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. 2019 Jun 11:10:653.
doi: 10.3389/fphar.2019.00653. eCollection 2019.

Quantitative Proteomic Analysis Reveals the Sites Related to Acetylation and Mechanism of ACY-1215 in Acute Liver Failure Mice

Wen-Bin Zhang  1 , Hai-Yue Zhang  1 , Yao Wang  1 , Fang-Zhou Jiao  1 , Lu-Wen Wang  1 , Zuo-Jiong Gong  1
Affiliations

Quantitative Proteomic Analysis Reveals the Sites Related to Acetylation and Mechanism of ACY-1215 in Acute Liver Failure Mice

Wen-Bin Zhang et al. Front Pharmacol. .

Abstract

Background: ACY-1215 is a well-known selective histone deacetylase 6 (HDAC6) inhibitor, and it has been considered as a potential therapeutic drug in inflammatory diseases, including acute liver failure (ALF). However, little is known about the impact of ACY-1215 treatment on histone lysine acetylation and proteome in ALF. In this study, we aim to investigate whether ACY-1215 has inhibitory effects and mechanism on the necrosis of hepatocytes; moreover, the impact of ACY-1215 treatment on histone lysine acetylation still needs further elucidation. Methods: Male C57/BL6 mice were divided into normal, model, and ACY-1215 groups. ACY-1215 (25 mg/kg) and same amounts of saline were injected intraperitoneally to the mice before the establishment of ALF model induced by lipopolysaccharide (LPS) (100 µg/kg) combined with D-gal (400 mg/kg). All animals were sacrificed after 24 h. In this study, detection programs, including quantitative proteomic analysis, transmission electron microscopy (TEM) micrographs, pathological staining, protein expression, the detection of reactive oxygen species (ROS) as well as glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) measurement. Results: The function of liver and the necrosis of hepatocytes in ALF mice were significantly normalized by ACY-1215 pretreatment. The quantitative proteomic analysis revealed that ACY-1215-restrained oxidative phosphorylation normalized the function respiratory electron-transport chain in the mitochondria. Moreover, pretreatment of ACY-1215 not only normalized the structure of mitochondria but also inhibited the generation of reactive oxygen species (ROS). Conclusions: ACY-1215 was able to inhibit necrosis of hepatocytes in ALF mice through regulating the mitochondrial-mediated oxidative stress, and we identified the common sites related to acetylation level.

Keywords: ACY-1215; acute liver failure; histone deacetylase 6; necrosis; quantitative proteomic analysis.

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Figures

Figure 1
Figure 1
Experimental strategy for quantification of histone lysine acetylation and protein expression in acute liver failure (ALF) mice upon ACY-1215 treatment.
Figure 2
Figure 2
The effect of ACY-1215 treatment of the necrosis of hepatocytes and the impaired liver function in ALF mice. (A) The results of the hematoxylin and eosin (HE) staining (original magnification ×100) showed that ACY-1215 could alleviate necrotic changes and preserve the architecture of liver. (B) The results of terminal deoxynucleotidyl transferase (TdT)-mediated 2'-Deoxyuridine 5'-Triphosphate (dUTP) Nick-End Labeling (TUNEL) showed the effect of ACY-1215 on the necrosis of hepatocytes. (C) Treatment with ACY-1215 normalized the activities of glutamic oxaloacetic transaminase (GOT). (D) Treatment with ACY-1215 normalized the activities of glutamate pyruvic transaminase (GPT). *P < 0.05 as compared with the corresponding normal group. # P < 0.05 vs model group.
Figure 3
Figure 3
Enrichment and clustering analysis of the quantitative proteomics data sets based on gene ontology (GO) annotations. Quantifiable proteins were classified by gene ontology annotation based on three categories: (A) biological process; (B) cellular compartment, (C) molecular function, (D) the kyoto encyclopedia of genes and genomes (KEGG) pathway, and (E) the protein domain.
Figure 4
Figure 4
The impacts of ACY-1215 treatment in oxidative phosphorylation in ALF mice. (A) and (B) Compared with the ALF mice, the subcellular localization and distribution of the up-regulated proteins in ACY-1215 treatment mice. (C) and (D) Compared with the ALF mice, the subcellular localization and distribution of the down-regulated proteins in ACY-1215 treatment mice. (E) Compared with the normal mice, the protein–protein interaction network analyses for the down-regulated oxidative phosphorylation proteins in ALF mice. (F) Compared with the ALF mice, the protein–protein interaction network analyses for the up-regulated oxidative phosphorylation proteins in ALF mice with treatment of ACY-1215.
Figure 5
Figure 5
The effects of ACY-1215 on the mitochondria-dependent reactive oxygen species (ROS) of apoptosis. (A) Representative transmission electron microscopy TEM images of macrophages on indicated groups. (B) The ROS generation in liver tissues was detected by using 2′,7′-dichlorofluorescein diacetate (DCFH-DA). (C) The representative expression of Bax and Bcl-2 were analyzed by western blotting. (D) The quantitative blots of Bax and Bcl-2 in the indicated groups. *P < 0.05 as compared with the corresponding normal group. # P < 0.05 vs model group.
Figure 6
Figure 6
ACY-1215 treatment changed the histone lysine acetylation profiling. The extracted core histones was subject to SDS/PAGE followed by western blotting analysis to exam core histone site-specific lysine acetylation changes in H2A (A) and H2B (B) using indicated histone site-specific lysine acetylation antibodies.
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