Preparation of Lactucin
Lactucin was prepared by our research team: the preparation method was referred to the literature[23]. And the discovery process of Lactucin is shown in Fig. 2 and Fig. 3.
Cell Culture and Processing
Rat hepatic stellate cells (HSC-T6) were donated by Xinjiang Uygur Autonomous Region Medical Research Institute. Cells were routinely cultured in DMEM high-glucose medium (HyClone, Logan, Utah, United States) containing 10% v/v fetal bovine serum (FBS, Gibco, United States) and 1% penicillin-streptomycin (HyClone, Logan, Utah, United States) at 37 ℃ and 5% CO2, and subcultured every 2–3 days to maintain logarithmic growth. Different concentrations of Lactucin solution were prepared using Dimethyl sulfoxide (DMSO) (Beijing Solarbio Technology Co., Beijing, China) and added to the cell culture medium. The percentage of DMSO (Solarbio) in the cell culture medium was 0.1% (v/v).
Effect of Lactucin on the Viability of HCS-T6 Cells
The effect of Lactucin on the viability of HSC-T6 cells was determined by MTT (Solarbio). HSC-T6 cells were inoculated in 96-well plates at a density of 1×105 cells and incubated overnight, and then the cells were treated with medium containing different concentrations (Fig. 4A) of Lactucin for 24 h. Then the culture medium was carefully aspirated and fresh medium containing 10% MTT solution was added, and the culture was continued for 4 h. Then the culture medium was aspirated and 110 µL of Formazan solution was added to each well, and the wells were shaken at low speed for 10 min, and then the absorbance (OD) of each well was measured at 490 nm by an enzyme marker (Thermo Fisher, United States). And the cell viability was calculated by the following equation: Cell Survival Rate (%) = (OD1 / OD0) × 100%. The OD1 indicates the absorbance value of cells in each treatment group, and the OD0 indicates the absorbance value of normal cultured cells.
Effect of TGF-β1 cell growth factor on the viability of HSC-T6 cells
HSC-T6 cells were inoculated in 96-well plates at a density of 1×105 cells and incubated overnight, then medium solutions containing different concentrations (Fig. 4B, C) of Lactucin were added, while TGF-β1 cell growth factor at a final concentration of 2 µg/L was added to each well to co-stimulate the cells for 24 h and 48 h, and then the cell viability of HSC-T6 was measured by MTT assay.
Detection of the effect of Lactucin on apoptosis of HSC-T6 cells by flow cytometry
After co-stimulation of Lactucin and TGF-β1 for 48 h, cells of each experimental group were digested with EDTA-free trypsin (Gibco, United States) and 5×105 cells were collected. The cells were washed twice using PBS and 500 µL of EDTA-free trypsin was added to each well to digest the cells. The cells were then collected and 195 µL of cell binding solution, 5 µL of AnnexinV-FITC, and 10 µL of propidium iodide (PI) were added to each tube. Finally, the cells were incubated for 30 min under protection from light and detected by flow cytometry (BD FACSCalibur, United States). The experiment was repeated three times to take the average value to calculate the apoptosis rate.
Apoptosis rate (%) = number of apoptotic cells/(number of apoptotic cells + number of normal cells)×100%.
Cell immunofluorescence Assay
Double immunofluorescence staining method was used. Pretreated HSC-T6 cells from each group were digested with trypsin and transferred to cell crawl sheets in equal amounts. Lactucin and TGF-β1 were used to co-stimulate the cells for 48 h, and then the cells were fixed with 4% paraformaldehyde. After washing, 0.3% Triton X-100 was added and incubated for 10 min. The cells were then blocked with 5% bovine serum albumin (BSA) for 1 h. Primary antibodies including Stat3 (1:1000, Cell Signaling Technology, United States), p-Stat3 (1:1000, Cell Signaling Technology, United States) were incubated overnight at 4℃. Then cells were incubated with Alexa Fluor 594 or Alexa Fluor 488-coupled secondary antibody (1:1000, Proteintech) for 1 h at room temperature. Finally, the cells were incubated with 4, 6-diamidino-2-phenylindole (DAPI, leagene) for 15 min and the staining was observed using laser confocal microscopy (Nikon, Japan). This part of the experiment was entrusted to Wuhan Xavier Biotechnology Co.
