Front. Cell. Infect. Microbiol.Frontiers in Cellular and Infection MicrobiologyFront. Cell. Infect. Microbiol.2235-2988Frontiers Media S.A.10.3389/fcimb.2025.1653725Original ResearchUrantide alleviates lipopolysaccharide/D-galactosamine-induced acute liver failure through upregulating carboxylesterase1f in miceYangWanhua†Writing – original draftBianShu†Writing – original draftLiuLiangming*Writing – review & editingSongjiang Hospital Affiliated to Shanghai Jiaotong University School of Medicine, Shanghai, ChinaCorrespondence: Liangming Liu, liuliangming@shsmu.edu.cn
These author shave contributed equally to this work and share first authorship
To investigate the effect of urotensin II(UII)/UII receptor(UT) on hepatic carboxylesterase1f(Ces1f) expression in acute liver failure(ALF) mice.
Methods
ALF was induced in male Balb/c mice using lipopolysaccharide (LPS)/D-galactosamine (D-GalN) i.p. after a tail vein injection of Urantide, a specific antagonist of UT receptor. Liver tissues were collected at 0-12h to detect UII and Ces1f mRNA levels in ALF mice. Mice were divided into four groups (n=6 each group): (A) Urantide(-), LPS/D-GalN(-), (B) Urantide(+), LPS/D-GalN(-), (C) Urantide(-), LPS/D-GalN(+), and (D) Urantide(+), LPS/D-GalN(+). Liver tissues were collected at 6 h after a challenge of LPS/D-GalN. Time-depended levels were observed of hepatic UII and Ces1f mRNA over a period of 12 hours after LPS/D-GalN challenge. Liver injury was evaluated via hematoxylin-eosin (HE) staining, serum alanine aminotransferase(ALT)/aspartate aminotransferase(AST), and the mRNA and protein expression levels of Ces1f and UII expression were determined by fluorescence in situ hybridization(FISH), quantitative real-time PCR (qPCR) and Western blotting (WB), respectively.
Results
After LPS/D-GalN injection, hepatic UII mRNA rose at 2 h (P < 0.05 vs 0 h), reached the peaked level at 6 h, and the level began to degrade at 8 h, but remained higher than at 0 h (P < 0.05, 10 h vs 0 h) till 12 h (P>0.05, 12 h vs 0 h); while hepatic Ces1f mRNA decreased at 6 h (P < 0.05 vs 0 h), reached the lowest level at 10 h, but began to rise at 12 h (P>0.05, 12 h vs 10 h). In addition, urantide pretreatment inhibited the up-regulated expressions of hepatic UII mRNA and protein, whereas increases the down-regulated expressions of hepatic Ces1f mRNA and protein induced by LPS/D-GalN attack at 6 h.And serum ALT and AST levels were significantly decreased, whereas hepatic inflammatory injury improved via urantide injection in LPS/D-GalN-induced ALF mice.
Conclusion
Ces1f maybe negatively regulated by UII/UT signal in LPS/D-GalN-induced ALF.
acute liver failureurotensin IIUII receptorCes1fmouseThe authors declared that financial support was received for work and/or its publication. The research was mainly funded by National Natural Science Foundation of China (No.81770612, 81070357) and Science and Technology Research of Songjiang District Science and Technology Commission in Shanghai (No.2023SJKJGG027).section-at-acceptanceClinical and Diagnostic Microbiology and ImmunologyIntroduction
Acute liver failure (ALF) is an acute onset condition characterized by severe liver damage caused by multiple factors, leading to significant impairment or decompensation of the liver’s synthetic, detoxification, metabolic, and biotransformation functions. As the body’s largest immune organ, hepatic damage involves immune-mediated inflammatory mechanisms exerted by hepatic non-parenchymal on parenchymal cells underlying the onset and progression of ALF (Wu et al., 2010). In the liver, Kupffer cells (KC), the liver’s resident macrophages, are the most important non-parenchymal cells, playing a pivotal role in the innate immue. An innate activation is the most early event triggering hepatic inflammatory injury (Triantafyllou et al., 2018a, Triantafyllou et al., 2018b; Antoniades et al., 2014). Under the stimulation of liver injury factors (including lipopolysaccharide (LPS)), KCs are activated and undergo M1 polarization (inflammatory phenotype), releasing large amounts of pro-inflammatory cytokines (such as tumor necrosis factor α(TNF-α), interleukin 1β (IL-1β), interferon γ (IFN-γ), interleukin 6 (IL-6), etc.) and chemokines (e.g.,C-X-C motif chemokine ligand 1 (Cxcl1), C-C motif chemokine ligand (Ccl3), C-C motif chemokine receptor-like 2 (Ccrl2), etc.), working in concert with acquired immune cells to eliminate common pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the body (Parlar et al., 2023; Li et al., 2023).
