Reagents
JFG was provided by Shandong NewTimes Pharmaceutical Co., Ltd. (gyzz z37020357, batch No.:8022009004). Con A was purchased from Sigma-Aldrich (St. Louis, Mo, USA. Cat#C2010). Tumor necrosis factor α (TNF-α) ELISA kit was purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd (Nanjing, China. Cat#H052-1). An interleukin (IL) -1β (Cat#ml1063132-2), IL-4 (Cat#ml064310), IL-6 (Cat#ml1002293-2), IL-10 (Cat# ml1002285-2) and interferon (IFN)-γ (Cat#ml002277) ELISA kit was purchased from mlbio company (Shanghai, China). Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) apoptosis detection kit were purchased from KeyGEN BioTECH (Nanjing, China. Cat#KGA7071). The antibodies MyD88 (myeloid differentiation factor 88) (Cat#4283), TRAF6 (TNF receptor associated factor 6) (Cat#67591), NF-κB (nuclear transcription factor-κB) (Cat#8242), p-NF-κB (Cat#80477), p-38 (Cat#14451), p-p38 (Cat#4092), ERK (Cat#4695), p-ERK (Cat#4370), JNK (Cat#9252), p-JNK (Cat#9255), Bcl-2 (Cat#3498), Bax (Cat#5023), Cleaved-Caspase-3 (Cat#9664) and anti-GAPDH (Cat#5174) were purchased from Cell Signaling Technology (Boston, MA, United States). The antibody TLR4 (Toll-like receptor4) (Cat#ab218987), anti-CS (Citrate-Synthase) (Cat#ab129095), anti-ACLY (ATP citrate lyase) (Cat#ab40793), anti-IDH1 (Isocitrate Dehydrogenase) (Cat#ab172964), anti-SUCLG1 (Succinate-CoA Ligase) (Cat#ab97867), anti-SUCLA2 (Succinate-CoA Ligase ADP) (Cat#ab202582) were purchased from Abcam company (Shanghai China). The BCA (Bicinchoninic acid) Protein Assay Kit and Western ECL Substrate kit and HRP rabbit secondary antibody and RIPA lysate buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China). IAA (Iodoacetamide), UA (Urea), SDS (Sodium dodecyl sulfate), DTT (Dithiothreitol), Tris HCl (Triaminomethane Hydrochloride) and formic acid (FA) were purchased from sigma Aldrich (St. Louis, Mo, USA). ACN (Acetoniteile, LC-MS) and H2O (LC-MS) were from ANPEL laboratory technologies (Shanghai, China). Trypsin was purchased from Promega (Madison, WI, USA).
Preparation of JFG
The ingredients of JFG include eleven kinds of herbs: Jing Jie (Schizonepeta tenuifolia), Fangfeng (Saposhnikovia divaricata), Chai Hu (bupleurum), Chuan Qiong (Ligusticum chuanxiong), Qiang Huo (notopterygium root), Du Huo (angelica root), Qian Hu (Peucedanum praeruptorum), Fu Ling (Poria cocos), Jie Geng (Platycodon grandiflorum), Zhi Ke (Citrus aurantium) and Gan Cao (Glycyrrhiza uralensis) at a ratio of 3:3:3:3:3:3:3:3:3:3:1, respectively (dry weight). All herbs are phytochemically identified according to the Pharmacopoeia of the People's Republic of China (2015 edition). The clinical dosage of JFG is 15×3 g/day. Conversion method driven by body surface area coefficient based on mice (mouse to human body surface area ratio = 0.0026) and humans. They are calculated as follows: mouse: 45 g/day (human dose) ×0.0026/0.03 kg (mouse body weight) = 4.0 g/kg/day. All herbs were obtained from Shandong NewTimes Pharmaceutical (Linyi, China) and each herb was authenticated by the State Key Laboratory of Generic Manufacture Technology of Chinese Traditional Medicine, Lunan Pharmaceutical Group (Linyi China). The material basis of JFG efficacy was researched in detail by GC-MS and UPLC-Q-Exactive MS techniques as previously described [25]. In other words, Extract the volatile oil from JFG according to the method for determination of volatile oil in Chinese Pharmacopoeia 2020 (general rule 2204 a method) to obtain the GC test sample solution. In addition, JFG sample is filtered with 0.22µm microporous filter membrane after methanol ultrasound to obtain liquid test sample solution. Subsequently, perform quality control analysis on the above extracted test solution.
Animal and experimental design
ICR mice were purchased from Jinan Pengyue experimental animal breeding Co., Ltd (24 ~ 26g, 6 weeks, Male). The mice were housed under specific pathogen-free environment with circulated 12-hour light-dark and were administered standard aliment and water ad libitum. All experimental procedures were performed by the Animal Ethics Committee of Lunan Pharmaceutical company and in accordance with Guidelines for the Care and Use of State Key Laboratory of generic technology of traditional Chinese Medicine (linyi, Shandong, China).
