Introduction

Biliary atresia (BA), which appears in the neonatal period, is a severe hepatobiliary disease that affects both the intrahepatic and extrahepatic biliary systems. If untreated, patients develop rapidly progressive liver fibrosis, leading to portal hypertension and end-stage liver disease, invariably resulting in death within the first two years of life.1 Although Kasai portoenterostomy is the preferred treatment, many children still need liver transplantation for long-term survival.2 The etiopathogenesis of BA has been the subject of extensive research for years but yet to be fully clarified, making it difficult to improve treatment strategy.1 Thus, exploring new mechanisms and finding effective therapeutic targets is urgently needed for BA.

Epidermal growth factor (EGF) is a member of the growth factor family. The activation of the EGF/EGF receptor (EGFR) axis has been thoroughly studied in multiple biological processes, including cell growth, proliferation, and differentiation. In addition, EGF participates in many liver pathological processes, such as inflammation, fibrosis, and tumor metastasis.3,4,5,6 However, studies on EGF in BA patients are scarce, and most of them only measured serum levels, with no further investigation into the mechanisms.7,8,9

During liver fibrosis and cirrhosis, the upregulated EGFR was found in biliary epithelial cells, hepatocytes, and hepatic stellate cells (HSCs).4,6 EGF, as one of its main ligands, may have changed some processes of these cells in liver fibrosis of BA. Epithelial-mesenchymal transition (EMT), a cellular process crucial to embryonic development and tissue reconstruction, is also a promoting factor of liver fibrosis.10 Through EMT, the biliary epithelial cells lost their epithelial features and transformed into myofibroblasts, participating in fibrosis.11 In addition, the EMT process can be regulated by EGF in fibrotic diseases.12,13 As a result, we speculated that EGF could influence the progression of liver fibrosis in BA patients by promoting EMT of biliary epithelial cells.

This study detected the EGF levels in serum and liver samples of BA children and analyzed their relationship with liver fibrosis, bile duct proliferation, and the EMT process. Then, the mechanisms behind these associations were explored in the EGF-treated human intrahepatic biliary epithelial cell line (HIBEpic). Furthermore, we examined EGF’s effects on the hepatocyte line (L-02) and the hepatic stellate cell line (LX-2) in light of its extensive biological and pathological effects. Furthermore, we used bile duct ligation (BDL) mice to investigate the effects of blocking the EGF signaling pathway on liver fibrosis and explore its therapeutic value for BA children.

Material and Methods

Human serum and liver specimens

From January 2019 to January 2020, liver samples of 23 children with BA and 9 with choledochal cysts (CC) were obtained during surgery. Moreover, serum samples of 23 children with BA, 5 with cholestasis, and 20 age-matched healthy infants as healthy control (HC) were collected before surgery. Serum samples and some liver samples were stored in a refrigerator at −80 °C, and the remaining liver samples were soaked in paraformaldehyde. Guardians of all enrolled children provided written informed consent. Sample collection and use were approved by the Ethics Committee at the Tianjin Children’s Hospital, Tianjin, China (Ethics Approval Number: L202011).14

Cell lines

HIBEpic, L-02, and LX-2 cells were purchased from BeNa Culture Collection (BNCC, Beijing, China). Cells were cultured in RPMI 1640 medium or DMEM with high glucose, containing 100 μg/mL streptomycin, 100 U/mL penicillin, and 10% fetal bovine serum. The incubator was maintained under saturated humidity, in 5% CO2 at 37 °C. Cells were routinely passaged every 3 to 5 days, and 0.25% trypsin was used for cell digestion. All experiments were performed in the exponential growth phase of cells. The recombinant human EGF (Bioss, Beijing, China) was added to the medium of HIBEpic, L-02 and LX-2 cells at the concentrations of 0, 25, 50 and 100 ng/mL to detect its effects on these three cell lines. KO-947 (MedChemExpress, New Jersey) was used to specifically inhibit ERK1/2 signaling pathway in HIBEpic and L-02 cell lines.

