Cell culture and Reagents
Human intrahepatic biliary epithelial cell lines (HIBECs) and Human monocyte cell line (THP-1) were obtained from the Center of Hepato-Pancreato-Biliary Surgery in the First Affiliated Hospital of Sun Yat-sen University. Both HIBECs and THP-1 cells were cultured in RPMI-1640 medium (Gibco, Carlsbad, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, USA) with 5% CO2 at 37°C. THP-1 cells were treated with 200 ng/mL of phorbol-12-myristate-13-acetate (PMA) (HY-18739, Med Chem Express, USA) for 48 h and then treated with 20 ng/mL of IL-4 (NBP2–35131, NOVUS, USA) to generate M2 polarization or treated with 100 ng/ml of LPS (HY-D1056, Med Chem Express, USA) to generate M1 polarization.
Recombinant protein MBP and MBP-CsGRN were obtained from our previous study [14]. The CsGRN fragment was amplified using forward primers 5′-ATA AGG ATC CCG GAG CAC AGGTGTAG-3′ (EcoR I) and reverse primers 5′-CGC GGA TCC TGT AAA TATA ACC AGA CTT G-3′ (BamH I), under the following conditions: 30 s at 94°C for denaturation, 30 s at 60°C for annealing, and 1 min at 72°C for extension for 30 cycles. Moreover, CsGRN was cloned into the PCDH-luc vector plasmid (ID: CS-HLUC-pCDH, GeneCopoeia, China) to constructed the recombinant plasmid PCDH-luc-CsGRN. The recombinant plasmids were purified with the endotoxin-free Maxiprep kit (Qiagen, USA) and then transformed into E. coli DH5α to amplify (Promega, USA). Finally, the recombinant plasmid then extracted with E.Z.N.A.® Plasmid Mini Kit I (Omega, USA).
Animal Studies
BALB/c wild-type mice aged 6–8 weeks were purchased from the Laboratory Animal Center of Sun Yat-sen University. The following animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC), Sun Yat-sen University (NO. SYSU-IACUC-2022-000370). Each mouse was intravenously injected with 2 ml saline dissolved of 20 µg PCDH-luc vector or 20 µg PCDH-luc-CsGRN plasmid, respectively. The plasmid solution was rapidly injected through the lateral tail vein within 8 s. 5 mice per group. The PCDH-luc vector was used as control. Check the mice once a week to determine whether they are suffering from abdominal distension or other health problems. 4 weeks after the injection, all mice were sacrificed using anesthesia at the end of the experiment. The serum and proteins from liver tissues were extracted for following analyses.
Detection of the location of PCDH-luc- Cs GRN in mice
The location of injective CsGRN-plasmid in mice was detected by IVIS Spectrum Imaging System (IVIS 100; Xenogen, Alameda, CA). Briefly, 4 weeks after injection of PCDH-luc vector or PCDH-luc-CsGRN, animals were anesthetized. After treated with 100 µl of D-Luciferin (HY-12591A, Med Chem Express, USA) solution (10 mg/ml) through intraperitoneal injection, the fluorescence imaging of injective CsGRN-plasmid was observed and analyzed by IVIS Spectrum Imaging System.
Establishment Of Co-culture System
Transwell chambers (0.4/8 µm PET, Millipore) were used for cell co-cultivation. After preconditioning the HIBECs for 24 h with 10µg/ml of MBP-CsGRN, the supernatant was collected and then used to create the co-culture system with THP-1 cells. HIBECs were cultured in the lower chambers. THP-1 cells were into the upper chambers. THP-1 group was as the negative control. THP-1 cells treated with 10µg/ml MBP-CsGRN were as the THP-1-MBP-CsGRN group. THP-1 cells treated with the supernatant of HIBECs in MBP-CsGRN group were as the THP-1-HIBEC-MBP-CsGRN group. THP-1 cells treated with 200ng/ml of PMA for 48 h and 20ng/ml of IL-4 for 48 h were as the THP-1-PMA/IL-4 group.
Measurement Of Cell Proliferation
Cell proliferation was estimated by employing EdU-488 incorporation assay kit (C0071S, Beyotime, China) and Colony Formation assay, respectively. The treated cells were labeled with EdU-488 for 2h following the product protocol. DAPI was utilized in staining the nuclei for 10 min. The number of cells with EdU-488 incorporation was observed by using fluorescence microscope (Leica DMI8, Wetzlar, Germany) and analyzed by image J software (NIH, USA). The colony formation assay was used to analyze the long-term effects of CsGRN on HIBECs proliferation. Briefly, 1000 cells were cultivated in 24-well plates and treated with 10µg/ml of recombinant MBP-CsGRN proteins or MBP proteins for 14 days. Subsequently, the fixed cells were then stained by crystal violet after 30 min in 4% paraformaldehyde. After washing with ddH2O for 3 times, the colony number was determined by Image J software (NIH, USA).
Measurement Of Cell Migration
Cell migration was detected by wound-healing assay and transwell assay, respectively. For the migration effect of CsGRN on HIBECs, HIBECs cells were inoculated into 6-well plates with 80% density and wounds were created into the center of the plates with 10-L plastic pipettes. Then the cells were treated with 10µg/ml of recombinant MBP-CsGRN proteins or MBP proteins for 24 h, followed by observing and imaging with light microscope (Leica DMI3000B, Wetzlar, Germany). Moreover, treated 5.0 × 104 HIBECs cells were cultured in 24-well transwell upper chambers (Costar, New York, USA) for 24 h, while medium containing 10% FBS was added into the lower chambers. The migrant cells were then stained with 10% crystal-violet (Sigma-Aldrich, St. Louis, USA) for 10 min and were counted with light microscope (Leica DMI3000B, Wetzlar, Germany).
