Materials and Methods
Animal Studies
The C57BL/6 J female/male mice weighed 20 ± 2 g and aged 6–8 weeks old were obtained from Xi’an Jiaotong University’s Animal Experimentation Centre. All mice were housed in a pathogen-free environment with a temperature range of 23 ± 2 °C, humidity between 50 and 70%, light and dark cycles lasting 12 h, and unlimited access to food and water. The animal care and experimental procedures underwent approval from the Xi’an Jiaotong University Animal Experiment Ethics Committee and Authority, as evidenced by Approval No. 2022–390.
To establish the ISO + ALF group, ISO (10 mg/kg) was administered intraperitoneally 1 h prior to LPS/D-GalN injection, corresponding to published dosages for liver disease [
10,
13]. In addition, we conducted experiment with different doses of mere isoproterenol, 0, 10, and 100 mg/kg, injected into mouse. These experiments preliminarily suggested that ISO had no significant impact on liver structure or hepatotoxicity (Fig.
S1).
According to established procedures [
14], the ALF model was induced in C57BL/6 J mice by intraperitoneal administration of LPS (30 mg/kg, Sigma, USA) and D-galactosamine (600 mg/kg, Sigma, USA). The mice were monitored in a controlled environment for a maximum of 12 h. Afterward, all the mice were humanely euthanized, and their liver tissue and serum samples were collected for further analysis of the results.
Cell Culture and Treatment
THP-1, a cell line of human macrophages, was acquired from the National Collection of Authenticated Cell Cultures of China. The cells were grown in 1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin added. The incubator was kept in an environment with 5% CO2 at 37 °C. THP-1 cells were exposed to LPS (20 ng/ml; Sigma) for 2 h in order to cause inflammation. Two hours before LPS stimulation, ISO (at a dose of 50 µM; MCE) was given after being dissolved in DMSO. Following the extraction of cell lysis and collection of mRNA, the samples were collected and kept at −80 °C until they were needed.
NPCs Isolation
According to previously published protocols [
15,
16], hepatic non-parenchymal cells (NPCs) were obtained from liver tissues by collagenase type IV digestion (0.05 mg/mL) and gradient centrifugation. In brief, liver tissues were infused with perfusion buffer for up to 20 min then perfused with collagenase until digested. Collect the cell suspension and filter into 50-ml plastic tubes through a gauze-lined plastic funnel. Add more stop solution to the sample of liver tissue until a final volume of 500 ml is reached. Centrifuge the cell suspension at 50× g for 5 min at 4 °C for subsequent isolation of non-parenchymal cells, collect the supernatant. Wash the cell pellet with PBS and collect for hepatocyte. Centrifuge the harvested supernatant at 72× g for 5 min at 4 °C to remove any remaining red blood cells and liver cells. Pool the supernatants and centrifuge them twice. Combine both pellets and resuspend them in HBSS. Prepare a 25% and a 50% density gradient by mixing the density gradient solution with PBS for density gradient centrifugation. Carefully place the 25% density gradient solution on top of the layer containing the 50% density gradient solution. Then, add the NPC suspension slowly on top of the 25% density gradient solution to ensure a clear separation between the layers is achieved. Centrifuge the cell suspension on the density gradient at 1800× g for 20 min at 4 °C without applying any brake. Remove dead cells and cell debris from the topmost layer. The non-parenchymal cells can be found in the interphase between the 25 and 50% density gradient layers. Collect the cell suspension using the dual centrifugation step described above and wash them with Hank’s balanced salt solution (HBSS) and centrifuge. Store the collected NPCs at −80 °C until further use.
Protein Extraction and Digestion for Proteomics Analysis
After isolating the sample, liquid nitrogen was served to grind the cells into powder, and the powder was then transferred to a 5-mL centrifuge tube. Following this, four volumes of lysis buffer (8 M urea, 1% protease inhibitor cocktail) were added to the cell powder and subjected to three rounds of ultrasonication on ice using a high intensity ultrasonic processor (Scientz). In the case of PTM experiments, inhibitors were additionally included in the lysis buffer, for example. For acetylation, a concentration of 3 µM TSA and 50 mM NAM was utilized, while for phosphorylation, 1% phosphatase inhibitor was added. Centrifugation at 12,000 g at 4 °C for 10 min effectively eliminated the remaining debris. Afterward, the liquid above the sediment was gathered and the amount of protein was measured using the BCA kit as per the instructions supplied by the manufacturer.
