Cell viability: Normal HepG2 cells, as well as HepG2 cells transfected with siPINK1 or siNC, were exposed to APAP conditions with or without CGA for 24 h, and cell viability was measured by MTT assay. (E) Representative Western blotting images of PINK1, Parkin, LC3, and p62 in HepG2 cells. GAPDH was used as an internal standard. (F) Quantitative analysis of PINK1, Parkin, LC3II/LC3I, and p62 in HepG2 in different groups by Bio-Rad Quantity One (n = 3). ^# p < 0.05, ^## p < 0.01 vs. Ctrl group; * p < 0.05, ** p < 0.01 vs. APAP group.

Figure 7

Graphical summary of the results. CGA protects against acetaminophen-induced hepatotoxicity in mice through activating PINK1-dependent mitophagy to inhibit apoptosis.