Inhibition RIP1 prevents acute liver failure by suppressing hepatic apoptosis and attenuating the secretion of TNF-α from macrophages

Abstract

Background and aims

Acute liver failure (ALF) is a rapidly progressing clinical syndrome with a high mortality rate and limited treatment options. In this study, we used the RIP1 kinase inhibitor necrostatin-1 (Nec-1) to explore the effect and mechanism of RIP1 in lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced ALF.

Results

Nec-1 pretreatment significantly ameliorated ALF, as evidenced by reduced hepatic necrosis and serum alanine aminotransferase levels. Additionally, Nec-1 administration alleviated LPS/GalN-induced hepatocyte apoptosis in liver tissues. Further in vitro experiments revealed that Nec-1 inhibited the secretion of TNF-α from macrophages and reduced TNF-α-induced hepatocyte apoptosis.

Conclusions

Inhibition of RIP1 effectively alleviated LPS/GalN-induced ALF by reducing hepatic apoptosis and attenuating the secretion of TNF-α from macrophages, suggesting its potential as a therapeutic agent for ALF patients.

Introduction

Acute liver failure (ALF) is a life-threatening hepatic disease characterized by acute exacerbation of liver function and 50% mortality, largely due to limited treatment options [1, 2]. Both infectious or noninfectious factors, such as viruses, drugs, poisonous substances, and hereditary or autoimmune diseases, can induce ALF [3]. Except for liver transplantation, no curative treatment modalities are available for ALF therapy because of rapid disease progression [4].Therefore, there is an urgent need to discover new therapeutic targets to improve the survival rate of patients with ALF.

Systemic inflammatory response syndrome and extensive hepatocyte death are two features involved in the pathogenesis of ALF. During the acute inflammatory response phase of ALF, macrophage activation is a key step that determines the inflammatory response via the release of the important cytokine tumour necrosis factor alpha (TNF-α), further aggravating liver damage [5]. TNF-α is a proinflammatory cytokine that coordinates tissue homeostasis by regulating cytokine production, cell survival, and cell death. The TNF-α level is strongly positively associated with ALF severity [6]. However, systemic administration of TNF-α therapies may lead to immune disorders, which makes targeting key downstream nodes in the TNF signal transduction pathway highly important.

Receptor-interacting protein kinase 1 (RIP1) is an important downstream target of TNF and ligands of the Toll-like receptor family that regulates multiple cellular pathways involved in regulating inflammation and cell death [7]. The serine/threonine kinase RIP1 harbours an N-terminal kinase domain, a 250-amino acid-long linker region, a C-terminal RIP homotypic interaction motif, and death domain and has important kinase-dependent and scaffolding functions. The kinase activity of RIP1 is critical for regulating both inflammation and apoptotic and necrotic cell death while scaffolding function mediates NF-κB activation and cell survival [8, 9]. Mice with Rip1 gene knockout die after birth, whereas point mutant mice with inactivated RIP1 kinase can survive and are protected against TNF-α-induced cell death and inflammatory diseases [10]. Inhibiting RIP1 kinase activity can play a protective role in a series of inflammatory diseases, such as heart and kidney ischaemia‒reperfusion injury, dermatitis, psoriasis, and septic shock, making RIP1 kinase a promising therapeutic target for human diseases [11]. For example, RIP1 kinase was markedly activated in human nonalcoholic steatohepatitis, and kinase activation occurred mainly in liver macrophages [12]. However, the role of RIP1 in ALF remains elusive.

Materials and methods

Animal model

C57BL/6 J male mice aged six-week-old were used. All the animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Zhejiang. ALF was induced in the mice via intraperitoneal (i.p.) injection of LPS (40 μg/kg) and GalN (800 mg/kg). The control mice received saline injections. Nec-1 (5 mg/kg) was injected i.p. 30 min before LPS/GalN. We determine the dose selection for Nec-1 by referring to the following articles (10.1016/s0006-291x(02)00789–1, 10.1080/03009740410005025, 10.1016/j.biopha.2017.09.063, 10.1038/cddis.2012.176). Serum and liver samples were harvested for subsequent analysis 6 h after LPS/GalN injection. TNF-α in serum was measured by Enzyme linked immunosorbent assay kit (ELISA). The levels of apoptosis pathway related protein caspase 3 and cleaved casp-3 in mouse liver tissues were detected by Western blot. LPS (L4391), GalN (G0500), and Nec-1 (N9037) were purchased from Sigma‒Aldrich.

