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. 2023 Nov 28;16(1):82–93. doi: 10.1016/j.chmed.2023.07.003

Chuanxiong Rhizoma extracts prevent liver fibrosis via targeting CTCF-c-MYC-H19 pathway

Yajing Li a, Fanghong Li b, Mingning Ding a, Zhi Ma a, Shuo Li b, Jiaorong Qu a, Xiaojiaoyang Li a,⁎
PMCID: PMC10874761  PMID: 38375042

Abstract

Objective

Hepatic fibrosis has been widely considered as a conjoint consequence of almost all chronic liver diseases. Chuanxiong Rhizoma (Chuanxiong in Chinese, CX) is a traditional Chinese herbal product to prevent cerebrovascular, gynecologic and hepatic diseases. Our previous study found that CX extracts significantly reduced collagen contraction force of hepatic stellate cells (HSCs). Here, this study aimed to compare the protection of different CX extracts on bile duct ligation (BDL)-induced liver fibrosis and investigate plausible underlying mechanisms.

Methods

The active compounds of CX extracts were identified by high performance liquid chromatography (HPLC). Network pharmacology was used to determine potential targets of CX against hepatic fibrosis. Bile duct hyperplasia and liver fibrosis were evaluated by serologic testing and histopathological evaluation. The expression of targets of interest was determined by quantitative real-time PCR (qPCR) and Western blot.

Results

Different CX extracts were identified by tetramethylpyrazine, ferulic acid and senkyunolide A. Based on the network pharmacological analysis, 42 overlap targets were obtained via merging the candidates targets of CX and liver fibrosis. Different aqueous, alkaloid and phthalide extracts of CX (CXAE, CXAL and CXPHL) significantly inhibited diffuse severe bile duct hyperplasia and thus suppressed hepatic fibrosis by decreasing CCCTC binding factor (CTCF)-c-MYC-long non-coding RNA H19 (H19) pathway in the BDL-induced mouse model. Meanwhile, CX extracts, especially CXAL and CXPHL also suppressed CTCF-c-MYC-H19 pathway and inhibited ductular reaction in cholangiocytes stimulated with taurocholate acid (TCA), lithocholic acid (LCA) and transforming growth factor beta (TGF-β), as illustrated by decreased bile duct proliferation markers.

Conclusion

Our data supported that different CX extracts, especially CXAL and CXPHL significantly alleviated hepatic fibrosis and bile duct hyperplasia via inhibiting CTCF-c-MYC-H19 pathway, providing novel insights into the anti-fibrotic mechanism of CX.

Keywords: Chuanxiong Rhizoma, cholangiocytes, ductular reaction, Ligusticum chuanxiong Hort., liver fibrosis, lncRNA H19

1. Introduction

Liver fibrosis is a wound-healing response and is characterized by the deposition of extracellular matrix (ECM), which has been widely considered as a conjoint consequence of almost all chronic liver diseases (Zhang, Liu, Wang, & Sui, 2020). If left untreated, the continuum of fibrotic liver abnormalities will further progress to cirrhosis and even hepatocellular carcinoma (Parola & Pinzani, 2019). Among the pathogenic factors of liver fibrosis, the hepatic retention of bile acids (BAs) and excessive ductular reaction (DR) can lead to multiple strictures and obstructing of both intra-and extra-hepatic bile ducts, which will develop into more aggressive hepatobiliary diseases clinically named as primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) (Aseem et al., 2021). In the bile duct ligation (BDL)-induced experimental model, cholangiocytes are sensitive to overloaded BAs or intracellular pathogenic molecules secreted by diverse hepatic cells and then proliferate significantly more rapidly to participate in the progression of liver fibrosis (Huang et al., 2016). While, ursodeoxycholic acid (UDCA), a capital FDA-approved pharmacotherapy for PBC or liver fibrosis complications, may stimulate several side effects including diarrhea and additional urticaria in the clinical application (Hirschfield et al., 2021). Thus, there is an unmet need to discover novel anti-fibrotic drugs targeting hyperproliferative or injured cholangiocytes.

