FTY720 attenuates APAP‑induced liver injury via the JAK2/STAT3 signaling pathway
- Authors:
- Published online on: March 17, 2022 https://doi.org/10.3892/ijmm.2022.5123
- Article Number: 67
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Copyright: © He et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Acetaminophen (APAP) is a widely used analgesic and antipyretic drug (1). Although APAP is generally safe and effective, excessive APAP use can cause severe liver injury that eventually develops into liver failure (2). The APAP-induced liver injury (AILI) animal model displays characteristics similar to those observed in patients and has been widely used to study the mechanisms underlying this condition in order to identify potential therapeutic intervention strategies (3,4). The main pathogenic mechanism of AILI is that excessive APAP produces toxic metabolites of N-acetyl-p-benzoquinone imine (NAPQI) through cytochrome P450 (CYP) metabolism (mainly CYP2E1). The accumulated NAPQI depletes the intracellular glutathione levels. Any glutathione that is not bound to NAPQI can then bind with mitochondrial membrane proteins, leading to mitochondrial permeability transition. This can directly impair mitochondrial function in hepatocytes, causing lipid peroxidation, oxidative stress and DNA fragmentation, thus resulting in hepatocyte necrosis (5-7). In addition, it has also been shown that, following APAP treatment, a large number of neutrophils infiltrate the site of injury, further aggravating liver damage (8).
Fingolimod (FTY720) is a novel type of immunosuppressive drug derived from the ascomycete Cordyceps sinensis (9). Different from conventional immunosuppressive compounds, such as cyclosporine, tacrolimus and sirolimus, FTY720 acts by inducing lymphocyte apoptosis and homing (10). FTY720 is a sphingosine-1-phosphate (S1P) analogue, which can be phosphorylated by sphingosine kinase. Phosphorylated (p)-FTY720 is transported by S1P transporter proteins across the membrane to the outside of the cell and competitively binds to the S1P receptor, thus antagonizing downstream S1P signaling (11).
The JAK2/STAT3 signaling pathway can be activated by various cytokines and participates in numerous important biological processes, such as cell proliferation, differentiation and immune regulation (12-14). Previous studies have found that hepatocyte necrosis was closely related to the JAK2/STAT3 signaling pathway (15-17). However, it is unclear whether FTY720 exerts its immunosuppressive effect through the JAK2/STAT3 signaling pathway. Therefore, in order to further explore the preventive and therapeutic effect of FTY720 in vivo, an AILI animal model was used. The specific mechanism underlying the effect of FTY720 on the liver was evaluated by examining serum liver function markers and pathological changes in hepatocytes (18-20).
Materials and methods
Experimental animals and experimental design
Male C57BL/6 mice (8-9 weeks of age) were purchased from the Animal Experimental Center of China Medical University (Shenyang, China) and housed in a pathogen-free environment (temperature, 23±2°C; humidity, 55±5%) with a 12-h light/dark cycle and free access to food and water. Mice weighing 18-22 g (a total of 192 mice; 64 mice used for 3 cycles of experiments) were randomly separated into four groups after overnight fasting as follows: i) Control group, mice were administered 200 μl PBS; ii) FTY720 group, mice received 5 mg/kg FTY720 [Cayman Chemical Company; molecular formula, C19H34NO5P (Fig. 1A); molecular weight, 387.5; purity ≥98%; solubility in chloroform, 0.5 mg/ml; slightly soluble in DMSO)] intraperitoneally (i.p.); iii) APAP group, mice received 300 mg/kg APAP (MilliporeSigma) i.p.; and iv) FTY720 + APAP group, mice were administered FTY720 again after APAP challenge for 30 min. Mice were anesthetized with 200 mg/kg ketamine and 10 mg/kg xylazine i.p., then euthanized 6 h (n=8/group) or 12 h (n=8/group) after APAP injection. Serum was collected and livers were harvested. In addition, AG490 was used to inhibit the JAK2/STAT3 signaling pathway in mice. Mice weighing 18-22 g (a total of 120 mice; 30 mice used for 3 cycles of experiments) were randomly separated into four groups after overnight fasting as follows: Each group received 300 mg/kg APAP i.p; i) Control group, mice were administered 200 μl PBS; ii) FTY720 group, mice received 5 mg/kg FTY720; iii) AG490 group, mice received 50 μM AG490; and iv) FTY720 + AG490 group, mice were administered FTY720 and AG490 again after APAP challenge for 30 min. Mice were anesthetized with 200 mg/kg ketamine and 10 mg/kg xylazine i.p., then euthanized 6 h (n=10/group) or 12 h (n=8/group) after APAP injection. Serum was collected and livers were harvested. The experimental protocols were approved (approval no. CMU2021076) by the Institutional Animal Care and Use Committee and conformed to the Guidelines for the Care and Use of Laboratory Animals of China Medical University (Shenyang, China).
