Skip to main content
NCBI home page
BMC Gastroenterology logoLink to BMC Gastroenterology
. 2026 Mar 4;26:221. doi: 10.1186/s12876-026-04691-y

α-AMA induced liver failure is associated with elevation of HMGB1 lactylation modification in mice

Shaofang Huang 1, Jian Tao 1, Hui Xiao 1, Shuling Wu 1, Wei Ye 1, Shipeng Huang 1,✉
PMCID: PMC13067735  PMID: 41781906

Abstract

Background

Acute liver failure (ALF) induced by poisonous mushrooms represents the most significant threat to humans from mushroom poisoning, primarily attributable to α-amanitin (α-AMA). This study aimed to investigate the mechanism of α-AMA in liver failure.

Methods

Mice were injected intraperitoneally with α-AMA to induce liver failure in vivo. Liver tissues were stained with H&E, Masson and Sirius Red to assess pathological changes in liver tissue. Human hepatocytes (L02) were cultured with α-AMA. Cell viability and apoptosis were detected by CCK-8 and flow cytometry. Immunohistochemistry, immunofluorescence, qRT-PCR and western blotting were used to detect HMGB1 expression. Co-IP combined with western blot assay was used to detect the level of HMGB1 lactylation in cells. In addition, the levels of inflammatory factors were determined using enzyme-linked immunosorbent assay (ELISA).

Results

Compared with the control group, mice induced with α-AMA exhibited significant hepatocyte edema, increased interstitial inflammatory cell infiltration, and elevated fibrosis levels. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) were also markedly elevated in α-AMA-treated mice. α-AMA treatment significantly increased hepatic lactate levels and dramatically upregulated HMGB1 expression. Furthermore, in vitro experiments confirmed that α-AMA dose-dependently enhanced both HMGB1 expression and its lactylation level. Critically, α-AMA-induced cellular injury could be effectively reversed by interfering with HMGB1 expression and lactylation.

Conclusion

α-AMA elevated the expression of HMGB1 by promoting the lactated modification level of HMGB1, which in turn induced hepatocellular injury and aggravated liver failure.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12876-026-04691-y.

Keywords: Α-amanitin, Liver failure, HMGB1, Lactylation, Proliferation, Apoptosis

Background

Poisoning caused by the consumption of poisonous mushrooms is one of the most serious types of food poisoning in China and the world. Among all the fatal cases of mushroom poisoning, the proportion of the cases caused by Amanitin phalloides is as high as 95% [1]. It is also known as the death cap and is considered the most toxic mushroom species in the world [2, 3]. Acute liver failure, rhabdomyolysis, acute renal failure and hemolysis caused by poisonous mushrooms are the most significant threats of mushroom poisoning to the Chinese population [4–6]. Its content of α-amanitin (α-AMA) is a major cause of severe liver and kidney damage from mushroom poisoning [7]. α-AMA is absorbed into the liver through the intestine, which targets the liver and causes liver damage. This toxin is not metabolized in the liver but is excreted in large quantities by the kidneys through urine, thereby damaging kidney tissues [8]. The mechanism of α-AMA-induced liver injury is currently unclear and still needs to be explored.

High mobility group box 1 (HMGB1) is an abundant nuclear protein in the HMGB subfamily that is expressed in multiple organs [9]. A previous study has found that HMGB1 is elevated in LPS-induced liver injury [10]. HMGB1 is induced to translocate into the cytoplasm when HMGB1 is very close to or located in regions that have been post-translationally modified with nuclear localization sequences. Ultimately, it can lead to the release of HMGB1 during inflammation [11–13]. Additionally, serum lactate has been considered a biomarker of prognosis in liver failure. Elevated serum lactate levels are positively associated with liver failure mortality [14]. In patients with hepatic failure or significant hepatic hypoperfusion, serum lactate levels may be elevated due to the inability of the liver to metabolize the additional lactate load [15, 16]. However, the role of lactate in the acetylation and release of HMGB1 has been reported in liver failure, especially α-AMA-induced liver failure [6]. In addition, oxygen glycolysis has been reported to stimulate HMGB1 acetylation in macrophages [16]. Thus, HMGB1 may play a role in α-AMA-induced liver failure. This work aims to investigate the molecular mechanism of α-AMA in causing liver failure and provides new therapeutic targets and strategies for its treatment.

Methods

Animals

All animal procedures were conducted in accordance with the Animal Care and Use Committee guidelines of The First Affiliated Hospital of Nanchang University and adhered to the ARRIVE guidelines for reporting animal research. The study protocol was approved by the Institutional Animal Ethics Committee of The First Affiliated Hospital of Nanchang University (approval number: CDYFY-IACUC-202304QR051). Male BALB/c mice weighing 20 ± 2 g at 7 weeks of age were purchased from Huafukang Biotechnology Co., Ltd (Beijing, China) and housed under SPF conditions with constant temperature (21–26 °C) and constant humidity (40–70%) with free access to food and water. All efforts were made to minimize pain, distress, and the number of animals used. At the end of the study, mice were humanely euthanized using an overdose of pentobarbital sodium (150 mg/kg, intraperitoneally), in compliance with the AVMA Guidelines for the Euthanasia of Animals (2020 edition). Death was confirmed by cessation of heartbeat and respiration.