Increasing studies commit to clarify the underlying mechanisms of DR pathogenesis. H19, an imprinted and maternally expressed gene, is one of a few long noncoding RNAs (lncRNAs) conserved between human and mouse, playing a key role in the regulation of cell proliferation and differentiation (Li & Liu, 2020). We previously demonstrated that H19 was dramatically upregulated in the cholangiocytes of carbon tetrachloride (CCl4)-induced cirrhosis and BDL-induced fibrosis mouse models and was positively associated with disease progression (Li et al., 2017). Indeed, H19 transcription can be regulated by serval transcription factors and organizer of genome spatial. For instance, c-MYC bind to H19 DNA sequence elements called E-boxes close to the imprinting control region (ICR) with its obligate heterodimerization partner Max, thus positively modulating H19 transcription (Barsyte-Lovejoy et al., 2006). CCCTC binding factor (CTCF), as another master organizer of genome spatial organization, regulates the imprinted genes H19 by organizing chromatin at the H19 locus (Freschi et al., 2021). Meanwhile, the transcriptional regulation of c-myc was mediated by a long-distance CTCF dependent enhancer-promoter interaction (Hyle et al., 2019). Notably, c-MYC and CTCF may also be involved in hepatic fibrosis by affecting phosphatidylinositol 3-kinase (PI3K)/AKT pathway (Liu et al., 2021, Salgado-Albarran et al., 2022). But until now, the regulation of c-MYC and CTCF in H19-related liver fibrosis is not well characterized and whether these targets could be prevented by any pharmacological therapies is unknown.

Traditional Chinese medicine (TCM), clinically used for thousands of years and generally considered to be safe, is a promising treatment against multiple chronic digestive diseases. Chuanxiong Rhizoma (Chuanxiong in Chinese, CX), the dried rhizome of Ligusticum chuanxiong Hort., has been demonstrated to prevent cerebrovascular, gynecologic and hepatic diseases via either using alone or in combination with other herbs (Rai et al., 2019, Wang et al., 2022, Wang et al., 2020). In clinical and experimental research, TCM formulas containing CX like Si-Wu-Tang and herb-pair of Angelicae Sinensis Radix and CX were reported to improve hepatitis and liver fibrosis via restoring multicellular function in multiple pathways (Ma et al., 2022, Wu et al., 2021). Furthermore, we previously found that tetramethylpyrazine (TMP), a representative bioactive ingredient derived from CX, ameliorated liver fibrosis via preventing the transfer of mitochondrional DNA from hepatocytes to hepatic stellate cells (HSCs) (Li et al., 2022). So far, hundreds of chemicals have been identified in CX and mainly belong to primary groups, including but not limited to alkaloids, phenolic acids and phthalides and the therapeutic promise of different extracts of CX has been emphasized (Huang et al., 2016). These studies encourage us to explore which extracts of CX play a major therapeutic role in relieving DR and whether the protection of CX against liver fibrosis are associated with the regulating of CTCF-c-MYC-H19 pathway.

In this study, our data supported that different CX extracts, especially for alkaloid and phthalide extracts of CX (CXAL and CXPHL) alleviated hepatic fibrosis and cholangiocyte hyperproliferation via inhibiting CTCF-c-MYC-H19 pathway both in vivo and in vitro, providing novel insights into the anti-fibrotic mechanism of CX.

2. Materials and methods

2.1. Materials

The traditional Chinese herb CX were purchased from Beijing Tongrentang (Group) Co., Ltd (China) and were performed by Dr. Bing Xu from the Beijing University of Chinese Medicine. Ferulic acid (A33754) and TMP (A45751) were purchased from Innochem (Beijing, China). Senkyunolide A (PS2132) was purchased from Push Bio-Technology (Chengdu, China). Human TGF-β1 (CA59) and lithocholic acid (LCA) (434-13-9) were purchased from Novoprotein Scientific Inc. (Shanghai, China). Chlorogenic acid (B20782) and taurocholic acid (TCA) (S30708) were purchased from Yuanye BioTechnology (Shanghai, China).

2.2. Separation and identification of different CX extracts

The four different extracts of CX including CXAE, CXAL, CXPA and CXPHL were prepared as followed: (1) CXAE: CXs were sliced and soaked for 1 h. The mixture was extracted twice with eight volumes of distilled water using the condensation reflux method. The aqueous extract was concentrated with a rotary evaporator, filtered twice, and stored at −80 °C. (2) CXAL: The CXs were crushed and extracted twice with 15 times the amount of 80% ethanol, refluxing for 2.5 h each time. (3) After weighting, CXs were extracted twice with the 15 times amount of 80% (volume percentage) ethanol through a reflux extraction device. (4) The smashed air-dried CX was fully soaked in 95% ethanol for more than 1 h, and extracted with 15-fold volume of 95% ethanol using diacolation method. The condition for high performance liquid chromatography (HPLC) was provided in the Supplementary files.