In ALF mice, KCs express Urotensin II (UII) at high levels. UII is a cyclic polypeptide molecule containing 11 amino acids with neurohormone-like biological activity.It plays a significant role in various pathophysiological activities, such as regulating immune function and participating in the regulation of hypertension and renal fibrosis (Sun and Liu, 2019). The UII receptor (Urotensin receptor, UT) is a specific orphan G protein-coupled receptor that binds specifically to UII to mediate signal transduction. Cells expressing UII and UT include endothelial cells, hepatobiliary epithelial cells, and KCs in the liver (Sun and Liu, 2019). Research confirms that UII primarily mediates hepatic immune-inflammatory injury by activating the toll-like receptor 4 (TLR4) signaling pathway and releasing its downstream proinflammatory cytokines, including TNFα and IL-1β (Liang et al., 2013). The hepaticellular distribution patterns of UII and UT show remarkable consistency, suggesting potential regulation by autocrine and paracrine mechanisms. As the liver’s innate immune cells, KCs have been identified as the primary cellular source of UII/UT and inflammatory cytokine expression. Urantide, a specific antagonist of UT, can effectively inhibit the UII/UT signal and the release of these inflammatory mediators (Yu et al., 2020).
During ALF, hepatic metabolism is significantly impaired, including drug metabolism and lipid metabolism. Carboxylesterase 1 (Ces1), as a primary metabolic enzyme in the liver, is synthesized and highly expressed in the liver (Collins et al., 2022). It promotes the metabolism of various environmental toxins, carcinogens, and drugs (e.g., clopidogrel, sacubitril/valsartan, oseltamivir) (Liu et al., 2024) and participates in the transport and metabolism of cholesterol esters and free fatty acids (Satoh et al., 2002; Sanghani et al., 2009; Satoh and Hosokawa, 1998). Ces1f, a subtype of carboxylesterase 1, exhibits hydrolytic activity toward triglycerides and retinol (RE) (He et al., 2019; Crow et al., 2010; Mangum et al., 2018). Study indicate Ces1f plays an important role in liver X receptor α(LXRα) protection of hepatic damage (Becares et al., 2019). Our reporter showed that KCs expressed Ces1f, and hepatic damage aggravated due to Ces1f gene knockdown in the liver. It suggests Ces1f has hepatic protection against immune infammation via KC activation. However, the effect of UII/UT on Ces1f remains unclear.
This study aims to investigate <italic>Ces1f</italic> gene expression in LPS/D-GalN-induced ALF mice model using UT antagonist urantide, thereby further elucidating the pathophysiological mechanisms through which the UII/UT system mediates ALF development.Materials and methodsReagents
Lipopolysaccharide (LPS) and D-galactosamine (D-GalN) were purchased from Sigma-Aldrich (St. Louis, MO,USA). Urantide was obtained from Peptides International (Tokyo, Japan). The SYBR Green PCR Master Mix and reverse transcription kit were acquired from TaKaRa Bio Inc. (Tokyo, Japan). Trizol reagent was procured from Thermo Fisher Scientific (Shanghai, China). All PCR primers were commercially synthesized by Shanghai Bioengineering Co. Ltd.(Shanghai, China).
Animals experiments
Healthy male Balb/c mice, aged 6–8 weeks, specific pathogen-free (SPF) grade, weighing 20–22 g, were obtained from the Experimental Animal Center of Shanghai First People’s Hospital Affiliated to Shanghai Jiao Tong University (License No.SCXK (Hu) 2018-0004). The mice were housed under controlled environmental conditions: temperature (20–22 °C), noise (<50 dB), humidity (40–80%), and a 12/12-hour light/dark cycle. They were provided with ad libitum access to food and water. Before the experiment, the mice were fasted for 12 hours with free access to water. Animal experimental protocols were approved by the Animal Welfare and Ethics Committee of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine (Approval No. ACE-001-2024-R1).
A total of 42 healthy male Balb/c mice were intraperitoneally administered LPS (50 μg/kg) combined with D-GalN (800 mg/kg). Liver tissue samples were collected at specified time points (0, 2, 4, 6, 8, 10, and 12 h; n=6 per time point) for analysis of UII mRNA and Ces1f mRNA expression levels.An additional cohort of 24 healthy male Balb/c mice were randomly allocated into four experimental groups (n=6 per group): Group A: urantide(-) LPS/D-GalN(-); Group B: urantide(+) LPS/D-GalN(-); Group C: urantide(-) LPS/D-GalN(+); Group D: urantide(+) LPS/D-GalN(+). The ALF mice model was established according to established protocols (Sanghan et al.,2009) through intraperitoneal injection of LPS (50 μg/kg) and D-GalN (800 mg/kg) dissolved in 0.2 mL of 0.9% sodium chloride solution. Control animals received an equivalent volume (0.2 mL) of 0.9% sodium chloride solution alone. For pretreatment groups, urantide (0.6 mg/kg dissolved in 0.9% sodium chloride) was administered via tail vein injection 30 minutes prior to LPS/D-GalN or saline administration. Liver tissue specimens and serum samples were collected 6 hours post-treatment.
