Human HSCs

Human HSCs LX-2 (SCSP-527) were acquired from the National Collection of Authenticated Cell Cultures (Shanghai, China), and their identity was verified through STR. These cells were maintained in DMEM plus 2% fetal bovine saline at 37 °C with 5% CO2. The medium was renewed every 2–3 d, and the cells were subcultured at a 1:3 ratio every 3–4 d. Cells from passages 3 to 5 were selected for subsequent assays.

Stable transfection of genes in cells was achieved using lentiviral vectors. These vectors encapsulate short hairpin (sh) RNAs targeting LPAR3 and TEAD4 (shLPAR3 #1, 2, 3 and shTEAD4 #1, 2, 3), overexpression vector for TEAD4 and LPAR3 (Vector-TEAD4 and Vector-LPAR3), and negative control (NC) (shScramble and Vector-NC). The lentiviral vectors were obtained from VectorBuilder (Guangzhou, Guangdong, China) at a MOI of 5. Stable cells with selected using puromycin.

HSC activation was induced by transforming growth factor-beta 1 (TGF-β1) guided by a previous report (Kong et al. 2023). Briefly, LX-2 cells were starved in serum-free medium for 18 h and induced by 10 ng/mL recombinant human TGF-β1 (M9391, AbMole BioScience, Houston, TX, USA) in serum-free medium for 2 d. Cells treated with PBS were set to controls. The p38 MAPK pathway activator chromium picolinate (CrPic, HY-125588, MedChemExpress [MCE], Monmouth Junction, NJ, USA) at 100 nM (Wang and Yao 2009), or the PI3K/AKT activator 740 Y-P (HY-P0175, MCE) at a concentration of 20 μM (Yu et al. 2022), were used to treat LX-2 cells in conjunction with TGF-β1, with DMSO solution used as the control.

Cell immunofluorescence staining

The treated LX-2 cells were fixed with 4% neutral formaldehyde at 4 °C for 15 min and permeabilized with 0.3% Triton X-100 at 22–25℃ for 5 min. The cells were sealed with 5% normal goat serum at 37 °C for 0.5 h and probed with antibodies against α-SMA and Collagen I and with the secondary antibody at 37℃ for 60 min. The nuclei were stained using a DAPI solution at 22–25℃ for 30 min. The mean fluorescence intensity (MFI) of the staining was analyzed using Image J software. Details of the antibodies used are provided in Table 1.

Table 1 Antibodies used in immunofluorescence and WBActin detection

Filamentous actin (F-actin) in LX-2 cells was fluorescently labeled using CellMask Green Actin Tracking Stain (A57243, Invitrogen, Thermo Fisher Scientific, Rockford, IL, USA). Briefly, after fixing and being permeabilized, the LX-2 cells were incubated with 1X Actin Tracking staining solution at 22–25℃ for 15 min. The nuclei were counterstained with DAPI. MFI was quantitatively analyzed using Image J.

RT-qPCR

The cDNA was synthesized using extracted total RNA from LX-2 cells with the StarScript II RT Kit (A214-10, GenStar BioSolutions, Beijing, China). qPCR analysis was conducted using the RealStar Fast SYBR qPCR Mix (A301, GenStar BioSolutions) on the API 7500 qPCR system (Thermo Fisher Scientific). Relative mRNA expression, normalized to GAPDH, was evaluated using the 2−ΔΔCt method. As indicated in Table 2, the sequences for the forward and reverse primers are enumerated.

Table 2 Primers used in qPCRWestern blot (WB)

Total protein from LX-2 cells was isolated using RIPA lysis buffer. After determining protein concentration using the bicinchoninic acid method, equivalent amounts of protein samples were separated by 10% SDS-PAGE and loaded onto PVDF membranes. The membranes were sealed with 5% BSA at 22–25℃ for 1 h and probed with primary antibodies overnight at 4 °C and with goat anti-rabbit IgG (1:10,000, ab6721, Abcam) at 22–25℃ for 2 h. Band signals were developed using ECL, and protein bands were normalized to the loading control Cyclophilin A and analyzed using Image J software.

Collagen gel contraction assay

The contractile activity of HSCs was evaluated using the collagen gel contraction assay as previously described (Bujak et al. 2015). Briefly, stably transfected LX-2 cells (1 × 105) were resuspended in serum-free DMEM and supplemented with type I collagen solution (C3867, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) to prepare a cell suspension containing 500 μL of collagen (1 mg/mL). The mixture was incubated at 37 °C for 1 h in a 24-well plate. Using a sterile pipette tip, the gel was detached from the surface of the well, followed by treatments with TGF-β1, CrPic, or 740 Y-P. After 48 h, the gel area in each well was quantified relative to the original gel area (0 h) using Image J.