Methanol was used as the reference substance solution, and the concentration of reference liquid including chlorogenic acid, ferulic acid, tetramethylpyrazine and senkyunolide A were 0.2 mg/mL. The compounds of CX were analyzed by HPLC and performed on a ACQUITY-HPLC-BEH-C18 column (2.1 mm × 150 mm, 1.7 μm). The mobile phase used were (A) a mixture of 0.1% formic acid aqueous solution and (B) acetonitrile. The gradient elution program of CX AEs was as follows: 0–0.5 min, 5% A; 0.5–8 min, 5%–45% A; 8–10 min, 45% A; 10–11 min, 45%–48% A; 11–16 min, 48%–50% A; 16–18.5 min, 50%–60% A; 18.5–20 min, 60%–75% A; 20–22 min, 75%–90% A; 22–23 min, 90%–5% A; 23–25 min, 5% A. The total speed was 0.2 mL/min and 2 μL samples were injected each time. The detection wave was 295 nm. The mobile phase used for CX Als were (A) a mixture of ddH2O solution and (B) acetonitrile. The gradient elution program of CXAE was as follows: 0–10 min, 5%–20% B; 10–22.5 min, 20%–40% B; 22.5–37.5 min, 40% B; 37.5–42.5 min, 40%–70% B; 42.5–60 min, 70%–80% B; 60–70 min, 80%–100% B. The total speed was 0.3 mL/min, and 2 μL samples were injected each time. The detection wave was 280 nm. As for CXPA and CXPHL, the mobile phase was (A) 1% formic acid aqueous solution and (B) acetonitrile. The gradient elution program was as follows 0–0.2 min (5% A), 0.2–7.5 min (5% A–80% A), 7.5–8.0 min (80% A–100% A), 8.5–9.0 min (100% A–5% A), 9.0–10.0 min (5% A). The total speed was 0.4 mL/min, and 2 μL samples were injected each time. The detection wave was 320 nm at 35 °C. The content of Senkyunolide I and Senkyunolide A were detected at 280 nm, and the content of chlorogenic acid, ferulic acid, ferulic acid methyl ester and ligustilide Z were detected at 320 nm.

2.3. Animal studies

Male and female C57BL/6J mice (nine weeks old, 24–25 g) were purchased from SIBEIFU Biotechnology Co., Ltd. (Beijing, China). All mice were kept under 12 h light/ 12 h dark cycle at a consistent temperature (22 ± 2) °C and provided with standard chow with access to sterile water ad libitum. After one week of acclimation, mice were randomly divided into six groups (n = 8): sham group, BDL group, BDL + CXAE group, BDL + CXAL group, BDL + CXPA group, and BDL + CXPHL group. Our preliminary results indicated that 50 mg/kg was the most effective for CXAE extracts against cholestasis. According to the extraction yield (CXAL: 19.82%, CXPA: 33.41% and CXPHL: 36.89%), we prepared an administration dose of 10 mg/kg (CXAL), 17 mg/kg (CXPA) and 18 mg/kg (CXPHL). Mice in sham and BDL groups were subjected to sham operation or BDL surgery for 7 d (Liu et al., 2019), which was a well-accepted thereby to construct liver fibrosis mice. Mice in the other four groups were pre-administrated with CX extracts for 2 d, then subjected to BDL surgery and administrated with CX extracts for another 4 d after 3 days of post-operation. After being sacrificed at corresponding times, mouse serum was collected and liver samples were harvested and stored in a −80 °C freezer. All animal studies and procedures were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine (BUCM-4–20200730023160).

2.4. Network pharmacology-based prediction of effects of CX on liver fibrosis

First, all natural ingredients of CX were obtained from TCM system pharmacology database and literatures (TCMSP, https://tcmspw.com/tcmsp.php). Furthermore, we selected out the potential active compounds by oral bioavailability (OB) and drug-likeness (DL) (threshold values set to be: OB > 30% and DL > 0.18). Only components meeting the threshold value were considered as potential active ingredients. The most probable biological targets were obtained from Swiss Target Prediction (https://swisstargetprediction.ch) and further screened by searching Uniprot (https://www.uniprot.org) for human normalization. The targets related to ‘liver fibrosis’ were obtained from Online Mendelian Inheritance in Man database (OMIM, https://omim.org) and Gene Cards (https://www.genecards.org). Common targets were selected but other targets were removed. In this study, targets obtained from network pharmacology were introduced into String data (https://string db.org) and Cytoscape software. In addition, we further analyzed the relationship between the selected targets by establishing a protein–protein interaction (PPI), performing Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis.

