Introduction
Herbs and their components account for 25.3% of drug-induced liver injuries,1 with a higher prevalence observed in Eastern countries. Pyrrolizidine alkaloids (PAs)-induced hepatotoxicity is an increasing concern in clinical practice,2 due to the detection of PAs in common dietary items.3 Pyrrolizidine alkaloids-induced liver injury (PA-ILI) is characterized by hepatic sinusoidal obstruction, which can be partly attributed to platelet aggregation.4,5 Importantly, PA-ILI patients present with a reduced count of peripheral platelets,6 indicating systemic hematological changes during the progression of PA-ILI that resemble the pathological conditions seen in acute liver failure.7
Von Willebrand factor (VWF) plays a crucial role in regulating platelet accumulation and clearance.8 Blocking VWF reduces hepatic platelet accumulation and promotes liver repair in cases of acetaminophen-induced liver injury.8 The presence of VWF is regulated by metalloproteinases, particularly a disintegrin and metalloproteinase with a thrombospondin type I motif member 13 (ADAMTS13).9 Studies have shown that an increased VWF/ADAMTS13 ratio may correlate with poor outcomes in patients with acute liver injury10 and hepatic carcinoma.11 This suggests that alterations in the VWF/ADAMTS13 ratio could regulate hepatic platelet activity in PA-ILI. Therefore, this study investigated the roles of ADAMTS13 and VWF in PA-ILI in both mice and patients, focusing specifically on platelet accumulation in the liver.
Methods
Establishment of a mouse model
Experiments were conducted on eight-week-old mice (male, C57BL/6J mice, Beijing Vital River Laboratory, Beijing, China) selected randomly. All procedures involving animals complied with the Guide for the Care and Use of Laboratory Animals (8th edition, revised 2011) by the US National Institutes of Health and were approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University (Shanghai, China; Approval No. 2020-006).
To induce PA-ILI, monocrotaline (MCT; C101556, 400 mg/kg; Aladdin, Shanghai, China), a toxic PA, was administered via intragastric gavage for six days. A 0.9% sodium chloride solution served as the solvent control.12 To assess platelet accumulation’s involvement in PA-ILI, Fraxiparine (0.1 mL/10 kg), a low-molecular-weight heparin (LMWH), was administered subcutaneously on day (D) 4 and D5. The role of ADAMTS13 was evaluated by injecting recombinant ADAMTS13 (1 µg dissolved in 100 µL endotoxin-free PBS) via the tail vein on D4 and D5 (Fig. 1A). Liver and blood samples were collected for analysis.
Patients and samples
Demographic and laboratory data from confirmed drug-induced liver injury cases, hospitalized from 2012 to 2019 at Zhongshan Hospital, Fudan University, were collected.
To diagnose PA-ILI, the Nanjing criteria were applied in this study.13 Non-PA-ILI patients were also included (i.e., those with a history of taking medications not containing PAs within the past three months and a Roussel Uclaf Causality Assessment Method score greater than three). Patients diagnosed with viral infections, autoimmune diseases, alcohol abuse, fatty liver, heart failure, Budd-Chiari syndrome, congenital liver disease, or liver carcinoma were excluded from both the PA-ILI and non-PA-ILI groups. Patients lacking information on their medications were also excluded.14 The 5-point scale from the Drug-Induced Liver Injury Network (DILIN) was used to assess the severity of liver injury.
Cancer-adjacent normal tissue samples from patients with liver malignancies served as the control group. Hematoxylin and eosin staining of these liver tissues showed no signs of congestion, sinusoidal dilation, or inflammatory response, and liver cell structures appeared neatly arranged. Liver biopsy samples from PA-ILI and non-PA-ILI cases, as well as adjacent non-tumor liver tissues from hepatic cancer patients (control group), were collected [Zhongshan Hospital, Fudan University, Approval No. B2018-070(2)]. Patient plasma samples were stored at −80°C for ADAMTS13 detection.
