Peptidoglycan isolated from the fruit of Lycium barbarum alleviates liver fibrosis in mice by regulating the TGF-β/Smad7 signaling and gut microbiota

Abstract

The hepatoprotective effect of the fruit of Lycium barbarum has been documented in China over millennia. Lycium barbarum polysaccharides (LBPs) were the first macromolecules reported to mitigate liver fibrosis in carbon tetrachloride (CCl₄)-treated mice. Herein, a neutral peptidoglycan, named as LBPW, was extracted from the fruit of Lycium barbarum. In this study, we investigated the hepatoprotective mechanisms of LBPW. CCl₄-induced liver fibrosis mice were administered LBPW (50, 100, 200 mg ·kg^–1 ·d^–1, i.p.) or (100, 200, 300 mg· kg^–1 ·d^–1, i.g.) for 6 weeks. We showed that either i.p. or i.g. administration of LBPW dose-dependently attenuated liver damage and fibrosis in CCl₄-treated mice. Pharmacokinetic analysis showed that cyanine 5.5 amine (Cy5.5)-labeled LBPW (Cy5.5-LBPW) could be detected in the liver through i.p. and i.g. administration with i.g.-administered Cy5.5-LBPW mainly accumulating in the intestine. In TGF-β1-stimulated LX-2 cells as well as in the liver of CCl₄-treated mice, we demonstrated that LBPW significantly upregulated Smad7, a negative regulator of TGF-β/Smad signaling, to retard the activation of hepatic stellate cells (HSCs) and prevent liver fibrosis. On the other hand, LBPW significantly boosted the abundance of Akkermansia muciniphila (A. muciniphila) and fortified gut barrier function. We demonstrated that A. muciniphila might be responsible for the efficacy of LBPW since decreasing the abundance of this bacterium by antibiotics (Abs) blocked the effectiveness of LBPW. Overall, our results show that LBPW may exert the hepatoprotective effect via rebalancing TGF-β/Smad7 signaling and propagating gut commensal A. muciniphila, suggesting that LBPW could be leading components to be developed as new drug candidates or nutraceuticals against liver fibrosis.

Introduction

Hepatic fibrosis is an outcome of the dysregulated wound-healing response of the liver to sustained chronic liver injury of any etiology and is characterized by the excessive accumulation of extracellular matrix (ECM) [1]. Liver fibrosis has increased morbidity and mortality worldwide [2, 3], and the severity of fibrosis is a determinant of outcomes in patients with chronic liver diseases (CLDs) [2, 4, 5]. Without effective intervention, liver fibrosis can lead to cirrhosis and, in some cases, hepatocellular carcinoma, for which effective therapy is lacking other than liver transplantation [6]. Although belapectin [7], selonsertib [8], obeticholic acid (OCA) [9–12], and other candidate drugs have been evaluated under clinical trials for liver diseases with fibrosis, they either fail to ameliorate fibrosis or have relatively serious side effects. Although resmetirom has been recently approved for the treatment of metabolic dysfunction-associated steatohepatitis (MASH) with fibrosis (stage F2 to F3) [13], the development of direct drugs for the treatment of liver fibrosis is still a major unmet need.

In the fibrotic liver, activated hepatic stellate cells (HSCs) are believed to be the major source of fibrogenic myofibroblasts, which highly express α-smooth muscle actin (α-SMA) and produce ECM [14]. Consequently, the inhibition of HSC activation is a pivotal strategy for alleviating liver fibrosis. Transforming growth factor-β1 (TGF-β1) is generally considered the most potent profibrotic cytokine [15]. TGF-β1 binds to TGFβR2, which then phosphorylates TGFβR1. Subsequently, TGFβR1 phosphorylates Smad2 and Smad3. These phosphorylated Smads form a heteromeric complex with Smad4 and enter the nucleus, where they promote the transcription of target genes during HSC activation, including profibrotic genes [16]. In contrast, Smad7 acts as a negative regulator to antagonize TGF-β/Smad signaling [17, 18] by blocking the interaction and phosphorylation of Smad2 and Smad3 by TGFβRI [18] and promoting the ubiquitination [19] and the dephosphorylation of TGFβRI [20]. Thus, Smad7 plays a positive role in liver fibrosis since its overexpression inhibits the activation of HSCs and liver fibrosis [21], and disruption of the Smad7 gene enhances liver fibrogenesis in vivo and in vitro [22]. In addition, the overexpression of Smad7 in mice also results in reduced fibrogenesis in other organs [23–25]. Notably, pharmaceutical agents that increase the expression of Smad7 retard liver fibrosis [26–28]. Hence, increasing the expression of Smad7 is a promising strategy for preventing hepatic fibrosis.

