
Liver fibrosis is the most common chronic liver disease, which occurs due to various reasons such as viral infection, alcohol abuse, and nonalcoholic steatohepatitis (NASH) (Bataller and Brenner, 2005). These factors give rise to hepatocyte injury and subsequently, the deposition of extracellular matrix components (ECM) (Arriazu
Apoptosis-signal regulating kinase 1 (ASK1) is known as a member of mitogen-activated protein kinase family (MAPK), which is involved in severe human diseases including inflammatory diseases, cancer, and liver diseases (Noureddin
Selonsertib is a first-in-class, small molecule inhibitor of ASK1, which is characterized by anti-fibrotic and anti-inflammatory properties, was in clinical trials for the treatment of NASH (Loomba
HSC-T6 and LX-2 cell lines, immortalized human HSCs were donated from Professor S. L. Friedman (Liver Disease Research Center of San Francisco General Hospital, CA, USA). The cells were routinely cultured with complete Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Rockville MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco) and 1% antibiotic/antimycotic solution. The cultures were maintained at 37°C in a CO2 incubator with a controlled humidified atmosphere composed of 95% air and 5% CO2.
Cells were seeded at a confluence of 3−5×103 cells/well in 96-well plates and incubated for 24 h. Subsequently, the media were discarded and the cells were treated with either DMSO or diverse concentrations (0.5-100 μM) of selonsertib. After 48 h, 10% of an MTT solution (2 mg/mL) was added to each well, and then the cells were incubated for another four h at 37°C. The formed formazan crystals were dissolved in DMSO (200 μL/well) shaking for 5-10 min. Absorbance was then measured by a microplate reader at 540 nm. Quadruplet replicated wells were evaluated for each analysis.
Proliferation of cells was demonstrated by growth curves using JULITM Stage real-time image recording system. This program automatically captured cell morphologies in every configured time point through equipped camera in the CO2 incubator, which quantified and graphed data.
Annexin V and propidium iodide (PI) staining was carried out using Annexin V-APC apoptosis detection kit I (BD pharmingenTM, San Diego, CA, USA). HSC-T6 and LX-2 cells (2×105 cells/mL) were exposed to selonsertib (10-50 μM) for 48 h, and then cellular apoptosis was detected by fluorescent staining. Prepared cells were incubated in Annexin V-APC and PI solution for 15 min in the dark, and the stained cells were instantly analyzed using a FACS verse flow cytometer (BD Biosciences, San Jose, CA, USA).
Assays were conducted with the ApopTag® peroxidase
HSC-T6 and LX-2 cells were washed with DPBS and lysed with RIPA buffer (Biosesang, Seongnam, Korea) containing 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH 7.5), and 2 mM EDTA (pH 8.0). Additionally, Xpert protease inhibitor and phosphatase inhibitor Cocktail (genDEPOT, Katy, TX, USA) were mixed with the buffer, followed by centrifugation at 15,000 rpm for 30 min. Approximately 30 μg of total protein lysate was separated in SDS-polyacrylamide gel electrophoresis (PAGE), which was then transferred to polyvinylidene fluoride (PVDF) membrane using the wet transfer kit (Bio-Rad Laboratories, Berkeley, CA, USA). The membrane was blocked with 5% skim milk in PBS and 0.5% of Tween 20 (PBS-T), followed by overnight incubation at 4°C with primary antibodies that were diluted in PBS-T containing 5% BSA. Anti-p-ASK1 (catalog no. 3765S), anti-p-p38 (catalog no. 4631S), anti-p-JNK (catalog no. 9251S), and anti-JNK (catalog no. 9252S) were purchased from Cell Signaling Technology (Beverly, MA, USA); anti-ASK1 (catalog no. 107921) from Genetex (Alton Pkwy Irvine, CA, USA); anti-p38 (catalog no. 4631S) from Santa Cruz Biotechnology (Dallas, TX, USA); anti-collagen I (catalog no. ab34710), anti-fibronectin (catalog no. ab2413) from Abcam (Cambridge, UK); anti-α-SMA (catalog no. F3777), anti-β-Actin (catalog no. A5441) from Sigma-Aldrich (St. Louis, MO, USA). The secondary antibodies were from Cell Signaling Technology, and the blots were visualized using an enhanced chemiluminescence system (Bio-Rad Laboratories).