High-throughput RNA sequencing (RNA-seq) and bioinformatic analysis
High-throughput RNA-seq was conducted using the Tusanqi-induced liver injury model. Tusanqi is widely utilized in traditional Chinese medicine, but it may cause liver injury15,16 due to its content of four PAs: senecionine, seneciphylline, seneciphylline N-oxide, and senecionine N-oxide. According to our previous study, Tusanqi-induced liver injury in mice was also applied in this study. FASTQ data were analyzed using fastp,17 HISAT2,18 and htseq-count19 programs. Differentially expressed genes (DEGs) were identified using the “DEseq2” package in R,20 when their fold change > 1 and adjusted p-value < 0.05. The “ClusterProfiler” package21 was used to explore the biological processes of DEGs. The STRING online database (https://www.string-db.org/ ) was used to predict potential correlations between VWF and DEGs.22 RNA-seq data were deposited in the Gene Expression Omnibus database (GSE171874).
Measurement of mouse liver injury
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) kits were obtained from Wuhan Servicebio Culture Consulting Co., Ltd. (Wuhan, China). Paraffin-embedded liver tissues underwent hematoxylin and eosin staining. To assess liver necrotic areas, at least two sections from the left lateral lobe of each mouse and patient sample were evaluated and quantified by a blinded technician using ImageJ software. Liver sections were scanned using the Zeiss Scan Z1 system (Carl Zeiss AG, Oberkochen, Germany).
Immunofluorescence staining
Fibrinogen, ADAMTS13, VWF, and CD41 were detected by immunofluorescence staining. Antigen retrieval was conducted at 100°C for 15 m in Tris-EDTA (pH 9.0). After blocking with 10% normal goat serum at room temperature for 1 h, antibodies against CD41 (ab134131, 1:50 for mouse samples and 1:200 for human samples; Abcam, Cambridge, UK), VWF (ab11713, 1:200; Abcam), and fibrinogen gamma (FGG; 15841-1-AP, 1:100; Proteintech, Wuhan, China) were incubated at 4°C overnight. Corresponding secondary antibodies were applied the following day. Fluorescence images were obtained using the Zeiss Scan Z1 system and analyzed with ImageJ software. Positive areas of platelet accumulation and VWF were quantified in three to four randomly selected images per sample.
ELISA
Plasma ADAMTS13 levels were measured using a Human ADAMTS13 Quantikine ELISA kit (DADT130; R&D Systems, Minneapolis, MN, USA) per the manufacturer’s instructions.23
RNA extraction and real-time PCR analysis
Total RNA was extracted from mouse liver tissues using a Total RNA Kit (DP419; Tiangen, Beijing, China) and synthesized into cDNA using the Superscript II reverse transcriptase kit (TOYOBO, Tokyo, Japan). Quantitative PCR amplification was conducted using a Roche LightCycler® 480 II Real-time PCR system and SYBR Green (Tiangen). Primer sequences for GAPDH and ADAMTS13 are presented in Supplementary Table 1.
Western blot analysis
Protein concentrations of cell and tissue lysates were determined using a BCA protein assay kit (Beyotime, Shanghai, China). Proteins were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and primary antibodies were used to detect caspase-3 (9665S, 1:1,000; Cell Signaling Technology, Danvers, MA, USA), cleaved caspase-3 (9661T, 1:1,000; Cell Signaling Technology), FGG (15841-1-AP, 1:1,000; Proteintech), ADAMTS13 (A8482, 1:1,000; ABclonal, Woburn, MA, USA), and β-actin (1:5,000; Sigma-Aldrich, St. Louis, MO, USA). Protein expression was quantified using ImageJ software.
Statistical analysis
Data were analyzed using GraphPad Prism 9.0 software (GraphPad Software Inc., La Jolla, CA, USA). Student’s t-test was applied to determine statistical significance, with data presented as mean ± standard deviation. A p-value < 0.05 was considered statistically significant.
Results
Hepatic platelet accumulation and increased VWF expression were observed in PA-ILI mice and patients
Apparent hepatocyte death and congestion were noted in mouse livers stimulated with MCT (Supplementary Fig. 1). The TUNEL assay showed increased apoptotic areas in MCT-induced mice (Supplementary Fig. 1). Accumulation of platelets, as indicated by CD41 staining (also known as platelet glycoprotein [GP] IIb/IIIa), was found in the MCT-stimulated livers but not in the control mice (Fig. 1B). Given that VWF regulates platelet adhesion and accumulation through GPIb/V/IX and CD41,24 further experiments demonstrated that VWF expression was upregulated in the MCT-induced mouse model (Fig. 1C).
In PA-ILI patients, pathological examination revealed hepatic sinus dilatation and congestion in the liver (Supplementary Fig. 2A). CD41 expression was significantly elevated in PA-ILI patients compared to the control group (p = 0.0004) and non-PA-ILI patients (p = 0.0006) (Fig. 2A). Moreover, VWF protein expression was significantly increased in PA-ILI patients compared to non-PA-ILI patients (p = 0.0105) and the control group (p = 0.0404) (Fig. 2B).