2.5. Biochemical assays

Serum was collected from blood sample after animal sacrificed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-BIL) and gamma-glutamyl transferase (γ-GT) were measured by ALT assay kit (C009-2-1), AST assay kit (C010-2-1), T-BIL assay kit (C019-1-1) and γ-GT assay kit (C017-2-1).

2.6. Histopathology staining

Mice were sacrificed and the livers were immobilized with 4% FPA and embedded in paraffin. Tissues were cut into 4.5-μm sections and stained with hematoxylin and eosin (H&E) and Masson trichrome staining as previously described (Li, Li, Xue, Wang, & Li, 2022). After dewaxing and rehydration, tissue sections were stained with Van Gieson's Stain Kit (RS3930) and imaged with Aperio Versa (Leica, Wetzlar, Germany).

2.7. Cell culture of HIBECs

Human biliary epithelial cells (HIBECs) were obtained from ATCC and cultured with Minimum eagle’s medium (MEM) containing 10% fatal bovine serum (FBS), penicillin G (100 U/mL) and streptomycin (100 μg/mL) in an incubator with a humidified atmosphere of 5% CO2 at 37 °C for further experiments.

2.8. Quantitative real-time PCR (qRT-PCR)

Total RNA in HIBECs and liver tissues in mice was extracted with Fast Pure Cell/ Tissue Total RNA isolation kit (RC101-01, Vazyme Biotech) and was reverse-transcribed with HiScript III RT SuperMix (R323-01, Vazyme Biotech). The mRNA levels of targeted genes were detected by AceQ Universal SYBR qPCR Master Mix (Q511-02, Vazyme Biotech) and normalized with Hprt1 as previously described (Zeng, Yuan, Cai, Tang, & Gao, 2021).

2.9. Western blotting

Total proteins of livers and cell samples were extracted with RIPA buffer (Beyotime, China). After being resolved in 10% SDS-PAGE gel, equal proteins were transferred to the PVDF membrane, blocked 5% milk at room temperature for 1 h, and incubated in specific primary antibodies at 4 °C overnight. After rinsing with TBST for three times, different targeted proteins were incubated with corresponding secondary antibodies, and were further detected on a Bio-Rad Gel Doc XR + Imaging System (CA, USA) and analyzed by Quantity-One software.

2.10. Statistical analysis

All data are repeated at least three times and presented as mean ± SEM. GraphPad Prism 8 (Graph-Pad, San Diego, CA) was used to perform a one-way ANOVA to compare differences in results between multiple groups. P values < 0.05 were considered as statistically significant.

3. Results

3.1. Chemical characterization of different extracts of CX

To describe chemical characterization of CX, we established HPLC methodology and identified a total of 10 constituents of aqueous of CX (CXAE) (Fig. 1A and Table S1), and we selected four chemical markers, including TMP (the specific representative component of CX) (Fig. 1B), senkyunolide A (the most abundant ingredient of CX) (Fig. 1D), ferulic acid (FA, the quality control component of CX as described in Chinese Pharmacopoeia) (Fig. 1C) and chlorogenic acid (CA) (Fig. 1E). Based on the representative compounds identified in CXAE, we subsequently prepared and characterized different CX extracts including alkaloid, phenolic acid and phthalide extracts of CX (CXAL, CXPA and CXPHL). Subsequently, we prepared ‘Standard Mixture’ (SM) (different standards containing TMP, FA, senkyunolide A and CA) to characterize the different CX extracts (Fig. 1F). Based on this method, we confirmed the CXAL by labeling TMP (Fig. 2A and 2B) and identified the CXPA by FA (Fig. 2C and 2D). Meanwhile, the CXPHL was identified by senkyunolide A in Fig. 2E and 2F. Above all, the preparation and identification of different CX extracts including CXAE, CXAL, CXPA and CXPHL laid the ground for further research both in vivo and in vitro.

Fig. 1.

