PAE was obtained from Ganlong capsules, produced by Saino Pharmaceuticals (Kunming Saino Pharmaceutical Co., Ltd., State Pharmaceutical Approval No. Z20050113). The capsules contain a brownish powder, with PAE as the primary component. Dissolve the powder in ddH₂O using ultrasonic solubilization and store it protected from light. Prepare a master batch at a final concentration of 30 mg/mL and store at 4°C.

HSC-T6 and LX2 cells were grown in DMEM with 10% FBS (ExCell, FSP500) and 1% penicillin-streptomycin (Gibco, 15140122) at 37°C in a 5% CO₂ environment. The cells were purchased from Procell in Wuhan, China. After the cells reached logarithmic growth, they were co-treated for 48 h with different doses of PAE and TGFβ (5 ng/mL, MCE, China).

After 24 h of incubation, cells were transferred to 96-well plates. There was a control group, a TGFβ group with a concentration of 5 ng/mL, and PAE groups with concentrations ranging from 0.3125 mg/mL to 20 mg/mL (0.3125 mg/mL, 0.625 mg/mL, 1.25 mg/mL, 2.5 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL), spread throughout seven different levels. Every PAE treatment group received 5 ng/mL of TGFβ. Following 48 h, 110 µL of basal medium and CCK-8 reagent (TargetMol, America) were added to each well in a 10:1 ratio to replace the old medium. The OD was assessed at 450 nm with an enzyme counter following a 2-h incubation period. Each group was independently repeated at least three times. Inhibitory concentration values (IC₂₀, IC₄₀, IC₅₀) for the CCK-8 assay were derived using SPSS 18.0 and expressed as mean ± standard deviation (SD).

An in vitro kit, Cell-light™ EdU Apollo567, was used to measure cell proliferation (RiboBio, China). The cells were cultured in a Petri dish, exposed to PAE and TGFβ for a duration of 48 h, stained with the EDU kit, and then photographed using fluorescence microscopy. Using ImageJ software, the cells that tested positive were analyzed. EdU incorporation assays were conducted on three independent biological replicates.

1 × 10⁵ cells/mL were inoculated into Transwell inserts (100 µL per well). After 48 h, the inserts were transferred to wells containing 1 mL of 4% paraformaldehyde for fixation. The contents of the upper chamber were drained, and 200 µL of 4% paraformaldehyde was added, which was left for 30 min. Afterward, the inserts were placed in wells containing 500 µL of crystal violet staining solution for 15 min. Transwell migration assays were performed on three independent biological replicates, with the inserts subsequently examined under a microscope and photographed.

At approximately 80% confluence, a 20 µL pipette tip was used to create a horizontal scratch. After sterilising the cells three times with PBS, the media was changed to fresh serum-free medium. The migration distance was measured by taking images at 0, 12, and 24 h and examining them under a microscope. Wound healing assays were conducted on three independent biological replicates.

Antibodies employed included ITGA4 (Bioss, bs-0641R), α-SMA (Proteintech, 14395-1-AP), COL1 (Proteintech, 14695-1-AP), TNFα (Affinity, AF7014), IL-6 (Affinity, DF 6087), ECA (Bioss, bs-10009R), NCA (Bioss, bs-1172R), Snail (CST, 3879T), Vim (CST, 5741S), TGF-β1 (Abcam, ab92486), FAK (CST, 71433S), PI3K (Abcam, ab191606), AKT (CST, 4691L), P-FAK (CST, 3284), P-PI3K (Abcam, ab278545), P-AKT (CST, 4060s), β-actin (Proteintech, 60008-1-Ig), and GAPDH (Proteintech, 60004-1-lg). Western blot analyses were conducted using at least three independent biological replicates.

Two groups of LX2 cells were analyzed: a TGFβ-treated group (5 ng/mL) and a TGFβ + PAE-treated group (6.120 mg/mL), with three samples in each group, designated as TGF21-23 and TGF31-33, respectively. All data have been uploaded to NCBI with a BioProject accession number of PRJNA1177452 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1177452 100).