Animals and models

Eight-week-old male C57BL/6 mice were obtained from Vital River Laboratories (Beijing, China). All experimental protocols were approved by Tianjin Medical University’s Committee on the Ethics of Animal Experiments. The mice had free access to standard food and water and were housed at 25 °C and 12/12 h light/dark cycle. They were randomly assigned to 4 groups. Five mice were sham-operated as a control group. Five mice were administrated with normal saline by tail-vein injection every two days after bile duct ligation (BDL) as the BDL group. Five mice were administered with mouse immunoglobin (IgG, 4 μg/20 g, Sigma Aldrich, St. Louis, MO) by tail-vein injection as BDL + IgG group. In addition, five mice were administered with EGF antibodies (4 μg/20 g, Sigma Aldrich, St. Louis, MO) by tail-vein injection every other day after BDL until day 14 as the BDL + EGF Ab group. Mice were anesthetized and placed on a heating pad for BDL procedure. Briefly, a midline incision in the upper abdomen was made. Then, the common bile duct was identified, isolated, and ligated with silk. Isolation, but not ligation of the common bile duct was performed on the sham-operated mice. Liver and serum samples were collected subsequently.

Enzyme-linked immunosorbent assay (ELISA)

Serum levels of EGF were detected using EGF ELISA Kit (RENJIEBIO, Shanghai, China) according to the manufacturer’s protocols. Briefly, the standards or serum samples (50 μL/well) were added to the microplates, followed by the addition of peroxidase-conjugated anti-EGF polyclonal antibodies (100 μL/well), and then incubated for 1 h at 37 °C. After washing three times with 350 μL wash buffer, the peroxidase-specific substrates (substrate A 50 μL/well, substrate B 50 μL/well) were added and incubated for 15 min at 37 °C. Finally, a stop solution (50 μL/well) was added to the microplates to stop the peroxidase reaction and achieve a color intensity proportional to the concentration of EGF at 450 nm.

Immunohistochemistry (IHC)

Paraformaldehyde-fixed liver tissues were embedded in paraffin and cut into 4 μm sections. Serial sections were dewaxed, rehydrated, and pretreated with a heat-induced antigen retrieval technique. Next, the prepared sections were blocked with bovine serum and incubated with primary antibodies overnight at 4 °C. The primary antibodies included: Cytokine 19 (CK19, Abcam, Cambridge, UK), N-cadherin (Cell Signaling Technology, CST, Boston, MA), phosphorylated extracellular regulated protein kinases 1/2 (p-ERK1/2, CST), EGF (Abcam). Next, sections were incubated with horseradish peroxidase (HRP) labeled goat anti-mouse/rabbit IgG (Zhongshan Golden Bridge Bio-technology, ZSGB, Beijing, China) for 30 min at room temperature. Then, sections were visualized using a diaminobenzidine (DAB) kit (ZSGB), followed by counterstaining with hematoxylin. Finally, the quantitative analysis of the positive areas was performed using Image-Pro Plus 6.0.

Immunofluorescence (IF)

Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Paraffin-embedded liver tissues were sectioned, deparaffinized, rehydrated, and heated with boiling citrate buffer. After being blocked with normal serum for 60 min at room temperature, the cells or liver tissue sections were labeled with N-cadherin, E-cadherin (CST), phospho-EGF receptor (p-EGFR, Bioss), CK19, and α-SMA (Bioss) antibodies overnight at 4 °C and incubated with a suitable fluorophore-conjugated secondary antibody (Bioss) for 30 min at room temperature. The nuclei were counterstained with DAPI (ZSGB). Furthermore, the cells/liver sections were observed by fluorescence microscopy.

Masson’s trichrome and Sirius red staining

Paraffin-embedded liver tissues were cut into 4 μm sections. The sections were deparaffinized and stained with Masson’s trichrome (SenBeiJia Biological Technology, Sbjbio, Nanjing, China) and Sirius red (Sbjbio) to visualize extracellular matrix deposition. The relative collagen content was analyzed by the collagen area based on the whole area of the liver sections using Image-Pro Plus 6.0.