Sixty ICR mice were randomly divided into control group (C), model group (M) and JFG group (JFG). In addition to the control group, the autoimmune hepatitis model was established by intravenous injection of 10 mg / kg Con A in the model group and JFG group. Mice in the JFG group were administered JFG (4 g/kg/d) via intragastric gavaged (i.g.) 1 hour after Con A injection. The mice in the control and model groups were administered the same amount of purified water.
The mortality of mice within 24 hours after Con A injection was counted. Then, serum and liver tissue samples were randomly collected after anesthesia with 1% pentobarbital solution (Sigma-Aldrich). The liver was fixed with paraformaldehyde solution for pathological observation and the remaining liver tissue was stored in the − 80°C for further analysis.
Serum biochemical analysis
The serum levels of ALT, AST and LDH were detected by BS-800 automatic biochemical analyzer (Shenzhen Mairui Biomedical Electronics Co., Ltd.) as mentioned above.
Histopathological analysis
The liver tissues were dehydrated and embedded in paraffin (Tissue TEK-® TEC™5 Sakura Fine tek, Japan). The tissues (5µm) were stained with H&E by automatic staining machine (Leica st5020). Then, the histopathological evaluation and images were observed using a light microscope.
TUNEL staining
Paraffin fixed liver sections were stained with TUNEL apoptosis detection kit. The liver tissues were first fixed in 4% paraformaldehyde, dehydrated s and embedded in paraffin wax. These tissues were cut into small pieces and mounted on glass slides. After that, endogenous peroxidase activity was inhibited with 5% hydrogen peroxide. The slides were washed with distilled water and stained with DAPI and the apoptosis was gained under light microscope after flushed with PBS.
Evaluation of inflammatory cytokines
Mice liver tissue was homogenized in PBS. The obtained tissue homogenate was centrifuged (12,000 × g, 4℃, 10 min), the supernatant was transferred to the centrifuge tube. The levels of cytokines such as TNF-α, IL-1β, IL-4, IL-6, IL-10 and IFN-γ in liver tissue were measured through the instructions of ELISA kits.
Western blot
Liver tissue was homogenized and lysed with RIPA lysis buffer to extract the proteins. The total protein concentration was measured used the BCA. 12% polyacrylamide gel was used for SDS-PAGE (SDS-polyacrylamide gel electrophoresis). The proteins on the gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad) via electroblotting. The protein in the PVDF membrane was blocked in PBS solution containing 5% skim milk powder and 0.1% Tween 20 (PBST) for 2 h at room temperature. Subsequently, the isolated proteins and probes were immunoblotted with the following primary antibodies overnight at 4℃: TLR4 (1:1000), MyD88 (1:1000), TRAF6 (1:1000), NF-κB P65 (1:1000), p-NF-κB P65 (1:1000), p-38 (1:1000), p-p38 (1:1000), ERK (1:1000), p-ERK (1:1000), JNK (1:1000), p-JNK (1:1000), Bcl-2 (1:1000), Bax (1:1000), Cleaved-Caspase-3 (1:1000), anti-CS (1:1000), anti-ACLY (1:1000), anti-IDH1 (1:1000), anti-SUCLG1 (1:1000), anti-SUCLA2 (1:1000) and anti-GAPDH (1:1000). The samples were washed with PBST and incubated with HRP rabbit secondary antibody (1:5000) at room temperature for 1 hour. The immunoreactive protein was observed by ECL kit. The protein band intensity was scanned by chemiluminescence imaging system (Shanghai Qinxiang Scientific Instrument Co., Ltd.) and the gray value was detected by image J (1.51, National Institutes of Health, Bethesda, Maryland, USA). The proteins were normalized against GAPDH.
Proteomic analysis
The liver tissue samples were collected. After washing with PBS, the RIPA working solution was added and fully mixed. The experimental material was ground on ice for 4 min and repeated three times. Transfer all samples to a new EP tube after centrifuge (4℃, 12,000 rpm, 10min). The sample was sonicated on ice for 10 min and then centrifuged (4℃, 12,000 rpm, 10min) to extract the supernatant. The concentration of extracted protein was determined by BCA method, the standard curve was fitted according to the standard protein, and the protein concentration of corresponding samples was calculated. The protein was used for subsequent detection after acetone precipitation, re-dissolution, reduction, alkylation, enzymatic hydrolysis, SDC removal and peptide desalination.
For each sample, 2 µg of total peptides were separated and analyzed with a nano UPLC (EASY-nLC1200), which was coupled with Q active HFX Orbitrap instrument (Thermo Fisher Scientific) with nano electrospray ion source. Separation was performed using a reversed phase column (100 µm ID ×15 cm, Reprosil-Pur 120 C18 AQ, 1.9 µm, Dr. Maisch). The mobile phase is H2O containing 0.1% FA, 2% ACN (phase a) and 80% ACN, 0.1% FA (phase B). The samples were separated at a flow rate of 300 nl/min and gradient of 120 min. Gradient B: 2–5% for 2 min, 5–22% for 88 min, 22–45% for 26 min, 45–95% for 2 min, 95% for 2 min.
