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. 2026 May;13(27):e21818.
doi: 10.1002/advs.202521818. Epub 2026 Feb 28.

Artesunate Ameliorates APAP-induced Liver Injury by Promoting NEDD4L-Mediated Ubiquitination and Degradation of TXNIP

Zhe Zhang  1 , Hongyi Zhang  1 , Haixiang Guo  1 , Xiwen Zhang  1 , Yidan Wang  1 , Baoyin Wang  1 , Tong Wang  1 , Guokun Zhao  1 , Qing Zhang  1 , Fei Gao  1 , Shuang Liang  1 , Hao Jiang  1 , Yu Ding  1 , Xiliang Du  2 , Jiabao Zhang  1 , Yi Zheng  1 , Xinwei Li  2 , Bao Yuan  1
Affiliations

Artesunate Ameliorates APAP-induced Liver Injury by Promoting NEDD4L-Mediated Ubiquitination and Degradation of TXNIP

Zhe Zhang et al. Adv Sci (Weinh). 2026 May.

Abstract

Liver injury can lead to severe acute liver failure and even death in patients. Artesunate (ART), which is a derivative of artemisinin that has been approved by the FDA for the treatment of malaria, has significant regulatory effects on cell death and inflammation. In this study, we found that ART exerts a protective effect on various preclinical animal models of liver injury, including mouse models of liver injury induced by APAP, CCl4, and Con A. Mechanistically, CETSA, DARTS and SPR indicate that ART directly binds to the LYS653 and ASP837 residues within the HECT domain of NEDD4L, and enhances the interaction between NEDD4L and the substrate TXNIP, promoting the ubiquitination and proteasomal degradation of TXNIP, ultimately alleviating APAP-induced liver injury. Furthermore, the overexpression of TXNIP as well as the global knockout or liver-specific knockdown of NEDD4L eliminates the effect of ART on alleviating liver injury. These data suggest that the NEDD4L-TXNIP axis participates in the development of liver injury and highlight the potential of ART to be used in the clinical treatment of liver injury.

