2.1 Chemicals and reagents

Dulbecco’s Modified Eagle Medium (DMEM) was from Invitrogen, USA. Fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco, USA. Asperuloside (ASP, CAS#14259-45-1, HPLC ≥ 98%) (Fig. 1) was obtained from Yuanye, China. Radio immunoprecipitation assay (RIPA) lysis buffer, 1% protease inhibitor, phosphorylase inhibitor, 5% bovine serum albumin (BSA), Tris-buffered saline with Tween (TBST), phosphate-buffered saline (PBS), and 0.1% crystal violet were purchased from Beyotime Biotechnology, China. Lipofectamine 2000 (11668-019), BCA protein assay kit (A53226), and Trizol were provided by Thermo Fisher Scientific, USA. The PrimeScript RT kit was purchased from Takara, Japan, and the SYBR GREEN kit from Yeasen, China. Cell matrix type I-A was purchased from Nitta Gelatin, Japan.

Fig. 1

The chemical structure of ASP (Drawn by chemical draw software, version 20)

2.2 Cell culture and treatment2.2.1 Cell culture and identification

CAFs and normal fibroblasts (NFs) were purchased from SAIOS Biotechnology Co., Ltd (Wuhan, China), which were originated from pancreatic cancer tissue and normal pancreatic tissue, respectively. The cells were cultured in the Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 2% penicillin and streptomycin under the condition of 5% CO2 and 37 °C. After that, the cell morphology was observed by an electron microscope (ECLIPSE E100, Nikon, Japan) and the images were collected. Further, western blot was used to detect the expression of fibroblast-specific markers α-smooth muscle actin (α-SMA), fibroblast activating protein 1 (FAP-1), and vimentin in CAFs and NFs.

2.2.2 Cell viability assay

Cell viability was measured by the CCK-8 assay [20]. After inoculation of CAFs and NFs in 96-well plates at a density of 4000 cells per well for 24 h, the supernatant was removed and a complete medium with different ASP concentrations was added. After further incubation for 24 h, the supernatant was aspirated and complete medium containing 10% CCK8 was added, and continued to incubate protected from light for 1 h. The absorbance at 450 nm was then detected in a microplate reader (ELx800, BioTek, USA).

2.2.3 Cell transfection

CAFs in the logarithmic growth phase were collected, digested, and seeded into cell culture dishes. When cell confluence reached 60–70%, transfection was performed. The pcDNA3.1-ATF6 (ATF6 over-expression plasmid) and the negative control pcDNA3.1-vector plasmid were designed and synthesized by BGI Genomics (China). These plasmids were transfected into CAFs using Lipofectamine 2000 (11668-019, Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer’s instructions, ensuring efficient gene delivery and expression.

2.2.4 Experimental grouping

To explore the effect of different concentrations of ASP on the activation of CAFs, CAFs were divided tinto four groups: 0 mM group: CAFs were treated with 0 mM of ASP; 1 mM group: CAFs were treated with 1 mM of ASP; 3 mM group: CAFs were treated with 3 mM of ASP; 5 mM group: CAFs were treated with 5 mM of ASP. To clarify the role of ATF6, CAFs were divided into four groups: Control group: CAFs were not subjected to any treatment; ASP group: CAFs were treated with ASP (5 mM) for 24 h; ASP + vector group: CAFs were treated with ASP (5 mM) for 24 h after transfection with pcDNA3.1-vector; ASP + ATF6 group: CAFs were treated with ASP (5 mM) for 24 h after transfection with pcDNA3.1-ATF6 [21].

2.2.5 Western blot

The cells were lysed with radio immunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor and phosphorylase inhibitor. After centrifugation, the supernatant was collected. Next, a bicinchoninic acid (BCA) protein assay kit (A53226, Thermo Fisher Scientific, Rockford, IL, USA) was used to determine the protein concentration. After that, the total protein was mixed with 5 × loading buffer (P1040, Solarbio, Beijing), heated for 10 min, and denatured. Subsequently, the protein samples were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was cut into sections prior to hybridization with antibodies to focus on specific protein regions of interest.Then, the membrane was sealed with 5% bovine serum albumin (BSA) at room temperature. Upon 1 h, the membrane was incubated overnight at 4 ℃ with primary antibodies as follows: [FAP (1: 1000, ab207178, Abcam), vimentin (1: 1000, ab92547, Abcam), α-SMA (1: 1000, ab7817, Abcam), ATF6 (1:1000, ab37149, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1: 1000, ab8245, Abcam). Subsequently, the membrane was washed with TBST, and the second antibody (1: 5000, ab205718/ab205719, Abcam) was added for another 1 h of incubation at room temperature. After that, the chemiluminescent liquid (WBAVDCH01, Sigma, MO, USA) was dripped evenly on the membrane, and an imager (Amersham Imager 600, USA) was employed for scanning and collecting images. Besides, Image J (version 1.8.0, NIH, USA) was used for gray- level analysis. Finally, data were normalized using GAPDH as a reference, and the relative expression of target proteins was quantified.

2.2.6 Reverse transcription-quantitative polymerase chain reaction

Total RNA was extracted from each group of cells using Trizol (Thermo Fisher Scientific, USA) and the concentration was quantified through a NanoDrop 2000c spectrophotometer. The extracted total RNA was reversely transcribed into cDNA using the PrimeScript RT kit (Takara, Japan). Next, cDNA was used to detect the expression of target genes according to the instructions of the SYBR GREEN kit (Yeasen, China) on the Thermal Cycler Dice® Real-Time System. The procedure was conducted using a Real-time fluorescence quantifier (Bio-rad, USA) and data was analyzed using the 2−ΔΔCt method with GAPDH as the internal control gene. The primer sequences are shown in Table 1.

Table 1 The primer sequences of reverse transcription-quantitative polymerase chain reaction2.2.7 Collagen gel contraction assay

A collagen gel contraction assay was performed to check the contraction of cells [22]. In brief, cells (3 × 105) were mixed with 1 ml cell matrix type I-A (Nitta Gelatin, Osaka, Japan) and seeded in 12-well plates. The gel mixture was incubated at 37 °C for 30 min to polymerize the gel. Then, 1 mL serum-free medium was added, and the gel was separated from the well wall with the pipette tip. After incubation for 48 h, the wells were imaged. Subsequently, Image J software (version 1.8.0, NIH, USA) was utilized to measure the gel surface area and calculate the gel contraction percentage [the gel contraction percentage = (gel surface area/well surface area) × 100%].

2.2.8 Transwell assay

Firstly, 100 μL cell suspension (1 × 105 cells/mL) was seeded into Transwell chambers, and the lower chamber was supplemented with 500 μL DMEM containing 10% FBS. After 24 h of culture, the Transwell chamber was taken out and washed twice with phosphate buffered saline (PBS). Then, the cells on the upper chamber surface were wiped off with a cotton swab. After methanol fixation for 30 min, the cells were stained using 0.1% crystal violet for 20 min. Finally, the cells were observed by an inverted microscope (ECLIPSE E100, Nikon, Japan), and five fields of view were randomly selected to photograph and count.

2.3 Statistical treatment

SPSS21.0 (IBM SPSS Statistics, Chicago, IL, USA) was used for statistical analysis. The data were expressed by mean ± standard deviation (SD). An independent sample t- test was used for comparison between two groups, and one-way analysis of variance (ANOVA) for comparison among multiple groups. P < 0.05 was considered as the criterion for evaluating the statistically significant difference.