Fig. 1

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HPLC spectrum profiles of representative extracts in CXAE. HPLC spectrum profile of CXAE (A), TMP (B), FA(C), SenA (D), CA (E) and CXAE + SM (F). CXAE, aqueous extract of CX; TMP, tetramethylpyrazine; FA, ferulic acid; SenA, senkyunolide A; CA, citric acid; CXAE + SM, aqueous extract of CX plus standard mixture.

Fig. 2.

Fig. 2

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Identified of three extracts in CX by HPLC. (A) HPLC spectrum profile of CXAL. (B) HPLC spectrum profile of CXAL + SM. (C) HPLC spectrum profile of CXPA. (D) HPLC spectrum profile of CXPA + SM. (E) HPLC spectrum profile of CXPHL. (F) HPLC spectrum profile of CXPHL + SM. CXAL: alkaloid extract of CX; CXAE + SM: alkaloid extract of CX plus standard mixture; CXPA: phenolic acid extract of CX; CXPA + SM: phenolic acid extract of CX plus standard mixture; CXPHL: phthalide extracts of CX; CXPHL + SM: phthalide extracts of CX plus standard mixture.

3.2. Network pharmacology prediction of CX against liver fibrosis

In order to clarify the influence of CX on liver fibrosis, the network pharmacology-based prediction was performed. In this study, a total of 77 ingredients of CX were extracted from TCMSP and 12 candidate components satisfied the rules of OB > 30% and DL > 0.18. In addition, 2047 known targets associated with liver fibrosis were screened from DrugBank database and 183 targets were derived from OMIM database. After eliminating the redundancy, 1884 targets related to liver fibrosis were retained. We obtained 42 overlap targets by merging the candidates targets of CX and liver fibrosis (Fig. 3A). As shown in Fig. 3B, we calculated the main targets and established the potential connection between the targets. To scientifically unveil the underlying mechanism of CX on liver fibrosis, PPI network was obtained from Cytoscape software (Fig. 3C). Total 29 nodes and 128 edges were embodied in the network. Furthermore, in order to further clarified the categories of major hubs in the biological process (BP), molecular function (MF) and cellular components (CC), KEGG enrichment analysis was carried out on the major proteins, and the top 20 terms (P < 0.05) were selected as significant entries based on the P value (Fig. 3D). The KEGG categorical results indicated that majority of hubs were enriched in the BP, mainly including cellular response to inorganic substance, toxic substance and reactive oxygen species. Based on the KEGG enrichment analysis, we further established ‘target-pathway’ network to demonstrate the relationship between overlap targets and potential pathways through polychrome lines (Fig. 3E). Consistent with KEGG enrichment analysis results, the target proteins or genes involved in hyperproliferation and inflammatory response were identified, and among these targets, c-myc, CTCF, Cyclin D1 (CCND1), Cyclin D2 (CCND2), epithelial cell adhesion molecule (EpCAM), PI3K, Bcl2 and FOS were considered as the central link of the anti-fibrotic effects of CX, notably, most of which were also related with the regulation of H19 or its-mediated function (Fig. 3F).

Fig. 3.

Fig. 3

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Network pharmacological analysis. (A) Venn diagrams of potential targets of ‘CX’ and ‘liver fibrosis’. (B) Network of potential targets of CX and liver fibrosis analyzed by STING. The node size represented the connectivity degree, and thickness of the edges represented the combine score between targets. (C) PPI network. (D) KEGG analysis of potential targets of ‘CX’ and ‘liver fibrosis’. (E) ‘Targets-pathway’ network diagram. (F) ‘Potential Targets’ diagram.

3.3. CX extracts ameliorate BDL-stimulated liver fibrosis in mice

To examine and compare the protection of CX extracts, we established BDL mouse model as described in ‘Methods’ (Fig. 4A) and observed that BDL significantly induced cholestatic liver fibrosis, as demonstrated by increasing serum ALT, AST, T-BIL and γ-GT, which were subsequently decreased by CXAE, CXAL and CXPHL but not CXPA (Supplementary Fig. S1 and Fig. 4B). As shown in Fig. 4C, liver histopathology study illustrated that CXAE, CXAL and CXPHL reversed BDL-induced inflammation cell infiltration, diffuse severe bile duct hyperplasia, portal edema and mild portal infiltrates as demonstrated by HE and Masson’s trichrome staining. Furthermore, collagen deposition was significant decreased in CXAE, CXAL and CXPHL-treated groups by Van Gieson staining. In addition, the mRNA levels of liver fibrotic markers including Fibronectin and Lox12 were markedly elevated by BDL and subsequently reversed by CX extracts including CXAE, CXAL and CXPHL (Fig. 4D). During BDL-stimulated liver fibrosis, overloaded BAs stimulated cholangiocyte proliferation as illustrated by increasing mRNA levels of Ccnd1, Ccnd2, Epcam and proliferating cell nuclear antigen (Pcna). While, CXAE, CXAL and CXPHL decreased the expression of these genes (but only CXPHL decreased Epcam expression) and thus ameliorated cholangiocyte proliferation in liver fibrosis (Fig. 4E).