Biochemical assays
Frozen serum samples from each experimental group were retrieved from -80 °C storage and thawed at room temperature. The serum was then diluted to an appropriate volume with 1× phosphate-buffered saline (PBS). Subsequently, the samples were sent to the Clinical Laboratory of Songjiang District Central Hospital (Shanghai, China) for automated biochemical analysis. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured using a fully automated biochemical analyzer. The obtained values were multiplied by the corresponding dilution factor to determine the actual serum ALT and AST concentrations.
Patholigical examination
The tissue microarray sections were sequentially immersed in xylene and graded ethanol solutions for deparaffinization and rehydration. Following antigen retrieval, immunohistochemical staining was performed using 3,3’-diaminobenzidine (DAB) as the chromogen with hematoxylin counterstaining. The stained sections were then dehydrated through an ethanol gradient, cleared in xylene, and mounted with neutral balsam under cover slips. All histological assessments were conducted in a blinded manner by certified pathologists from our institution, who randomly selected 8–10 high-power fields (×400 magnification) for evaluation under light microscopy.
For RNA isolation, 50 mg of liver tissue was homogenized in 1 mL of Trizol reagent (Takara R036A) following the manufacturer’s protocol, with all procedures performed on ice to prevent RNA degradation. The extracted total RNA was resuspended in 50 μL of RNase-free water, and its concentration and purity were spectrophotometrically determined. All samples exhibited A260/A280 ratios >1.8, indicating high RNA purity. First-strand cDNA synthesis was performed using 500 ng of total RNA with 2 μL of 5× PrimeScript RT Master Mix in a 10 μL reaction volume (adjusted with DEPC-treated water). The reverse transcription conditions were: 37 °C for 90 s, 85 °C for 5 s, followed by indefinite hold at 4 °C. The resulting cDNA was stored at -20 °C until use.Gene-specific primers (designed and synthesized by ShanghaiBioengineering Co.) with their corresponding sequences and product lengths are listed in Table 1. Quantitative real-time PCR was performed using the SYBR Green system(Takara RR420) in a laminar flow hood with reagents maintained on ice. The 20 μL reaction mixture contained:10 μL of TB Premix Ex Taq (2×),0.4 μL each of forward and reverse primers,0.4 μL of ROX Reference Dye II (50×),2 μL of cDNA template,RNase-free water to final volume.Amplification was conducted on an ABI7500 instrument using a two-step protocol:Initial denaturation: 95°C for 15 min (1 cycle);Amplification: 40 cycles of 94°C for 20 s and 60°C for 34 s.GAPDH served as the endogenous reference gene. Following amplification, CT values were obtained and relative mRNA expression levels were calculated using the 2-ΔΔCT method.
Fresh liver tissues were immersed in fixative solution for ≥24 h at 4°C,then dehydrated through a graded ethanol series (70% to 100%),cleared in xylene, and infiltrated with paraffin (58–60°C) using an automated tissue processor. Tissues were embedded in paraffin blocks. Briefly, molten paraffin was poured into molds, and tissues were oriented before cooling on a -20°C freezing stage. Solidified blocks were trimmed for sectioning. Serial sections (5μm thickness) were dried at 60°C for 45 min and then dewaxed in xylene (2×10 min) and rehydrated through a graded ethanol series (100% to 70%).Slides were treated with 3% hydrogen peroxide (H2O2) in methanol for 10 min at room temperature (RT) to block peroxidase activity. Target mRNA was exposed by incubating sections in citrate buffer (pH 6.0) at 95°C for 15 min. Sections were covered with prehybridization buffer and incubated at 37°C for 4 h in a humidified chamber to reduce nonspecific binding. Digoxigenin (DIG)-labeled Ces1f probes (100 ng/μL in hybridization buffer) were applied, and slides were hybridized at 60°C overnight (16–18 h).Stringency washes: 2× SSC (RT, 10 min) → 0.5× SSC (60°C, 15 min).Blocking: 5% bovine serum albumin (BSA) in PBS for 1 h at RT. Probes were detected with biotinylated anti-DIG antibody (1:500, Roche) for 1 h at RT. Nuclei were counterstained with DAPI (5 μg/mL, 2 min).Slides were cover slipped with antifade mounting medium (Thermo Fisher) and imaged under a Zeiss LSM 880 confocal microscope (20×/40×objectives).
