Paeoniflorin protects hepatocytes from APAP-induced damage through launching autophagy via the MAPK/mTOR signaling pathway

Abstract

Background: Drug-induced liver injury (DILI) is gradually becoming a common global problem that causes acute liver failure, especially in acute hepatic damage caused by acetaminophen (APAP). Paeoniflorin (PF) has a wide range of therapeutic effects to alleviate a variety of hepatic diseases. However, the relationship between them is still poorly investigated in current studies.

Purpose: This work aimed to explore the protective effects of PF on APAP-induced hepatic damage and researched the potential molecular mechanisms.

Methods: C57BL/6J male mice were injected with APAP to establish DILI model and were given PF for five consecutive days for treatment. Aiming to clarify the pharmacological effects, the molecular mechanisms of PF in APAP-induced DILI was elucidated by high-throughput and other techniques.

Results: The results demonstrated that serum levels of ALP, γ-GT, AST, TBIL, and ALT were decreased in APAP mice by the preventive effects of PF. Moreover, PF notably alleviated hepatic tissue inflammation and edema. Meanwhile, the results of TUNEL staining and related apoptotic factors coincided with the results of transcriptomics, suggesting that PF inhibited hepatocyte apoptosis by regulated MAPK signaling. Besides, PF also acted on reactive oxygen species (ROS) to regulate the oxidative stress for recovery the damaged mitochondria. More importantly, transmission electron microscopy showed the generation of autophagosomes after PF treatment, and PF was also downregulated mTOR and upregulated the expression of autophagy markers such as ATG5, ATG7, and BECN1 at the mRNA level and LC3, p62, ATG5, and ATG7 at the protein level, implying that the process by which PF exerted its effects was accompanied by the occurrence of autophagy. In addition, combinined with molecular dynamics simulations and western blotting of MAPK, the results suggested p38 as a direct target for PF on APAP. Specifically, PF-activated autophagy through the downregulation of MAPK/mTOR signaling, which in turn reduced APAP injury.

Conclusions: Paeoniflorin mitigated liver injury by activating autophagy to suppress oxidative stress and apoptosis via the MAPK/mTOR signaling pathway. Taken together, our findings elucidate the role and mechanism of paeoniflorin in DILI, which is expected to provide a new therapeutic strategy for the development of paeoniflorin.

Keywords: Acetaminophen; Cell death; Drug-induced liver injury; Natural products; Oxidative stress; Signal transduction.

Fig. 1

Biochemical and pathological parameters changes by PF on APAP-induced mice. The levels of liver index (A) and spleen index (B) are shown (n = 8). C Weight of mice during the whole experiment (n = 8). D–H Serum levels of ALT, AST, ALP, TBIL, and γ-GT in mice (n = 8). I Necrosis index of mice in each group. J H&E staining in mice (×100, ×400) (n = 3). The red arrow points to the area of hepatic lesion. Data are presented as mean ± SD. *p < 0.05 and **p < 0.01 versus control; #p < 0.05 and ##p < 0.01, versus APAP

Fig. 2

Effects of PF on oxidative stress with APAP stimulation. The levels of GSH (A), SOD (B), and MDA (C) are shown(n = 6). D Mean density of ROS. E Immunofluorescence of ROS (n = 3). F Western blotting images of CYP3A4 (n = 3). G Relative protein expression of CYP3A4. H, I The expression of CYP2E1 by Immunohistochemistry (×200, ×400) (n = 3). J Transmission electron microscope (n = 3). Data are presented as mean ± SD. *p < 0.05 and **p < 0.01, vs. control; #p < 0.05 and ##p < 0.01, vs. APAP

Fig. 3

Anti-apoptotic effects of PF in APAP-induced hepatic damage. A The apoptotic expression is shown by TUNEL staining (×100, ×400). The expression of caspase 9 (B) and caspase 3 (C) is shown in the different groups (×100, ×400). D The TUNEL positive rate in mice. E Western blotting images of BAD, BCL-2, and BAX in mice. F Western blotting images of caspase 9 and caspase 3 in mice. The relative protein expression of BAD (G), BCL-2 (H), BAX (I), caspase 9 (K), and caspase 3 (L) is depicted. J Ratio of BCL-2/BAX in mice. The red arrow points to positive expression areas. Data are presented as mean ± SD in each three samples in group. *p < 0.05 and **p < 0.01, versus control; #p < 0.05 and ##p < 0.01, versus APAP

Fig. 4

Changes in autophagy representative indices modulated by PF. A Transmission electron microscopy of autophagosomes. B Immunohistochemistry of LC3 and p62 in different groups (×200). C Western blotting images of LC3 and p62. The mean density of LC3 (D) and p62 (E) in immunohistochemistry. F Ratio of LC3II/LC3I in mice. G Relative protein expression of p62. The red arrow points to autophagosomes. Data are presented as mean ± SD in each three samples in group. *p < 0.05 and **p < 0.01, versus control; #p < 0.05 and ##p < 0.01, versus APAP

Fig. 5

Activation of autophagy by PF on APAP-induced mice. A Immunofluorescence of LC3. B Western blotting images of ATG7 and ATG5. C Mean density of LC3 in immunofluorescence. Relative protein expression of ATG7 (D) and ATG5 (E). Data are presented as mean ± SD in each three samples in group. *p < 0.05 and **p < 0.01, versus control; #p < 0.05 and ##p < 0.01, versus APAP

Fig. 6

Transcriptomic analysis of PF in improving APAP-induced liver injury. Volcano map of the control versus APAP groups (A) and HPF versus APAP groups (B). GO analysis of the control versus APAP groups (C) and HPF versus APAP groups (D). KEGG analysis of control verus APAP groups (E) and HPF versus APAP groups (F)

Fig. 7

Targeting of p38 by PF. A Molecular docking analysis of PF to p38. B RMSD of the p38–PF complex. C SASA of the p38–PF complex. D Rg of the p38–PF complex. E RMSF of the p38–PF complex. F Hydrogen bonding analysis of the p38-PF complex

Fig. 8

Regulation of PF on MAPK/mTOR signaling. A–C Relative gene expression of MAPK14, MAPK1 and MTOR. D Western blotting images of p38, p-p38, ERK, p-ERK, mTOR, and p-mTOR. E, F Ratio of p-p38/p38 and p-ERK/ERKin mice. G Western blotting images of ULK1 and p-ULK1. H, I Ratio of p-mTOR/mTOR and p-ULK1/ULK1 in mice. Data are presented as mean ± SD in each three samples in group. *p < 0.05 and **p < 0.01, versus control; #p < 0.05 and ##p < 0.01, versus APAP

Fig. 9

Schematic diagram of PF treatment on hepatic injury with APAP stimulation. Black arrows represent the development of hepatic injury generated by APAP, while red arrows suggest the mechanism of action of paeoniflorin in ameliorating APAP-induced liver injury