Fig. 4.

Fig. 4

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CX extracts ameliorate BDL-stimulated bile duct hyperplasia and liver fibrosis (mean ± SEM, n = 6). (A) Flowchart of animal experiments with associated photographic images of each group. (B) Serum T-BIL and γ-GT levels. (C) Representative images of H&E staining, Masson trichrome’s staining and Van Gieson immunohistochemistry staining of liver tissues. And the quantified data were obtained using Image J software. Scale bar = 50 μm. Relative mRNA levels of (D) Fn, Loxl2, (E) Ccnd1, Ccnd2, Epcam and Pcna were determined by qPCR and normalized using Hprt1 as an internal control. **P < 0.01, ***P < 0.001, vs sham group; #P < 0.05, ##P < 0.01 vs BDL group (n = 8).

3.4. CX extracts alleviated bile duct hyperplasia via suppressing CTCF-c-MYC-H19 pathway

Given the anti-fibrotic effects of CX extracts in vivo and network pharmacology prediction, we next explored whether the underlying mechanism of these protection was related with H19 and its related targets, c-myc and CTCF. Recently, we observed that herb pair containing CX significantly down-regulated the expression of H19, a hepatic fibrosis marker (J. Z. Wu et al., 2021) and similar suppressive effects on H19 were also found in different CX extracts (Fig. 5A). c-MYC promoted the transcription of the H19 via specifically binding directly to E-boxes close to the ICR. Meanwhile, CTCF sites at mammalian c-myc promoter and H19 imprinting control region, can regulate the transcription of H19 and c-myc (Liu et al., 2014, Ottema et al., 2021). Therefore, we detected the expression of c-Myc and Ctcf in different groups and found that CXAE, CXAL CXPA and CXPHL significantly decreased the mRNA expression of c-Myc and Ctcf induced by BDL (Fig. 5A). Consistently, immunofluorescence staining indicated that BDL significantly increased the expression of c-MYC, which was subsequently down-regulated by CXAE and CXPHL (Fig. 5B). Similarly, CXAE and CXPHL markedly down-regulated the expression and distribution of CTCF, as illustrated by immunofluorescence staining in Fig. 5C. Furthermore, the protein levels of c-MYC and CTCF were also significantly elevated by BDL and subsequently reversed by some CX extracts, especially for CXAL and CXPHL (Fig. 5D). Collectively, these results demonstrated that CX extracts, especially CXAL and CXPHL alleviated cholangiocyte proliferation via suppressing CTCF-c-MYC-H19 pathway.

Fig. 5.

Fig. 5

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CX extracts inhibit CTCF-c-MYC-H19 pathway in BDL-induced mice (mean ± SEM, n = 6). (A) Relative mRNA levels of H19, c-myc and Ctcf determined by qPCR and normalized using Hprt1 as an internal control. Representative images of c-MYC (B) and CTCF (C) staining of liver tissues. And the quantified data were obtained using Image J software. Scale bar = 50 μm. (D) Representative immunoblots against c-MYC, CTCF and β-actin in liver. **P < 0.01, ***P < 0.001 vs sham group; #P < 0.05, ##P < 0.01 vs BDL group (n = 8).