Liver fibrosis grading

METAVIR staging was used to grade liver fibrosis of BA.14 F0: No fibrosis; F1: Enlargement of portal tract but without septa formation; F2: Enlargement of the portal tract with rare septa formation; F3: Numerous septa without cirrhosis; F4: Cirrhosis.

Real-time polymerase chain reaction (RT-PCR)

The total RNA of liver samples from patients was prepared using a Trizol reagent (Invitrogen, Carlsbad, CA). The total RNA of cells was prepared using an RNA extraction kit (Omega Bio-Tek, Doraville, GA). The Reverse Transcription kit (Sangon Biotech, Shanghai, China) transcribed RNA into cDNA. RT-PCR was performed using SYBR Green fluorescence PCR kit (Sangon Biotech). The program was used for cDNA amplification: 1 cycle at 95 °C for 5 min followed by 40 cycles of heating at 95 °C for 10 s, annealing at 57 °C for 10 s, and extension at 72 °C for 10 s. The fold changes in mRNA of target genes were calculated using the 2-△Ct method. GAPDH was used as the internal control. The primer sequences for tested proteins are listed in Table 1.

Table 1 Nucleotide sequences of primers used for PCR.
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Western blotting (WB)

Protein extracts from total cells were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were then blocked with 5% nonfat dry milk and incubated overnight at 4 °C with primary antibodies. The primary antibodies were as follows: E-cadherin, N-cadherin, ERK1/2 (CST), p-ERK1/2, signal transducer and activator of transcription 3 (STAT3, CST), phospho-STAT3 (p-STAT3, CST), phospho-nuclear factor kappa-B p65 (p-NF-κB p65, CST), α-SMA, and β-actin (Abbkine, Wuhan, China). After being washed, the membranes were incubated with secondary antibodies (ABclonal, Wuhan, China) for 1 h at room temperature. Protein bands were visualized using chemiluminescence reagent (Advansta, Menlo Park, CA).

Cell proliferation assay

HIBEpic cells were plated in 96-well plates with 1.5 × 104 cells per well and were treated with human recombinant EGF (Bioss) or PBS. Cell proliferation was detected using a Cell Counting Kit-8 (CCK-8, Dojindo Molecular Technology, Kumamoto, Japan). After being treated for 24 h, 10 μL CCK8 solution was added to each well and incubated for 2 h at 37 °C. The absorbance was measured at 450 nm, and the result was revealed as optical density (OD).

Statistical analysis

SPSS 26.0 software was used for the statistical analysis. Data were presented as mean ± standard deviation. Comparisons for data meeting homogeneous-variance assumptions were determined using a two-tailed Student’s t-test or one-way ANOVA, followed by the Newman-Keuls test when the results of one-way ANOVA indicated significance. P < 0.05 was considered a statistically significant difference.

Results

EGF was highly expressed and correlated with the degree of liver fibrosis in BA

Basic information of all included subjects is displayed in Table 2, and there was no significant difference among the groups. Serum parameters of liver function and injury in children with BA, cholestasis and HC are illustrated in Table 3. BA children had higher levels of ALT, AST, GGT, ALP, TBIL, DB, and TBA than HC. In addition, the serum GGT levels were elevated significantly compared with CC patients.

Table 2 Baseline characteristics of all subjects.
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Table 3 Serum parameters of liver function and injury in children with BA, cholestasis and healthy controls.
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Serum levels of EGF were significantly higher in BA children than in HC and cholestasis. However, it did not reach statistical significance (P = 0.2199) (Fig. 1a). The mRNA expression of EGF was significantly increased in BA liver compared with CC (Fig. 1b). And the mRNA expression of EGF increased with the degree of liver fibrosis (Fig. 1c). Considering the high affinity between EGF and EGFR, we evaluated the p-EGFR levels in liver sections. IF staining demonstrated that the expression of p-EGFR was increased in biliary epithelial cells and hepatocytes in the BA liver (Fig. 1d). These results suggested that EGF was related to liver fibrosis. In addition, EGFR was activated in biliary epithelial cells and hepatocytes in BA.

