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 had not received any medication in the two weeks prior to sample collection participated in this study.
CCl4 (C112040) and olive oil (O108686) were purchased from Aladdin (Shanghai, China). The haematoxylin 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). Following antibodies were used: anti-TRPM8 (GTX54866, GeneTex, California, USA), anti-α-SMA (BM0002, Boster, Beijing, China), anti-COL1A1 (BA0325, Boster, Beijing, China), anti-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-/- 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 and all animal experiments were approved by the University Committee on Use and Care of Animals.
6-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 8 weeks (n = 5 per group). Two days after the last CCl4 injection, mice were sacrificed and specimens were collected.
6-week-old male WT and TRPM8-/- mice underwent ligation of the common bile duct (n = 5 per group) to mimic cholestatic liver fibrosis. 14 days after the BDL procedure, mice were sacrificed and specimens were collected.
For therapeutic studies, CCl4-injured C57BL/6J mice received either DMSO or 10 mg/kg M8-B hydrochloride intraperitoneal injection  every other day from the 7th to the 8th week (n = 5 per group), while BDL-injured mice received either DMSO or 10 mg/kg M8-B hydrochloride intraperitoneal injection every other day after 1 week of BDL treatment (n = 5 per group).
Formalin-fixed, paraffin-embedded 4 µm-thick liver sections were stained with either hematoxylin and eosin (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 shown. 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.
The expression of TRPM8, α-SMA, COL1A1, F4/80, CK19, S100A9 and HNF4α in the liver was 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 incubated 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 ℃ 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 haematoxylin for 10 min, dehydrated and sealed with neutral balsam before a light microscopy 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 levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using automatic biochemical analysers (Abbott, Chicago, USA). Human serum S100A9 was measured using a S100A9 ELISA kit (CSB-E11834h, CUSABIO, Wuhan, China) following manufacturer’s recommended procedure.
L02 cells were purchased from Cell Bank of Xiangya Medical School, Central South University. L02 cells were cultured for 24 h before 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. 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 . 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 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 supplementary Table S1. A melting curve of 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), 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. ImageJ was used to quantify the grey values for each target protein bands.
Data were expressed as mean ± SD and analyzed using GraphPad Prism 7. Statistical differences between two groups were analyzed by the unpaired Student t test with a two tailed distribution. Differences between multiple groups of data were analyzed by one-way ANOVA with Bonferroni correction. P-value of less than 0.05 was considered to be statistically significant.