Animal groups and treatment

Mice were randomly divided into 4 groups: Control, low dosage, medium dosage and high dosage groups. Mice were injected intraperitoneally with 0.23, 0.35, 0.53 mg/kg of α-AMA (Med Chem Express, Monmouth Junction, NJ, USA) following the previous study reported [17]. The sample size in each group was determined using the Resource Equation Method, aiming to maintain the error degrees of freedom (DF) in an analysis of variance (ANOVA) within the acceptable range of 10 to 20. For the two-group comparisons planned in this study, this method yielded a recommended sample size range of 6 to 11 animals per group. A sample size of eight mice per group was chosen to ensure sufficient statistical power while adhering to the 3R (Replacement, Reduction, Refinement) principles. The mice used were numbered, and a random number was generated for each animal using Excel rand () function or random number table, sorted in ascending order according to the random number, and evenly distributed to each group according to the serial number.

The inclusion criteria used in this study were as follows: the biochemical index ALT/AST increased significantly, TBIL increased ≥ 17.1 µmol/L every day, and there were obvious clinical symptoms (apathy, decreased activity, jaundice, decreased appetite, and may be associated with hepatic encephalopathy related manifestations); The exclusion criteria were as follows: the biochemical /pathological threshold was not reached (such as ALT < 3 times normal, PTA > 40%), or the symptoms were atypical, and severe non hepatic death occurred during modeling. The survival status of each group was as follows: in the Control and low dosage (0.23 mg/kg α-AMA) groups: no mice died; in the medium dosage (0.35 mg/kg α-AMA) group and high dosage (0.53 mg/kg α-AMA) group, 2 mice died and 6 survived. Finally, 6 mice in each group were selected for subsequent analysis. During the injection of α-AMA, the mice did not receive any drugs. Forty-eight hours after injection, mice were euthanized with 150 mg/kg pentobarbital sodium (intraperitoneally). Liver tissue and peripheral blood were taken from mice for further analysis.

Cell culture and treatment

Human embryonic hepatocytes HHL-5 were purchased from Edit Gene Biotechnology Co., Ltd (Guangzhou, China). HHL-5 cells were cultured in RPMI 1640 (R8758, Merck, Shanghai, China) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Merck) at 37℃ in a humidified atmosphere of 5% CO2. HHL-5 cells were treated with α-AMA at concentrations of 0.5, 1, 2, 5, 10 µM and 37℃ for 24 h. Additionally, HHL-5 cells were treated with 10 mM sodium lactate (HY-B2227B, Med Chem Express) at 37℃ for 6 h. HHL-5 cells were treated with 20 mM Sodium oxalate (379735, Merck) at 37℃ for 30 min. Sodium oxalate was used as an inhibitor of lactylation.

Cell transfection

Small interfering RNA specially targeting HMGB1 (si-HMGB1-1/2/3: 5’-GGA TTA CGC TGA CGA AAG AGA-3’; 5’-GCC ACA GAG TGC ATG TTC ACA-3’; 5’-GAT CTG TCC CTG CTG CTC TAA-3’) was constructed for HMGB1 silencing (Genepharm, Shanghai, China). Scrambled siRNA (si-NC: 5’-TAG CGC TGA GGC GTG CAA GCT GAT TCT TA-3’) served as control. Cells were transfected with si-HMGB1 or si-NC applying Lipofectamine™ 2000 reagent (Thermo Fisher Scientific, Waltham, MA, USA).

Cell viability assay

Cells were resuspended and inoculated in 96-well plates at a density of 5 × 104/100 µL. Five replicate holes were set up for each group. Cells were placed in a constant temperature incubator for 48 h. Cells were incubated with 10 µL CCK-8 reagent (Beyotime, Shanghai, China) for 1 h. Absorbance at 450 nm (A450) was measured using an enzyme labeler (ELX-800 BIOTEK, USA).

Histochemical analysis

Liver tissues were fixed with paraformaldehyde and embedded with paraffin. Paraffin Sect.  (4 μm) were dewaxed with xylene and dehydrated by 90%, 80% and 70% ethanol for 5 min. For hematoxylin and eosin (HE) staining, the sections were stained with hematoxylin for 5 min and counterstained with eosin for 1 min. For evaluation of hepatic fibrosis, Masson’s Trichrome Staining Kit (Beyotime) was used to stain liver tissues. Paraffin sections were stained with Weigert’s iron hematoxylin for 15 min. The sections were then differentiated with 1% hydrochloric acid alcohol for 30 s. After that, the sections were stained with 1% aniline blue for 5 min and then incubated with ponceau-acid fuchsin for 10 min. Applying the Modified Sirius Red Stain Kit (Solarbio, Beijing, China), the sections were stained with Weigert’s iron hematoxylin for 15 min and counterstained with Sirius Red reagent for 20 min. Following dehydration and clearing, the sections were sealed with neutral gum. Finally, they were observed under a normal light microscope.

Detection of relevant indicators

Liver tissue and serum samples were collected from each group of mice. Tissues were processed through a cryo-ultrasonic crusher. Then, the samples were centrifuged at 12,000 r. min− 1 for 5 min in a 4℃ low-temperature centrifuge. The supernatant was taken and tested for aspartate aminotransferase (AST) (MAK055-1KT, Merck), alanine aminotransferase (ALT) (mak052, Merck) and total bilirubin (TBIL) (BC5185, Solarbio) activity as well as lactate (BC2235, Solarbio) in the tissue according to the kit procedure. The levels of IL-6, TNF-α and HMGB1 in serum were detected by applying the Mouse IL-6 ELISA kit (EM30325S; WELLBIO; Shanghai, China), Mouse TNF-α ELISA kit (EM3311S; WELLBIO) and Mouse HMGB1 ELISA kit (EM30645S; WELLBIO).