Total RNA was extracted from LX2 and HSC-T6 cells using TRIzol reagent (Thermo Fisher, 15596018). cDNA was synthesized using the FastKing RT Kit (with gDNase). Gene-specific mRNA sequences were retrieved from PubMed, and primers were designed based on the CDS sequences using Beacon Designer 7.90. qRT-PCR analyses were performed with at least three independent biological replicates. The primer sequences included: β-actin (F: 5′-GCA​GGA​GTA​CGA​TGA​GTC​CG-3′; R: 5′-ACG​CAG​CTC​AGT​AAC​AGT​CC-3′); α-SMA (F: 5′-TCA​AGA​TTA​TTG​CTC​CTC-3’; R: 5′-CTG​GAA​GGT​AGA​TAG​AGA-3′); COLⅠ (F: 5′-AAG​AAG​ACA​TCC​CTG​AAG-3′; R: 5′-AGA​TAC​AGA​TCA​AGC​ATA​CA-3′); ITGA4 (F: 5′-TTG​AAG​ATA​TTG​CTA​TTG​G-3′; R: 5′-GAG​TAT​GTA​GAG​GAG​ATG-3′).

Genomeditech vectors (Shanghai, China) were used to achieve ITGA4 overexpression. Thermo Fisher, China’s Lipofectamine™ 3000 was used to transfect cells with ITGA4 plasmids. The ITGA4 plasmid was transfected for 24 h, followed by a treatment with TGFβ and PAE for an additional 48 h. Additional primer sequences for ITGA4 were GAPDH (F: 5′-TTG​CCC​TCA​ACG​ACC​ACT​TT-3′; R: 5′-TGG​TCC​AGG​GGT​CTT​ACT​CC-3′); ITGA4 (F: 5′-ATC​TCG​TCA​AC-CTT​CTC​A-3′; R: 5′-CAT​CTA​CAT​AGC​CAT​TAT​TAT​CTG-3′).

Data analysis was performed using GraphPad Prism 9.0. Results from three independent, repeated experiments are presented as mean ± standard deviation (SD). Statistical differences among various groups were determined through one-way ANOVA, with a threshold of P < 0.05 for statistical significance.

The effect of PAE on TGFβ-induced activation of HSCs were evaluated by measuring the viability of LX2 and HSC-T6 cells through the CCK8 assay. The data demonstrated that TGFβ at 5 ng/mL significantly increased the activation of HSC (Figures 1A, B). Conversely, PAE effectively decreased cell proliferation in a concentration-dependent manner. Statistically, the IC₂₀, IC₄₀, and IC₅₀ values for PAE were 2.29 ± 0.33 mg/mL, 4.53 ± 0.38 mg/mL, and 6.02 ± 0.34 mg/mL in LX2 cells, and 0.79 ± 0.10 mg/mL, 4.27 ± 0.34 mg/mL, and 8.89 ± 1.26 mg/mL in HSC-T6 cells, with the most significant effects observed at the IC₅₀ concentration for both cell types. The subsequent experiments were thus designed around these concentrations (IC₂₀, IC₄₀, and IC₅₀).

The Transwell assay demonstrated that PAE reduced the migration of activated HSC (Figure 1D). TGFβ increased the migration of LX2 and HSC-T6 cells, whereas PAE markedly reduced the number of migrating cells from these cell lines. In the Edu assay, it was observed that PAE inhibited the proliferation of HSCs stimulated by TGFβ (Figure 1C), indicating that PAE attenuates LF by suppressing both cell migration and proliferation.

RNA sequencing was conducted to ascertain the effects of PAE on activated HSCs (LX2), with three independent biological replicates. Correlation analysis confirmed that replicates clustered distinctly for each sample group (Figure 2A). Post-quality control, differential expression was determined, with significant upregulation and downregulation noted at an FDR < 0.05 and |log2 (FC)| > 0.5. This analysis identified 142 upregulated and 121 downregulated genes (Figures 2B, C).

Enrichment analysis using the Reactome database highlighted that downregulated genes were predominantly involved in the assembly of collagen fibrils (including COL1A2; COL5A3; COL9A2; LOXL4; MMP13), ECM proteoglycans (including BGN; COL1A2; COL5A3; COL9A2; LUM), collagen degradation (including COL1A2; COL5A3; COL9A2; LOXL4; MMP13), and collagen formation (COL1A2; COL5A3; COL9A2; MMP13), among other related processes (Figure 2D). Conversely, upregulated genes primarily participated in cytokine signaling, interleukin-10 signaling, and interferon α/β signaling within the immune system (Figure 2F).

The transcriptomic results revealed that genes associated with collagen fibers and inflammatory pathways were substantially downregulated under PAE’s influence, aligning with our observation that PAE mitigates hepatic fibrosis. Notably, ITGA4 was among the downregulated genes, corroborating previous findings from our group regarding its role in PAE-mediated inhibition of carbon tetrachloride-induced LF in rats. The precise mechanism by which ITGA4 contributes to PAE’s inhibition of LF warrants further investigation.