Liver histopathology
The mice were anesthetized with 200 mg/kg ketamine and 10 mg/kg xylazine i.p., then sacrificed by cervical dislocation. For histopathological examination, the liver tissues were fixed in 10% formalin at 25°C for 24 h, then embedded in paraffin, sectioned at 5-μm thickness and stained with H&E at 25°C for 10 min. To estimate the extent of necrosis, the specimen was observed under a light microscope, using ImageJ software (version 1.8.0; National Institutes of Health) to calculate the necrotic area.
Immunohistochemistry
Paraffin tissue sections of mouse livers were baked at 60°C for 1 h. Next, the samples were dewaxed in gradient xylene, and placed in water after dewaxing. Subsequently, 10 parts of distilled water to 1 part of 30% H2O2 was added, and then the sections were washed 3 times with distilled water at room temperature for 10 min each time. The sections were then immersed in 0.01 M citric acid buffer, heated to boiling at maximum power in a microwave (98-100°C), cooled (5-10 min), and repeated twice. The slices were cooled naturally at room temperature and washed 3 times with PBS for 5 min each time. Next, the sections were sealed with 5% BSA at room temperature for 20 min, and excess liquid was removed. The primary antibody (F4/80; 1:500; product no. 70076; Cell Signaling Technology, Inc.) was then added dropwise at 37°C for 1 h or 4°C overnight. The sections were then washed 3 times with PBS for 3 min each time. Subsequently, the secondary antibody (HRP-conjugated goat anti-mouse IgG; product no. 91196; 1:5,000; Cell Signaling Technology, Inc.) was added dropwise at 37°C for 15-30 min. The sections were then washed 3 times with PBS for 3 min each time. The streptavidin-biotin complex (SABC) was then added dropwise at 37°C for 30 min. Next, the sections were washed 3 times again with PBS for 5 min each time. The color developing agent was then added dropwise in 1 ml distilled water and mixed well. After the DAB chromogenic agent was prepared, it was added onto the sections at room temperature, and the reaction time was observed under a microscope (~5 min). The sections were then rinsed with tap water and then with distilled water. The sections were counterstained with hematoxylin for 2 min and then rinsed with tap water. Finally, the film was sealed by gradient dehydration and neutral resin. The film was observed and images were captured under a light microscope (OLYMPUS IX73; Olympus Corporation).
Plasma assay
Plasma alanine transaminase (ALT; cat. no. C009-2-1), aspartate transaminase (AST; cat. no. C010-2-1) and albumin (ALB; cat. no. A028-2-1; all from Nanjing Jiancheng Bioengineering Institute), and total bilirubin (TBIL) activities (cat. no. Hhzt-4576; Shanghai Huzheng Biology Pharmaceutical Co., Ltd.) were measured according to the manufacturer's protocol.
Myeloperoxidase (MPO) ELISA
The levels of MPO in the liver tissue were determined using an MPO ELISA kit [cat. no. ZC-38698; Shanghai Zhuo Cai Technology Co., Ltd. (ZCIBIO)] according to the manufacturer's protocol. Briefly, liver tissue samples were washed with PBS and homogenized in RIPA (cat. no. 89900; Thermo Scientific, Inc.) buffer using a tissue homogenizer (Ultra-Turrax). The homogenate was then centrifuged at 5,000 × g for 5 min at 4°C and immediately used for ELISA. The sample readings were obtained using an ELx800 universal microplate reader (Biotek Instruments, Inc.).
