Cyp4a12-mediated retinol metabolism in stellate cells is the antihepatic fibrosis mechanism of the Chinese medicine Fuzheng Huayu recipe

Ethics

All experimental procedures complied with the Guidelines for Experimentation of Shuguang Hospital, affiliated with Shanghai University of Traditional Chinese Medicine. The protocols were reviewed and approved by the Ethics Committee of the institution.

Drug preparation and identification

The FZHY recipe consists of six crude herbs with a 1-day dose for adults: 8.0 g of Radix Salviae Miltiorrhizae, 4.0 g of Fermentation Mycelium Powder, 2.0 g of Fructus Schisandrae Chinensis, 2.0 g of Semen Persicae, 2.0 g of Pollen Pini, and 6.0 g of Gynostemma Pentaphyllammak. The FZHY powder was purchased and major ingredients were determined by Shanghai Sundise Medicine Technology Development Co. Ltd. for quality control as previously described [14] (Additional file 1: Figure S1).

Mice

Male C57BL/6 mice weighing 25 ± 1 g were supplied by Beijing Vital River Laboratory Animal Technology. The mice were fed in the Experimental Animal Center of Shanghai University of Traditional Chinese Medicine (Licence No: SCXK[Hu]2020-0009) and were acclimatized to the animal centre conditions for 7 days before the experiments. The mice were given rodent laboratory chow and water ad libitum and maintained under controlled conditions with a temperature of 22–25 °C, relative humidity of 46–52%, and a 12/12-h light/dark cycle (lights on at 7:00 am). Our experiments were conducted in accordance with the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (Licence No: SZY201804008).

Carbon tetrachloride (CCl 4 ) inductionof liver fibrosis

For liver fibrosis, mice were intraperitoneally injected with 10% CCl4/olive oil 2 ml/kg body weight three times a week, for 6 weeks [15]. Following pentobarbital sodium anaesthesia, all mice were sacrificed 24 h after the last injection. Serum and liver samples were harvested. For histology, tissue specimens were fixed in buffered formalin and embedded in paraffin wax (Leica, 39,601,006, USA). For fluorescence, liver specimens were embedded in OCT compound (Sakura, 4583, USA). Serum and liver samples were kept frozen at −70 °C until assayed.

Animal groups and experimental design

For evaluation of the effect of Fuzheng Huayu Recipe (FZHY) against liver fibrosis, mice were randomly divided into 4 groups (12 mice per group): normal control, FZHY control, model control and FZHY treatment. From the third week of CCL4 injection, In the FZHY control and the FZHY treatment groups were orally administered Fuzheng Huayu Recipe at 5.6 g/kg daily, once a day for 4 weeks, which was equivalent to the dosage of a 60 kg adult. In the normal control and model control groups, mice were treated with ddH2O by gavage.

Measurements of serum liver function and hydroxyproline content

The activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were quantitated by following the instructions provided by the manufacturer (Nanjing JianCheng Bioengineering Institute, Nanjing, China), including standardization. Levels of hydroxyproline (Hyp) in tissue were measured by Jamall’s method [16].

Histopathological assessment of liver injury

Liver specimens were fixed in 10% formaldehyde solution and dehydrated in a graded alcohol series, embedded in paraffin blocks, and cut into 4 μm thick slices. For histopathological examination, slices were stained by using the standard procedure of haematoxylin and eosin (H&E). For investigation of the hepatic collagen deposition, Sirius red polarization staining was performed and red colour staining was considered to indicate collagen deposition. Images were analysed with a light microscope (Olympus BX40, Japan).