Western blotting
Approximately 20 mg of liver tissue was placed in a 1.5 mL tube. Added 1 mL of RIPA buffer supplemented with 50 μL of protease inhibitor cocktail (Roche). Tissue was mechanically disrupted using three 3-mm stainless steel beads in a tissue homogenizer (60 Hz, 2 min), followed by incubation on ice for 30 min to complete lysis. Lysates were centrifuged at 12,000 × g, 4°C for 10 min. Supernatants were collected and stored at −80°C until use. Protein concentration was determined using a BCA assay, following the manufacturer’s protocol. Samples were mixed with 4× Loading buffer and heated at 95°C for 5 min. Equal amounts of protein (20–30 μg/lane) were resolved on 12% polyacrylamide gels at 100 V for 90 min. Proteins were electrophoretically transferred to nitrocellulose membranes. Membranes were incubated in 5% non-fat milk in TBST for 1 h at RT. Rabbit monoclonal anti-Ces1f (1:1000, Abmart) and Rabbit monoclonal anti-UII (1:1000, Santa Cruz)Diluted in 5% BSA/TBST; incubated overnight at 4 °C with gentle agitation. HRP-conjugated goat anti-rabbit IgG (1:2000, beyotime) in 5% milk/TBST, 1 h at RT. Membranes were incubated with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) for 1 min and imaged using a Bio-Rad ChemiDoc™ MP System. Antibodies were removed with stripping buffer at RT for 10 min. Reblocked and reprobed for β-actin (1:10000, beyotime) or GAPDH (1:10000, beyotime) as loading controls. Band intensities were quantified using ImageJ 1.4.3.67 (NIH).
Statistical analysis
Quantitative data were expressed as mean ± standard deviation (SD) and analyzed using SPSS 20.0 software (IBM Corp., USA). For comparisons between two groups, independent samples t-test was used for parametric data, while the Mann-Whitney U test was applied for non-parametric data. Multiple group comparisons were performed using one-way ANOVA followed by appropriate post hoc tests (Tukey’s or LSD) when significance was detected; when the assumption of homogeneity of variance was violated (as assessed by Levene’s test), the Kruskal-Wallis test was employed instead. The P-value < 0.05 was considered statistically significant for all analyses.
ResultsTime-dependent expression of hepatic <italic>UII</italic> mRNA in LPS/D-GalN-induced ALF mice
Following LPS/D-GalN administration, hepatic UII mRNA expression exhibited a time-dependent dynamic pattern, with relative expression levels of 1 (baseline), 2.62 ± 0.61, 4.09 ± 0.71, 10.38 ± 1.49, 9.15 ± 0.88, 2.56 ± 0.57, and 1.46 ± 0.52 at 0, 2, 4, 6, 8, 10, and 12 h post-treatment, respectively. Statistical analysis revealed a significant upregulation as early as 2 h (P < 0.01 vs 0 h), peaking at 6 h (10.38 ± 1.49, P < 0.01) and remaining elevated at 8 h. Expression levels subsequently declined, showing significant reduction at 10 h (P < 0.05 vs 0 h) and returning to baseline by 12 h (P = 0.435 vs 0 h) (Figure 1). This biphasic response suggests rapid activation and subsequent resolution of UII signaling following inflammatory challenge.
Hepatic UII mRNA Expression in LPS/D-GalN-Induced ALF Mice at Different Time Points. The vertical axis represents the relative expression level of UII 2-△△ct, *Compared to 0 h P<0.05, #Compared to 8 h P<0.05.
Bar graph showing UII relative expression levels over time. Time points are 0h, 2h, 4h, 6h, 8h, 10h, and 12h. Expression peaks at 6h, with a significant decrease by 12h. Asterisks and hash marks denote statistical significance.Time-dependent expression of hepatic <italic>Ces1f</italic> mRNA in LPS/D-GalN-induced ALF mice
Following LPS/D-GalN challenge, hepatic Ces1f demonstrated a progressive downregulation, with relative expression levels measuring 1 (baseline), 0.85 ± 0.10, 0.92 ± 0.09, 0.58 ± 0.05, 0.43 ± 0.11, 0.26 ± 0.04, and 0.41 ± 0.04 at 0, 2, 4, 6, 8, 10, and 12 h post treatment, respectively. Statistical analysis revealed this suppression became statistically significant by 6 h (P < 0.01 vs 0 h), reaching a nadir at 10 h (0.26 ± 0.04), remained during the observed time window (all P < 0.05 vs baseline). Notably, the expression levels began to rise at 12 h (0.41 ± 0.04, P < 0.05 vs 10 h). These findings demonstrate that Ces1f downregulation (1) initiates till 6 h, (2) correlates temporally with ALF progression, and (3) exhibits a statistically reversion at the end of the observed timeframe (Figure 2), suggesting its expression kinetics may serve as an indicator of hepatic metabolic dysfunction or recovery during inflammatory injury.