For the process of digestion, the protein solution underwent reduction using a 5 mM dithiothreitol solution for a duration of 30 min at a temperature of 56 °C. Subsequently, the solution was alkylated with an 11 mM iodoacetamide solution for a duration of 15 min at room temperature in a dark environment. To decrease the concentration of urea in the protein sample to less than 2 M, it was diluted by the addition of a 100 mM TEAB solution. Following this, trypsin was introduced at a trypsin-to-protein mass ratio of 1:50 for the initial overnight digestion and at a ratio of 1:100 for a subsequent 4-h digestion. Finally, the peptides were purified through the usage of a C18 solid-phase extraction column.
TMT Labeling and HPLC Fractionation
During the TMT labeling procedure, the tryptic peptides were first dissolved in a 0.5 M TEAB solution. Then, each individual peptide channel was labeled using the appropriate TMT reagent (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions, and it was incubated for 2 h at room temperature. Five microliters of each sample was mixed, purified, and then exposed to MS analysis in order to evaluate the labeling efficiency. After the efficacy of the labeling was evaluated, 5% hydroxylamine was added to the samples to quench them. The mixed samples were then dried by vacuum centrifugation after being desalted using a Phenomenex Strata X C18 SPE column.
The sample was subjected to HPLC grading, wherein it was fractionated into distinct fractions using a high pH reverse-phase HPLC method employing an Agilent 300 Extend C18 column (consisting of 5 µm particles, with an inner diameter of 4.6 mm and a length of 250 mm). In summary, peptides were separated by employing a gradient of acetonitrile ranging from 2 to 60% in a 10 mM ammonium bicarbonate solution at pH 10, over a duration of 80 min, resulting in the generation of 80 fractions. Subsequently, the peptides were consolidated into 9 fractions and subsequently dried through vacuum centrifugation.
LC–MS/MS Analysis
The tryptic peptides were dissolved in solvent A, which consisted of 0.1% formic acid and 2% acetonitrile in water. Subsequently, they were loaded on a reversed-phase analytical column that was 25 cm in length and had an inner diameter of 75 µm. The peptides were isolated using a gradual change in solvent B (0.1% formic acid in 90% acetonitrile) over a period of 60 min, followed by a transition from 25 to 35% in 22 min and a rapid increase to 80% in 4 min. The concentration was maintained at 80% for the final 4 min, while the flowrate remained constant at 450 nL/min on an EASY-nLC 1200 UPLC system (Thermo Fisher Scientific). The peptides that had been separated were examined using a nano-electrospray ion source in QExactiveTM HF-X (Thermo Fisher Scientific). A voltage of 2.0 kV was applied for electrospray. For a scan range of 350–1600 m/z, the complete MS scan resolution was adjusted to 60,000. 30 s dynamic exclusion was used to select the top 20 most prevalent precursors for subsequent MS/MS analyses. The fragmentation of HCD was carried out with a collision energy (NCE) of 28%. Fragments were identified in the Orbitrap with a resolution of 30,000. The initial mass was fixed at 100 m/z. The AGC target for automatic control was established at 1E5, with a threshold of 3.3E4 for intensity and a maximum injection time of 60 ms.
Identification and Quantitation of Proteins
MaxQuant search engine (v.1.6.15.0) was utilized to process the above LC–MS/MS data. The human SwissProt database (20422 entries) was used to match tandem mass spectra. In the initial search, the tolerance for precursor ions was set at 20 ppm, while it reduced to 5 ppm in the primary search. Additionally, the tolerance for fragment ions was defined as 0.02 Da. Carbamidomethyl on Cys was referred to as a fixed modification, while acetylation on protein N-terminal and oxidation on Met were referred to as variable modifications. The adjustment of FDR was made to less than 0.001.
Gene Set Enrichment Analysis
Using gene set enrichment analysis (GSEA) with the gene sets of mh.all.v2022.1 and Mm.symbols.gmt as references, the potential pathway from selected gene lists was examined. Protein sets input was classified as highly enriched if they had a P value < 0.05, a normalized enrichment score (NES) > 1 or < −1, and a False Discovery Rate (FDR) < 0.25. Data visualization was created using the clusterProfiler and ggplot2 packages in R (version 4.2.1).