Fibrogenesis is a multiorgan reaction in which the gut microbiota has been shown to closely correlate with the occurrence and progression of liver fibrosis [29–32]. The commensal gut microbiota can maintain liver homeostasis and prevent liver fibrosis [33], and gut dysbiosis is associated with fibrosis severity [34–39]. Dysfunction of the gut barrier accelerated by microbial dysbiosis facilitates the translocation of bacteria and their products and, ultimately, aggravates liver fibrogenesis [40]. Akkermansia muciniphila (A. muciniphila) is a gram-negative, obligate anaerobe that consumes host-derived mucins and human milk oligosaccharides to thrive in the human intestine [41–43]. A lower abundance of A. muciniphila has been observed in individuals with CLDs [44, 45], and the recovery or supplementation of A. muciniphila is beneficial for preventing alcoholic liver disease [44] and liver fibrosis [46]. The efficacy of A. muciniphila might be attributed to its ability to produce propionate and outer membrane proteins to promote mucus thickening and gut barrier function [47]. Overall, we speculated that A. muciniphila might play an important role in ameliorating hepatic fibrosis.

The fruit of Lycium barbarum is documented in Shen Nong’s Materia Medica as having the ability to nourish the liver. Although the hepatoprotective effect of the fruit of L. barbarum has been recorded over millennia, the pharmacodynamic material basis and molecular mechanisms are still not clear. L. barbarum polysaccharides (LBPs) were the first macromolecules reported to mitigate liver fibrosis in carbon tetrachloride (CCl₄)-treated mice [48]. In this study, a neutral peptidoglycan, named LBPW, was extracted from the fruit of L. barbarum. We thus hypothesized that LBPW might have hepatoprotective potential. However, how LBPW protects the liver is largely unknown.

We addressed this question by intraperitoneally and intragastrically administering LBPW to CCl₄-induced liver fibrosis model mice to evaluate its effectiveness. The tissue distribution of cyanine 5.5 amine (Cy5.5)-labeled LBPW (Cy5.5-LBPW) was assessed after i.p. and i.g. administration via near infrared (NIR) imaging. We employed RNA sequencing and siRNA-mediated knockdown of Smad7 in the human stellate cell line LX-2 to investigate the potential mechanism by which LBPW inactivates HSCs. Moreover, the contribution of the gut microbiota to the beneficial effects of LBPW on liver fibrosis was evaluated via 16S rRNA gene sequencing, metagenomics sequencing, an assessment of gut barrier function and antibiotic treatment. Our results may provide evidence for the development of a novel type of molecule based on the fruit of L. barbarum as a drug candidate or nutraceutical for the treatment or prevention of liver fibrosis.

Materials and methods

Preparation of LBPW

The crude polysaccharide, designated LBP, was initially extracted from 5 kg of dried L. barbarum (purchased from Ningxia, China) through seven cycles of boiling water treatment. The resulting extract was subsequently concentrated and dialyzed against running water for 48 h using dialysis bags (Shanghai Green Bird Company, Shanghai, China) (MWCO 3500 Da). Following centrifugation, the supernatant was further precipitated with four volumes of 95% ethanol and stayed overnight. After centrifugation, the ethanol was evaporated in a drying oven at 60 °C, followed by redissolution and freeze-drying [49]. A total of 561 g of crude polysaccharide (LBP) was obtained via water extraction and ethanol precipitation from 7.6 kg of dry raw medicine, yielding 7.4%.

The crude fraction of LBP was further separated using double-distilled (dd) water. Specifically, 50 g of LBP powder was dissolved in 400 mL of dd water, followed by precipitation. The resulting supernatant was then loaded onto a DEAE Sepharose™ Fast Flow column (10 cm × 100 cm, GE Healthcare, China) at an elution rate of 100 mL/min. Subsequent isolation of LBPW with water serving as the mobile phase resulted in the acquisition of 10 g samples, with a yield of 1.8%.