Data dependent acquisition (DDA) was performed in profile and positive mode with Orbitrap analyzer at a resolution of 120,000 (@200 m/z) and m/z range of 350 1600 for MS1; For MS2, the resolution was set to 15,000 with a dynamic first quality. The automatic gain control (AGC) target for MS1 was set to 3E6 with max IT 50 ms, and 1E5 for MS2 with max IT 110 ms. The top 20 strongest ions were fragmented by HCD with normalized collision energy (NCE) of 27%, and isolation window of 1.2 m/z. The dynamic exclusion time window was 45 s, the single charged peaks and peaks with charge exceeding 6 were excluded from the DDA program.
Vendor’s raw MS files were processed using Proteome Discoverer (PD) software (Version 2.4.0.305) and the built-in Sequest HT search engine. MS spectra lists were searched against their species-level UniProt FASTA databases (uniprot-Mus + musculus-10090-2021-8. fasta), Carbamidomethyl [C] as a fixed modification, Oxidation (M) and Acetyl (Protein N-term) as variable modifications. Trypsin was used as proteases. A maximum of 2 missed cleavage(s) was allowed. The false discovery rate (FDR) was set to 0.01 for both PSM and peptide levels. Peptide identification was performed with an initial precursor mass deviation of up to 10 ppm and a fragment mass deviation of 0.02 Da. Unique peptide and Razor peptide were used for protein quantification and total peptide amount for normalization. All the other parameters were reserved as default.
The screening criteria of differentially expressed proteins were p-value < 0.05 and fold change ≤ 0.67 or fold change ≥ 1.5. It was visualized by volcanic-map and heat-map. The enrichment analysis of differentially protein expressed signal pathway was carried out through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases.
Metabonomics analysis
The differential metabolites in the three groups were screened by liquid chromatography-mass spectrometry (LC-MS).
The liver tissues were quenched with liquid nitrogen and put into cryopreservation tube. 80% methanol was added in the ratio of 1:3 (w:v) and then homogenized and broken. The supernatants were concentrated and dried after centrifugation (12000 rpm, 10 min, 4°C). Then dissolved them with 200 µL 80% methanol. The samples were placed in an inner liner for LC-MS analysis. All samples were mixed in the same volume to ensure quality control (QC) in order to take stock of the stability and repeatability of the system during sample collection.
Chromatographic separation was performed on Thermo Scientific LTQ oribitrap high-resolution mass spectrometer (Thermo Fisher Company, Shanghai, China) to analyze the metabolic characteristics of each tissue sample. The positive and negative modes were analysis by the chromatographic column with HSS T3 chromatographic column (waters, 2.1mm × 100mm, 1.7 µm). The mobile phase was consisted of mobile phase A (0.1% formic acid) and mobile phase B (methanol). The flow rate was constant at 0.2ml/min, the injection volume was 2.0 µL and the column temperature was 40°C. Separation process as follows: 0 ~ 2 min, 2% B; 2 ~ 3 min, 2%~35% B; 3 ~ 28 min, 35%~98% B; 28 ~ 32 min, 98%~2% B; 32 ~ 34 min, 2%~2% B.
Positive and negative mode electrospray ionization (ESI) technology was used to carry out MS scanning. The scanning ranged from 400 m/z to 1000 m/z with a resolution of 70000. The scanning was range from 400 m/z to 1000 m/z with a resolution of 70000. The voltage of positive and negative ion source was 4 and 3.5kV respectively. The capillary heating temperature of the two ions was set to 300℃. The sheath gas pressure and auxiliary gas pressure were 40 and 20 psi (temperature: 350℃, maximum isolation time: 50ms). Quality control tests was conducted on every six samples to verify the accuracy of the data during the analysis.
The chromatographic peaks were quantified by compound discover software. SIMCA software (14.1, umetrics) was used for principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to visualize the metabolite data results. PCA was used to analyze the expression of differential variation within and between groups. Variable Importance in Projection (VIP) ≥ 1 in the OPLS-DA model and the p-values (p < 0.05) according to t-test were considered as a potential differential metabolic marker. For these metabolic biomarkers, metaboanalyst website (www.metaboanalyst.ca) was used to analyze the metabolic pathway and the relevant metabolic action mechanism [26–27].
Statistical analysis
The experimental data of each group were statistically analyzed by GraphPad Prism 8.0.2 statistical software (La Jolla, California, United States) and shown as the mean ± standard error of mean (SEM). Statistical significance was evaluated the differences between multiple groups and two groups followed by the one-way analysis of variance (ANOVA) and student’s t-test. Results with a value of * p and # p < 0.05 were regarded as statistically significant.