Immunohistochemistry

Mouse liver tissues were fixed with paraformaldehyde and embedded with paraffin. The sections were incubated with 3%H2O2 and blocked with goat serum. After that, the sections were incubated with anti-HMGB1 (ab79823; Abcam; Cambridge, MA, USA) at 4 °C overnight, and then stained with HRP-IgG (ab6721; Abcam). Sections were color-developed using DAB. The expression of HMGB1 was observed under an optical microscope.

Immunofluorescence

Cells were fixed in 4% paraformaldehyde for 15 min. The sections were incubated with 3%H2O2 and blocked with goat serum. The sections were incubated with anti-HMGB1 (ab18256; Abcam) at 4 °C overnight and then stained with Alexa Fluor® 488-IgG (ab150077; Abcam) at 37 °C for 2 h. The nuclei were dyed again with DAPI. After blow-drying, the film was sealed with an anti-fluorescence bursting agent and the image was acquired under a fluorescence microscope.

Western blotting

Mouse liver tissue and human hepatocyte samples were collected. Total tissue and cellular proteins were extracted by adding an appropriate amount of RIPA lysate. The BCA method was used to determine the protein concentration. Proteins were separated by SDS-PAGE electrophoresis after thermal denaturation and then transferred to PVDF membranes. PVDF membranes were blocked with 5% skimmed milk/TBST mixture at room temperature for 2 h. After the membrane was washed with TBST, incubated with HMGB1 (MA5-17278, Thermo Fisher Scientific), cleaved caspase-3 (PA5-114687, Thermo Fisher Scientific), Bcl-2 (138800, Thermo Fisher Scientific), BCL-XL (MA5-15142, Thermo Fisher Scientific), RIPK3 (A5431, Abclonal), PCNA (14-9910-82, Thermo Fisher Scientific) and Ki67 (MA5-14520, Thermo Fisher Scientific) antibodies at 4 °C overnight. The membrane was washed and stained with the secondary antibody HRP-IgG (ab6721; Abcam) at 37 °C for 2 h. Histone H3 and β-actin served as loading control. ECL luminescent solution was used to develop WB bands. The bans were analyzed by ImageJ software.

Co-Immunoprecipitation (Co-IP)

HMGB1 antibody was incubated with protein A + G Beads for 8 h at 4 °C. Antibody-conjugated Beads were added to the cellular protein lysate and co-incubated overnight to obtain the target protein. The target protein was separated from the beads. The results of immunoprecipitation were verified by western blotting.

Quantitative real-time PCR (qRT-PCR)

Total RNA extraction from hepatocytes was performed by TRIzol lysis. RNA integrity was examined by 1.5% denaturing agarose gel electrophoresis. RNA concentration and purity were determined using an OD260/OD280 ratio employing a nucleic acid analyzer. The cDNA was synthesized applying PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, Beijing, China) and stored at -20℃. Relative mRNA expression levels were determined by fluorescence quantitative PCR utilizing TB Green® Premix Ex Taq™ (Tli RNaseH Plus) (Takara). The internal reference primers and gene-specific primers were designed by Primer 5.0 software. Primers were synthesized by Shanghai Jerry Biotechnology Co., Ltd.

Flow cytometry

Hepatocytes were incubated in culture flasks for 24 h, and the apoptosis rate of hepatocytes was detected by V-APC/7AAD Apoptosis Kit (MULTI SCIENCES, Shanghai, China). Cells were washed with pre-cooled PBS 3 times and the supernatant was discarded. Cells were co-stained at 28℃ for 15 min and incubated on ice for 15 min. The apoptosis rate was detected by employing flow cytometry (BD, FAC SCalibur, NY, USA). Data were analyzed by CELLQUESTPRO software (BD).

Statistical analysis

The experimental assessments were conducted by double blind design: investigators blinded to group allocation carried out the experiment and researchers blinded to the experiment operation carried out pathological evaluation, so as to ensure the authenticity of the experimental results. Animals with unsuccessful model establishment were excluded from final analysis. The data were analyzed by one-way ANOVA with SPSS 22.0 software, with LSD post hoc analyses. Results were statistically significant at P < 0.05, P < 0.01, and P < 0.001 and highly significant at P < 0.001. All experiments were performed at least three times independently and the data were expressed as mean ± standard deviation (x ± s).

Results

Liver failure in mice was induced by α-AMA

To explore the function role of HMGB1 in liver failure, a mouse model of liver failure was constructed by administrating with α-AMA. The pathological changes of liver tissues were examined by H&E staining. It was found that α-AMA caused significant hepatocyte edema, cytoplasmic vacuolation and interstitial inflammatory cell infiltration. Hepatocyte morphological damage was increased with increasing dosages of α-AMA (Fig. 1A). Fibrosis of liver tissues was evaluated by performing Masson and Sirius red staining, α-AMA treatment elevated fibrosis of liver tissues in mice in a dosage-dependent manner (Fig. 1B-C). Then, serum liver function indicators were evaluated. ALT, AST and TBIL levels were significantly elevated in mice in the presence of α-AMA (0.23, 0.35, 0.53 mg/kg) (Fig. 1D-F). α-AMA treatment caused a significant increase in lactate levels in the liver tissue of mice, and their levels increased progressively with increasing dosage of α-AMA (Fig. 1G). It indicated that α-AMA-treated mice appeared a bilirubin-transaminase separation and liver function deteriorated. Additionally, α-AMA treatment elevated the levels of IL-6, TNF-α, IL-10 and HMGB1 in the serum of mice in a dosage-dependent manner, as determined by ELISA (Fig. 1H-I). The expression of HMGB1 in liver tissue was further examined by immunohistochemistry. It was found that hepatic lactate metabolism was weakened with increasing concentrations of α-AMA, and high levels of lactate elevated the expression of HMGB1 in the liver tissues of mice (Fig. 1J). Western blot experiment also revealed that the expression of HMGB1 was elevated in the liver tissues of mice in the presence of α-AMA (Fig. 1K). Thus, Liver failure in mice was induced by α-AMA.