HSC-T6 and LX-2 cells were seeded on 18-mm cover glasses in DMEM at confluence of 1×105 cells/well and incubated for 24 h. The cells were then treated with selonsertib for 12-24 h and then rinsed with PBS, followed by fixation in ice-cold acetic acid/ethanol mixture. After washing thrice, the fixed cells were incubated for 1 h in CAS blocking solution (Life Technology, Seoul, Korea) to prevent non-specific binding of antibodies, and then subjected to the incubation with indicated primary antibodies at 4°C overnight. Next, after washing, the cells were cross-reacted with Alexa 488 and 594-conjugated goat anti-mouse or anti-rabbit secondary antibodies for 1 h at room temperature. Nuclei were counterstained with 4’,6’-diamidino-2-phenylindole (DAPI) in the dark for 30 min at room temperature. The slides were sufficiently rinsed, then mounted with fluorescence mounting medium (DAKO, CA, USA). All the samples were examined with a confocal laser microscope (Olympus, Tokyo, Japan) at 488 and 568 nm.
Animal care and experiments were accomplished in compliance with the Guide for Animal Experiments edited by the INHA Institutional Animal Care and Use Committee (INHA IACUC 190315-628) of the Medical School of Inha University. The animals were housed in an air-conditioned room at 25°C with a dark/light cycle for 12 h. All animals received humane care during the study with unlimited access to food and water. Forty male six week-old Sprague-Dawley rats were obtained from Orient-Bio Laboratory Animal Research Center Co., Ltd. (Seongnam, Korea), and were divided into four groups (control, dimethylnitrosamine (DMN), selonsertib 10 or 50 mg/kg). Liver fibrosis was induced by intraperitoneal injections of 10 mg/kg DMN thrice a week for four weeks. Selonsertib dissolved in 0.5% methylcellulose (MC) was administered orally for five days a week for three weeks. Control animals were treated with only vehicles; saline and 0.5% MC solution equivalently. After the final administration, all rats were sacrificed under anesthesia with ketamine.
Liver sections were fixed in 10% formaldehyde solution, and then processed using a paraffin embedding manner. Sections about 4 μm thick were stained with hematoxylin and eosin (H&E) for routine histology, and Sirius Red staining for collagen type I/III. The degree of hepatic fibrosis was evaluated by the Knodell scores of fibrosis, which is a four-grade derived from the Metavir scoring method; the area of fibrosis was measured by image analysis. The histopathological scores were as follows: 0, no fibrosis; I, perivenular or pericellular fibrosis; II, septal fibrosis; III, incomplete cirrhosis; and IV, complete cirrhosis. The degree of fibrosis was estimated as the mean of several fields.
Fresh sera were immediately separated from the blood of sacrificed rats. Cytokine levels of IFN-γ and TNF-α were measured by sandwich enzyme-linked immunoassay (ELISA). Briefly, this examination was conducted using the appropriate capture and detection antibodies (R&D system, Minneapolis, MN, USA), which is applied with a visualizing system of streptavidin-conjugated HRP and TMB microwell peroxidase substrate (Vector Laboratories, CA, USA).
Immunohistochemical staining analysis was performed using formalin-fixed and deparaffinized tissue sections as previously described (Ikejima
Data were expressed as mean ± SD, and statistical analysis was performed using an ANOVA and an unpaired Students
To investigate the effect of seonsertib on the proliferation/growth of HSCs, two cell lines (HSC-T6 and LX-2) were exposed to various concentrations of selonsertib for 48 h. Selonsertib treatment decreased cell viability in both HSC lines in a dose-dependent manner (Fig. 1A). Furthermore, the selonsertib treatment significantly and dose-dependently decreased the proliferative ability of HSCs and caused changes in the cell morphologies. Notably, it inhibited cell growth by 80-90% at a concentration of 50 μM (Fig. 1B, 1C).