Hepatic Ces1f mRNA Expression in LPS/D-GalN-Induced ALF Mice at Different Time Points. The vertical axis represents the relative expression level of Ces1f2-△△ct, *P < 0.05 vs 0 h, #P < 0.05 vs 10 h.
Bar chart displaying Ces1f relative expression levels (2^-ΔΔCt) over time from zero to twelve hours. Expression peaks at zero hours and declines steeply, reaching the lowest point at ten hours, with slight recovery at twelve hours. The legend indicates color coding for each time point. Asterisks denote significant differences, and a hash mark indicates additional significance.Hepatic <italic>UII</italic> mRNA expression in mice challenged by LPS/D-GalN via urantide pretreatment
Quantitative analysis of hepatic UII mRNA expression revealed significant differences among experimental groups, with relative expression levels of 1.00 (Group A), 1.08 ± 0.33 (Group B), 9.62 ± 2.31 (Group C) and 5.61 ± 1.3 (Group D). Statistical evaluation demonstrated that: (i) LPS/D-GalN-challenged Group C exhibited a 9.6-fold upregulation compared to controls (P < 0.01 vs Groups A/B); (ii) no significant difference existed between untreated controls (Group A vs B, P = 0.99); and (iii) urantide pretreatment in Group D significantly attenuated this drug induction by 41.7% versus Group C (P < 0.05) (Figure 3). These results establish that: (1) UII is robustly upregulated during ALF, (2) basal UII expression remains stable under physiological conditions, and (3) pharmacological UT receptor blockade partially reverses inflammation-induced UII overexpression, supporting the involvement of UII/UT signaling in ALF pathogenesis.
Expression of UII mRNA in liver tissue of mice treated with LPS/D-GalN at 6h. (A) Urantide(-)LPS/D-GalN(-); (B) Urantide(+)LPS/D-GalN(-); (C) Urantide(-)LPS/D-GalN(+); (D) Urantide(+)LPS/D-GalN(+); *P < 0.01 vs group A; #P < 0.05 vs group C;The vertical axis represents the relative expression levels of UII 2-△△ct.
Bar graph illustrating UII relative expression (2^-ΔΔCt) across four groups labeled A, B, C, and D. Group C shows the highest expression, followed by D, while A and B have low values. Significant differences are indicated by asterisks for C and both asterisk and hash for D. A color legend is on the right.Hepatic <italic>Ces1f</italic> mRNA expression in mice challenged by LPS/D-GalN via urantide pretreatment
The relative expression of hepatic Ces1f mRNA exhibited significant variations across experimental groups (Figure 4), with values of 1.00 (Group A), 0.91 ± 0.31 (Group B), 0.44 ± 0.12 (Group C), and 1.14 ± 0.40 (Group D). Key findings include: (1) Group C showed a 56% reduction in Ces1f expression compared to Group A (P < 0.01), demonstrating LPS/D-GalN’s potent suppressive effect; (2) No significant difference existed between control Groups A and B (P = 0.861), confirming urantide’s neutral effect under physiological conditions; and (3) Group D exhibited a 2.6-fold higher expression than Group C (P < 0.01), with levels similar to Group A (P = 0.372), indicating urantide’s capacity to completely rescue Ces1f suppression during ALF. These results demonstrate that UII/UT system activation mediates Ces1f downregulation in ALF, while its pharmacological inhibition restores normal expression patterns, suggesting Ces1f as both a biomarker and potential therapeutic target in acute liver injury.
Expression of Ces1f mRNA in liver tissue of mice treated with LPS/D-GalN at 6h. (A) Urantide(-)LPS/D-GalN(-); (B) Urantide(+)LPS/D-GalN(-);(C) Urantide(-)LPS/D-GalN(+); (D)Urantide(+)LPS/D-GalN(+);*P<0.01 vs group A; #P<0.01 vs group C;The vertical axis represents the relative expression level of Ces1f 2-△△ct.