Determination of the homogeneity and monosaccharide composition

The homogeneity of LBPW was determined via high-performance gel permeation chromatography (HPGPC) (Agilent Technologies, Inc., USA), which employs two tandem columns, KS-804 and KS-802 (Shodex Co., Tokyo, Japan) [50]. LBPW presented an evident peak on HPGPC with a molecular weight of 4.7 kDa, and a distinct peak at 280 nm was detected with an ultraviolet (UV) detector (Supplementary Fig. 1a, b). The monosaccharide composition was determined via high-performance liquid chromatography using the 1-phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivatization method [51, 52]. LBPW consisted primarily of 16.37% mannose, 33.82% glucose, 19.19% galactose, 20.41% arabinose, and 8.54% xylose (Supplementary Fig. 1d, e).

Animal experiments

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Shanghai Institute of Materia Medica (Shanghai, China). Eight-week-old male C57BL/6J mice weighing 20 ± 2 g were housed in specific pathogen-free facilities on a 12 h light/12 h dark cycle with ad libitum access to food and water. For the CCl₄-induced liver fibrosis model, the mice were intraperitoneally injected with 10% CCl₄ diluted with olive oil at a dose of 2 mL/kg 3 times a week for eight weeks. The mice in the control group were injected with the same volume of olive oil 3 times a week for eight weeks.

Animal Experiment 1: Intraperitoneal injection of LBPW

As shown in Fig. 1a, after 2 weeks, the mice in the control group were injected intraperitoneally with olive oil and 10 mL ·kg^–1 ·d^–1 of normal saline (n = 7). Mice injected intraperitoneally with CCl₄ for 2 weeks were divided into five groups (n = 7) and treated for 6 weeks: (1) CCl₄: mice were injected intraperitoneally with CCl₄ along with 10 mL ·kg^–1 ·d^–1 normal saline; (2) CCl₄ + 50 mg ·kg^–1 ·d^–1 LBPW: mice were injected intraperitoneally with CCl₄ along with 50 mg ·kg^–1 ·d^–1 LBPW; (3) CCl₄ + 100 mg ·kg^–1 ·d^–1 LBPW: mice were injected intraperitoneally with CCl₄ along with 100 mg ·kg^–1 ·d^–1 LBPW; (4) CCl₄ + 200 mg ·kg^–1 ·d^–1 LBPW: mice were injected intraperitoneally with CCl₄ along with 200 mg ·kg^–1 ·d^–1 LBPW; and (5) CCl₄ + 30 mg ·kg^–1 ·d^–1 OCA (Dalian Meilun Biotech Co. Ltd, China; Cat: MB6084): mice were injected intraperitoneally with CCl₄ and i.g. administered 30 mg ·kg^–1 ·d^–1 OCA. Notably, LBPW was dissolved in normal saline, and OCA was dissolved in 0.5% sodium carboxymethyl cellulose (CMC-Na), both of which were administered to mice at a volume of 10 mL ·kg^–1·d^–1. Mice were sacrificed after the daily i.p. injection of LBPW or i.g. administration of OCA for 6 weeks. Serum samples were collected for biochemical analyses, and the liver samples were collected for hematoxylin and eosin (H&E) staining, Sirius red staining, immunohistochemical (IHC) staining, Western blotting and the detection of the hydroxyproline content.

Fig. 1. i.p. and i.g. treatment of LBPW ameliorates CCl₄-induced hepatic injury in mice.

a The schematic diagram of the animal test by i.p. injection of LBPW in mice (biological replicates, n = 7). Serum ALT (b) and AST (c) levels from mice in Animal Experiment 1 were detected by biochemical assay kits. d Representative morphology and H&E staining (scale bar = 500 μm) of the liver from mice in Animal Experiment 1. e The schematic diagram of the animal test by i.g. treatment of LBPW in mice (biological replicates, n = 6 or 7). Serum ALT (f) and AST (g) levels from mice in Animal Experiment 2 were detected by an automatic biochemical analyzer. h Representative morphology and H&E staining (scale bar = 500 μm) of the liver from mice in Animal Experiment 2. Ctr, control; i.p., intraperitoneal injection; q.d., quaque die; i.g., intragastric administration. Data are presented as the mean ± SD. The P values were calculated by one-way ANOVA followed by Dunnett’s multiple-comparison test.