Isolation and culture of primary liver cells
Mouse hepatocytes were isolated using a two-step collagenase perfusion method, as previously described (21). The cells were re-suspended in high-glucose DMEM (HyClone; Cytiva), then filtered using a 100-μm pore size cell strainer to remove undigested tissue fragments and connective tissue. The samples were then centrifuged at 50 × g for 3 min at 4°C. The supernatant was discarded and the cells were then re-suspended in 20 ml high-glucose DMEM. An equal volume of 90% Percoll (Cytiva) was added and mixed. The samples were then centrifuged at 200 × g for 10 min at 4°C. The dead cells at the top of the gradient were discarded, and the cell pellet was re-suspended in 30 ml high-glucose DMEM. The centrifugation (200 × g for 3 min at 4°C) was then repeated. The isolated hepatocytes were cultured in DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.) at 37°C. Cell viability was determined using 0.4% Trypan blue staining at room temperature for 5 min.
PI staining
Primary liver cells were cultured in a 24-well culture plate for 24 h, and then stimulated with APAP (concentration, 10 mM) or FTY720 (concentration, 5 μM), or APAP + FTY720 combined for 6 h, and dead cells were stained with 1 μg/ml PI (cat. no. P4170; Sigma-Aldrich; Merck KGaA) for 5 min. The nuclei were then stained with DAPI at room temperature for 5 min. Images were obtained using Nikon eclipse TE 2000-S fluorescence microscope (Nikon Corporation). The binary processing function in ImageJ software (version 1.8.0) was used to process immunofluorescence images and measure the fluorescence parameters of cells.
Preparation of murine livers for laser scanning intravital microscopy
The mice were anesthetized with 200 mg/kg ketamine and 10 mg/kg xylazine (22). A midline and lateral incision along the costal margin to the midaxillary line was used to expose the liver. The mouse was placed in the right lateral position on the heating plate, and the ligaments connecting the liver to the diaphragm were transected to allow the liver to become exteriorized onto a glass coverslip. The blood flow was maintained. To prevent dehydration, exposed abdominal tissues were covered with a saline-soaked gauze. Neutrophils of mice at normal body temperature were stained using a rhodamine-conjugated anti-Ly-6G antibody (1:200; cat. no. PE-65140; ProteinTech Group, Inc.) for 30 min. Intravital microscopy was performed using an LSM-880 inverted microscope (Zeiss AG).
Cell viability measurement
Hepatocytes were seeded at a density of 2×103 cells/well and treated with APAP (10 mM) and FTY720 (5 μM) for 6 h in a 96-well plate incubated at 37°C with 5% CO2. A 10-μl volume of Cell Counting Kit-8 (Dojindo Laboratories, Inc.) solution was added to the cells, which were then incubated at 37°C with 5% CO2 for 2 h. The optical density was measured at a wavelength of 450 nm.
Reverse transcription-quantitative PCR (RT-qPCR)
Total RNA was extracted from liver tissue in TRIzol® (Takara Bio, Inc.) according to the manufacturer's protocol. First-strand cDNA synthesis was carried out using a reverse transcription kit (Takara Bio, Inc.) according to the manufacturer's protocol. SYBR® Green was used to set up the PCR using an CFX 96 Touch System (Bio-Rad Laboratories, Inc.) according to the manufacturer's instructions. The amplification conditions included 32 cycles at 95°C for 20 sec, 57.2°C for 30 sec, 72°C for 30 sec and 95°C for 2 min. qPCR was carried out using the following primers: TNF-α forward, 5′-ACG GCA TGG ATC TCA AAG