Fig. 1.

Fig. 1

Open in a new tab

Liver failure in mice was induced by α-AMA

Mice were injected α-AMA to induce liver failure. (A) The pathological change of liver tissues was examined by H&E staining. (B-C) Fibrosis of liver tissue was assessed by Masson and Sirius Red staining. (D-F) Detection of serum liver function indices ALT, AST and TBIL in mice. (G) Lactic acid content in liver tissues was detected. (H-I) The levels of IL-6, TNF-α, IL-10 and HMGB1 in the serum of mice by ELISA. (J) The expression of HMGB1 in liver tissue was detected by immunohistochemistry. (K) The expression of HMGB1 in liver tissue was determined by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group

α-AMA induced damage to hepatocytes

HHL-5 cells were treated with α-AMA to mimic liver failure in vitro. The results of CCK-8 assay are presented in Fig. 2A. It was found that cell viability of HHL-5 cells was inhibited by α-AMA treatment in a dosage-dependent manner. Results of flow cytometry showed that α-AMA treatment elevated apoptosis of HHL-5 cells, especially 10 µM α-AMA (Fig. 2B). Then, the expression of apoptosis-associated proteins cleaved caspase-3, Bcl-2, BCL-XL, necrotic apoptosis-related protein RIPK3 and proliferation-associated proteins PCNA, Ki67 were examined by western blotting. The results showed that the up-regulation of Caspase-3 and the down-regulation of Bcl-2, PCNA and Ki67 were dose-dependent after α-AMA treatment of HHL-5 cells (Fig. 2C, E). The western blotting results of BCL-XL and RIPK3 showed the lowest level of expression of BCL-XL and the highest level of expression of RIPK3 at a concentration of 5 µM (Fig. 2D). These data indicated that α-AMA induced damage to hepatocytes.

Fig. 2.

Fig. 2

Open in a new tab

Toxicity of α-AMA to hepatocytes

HHL-5 cells were treated with 0.5, 1, 2, 5, 10 µM α-AMA. (A) Cell viability was assessed by CCK-8 assay. (B) Cell apoptosis was detected by flow cytometry. (C-E) The expression levels of Cleaved caspase-3, Bcl-2, BCL-XL, RIPK3, PCNA and Ki67 were determined by western blotting. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group

Lactate and HMGB1 expression levels were increased in α-AMA-induced hepatocyte injury

The levels of lactate in HHL-5 cells were examined. As shown in Fig. 3A, the lactate content was increased with the dosage of α-AMA (Fig. 3A). The expression of HMGB1 was detected by western blotting. The results showed that the expression of HMGB1 was increased in HHL-5 cells following treatment of α-AMA (Fig. 3B). Results of immunofluorescence also demonstrated that HMGB1 expression was increased in HHL-5 cells in the presence of α-AMA. HMGB1 was expressed in both cytoplasm and nucleus. With the increasing concentration of α-AMA, HMGB1 was increased in the cytoplasm and decreased in the nucleus (Fig. 3C).

Fig. 3.

Fig. 3

Open in a new tab

Lactate and HMGB1 expression levels were increased in α-AMA-induced hepatocyte injury

HHL-5 cells were treated with 0.5, 1, 2, 5, 10 µM α-AMA. (A) Lactate content in cells was detected. (B) The expression of HMGB1 was detected by western blotting. (C) The level of HMGB1 was determined by immunofluorescence. *P < 0.05, **P < 0.01, ***P < 0.001 vs. control group

α-AMA induced hepatocyte injury by promoting HMGB1 expression

HMGB1 was silenced in HHL-5 cells treated with 5 µM α-AMA to determine the functional role of HMGB1 in α-AMA-induced liver failure. The expression of HMGB1 was detected by qRT-PCR and western botting. The results showed that the expression of HMGB1 was deceased in HHL-5 cells after si-HMGB1 interference treatment, especially si-HMGB1-3. Therefore, si-HMGB1-3 was used for knockdown of HMGB1 in subsequent experiments (Fig. 4A-B). Moreover, the influence of HMGB1 deficiency on cell viability and apoptosis of HHL-5 cells was determined by CCK-8 and flow cytometry. Compared with the α-AMA + si-NC group, HMGB1 silencing elevated cell viability of HHL-5 cells (Fig. 4C). Similarly, apoptotic cells were decreased in α-AMA-treated HHL-5 cells following transfection of si-HMGB1 (Fig. 4D). Additionally, HMGB1 inhibition reduced the levels of lactate in α-AMA-treated HHL-5 cells (Fig. 4E). Thus, α-AMA induced hepatocyte injury by elevating HMGB1 expression.

Fig. 4.