Immunohistochemical analysis

Immunohistochemical analysis was performed to detect alpha smooth muscle actin (α-SMA) expression in liver tissues. In brief, paraffin-embedded sections were deparaffinized in xylene twice for 10 min, rehydrated in graded concentrations of ethanol (100%, 95%, 90%, 80% and 70%) and then were submerged in water. For antigen retrieval, the whole sections were submerged in citrate antigenic retrieval buffer and microwaved. They were then treated with 3% hydrogen peroxide in methanol to inactivate the endogenous enzymes. Sequentially, the sections were incubated with 5% bovine serum albumin (BSA) for 20 min at room temperature to block nonspecific binding. Sections were incubated with α-SMA (1:200, CST, D4K9N, USA) antibody overnight at 4 °C. After washing with TBST, tissue sections were treated with secondary anti-rabbit antibody (Boster, SA1028, China), followed by incubation with conjugated horse-radish peroxidase–streptavidin. Tissue sections were then counterstained with haematoxylin, dehydrated and mounted.Additional file: As per journal requirements, every additional file must have a corresponding caption. In this regard, please be informed that the caption of Additional file [1, 2] was taken from the additional e-file itself. Please advise if action taken appropriate and amend if necessary.Yes, the action taken is appropriate, thank you.

Samples for RNA sequencing

RNA sequencing was conducted using homogenized liver tissue (three replicates each for the normal control, model control, and FZHY treatment). Total RNA was extracted using TRIzol reagent (Qiagen, Germany) according to the manufacturer’s instructions. RNA concentration, integrity, and quality were assessed using an Agilent Bioanalyzer system (Agilent, USA), with criteria for RIN values > 7 and 28 S:18 S ratio > 0.7. cDNA library construction using the Illumina TruSeqTM RNA Sample Prep Kit method. Subsequently, the Illumina NovaSeq 6,000 platform (LC Science, United States) was employed for quantification and sequencing according to a standard sequencing protocol. Quality control of raw sequencing data was performed using Trimmomatic software to generate clean data. Clean data were mapped to the mouse reference genome using HISAT2 (Rattus_norvegicus, version Rnor_6.0). Next, the gene FPKM expression value was quantified using cufflinks software. The read counts were normalized using DESeq2 software, the fold difference was calculated, and the significance of the difference was tested using the nbinomTest function, which used the default filter conditions (p-adjust < 0.05 and |log2 FC| ≥ 1).

Quantification of retinoids by HPLC

Retinol metabolites were quantified in a blinded manner. All samples were frozen at collection and stored at −70 °C until extraction. Liver tissues were homogenized in saline and subjected to a 2-step liquid-liquid extraction under yellow lights as described previously. Levels of retinoic acid were determined by LC-MRM3 on a Shimadzu Prominence UFLC XR liquid chromatography system coupled to an AB Sciex 5500 QTRAP hybrid triple-quadrupole mass spectrometer using atmospheric pressure chemical ionization operated in positive ion mode. Retinol and retinal were quantified via HPLC-UV. Retinoic acid, retinol, and retinal were normalized per gram of tissue.

Real-time fluorescence quantitative polymerase chain reaction

Total RNA was extracted using TRIzol Reagent (Sangon Biotech, Shanghai, China) in accordance with the manufacturer’s protocol. After the concentration of RNA was determined, 500 ng of total RNA was used as the template for reverse transcription into single-stranded cDNA by a Reverse Transcription Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). Real-time fluorescence quantitative polymerase chain reaction was performed using SYBR Premix Ex Taq (Tli RNaseH Plus; TaKaRa) and a ViiA 7 Real-Time PCR System (ABI, Carlsbad, CA, USA). The β-actin gene was amplified as an internal control, and the primer sequences are listed in Table 1. The relative gene quantities compared with β-actin were calculated through the 2−∆∆CT method.Author contributions: Journal standard instruction requires the statement [All authors read and approved the final manuscript.] in the [Author contributions] section. This was inserted at the end of the paragraph of the said section. Please check if appropriate.We have verified it and resolved the issue, thank you.