The protein list was analyzed by using DEqMS package in R to determine differentially expressed proteins (DEPs). DEPs were expressed with a
P value < 0.05, fold change > 1.5 or < 0.75 and false discovery rate (FDR) < 0.01 within each group. GO (
https://www.geneontology.org) and KEGG (
https://www.genome.jp/kegg/) enrichment analysis was conducted to analyze the biological functions of DEPs through DAVID Functional Annotation Dataset (
ncifcrf.gov) [
17,
18]. GO enrichment analysis was performed for three ontologies, including biological process, molecular function, and cellular component. A significant GO term or pathway was identified with a threshold of protein number more than two and
P value less than 0.05. Protein-protein interaction (PPI) analysis was constructed by using the String (
https://string.embl.de/) database and Cytoscape (
https://www.cytoscape.org/) software. Furthermore, we utilized ClusterViz (V. 1.0.3, Bochum, Germany) in Cytoscape to visualize the clustering analysis based on the molecular complex detection (MCODE) algorithm. We established the criteria as a degree cut-off of 2 and a node score cut-off as 0.2, for the MCODE algorithm, with a maximum depth as 100 k-score as 2 and high confidence as no less than 0.7. Additionally, rank abundance curves, clustering heatmaps, bubble plots, and Venn diagrams were carried out by using the R package in version 4.2.1. Specially, a hierarchical K-means cluster was used to analyze DEP heatmaps [
19].
RNA Extraction and qRT-PCR Analysis
RNA extraction and quantitative real-time PCR (qRT-PCR) were conducted following previously established protocols. Briefly, total RNA was extracted from frozen liver or cultured cells using TRIzol reagent (Thermo, Life Technologies, Carlsbad, CA, USA). The concentration and quality of the RNA were assessed, and mRNA was subsequently reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Rockford, AL, USA). Subsequently, the relative levels of mRNA transcripts were determined through qRT-PCR analysis using the SYBR Premix ExTaq™II kit (TaKaRa, Dalian, China) on an iQTM multicolor real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The 2−ΔΔCT method was applied to calculate the relative expression of each gene using either actin or Glyceraldehyde-3-phosphate dehydrogenase as internal standards. Three technical replicates and three biological replicates were used for each PCR reaction.
Western Blotting
RIPA lysis buffer supplemented with protease inhibitor cocktail tablets (Roche) and phosSTOP phosphatase inhibitor cocktails (Roche) was prepared to extracts total protein from cells or liver tissues. After being quantified using BCA assay, equal quantities of protein (30–50 µg) were loaded onto SDS/PAGE gels, separated, and then transferred onto PVDF membranes. After that, the membranes were blocked for 1 h at room temperature in a blocking buffer containing 5% non-fat milk. After an overnight incubation at 4 °C with certain primary antibodies, the blocked membranes were treated with secondary antibodies that were conjugated with HRP. Finally, the blots were visualized using ImageQuant LAS-4000 (Fujifilm) on a standard exposure setting and quantified using ImageJ (
rsb.info.nih.gov/ij).
Histological Examination
Samples of liver tissue taken from each group were immediately preserved in paraformaldehyde and then covered in paraffin. Using a microtome, tissues were divided into 4–5-µm sections and placed on glass microscope slides. After routine staining with hemotoxylin-eosin (H&E), two independent researchers analyzed the slides using light microscopy (DIALUX 20 EB).
Flow Cytometry
The surface marker on THP-1 cells in both the resting (M0) and M1 polarized states was examined by using flow cytometry. The cells were lightly scraped after the 24-h stimulation of LPS or ISO, freshened three times with PBS, and then transferred into FACS tubes. The M0 and M1 phenotypes were then identified by adding surface CD40 (FITC, Biolegend) and CD14 (Pacific blue, Biolegend) fluorochrome-labeled monoclonal antibodies. Following a 4-h incubation period at 4 °C, the cells were subjected to three PBS washes before being analyzed using a BD Biosciences FACSCanto II flow cytometer. Each sample was recorded with at least 20,000 events. Each group was measured using the FlowJo program (Version 10.0), and at least three duplicates of each group were done.
Statistical Analysis
Statistical data is presented as means ± standard deviation. Statistical differences among various treatment groups were conducted using one-way analysis of variance (ANOVA) with Tukey’s test in SPSS 13.0 (Chicago, IL, USA) or GraphPad Prism 5. Statistical significance was ascertained by considering two-tailed P value with a threshold of < 0.05.