Fig. 4

Open in a new tab

α-AMA induced hepatocyte injury by promoting HMGB1 expression

HHL-5 cells were treated with 5 µM α-AMA and then transfected with si-HMGB1 or si-NC. (A) The expression of HMGB1 was detected by qRT-PCR. (B) The expression of HMGB1 was determined by western blotting. (C) Cell viability was assessed by CCK-8 assay. (D) Cell apoptosis was detected by flow cytometry. (E) Lactate content in cells was detected. *P < 0.05, **P < 0.01, ***P < 0.001 vs. α-AMA + si-NC group

α-AMA-induced cellular damage was reversed by inhibiting HMGB1 lactylate

HHL-5 cells were treated with sodium lactate to mimic high lactylation conditions. Compared with the control group, the levels of lactate were elevated in HHL-5 cells following sodium lactate or α-AMA treatment (Fig. 5A). The HMGB1 protein levels in the cells were examined by western blotting. It was found that HMGB1 expression was significantly elevated in HHL-5 cells in the presence of sodium lactate or α-AMA (Fig. 5B). Both sodium lactate and α-AMA treatment elevated the expression of HMGB1 in the cytoplasm and reduced the expression of HMGB1 in the nucleus of HHL-5 cells (Fig. 5C). Furthermore, the level of HMGB1 lactylation in HHL-5 cells was detected by Co-IP combined with western blot assay. The results showed that the level of HMGB1 lactylation in HHL-5 cells was increased by the sodium lactate and α-AMA treatment. The level of HMGB1 lactylation was significantly higher in α-AMA-treated HHL-5 cells than in sodium lactate-treated HHL-5 cells (Fig. 5D). Additionally, sodium oxalate was utilized as an inhibitor of lactylation. The level of HMGB1 lactylation was increased in α-AMA-treated HHL-5 cells, which was reversed by sodium oxalate treatment (Fig. 5E). Then, cell viability and apoptosis of HHL-5 cells were assessed by CCK-8 and flow cytometry. α-AMA treatment caused a decrease in cell viability and led to an increase in apoptosis of HHL-5 cells. The influence conferred by α-AMA treatment was abolished by sodium oxalate treatment (Fig. 5F-G). All these data indicated that α-AMA-induced cellular damage was reversed by inhibiting HMGB1 lactylation.

Fig. 5.

Fig. 5

Open in a new tab

α-AMA-induced cellular damage was reversed by inhibiting lactylation

HHL-5 cells were treated with 10 mM sodium lactate, 2 µM α-AMA or 5 µM α-AMA. (A) The lactic acid content in cells was detected. (B) The HMGB1 in cell was assessed by western blotting. (C) The expression of HMGB1 in the nucleus and cytoplasm was determined by western blotting. (D) The level of HMGB1 lactylation was detected by Co-IP combined with western blotting. HHL-5 cells were treated with 5 µM α-AMA or combined with 20 mM sodium oxalate. (E) The level of HMGB1 lactylation was detected by Co-IP combined with western blotting. (F) Cell viability was assessed by CCK-8 assay. (G) Cell apoptosis was detected by flow cytometry. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Control group; #P < 0.05 vs. Sodium lactate. ###P < 0.001 vs. 5 µM α-AMA

Discussion

Wild mushrooms in China are rich in resources and come in many varieties [18]. Poisonings due to accidental ingestion of wild poisonous mushrooms occur frequently [19]. α-AMA is a major contributor to deaths from mushroom poisoning worldwide [20]. The liver is the main target organ for α-AMA poisoning [21]. α-AMA is rapidly absorbed in the intestinal lumen, reaches the liver tissue, and is presented in the hepatic and renal circulation [22]. Therefore, exploring the mechanism of action of α-AMA on liver failure can help to investigate the methods of α-AMA detoxification. In this work, we found that α-AMA treatment notably elevated the levels of ALT, AST and TBIL in mice in a dosage-dependent manner. Clinical evidence shows that ALT and AST are sensitive indicators of hepatocellular injury and its extent [23–25]. TBIL reflects hepatic excretory function and detects hepatic excretory clearance capacity for certain endogenous or exogenous high-uptake substances [17]. Thus, α-AMA treatment induced significant liver failure in mice. α-AMA targets the liver, but this toxin is not metabolized in the liver. It is ultimately excreted in large quantities by the kidneys through the urine [26]. A previous study has found that α-AMA treatment elevates oxidative stress and nephrotoxicity in mice [27]. α-AMA induces apoptosis of Hepa1-6 cells in a time and concentration-dependent manner [28]. In this work, we also confirmed the cytotoxicity of α-AMA on HHL-5 cells. α-AMA treatment elevated apoptosis and inhibited cell viability of HHL-5 cells, which may contribute to an increase in the expression of HMGB1.

Lactate is a by-product of glycolysis in normal cells under hypoxic conditions and has long been considered a metabolic waste product [29]. Serum lactate has been considered a biomarker for the prognosis of liver failure. Elevated serum lactate levels are positively associated with liver failure mortality [30]. This work found that the serum levels of lactate were notably increased in α-AMA-treated mice. It indicated that α-AMA induced liver failure in mice by elevating the serum levels of lactate. Moreover, previous studies have reported a novel post-translational modification of proteins using lactic acid as a substrate-protein lactylation. It also reveals that lactylation is an important means by which lactate regulates cellular life activities [31, 32]. A previous study has showed that lactate is able to induce lactated modifications of HMGB1 in the mouse macrophage cell line RAW 264.7 [33]. In the present work, we found that α-AMA treatment promoted the levels of lactate and HMGB1 in HHL-5 cells. Sodium lactate elevated the expression and lactylation of HMGB1 in HHL-5 cells, which is consistent with the effect of α-AMA on HMGB1 expression. Thus, α-AMA induced hepatocyte injury by promoting HMGB1 expression.