Fig. 1: Serum levels and liver mRNA expression of EGF were higher in BA children, and the latter was related to liver fibrosis, and p-EGFR levels were elevated in the BA liver.
figure 1

Serum samples of HC (n = 20) and children with BA (n = 20) and cholestasis (n = 5), liver samples of children with BA (n = 23) and CC (n = 23) were collected. a Compared with HC, the serum levels of EGF were significantly elevated in BA. b The EGF mRNA expression in BA liver was significantly increased compared with CC. c EGF mRNA expression increased with the degree of liver fibrosis. *P < 0.05; ***P < 0.001. d IF demonstrated that the p-EGFR was positive in both biliary epithelial cells (long arrow) and hepatocytes (short arrow), and co-localization of CK19 and p-EGFR was present in biliary epithelial cells of BA. (Magnification: ×400).

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EMT and proliferation of biliary epithelial cells were present in BA liver

Since EGF has been shown to promote EMT, we looked for EMT marker proteins in liver sections. Through IHC and IF staining with N-cadherin and CK19 in liver samples, we found that the expression of mesenchymal cell marker N-cadherin was increased in proliferated biliary epithelial cells of BA liver; However, this phenomenon was not found in CC liver (Fig. 2a, b). IF staining of E-cadherin and CK19 depicted that the expression of E-cadherin in cholangiocytes was decreased in BA liver compared with CC liver (Fig. 2c). These results indicated that EMT might occur in biliary epithelial cells of BA liver. In addition, IHC staining with CK19 also showed a significant increase of biliary epithelial cells in BA liver compared with CC liver (Fig. 2d). Our quantitative analysis found that the number of bile ducts increased with the degree of liver fibrosis (Fig. 2e).

Fig. 2: EMT and proliferation of biliary epithelial cells present in BA.
figure 2

a IHC staining of serial liver sections revealed that N-cadherin presented in biliary epithelial cells (black arrow) of the BA liver. (Magnification: left panel: ×200; right panel: ×400) b IF revealed that the co-localization of CK19 and N-cadherin was presented in the biliary epithelial cells (white arrow) of the BA liver. (Magnification: ×400). c IF depicted that the expression of E-cadherin was reduced in the BA liver’s biliary epithelial cells (white arrow) compared with the CC liver. (Magnification: ×400). d IHC staining with CK19 of liver sections displayed that biliary epithelial cells increased in the BA liver. (Magnification ×100). e Quantitative analysis of CK19 in liver tissues. CK19 AOD (average optical density) value increased with the degree of liver fibrosis. ns: P > 0.05; ***P < 0.001; ****P < 0.0001.

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EGF-induced EMT and proliferation of HIBEpic cells

To evaluate the relevance between EGF and the above changes in biliary epithelial cells, we examined the effects of EGF intervention on EMT and proliferation of biliary epithelial cells in vitro. After treating HIBEpic cells with EGF at concentrations ranging from 0 to 100 ng/mL for 24 h, N-cadherin protein and mRNA levels increased in a concentration-dependent manner, while E-cadherin decreased (Fig. 3a–c). IF staining demonstrated the same trend that the expression of epithelial cell marker CK19 was down-regulated, and the expression of the mesenchymal cell marker N-cadherin was upregulated after EGF treatment (Fig. 3d). CCK-8 assay demonstrated that EGF also induced HIBEpic cells proliferation in a concentration-dependent manner (Fig. 3e). These results indicated that EGF could induce EMT and proliferation of HIBEpic cells in vitro.