Bar chart showing Ces1f relative expression levels (2^−ΔΔCt) across four groups: A, B, C, and D. Group C has the lowest expression, marked by an asterisk, and group D has the highest, marked with a hashtag. Error bars indicate variability.FISH Assay for the localization and expression levels of <italic>Ces1f</italic> mRNA in the liver
Fluorescence in situ hybridization (FISH) analysis demonstrated that Ces1f mRNA was predominantly localized in the cytoplasmic compartment with negligible nuclear staining across all experimental groups. Quantitative fluorescence intensity revealed striking differences in expression levels among groups: Group A (214,708.99 ± 34,817.92), Group B (209,717.48 ± 30,330.57), Group C (19,736.74 ± 3,059.74) and Group D (185,306.75 ± 18,052.25). Statistical analysis showed Group C exhibited significantly lower expression than Groups A, B and D (all P < 0.01), while Group D demonstrated a remarkable 9.4-fold increase versus Group C (P < 0.01), with no difference observed between Groups A and B (P = 0.755). These results clearly indicate that: (1) ALF induces profound suppression of cytoplasmic Ces1f expression; (2) Urantide pretreatment effectively restores Ces1f to near-physiological levels; and (3) The UII/UT system specifically modulates Ces1f expression under pathological conditions without affecting basal levels, suggesting its role as a stress-responsive regulatory mechanism in hepatocytes (Figure 5). The subcellular localization pattern further supports Ces1f’s functional role in cytoplasmic metabolic processes that are disrupted during acute liver injury.
FISH Assay for the Localization and Expression Levels of Ces1f mRNA in the Liver. (A) Urantide(-)LPS/D-GalN(-); (B) Urantide(+),LPS/D-GalN(-); (C) Urantide(-),LPS/D-GalN(+); (D) Urantide(+),LPS/D-GalN(+);*P < 0.01 vs group A; #P < 0.01 vs group C.
Fluorescence microscopy images showing Ces1f expression with red signals, and a merged image with blue counterstaining, across four samples labeled A, B, C, and D. Below, a bar graph compares the FISH IOD sum. Sample C shows a significant decrease indicated by an asterisk, while sample D shows a notable increase indicated by a hash mark.Expression of hepatic UII and Ces1f proteins in mice challenged by LPS/D-GalN via urantide pretreatment
Western blot analysis revealed significant alterations of hepatic UII and Ces1f protein expression across experimental groups (Figure 6). Statistical analysis shows the levels of UII protein in mouse liver among groups:Group A (0.8043 ± 0.1161), Group B (0.7231 ± 0.1061), Group C (1.046 ± 0.1817), and Group D (0.8043 ± 0.1233). The UII protein expression in Group C showed a marked increase compared to Group A (normal control) (P < 0.05), while urantide pretreatment in Group D significantly attenuated this upregulation relative to Group C (P < 0.05). Notably, no difference in UII expression was observed between Groups A and B (P = 0.7276). Statistical analysis shows the levels of Ces1f protein in mouse liver among groups:Group A (1.268 ± 0.1730), Group B (1.104 ± 0.34446), Group C (0.7813 ± 0.3829), and Group D (1.279 ± 0.2818). Ces1f protein expression demonstrated an inverse pattern, with Group C exhibiting significant downregulation versus Group A (P < 0.05), and Group D showing restored expression levels compared to Group C (P < 0.05). The similar Ces1f expression between Groups A and B (P = 0.6784) confirmed that urantide administration does not affect basal protein levels in normal hepatocytes. These protein-level findings corroborate our mRNA expression data, demonstrating that: (1) ALF induces coordinated upregulation of UII and downregulation of Ces1f; (2) Urantide effectively normalizes these pathological alterations; and (3) The UII/UT signal blocker specifically modulates Ces1f expression during inflammatory injury without influencing physiological homeostasis, highlighting its potential as a therapeutic target for acute liver failure.
Western blotting detection of UII and Ces1f protein expression in liver tissue. (A) Urantide(-),LPS/D-GalN(-); (B) Urantide(+),LPS/D-GalN(-); (C) Urantide(-),LPS/D-GalN(+); (D) Urantide(+),LPS/D-GalN(+);*P < 0.05 vs group A; #P < 0.05 vs group C.