Animal Experiment 2: Intragastric administration of LBPW

As shown in Fig. 1e, after 2 weeks, the mice in the control group were injected intraperitoneally with olive oil and i.g. administered 10 mL ·kg^–1 ·d^–1 normal saline (n = 6). Mice injected intraperitoneally with CCl₄ for 2 weeks were divided into five groups and treated for 6 weeks: (1) CCl₄ (n = 6): mice were injected intraperitoneally with CCl₄ and i.g. administered 10 mL ·kg^–1·d^–1 normal saline; (2) CCl₄ + 100 mg ·kg^–1 ·d^–1 LBPW (n = 6): mice were injected intraperitoneally with CCl₄ and i.g. administered 100 mg ·kg^–1 ·d^–1 LBPW; (3) CCl₄ + 200 mg ·kg^–1 ·d^–1 LBPW (n = 7): mice were injected intraperitoneally with CCl₄ and i.g. administered 200 mg ·kg^–1 ·d^–1 LBPW; (4) CCl₄ + 300 mg ·kg^–1 ·d^–1 LBPW (n = 7): mice were injected intraperitoneally with CCl₄ and i.g. administered 300 mg ·kg^–1 ·d^–1 LBPW; and (5) CCl₄ + 30 mg ·kg^–1 ·d^–1 OCA (MedChem Express, USA; Cat: HY-12222) (n = 6): mice were injected intraperitoneally with CCl₄ and i.g. administered 30 mg ·kg^–1 ·d^–1 OCA. Mice were sacrificed after the daily i.g. administration of LBPW or OCA for 6 weeks. Serum samples were collected for biochemical analyses. Liver samples were collected for H&E staining, Sirius red staining, IHC staining and Western blotting. The cecal contents were collected for sequencing of the full-length 16S rRNA gene to assess diversity and for metagenomics sequencing. Ileum samples were collected for IHC staining and transmission electron microscopy (TEM). Colon specimens were collected for H&E staining, IHC staining and Western blotting.

Animal Experiment 3: Antibiotic treatment

As shown in Fig. 7a, after 2 weeks, the mice in the control group were injected intraperitoneally with olive oil and i.g. administered 10 mL ·kg^–1 ·d^–1 normal saline (n = 6). Mice that were injected intraperitoneally with CCl₄ for 2 weeks were divided into five groups (n = 6) and treated for 6 weeks: (1) CCl₄: mice were injected intraperitoneally with CCl₄ and i.g. administered 10 mL ·kg^–1 ·d^–1 normal saline; (2) CCl₄ + antibiotics (Abs): mice were injected intraperitoneally with CCl₄, i.g. administered 10 mL ·kg^–1 ·d^–1 normal saline, and administered antibiotics in the drinking water; (3) CCl₄ + 300 mg ·kg^–1 ·d^–1 LBPW: mice were injected intraperitoneally with CCl₄ and i.g. administered 300 mg ·kg^–1 ·d^–1 LBPW; (4) CCl₄ + antibiotics + 300 mg ·kg^–1 ·d^–1 LBPW: mice were injected intraperitoneally with CCl₄, i.g. administered 300 mg ·kg^–1 ·d^–1 LBPW, and administered antibiotics in the drinking water; (5) CCl₄ + 30 mg ·kg^–1 ·d^–1 OCA (MedChem Express, USA; Cat: HY-12222): mice were injected intraperitoneally with CCl₄ and i.g. administered 30 mg ·kg^–1 ·d^–1 OCA. For the antibiotic treatment, the mice were administered drinking water containing 1 g/L ampicillin (Sigma‒Aldrich, Germany; Cat: A9518), 0.25 g/L vancomycin (Dalian Meilun Biotech Co. Ltd, China; Cat: MB1260-2) and 0.2 g/L spectinomycin (Dalian Meilun Biotech Co. Ltd, China; Cat: MB1497) during LBPW administration, and the water supply was renewed every 3 days. Mice were sacrificed after the daily i.g. administration of LBPW or OCA for 6 weeks. Serum samples were collected for biochemical analyses. Liver samples were collected for H&E staining, Sirius red staining and Western blotting. The cecal contents were collected to sequence the full-length 16S rRNA gene to assess diversity. Colon specimens were collected for H&E staining, IHC staining and Western blotting.