Discussion
The injection of LPS/D-GalN is a common approach to create a mouse model of ALF [
21,
22]. Agreed with previous models, our study shows that the liver tissue from LPS/D-GalN treated mice exhibited more pronounced inflammation and hyperemia than that in healthy mice, and this was associated with poor survival and liver dysfunction. To identify related molecule mechanisms involved in ALF progression, we isolated proteins from liver non-parenchymal tissue for consequent TMT analysis.
Our present study was the first to demonstrate that the prior administration of SNS agonist, ISO, showed effective prevention against the hepatotoxicity and mortality caused by LPS/D-GalN by decreasing levels of ALT and necrosis. The mechanism underlying the protective effects of ISO appears to be due to shield non-parenchymal hepatocytes from LPS/D-GalN-induced toxicity and inflammation. Additional research needed to prolong the investigation whether a delay administration of ISO still produced protective affection.
ALF is a severe liver injury accompanied acute inflammation, and many efforts have been taken to develop therapies to mitigate its condition. Numerous evidences have suggested that the liver is intricately innervated by the sympathetic nervous system (SNS) to control innate immunity and liver fibrosis. Activation of beta-adrenergic receptors by neurotransmitters has been known to contribute to the regulation of SNS on normal and pathogenesis of liver disease diseased liver function. As a non-selective beta-adrenergic receptor agonist, ISO has been revealed to induce the proliferation of progenitor stem liver cells and alleviate the progression of acetaminophen-induced liver injury [
12]. Furthermore, beta-adrenergic receptors were able to accelerate the proliferation of parenchymal hepatocytes to mitigate the development of liver failure. However, during the pathogenesis of acute liver failure, inflammatory mechanisms are be reckoned with and will certainly impact the liver damage. Non-parenchymal liver tissues, such as Kupffer cells, hepatic stellate cells, and endothelial cells, are important components contributing to inflammation. However, no studies have reported the dynamic mechanisms of protein molecules in these non-parenchymal liver cells especially during the ISO treatment of ALF [
11]. In our present study, ISO was pre-injected into mice before LPS/GalN induction and protected the mice from acute liver damage, as evident by the morphology of microscopical observations, which effectively reduced the inflammatory invasion and mortality of ALF mice, and was consistent with the impact of ISO in other animal model of ALF. We isolated proteins from non-parenchymal liver cells for TMT proteomic analysis to make a thorough understanding of the protein functions. Quality control of our proteomic data indicated the successful isolation. Interestingly, the amount of protein in the comparison of the ISO
vs. ALF group was comparable to that in the comparison of the control
vs. ALF group. Moreover, over 50% of the DEPs in the ISO
vs. ALF group comparison were shared with the control
vs. ALF group comparison, and their fold changes had similar tendency. Altogether, these observations confirmed the effect of ISO treatment in alleviating the development of ALF.
Lipopolysaccharide (LPS) is a commonly encountered pathogen-associated molecular pattern that is discharged by Gram-negative bacteria and can stimulate efficacious innate immune responses towards subsequent infections [
14,
23]. Recognition of lipopolysaccharide pattern by Toll-like receptor (TLR) contributes to activation of multiple signaling components,
e.g., NF-κB and MAPK, and the subsequent production of pro-inflammatory cytokines. The intraperitoneal application of lipopolysaccharide (LPS) combing with other hepatotoxins is a general experimental approach to achieve acute liver injury model [
23]. After administering LPS/D-GalN, we screened out the top 10 upregulated and downregulated proteins in non-parenchymal liver cells through comparison with an ISO pretreated mouse model. We then validated the ranking order of these proteins by comparing with control mice. Human zymogen granule protein 16 (ZG16), mitochondrial ribosomal protein large 24 (MRPL24), peptidoglycan recognition protein 2 (PGLYRP2), carboxypeptidase A1 (CPA1), pancreatic alpha-amylase (AMY2), and serpin I2 (SERPINI2) were also highly expressed in LPS/D-GalN mice when compared to the group treated with PBS. ZG16 is highly expressed in mucus-secreting cells, plays a role in pathogen identification by binding to glycosaminoglycans, and has potential functions in promoting local T-cell immunity [
24]. Mitochondrial ribosomal protein has been shown to be a contributor to primary deficiencies in mitochondrial respiratory chain activity as well as other inflammatory disorders. Mutations in MRPL24 genes may result in cerebellar atrophy and intellectual disability [
25]. PGLYRP2, a protein of host defense highly conserved in mammals that detect bacterial cells, is mainly expressed in the liver [
26]. What’s more, PGLYRP2 gene expression was significantly increased in bone marrow-derived macrophages [
26]. Carboxypeptidases, a large group of enzymes that cleave one or two amino acids from the carboxy terminus of proteins or peptides, may be involved in antigen processing and may be associated with the production or regulation of several pro-inflammatory mediators [
27,
28]. Serpins, also recognized as serine protease inhibitors, utilize a unique and irreversible mechanism differing from typical protease inhibitors. They are linked to inflammatory diseases, including COPD and chronic lung diseases, and exhibit an increase in expression [
29]. Consequently, these functions of upregulated proteins in our study inferred that ISO had the capability to suppress pathogen recognition and decrease pro-inflammatory progression of live.