Keywords: NEDD4L; TXNIP; artesunate; liver injury.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
Artesunate (ART) alleviates APAP‐induced liver injury. (A) Schematic diagram of the experimental procedure. The mice were fasted for 16 h, then intraperitoneally injected with 400 mg/kg APAP, and 1 h later, they were injected with ART. The samples were collected 24 h later (n = 8). (B,C) The levels of ALT and AST in the serum of the mice (n = 8). (D–F) The levels of TNF‐α, IL‐6, and IL‐1β in the serum of the mice (n = 8). (G) LDH levels in the serum of the mice (n = 8). (H) Representative images of H&E and TUNEL staining of the livers of the mice. (I) Quantification of necrotic area in the livers of the mice (n = 8). (J) Schematic diagram of the experimental procedure. The mice were fasted for 16 h, then intraperitoneally injected with 400 mg/kg APAP, and 10 h later, they were injected with ART. The samples were collected 24 h later (n = 8). (K,L) The levels of ALT and AST in the serum of the mice (n = 8). (M) Representative images of HE‐stained livers from the mice. (N) Quantification of the necrotic area in the livers of the mice (n = 8). (O) LDH levels in the serum of the mice (n = 8). (P) Schematic diagram of the experimental procedure. The mice were fasted for 16 h, then intraperitoneally injected with 650 mg/kg APAP, and 10 h later, they were injected with ART (n = 10). (Q) Survival curve of mice treated with ART after the administration of a lethal dose of APAP (n = 10). Data are presented as mean ± SEM. ** P  < 0.01.
FIGURE 2
FIGURE 2
ART inhibits liver injury by downregulating TXNIP. (A) Volcano plot of differentially expressed proteins after ART treatment analyzed by proteomic sequencing. (B) Heatmap of the top ten differentially expressed proteins after ART treatment (n = 4). (C) Protein expression of TXNIP in the livers of mice after ART treatment (n = 3). (D) TXNIP protein expression in mouse primary hepatocytes treated with different concentrations of ART. (E,F) Immunofluorescence images and quantitative analysis of TXNIP in the liver after ART treatment (n = 8). (G,H) Protein levels of p‐ASK1/ASK1 in the livers of mice after ART treatment (n = 3). (I–K) Immunofluorescence images and quantitative analysis of NLRP3 and ASC in the liver after ART treatment (n = 8). (L,M) Relative mRNA expression of Nlrp3 and Asc in the livers of mice after ART treatment (n = 8). (N–P) Protein levels of NLRP3 and ASC in the livers of mice after ART treatment (n = 3). (Q,R) Flow cytometry detection of the level of apoptosis in mouse primary hepatocytes treated with ART after overexpression of TXNIP and quantitative analysis of the results (n = 6). (S–U) ALT, AST, and LDH levels in the culture medium of mouse primary hepatocytes treated with ART after TXNIP overexpression (n = 6). (V) Viability of mouse primary hepatocytes treated with ART after TXNIP overexpressed (n = 6). Data are presented as mean ± SEM. *P  <  0.05, **P  < 0.01.
FIGURE 3
FIGURE 3
TXNIP overexpression abolishes the protective effect of ART on liver injury. (A) Schematic diagram of the experimental procedure. Eight‐week‐old WT mice were treated via tail vein injection with AAV‐Txnip or AAV‐NC. Four weeks after treatment, the mice were fasted for 16 h and then intraperitoneally injected with 400 mg/kg APAP. One hour later, the mice were treated with ART, and samples were collected 24 h later (n = 8). (B,C) The levels of ALT and AST in the serum of the mice (n = 8). (D–F) The levels of TNF‐α, IL‐6, and IL‐1β in the serum of the mice (n = 8). (G) LDH levels in the serum of the mice (n = 8). (H) H&E staining, F4/80 staining, and TUNEL staining of the livers of the mice. (I,J) Quantitative analysis of necrotic area and TUNEL‐positive cells in the livers of the mice (n = 8). (K–N) Relative mRNA expression of the proinflammatory genes in the livers of the mice (n = 8). (O–Q) Protein levels and quantitative analysis of NLRP3 and ASC in the livers of the mice (n = 3). Data are presented as mean ± SEM. * P < 0.05, ** P  < 0.01.
FIGURE 4
FIGURE 4
ART regulates the degradation of the TXNIP protein through NEDD4L. (A) The protein level of TXNIP in HepG2 cells treated with ART after being treated with different concentrations of CHX. (B) The protein level of TXNIP in HepG2 cells treated with DMSO, MG132 or CQ after being treated with ART. (C) The ubiquitination level of TXNIP in HepG2 cells treated with different concentrations of ART. (D) TXNIP was transiently overexpressed in HepG2 cells. Proteins interacting with TXNIP were identified by IP‐MS. (E) The protein level of TXNIP in HepG2 cells treated with ART after NEDD4L was knocked down (n = 3). (F) Endogenous Co‐IP assay to evaluate the interaction between NEDD4L and TXNIP in HepG2 cells treated with ART. (G) Exogenous Co‐IP assay to evaluate the interaction between NEDD4L and TXNIP in HepG2 cells treated with ART. (H) Fluorescence colocalization of TXNIP and NEDD4L in HepG2 cells treated with ART. (I) The protein level of TXNIP in HepG2 cells transfected with NEDD4L and treated with MG132 (n = 3). (J) The ubiquitination level of TXNIP in HepG2 cells treated with ART after NEDD4L was knocked down. (K) Co‐IP assay to determine the interaction between TXNIP (WT) or TXNIP (MUT) and NEDD4L in HepG2 cells treated with ART. (L) The protein level of TXNIP in HepG2 cells treated with ART after the mutation of TXNIP.