Fig. 7. The reduced abundance of A. muciniphila negates the protective effects of LBPW on liver damage and fibrosis in mice.

a Schematic diagram of Animal Experiment 3. b The abundance of A. muciniphila in the cecum was accessed by 16S rRNA gene sequencing. c Serum ALT level was measured by the biochemical assay kit. d Serum AST level was measured by the biochemical assay kit. e Representative morphology and H&E staining (scale bar = 500 μm) of the liver. f Representative Sirius red staining of the liver (scale bar = 100 μm). g Quantification of Sirius red staining in (f) by ImageJ. h The expression of α-SMA in the liver was detected by Western blotting. i Quantification of the expression of α-SMA in (h) by KwikQuant Image Analyzer. Ctr, control; Abs, antibiotics cocktail; q.d., quaque die; i.g., intragastric administration. Data are presented as the mean ± SD. In b, the P value was calculated by the Wilcoxon rank-sum test. In c, d, g and i, the P values were calculated by one-way ANOVA with Benjamini–Hochberg adjustment.

Cell culture and cellular fibrosis model construction

The human HSC line LX-2 was purchased from the BeNa Culture Collection (China; Cat: BNCC337957), and the human hepatocyte cell line LO₂ was purchased from the Cell Bank of the Chinese Academy of Sciences (China). Both cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, USA; Cat: A5669701), 100 units/mL penicillin and 100 μg/mL streptomycin. The liver fibrosis cell model was constructed with LX-2 cells within 20 passages, and the model was generated via an incubation with 10 ng/mL TGF-β1 (SinoBiologicol, China; Cat: 10804-HNAC) for 48 h.

Protein extraction and Western blotting

LX-2 cells were preincubated with 0.25 mg/mL, 0.5 mg/mL or 1.0 mg/mL LBPW for 1 h. Then, the cells were stimulated with 10 ng/mL TGF-β1 for 48 h. For this process, the cells were treated with 10 μM OCA as a positive control. Total protein was extracted from LX-2 cells using RIPA lysis buffer (Absin, China; Cat: abs9229) supplemented with phosphatase inhibitors (Selleck, USA; Cat: B15001) and the protease inhibitor (Selleck, USA; Cat: B14001). Total proteins were extracted from liver and colon tissues with a total protein extraction kit (Sangon Biotech, China; Cat: C510003). Tris-HCl polyacrylamide gels were used with Tris-glycine running buffer. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose (NC) membranes. The membranes were subsequently blocked with 5% bovine serum albumin or skim milk, followed by an incubation with primary antibodies at 4 °C overnight. After washing, horseradish peroxidase-conjugated secondary antibodies were used for immunoblotting. Bands were visualized using chemiluminescence (ECL) reagents and the KwikQuant digital Western blotting detection system. The primary antibodies used in this study are listed in Supplementary Table 2.

Biochemical analysis

The serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the mice intraperitoneally injected with LBPW and in the mice treated with antibiotics were measured using an ALT Kit (Nanjing Jiancheng, China; Cat: C009-2-1) and an AST Kit (Nanjing Jiancheng, China; Cat: C010-2-1), respectively. The serum ALT and AST levels in the mice intragastrically administered LBPW were measured using an automatic biochemical analyzer (Sysmex, Japan).

Small interfering RNA (siRNA)-mediated Smad7 knockdown

siRNAs targeting human Smad7 and the negative control were synthesized by Genomeditech (Shanghai, China), and the sequences are listed in Supplementary Table 3. LX-2 cells were plated into six-well plates in RPMI-1640 medium supplemented with 10% FBS but no antibiotics, which was termed transfection medium, for 18 h before transfection. Subsequently, the cells (70%–80% confluence) were transfected with 3 μL of 20 μM siRNA per well using 5.6 μL of Lipofectamine^™ 3000 (Invitrogen, USA; Cat: L3000015), and the siRNA–lipid complexes were prepared in Opti-MEM (Gibco, USA; Cat: 31985070). At 6 h after transfection, the medium was replaced with fresh transfection medium, and the cells were stimulated with 10 ng/mL TGF-β1 with or without 1 mg/mL LBPW for an additional 36 h and then harvested for Western blotting.