Apoptotic cell death by selonsertib was evaluated by double staining with Annexin V-APC and PI using flow cytometry. Subsequent detection of cellular changes was analyzed after treatment with 10, 25, and 50 µM of selonsertib. As shown in Fig. 2A, the distribution of the cell population toward apoptosis was gradually increased in proportion to the concentration. We also identified the apoptotic effect of selonsertib by characterizing nuclear morphology with TUNEL staining. Selonsertib induced acquisition of the morphological features of apoptotic cells such as nuclear condensation and the formation of perinuclear apoptotic bodies in a dose-dependent manner (Fig. 2B). Additionally, the expression of cleaved caspase-3 (red) was increased by selonsertib treatment in both cell lines, which was assessed by quantification (Fig. 2C). These results indicated that selonsertib prevented HSC activation through induction of apoptotic cell death.
ASK1 is closely related to the induction of liver disease through activation of the MAPK signaling pathway, including p38 and JNK (Kovalic
The effects of selonsertib on the body weight and liver weight of the rats are shown in Fig. 4. Treatment with DMN caused a significant decrease in both body weight and liver weight compared to control group. However, oral administration of selonsertib (50 mg/kg) decreased the loss by 20-30% (Fig. 4). Moreover, liver fibrosis induced by DMN in rats was accessed by two histological methods: H&E and Picro-Sirius Red staining. Liver tissues of the control group showed typical architecture, whereas the DMN group displayed extensive and severe hemorrhagic necrosis, disruption of tissue architecture, along with infiltration of inflammatory cells. These alterations were remarkably reduced in the selonsertib group. Moreover, Picro-Sirius Red staining was conducted to identify collagen accumulation. In the control group, collagen was observed only in the periportal area, but the DMN group showed increased collagen and displayed bundles of collagen fibers surrounding the lobules, forming large fibrous septa. The thickening of these collagen fiber bundles was markedly reduced in the selonsertib group (Fig. 5A, 5B). The histopathological fibrosis scores and Sirius red staining index confirmed that the liver fibrosis was significantly reduced by selonsertib treatment, as compared to those in the DMN group. Besides, selonsertib decreased the levels of IFN-γ and TNF-α in the serum of DMN-treated rats (Fig. 5C).
We further determined whether selonsertib could inhibit the expression of profibrotic mediators such as α-SMA and collagen by suppression of ASK1 phosphorylation in DMN-induced liver fibrosis model (Fig. 6). The DMN group showed high expression levels of α-SMA, collagen, vimentin, and fibronectin in the periportal fibrotic areas, central veins, portal tracks, and fibrous septa. However, these fibrosis-related markers were weakly expressed in the selonsertib-treatment groups. Additionally, selonsertib inhibited the expression of matrix metalloprotease (MMP), which plays an important role in mediating HSC proliferation and activation, potentially by regulating ECM turnover. Furthermore, we investigated whether selonsertib inhibited ASK1/MAPK signaling. Expression of p-ASK1, p-p38, and p-JNK were amplified by DMN induction, whereas it was significantly diminished after selonsertib treatment (10 and 50 mg/kg). Taken together, our results demonstrated that selonsertib exerts potent effects against DMN-induced liver fibrosis models.
Liver fibrosis is a common consequence of chronic liver injury, leading to cirrhosis and hepatocellular carcinoma (Baffy, 2013). Activation of HSCs plays an essential role in this process, which is characterized by fibrogenic potential and high proliferative index (Zhang
Since HSCs are the primary source of ECM that play a pivotal role in liver fibrogenesis, they attracted attention as critical target cells for antifibrotic therapy (Higashi
Based on these results, the effect of selonsertib on apoptosis was anticipated in HSCs and animal models with DMN-induced liver fibrosis. Several studies have reported that activated HSCs increase ECM-related proteins, and in turn, the ECM itself regulates cell proliferation and apoptosis (Priya and Sudhakaran, 2008; Wang
Our data also revealed that selonsertib inhibited the ASK1/MAPK signaling pathway by decreasing the expression of p-p38 and p-JNK in both HSCs and DMN-induced liver fibrosis models. Given that phosphorylated p38 and JNK are known to increase expression of collagen (Tsukada
In conclusion, the current study demonstrated that selonsertib markedly attenuated the development of liver fibrosis by blocking ASK1/MAPK signaling both
This research was supported by National Research Foundation of Korea (2018R1A2A1A05077263, 2019M3E5D1A020 69621, 2014009392).
The authors declare that they have no competing interests.
![]() |
![]() |