Fig. 3: EGF induced EMT and proliferation of HIBEpic cells.
figure 3

a After treatment with EGF for 24 h, protein expression of N-cadherin increased in a concentration-dependent manner, and E-cadherin decreased. b, c EGF treatment decreased the mRNA expression of E-cadherin (CDH1) and increased that of N-cadherin (CDH2) in a concentration-dependent manner. ns: P > 0.05; ***P < 0.001; ****P < 0.0001. d IF staining displayed that the fluorescence intensity of CK19 decreased, while that of N-cadherin increased after EGF treatment. (Scale bar: 200 μm). e CCK-8 assay demonstrated that EGF promoted the proliferation of HIBEpic cells in a concentration-dependent manner. ns: P > 0.05; ***P < 0.001; ****P < 0.0001.

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EGF promoted EMT process by activating ERK1/2 pathway in HIBEpic cells

To identify the downstream of EGF, we evaluated several closely related signaling pathways such as ERK1/2, NF-κB and STAT3 in HIBEpic cells. After intervention with EGF, the expression of p-ERK1/2 and p-NF-κB p65 was increased (Fig. 4a). Then, we verified this result in liver samples through IHC by showing the elevated levels of p-ERK1/2 and p-NF-κB p65 in BA liver (Fig. 4g). According to previous reports, both ERK and NF-κB could regulate EMT associated transcription factors. However, ERK was the key downstream mediator that EGFR activates in response to growth factors.15 So, we chose ERK for the following study. We used KO-947, a specific ERK1/2 inhibitor, to verify the effects of ERK1/2 on EGF-induced EMT. Results demonstrated that the addition of KO-947 inhibited the EGF-induced elevation of p-ERK1/2 (Fig. 4b). The increased expression of N-cadherin and decreased expression of E-cadherin induced by EGF was also inhibited with the KO-947 treatment (Fig. 4c–e). Likewise, the IF staining displayed that CK19 was down-regulated and N-cadherin was up-regulated by EGF treatment, and the opposite was observed after KO-947 was added (Fig. 4f). These findings, together with the findings in BA liver, suggested that EGF may promote EMT of biliary epithelial cells in BA through EGF/EGFR-ERK1/2 pathway.

Fig. 4: EGF promoted the EMT process by activating the ERK1/2 pathway in HIBEpic cells.
figure 4

a Protein levels of p-ERK1/2 and p-NF-κB p65 increased 45 min after treatment with EGF. t-: total; p-: phosphorylated. b Protein levels of p-ERK1/2 increased by EGF treatment alone, and decreased after adding KO-947. c Protein levels of N-cadherin increased after EGF treatment, and decreased with the addition of KO-947. Opposite changes of E-cadherin were observed. d, e mRNA expression of E-cadherin (CDH1) decreased after EGF treatment and increased after KO-947 was added. Changes in N-cadherin (CDH2) were the opposite. *P < 0.05; ****P < 0.0001. f IF staining depicted that KO-947 inhibited the decreased fluorescence intensity of CK19 and increased fluorescence intensity of N-cadherin induced by EGF. (Scale bar: 100 μm) g IHC staining displayed that the expression of p-ERK1/2 and p-NF-κB p65 were significantly higher in biliary epithelial cells (black arrow), and slightly increased in hepatocytes of BA liver, compared with CC. (Magnification: left panel: ×200; right panel: ×400).

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EGF promotes the expression of IL-8 in L-02 cells by activating the ERK1/2 pathway

Other than biliary epithelial cells, the increased p-EGFR and p-ERK1/2 were also detected in hepatocytes of BA liver (Figs. 1d, and 4g). And according to previous reports, p-EGFR also expressed in activated HSCs.4 We intended to examine its effects on these cell lines in vitro. Given the higher expression of IL-8 in BA and its correlation with EGF, we further explored the relationship between IL-8 and EGF in these three cell lines.16,17,18 After intervention with EGF, the expression of IL-8 increased only in L-02 but not in LX-2 and HIBEpic cells (Fig. 5a). Moreover, EGF promoted IL-8 expression in a concentration-dependent manner (Fig. 5b). We used KO-947 to treat L-02 cells to see if EGF induced IL-8 production via the ERK1/2 pathway. Results indicated that KO-947 inhibited the overexpression of IL-8 induced by EGF in L-02 cells (Fig. 5c). We concluded that EGF promoted IL-8 expression in L-02 cells via the ERK1/2 pathway.