AC-3′ and reverse, 5′-AGA TAG CAA ATC GGC TGA CG-3′; IL-6 forward, 5′-ACA ACC ACG GCC TTC CCT AC-3′ and reverse, 5′-TCC ACG ATT TCC CAG AGA ACA-3′; IL-1β forward, 5′-TGT CTT GGC CGA GGA CTA AGG-3′ and reverse, 5′-TGG GCT GGA CTG TTT CTA ATG C-3′; C-C motif chemokine receptor (CCR)2 forward, 5′-TGG TAA ATT CTT CAG CTT TTC C-3′ and reverse, 5′-TCC ACA ACC TGA TAA AGC CTC C-3′; CCR5 forward, 5′-CTG GCC ATC TCT GAC CTG TTT TTC CTC C-3′ and reverse, 5′-CAG CCC TGT GCC TCT TCT TCT CAT TTC-3′; C-X-C motif chemokine ligand (CXCL)9 forward, 5′-TCC TTT TGG GCA TCA TCT TC-3′ and reverse, 5′-TTC CCC CTC TTT TGC TTT TT-3′; C-X-C motif chemokine receptor (CXCR)2 forward, 5′-ATG CCC TCT ATT CTG CCA GAT-3′ and reverse, 5′-GTG CTC CGG TTG TAT AAG ATG AC-3′; catalase (CAT) forward, 5′-AGG CTC AGC TGA CAC AGT TC-3′ and reverse, 5′-ATG GAG AGA CTC GGG ACG AA-3′; glutathione peroxidase (GSH-PX) forward, 5′-TAC AAC ATG TCG GAC CCA CG-3′ and reverse, 5′-CCA GGT GGA ATG AGG GCA AT-3′; total superoxide dismutase (T-SOD) forward, 5′-GCC CAA ACC TAT CGT GTC CA-3′ and reverse, 5′-AGG GAA CCC TAA ATG CTG CC-3′; and GAPDH forward, 5′-TGC AGT GGC AAA GTG GAG ATT-3′ and reverse, 5′-TCG CTC CTG GAA GAT GGT GAT-3′. qPCR assays were conducted in triplicate for each sample, and the expression levels were calculated using the 2−ΔΔCq method (23). GAPDH was used as the housekeeping gene.
Western blot analysis
Hepatocytes were cultured in six-well plates and exposed to APAP treatment or FTY720/PBS. The cells were lysed using RIPA lysis buffer with 1 mM PMSF and 1 mM phosphatase inhibitors for 30 min on ice, then centrifuged at 500 × g for 15 min at 4°C. The supernatant was diluted in SDS-PAGE loading buffer (the proteins were determined using BCA kits), boiled at 100°C for 10 min, and then 20 μg of protein was loaded on 8-12% gels and transferred to a PVDF membrane (MilliporeSigma). The membranes were blocked with 5% BSA in PBS with Tween-20 (1:2,000 in PBS) for 1 h at room temperature, then incubated with primary antibodies against phosphorylated (p)-JAK2, JAK2, p-STAT3, STAT3, BAX, BCL-2 and GAPDH overnight at 4°C. The membranes were washed 3-4 times with PBST, incubated with HRP-conjugated goat anti-mouse IgG secondary antibody (cat. no. 91196; 1:5,000; Cell Signaling Technology, Inc.) for 1-2 h at room temperature and washed again 3-4 times with PBST. The protein bands were visualized using enhanced chemiluminescence reagent (PerkinElmer, Inc.). A western blotting imaging system (Model, JP-K300; Shanghai Jiapeng Technology Co., Ltd.) was used to obtain all images. Densitometric analysis was performed using ImageJ software version 1.8.0. The list of antibodies is presented in Table I.
ROS staining
Primary hepatocytes were cultured in a 24-well culture plate for 24 h, and then stimulated with APAP (concentration, 10 mM) or FTY720 (concentration, 5 μM), or APAP + FTY720 + AG490 (concentration, 5 μM; cat. no. CSN13724; CSNpharm, Inc.) combined at 37°C for 6 h. The primary hepatocytes were stained with 10 μmol/ml ROS-DHE (cat. no. D6883; Sigma-Aldrich; Merck KGaA) in a dark incubator at 37°C for 30 min. ROS levels were deter- mined by measuring the fluorescence under a fluorescence microscope (Olympus Corporation) and analyzed using ImageJ software (version 1.8.0).
Statistical analysis
The data are presented as the mean ± SD. Data were analyzed using unpaired two-tailed Student's t-tests. Statistical analysis was carried out using SPSS software (version 23.0 for Windows; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.