Table 1 Primers for RT‒PCR RNA in situ hybridization

RNA in situ hybridization was performed as follows. Briefly, liver samples were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at 4 °C overnight. Then the samples were embedded in Paraplast X-Tra (McCormic Scientific) and sectioned at a thickness of 8 μm using a microtome (Thermo Scientific). Sections were transferred to microslides and dried overnight at 42 °C on a slide warmer. Sections were dewaxed and incubated in pretreatment solution A for 30 min at room temperature to remove endogenous enzymes and then boiled in prereaction solution B containing citrate for 5 min. The mRNA nucleic acid to be tested was fully exposed by protease digestion at 40 °C for 10 min, and the gene-specific probe was incubated in the probe hybridization solution at 40 °C for two hours. After washing to remove unhybridized probes, signal amplification was performed using cascade signal amplification technology based on nucleic acid-protein hybridization (Chinese Patent No. ZL202110575231.5). Finally, fast red substrate was added for alkaline phosphatase-based colour reaction, and the position of target RNA is displayed as red dots or patch staining. The mRNA in situ detection of each gene used the PinpoRNATM RNA in situ hybridization detection kit (Guangdong Pinpoease Biotechnology product number PIT0001, https://www.pinpoease.com) manual operation detection. The probes used in PinpoRNA technology were designed with multiple sets of short probes for RNA detection (China Patent No. ZL202110581853.9) to ensure the specificity of the probes. The detection signal could only be displayed when it was combined with the mRNA to be detected at the same time. The designed probe covers the 283–747 base region of Cyp4a12a (Cat. No. IB1-00166).

Immunofluorescence staining

The liver tissues embedded in OCT compound were cut into frozen sections at 10 μm in thickness. Then the sections were fixed in cold acetone for 30 min and treated with 0.1% Triton X-100 for 2 min at 4℃. The sections were blocked with normal donkey serum for 30 min at room temperature, before incubation with the α-SMA or retinoic acid early inducible gene 1 (Rae-1) (1:20, R&D, AF1136, USA). The tissues were then stained with Cy3 or FITC-conjugated secondary antibodies. After washing, DAPI (1:2000, Abcam, ab228549, UK) was used to stain the nuclei of the tissue. Images were taken with an Olympus confocal microscope, and semiquantitative analysis was performed using Image Pro Plus software.References: As per pubmed findings, citation details [Page no] for Reference [26] have been inserted. Kindly check and confirm the inserted details.We have conducted a thorough recheck of the reference and can confirm that it is in order. Thank you.

Primary hepatocyte and HSC isolation, culture, and identification

Hepatocytes and HSCs were isolated from C57BL/6 mice by perfusing the liver through the inferior vena cava. The liver was perfused with EGTA buffer (136.89 mM NaCl, 5.37 mM KCl, 0.64 mM NaH2PO4.H2O, 0.85 mM Na2HPO4, 9.99 mM HEPES, 4.17 mM NaHCO3, 0.5 mM EGTA, and 5 mM glucose (pH 7.35–7.4)) at a rate of 5 mL/min for 2 min, followed by enzyme buffer (136.89 mM NaCl, 5.37 mM KCl, 0.64 mM NaH2PO4.H2O, 0.85 mM Na2HPO4, 9.99 mM HEPES, 4.17 mM NaHCO3, and 3.81 mM CaCl2.2H2O (pH 7.35–7.4)) containing 0.4 mg/mL pronase (Roche Diagnostics, Indianapolis, IN, USA) at a rate of 5 mL/min for 5 min, and then enzyme buffer containing 0.193 U/mg collagenase (Roche Diagnostics) at a rate of 5 mL/min for 7 min. After perfusion, the liver was shaken for 25 min at 37 °C and filtered through a 70 μm nylon mesh, and hepatocytes were pelleted by centrifugation at 500 rpm for 1 min at 4ºC. The supernatant was transferred and centrifuged at 580× g for 10 min at 4 °C. Pelleted HSCs were resuspended in Gey’s balanced salt solution (GBSS) (Sigma-Aldrich), gently overlaid with a gradient of Nycodenz (Alere Technologies, 1,002,424, USA) prepared with GBSS using a pipette, and then centrifuged at 1380 g for 17 min at 4 °C without braking. HSCs present in a thin white layer at the interface between Nycodenz and GBSS were harvested and washed with Hank’s balanced salt solution. The hepatocytes were plated in 1640 containing 20% foetal bovine serum (FBS), 10− 8 M dexamethasone, and 10− 8 insulin and HSCs were plated in DMEM containing 20% FBS.

