Human serum and liver samples
Serum samples were obtained from 15 patients with clinically diagnosed cirrhosis and 10 healthy donors. Liver tissues were obtained from 10 patients with hepatic hemangioma (non-fibrotic samples) and 40 patients with cirrhosis who underwent liver transplantation at the Second Xiangya Hospital of Central South University. All study related experimental protocols were under the ethical guidelines of the 1975 Declaration of Helsinki Principles and were approved by the Ethics Committee of the Second Xiangya Hospital of Central South University. Written informed consent was obtained from all human subjects. Healthy donors with no previously known liver abnormality and who had not received any medication in the two weeks prior to sample collection participated in this study.
Reagents
CCl4 (C112040) and olive oil (O108686) were purchased from Aladdin (Shanghai, China). The hematoxylin and eosin (H&E) staining kit (G1120), Sirius Red staining kit (G1471), and Masson’s trichrome staining kit (G1340) were purchased from Solarbio (Beijing, China). TRIzol reagent was purchased from Invitrogen (Thermo Fisher SCIENTIFIC, MA, USA). A Universal Two-Step Test Kit (PV-9000) was purchased from ZsBio (Beijing, China). The following antibodies were used: anti-TRPM8 (GTX54866, GeneTex, California, USA), anti-α-smooth muscle actin (α-SMA) (BM0002, Boster, Beijing, China), anti-collagen type I (COL1A1) (BA0325, Boster, Beijing, China), anti-cytokeratin 19 (CK19) (GB11197, Servicebio, Wuhan, China), anti-β-actin (60008-1-AP, Proteintech, Rosemont, USA), anti-F4/80 (28463-1-AP, Proteintech, Rosemont, USA), anti-S100A9 (14226-1-AP, Proteintech, Rosemont, USA), and anti-HNF4α (ab41898, Abcam, Cambridge, UK).
Animals and animal experiments
WT and TRPM8−/− (systemic knockout) C57BL/6J mice were obtained from Jackson Laboratory (ME, USA). All mice received humane care throughout experiments according to the guidelines of Central South University. All animal experiments were approved by the University Committee on Use and Care of Animals.
CCl4 is a hepatotoxic substance, and the CCl4-induced liver fibrosis model is one of the most common toxic liver fibrosis models. Six-week-old male WT and TRPM8−/− mice were given intraperitoneal injections of CCl4 to mimic toxic-induced fibrosis (1.0 mL/kg body weight, 1:4 dissolved in olive oil) or vehicle (olive oil) twice a week for eight weeks (n = 5 per group). Two days after the last CCl4 injection, mice were sacrificed and specimens collected.
Persistent cholestasis can damage the liver, and the BDL-induced liver fibrosis model is an ideal model to study cholestatic liver fibrosis. Six-week-old male WT and TRPM8−/− mice underwent ligation of the common bile duct (n = 5 per group) to mimic cholestatic liver fibrosis. Fourteen days after the BDL procedure, mice were sacrificed and specimens collected.
For therapeutic studies, CCl4-injured C57BL/6J mice received DMSO, 10 mg/kg M8-B hydrochloride, or 10 mg/kg WS-12 intraperitoneal injection [22] every other day from the 7th to the 8th week (n = 5 per group), while BDL-injured mice received either DMSO, 10 mg/kg M8-B hydrochloride, or 10 mg/kg WS-12 intraperitoneal injection every other day after one week of BDL treatment (n = 5 per group).
Histology analysis
Formalin-fixed, paraffin-embedded 4 μm thick liver sections were stained with either H&E for cell morphology characterization or Sirius Red and Masson’s trichrome for quantitative assessment of liver fibrosis severity. Axio Scope A1 light microscopy (Carl Zeiss, Germany) was used to acquire digital images from random fields, and the most representative views of the sections are presented. Cell morphology and the severity of liver fibrosis were evaluated by experienced pathologists while blinded to the treatment groups.
Transmission electron microscopy
Liver tissue obtained from CCl4-treated mice was thin-sectioned first followed by 2.5% glutaraldehyde fixation in phosphate-buffered saline by perfusion via the inferior vena cava. Transmission electron microscopy was performed as described previously [23].
Immunohistochemistry (IHC)
The expressions of TRPM8, α-SMA, COL1A1, F4/80, CK19, S100A9, and HNF4α in the liver were evaluated using immunohistochemical staining. Briefly, the tissue sections were deparaffinized, hydrated, and incubated in 3% hydrogen peroxide, to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) for 20 min, and then incubation with primary antibodies against TRPM8 (1:200 dilution), α-SMA (1:500 dilution), COL1A1 (1:500 dilution), F4/80 (1:1000 dilution), CK19 (1:1000 dilution), S100A9 (1:400 dilution) or HNF4α (1:500 dilution) antibodies at 4 °C overnight. A universal two-step test kit was used to treat the samples after they had been incubated with appropriate primary antibodies. Finally, sections were counterstained with hematoxylin for 10 min, dehydrated and sealed with neutral balsam before a light microscope equipped (Olympus, Hamburg, Germany) with a digital camera was used to photograph the regions of interest. Three images at randomly selected locations were acquired for each tissue section.
Serum biochemistry
Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using automatic biochemical analyzers (Abbott, Chicago, USA). Human serum S100A9 was measured using a S100A9 ELISA kit (CSB-E11834h, CUSABIO, Wuhan, China) following the manufacturer’s recommended procedure.
Cell experiments
L02 cells were purchased from the Cell Bank of Xiangya Medical School, Central South University. L02 cells were cultured for 24 h before being exposed to TRPM8 siRNA, S100A9 siRNA (RiboBio, Guangzhou, China), or S100A9 plasmid (GeneChem, Shanghai, China). Lipofectamine™ 3000 transfection reagent was used to treat L02 cells following the manufacturer’s instructions. The transfection medium was replaced with complete culture medium after 6 h. Cells were collected for further investigations after 48 h.
Primary mouse hepatocytes were prepared from male WT and TRPM8−/− mice following the well-established collagenase perfusion method [24]. After purifying the primary mouse hepatocytes, cells were collected for further investigations.
qRT-PCR and RNA-seq analysis
Total RNA was extracted from frozen liver tissue using TRIzol reagent following the manufacturer’s instructions. Relative mRNA expression levels were calculated using the relative quantification method (2−ΔΔCT). All used PCR primer sequences can be found in Additional file 6: Table S1. A melting curve for each amplicon was determined to verify its specificity. Genes were normalized to β-actin as an internal control. Liver RNA samples were submitted for RNA-seq analysis at Annoroad Gene Technology Co., Ltd.
Western blot analysis
Proteins in lysates were resolved by SDS–PAGE electrophoresis and transferred to PVDF membrane (Millipore Corporation, MA, USA). The blotted membrane was blocked and immunoblotted with TRPM8 (1:500 dilutions), α-SMA (1:500 dilutions), COL1A1 (1:500 dilutions), S100A9 (1:500 dilutions), and HNF4α (1:1000 dilutions) primary antibodies at 4 ℃ overnight. β-actin (1:5000 dilution) was used as a control. After washing with TBST, the membranes were incubated with secondary antibodies (1:5000 dilution) for 1 h at room temperature, followed by detection with enhanced chemiluminescence system. Image J was used to quantify the grey values for each target protein band.
Statistical analysis
Data were expressed as mean ± SD and analyzed using GraphPad Prism 7. Statistical differences between two groups were analyzed by the unpaired t-test with a two-tailed distribution. Differences between multiple groups of data were analyzed by one-way ANOVA with Bonferroni correction. A P-value of less than 0.05 was considered to be statistically significant.