Aminotransferase measurement

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were analysed via the standard analyser DRI—CHEM 4000ie (FUJIFILM).

Liver histology and TUNEL staining

Fresh livers were fixed in 4% paraformaldehyde and embedded in paraffin. Sections were stained with haematoxylin and eosin (H&E) and observed via a Pannoramic P250 FLASH III. Hepatic apoptosis was measured via a TUNEL fluorescence apoptosis detection kit (Nanjing Novizan Biotech Co., Ltd.). Imaging was performed via a TCS SP8 confocal microscope (Leica).

Cell culture and stimulation

The mouse leukemic macrophage cell line RAW264.7 and human hepatocyte cell line L02 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 1% penicillin‒streptomycin (Gibco) in a humidified atmosphere with 5% CO2 at 37 °C. The RAW264.7 cells were stimulated with Nec-1 (25 μM) for 1 h first and then stimulated with LPS (100 ng/mL) for 6 h. The supernatant of the cell culture medium was collected to determine the TNF-α concentration. After L02 cells were treated with Nec-1 (50 μM) for 1 h, TNF-α (10 ng/mL) and ACTD (0.2 μM) were added, and the mixture was stimulated for 6 h. Annexin V-FITC/PI double-staining flow cytometry was used to observe the degree of L02 cells apoptosis. Proteins were extracted from L02 cells 3 h and 6 h after stimulation with TNF-α (10 ng/mL) and ACTD (0.2 μM), respectively, to detect cleaved casp-3, and the protein bands were visualised via the ChemiScope Western blot Imaging System (Clinx Science Instruments Co., Ltd.).

Flow cytometric analysis of apoptosis

Cell death was assessed using an Accuri C6 flow cytometer (BD Biosciences, CA, USA). After the human hepatocyte cell line L02 was treated with Nec-1 (50 μM) for 1 h, TNF-α (10 ng/mL) and ACTD (0.2 μM) were added, and the mixture was stimulated for 6 h. We determined the dose selection for Nec-1 in cell models by for Nec-1 by referring to the following articles (10.1016/s0006-291x(02)00789–1, 10.1080/03009740410005025, 10.1016/j.biopha.2017.09.063, 10.1038/cddis.2012.176). After 6 h, the cells were washed, trypsinized, resuspended in binding buffer, and then stained for 15 min with Annexin V-FITC and propidium iodide (PI) (BD Pharmingen, CA, USA). The proportions of apoptotic cells, including early apoptotic and late apoptosis were determined by the percentages of Annexin V-positive/PI-negative cells and Annexin V-positive/PI-positive cells, respectively.

Western blot

The cell pellets and liver tissues were lysed on ice with ice-cold radioimmunoprecipitation (RIPA).

RIPA buffer (Beyotime Biotechnology) containing a protease inhibitor cocktail (Roche) for 30 min before centrifugation at 12000 rpm for 20 min at 4 °C. The soluble protein concentrations in the lysates were subsequently determined by using a BCA protein assay kit (Thermo Scientific, Fremont, CA, USA). Western blot analysis was performed as previously described [13]. The following antibodies were used: anti-caspase-3, anti-cleaved casp-3 (9662S, 9661S, Cell Signaling Technology, Danvers, MA, USA), and anti-GAPDH (5174, Cell Signaling Technology, Danvers, MA, USA).

Statistical analysis

The data are presented as the means ± SEMs. Statistical differences between two groups were tested via the conventional Student's t test with GraphPad Prism 7 software. P < 0.05 was considered statistically significant.

Results

Inhibition of RIP1 prevents liver injury and inflammatory responses

We used the RIP1 kinase inhibitor Necrostatin-1 (Nec-1) to explore the role and mechanism of RIP1 in LPS/GalN-induced ALF [14]. The mice were treated with 5 mg/kg Nec-1 30 min prior to the administration of LPS/GalN and then examined at 6 h post-treatment. As shown in Fig. 1A, liver hepatocytes were radially arranged around the central vein in the liver lobules in the normal control group, the liver lobule structure was damaged, with obvious necrosis, a large amount of congestion, swollen cells in the necrotic area, and the disappearance of cell structures in the mice challenged with LPS/GalN. LPS/GalN induced significant liver injury, as indicated by dramatically elevated serum ALT and AST accompanied by massive hepatic necrosis, while pretreatment with Nec-1 resulted in a remarkable reduction in serum ALT and AST levels and significantly improved pathological liver damage, as shown in Fig. 1B. Moreover, the LPS/GalN-induced increase in the production of TNF-α in the serum was significantly attenuated in mice pretreated with Nec-1(Fig. 1C), which suggests that pretreatment with RIP1 inhibition can alleviate LPS/GalN-induced liver injury and inflammatory responses.