Results
FTY720 alleviates APAP-induced liver injury
The serum levels of ALT and AST were upregulated 6 and 12 h following treatment with APAP (Fig. 1B), which indicated that excessive APAP could induce liver injury. Additionally, APAP treatment resulted in the destruction of liver tissue structure. In fact, as evidenced by H&E staining, the area of liver injury increased significantly (Fig. 1C). However, FTY720 significantly reduced the serum levels of AST and ALT at 6 and 12 h following treatment with APAP (Fig. 1D). H&E staining also indicated that FTY720 significantly reduced the area of liver injury at 6 and 12 h following treatment with APAP (Fig. 1E). These data indicated that FTY720 could alleviate the liver damage induced by APAP.
FTY720 reduces APAP-induced inflammation
Previous studies (24-26) have demonstrated that APAP-induced hepatocyte necrosis causes local inflammation and activates cells to express adhesion molecules, which promotes the recruitment of immune cells and the release of pro-inflammatory factors and chemokines, thereby aggravating liver injury.
The expression of MPO in the FTY720 + APAP group was significantly reduced compared with the APAP group (Fig. 2A). Furthermore, the effect of FTY720 on neutrophil and macrophage recruitment in APAP-induced liver injury was examined. Compared with the APAP group, macrophage recruitment was significantly reduced in the FTY720 + APAP group (Fig. 2B). In vivo imaging was used to directly observe the dynamic recruitment of neutrophils under different treatment conditions. A large number of neutrophils was recruited and adhered to the liver in APAP-treated mice. However, in the FTY720 + APAP group, recruitment and adhesion of neutrophils to the liver was reduced. Neutrophils were not recruited in mice treated with PBS or FTY720 alone (Videos SI-IV). In addition, FTY720 significantly reduced the expression levels of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1β in damaged liver tissue (Fig. 2C). Similarly, FTY720 significantly reduced the mRNA expression of the chemokines and chemokine receptors CCR2, CCR5 and CXCL9 (Fig. 2C). These findings indicated that FTY720 reduces APAP-induced immune cell recruitment and release of pro-inflammatory cytokines and chemokines, thereby attenuating AILI.
FTY720 can activate JAK2/STAT3 signaling and inhibit apoptosis and inflammation
Previous studies have suggested that the JAK2/STAT3 signaling pathway is involved in important biological processes, such as cell proliferation, differentiation, apoptosis and immune regulation (12-14). The effect of FTY720 on the JAK2/STAT3 signaling pathway was therefore examined. APAP + FTY720 co-treatment significantly activated the JAK2/STAT3 signaling pathway compared with APAP treatment alone (Fig. 3A). Furthermore, the BAX protein expression levels were downregulated, while those of BCL-2 were upregulated in the APAP + FTY720 group compared with the APAP treatment group (Fig. 3B). In addition, FTY720 significantly reduced the activation of p65 in liver tissue compared with APAP treatment (Fig. 3C). These results indicated that FTY720 could inhibit apoptosis in hepatocytes.
FTY720 can directly protect hepatocytes against APAP-induced hepatotoxicity
To confirm whether FTY720 has a direct protective effect on hepatocytes, primary hepatocytes were treated with APAP, either alone or with FTY720. At the 6-h time-point, APAP induced hepatocyte apoptosis; however, FTY720 reversed this effect (Fig. 4A). Compared with APAP treatment, the viability of primary hepatocytes co-treated with APAP and FTY720 was increased (Fig. 4B). In addition, the JAK2/STAT3 signaling pathway was activated in primary hepatocytes co-treated with APAP and FTY720 (Fig. 4C). It was also observed that FTY720 downregulated the expression of the pro-apoptotic protein BAX and increased that of the anti-apoptotic protein BCL-2 (Fig. 4D). Thus, consistent with the in vivo results, FTY720 reduced hepatocyte apoptosis.
Inhibition of JAK2/STAT3 signaling attenuates the effect of FTY720 on hepatocyte apoptosis
AG490 (50 μM; 48 h), an inhibitor of the JAK2/STAT3 signaling pathway, was used in mice following the administration of APAP, either alone or together with FTY720 (Fig. 5A). In both groups, the inhibition of JAK2/STAT3 signaling resulted in a significant increase in ALT and AST (Fig. 5B). H&E staining also demonstrated that, following the inhibition of the JAK2/STAT3 signaling pathway, the area of damaged liver tissue significantly increased in both groups (Fig. 5C). In addition, neutrophil recruitment and the survival rate were also significantly increased (Fig. 5D and E, respectively). These data suggested that the JAK2/STAT3 signaling pathway may mediate the effects of FTY720 on hepatocytes.