HMGB1 is an abundant nuclear protein in the HMGB subfamily and is expressed in various organs [34]. HMGB1 is involved in various cellular activities in the cell, such as nucleosome construction, DNA replication, repair, cell differentiation, gene expression and regulation [35]. Extracellularly, HMGB1 mediates inflammatory responses, promotes tumor growth and has a wide range of biological effects [36]. In this study, HMGB1 is key to α-AMA-induced liver failure. We found that α-AMA treatment enhanced the expression of HMGB1 in liver failure mice. However, HMGB1 expression was less in 0.53 mg/kg α-AMA-induced liver failure mice than that in 0.35 mg/kg α-AMA-induced liver failure mice. As the concentration of α-AMA increased, the lactate metabolism ability of liver tissues weakened. Higher levels of lactate caused an increase in HMGB1 expression in mice. A high dosage of 0.53 mg/kg α-AMA may lead to liver failure, the ability of liver tissue to synthesize HMGB1 protein may be inhibited. Additionally, α-AMA treatment elevated the levels of lactate in liver failure mice and HHL-5 cells. HMGB1 knockdown reduced the levels of lactate in α-AMA-treated HHL-5 cells. It indicated that lactate is not only upstream, but also downstream the HMGB1 pathway. Thus, α-AMA-induced liver failure in mice by elevating the expression and lactylation of HMGB1. Wang et al. have confirmed that TNF-α/HMGB1-mediated inflammatory response promotes the occurrence of pyroptosis, which contribute to accelerated acute liver failure and acute kidney injury [37]. HMGB1 may be a prognostic biomarker for acute kidney injury among hepatitis B virus-related acute-on-chronic liver failure patients [38]. All these data demonstrated that HMGB1 is a promising therapeutic target for acute liver failure [39].

The animal model employed in this study is well-established, reliable, easy to implement, and cost-effective. However, this modeling approach also has certain limitations, such as potential animal mortality during the modeling process, which may reduce the final sample size and compromise the robustness and statistical power of the results. In future studies of a similar nature, we plan to increase the number of experimental animals, enhance close monitoring of animal pain and distress levels, and implement humane endpoints when necessary to ensure an adequate sample size for reliable subsequent analyses. Moreover, there are also many shortcomings in this study. α-AMA is toxic to both the liver and kidneys [8]. Acute liver failure patients have moderate (if any) portal hypertension, thrombocytopenia and increased International Normalized Ratio. Therefore, clinicians consider acute liver failure patients may face a high risk of bleeding complications [40]. This work only investigated the toxic effects of α-AMA on the liver tissues, and its impact on the kidneys and hematopoietic function was not explored. Whether α-AMA affects renal injury and hematopoietic function by regulating HMGB1 needs further investigation. Furthermore, whether HMGB1 can serve as a biomarker for liver failure still requires extensive basic and clinical research.

Conclusion

In summary, this work demonstrated that α-AMA induced liver failure by elevating lactylation of HMGB1. This study suggests that HMGB1 may be a potential target for liver failure treatment.

Supplementary Information

Supplementary Material 1. (970.7KB, pdf)

Acknowledgments

Not applicable.

Abbreviations

α-AMA

Alpha-amanitin

ANOVA

Analysis of variance

ALF

Acute liver failure

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

CDC

Chinese Centre for Disease Control and Prevention

DF

Degrees of freedom

ELISA

Enzyme-linked immunosorbent assay

HHL-5

Human embryonic hepatocytes

HMGB1

High mobility group box 1

TBIL

Total bilirubin

Authors’ contributions

Shaofang Huang and Jian Tao: conceptualization, data curation, formal analysis, writing-original draft preparation; Hui xiao and Shuling Wu: conceptualization, methodology, writing-original draft preparation; Wei Ye and Shipeng Huang: supervision, writing-review & editing, funding. All authors read and approved the final manuscript.