RNA sequencing

LX-2 cells were stimulated with 10 ng/mL TGF-β1 for 48 h as the cModel group and preincubated with 1 mg/mL LBPW for 1 h, followed by the addition of 10 ng/mL TGF-β1 for 48 h as the cHdose group. The RNA was isolated using TRIzol reagent (Sangon Biotech, China; Cat: B511311), and its concentration, quality and integrity were determined using a Qubit 2.0 spectrophotometer (Invitrogen, USA) and gel electrophoresis. The RNA-seq libraries were prepared using the MaxUp Dual-mode mRNA Library Prep Kit (Yeasen, China; Cat: 12301ES96) and sequenced on an Illumina NovaSeq 6000 instrument by Shanghai Sangon Biotech Co. Ltd (Shanghai, China).

16S rRNA gene sequencing

High-throughput sequencing was performed using the DNA extracted from the cecal contents of the mice in the control, model, Hdose, CCl₄ + LBPW and CCl₄ + LBPW + antibiotics groups. The DNA library was prepared with the primer pair 27 F (5′-AGRGTTYGATYMTGGCTCAG-3′) and 1492R (5′-RGYTACCTTGTTACGACTT-3′) targeting the full-length 16S rRNA gene and sequenced on a PacBio Sequel IIe System (Pacific Biosciences) by Majorbio Biopharm Technology Co. Ltd (Shanghai, China). The data were processed via SMRTLink (v.11.0) to obtain high-fidelity (Hifi) reads, which were denoised using the DADA2 plugin in QIIME2 to obtain amplicon sequence variants (ASVs). The taxonomic assignment was accomplished using the vsearch taxonomic classifier and annotated with the Silva 16S rRNA database (v.138). The analyses were performed on the Majorbio Cloud Platform (https://cloud.majorbio.com), and the details of the database and software are listed in Supplementary Table 4.

H&E staining, Sirius red staining and immunohistochemistry

Mouse liver, ileum and colon samples were fixed with 4% paraformaldehyde, embedded in paraffin, cut into 4 μm thick sections and mounted on glass slides. H&E staining, Sirius red staining and immunohistochemistry were subsequently performed by Wuhan Servicebio Technology Co., Ltd (China) using standard protocols. Whole fields of the sections were scanned with a NanoZoomer 2.0-HT slide scanner (Hamamatsu, Japan).

Transmission electron microscopy (TEM)

Fresh mouse ileum tissues cut into 1–2 mm³ pieces were immersed in an electron microscopy fixative solution (Wuhan Servicebio Technology Co., Ltd, China; Cat: G1102), fixed for 2 h at room temperature in the dark, and then transferred to 4 °C for preservation. The subsequent steps were conducted by Wuhan Servicebio Technology Co., Ltd (China).

Synthesis and characterization of fluorescein-labeled LBPW

LBPW was labeled with Cyanine5.5 amine (Cy5.5) and purified using Sephadex G50 [53]. Specifically, 100 mg of LBPW was dissolved in 50 mL of 2-morpholinoethanesulfonic acid (MES) at pH 6, then 75 mg of 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) and 80 mg of N-hydroxysuccinimide (NHS) were added. The mixture was activated for 30 min with full stirring, followed by adjusting the pH with an NaOH solution. Thus, Cy5.5 was utilized to label LBPW, and the mixture was kept for 48 h in the dark at room temperature. After dialysis and purification with a Sephadex G50 chromatography column, the purified marker, designated Cy5.5-LBPW, was finally pooled and freeze-dried.

Near-infrared imaging studies

Male BALB/c mice (20 ± 2 g) were selected and intragastrically or intraperitoneally administered 0.2 mL of Cy5.5-LBPW at concentrations of 2 mg/mL or 1 mg/mL, respectively, at different time points. At 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h and 12 h after administration, the mice were sacrificed by cervical dislocation and immediately placed on a small animal live imaging device for imaging. After that, the mice were dissected, and the gastrointestinal tract and organs of the mice were removed and neatly placed on black cardboard for near-infrared imaging (near-infrared small animal live imaging device: Pearl Trilogy, Li-COR Bioscience, USA). Image Studio was used for data analysis. Relative fluorescence intensity of isolated tissue = total fluorescence intensity of isolated tissue/imaging area of the isolated tissue [54].