Mouse model of cirrhotic PHT

Male CD-1 (ICR) IGS Mice (weighing 25–32 g, 6 weeks old, Vital River, Beijing, China) were housed at a temperature of (23 ± 2) ℃, relative humidity of 52% ± 3.0%. All mice were kept under an ad libitum supply of food and water and a 12/12 h day-night rhythm. Before the experiment, mice were acclimatized for one week. All animal experiments were approved by the Animal Ethics Committee of the Fifth Medical Center of Chinese PLA General Hospital (approval number: P-SL-2023–366) and followed the Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, MD, USA).

Following previous literature (Zhang et al. 2021), a mouse model of cirrhotic PHT was established by injection of a mixture of 5 mL/kg carbon tetrachloride (CCl4) and olive oil (1:4 [v/v]) three times per week for 6 weeks. Mice injected with olive oil (vehicle) alone served as controls. Each group contained 6 mice. For artificial gene interference, PHT mice were given 100 μL of lentiviral vectors via tail vein injection during the first CCl4 injection. These lentiviral vectors included shScramble (n = 12, divided into two experiments), shLPAR3 (n = 6), shTEAD4 (n = 6), shTEAD4 + Vector-NC (n = 6), and shTEAD4 + Vector-LPAR3 (n = 6).

Six weeks after the CCl4 treatment, the portal pressure of mice was evaluated under anesthesia (1.5% isoflurane) using a 24-G catheter inserted into the colonic vein. Subsequently, the mice were euthanized, and blood and liver samples were harvested for subsequent use.

Assessment of liver injury markers

The mouse blood samples were centrifuged at 1000 g at 4℃ for 15 min. The ALT and AST levels in serum samples were determined using mouse ALT (CSB-E16539m, CUSABIO Biotech Co., Ltd., Wuhan, Hubei, China) and AST (CSB-E12649m, CUSABIO) ELISA Kits.

Histological staining of the mouse liver

Mouse liver tissues were fixed, paraffined, and sectioned at 5 μm. Pathological changes in the tissue sections were assessed using a HE stain kit. The Masson's and Sirius Red stain kits (all from Solarbio, Beijing, China) were used to examine collagen deposition in the liver. Image J was used for quantification.

Immunohistochemistry (IHC)

After deparaffinization and hydration, the prepared mouse liver tissue Sects. (5 μm) were subjected to antigen retrieval. After blocking, the sections were probed with the Myosin IIA antibody (1:500, ab238131, Abcam) at 22–25℃ for 2 h, followed by incubation with IgG (Alexa Fluor 488) (1:1000, ab150077, Abcam) at 22–25℃ in darkness for 1 h. Additionally, fluorescent primary antibodies were added as needed and incubated in the dark for 1 h. These antibodies included AbBy Fluor 488-conjugated anti-LPAR3 (1:200, bs-2882R-BF488, Bioss Antibodies, Woburn, MA, USA), anti-phospho-p38 MAPK (1:200, bs-23251R-BF488, Bioss), anti-phospho-PI3K (1:200, bs-5571R-BF488, Bioss), anti-phospho-AKT (1:200, bs-12456R-BF488, Bioss), anti-TEAD4 (1:200, bs-9603R-BF488, Bioss), and Alexa Fluor 647-conjugated anti-α-SMA (1:200, ITT5797-647, G-Biosciences, St Louis, MO, USA). Subsequently, the nuclei were counterstained with DAPI, and the mean fluorescence intensity or positive staining area was evaluated using Image J software.

ChIP-qPCR

The ChIP kit (Bes5001, BersinBio, Guangzhou, Guangdong, China) was used. LX-2 cells were added with 1% paraformaldehyde at 22–25℃ for 10 min of DNA–protein crosslinking, which was quenched by the addition of glycine. The cells were lysed and then subjected to ultra-sonification for 15 min to generate DNA fragments at 200 bp ~ 600 bp. The samples were centrifuged at 1,3000 g at 4℃ for 10 min to collect the supernatant. The supernatant was divided into 0.8 mL (IP) and 0.1 mL (Input) portions, and the IP portion was added with the TEAD4 antibody (1:50, 12,418–1-AP, ProteinTech) or normal rabbit IgG for IP reaction. The immunoprecipitated complexes were collected, eluted, and de-crosslinked. The DNA was then collected and purified. The abundance of LPAR3 promoter was analyzed using qPCR analysis, and the results were presented as a percentage of the Input group.

Dual-luciferase assay

The LPAR3 promoter fragment (chr1:84,893,084–84893420) was subcloned into the pGL3-Basic luciferase vector (Promega Corporation, Madison, WI, USA) to construct the LPAR3 promoter luciferase reporter vector. This construct was co-transfected into LX-2 cells, either overexpressing TEAD4 or control cells, along with a Renilla luciferase reporter plasmid for normalization purposes for 48 h. The luciferase activity was measured.

Statistical analyses

All experimental data are expressed as the mean ± SEM. Statistical analyses were performed using Prism 10.4.2 software (GraphPad, La Jolla, CA, USA). Differences between the two groups were compared using the unpaired t-test. For cases where multiple groups were included, one-way ANOVA was applied, as appropriate, followed by Tukey's test. A p-value less than 0.05 was considered statistically significant.