Fig. 1.

Nec-1 alleviates LPS/GalN-induced liver damage in mice. Six-week-old male C57BL/6 mice were randomly divided into a normal control group (Control), a model group (LPS/GalN), and a Nec-1 intervention group (Nec-1). The mice in the LPS/GalN group were intraperitoneally injected with LPS (40 μg/kg) or GalN (800 mg/kg). In the Nec-1 group, Nec-1 (5 mg/kg) was administered 30 min before LPS/GalN injection. Six hours after LPS/GalN injection, the mice were sacrificed under anaesthesia, and serum and liver specimens were collected for biochemical and liver pathological examinations. A H&E staining of mouse livers. B Serum ALT and AST levels in the mice. C Serum samples were harvested at 6 h after LPS/GalN injection to test the level of TNF-α. The data are presented as the means ± standard errors of the means, and statistical analysis was performed using t tests. (**P < 0.01, ***P < 0.001, ****P < 0.0001). LPS lipopolysaccharide, GalN D-galactosamine, Nec-1 necrostatin-1 (a RIP1 kinase inhibitor), H&E haematoxylin and eosin, AST aspartate aminotransferase, ALT alanine aminotransferase

RIP1 inhibition prevents hepatocyte apoptosis

RIP1 is located at a key position in the cell death receptor signalling pathway. After the death receptor is activated by TNF-α, RIP1 can form a protein complex with Fas-associated protein with a novel death domain (FADD) and cysteine aspartic acid protease 8 (Caspase 8), which contains death domains, to activate the apoptotic pathway[15]. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) staining is a commonly used method for detecting apoptosis. As expected, LPS/GalN administration markedly increased hepatic apoptosis in liver tissues, as indicated by the increased proportion of TUNEL-positive cells, whereas Nec-1 pretreatment markedly reduced the proportion of apoptotic liver cells (Fig. 2A). Quantitative analysis confirmed the changes in hepatic apoptosis levels caused by pretreatment with Nec-1 (Fig. 2B).

Fig. 2.

Nec-1 inhibits hepatocyte apoptosis in mice with ALF. Six-week-old male C57BL/6 mice were randomly divided into a normal control group (Control), a model group (LPS/GalN), and a Nec-1 intervention group (Nec-1). The mice in the LPS/GalN group were intraperitoneally injected with LPS (40 μg/kg) or GalN (800 mg/kg). In the Nec-1 group, Nec-1 (5 mg/kg) was administered 30 min before LPS/GalN injection. Six hours after modelling, the liver was harvested for TUNEL staining. TUNEL staining was used to detect the proportion of apoptotic cells in liver tissues (A, B). C Proteins were extracted from liver tissues to detect caspase 3 and cleaved casp-3. Data are presented as the mean ± standard error of the mean, and statistical analysis was performed using t tests. (*P < 0.05, **P < 0.01, ***P < 0.001). TUNEL Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling, cleaved casp-3 Cleaved caspase- 3

As the key node in the TNFα-triggered apoptosis pathway, caspase 3 was also investigated, as shown in Fig. 2C, and the results indicated that caspase 3 was markedly cleaved during LPS/GalN administration. Nec-1 pretreatment markedly blocked caspase 3 activation. These results indicate that inhibiting RIP1 kinase reduces hepatocyte apoptosis in mice with ALF induced by LPS/GalN.

Nec-1 reduces TNF-α secretion by macrophages and inhibits TNF-α-induced hepatocyte apoptosis

