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. 2020 Feb 28;11:162.
doi: 10.3389/fphar.2020.00162. eCollection 2020.

Fisetin Prevents Acetaminophen-Induced Liver Injury by Promoting Autophagy

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Free PMC article

Fisetin Prevents Acetaminophen-Induced Liver Injury by Promoting Autophagy

Jiaqi Zhang et al. Front Pharmacol. .
Free PMC article

Abstract

Acetaminophen (APAP) overdose is a leading cause of drug-induced acute liver failure in clinical and hospital settings. Fisetin (FST) is a phenolic compound derived from natural products such as fruit and vegetables. Our research investigated the protective mechanisms of FST in APAP-induced hepatic injury in vitro and vivo. Assessment of mouse serum levels of alanine/aspartate aminotransferases (ALT/AST), liver myeloperoxidase (MPO) activity, malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species (ROS) demonstrated the protective effects of FST toward APAP-induced liver injury. FST also reversed an APAP-induced decrease in mouse L-02 cell line viability. Our results also showed that FST significantly promoted APAP-induced autophagy and inhibited inflammasome activation both in vivo and in vitro. We also found that silencing ATG5, using si-ATG5, reduced the protective effects of FST against APAP-induced hepatotoxicity and reversed the effects on autophagy. Finally, we used the autophagy inhibitor, 3-methyladenine (3-MA) to validate the involvement of autophagy in FST against APAP-induced hepatotoxicity in vitro. We demonstrated that FST prevented APAP-induced hepatotoxicity by increasing ATG5 expression, thereby promoting autophagy and inhibiting inflammasome activation.

Keywords: ATG5; acetaminophen; autophagy; fisetin; liver injury.