To further explore the anti-fibrotic effect of PAE on activated HSCs, both WB and QPCR analyses were employed. These analyses revealed elevated levels of α-SMA and COL1 proteins and mRNAs in TGFβ-stimulated LX2 and HSC-T6 cells compared to controls, signifying the activation by TGFβ (Figures 3A–D). Treatment with PAE led to a reduction in α-SMA and COL1 at both the transcriptional and translational levels (Figures 3A–D), with the most notable decrease observed at the IC₅₀ concentration of PAE. Additionally, PAE also lowered the expression of inflammatory markers TNFα and IL-6 in these cells (Figures 3A, B). Furthermore, while ITGA4 was highly expressed in these cells, PAE significantly decreased its expression (Figures 3A–D).

Further WB experiments assessed changes in EMT indicators. Expression of NCA, Snail, and Vim increased under TGFβ treatment compared to controls, while PAE inhibited their expression (Figures 3E, F). These results suggest PAE’s anti-fibrotic action through the inhibition of collagen, inflammatory factors, and EMT expression. Importantly, PAE was found to reduce the high expression of ITGA4 in TGFβ-induced HSCs.

Using an overexpressed ITGA4 plasmid, the effects on LF and its interaction with PAE’s inhibitory action were explored. Further investigation into the role of ITGA4 in LF was conducted through QPCR and WB. These assays confirmed an increase in ITGA4 expression at both mRNA and protein levels in LX2 cells following transfection with the ITGA4 plasmid, in contrast to cells transfected with an empty vector or the untreated group (Figures 3G, H).

The impact of ITGA4 on LX2 cell proliferation was evaluated using CCK8 and Edu assays. CCK8 results indicated that ITGA4 increased cell viability and promoted proliferation compared with the empty plasmid control (NC) group (Figure 4A). Furthermore, ITGA4 enhanced LX2 cell viability more than TGFβ treatment alone, suggesting its significant role in enhancing cell proliferation (Figure 4A). This was also supported by the Edu assay, where ITGA4 significantly increased the proliferation of activated LX2 cells, counteracting PAE’s inhibitory effect (Figure 4B). Additionally, both the Transwell migration and scratch assays showed that ITGA4 enhanced the migration of activated LX2 cells and reversed PAE’s inhibitory effects on their migration (Figures 4C, D). Compared with the TGFβ group, ITGA4 significantly promoted LX2 cell migration, indicating that the ability of ITGA4 to promote migration not only reversed the inhibitory effect of PAE but also exceeded the ability of TGFβ to promote cell activation. This highlights ITGA4’s substantial role in promoting the proliferation and migratory abilities of LX2 cells.

The study also included an assessment of α-SMA, COL1, and EMT markers via the WB assay to elucidate the regulatory role of ITGA4 in the pathogenesis of LF. The findings revealed that in TGFβ-treated HSCs, overexpression of ITGA4 reversed PAE’s inhibition of fibrosis. Specifically, in PAE-treated cells, ITGA4 promoted the expression of α-SMA, COL1, ECA, and Snail, while it inhibited the expression of NCA and Vim (Figures 5A, B). Although both PAE and ITGA4 suppressed TGF-β1 expression, the difference between their effects was not statistically significant (Figure 5B). The promotional effect of ITGA4 on these markers counteracted the inhibitory impact of PAE, underscoring ITGA4’s role in exacerbating hepatic fibrosis. These results further support that ITGA4 counteracts the inhibitory effect of PAE on hepatic fibrosis, highlighting ITGA4’s role in promoting this condition.

Transcriptomic analyses indicated that PAE’s inhibition of hepatic fibrosis involved the ECM pathway (Figure 2E). To validate this, the PI3K-AKT pathway was examined. Results showed that PAE suppressed the expression and phosphorylation of FAK, PI3K, and AKT in TGFβ-activated LX2 cells, whereas ITGA4 overexpression enhanced these expressions and phosphorylations. However, the expression levels of FAK, PI3K, and AKT were comparable across groups (Figure 5C). These findings suggest that PAE’s inhibition of LF in LX2 cells occurs through the ITGA4/FAK/PI3K/AKT pathway, emphasizing the critical role of ITGA4 inhibition in treating LF.