Fig. 5: EGF promoted IL-8 expression in L-02 cells by activating the ERK1/2 pathway, and EGF activated LX-2 cells.
figure 5

a Expression of IL-8 mRNA was elevated in L-02 cells after EGF intervention. b mRNA expression of IL-8 increased with the increase of EGF concentration. c KO-947 inhibited the overexpression of IL-8 induced by EGF. ns: P > 0.05; ***P < 0.001; ****P < 0.0001. After treating LX-2 cells with EGF for 24 h. d–g qRT-PCR showed mRNA expression of ACTA2, COLI1A1, COLI1A2 and S100A4 was upregulated. **P < 0.01; ***P < 0.001. h EGF promoted α-SMA expression in LX-2 cells in a concentration-dependent manner. i IF staining displayed that EGF promoted α-SMA expression in LX-2 cells. (Scale bar: 100 μm).

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EGF activated LX-2 cells

HSC activation is the linchpin of liver fibrosis and is regulated by many factors. We wondered if EGF is involved in the activation of HSCs. Therefore, we investigated the expression of α-SMA, collagen I, and S100A4 in LX-2 cells after intervention with EGF for 24 h. The results of qRT-PCR revealed increased expression of ACAT2, COLI1A1, COLI1A2, and S100A4 (Fig. 5d–g). In addition, WB and IHC depicted increased levels of α-SMA (Fig. 5h, i). These data indicated that EGF activated HSCs in vitro.

EGF antibody injection alleviated Liver fibrosis in BDL mice

Considering the effects of EGF on biliary epithelial cells, hepatocytes, and HSCs, we wondered if EGF neutralization antibodies could attenuate liver fibrosis in vivo. Since the pathological manifestations of BA are closely related to cholestasis caused by extrahepatic biliary occlusion, we used BDL mice as a disease model for our in vivo experiments.

Masson’s trichrome and Sirius red staining presented marked liver fibrosis in the portal area 2w after BDL surgery (Fig. 6a). Furthermore, the IHC staining revealed higher levels of EGF and its downstream molecular p-ERK1/2 in the liver of BDL group mice than in the sham-operated group (Fig. 6a). After EGF antibody injection, levels of EGF and p-ERK1/2 reduced (Fig. 6a). Masson’s trichrome and Sirius red staining also depicted a decreased collagen deposition in the portal area in the liver of BDL + EGF Ab group mice (Fig. 6a). Serum levels of liver enzymes demonstrated the same trends (Fig. 6b–d). The quantitative analysis of Sirius Red staining confirmed the treatment effect of the EGF neutralization antibody (Fig. 6e). Furthermore, we performed the immunofluorescence staining of N-cadherin and E-cadherin to detect the EMT process in intrahepatic cholangiocytes. Results revealed that the expression of N-cadherin increased and E-cadherin decreased in BDL mice. The EGF antibody injection reversed this phenomenon (Fig. 6f). These results suggested that the EGF neutralization antibody could alleviate liver fibrosis in BDL mice by decreasing the phosphorylation of ERK1/2 and mitigating the EMT process.

Fig. 6: Liver fibrosis alleviated, and the levels of EGF and p-ERK1/2 and the EMT process were decreased after EGF antibody injection in BDL mice.
figure 6

a EGF and p-ERK1/2 levels were increased, and marked liver fibrosis and collagen deposition were presented in the liver of BDL and BDL + IgG group mice (n = 5), compared with control group mice (n = 5). Furthermore, liver fibrosis and collagen deposition improved in BDL + Ab group mice, with decreased EGF and p-ERK1/2 levels compared with the BDL and BDL + IgG group. (Magnification: Sirius red and Masson trichrome staining: 40X; EGF and p-ERK1/2 IHC: ×100). b–d Serum levels of ALT, AST, and ALP were elevated in BDL mice and reduced after EGF antibody treatment. e Positive area of Sirius Red staining revealed the same trend. f IF revealed that the expression of E-cadherin in biliary epithelial cells decreased after bile duct ligation and increased after EGF neutralization antibody treatment, while N-cadherin depicted opposite results. (Magnification: ×200).