Western blot results and bar graphs show the expression levels of UII and Ces1f proteins. UII is detected at 14 kDa with GAPDH as a 36 kDa loading control. Ces1f is detected at 61 kDa with β-actin as a 43 kDa loading control. Bar graphs to the right illustrate the UII/GAPDH and Ces1f/β-actin ratios for samples labeled A through D, showing variations in expression levels marked by an asterisk and hash symbol indicating statistical significance.The levels of serum ALT and AST in mice challenged by LPS/D-GalN via urantide pretreatment
Serum biochemical analysis revealed marked differences in hepatic injury markers across experimental groups. The ALT levels were 94.63 ± 10.08 U/L (Group A), 85.13 ± 9.72 U/L (Group B), 12510.75 ± 2064.49 U/L (Group C), and 5593 ± 1316.53 U/L (Group D), while corresponding AST levels measured 380 ± 39.70 U/L, 309 ± 32.90 U/L, 8495 ± 1308.42 U/L, and 3455.25 ± 535.98 U/L, respectively. Statistical comparisons demonstrated that both LPS/D-GalN-challenged groups (C and D) exhibited significantly elevated ALT and AST levels compared to control groups (A and B) (P < 0.01). Notably, urantide pretreatment in Group D resulted in substantially attenuated transaminase elevations relative to untreated ALF mice (Group C), with reductions of 55.3% for ALT and 59.3% for AST (both P < 0.01) (Figure 7). These findings demonstrate that: (1) LPS/D-GalN successfully induced severe hepatocellular injury, and (2) UII/UT system blockade significantly ameliorates this damage, suggesting the therapeutic potential of urantide in acute liver failure.
The levels of serum ALT and AST in LPS/D-GalN-Induced ALF Mice after urantide. *P < 0.01 vs group A; #P < 0.01 vs group C.
Bar graph comparing ALT and AST levels in IU/L across four categories labeled A, B, C, and D. Categories A and B show minimal enzyme levels. Category C shows a significant increase, with ALT reaching nearly 18,000 IU/L and AST around 12,000 IU/L. Category D shows reduced levels, with ALT and AST both under 10,000 IU/L. Asterisks and hashtags denote statistical significance. Black bars represent ALT, and gray bars represent AST.Hepatic Histopathological changes in mice challenged by LPS/D-GalN via urantide pretreatment
Histopathological examination demonstrated distinct morphological changes across experimental groups (Figure 8). Control groups (A and B) maintained normal hepatic architecture, with intact lobular structure and morphologically normal hepatocytes. In contrast, Group C (LPS/D-GalN-only) exhibited severe parenchymal destruction characterized by: (1) disrupted lobular architecture, (2) extensive necroinflammatory foci, (3) marked hepatocellular necrosis, (4) dense inflammatory infiltrates, and (5) pronounced sinusoidal congestion. Notably, urantide-pretreated Group D showed significant histological improvement compared to Group C, with attenuated sinusoidal congestion and reduced hepatocellular necrosis, indicating partial protection against LPS/D-GalN-induced injury through UII/UT system blockade. These findings correlate with the observed biochemical improvements and suggest that urantide modulates both inflammatory and cytotoxic components of ALF pathogenesis.
Histopathological changes in liver tissue of mice treated with LPS/D-GalN for 6 hours. (A) Urantide(-),LPS/D-GalN(-); (B) Urantide(+),LPS/D-GalN(-); (C) Urantide(-),LPS/D-GalN(+); (D) Urantide(+),LPS/D-GalN(+); (HE staining, x200).Scale bar=100 μm.
Microscopic images of liver tissue slices labeled A, B, C, and D. Each image shows varying degrees of liver cellulature and structure in pink and purple hues, with some circular and elongated white spaces indicating blood vessels or ducts. Scale bars indicate magnification level at 100 micrometers.Discussion
UII, a neurohormone-like peptide primarily localized in KCs within the liver, has been identified as a key mediator in inflammatory diseases (Sun and Liu, 2019). Its biological effects are mediated through the UII/UT system, formed by binding to its specific receptor UT (Segain et al., 2007; Tomiyama et al., 2015). The uncontrolled release of proinflammatory cytokines from KCs leads to massive hepatocyte necrosis, constituting the primary pathophysiological mechanism of ALF (Maiwall et al., 2024).
Prior studies revealed that in ALF, both UII/UT levels and inflammatory cytokines (such as IL-6 and TNF-α) are significantly elevated (Liang et al., 2013; Leifeld et al., 2010). Mechanistically, UII/UT upregulation activates TLR4/p38-mitogen-activated protein kinase(p38-MAPK) and nuclear factor κB(NF-κB) signaling pathways, inducing excessive secretion of inflammatory mediators like IL-6 and TNF-α (Liu et al., 2015). Our studies further showed that serum UII secretion is an earlier event than TNF-α in ALF (Liu et al., 2015). It suggests that the UII/UT system induce hepatic injury by initiating the inflammatory cascade in ALF. Thus, the UII/UT system serves as a key trigger in LPS/D-GalN-induced ALF development. Previous reportor demonstrated that urantide protected against animals’ death and alleviated hepatic inflammatory injury by blocking UII/UT signaling in LPS/D-GalN-challenged ALF mice (Liang et al., 2013). In this experiment, hepatic inflammatory remission was again observed, including the decrease of serum ALT/AST and the improvement of hepatic tissue injury due to urantide pretreatment in ALF mice.