FIGURE 5
FIGURE 5
Knockout of NEDD4L inhibits the protective effect of ART on liver injury. (A) Schematic diagram of the experimental procedure. Eight‐week‐old male Nedd4l−/− mice were fasted for 16 h and then intraperitoneally injected with APAP. One hour later, the mice were treated with ART, and samples were collected 24 h later (n = 8). (B,C) The levels of ALT and AST in the serum of the mice (n = 8). (D–F) The levels of TNF‐α, IL‐6, and IL‐1β in the serum of the mice (n = 8). (G) LDH levels in the serum of the mice (n = 8). (H) HE staining, F4/80 staining, and TUNEL staining of the livers of the mice. (I,J) Quantitative analysis of necrotic area and TUNEL‐positive cells in the livers of the mice (n = 8). (K–N) Relative mRNA expression of the proinflammatory genes in the livers of the mice (n = 8). (O‒Q) Protein levels and quantitative analysis of NLRP3 and ASC in the livers of the mice (n = 3). Data are presented as mean ± SEM. ** P < 0.01.
FIGURE 6
FIGURE 6
Liver‐specific knockdown of NEDD4L inhibits the protective effect of ART on liver injury. (A) Schematic diagram of the experimental procedure. The specific knockdown of NEDD4L in the livers of the mice was carried out by tail vein injection of AAV‐shNedd4l. Four weeks after the injection, the mice were fasted for 16 h and then intraperitoneally injected with APAP. One hour later, the mice were treated with ART, and samples were collected 24 h later (n = 8). (B,C) The levels of ALT and AST in the serum of the mice (n = 8). (D–F) The levels of TNF‐α, IL‐6, and IL‐1β in the serum of the mice (n = 8). (G) LDH levels in the serum of the mice (n = 8). (H) HE staining, F4/80 staining, and TUNEL staining of the livers of the mice. (I,J) Quantitative analysis of necrotic area and TUNEL in the livers of the mice (n = 8). (K–N) Relative mRNA expression of the proinflammatory genes in the livers of the mice (n = 8). (O–Q) Protein levels and quantitative analysis of NLRP3 and ASC in the liver (n = 3). Data are presented as mean ± SEM. ** P < 0.01.
FIGURE 7
FIGURE 7
NEDD4L is responsible for the protective effects of ART on liver injury. (A) Schematic diagram of the experimental procedure. AAV‐Nedd4l was injected into Nedd4l−/− mice by tail vein, successfully restoring the specific expression of NEDD4L in the liver. Four weeks after the injection, the mice were fasted for 16 h and then intraperitoneally injected with APAP. One hour later, the mice were treated with ART, and samples were collected 24 h later (n = 8). (B,C) The levels of ALT and AST in the serum of the mice (n = 8). (D–F) The levels of TNF‐α, IL‐6, and IL‐1β in the serum of the mice (n = 8). (G) LDH levels in the serum of the mice (n = 8). (H) HE staining, F4/80 staining, and TUNEL staining of the livers of the mice. (I,J) Quantitative analysis of necrotic area and TUNEL‐positive cells in the livers of the mice (n = 8). (K–N) Relative mRNA expression of the proinflammatory genes in the livers of the mice (n = 8). (O–Q) Protein levels and quantitative analysis of NLRP3 and ASC in the livers of the mice (n = 3). Data are presented as mean ± SEM. * P  < 0.05, ** P  < 0.01.
FIGURE 8
FIGURE 8
NEDD4L is a direct target of ART. (A) CETSA was used to measure the binding ability of ART to NEDD4L in HepG2 cells. (B) DARTS was used to measure the binding ability of ART to NEDD4L in HepG2 cells (n = 3). (C) A detailed presentation of the stable three‐dimensional structure and binding sites of the binding sites between ART and NEDD4L based on molecular dynamics simulation. (D) SPR was used to detect the interaction between ART and NEDD4L. (E) Fluorescence colocalization of FITC‐ART and mCherry‐NEDD4L in HepG2 cells. (F) CETSA was used to measure the binding ability of ART to the NEDD4L mutant in HepG2 cells. (G) DARTS was used to measure the binding ability of ART to the NEDD4L mutant in HepG2 cells (n = 3). (H) Co‐IP was used to determine the interaction between NEDD4L (WT) or NEDD4L (MUT) and HA‐TXNIP in HepG2 cells treated with ART. (I) The protein level of TXNIP in HepG2 cells treated with ART after mutation of NEDD4L.
FIGURE 9
FIGURE 9
Working model of ART in liver injury. ART directly targets NEDD4L, enhances the interaction between NEDD4L and TXNIP, and promotes the ubiquitination and degradation of TXNIP, thereby alleviating liver injury.
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