Reduction in platelet accumulation with the improvement of hepatic function
LMWH, a traditional anticoagulant targeting factors IIa (thrombin) and Xa, has been reported to reduce platelet deposition.25,26 In the present study, LMWH treatment significantly reduced CD41 expression in MCT-induced mice (p = 0.0460) (Fig. 3A). Furthermore, LMWH treatment reduced the hepatic necrotic area (p = 0.0008) (Fig. 3B) and significantly improved plasma ALT and AST levels in MCT-induced mice (ALT: MCT vs. MCT+LMWH, 350.2455 ± 284.5684 vs. 40.9105 ± 10.2209 U/L, p = 0.0252; AST: MCT vs. MCT+LMWH, 283.8675 ± 130.7658 vs. 78.0098 ± 13.8228 U/L, p = 0.0204).
These data indicate that hepatic platelet accumulation and VWF upregulation occurred in both PA-ILI mouse and patient groups. Preventing these changes led to reversals of these effects and improvement in function.
Activation of the coagulation cascade is one of the mechanisms leading to platelet aggregation.27 However, in the present study, hepatic expression of FGG was unchanged in PA-ILI patients compared to both the control group and non-PA-ILI patients (Supplementary Fig. 2B), indicating that the coagulation cascade was not activated in PA-ILI patients.
Prediction of ADAMTS13 as a regulator of VWF in PA-ILI by bioinformatic analysis
RNA-seq identified 1,209 DEGs, with 631 upregulated and 578 downregulated genes in the PA-ILI group compared to the control group (Fig. 4A and B). Biological processes related to wound healing, hemostasis, coagulation, and platelet-related pathways were enriched (Fig. 4C and D). To explore potential regulators of VWF and platelet accumulation, VWF-related proteins (Fig. 4E), sorted upstream and downstream by the STRING22 were compared with DEGs associated with these biological processes. This analysis identified three genes: Selp (Selectin P), which was upregulated, and F8 (Coagulation Factor VIII) and ADAMTS13, which were both downregulated (Fig. 4F).
Reduced expression of ADAMTS13 protein regulates VWF upregulation in PA-ILI
F8, a stable coagulation factor, is activated by separating from the VWF molecule under pathological conditions.28 Given that Selp protein was not detected in the liver, attention was directed toward ADAMTS13, a circulating metalloproteinase that modulates VWF activity.29
Hepatic ADAMTS13 levels were reduced in the PA-ILI group compared to the control group (p = 0.0310) and non-PA-ILI patients (p = 0.0228) (Fig. 5A). Plasma ADAMTS13 levels were also downregulated in the PA-ILI group compared to non-PA-ILI patients (p < 0.0001) (Fig. 5B). Furthermore, peripheral platelet count (p = 0.0120) and plasma ADAMTS13 levels (p = 0.0015) were significantly lower in cases of severe liver injury (grade 3-4) than in mild liver injury cases (grade 1-2) (Fig. 5B).
In the mouse model, plasma ADAMTS13 levels were decreased in the MCT group compared to the control group (Fig. 5C). Both mRNA (Fig. 5D) and protein (Fig. 5E and F) expression levels of ADAMTS13 in the liver were reduced, while an upregulation of ADAMTS13 protein was observed in kidney tissue, with no significant changes noted in lung tissue (Fig. 5C). Additionally, there was a gradual decrease in ADAMTS13 protein levels over time (72 h, 96 h, and 144 h) under MCT influence, while both cleaved and total caspase 3 protein levels were elevated (Fig. 5E).
Administration of rADAMTS13 significantly restored plasma levels and protein expression of ADAMTS13 in the liver of MCT mice (Fig. 6A), reduced liver necrotic area (Fig. 6B and C), and decreased CD41 (p = 0.0005) and VWF (p = 0.0223) protein expression levels (Fig. 6D). Liver abnormalities were evident, although blood abnormalities were not yet apparent, as shown in Supplementary Figure 3.
Discussion
The present study found that downregulation of ADAMTS13 led to increased platelet accumulation in the liver, which could be involved in the pathogenesis of PA-ILI. The severity of liver injury was related to both peripheral platelet count and ADAMTS13 deficiency. Preventing platelet accumulation with rADAMTS13 reduced cell necrosis and restored liver function.