Funding

This work was supported by Science and Technology Research Project of Education Department of Jiangxi Province (No. GJJ2200127).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The study protocol was approved by the Institutional Animal Ethics Committee of The First Affiliated Hospital of Nanchang University (approval number: CDYFY-IACUC-202304QR051).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Liu Y, Li S, Feng Y, Zhang Y, Ouyang J, Li S, Wang J, Tan L, Zou L. Serum metabolomic analyses reveal the potential metabolic biomarkers for prediction of amatoxin poisoning. Toxicon: Official J Int Soc Toxinology. 2023;230:107153. [DOI] [PubMed] [Google Scholar]
  • 2.Liu J, Chen Y, Gao Y, Walline JH, Lu X, Yu S, Zhao L, Ge Z, Li Y. N-acetylcysteine as a treatment for amatoxin poisoning: a systematic review. Clin Toxicol (Phila). 2020;58(11):1015–22. [DOI] [PubMed] [Google Scholar]
  • 3.Diaz JH. Amatoxin-Containing mushroom poisonings: Species, Toxidromes, Treatments, and outcomes. Wilderness Environ Med. 2018;29(1):111–8. [DOI] [PubMed] [Google Scholar]
  • 4.Garcia J, Costa V, Carvalho A, Baptista P, de Pinho P, de Lourdes Bastos M, Carvalho F. Amanita phalloides poisoning: mechanisms of toxicity and treatment. Food Chem Toxicology: Int J Published Br Industrial Biol Res Association. 2015;86:41–55. [DOI] [PubMed] [Google Scholar]
  • 5.Wu ZLH, Zhang Y, Ding C, Zhao W, Dai J, Yu C, Yao Q, Zheng F, Fan J, Sun C. Liver transcriptome analyses of acute poisoning and recovery of male ICR mice exposed to the mushroom toxin α-amanitin. Arch Toxicol. 2022;96:1751–66. [DOI] [PubMed] [Google Scholar]
  • 6.Wang M, Chen Y, Guo Z, Yang C, Qi J, Fu Y, Chen Z, Chen P, Wang Y. Changes in the mitochondrial proteome in human hepatocytes in response to alpha-amanitin hepatotoxicity. Toxicon: Official J Int Soc Toxinology. 2018;156:34–40. [DOI] [PubMed] [Google Scholar]
  • 7.Garcia J, Costa V, Carvalho A, Silvestre R, Duarte J, Dourado D, Arbo M, Baltazar T, Dinis-Oliveira R, Baptista P, et al. A breakthrough on amanita phalloides poisoning: an effective antidotal effect by polymyxin B. Arch Toxicol. 2015;89(12):2305–23. [DOI] [PubMed] [Google Scholar]
  • 8.Xue J, Lou X, Ning D, Shao R, Chen G. Mechanism and treatment of α-amanitin poisoning. Arch Toxicol. 2023;97(1):121–31. [DOI] [PubMed] [Google Scholar]
  • 9.Ni Y, Chen H, Nie H, Zheng B, Gong Q. HMGB1: an overview of its roles in the pathogenesis of liver disease. J Leukoc Biol. 2021;110(5):987–98. [DOI] [PubMed] [Google Scholar]
  • 10.Zhang X, Su C, Zhao S, Li J, Yu F. Combination therapy of Ulinastatin with thrombomodulin alleviates endotoxin (LPS) - induced liver and kidney injury via inhibiting apoptosis, oxidative stress and HMGB1/TLR4/NF-κB pathway. Bioengineered. 2022;13(2):2951–70. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Matsuura R, Komaru Y, Miyamoto Y, Yoshida T, Yoshimoto K, Hamasaki Y, et al. Different biomarker kinetics in critically ill patients with high lactate levels. Diagnostics (Basel Switzerland). 2020;10(7):454. [DOI] [PMC free article] [PubMed]
  • 12.Bonaldi T, Talamo F, Scaffidi P, Ferrera D, Porto A, Bachi A, Rubartelli A, Agresti A, Bianchi M. Monocytic cells hyperacetylate chromatin protein HMGB1 to redirect it towards secretion. EMBO J. 2003;22(20):5551–60. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Ito I, Fukazawa J, Yoshida M. Post-translational methylation of high mobility group box 1 (HMGB1) causes its cytoplasmic localization in neutrophils. J Biol Chem. 2007;282(22):16336–44. [DOI] [PubMed] [Google Scholar]
  • 14.Cardoso F, Abraldes J, Sy E, Ronco J, Bagulho L, Mcphail M, Karvellas C. Lactate and number of organ failures predict intensive care unit mortality in patients with acute-on-chronic liver failure. Liver International: Official J Int Association Study Liver. 2019;39(7):1271–80. [DOI] [PubMed] [Google Scholar]
  • 15.Shin W, Kim Y, Bang J, Cho S, Han S, Hwang G. Lactate and liver function tests after living donor right hepatectomy: a comparison of solutions with and without lactate. Acta Anaesthesiol Scand. 2011;55(5):558–64. [DOI] [PubMed] [Google Scholar]
  • 16.Yang L, Xie M, Yang M, Yu Y, Zhu S, Hou W, Kang R, Lotze M, Billiar T, Wang H, et al. PKM2 regulates the Warburg effect and promotes HMGB1 release in sepsis. Nat Commun. 2014;5:4436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Zhao J, Cao M, Zhang J, Sun Q, Chen Q, Yang Z. Pathological effects of the mushroom toxin alpha-amanitin on BALB/c mice. Peptides. 2006;27(12):3047–52. [DOI] [PubMed] [Google Scholar]
  • 18.Liu Q, Sun L, Ding Y, Zhuang Y. Chemical composition, health benefits, food processing effects and applications of Boletus: a review. Crit Rev Food Sci Nutr. 2024;64(29):10812–34. [DOI] [PubMed]
  • 19.Patra A, Mukherjee A. Mushroom mycetism - A neglected and challenging medical emergency in the Indian subcontinent: A road map for its prevention and treatment. Toxicon: Official J Int Soc Toxinology. 2022;217:56–77. [DOI] [PubMed] [Google Scholar]
  • 20.Xu Y, Wang S, Leung C, Chen H, Wang C, Zhang H, Zhang S, Tan Y, Wang H, Miao L, et al. : α-amanitin induces autophagy through AMPK-mTOR-ULK1 signaling pathway in hepatocytes. Toxicol Lett. 2023;383:89–97. [DOI] [PubMed] [Google Scholar]