Quantification and statistical analysis

All the data are presented as the mean ± SD. The membranes were analyzed using ImageJ or KwikQuant Image Analyzer 5.9. Six images from each sample were randomly selected to quantify the Sirius red staining and IHC staining using ImageJ. Statistical analyses were performed with GraphPad Prism 9.5.1. The sample distribution was determined using the Kolmogorov‒Smirnov normality test or Shapiro‒Wilk normality test. The homogeneity of variance was tested using Bartlett’s test or the Brown–Forsythe test. A nonparametric test was used to compare two independent samples with the Wilcoxon rank-sum test. Comparisons of means among more than two groups were performed with one-way analysis of variance (ANOVA) or Welch’s ANOVA (for unequal variance) followed by multiple comparison tests. P values <0.05 were considered statistically significant. All the bar graphs were created with GraphPad Prism 9.5.1, and the images were combined into figures using Adobe Illustrator 2023 software.

Results

LBPW ameliorates CCl₄-induced hepatic injury in mice

The toxin CCl₄ was administered to induce liver fibrosis in mice and the effect of LBPW on liver fibrosis in vivo was assessed. The mice were injected intraperitoneally with CCl₄ for 2 weeks, followed by 6 weeks of CCl₄ injections along with daily i.p. injections of LBPW at doses of 50 mg/kg, 100 mg/kg or 200 mg/kg (Fig. 1a). The hepatoprotective effect of LBPW on mice was evaluated first. We observed that the serum ALT and AST levels were significantly decreased, resulting in a 63% reduction in ALT level and a 57% reduction in AST level after the administration of a daily dose of 200 mg/kg LBPW (Fig. 1b, c). In addition, the serum level of alkaline phosphatase (ALP), another indicator of liver function, was also significantly decreased after LBPW treatment (Supplementary Fig. 3a). The results of H&E staining of liver tissues revealed that, in the control group, hepatocytes within each liver lobule were arranged in cords radiating outward from the central vein (Fig. 1d). Conversely, livers from CCl₄-treated mice presented architectural distortion, hepatocyte damage and pronounced inflammatory cell infiltration (Fig. 1d). Compared with the CCl₄-induced model group, LBPW markedly improved liver architecture, reduced hepatocyte damage and decreased inflammatory cell infiltration (Fig. 1d). Moreover, LBPW improved the liver morphology, as evidenced by reduced graininess (Fig. 1d). Because the ability of L. barbarum fruit to nourish the liver in the clinic is achieved via oral administration, we investigated the efficacy of LBPW on CCl₄-induced liver injury in mice by intragastrically administering LBPW at daily doses of 100 mg/kg, 200 mg/kg or 300 mg/kg (Fig. 1e). Compared with those in the model group, the serum transaminase levels were markedly reduced after the i.g. administration of LBPW; a daily dose of 300 mg/kg reduced ALT level by 70% and AST level by 50% (Fig. 1f, g). Additionally, i.g. treatment with LBPW also preserved the liver morphology and architecture in mice (Fig. 1h). Taken together, these results suggested that LBPW might mitigate CCl₄-induced liver injury in mice following both i.p. and i.g. administration.

LBPW attenuates CCl₄-induced liver fibrosis in mice and restrains the activation of HSCs in vivo and in vitro

We next assessed the efficacy of LBPW for the treatment of liver fibrosis via i.p. administration. Sirius red staining of liver sections revealed that the livers from model mice presented substantial collagen deposition in both the central and portal regions, whereas LBPW reduced the amount of collagen and resulted in fewer fibrotic bridges (Fig. 2a, b). An assessment of the hydroxyproline (Hyp) content further revealed that the increase in collagen production was abated after the i.p. injection of LBPW (Supplementary Fig. 3b). Furthermore, the serum levels of four indicators of liver fibrosis, hyaluronic acid (HA), laminin (LN), procollagen-III (PC-III) and collagen-IV (IV-C), were significantly decreased upon the i.p. injection of LBPW (Supplementary Fig. 3c–f). In addition, the marked increase in α-SMA level in the HSCs of mice with CCl₄-induced fibrosis was blocked by treatment with LBPW, as indicated by IHC staining (Fig. 2c, d). Furthermore, a distinct LBPW-mediated decrease in the expression of α-SMA in the liver was also detected by Western blotting (Fig. 2e, f), confirming that LBPW inhibited the activation of HSCs in vivo. We then evaluated whether the i.g. administration of LBPW also promoted the regression of fibrosis in mice. The results of Sirius red staining revealed that the collagen content of LBPW-treated mice was significantly lower than that of model mice (Fig. 2g, h). Moreover, i.g. treatment with LBPW resulted in marked reductions in the expression of the collagen type I alpha-1 (COL1A1) and fibronectin 1 (FN1) proteins in the liver (Supplementary Fig. 3g, h). The above results revealed that the i.g. administration of LBPW reduced the severity of fibrosis. In addition, LBPW treatment led to a reduction in the number of HSCs stained with α-SMA compared with that in the model group (Fig. 2i, j). LBPW also decreased α-SMA expression, as shown by Western blotting (Fig. 2k, l). Overall, LBPW administered through both the i.p. and i.g. routes impeded fibrogenesis and the activation of HSCs in CCl₄-exposed mice.