NMRAL1, a redox-sensitive transcriptional regulator, was identified as one of the downregulated proteins in the comparison of ISO
vs. ALF. Decreased NMRAL1 levels resulted in increased nitric oxide (NO) production and reduced cell viability. Additionally, NMRAL1 has been shown to be a possible mechanism for controlling inflammatory signaling in LPS-treated cells [
30]. SF3A2 was one of the subunits SF3A complex that were required for mRNA splicing, and the SF3A mRNA splicing was suspended to inhibit by LPS induced MyD88 [
31]. αβ-hydrolase domain-containing 11 (ABHD11), a mitochondrial enzyme that controls OGDHc activity, is the master inhibitor of HIF-1α that plays a crucial role in activating inflammatory macrophage [
32,
33]. The mutant U2 small nuclear RNA auxiliary factor 1(U2AF1) leaded to elevated basal mRNA levels of IL-6 and IL-8, but not TNFα, when compared to the U2AF1-wild macrophage [
34]. NFU1 iron-sulfur cluster scaffold protein (NFU1) assembles [4Fe-4S] clusters and delivers them to target proteins. Iron–sulfur (Fe–S) clusters are required for numerous biological processes but its biogenesis machinery could be actively suppressed by TLR activation [
35,
36]. Therefore, according to the biological functions of these downregulated proteins, we can suspend ISO certainly increased the anti-inflammation mechanism in hepatic non-parenchymal tissues damaged by LPS/D-GalN.
When we submitted the differentially expressed proteins (DEPs) for functional enrichment analysis in the David dataset, we observed upregulation of hub proteins associated with COVID-19 pathway and/or prion disease (RPL30, RPL12, C1QA, C1QB, STAT3, MAPK14, CASP1, SOD1, CASP3) in cases of acute liver damage [
37,
38]. The alpha-subunit (PSMA) and the beta-subunit (PSMB) of the 20S proteasome complex in the KEGG pathway of prion disease had been identified as LPS-binding proteins [
39,
40], which was comprehensible for their upregulations in LPS/D-GalN-induced liver inflammation. Conversely, these proteins were alleviated in cases of ISO pretreated acute liver failure. Especially, the expression of pro-inflammatory proteins of STAT3, MAPK14, CASP1, SOD1 and CASP3, correlated to the activation of M1 macrophage [
41], was increased in ALF. It was expected that the core molecules related to COVID-19 pathway and prion disease would also be upregulated in the ALF model. Our
in vivo and
in vitro assays confirm that acute ISO stimulation reduced inflammatory response in non-parenchymal cells of ALF liver tissues, particularly in macrophages.
Following acute liver injury caused by LPS/D-GalN, the metabolic pathway was severely damaged, resulting in the disruption of the mitochondrial respiratory chain. The family members of cytochrome c oxidase (COX) and NADH dehydrogenase (complex I) are crucial complex proteins in the mitochondrial respiratory chain [
42,
43]. Patients with acute liver failure were found to have combined respiratory chain deficiency and reduced numbers of mitochondria [
43]. Furthermore, mitochondria are essential for the modulation of macrophage function. Indeed, macrophages stimulated with lipopolysaccharide (LPS) showed a rewired metabolism manifested by decreased mitochondrial respiration, inhibition of the TCA cycle, and upregulation of aerobic glycolysis [
44]. Thereafter, in terms of the mechanism, inhibited NAD + and the NAD + -dependent signaling pathway of metabolism contributed to liver damage in ALF [
45]. Enhancing mitochondrial function within the metabolic pathway could be targeted as part of a multifaceted therapeutic approach for various forms of liver disease. Furthermore, for the activation of peroxisome proliferator-activated receptor α (PPARα) pathway, which is a ligand-activated transcription factor that controlled lipid metabolism, it plays an important role in safeguarding the liver and reducing hepatocyte apoptosis in cases of ALF [
46].