  • 21.Garcia J, Carvalho A, das Neves R, Malheiro R, Rodrigues D, Figueiredo P, Bovolini A, Duarte J, Costa V, Carvalho F. Antidotal effect of cyclosporine A against α-amanitin toxicity in CD-1 mice, at clinical relevant doses. Food Chem Toxicology: Int J Published Br Industrial Biol Res Association. 2022;166:113198. [DOI] [PubMed] [Google Scholar]
  • 22.Wu Z, Li H, Zhang Y, Ding C, Zhao W, Dai J, Yu C, Yao Q, Zheng F, Fan J, et al. Liver transcriptome analyses of acute poisoning and recovery of male ICR mice exposed to the mushroom toxin α-amanitin. Arch Toxicol. 2022;96(6):1751–66. [DOI] [PubMed] [Google Scholar]
  • 23.Lin L, Tong Y, Lu Y. The characteristics of liver injury induced by amanita and clinical value of α-amanitin detection. Hepatobiliary Pancreat Dis International: HBPD INT. 2022;21(3):257–66. [DOI] [PubMed] [Google Scholar]
  • 24.Wu X, Zeng J, Hu J, Liao Q, Zhou R, Zhang P, Chen Z. Hepatoprotective effects of aqueous extract from Lingzhi or Reishi medicinal mushroom ganoderma lucidum (higher basidiomycetes) on α-amanitin-induced liver injury in mice. Int J Med Mushrooms. 2013;15(4):383–91. [DOI] [PubMed] [Google Scholar]
  • 25.Trabulus S, Altiparmak M. Clinical features and outcome of patients with amatoxin-containing mushroom poisoning. Clin Toxicol (Phila). 2011;49(4):303–10. [DOI] [PubMed] [Google Scholar]
  • 26.Garcia J, Costa VM, Carvalho A, Baptista P, de Pinho PG, de Lourdes Bastos M, Carvalho F. Amanita phalloides poisoning: mechanisms of toxicity and treatment. Food Chem Toxicology: Int J Published Br Industrial Biol Res Association. 2015;86:41–55. [DOI] [PubMed] [Google Scholar]
  • 27.Arici MA, Sahin A, Cavdar Z, Ergur BU, Ural C, Akokay P, Kalkan S, Tuncok Y. Effects of Resveratrol on alpha-amanitin-induced nephrotoxicity in BALB/c mice. Hum Exp Toxicol. 2020;39(3):328–37. [DOI] [PubMed] [Google Scholar]
  • 28.Gu X, Zhang L, Sun W, Liu K, Xu H, Wu P, Gui M, Qu W. Autophagy promotes α-Amanitin-Induced apoptosis of Hepa1-6 liver cells. Chem Res Toxicol. 2022;35(3):392–401. [DOI] [PubMed] [Google Scholar]
  • 29.Lin Y, Yan G, Wang M, Zhang K, Shu F, Liu M, Long F, Mao D. Crosstalk between lactic acid and immune regulation and its value in the diagnosis and treatment of liver failure. Open Life Sci. 2023;18(1):20220636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Horvatits T, Mahmud N, Serper M, Seiz O, Reher D, Drolz A, Sarnast N, Gu W, Erasmus H, Allo G, et al. MELD-Lactate predicts poor outcome in variceal bleeding in cirrhosis. Dig Dis Sci. 2023;68(3):1042–50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Henry C, Haller L, Blein-Nicolas M, Zivy M, Canette A, Verbrugghe M, Mézange C, Boulay M, Gardan R, Samson S, et al. Streptococcus thermophilusidentification of Hanks-Type kinase PknB-Specific targets in the phosphoproteome. Front Microbiol. 2019;10:1329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ruegsegger G, Manjunatha S, Summer P, Gopala S, Zabeilski P, Dasari S, Vanderboom P, Lanza I, Klaus K, Nair K. Insulin deficiency and intranasal insulin alter brain mitochondrial function: a potential factor for dementia in diabetes. FASEB Journal: Official Publication Federation Am Soc Experimental Biology. 2019;33(3):4458–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yang K, Fan M, Wang X, Xu J, Wang Y, Tu F, Gill PS, Ha T, Liu L, Williams DL, et al. Lactate promotes macrophage HMGB1 lactylation, acetylation, and Exosomal release in polymicrobial sepsis. Cell Death Differ. 2022;29(1):133–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Starkova T, Polyanichko A, Tomilin A, Chikhirzhina E. Structure and functions of HMGB2 protein. Int J Mol Sci. 2023;24(9):8334. [DOI] [PMC free article] [PubMed]
  • 35.Qian F, Xiao J, Gai L, Zhu J. HMGB1-RAGE signaling facilitates Ras-dependent Yap1 expression to drive colorectal cancer stemness and development. Mol Carcinog. 2019;58(4):500–10. [DOI] [PubMed] [Google Scholar]
  • 36.Martinotti S, Patrone M, Ranzato E. Emerging roles for HMGB1 protein in immunity, inflammation, and cancer. ImmunoTargets Therapy. 2015;4:101–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Wang Y, Zhang H, Chen Q, Jiao F, Shi C, Pei M, Lv J, Zhang H, Wang L, Gong Z. TNF-α/HMGB1 inflammation signalling pathway regulates pyroptosis during liver failure and acute kidney injury. Cell Prolif. 2020;53(6):e12829. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Liu Y, Yuan W, Fang M, Guo H, Zhang X, Mei X, Zhang Y, Ji L, Gao Y, Wang J, et al. Determination of HMGB1 in hepatitis B virus-related acute-on-chronic liver failure patients with acute kidney injury: early prediction and prognostic implications. Front Pharmacol. 2022;13:1031790. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Yamamoto T, Tajima Y. HMGB1 is a promising therapeutic target for acute liver failure. Expert Rev Gastroenterol Hepatol. 2017;11(7):673–82. [DOI] [PubMed] [Google Scholar]
  • 40.Stravitz RT, Ellerbe C, Durkalski V, Reuben A, Lisman T, Lee WM. Thrombocytopenia is associated with Multi-organ system failure in patients with acute liver failure. Clin Gastroenterol Hepatology: Official Clin Pract J Am Gastroenterological Association. 2016;14(4):613–e620614. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (970.7KB, pdf)

Data Availability Statement

No datasets were generated or analysed during the current study.

Articles from BMC Gastroenterology are provided here courtesy of BMC

RESOURCES