Histopathology
The mice livers were frozen in 10% formalin for 24 h at room temperature, dehydrated and transparentized, then embedded in paraffin and sectioned into 5µm-thick slices. Hematoxylin and eosin (H&E) staining were followed by immunohistochemistry (IHC) /immunofluorescence (IFA) analyses of the sections.
Immunohistochemistry
Tissue samples were sectioned at 5 µm-thick and incubated with 3% H2O2 at room temperature for 10 min, then blocked with 3% Bovine Serum Albumin (BSA) at 37°C for 30 min. The tissues were then incubated with primary antibodies at 4°C overnight and then followed with secondary antibodies at room temperature for 1h. The following primary antibodies were purchased from Cell Signaling Technology (CST, Boston, USA) and Abcam Shanghai Trading Corporation (Abcam, Shanghai, China): cytokeratin 19, MCP-1, CD206, INOS, IL6, p-JAK, p-STAT3, p-MEK and p-ERK. The HRP-conjugated secondary antibodies were purchased from Proteintech Group, USA. The immunohistochemical staining was imaged by the Caseviewer software (3DHISTECH, Hungary).
Immunofluorescence
The liver tissue specimen was fixed in 4% paraformaldehyde at room temperature for 10 min, permeabilized with 0.1% Triton X-100 (9002-93-1, Beijing Biotopped Science& Technology, China) for 10 min, and then incubated with 3% BSA in PBS at room temperature for 30 min. The tissues were then incubated with primary antibodies (CD68, 1:100; CD86, 1:100; CD163, 1:100, Abcam, Cambridge, UK) at 4°C overnight and in secondary antibodies (Alexa Fluor 488-conjugated goat anti-rabbit IgG; Alexa Fluor 594-conjugated goat anti-mouse IgG, Invitrogen, Waltham, USA) at room temperature for 1h. The nuclei were stained with DAPI (Abcam, Cambridge, USA) for 10min at room temperature. Each animal was imaged six times by the Confocal laser scanning microscopy (LSM780, Zeiss, Oberkochen, Germany). For each mouse liver, the average signal value of six images was represented as the real value.
Western Blot And Elisa
Western blot and ELISA
After cells or liver homogenates were harvested and washed in cold PBS twice, the proteins were extracted using Radio Immunoprecipitation Assay (RIPA) solution (Beyotime, Shanghai, China), and the protein concentration was measured using Bicinchoninic Acid Assay (BCA) protein assay kit (Thermo, Shanghai, China). Western blot was applied according to a previous study [14]. All primary antibodies were purchased from Cell Signaling Technology (CST, Boston, USA) as follows: anti-E-cadherin (1:2,000), anti-vimentin (1:2,000), anti-N-cadherin (1:2,000), anti-ZO-1 (1:2,000), anti-β-catenin (1:2,000), anti-IL6 (1:2,000), anti-COX2 (1:2,000), anti-MCP-1 (1:2,000), anti- p-JAK2 (1:2,000), anti-, p-STAT3 (1:2,000), anti-c-Myc (1:2,000), anti-p-MEK (1:2,000), anti-p-ERK (1:2,000), anti-STAT3 (1:2,000), anti-MEK (1:2,000), anti-ERK (1:2,000) and anti-GAPDH (1:2,000). In addition, ELISA kits (Multi Sciences, Hangzhou, China) were used to detect IL-6 levels in HIBECs and THP-1 co-culture medium.
Flow Cytometry Analysis
THP-1 cells and hepatic macrophages in each group were harvested and stained with APC/CY7 anti-mouse CD45 (103116, Biolegend, USA), PE anti-mouse CD11b (101208, Biolegend, USA), APC anti-mouse F480 (123116, Biolegend, USA), PC5.5 anti-mouse MHC-Ⅱ (107626, Biolegend, USA) and PC7 anti-mouse CD206 (141720, Biolegend, USA) antibodies at 4℃ for 30 min. Hepatic macrophages were detected in the mice by grinding the liver tissues gently, filtering the homogenate through 80µm nylon mesh filter, and isolating hepatic mononuclear cells with 40% and 80% Percoll (17-0891-01, GE Healthcare, UK). The isolated cells were stained with the above surface markers after being blocked with anti-mouse CD16/32 (TruStain FcX™ PLUS, BioLegend, USA) following red blood cell lysis (420301, BioLegend, USA). After staining, the cells were washed 3 times with PBS and resuspended with 200 µl of 10% BSA diluted by PBS, and analyzed by FACS with a BD FACS Aria II instrument (BD Science, USA). The data were analyzed using Flowjo software VX10 (TreeStar, Ashland, USA).
Quantitative Pcr
Total RNA from liver homogenates was extracted by using TRIzol solution according to the manufacturer protocol (Invitrogen, Carlsbad, USA). Quantitative PCR (q-PCR) kit (Vazyme, Nanjing, China) was used to quantify gene expression level. PCR conditions were performed as previously study[13]. The primer sequences for CsGRN are as follows: forward primer 5′-CGC GGA TCC TGT AAA TAT AAC CAG ACT TG-3′ and reverse primer 5′-TTA CTC GAG CGG AGC ACA GGT GTA GTG AT-3′.
Statistical analysis
All results are based on three independent experiments and presented as a mean ± SD. GraphPad Prism 8.0 software (San Diego, California, USA) was used for statistical analysis. Analyses of statistical differences were conducted using Student's t-test and ANOVA. Data were significant if: ns: not significant; *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001.