The role of platelet accumulation in liver disease remains controversial.30 Platelet aggregation promotes liver regeneration in liver transplantation patients,31 yet it deteriorates liver function in patients with hepatocellular carcinoma,32 non-alcoholic hepatitis,32 and ischemia–reperfusion liver disease. Contrary to a previous study,33 the present study revealed that alleviating hepatic platelet accumulation through LMWH or rADAMTS13 reduced PA-ILI, suggesting that platelet accumulation contributes to PA-ILI and leads to liver impairment.
ADAMTS13 cleaves VWF multimers by recognizing multiple A2 sites in the VWF domain. Recombinant ADAMTS13 has primarily been documented in the literature to exhibit no adverse effects on the liver.34,35 In our investigation, the group treated exclusively with rADAMTS13 showed no significant differences in ALT levels compared to the control group, with both groups maintaining low values. Although the ALT level in the group treated with both MCT and rADAMTS13 was slightly lower than in the MCT-only group, this difference was not statistically significant, potentially due to the prolonged half-life of ALT, which can extend up to 57 h (Supplementary Fig. 3). The downregulation of ADAMTS13 in both patients and the mouse model contributed to increased hepatic VWF protein expression and platelet accumulation in PA-ILI. Recombinant ADAMTS13 was able to reduce hepatic platelet accumulation and alleviate PA-ILI.
According to findings by Masahito Uemura et al.36 and Nancy A. Turner et al.,37 hepatic stellate cells may be the primary cellular source of ADAMTS13 in the human liver. Additionally, ADAMTS13 has been detected in vascular endothelial cells and human platelets.37 However, these results do not specifically address drug-induced liver injury. The precise origin of ADAMTS13 in liver injury associated with PA-ILI remains uncertain. We measured ADAMTS13 protein expression in cultured hepatic, stellate, and endothelial cells stimulated with MCT. After 24 h of stimulation, MCT significantly reduced ADAMTS13 expression in cultured hepatic cells, but not in stellate or endothelial cells (Supplementary Fig. 4). Based on these preliminary findings, hepatocytes may act as the principal source of ADAMTS13 in this disease context. Therefore, in PA-ILI, impaired hepatocytes produce less ADAMTS13, leading to increased VWF expression and platelet aggregation in a vicious cycle. Further validation of these findings will require additional investigations using primary cell cultures or other model systems.
Supporting information
Supplementary Table 1
qPCR primers used in this study.
(DOCX)
Supplementary Fig. 1
Liver tissue of mouse model.
Male C57BL/6J mice were given 400 mg/kg MCT or saline (i.p.), and samples were collected after 72 h, 96 h, and 144 h to establish the mouse liver injury model. Mice liver necrosis was determined by hematoxylin and eosin staining. Liver section apoptosis was determined by TUNEL assay. MCT, monocrotaline; H&E, Hematoxylin and eosin staining.
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Supplementary Fig. 2
Patient liver tissue and hepatic FGG labeling.
Paraffin sections from patients in three groups—control group (adjacent non-tumor liver tissue), PA-ILI group, and non-PA-ILI group—were obtained from Zhongshan Hospital. (A) Representative images of hematoxylin and eosin-stained liver sections in patients. (B) Representative images of hepatic FGG labeling (red). PA-ILI, pyrrolizidine alkaloids-induced liver injury; FGG, fibrinogen gamma; DAPI, 4′,6-diamidino-2-phenylindole.
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Supplementary Fig. 3
Plasma alanine aminotransferase (ALT) level and platelet (PLT) count in control, MCT, rADAMTS13, and MCT + rADAMTS13 group mice.
The ALT level and PLT count in the group treated with both MCT and rADAMTS13 were marginally lower than in the MCT-only group; however, no significant difference between these two groups was observed. MCT, monocrotaline.
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Supplementary Fig. 4
Representative western blots of ADAMTS13 and apoptosis-related proteins in L-02, sinusoidal epithelial, and LX-2 cells.
ADAMTS13, VWF, and apoptosis-related proteins in L-02, SEC, and LX-2 cells were detected by western blotting. L-02, SEC, and LX-2 cells were all treated with MCT for 24 h, 36 h, and 48 h. MCT, monocrotaline; VWF, von Willebrand factor; SEC, sinusoidal epithelial cell.
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