Inhibition of JAK2/STAT3 signaling attenuates the effect of FTY720 on oxidative stress
Oxidative stress resulting from APAP-induced hepatocyte apoptosis can aggravate liver injury. The oxidative stress level of primary hepatocytes decreased significantly following co-treatment with APAP and FTY720, although this effect was reversed by AG490 (Fig. 6A). Moreover, the mRNA expression levels of CAT, GSH-PX and T-SOD, which are markers of oxidative stress, increased significantly following FTY720 treatment and were significantly downregulated by AG490 (Fig. 6B). These data suggested that FTY720 inhibits APAP-induced oxidative stress by regulating the JAK2/STAT3 signaling pathway. In summary, APAP led to hepatocyte apoptosis and increased neutrophil and macrophage recruitment, further aggravating liver injury. FTY720 can reverse APAP-induced liver cell apoptosis, excessive oxidative stress injury and neutrophil and macrophage recruitment by the activating JAK2/STAT3 signaling pathway, thereby attenuating liver injury (Fig. 6C).
Discussion
The improper use of health products, such as prescription medicines, over-the-counter drugs and traditional Chinese medicines, can cause varying degrees of liver damage (27). The incidence of drug-induced liver injury (DILI) has been increasing in recent years (28). In the process of DILI, drugs and their metabolites combine with endogenous hepatocyte components to form new complexes, which activate hepatocytes and lead to hepatocyte injury or even death (29,30). In China, DILI accounts for ~10% of hospitalized patients with acute hepatitis. Clinical screening also suggests that 20-50% of the adults with elevated transaminase levels suffer from DILI (31). Therefore, DILI has become a common clinical liver disease.
It has been reported that excessive APAP intake leads to the accumulation of the toxic metabolite NAPQI in hepatocytes (32). The accumulation of NAPQI downregulates glutathione in hepatocytes; NAQPI also binds to outer mitochondrial membrane proteins (4,33). The covalent binding of NAPQI to mitochondrial proteins leads to oxidative stress and the opening of mitochondrial membrane permeability transition pore, which leads to reduced mitochondrial membrane potential, mitochondrial function and ATP production in hepatocytes. This results in lipid peroxidation, oxidative stress and DNA fragmentation, leading to the necrosis of hepatocytes (24). Following hepatocyte necrosis, a large number of damage-associated molecular patterns and chemokines are released, thereby activating Kupffer cells located in the liver and recruiting macrophages and neutrophils from the peripheral circulation to the site of liver injury (25,26). This results in aseptic inflammation, further aggravating liver injury (7,34). Taken together all aforementioned studies have revealed that APAP-induced liver injury is a very complex pathophysiological process, involving inflammation, apoptosis and neutrophil infiltration.