Western Blot analysis
HSC-T6 cells were inoculated at a density of 1×106 cells in a 6-well flat-bottom plate for 16 h. Cells were pre-protected with the corresponding concentration of Lactucin for 30 min, and then co-stimulated with TGF-β1 for 48 h. After washing cells twice with cold PBS, cells were collected by scraping, and 100 µL of radio immunoprecipitation assay (RIPA) lysis buffer (CoWin Biosciences Co., Beijing, China) was added, shaken thoroughly and placed in a refrigerator at 4 ℃, lysed for 30 min, and then centrifuged at 4 ℃ and 12000 g for 10 min, extracted the total cellular protein, and determined the protein concentration using bicinchoninic acid (BCA) protein concentration determination kit (Solarbio Life Sciences, Beijing, China). The proteins (50 µg) in each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF, Millipore, Billerica, MA, United States) membranes. The PVDF membranes were then incubated overnight at 4 ℃ with primary antibodies including TLR4 antibody (1:1000), Smad7 antibody (1:1000), Smad3 antibody (1:1000), all from (Cell Signaling Technology, United States). α-SMA antibody (1:2000) and TGF-β1 antibody (1:2000) are from (Boster Biological technology, WuHan, China), GAPDH antibody (1:2000, Beyotime Biotechnology, Shanghai, China). Then the membranes were then incubated for 1 h with horseradish peroxidase (HRP)-conjugated goat anti-rabbit/mouse IgG antibody (1:5000, Zhongshan Jinqiao Biotechnology Co., Beijing, China), and the immunoreactive proteins were visualized using the enhanced chemiluminescence reagent (ECL, Solarbio Technology Co., Beijing, China). The images were obtained using ChemiDoc™ Omnimaging System (Bio-Rad, Hercules, CA, United States) and developed using Image J (NIH, United States) Software for quantification.
Protocol of Animal Experiments in vivo
Ethical statement
The present study was conducted in compliance with the ARRIVE guidelines. All animal experiments were conducted in strict compliance with the Chinese Guidelines for Ethical Review of Laboratory Animal Welfare (GB/T 35892-2018) and approved by the Medical Ethics Committee of the First Affiliated Hospital of Shihezi University School of Medicine (Approval number: A2020-035-01). Every effort was made to minimize animal suffering and to limit the number of experimental mice. (The data presented here were collected from 60 mice).
Animals
Sixty SPF-grade male C57BL/6 mice weighting 22±2g purchased from Henan Skeleton Biotechnology Co., Ltd. with experimental animal production license number: SCXK (Yu) 2020-0005. They were housed in the same room in a 14 h-light 10 h-dark cycle in a controlled environment (temperature 22-25℃, humidity 55% ± 5). Mice were fed a standard rodent diet and had ad libitum access to water.
The mice were randomly divided into 6 groups of 10 mice each, namely normal control group, model group, Lactucin high dose intervention group (8 mg/kg), medium dose intervention group (4 mg/kg), low dose intervention group (2 mg/kg) and silymarin positive drug control group (4 mg/kg). After 7 days of adaptive feeding, a mouse liver fibrosis model was established by intraperitoneal injection of 10 % CCl4 oil solution twice a week for 6 weeks in all experimental groups except the normal control group. At the beginning of the seventh week, the intraperitoneal injection of CCl4 was stopped, and each dose group was gavaged with the corresponding test drug, and the blank control group and the model group were gavaged with an equal amount of saline once a day for 28 days. At the end of the experiment, and the blood samples were collected from the retro-orbital venous plexus of mice under gaseous anaesthesia. Serums were obtained after centrifugation of the blood at 1300g for 10 min and frozen at -80℃ until use. When blood collection was completed the mice were rapidly cervical dislocated and executed. Liver and colonic feces were collected from the mice immediately, and a small portion of fresh liver tissue samples were collected and fixed with 4% paraformaldehyde. The remaining tissues and freshly collected feces were rapidly frozen in liquid nitrogen and stored at -80℃ for tissue homogenization kit analysis and metabolomics analysis.
Determination of serum biomarkers
To determine liver function and biochemical parameters, according to the manufacturer's commercial kit instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), the activities of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (AKP), lactate dehydrogenase (LDH), γ-glutamate transferase (γ-GT) and hydroxyproline (HyP) in serum were determined by enzyme colorimetric method.