TNF-α is mainly secreted by macrophages and can cause hepatocyte damage, which may further exacerbate liver injury [16]. Therefore, we explored the role of RIP1 in macrophages. We used The mouse leukemic macrophage cell line RAW264.7 to investigate the effect of Nec-1 on TNF-α secretion by macrophages. We pretreated RAW264.7 cells with Nec-1 for 1 h, stimulated them with LPS for 6 h, and detected the TNF-α concentration in the culture medium supernatant via ELISA. The results revealed that Nec-1 significantly decreased the level of TNF-α secreted by LPS-activated macrophages (Fig. 3A). Then, we pretreated the human hepatocyte cell line L02 with Nec-1 for 1 h, added TNF-α and actinomycin D (ACTD) to stimulate the cells for 6 h to induce apoptosis, and detected the proportion of apoptotic L02 cells by flow cytometry. The results showed that Nec-1 significantly inhibited the TNF-α-induced apoptosis of L02 cells (Fig. 3B, C). The Western blot results also revealed that Nec-1 inhibited caspase 3 activation, further confirming that RIP1 mediates hepatocyte apoptosis in a kinase-dependent manner (Fig. 3D, E). These results suggest that Nec-1 can inhibit TNF-α-induced hepatocyte apoptosis by reducing TNF-α secretion by macrophages, thereby alleviating LPS/GalN-induced acute liver failure.

Fig. 3.

Nec-1 reduces TNF-α secretion by macrophages and inhibits TNF-α-induced hepatocyte apoptosis. A The mouse macrophage cell line RAW264.7 was stimulated with Nec-1 (25 μM) for 1 h in advance and then stimulated with LPS (100 ng/mL) for 6 h. The supernatant of the cell culture medium was collected to determine the TNF-α concentration (A). After the human hepatocyte cell line L02 was treated with Nec-1 (50 μM) for 1 h, TNF-α (10 ng/mL) and ACTD (0.2 μM) were added, and the mixture was stimulated for 6 h. Early apoptosis (Q4) and late apoptosis (Q2) were detected by Annexin V-positive/PI-negative cells, and Annexin V-positive/PI-positive cells were detected by flow cytometry to observe the degree of apoptosis in L02 cells (B, C). Proteins were extracted from L02 cells 3 h and 6 h after stimulation with TNF-α (10 ng/mL) and ACTD (0.2 μM), respectively, to detect cleaved casp-3 (D) and perform protein grayscale calculations (E). The data are presented as the means ± standard errors of the means, and statistical analysis was performed using t tests. (**P < 0.01, ***P < 0.001). Con Control, T TNF-α, A ACTD, N Nec-1, ACTD Actinomycin D

Discussion

ALF progresses rapidly and is a life-threatening clinical syndrome with a very high fatality rate [17]. The causes of ALF are complex and include hepatitis virus infections; chemical poisons or drugs; ischaemia; alcohol; radiation exposure; hereditary metabolic disorders; and autoimmune diseases [18]. There is a lack of effective treatment methods and drugs for ALF in clinical practice. Although artificial liver and liver transplantation therapies are the main clinical treatments for ALF [19], high medical expenses and a shortage of available donor livers limit their widespread clinical application. There is an urgent need to develop new therapeutic targets.

Macrophages represent key phagocytic cells in the innate immune system, and are essential for maintaining homeostasis and ensuring rapid responses to pathogenic stimuli. In liver, macrophages are the first cells to detect the presence of danger signals and are more susceptible to various external harmful substances, which may cause cell injury or even cell death [20]. ALF is associated with increased gut permeability, leading to increased LPS levels and endotoxemia in serum.By binding to LPS, macrophage was activated and released abundant inflammatory mediators such as TNF-α, interleukin(IL)−6, IL-1.., and further aggravating liver damage [21–23]. TNF-α is one of the most important cytokines contributing to regulate the inflammation process and development of liver disease [24].

TNF receptor activation affects multiple cellular responses that besides cytokine production also include cell survival and cell death. Regulated cell death including apoptosis, necroptosis, and pyroptosis is molecularly controlled type of cell death which is considered controllable even can be inhibited, it suggests that understanding the molecular machinery involved in cell death pathways may provide potential therapeutic strategies for acute liver failure [25–27]. The hepatocytes express high levels of death receptors like Fas, TNF-related apoptosis-inducing ligand (TRAIL) -R, TNFR on their surface, possibly leading high susceptibility of hepatocytes to TNF-α induced cell death [28].Apoptosis is a highly organized and genetically controlled type of cell death mediated by the recruitment of caspase 8 leading to initiation of apoptotic pathway in distinct extrinsic pathways [29]. Death receptor–mediated apoptosis is a key feature of many types of liver diseases. siTNF-α treatment can efficiently alleviate acute liver disease by reducing inflammatory cell infiltration and attenuating injured hepatocyte apoptosis [30]. However, systemic administration of TNF-α therapies may lead to immune disorders, which makes targeting key downstream nodes in the TNF signal transduction pathway highly important.