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Discussion

The present study identified the role of EGF in the liver fibrosis of BA patients. The main findings were as follows: (i) EGF was elevated in BA and correlated with liver fibrosis; (ii) EGF promoted EMT and proliferation of biliary epithelial cells of BA liver through EGF/EGFR-ERK1/2 signaling pathway; (iii) EGF promoted IL-8 expression in hepatocytes through the ERK1/2 pathway, and activated HSCs in vitro; (iv) EGF neutralization antibodies alleviated liver fibrosis in BDL mice.

First and foremost, we found elevated EGF and p-EGFR expression in BA, and EGF mRNA expression was related to the degree of liver fibrosis. The transcription of EGF was low in normal liver, but was elevated in bile duct epithelia in human cirrhotic and BDL mice liver.6 Furthermore, EGF was primarily synthesized by salivary and Brunner’s glands, and stored in granules and secreted into the gut, and to a lesser extent into the blood stream.19 The periportal hepatocytes took up more than 90% of EGF from vein in the first pass, and transport them into bile.20 So the increased EGF in BA liver may be produced by biliary epithelial cells themselves, or deposited in bile ducts, hepatocytes, even hepatic sinuses due to poor bile drainage. Likewise, Madadi-Sanjani et al.7 also demonstrated that multiple growth factors, including EGF, increased during Kasai operation. However, no correlation was discovered in the prognostic analysis. Vejchapipat et al.8 found that the post-Kasai serum levels of EGF were higher in children with good outcomes. These results revealed the complexity of its effects on liver fibrosis. EGF participates in the fibrotic and cirrhotic stages of BA. However, the Kasai operation may have significantly changed the microenvironment of liver cell populations. EGF may act as an initiator of liver regeneration/repair.8 However, evidence is still needed to confirm the dynamic changes in EGF expression and function.

Next, we evaluated EMT marker proteins in liver sections. EMT refers to polarized immotile epithelial cells losing adhesion-association junction proteins, such as E-cadherin, and gaining mesenchymal markers, such as N-cadherin.21 Studies have revealed that EGF promoted EMT in various fibrotic and tumor-related diseases such as primary sclerosing cholangitis, idiopathic pulmonary fibrosis, tubulointerstitial fibrosis, colorectal tumors, pancreatic tumors, hepatocellular carcinoma, and cholangiocarcinoma.12,13,22,23,24,25,26 The co-localization of CK19 and N-cadherin and the reduction of E-cadherin in CK19-positive cells in this study indicated that EMT might exist in the biliary epithelial cells of BA, which is consistent with previous studies.27,28,29,30 Furthermore, the proliferation of biliary epithelial cells was also observed in BA liver. Thus, the proliferating biliary epithelial cells and their EMT process may subsequently increase the number of myofibroblasts in the BA liver, resulting in accelerated fibrosis progression. Biliary epithelial cells are not only “victims” but also significant “participants” in the process of liver fibrosis in BA. However, whether and how EGF promotes EMT and proliferation of biliary epithelial cells in BA remains unknown.

Then, we focused on the effect of EGF on the EMT process in BA. We found that EGF treatment promoted EMT process and proliferation of HIBEpic cells via ERK1/2 pathway. Similar to our study, Clapéron et al.25 revealed that cholangiocarcinoma cell lines were dispersed and displayed a fibroblastic-like phenotype after EGF treatment, and these changes were abrogated by inhibiting STAT3 and mitogen-activated protein kinase/ERK kinase 1/2 (MEK1/2). Surahiet al.26 demonstrated that the EGF-enriched extracellular vesicles derived from senescent cholangiocytes of PSC patients could promote the proliferation of normal human cholangiocytes via the EGER-MEK1/2-ERK1/2 pathway. In addition, EGF could induce EMT in pancreatic ductal adenocarcinoma and lung cancer cells via the EGFR-ERK pathway.31,32 Furthermore, the higher levels of p-EGFR and p-ERK1/2 in biliary epithelial cells of BA confirmed the effects of the EGF/EGFR-ERK1/2 pathway on EMT and proliferation of biliary epithelial cells in BA.