Acute liver failure is a systemic inflammatory response comprising pro-inflammatory and anti-inflammatory components. It has been observed that CD38 deficiency may promote inflammation by activating Sirt1/NF-κB in macrophages [
47]. In patients with ALF, anti-inflammatory activity mediated by FcγRIIB-associated IgG1 is defective [
48]. Furthermore, Caveolin-1 negatively regulates inflammation by attenuating vascular injury caused by Low-Density Lipoprotein [
49,
50]. Fyn plays a role in the neurite outgrowth induced by angiotensin II type 2 receptors [
51]. Tyrosine kinase Fyn regulates iNOS expression stimulated by LPS [
52]. It is evident that certain pro-inflammatory molecules were rapidly exhausted and accompanied by a lack of anti-inflammatory mechanisms after LPS stress [
53,
54]. Thereafter, ISO may also be involved in regulating the systemic inflammatory response during acute liver injury caused by LPS/D-GalN in our present study.
Heme metabolism, fatty acid metabolism, and mitochondrial oxidation signaling pathways are crucial in anti-inflammatory macrophage polarization [
55‐
57]. ISO potentially affects the progression of ALF by regulating fatty acid metabolism and mitochondrial oxidation in monocytes/macrophages and promoting the upregulation of anti-inflammatory molecules to reduce ALF severity. Consequently, additional research must be conducted to ascertain the exact function of ISO in reducing ALF inflammation and whether this is attributed to a biological response from other liver cells rather than a direct impact on macrophages.
From the above discussion, it appears that isoproterenol plays a different role in acute liver injury than it does in inducing myocardial injury [
58]. A large amount of studies have shown that ISO causes a significant increase in inflammation in the field of cardia research. Therefore, we required to investigate whether isoproterenol had a different function in regulating macrophage activity in acute liver damage compared to the role in cardiac macrophages [
59].
Activated macrophages have two different phenotypes, namely M1 (classically activated) and M2 (alternatively activated), which are attributed to different stimuli. M1 macrophages are pro-inflammatory, essential in the host defense mechanism, while M2 are related to responses involving anti-inflammatory reactions and tissue remodeling [
41]. Since macrophages are the key cells involved in the systemic inflammation of ALF, we conducted an analysis of the 29 DEPs (differentially expressed proteins) associated with macrophage functions. Results from the PPI analysis using the STRING database showed that these DEPs were mostly enriched in macrophages. The proteins related to M1 macrophages were significantly decreased in the ISO-treated group, while the DEPs related to M2 macrophages were statistically increased. RT-qPCR confirmed the mRNA levels in the animal models. We further studied the effects of ISO treatment on inflammation regulation in a human macrophage cell line. Previous studies have shown that the β-adrenergic receptor agonist, isoproterenol, synergistically amplifies the anti-inflammatory role of cAMP pathway inducers, resulting in reduced expression of the key pro-inflammatory cytokine TNFα, and increased expression of the key anti-inflammatory cytokine IL-10 in macrophage cell lines [
60,
61]. Our results were consistent with this, showing that ISO could effectively reduce inflammation in ALF mice.
Currently, a probable explanation for the clear evidence of ISO’s protective effect on the liver is that the expansion of hepatic precursor cells leads to the release of Wnt ligands. These ligands safeguard hepatocytes from impairments [
11]. Nonetheless, it necessitates a reasonably prolonged duration to proliferate sufficient fresh hepatocytes from liver progenitor cells following acute ISO administration to obstruct the progression of damage. Other potential mechanisms, such as the metabolic and inflammation pathways, could contribute to a rapid alteration in biological functions under ISO treatment to limit acute liver hepatotoxicity. Previous studies have indicated the possible influence of the sympathetic nervous system on the immune response and associated inflammation [
62‐
64]. Thus, it is plausible that ISO treatment partially protects the liver by modulating the inflammatory process. Previous research indicates that the hepatic sympathetic nervous system plays a crucial role in preventing Fas-induced hepatic injury by means of antiapoptotic proteins [
27] and IL-6 [
65]. The observation of a significant difference in IL-6 expression following previous ISO treatment further supports our model outcomes.
However, we must acknowledge the limitations of our study. Although the proteomic data displayed a widespread impact of ISO on non-parenchymal cells, further analysis is necessary to determine its particular effects on specific types of these cells. Furthermore, more precise studies are required to comprehensively understand how ISO controls macrophage phenotype in Kupffer cells.