RIP1 is an important downstream target of TNF receptor family that regulates multiple cellular pathways involved in regulating inflammation and cell death [7]. Studies have shown that knocking down the Rip1 gene via antisense oligonucleotides can inhibit the activation of the c-Jun N-terminal kinase pathway and reduce acetaminophen (APAP)-induced cell necrosis [31]. Consistent with the abovementioned results, knocking down the Rip1 gene via small interfering RNA also has a protective effect on the liver [32]. However, RIP1 kinase was not related to liver damage in carbon tetrachloride and APAP models [33]. Therefore, we aimed to clarify whether RIP1 kinase-dependent functions are involved in mediating ALF. Compared with vehicle-treated mice, Nec-1-pretreated LPS/GalN-treated mice presented significant protection against liver injury, as determined by the serum ALT, AST, and TNF-α levels (Fig. 1B, C) and H&E staining (Fig. 1A). Moreover, Nec-1 administration ameliorated LPS/GalN-induced hepatocyte apoptosis, as indicated by TUNEL analysis and caspase 3 detection (Fig. 2A, B, C). Therefore, we concluded that RIP1 kinase plays an important role in ALF.

The kinase activity of RIP1 is critical for regulating both inflammation and apoptotic and necrotic cell death while scaffolding function mediates NF-κB activation and cell survival [8].After the death receptor is activated by TNF-α, RIP1 can form a protein complex with Fas-associated protein with a novel death domain (FADD) and caspase 8, which contains death domains, to activate the apoptotic pathway. When caspase 8 activity is reduced or absent, RIP1 can also form a complex with RIP3 and mixed lineage kinase domain-like proteins to cause programmed necrosis [34]. We demonstrated through in vitro experiments that Nec-1 can inhibit the secretion of TNF-α by macrophages and reduce TNF-α-induced hepatocyte apoptosis.

Some studies have shown that the membrane-permeable pancaspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (zVAD) significantly reduces hepatocyte apoptosis in the remaining liver and enhances the survival rate of rats after 95% hepatectomy [35]. zVAD can prevent an increase in postoperative serum liver enzymes and reduce liver dysfunction [35]. However, research on systemic inflammatory response syndrome (SIRS) has revealed that the use of apoptosis inhibitors exacerbates the symptoms of SIRS. Further research revealed that the use of zVAD promotes the activation of programmed necrosis [36]. Apoptosis inhibitors are associated with a risk of inducing programmed necrosis in nonparenchymal liver cells [37]. Together, our results revealed that inhibiting RIP1 kinase activity can reduce liver cell death and simultaneously reduce the release of TNF-α by macrophages. Therefore, RIP1 kinase can be used as an efficient drug target for the treatment of liver inflammatory diseases.

Conclusions

Our data revealed the protective effect of RIP1 inhibition on LPS/GalN-induced ALF via hepatic apoptosis and attenuation of the secretion of TNF-α from macrophages in this model. Our findings suggest that RIP1 may be a potential therapeutic target for inflammatory liver diseases.

Abbreviations

ALF

Acute liver failure

TNF-α

Tumour necrosis factor –α

RIP1

Receptor-interacting protein kinase 1

Nec-1

Necrostatin-1

LPS

Lipopolysaccharide

GalN

D-galactosamine

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

FADD

Fas-associated protein with a novel death domain

Caspase 8

Cysteine aspartic acid protease 8

TUNEL

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling

Caspase 3

Cysteine aspartic acid protease 3

ACTD

Actinomycin D

TRAIL

TNF-related apoptosis-inducing ligand

APAP

Acetaminophen

zVAD

Z-Val-Ala-DL-Asp-fluoromethylketone

SIRS

Systemic inflammatory response syndrome

H&E

Haematoxylin and eosin

Author contributions

Aichun Li performed the research and drafted the manuscript. Dahua Chen performed data analysis and interpretation. Jianwei Shen conceived of the study and supervised in its design and coordination. All the authors have read and approved the final manuscript.

Funding

This study was supported by the Doctoral Research Initiation Fund Project of Ningbo Medical Center Lihuili Hospital (2023BSKY-LAC). Provincial Natural Science Youth Fund(LQ20H030001).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

The animal study protocol was approved by the Animal Care and Use Committee (ACUC) of Ningbo University (SYXK(浙) 2024 002). The study adhered to the guidelines set by the committee. The Institutional Animal Ethics Committee of Ningbo University approved all the animal experiments.

Competing interests

The authors declare no competing interests.