In the present study, neutrophil infiltration into the liver was observed following intraperitoneal injection of APAP in mice. However, when FTY720 and APAP were co-administered, the number of neutrophils infiltrating the liver was significantly reduced compared with that of the APAP group. Another study has suggested that FTY720 induces neutrophil extracellular traps through an NADPH oxidase-independent pathway and that this compound could represent a potential antibacterial drug (35). FTY720 is an S1P analog and a potent S1P receptor modulator. It induces the internalization of S1P1 and consequently inhibits S1P activity. Previous studies have also confirmed that S1P prevents egress of hematopoietic stem cells from the liver to reduce fibrosis and suppresses TLR-induced CXCL8 secretion from human T cells (36,37). Liang et al (38) reported that CXCL9/10 possesses antifibrotic roles on liver non-parenchymal cells. In previous studies the depletion of peripheral T cells by FTY720 resulted in increased infiltration of innate immune cells concomitant to reduced T-cell infiltration and exacerbation of hypoxic-ischemic-induced brain injury, which indicates that neonatal T cells may promote endogenous neuroprotection in the term-born equivalent hypoxic-ischemic brain potentially providing new opportunities for therapeutic intervention (39,40). It may be hypothesized that FTY720 could directly affect the recruitment of neutrophils and macrophages that express S1P receptors. FTY720 is a novel type of immunosuppressant derived from the ascomycete Cordyceps sinensis, which plays an important role in immunosuppression by inducing lymphocyte apoptosis and homing (41,42). Furthermore, it has been reported that rat spleen cells cultured with FTY720 displayed the typical characteristics of apoptosis, including disappearance of microvilli on the cell surface, concentration of chromatin and the formation of apoptotic bodies (43). Human lymphocytes co-treated with FTY720 exhibit the same features. Nagahara et al (44) suggested that FTY720 induced lymphocyte apoptosis through changes in membrane permeability and the release of cytochrome c from cells. However, other studies have shown that the molecular mechanism of lymphocyte apoptosis was complex and resulted from a variety of factors (45,46). Due to the downregulation of the BCL-2 protein and upregulation of the BAX protein, it may be suggested that the BCL-2 family is involved in FTY720-induced lymphocyte apoptosis. In the present study, FTY720 reversed the BAX/BCL-2 ratio and APAP-induced oxidative stress. It has been demonstrated that FTY720 could inhibit the proliferation of glomerular mesangial cells by inducing cell cycle arrest and apoptosis, possibly via the regulation of the expression of cell cycle-related genes and BAX/BCL-2 (47). In addition, exosomes could ameliorate the morphology of neurons, reduce inflammatory infiltration and edema, decrease the expression of BAX and AQP-4, upregulate the expression of claudin-5 and BCL-2 and inhibit cell apoptosis (48).
In the present study, AILI led to hepatocyte apoptosis and upregulation of pro-inflammatory factors, indicative of activation of the innate immune system. IL family members (IL-6, -13 and -22) can effectively activate STAT3 during the process of liver repair. STAT3 is an essential signaling molecule that can directly or indirectly regulate the expression of important genes in the process of liver repair (49). STAT3 is mainly coupled with JAK tyrosine protein kinases and participates in the signal transduction process downstream of extracellular signals, such as ILs; with significant anti-apoptotic and pro-mitotic effects (50). It has also been found that pro-inflammatory factors can activate and phosphorylate JAK2, further induce STAT3 phosphorylation, thus participating in the regulation of gene transcription and mediating the expression of a variety of pro-inflammatory factors (51). Therefore, detecting the serum levels of IL-6, -13 and -22, as well as the expression of STAT3 and JAK2 in liver tissue can provide insight into the effect of FTY720 on AILI.
However, there is a limitation in the present study. FTY720 hydrochloride is an analog of sphingosine and a potent modulator of S1P receptors. FTY720 hydrochloride is phosphorylated by sphingosine kinases, particularly by SK2, and then binds S1PR1, 3, 4, and 5. FTY720 hydrochloride induces the internalization of S1P1, and consequently, inhibits S1P activity (52-54). S1P was not analyzed in the present study. Therefore, S1P could be analyzed in the future.
In conclusion, FTY720 could significantly ameliorate AILI. FTY720 was able to inhibit the metabolism of APAP oxidase and reduce the production of NAPQI; it could also inhibit the oxidative stress produced by mitochondrial permeability transition to reduce hepatocyte death. FTY720 also reduced the infiltration of neutrophils into the liver, thereby reducing AILI in mice. FTY720 may serve this protective role by regulating the JAK2/STAT3 signaling pathway. The present findings revealed a novel mechanism through which FTY720 attenuates AILI and provide a theoretical basis for the clinical treatment of AILI using this compound.
Supplementary Data
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions
XH and BC designed the study and carried out the experiments. KK, DP and YS analyzed the data, as well as wrote and edited the manuscript. All authors have read and approved the final manuscript and confirm the authenticity of all the raw data.
Ethics approval and consent to participate
Experiments were conducted under protocols approved (approval no. CMU2021076) by the Institutional Animal Care and Use Committee and conformed to the Guidelines for the Care and Use of Laboratory Animals of China Medical University (Shenyang, China).
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Acknowledgments
Not applicable.
Funding
No funding was received.
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