3.5. CX extracts especially CXPHL alleviate overloaded BAs-related DR in cholangiocytes via suppressing CTCF-c-MYC-H19 pathway

Under the stimulus of BAs, cholangiocyte injury may be an early event in BDL-induced hepatic fibrosis due to their specific physiological position (Chen et al., 2021). Among the toxic BAs, TCA and LCA are major components and have been used to mimic BDL-induced excessive BAs in vitro. Meanwhile, TGF-β is responsive increased and lead to cholangiocytes proliferation and HSC activation in BDL mice (Wu et al., 2021). As shown in Fig. 6A–C, it was demonstrated that TCA, LCA and TGF-β significantly promoted cholangiocyte proliferation in vitro by increasing mRNA levels of different DR markers (Epcam, Pcna and Ccnd2), which was reversed by CXAE, CXAL and CXPHL. Meanwhile, immunofluorescence staining of CK-19 indicated that CX extracts especially CXAE, CXAL and CXPHL significantly down-regulated TCA-, LCA- and TGF-β-stimulated DR in cholangiocytes (as shown by lower cytoplasmic expression) (Fig. 6D and Supplementary Fig. S2). Exactly, we have already confirmed that CX extracts suppressed bile duct hyperplasia in mice by targeting CTCF-c-MYC-H19 pathway (Fig. 5). Therefore, we subsequently detected the expression of the CTCF-c-MYC-H19 signaling in cholangiocytes and observed that CXAE, CXAL and CXPHL markedly decreased the mRNA expression of H19 and c-myc but gently down-regulated the Ctcf expression in vitro Fig. 7A–C. Consistently, as shown in Fig. 7D–F, the protein expression of CTCF and c-MYC were increased by TCA, LCA and TGF-β, which was subsequently down-regulated by CXAE, CXAL and CXPHL. Notably, CXPHL effectively decreased the expression of CTCF and c-MYC regardless of the stimulus, while CXAL may be more sensitive to stimulation of TCA and TGF-β. Overall, CX extracts especially CXAL and CXPHL alleviate overloaded BAs-related DR in cholangiocytes via suppressing CTCF-c-MYC-H19 pathway. Overall, different CX extracts, especially for CXAL and CXPHL inhibited cholangiocyte hyperproliferation and liver fibrosis (Fig. 8).

Fig. 6.

Fig. 6

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CX extracts alleviate DR in TCA-, LCA- and TGF-β-induced cholangiocytes. Relative mRNA levels of Epcam, Pcna and Ccnd2 were determined by qPCR and normalized using Hprt1 as an internal control in (A) TCA-, (B) LCA- and (C) TGF-β-treated HIBECs (mean ± SEM, n = 3). (D) Representative images of immunofluorescent staining of CK-19 in TCA-treated HIBECs. And the quantified data were obtained using Image J software. Scale bar = 50 μm. Statistical significance: ***P < 0.001 vs sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs BDL group.

Fig. 7.

Fig. 7

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CX extracts suppress CTCF-c-MYC-H19 pathway in cholangiocytes (mean ± SEM, n = 3). Relative mRNA levels of H19, c-Myc and Ctcf were determined by qPCR and normalized using Hprt1 as an internal control in (A) TCA-, (B) LCA- and (C) TGF-β-treated HIBECs. Representative immunoblots against c-MYC, CTCF and β-actin in (D) TCA-, (E) LCA- and (F) TGF-β-treated HIBECs. Statistical significance: **P < 0.01, ***P < 0.001 vs sham group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs BDL group.

Fig. 8.

Fig. 8

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Schematic diagram of proposed mechanisms of anti-fibrotic effects of CX extracts.

4. Discussion

Liver fibrosis represents a dynamic wound healing process and the hepatoprotection of CX in ameliorating fibrotic liver disorders have been demonstrated in clinical trials and basic research (J. Wang et al., 2022). In the current study, different CX extracts were isolated and the representative components of these extracts were identified through ‘Standard Mixture’ (Fig. 1, Fig. 2) then compared and investigated the anti-fibrotic effects of different CX extracts at the equivalent dose in liver fibrosis both in vitro and in vivo (Fig. 3, Fig. 4, Fig. 5, Fig. 6, Fig. 7). We found that CXAE, CXAL and CXPHL concurrently alleviated liver fibrosis to varying degrees, as illustrated by the suppression of serum T-BIL and γ-GGT, hepatocytes vacuolation, ECM accumulation. Interestingly, among these CX extracts, more obvious anti-fibrotic activity of CXAL and CXPHL has been observed, as demonstrated by the more significant inhibition of the levels of proliferation and DR markers both in vitro and in vivo. While, compared with CXAL, CXPHL may be the main contributor to the anti-fibrotic effects of CX, as illustrated by more obvious suppressive effects on the CTCF-c-MYC-H19 pathway. These events suggested that the hepatoprotective components in CX may be primarily derived from CXAL and CXPHL.