Figures

Figure 1
Figure 1
Fisetin (FST) prevents acetaminophen (APAP)-induced liver injury in mice. (A) The structure of FST. The molecular formula of FST is C15H10O6 and the molecular weight is 286.23 g/mol. (B) Serum alanine/aspartate aminotransferases (ALT/AST) levels at 6 h after APAP administration. (C) Representative images of H&E stained liver sections (×200 and ×400 magnification). (D) Liver myeloperoxidase (MPO) activity, (E) liver reactive oxygen species (ROS) levels, (F) liver malondialdehyde (MDA) activity, and (G) liver glutathione (GSH) levels. Data are expressed as mean ± SEM (n = 8). **P < 0.01, ***P < 0.001 compared to vehicle; ##P < 0.01, ### P< 0.001 compared to APAP.
Figure 2
Figure 2
Fisetin (FST) promotes autophagy and inhibits inflammasome activation. (A) Western blot of autophagy-related mouse liver proteins. (B) Quantification of Western blot. (C) A representative image of LC3 puncta (red) staining in liver tissue. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). (D) Quantification of liver tissue LC3 puncta-positive cells. (E) Western blot of inflammasome-related proteins. (F) Quantification of Western blot. (G) Mouse serum interleukin (IL)-1β and IL-18 levels as determined by ELISA. Data are expressed as mean ± SEM (n = 8). **P < 0.01, ***P < 0.001 compared to vehicle; ##P < 0.01, ###P < 0.001 compared to APAP.
Figure 3
Figure 3
Fisetin (FST) attenuates acetaminophen (APAP)-induced cytotoxicity by promoting autophagy and inhibiting inflammasome activation in L-02 cells. (A, B) Cell viability was assessed by MTT assay in response to APAP (10 mM) or N-acetyl-p-benzoquinoneimine (NAPQI) (200 μM) exposure for different periods in L-02 cells. (C, D) Cell viability was assessed by MTT assay in response to APAP (10 mM) or NAPQI (200 μM) exposure for 48 h in HepaRG cells. (E) Cell viability was assessed by MTT assay for different concentration of FST in L-02 cells. (F) Messenger RNA (mRNA) expression of ATG genes was determined by RT-PCR in the presence of APAP (10 mM) for 48 h. (G) Western blot of autophagy-related proteins in L-02 cells supplemented with APAP (10 mM) for 48 h. (H) Quantification of Western blot. (I, J) Representative immunofluorescence images of mRFP-GFP-LC3 in L-02 cells supplemented with APAP (10 mM) for 48 h. Representative profiles of autophagosomes (RFP+GFP+dots) and autolysosomes (RFP+GFP-dots) per cell section, assessed by confocal microscopy are shown and were quantified. (K, L) Autophagic vacuoles (autophagosomes) were assessed by transmission electron microscopy (TEM) plus APAP (10 mM) for 48 h in L-02 cells. Representative TEM images are shown and typical autophagosomes are marked with black arrows. Autophagosome numbers per cell were calculated by counting the number of double-membrane organelles in 10 cells. Data are expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared to control; # < 0.05, ### < 0.001 compared to APAP.
Figure 4
Figure 4
Fisetin (FST) inhibits inflammasome activation in L-02 cells. (A, B) Analysis and quantification of caspase-1 activation rates by FC with acetaminophen (APAP) (10 mM) for 48 h in L-02 cells. (C) Lactate dehydrogenase (LDH) release plus APAP (10 mM) for 48 h in L-02 cells. (D) Western blot of inflammasome-related proteins plus APAP (10 mM) for 48 h in L-02 cells. (E) Quantification of Western blot. Data are expressed as mean ± SEM (n = 3). ***P < 0.001 compared to control; ###P < 0.001 compared to APAP. Data are expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared to control; #P < 0.05, ###P < 0.001 compared to APAP.
Figure 5
Figure 5
Fisetin (FST) attenuates acetaminophen (APAP)-induced cytotoxicity via ATG5 in L-02 cells. (A, B) ATG5 messenger RNA (mRNA) expression was determined by real time (RT)-PCR and western blotting. (C) Cell viability was assessed by MTT assay in response to APAP exposure for different times in L-02 cells. (D) Western blot of autophagy-related proteins in cells plus APAP (10 mM) for 48 h. (E) Quantification of Western blot. (F, G) Representative immunofluorescence images of mRFP-GFP-LC3 in cells plus APAP (10 mM) for 48 h. Representative profiles of autophagosomes (RFP+GFP+ dots) and autolysosomes (RFP+GFP-dots) per cell section, as assessed by confocal microscopy are shown and were quantified. (H, I) Autophagic vacuoles (autophagosomes) were assessed by transmission electron microscopy (TEM) plus APAP (10 mM) for 48 h. Representative TEM images are shown and typical autophagosomes are marked with black arrows. Autophagosome numbers per cell were calculated by counting double-membrane organelles in 10 cells. Data are expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared to control or APAP; #P < 0.05, ###P < 0.001 compared to APAP or AAP+FST; $P < 0.05, $$P < 0.01, $$$P < 0.001 compared to APAP+FST.
Figure 6
Figure 6
Fisetin (FST) inhibits inflammasome activation via ATG5 in L-02 cells. (A, B) Analysis of caspase-1 activation rates by flow cytometry with acetaminophen (APAP) for 48 h and quantified. (C) Lactate dehydrogenase (LDH) release with APAP for 48 h. (D) Western blot of inflammasome-related proteins with APAP for 48 h. (E) Quantification of Western blot. Data are expressed as mean ± SEM (n = 3). ***P < 0.001 compared to control or APAP; ## < 0.01, ### < 0.001 compared to APAP or AAP+FST.
Figure 7
Figure 7
Inhibiting autophagy reverses the protective effects of fisetin (FST) in L-02 cells. (A) Cell viability was assessed by MTT assay in response to acetaminophen (APAP) (10 mM) exposure for different times in L-02 cells, pretreated with 3-methyladenine (3-MA) (5 mM) or rapamycin (RAPA) (100 nM) for 1 h. (B) Western blot of autophagy-related proteins in L-02 cells plus APAP (10 mM) for 48 h. (C) Quantification of Western blot. (D, E) Representative immunofluorescence images of mRFP-GFP-LC3 in L-02 cells plus APAP (10 mM) for 48 h. Representative profiles of autophagosomes (RFP+GFP+ dots) and autolysosomes (RFP+GFP-dots) per cell section, as assessed by confocal microscopy are shown and were quantified. (F, G) Autophagy vacuoles (autophagosomes) were detected by transmission electron microscopy (TEM) plus APAP (10 mM) for 48 h. Representative TEM images are shown and typical autophagosomes are marked with black arrows. Autophagosome numbers per cell were calculated by counting double-membrane organelles in 10 cells. Data are expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 compared to control or APAP; #P < 0.05, ###P < 0.001 compared to APAP or APAP+FST; $P < 0.05, $$P < 0.01, $$$P < 0.001 compared to APAP+FST.
Figure 8
Figure 8
Inhibiting autophagy reverses the inhibiting effect of fisetin (FST) on inflammasomes, in L-02 cells. (A, B) Analysis of caspase-1 activation rates by flow cytometry with acetaminophen (APAP) for 48 h and quantified. (C) Lactate dehydrogenase (LDH) release with APAP for 48 h. (D) Western blot of inflammasome-related proteins with APAP for 48 h. (E) Quantification of Western blot. Data are expressed as means ± SEM (n = 3). ***P < 0.001 compared to control or APAP; ## < 0.01, ### < 0.001 compared to APAP or AAP+FST.

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