Oil red O staining

HSCs were stained with oil red O reagents (Nanjing Jiancheng, D027-1-2, China) according to the manufacturer’s instructions and the red lipid droplets were observed using a light microscope.

Flow cytometry

After the mouse liver was removed, a mononuclear cell suspension was prepared by mechanical grinding. Percoll (Cytiva, 17,089,101, Sweden) was used for lymphocyte gradient separation. The cells were washed twice in PBS buffer containing 0.2% bovine serum albumin (BSA) and resuspended in PBS buffer for counting cell number with Trypan blue stain (Gibco, 15250-061, USA). Anti-mouse CD16/32 (eBioscience, 14-0161-86, United States) was used to block the Fc receptor. The following antibodies were used for immunophenotyping analysis of the samples: anti-mouse CD3-FITC (BD, 555,274, USA), anti-mouse NK1.1-APC (BD, 550,627, USA), and anti-mouse NKG2D-PE-Cy7(BD, 562,614, USA), Data were obtained by the flow cytometry (DxFLEX, Beckman Coulter, USA) and analysed using FlowJo 10.0 software.

Primary NK cell isolation, culture, and identification

Murine primary NK cells were isolated from Percoll-separated hepatic mononuclear cells by magnetic sorting using an NK cell isolation kit (Miltenyi,130-115-118, Germany). NK cells were sorted as cells that expressed NK1.1+CD3− using flow cytometry, and the purity was higher than > 95%. After purification, NK cells were cultured in a 96 round bottom wells in complete RPMI 1640 medium supplemented with 10% (v/v) foetal bovine serum (Hakata, HB-FBS-500, Japan), 100 units/ml penicillin(Sangon Biotech, A100339, China), 100 µg/ml streptomycin (Sangon Biotech, A100382, China) and interleukin-2 (IL-2, 10 U/ml, R&D,402-ML, USA).

Drug incubation

FZHY was initially dissolved as a concentrated stock solution in DMSO. For evaluation of the effect of FZHY on HSCs in vitro, HSCs were treated with 8 concentrations (6.25, 12.5, 25, 50, 100, 200, 400,800 µg/ml) of FZHY, for a period of 24 h.

Coculture of NK cells and HSCs

HSCs were cultured in adherent 96 flat bottom wells for 24 h. and the empty medium containing the FZHY drug was disturbed. After the drug-containing supernatant was discarded, primary NK cells were added to a 96-well plate (at a ratio of 10:1).

Cell viability assay

For the HSCs, cell viability assays were performed by using Cell Counting Kit-8 (CCK8, MCE, HY-K0301, USA) assays following the manufacturer’s suggestions.

Calculation of percent lysis

The CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega, G7570, USA) was used to calculate percent cocultured cell viability using the mean luminescence signal of a mixture of NK cells and HSCs (MeanMIX) minus the mean luminescence signal of NK cells (MeanNK) divided by the mean signal of HSCs (MeanHSC) according to the methods of a previous study [17].

Adenovirus transfection to silence genes

The recombinant adenovirus shuttle vector Hu6-MSC-CMV-EGFP for silencing CYP4a12a short hairpin RNA (shRNA) (AD-Cyp4a12a-RNAi) was constructed by GeneChem Co. Ltd (Shanghai, China). The optimal sequence for knockdown of CYP4a12a (5′-GGAACATCTTTCACCAGAATG-3′) was selected and the scrambled sequence (TTCTCCGAACGTGTCACGT) was used as a negative control. After 48 h of infection, HSCs with green fluorescence were recorded. The knockdown efficiency of shRNA was determined by real-time PCR.

Statistical analysis

All data were analysed by using PASW Statistics 18 software. Differences between the groups were assessed by nonparametric one-way analysis of variance (ANOVA) followed by the least significant difference (LSD) post hoc tests. Values in the text are means ± standard deviations (SD). Differences with p < 0.05 were considered to be statistically significant.

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