Indeed, researchers gradually pay attention to clarify the active compounds of the protection of different CX extracts. Network pharmacology, integrating system biology, pharmacology and computer analysis technology, has been used in the exploration of the complicated relationships among components, targets and diseases (Dai et al., 2020). Notably, more and more researchers have applied network pharmacology to predict the active components and potential targets of TCM and explore its effects and mechanisms against different diseases (Liu et al., 2022). Recently, network pharmacology has been used to explore the hepatoprotective of TCM, such as the molecular mechanism of Schisandra chinensis (Turcz.) Baill. against liver injury, the therapeutic mechanism of Danggui-Chuanxiong herb-pair on liver fibrosis (Wu et al., 2021). In the present study, based on the network pharmacology analysis, we focused on the cholangiocyte proliferation and CTCF-c-MYC-H19 relative signaling pathway, which has been overlooked for a long time. According to experimental analysis, we found that the differential protection of CX extracts may be attributed to different components and their regulation of different pathways. For example, representative components of CXAL and CXPHL, including TMP (Li et al., 2022, Wang, Xu, Yang, Chen, & Zhang, 2015), ligustilide (Yang & Xing, 2021), ligustchuane A (Wan et al., 2022) and ligustchuane B (Qin et al., 2022) have been demonstrated to respectively affect c-myc or H19, which may explain the phenomenon about the superimposed regulation of these two CX extracts on CTCF-c-MYC-H19 pathway. While the components in CXPA may suppress hepatic fibrosis via other pathways but not CTCF-c-MYC-H19. Overall, these events may lead to the differential protections of CX extracts in fibrotic liver injury.

Another interesting finding was that although both CXAL and CXPHL significantly suppressed liver fibrosis by targeting CTCF-c-MYC-H19 and DR, their regulation of individual targets is differential. Among these extracts, CXPHL significantly decreased the expression of H19, c-myc and CTCF, while other extracts may mainly affect c-myc, but not CTCF (Fig. 5, Fig. 7). H19 is predominantly expressed in cholangiocytes and play key roles in cholestatic liver injury that also has gradually become a recognized biomarker of liver fibrosis (Li et al., 2017). C-myc, a master transcriptional factor, regulates approximately 10%–15% of genes in the genome, and more importantly, playing critical roles in promoting H19 transcription by binding to the E-boxes in H19 DNA sequence (Wu et al., 2020). In addition, CTCF concurrently modulated the transcription of c-myc and H19, while CX extracts gently decreased the expression of CTCF. These events suggested that the regulation of CTCF on H19 transcription exist “two-pronged” effects. Therefore, we suspected that the more obvious anti-fibrotic effects of CXPHL may be attributed to the dual suppression of c-MYC and CTCF and subsequent inhibition of H19-related cholangiocyte proliferation. Exactly, as a recognized inhibitor of c-MYC, 10058-F4 has been devoted to the clinical therapeutics and basic research of hepatocellular carcinoma (Luo et al., 2021, Wu et al., 2020). While, the inhibitor of CTCF have not yet been not fully recognized, only a handful inhibitor is still in the preclinical, such as ZL 201910562893.1. These results also suggested that development of effective strategies targeting c-MYC or synergistic effects of Chinese medicine and specific target inhibitors will have great therapeutic significance for liver fibrosis and its complications.

5. Conclusion

Taken together, our results systemically indicated that different CX extracts significantly ameliorated liver fibrosis by disturbing CTCF-c-MYC-H19 pathway and alkaloid and phthalides isolated from CX were the most effective fractions. Overall, these data offered a novel therapeutic strategy for DR-associated liver fibrosis and contribute to the discovery of CX-associated therapeutic agents for hepatic fibrosis.

CRediT authorship contribution statement

XL conceived the original idea and supervised the study. YL prepared the manuscript and figures. YL, FL, MD, ZM, SL and JQ conducted all the experiments and performed data analysis. All data were generated in-house and no paper mill was used. All authors have approved the final manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This work was supported by the Beijing Municipal Science & Technology Commission (No. 7212174); the National High-Level Talents Special Support Program to XL; National Key Research and Development Program on Modernization of Traditional Chinese Medicine (No. 2022YFC3502100); National Natural Science Foundation of China (No. 82004045 and No. 82274186).

Footnotes

Appendix A

Supplementary data to this article can be found online at https://doi.org/10.1016/j.chmed.2023.07.003.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Supplementary data 1
mmc1.docx (299.6KB, docx)

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