It is demonstred that during ALF, p38 MAPK/c-jun n-terminal kinase(JNK) remains persistently activated (Guan et al., 2025), and overactivated p38 MAPK and JNK phosphorylate peroxisome proliferator-activated receptor alpha (PPARα), thereby suppressing the transcriptional activity of PPARα bound to the Ces1f gene promoter region (Fang and Judd, 2018; Zhang et al., 2024; Wen et al., 2019). Furthermore, we found that hepatic Ces1f expression is significantly downregulated in ALF (Zhao et al., 2024). Ces1f, as a member of the carboxylesterase family (Lian et al., 2016), is known to mediate xenobiotic detoxification (Quiroga et al., 2012; Yang et al., 2009) and ester metabolism (Hosokawa et al., 2008; Yang et al., 2007). In previous experimental, we further observed that Ces1f was mainly expressed in KCs, and hepatic injury exacerbated upon knocking down KC Ces1f gene in LPS/D-GalN-attacked mice (Zhao et al., 2024). However, it is unclear whether there is an association between the UII/UT signal and Ces1f expression in ALF.
In order to learn about the relationship between the above two, we analysed the time-dependent expression of hepatic UII and Ces1f genes in LPS/D-GalN-induced ALF in this experiment. We found that after LPS/D-GalN attack, hepatic UII mRNA rose rapidly, reached the peak within 6 h, and returned to normal levels at 12 h; while the Ces1f declined progressively from 6 h, but began to rise at 12 h. Our previous study indicated that 6 h is an important time point of hepatic injury, at which serum ALT/AST began to rise and hepatocellular death, inflammatory infiltration and hemorrhage appeared after LPS/D-GalN attacked (Liu et al., 2008). Therefore, the marked hepatic damage may originate from both UII/UT activation and Ces1f down-regulation. In addition, it seems that it can be deduced to a regulating effect of UII on Ces1f because hepatic UII expression is a significant earlier event than Ces1f in ALF.
To further know whether hepatic UII/UT signal has an influence on Ces1f, we assayed hepatic Ces1f expression at 6 h after LPS/D-GalN challenge using urantide pretreatment. In this experimental, we observed that urantide pretreatment reversed hepatic UII upregulation and Ces1f downregulation induced by LPS/D-GalN challenge in ALF mice. It suggests that UII/UT system can regulate hepatic Ces1f in ALF.
Using our established LPS/D-GalN-induced ALF model (Sanghani et al., 2009; Liu et al., 2013), we found that: 1. During the acute liver failure (ALF) phase, the time-dependent expression levels of UII and Ces1f mRNA exhibited opposite trends: UII mRNA began to increase 2 hours after LPS/D-GalN stimulation, peaked at 6 hours, remained elevated at 8 hours, and decreased to near-normal levels by 12 hours; conversely, Ces1f mRNA showed a significant decrease starting at 6 hours post-stimulation, reached its lowest point at 10 hours, and began to rise again by 12 hours. 2. Compared to controls, 6 hours after LPS/D-GalN treatment, the ALF model group exhibited upregulation of UII mRNA and protein levels in the liver, while Ces1f mRNA and protein levels in the liver were downregulated. 3. Following urantide administration, compared to the model group, the pretreatment ALF group showed downregulation of UII mRNA and protein levels in the liver, while Ces1f mRNA and protein levels in the liver were upregulated. This suggests that the UII/UT signaling system may exacerbate ALF by suppressing the expression of hepatic metabolic enzymes such as Ces1f. However, several questions remain unresolved: the mechanism by which UII/UT influences Ces1f remains unclear and requires investigation using primary hepatocytes. Future studies will further explore the downstream signaling pathways of UII/UT and its regulatory mechanisms on the transcription of hepatic metabolic enzymes.
Data availability statement
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.
Ethics statement
The animal study was approved by Animal Welfare and Ethics Committee of Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
WY: Writing – original draft. SB: Writing – original draft. LL: Writing – review & editing.
Acknowledgments
We would like to thank the support from Songjiang Hospital Affiliated to Shanghai Jiao Tong University School of Medicine and Shanghai Songjiang Clinical Medical College of Nanjing Medical University, and funding from National Natural Science Foundation of China.
Conflict of interest
The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The authors declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcimb.2025.1653725/full#supplementary-material
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Edited by: Jake J Wen, University of Texas Medical Branch at Galveston, United States
Reviewed by: Hanwei Jiao, Southwest University, China
Shruti Ahlawat, Shree Guru Gobind Singh Tricentenary University, India