Assay of IL-6, IL-1β and TNF-α in the liver
Approximately 1 g of liver tissue was weighed and 9 times ice normal saline was added, and the tissue was cut as much as possible with small ophthalmic scissors and ground into a 10 % tissue homogenate using a tissue homogenizer. The prepared homogenate was prepared with centrifuge at 4 ℃, 800 g, 15 min centrifugation for supernatant. The levels of IL-6, IL-1β and TNF-α in liver tissue were determined according to the enzyme-linked immunosorbent assay (ELISA) kit instructions (Shanghai Yaji Biological Co., Ltd., Shanghai, China).
Pathological evaluation of liver tissues
The liver tissues were fixed in 4 % paraformaldehyde. These tissues were subsequently dehydrated in a graded ethanol series (75-100 %) and embedded in paraffin wax. Tissue thickness was 4 μm in sections, and liver tissue was stained with hematoxylin and eosin (H&E) and Masson, digitally photographed by light microscopic at total magnifications of ×100.
Analysis of liver fibrosis-related protein expression in liver
Fresh mouse liver tissue was weighed 100 mg, 100 μL of cold RIPA lysis buffer was added, quickly ground using a tissue homogenizer, and then placed in a refrigerator at 4 ℃ for 30 min for lysis, centrifuged at 4 ℃ and 12000 g for 10 min to extract total tissue protein, and protein concentration was determined using a BCA protein concentration inhibition kit (Solarbio Life Sciences, Beijing, China). The proteins (50 μg) in each sample were separated by SDS-PAGE and transferred to PVDF membranes. The PVDF membranes were then incubated overnight at 4 ℃, including primary antibodies TLR4 (1:1000), Smad7 (1:1000), Smad3 (1:1000), α-SMA (1:2000), TGF-β1 (1:2000), Stat3 (1:1000), p-Stat3 (1:1000), GAPDH (1:2000). Then the membranes were then incubated for 1 h with HRP-conjugated goat anti-rabbit/mouse IgG antibody (1:5000), and the immunoreactive proteins were visualized using the ECL. The images were obtained using ChemiDoc™ Omnimaging System and developed using ImageJ Software for quantification.
Determination of SCFAs in feces
Fresh mouse feces was collected in a 2 mL centrifuge tube, 50 μL of 15 % phosphoric acid was added, then 100 μL of 125 μg/mL of internal standard (isocaproic acid) solution and 400 μL of ether were homogenized for 1 min, and the supernatant was centrifuged at 12000 g at 4 ℃ for 10 min. Then, the supernatant was taken for the determination of SCFAs using a Thermo TRACE 1310-ISQ LT gas chromatograph- mass spectrometer (GC-MS, Thermo, United States).
GC-MS detection: Chromatographic conditions: Agilent HP-INNOWAX capillary column (30 m×0.25 mm ID×0.25 μm); injection volumes was 1 μL with split injection, and split ratio was 10:1. Inlet temperature, ion source temperature, transmission line temperature and quadrupole temperature were 250 ℃, 230 ℃, 250 ℃ and 150 ℃, respectively. The program temperature was increased from 90 ℃ to 120 ℃ at 10 ℃/min, then to 150 ℃ at 5 ℃/min, and finally to 250 ℃ at 25 ℃/min for 2 min. The carrier gas was helium at a flow rate of 1.0 mL/min.
MS conditions: electron bombardment ionization (EI) source, SIM scanning mode, electron energy 70 eV.
Based on the assay results, targeted quantification of the detected samples was performed, and based on the quantification results, relevant data analysis was performed.
Growth effect of Lactucin on microorganisms
We measured the minimal inhibit concentration (MIC) of Lactucin on both S. aureus and E. faecalis at 250 μmol/L after preliminary experiments. 2 MIC concentrations of Lactucin were then used to co-culture the bacteria for 8 h. Bacteria were collected, washed three times with sterile PBS, fixed in 2.5 % glutaraldehyde fixative, embedded and sectioned, and observed using SEM and TEM to observe the morphology of bacteria and take pictures.
Statistical analysis
Quantitative data are expressed as the means±standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare differences among multiple groups, and unpaired Student’s t-test was used to analyze the significance between two groups. Statistical analyses were performed with IBM SPSS statistics version 22.0 (IBM, United States). GraphPad Prism 6.0 (GraphPad Software, United States) was used for image production. *P < 0.05, **P < 0.01 were used to denote statistical significance.