Nevertheless, the concept of EMT has recently became contentious. Lineage tracing research, for example, disproved EMT in biliary epithelial cells of liver fibrosis model mice.33,34,35 However, a case report of post-liver transplantation primary biliary cirrhosis (PBC) patients described that the EMT of biliary epithelial cells appeared before any other pathological manifestations of recurrent PBC, indicating that EMT of biliary epithelial cells may be an initial event.36 Moreover, the lineage tracing technique has its limitation, so the EMT in liver fibrosis still needs further exploration.

Other than biliary epithelial cells, EGFR ligands, like amphiregulin, have been reported to change the process of liver fibrosis in different mice models by regulating the apoptosis of hepatocytes or HSCs.37,38 Here we evaluated the effects of EGF on hepatocytes and HSCs. IL-8 mediates innate immune activation and regulates neutrophil recruitment and degranulation. Numerous studies have depicted that IL-8 expression was increased in BA children and was related to their outcomes.16,39,40,41 Bessho et al.42 identified 15 genes uniquely expressed in BA liver, including IL-8, and disruption of IL-8 homologs signaling in BA mice model resulted in improved living conditions. So, in addition to EMT, we also detected the IL-8 expression upon EGF treatment. We found that EGF induced IL-8 overexpression in L-02 cells and p-ERK1/2 also participated in this process, which was consistent with a previous study in lung cancer cells.17 These suggest that EGF may induce IL-8 overexpression in hepatocytes by phosphorylating ERK1/2, which promotes the chemotaxis of inflammatory cells in BA liver, aggravating its severity in another way.

Furthermore, we proved that EGF did activate LX-2 cells in vitro. In addition, it has been reported that levels of p-EGFR and p-ERK were increased in activated HSCs, and inhibition of EGFR phosphorylation reduced the number of activated HSCs and p-ERK levels.4,43 According to the above results, EGF/EGFR-ERK1/2 is also vital for the activation of HSCs.

Finally, in our BDL mice, EGF antibody injection decreased EGF and p-ERK levels, and reduced collagen deposition in liver tissue. Furthermore, the liver function indicators and EMT process were also improved after EGF antibody treatment. Consistent with our results, Xu HF et al.4 discovered that EGF neutralization antibodies attenuated liver fibrosis with decreased p-EGFR in myofibroblasts of BDL mice, but the downstream molecules have not been explored. Fuchs et al.43 used erlotinib, an EGFR inhibitor, to treat liver fibrosis mice model, and found out that erlotinib attenuated liver fibrosis with decreased p-EGFR and p-ERK, but this study could not exclude the effects of other EGFR ligands. These results conclude that the EGF/EGFR-ERK1/2 pathway may be an anti-fibrotic target in BA.

However, some limitations should be noted. First, we used livers of CC children as control group due to ethical constraints. Although mild, there is still some cholestatic liver injury, hence it is not a perfect control group for BA liver. Seconed, BDL can simulate partial intrahepatic bile duct reaction in children with BA, but it is not a standard biliary atresia model as the disease process is much more than just extra hepatic bile duct obstruction.

In summary, our data revealed the significant and distinctive overexpression of EGF in BA liver. EGF may promote EMT and proliferation of biliary epithelial cells, overexpression of IL-8 in hepatocytes, and activation of hepatic stellate cells through EGF/EGFR-ERK1/2 pathway, leading to aggravation of liver fibrosis in BA. EGF/EGFR-ERK1/2 inhibition may be a potential therapeutic target for BA.