Fig. 2. i.p. and i.g. treatment of LBPW mitigates CCl₄-induced liver fibrosis in mice and inhibits HSC activation in vivo and in vitro.

Representative Sirius red (scale bar = 100 μm) (a) and α-SMA IHC staining (scale bar = 100 μm) (c) conducted on liver sections from mice in Animal Experiment 1. b Quantification of Sirius red staining in (a) by ImageJ. d Quantification of α-SMA IHC staining in (c) by ImageJ. e The expression of α-SMA in the liver of mice from Animal Experiment 1 was detected by Western blotting. f Quantification of the expression of α-SMA in (e) by ImageJ. Representative Sirius red (scale bar = 100 μm) (g) and α-SMA IHC staining (scale bar = 100 μm) (i) conducted on the liver sections from mice in Animal Experiment 2. h Quantification of Sirius red staining in (g) by ImageJ. j Quantification of α-SMA IHC staining in (i) by ImageJ. k The expression of α-SMA in the liver of mice from Animal Experiment 2 was detected by Western blotting. l Quantification of the expression of α-SMA in (k) by ImageJ. m The expression of FN1 and α-SMA in LX-2 cells was detected by Western blotting. n Quantification of the expression of proteins in (m) by ImageJ. Ctr, control. Data are presented as the mean ± SD. In b, the P value was calculated by Welch’s ANOVA followed by Dunnett’s T3 multiple comparisons test. In d, f, h, j, l and n, the P values were calculated by one-way ANOVA followed by Dunnett’s multiple-comparison test.

Furthermore, we explored whether LBPW could restrain the activation of HSCs in vitro using the LX-2 cell line treated with TGF-β1. The results revealed that LBPW attenuated the synthesis of the α-SMA, COL1A1 and FN1 mRNAs (Supplementary Fig. 4a). Consistently, the protein expression levels of α-SMA and FN1 were strongly suppressed by LBPW (Fig. 2m, n). In addition, we detected the effects of LBPW on the viability of quiescent LX-2 and LO₂ using the cell counting kit-8 (CCK-8) regent and confirmed the safety of LBPW at concentrations ranging from 0.0625 to 1 mg/mL (Supplementary Fig. 4b, c). These data indicated that LBPW could inhibit the TGF-β1-induced activation of HSCs in vitro without causing cytotoxicity.

Taken together, these results suggest that LBPW attenuated CCl₄-induced liver fibrosis in mice following both i.p. and i.g. administration and restrained the activation of HSCs in vivo and in vitro.

Cy5.5-LBPW can be detected both in the liver and intestine

We next characterized the pharmacokinetics of LBPW. LBPW was labeled with Cy5.5 and subsequently tracked using near-infrared imaging to characterize its distribution in vivo after i.p. and i.g. administration. As shown in Fig. 3a–c, we first examined the distribution of Cy5.5-LBPW after i.p. administration. The fluorescence signals of the free Cy5.5 group were mainly concentrated at the abdominal administration site and in the gonad of the male mouse, and they gradually decreased. No distinct trend in the distribution of the free Cy5.5 was observed over time. After the i.p. administration of Cy5.5-LBPW, the fluorescence signal mainly accumulated in the abdomen and gonad of the male mouse. Cy5.5-LBPW began to spread after 30 min and was transported to other organs by 2 h. Fluorescence signals were observed in the heart and lungs after 6 h, and their intensity in all organs gradually decreased after 8 h. These results indicated that Cy5.5-LBPW had a prolonged and wide distribution in vivo, which was distinct from that of free Cy5.5 (Fig. 3a). Then, we performed ex vivo imaging of the main organs. Following the i.p. administration of Cy5.5, the fluorescent signals were predominantly distributed in the liver and kidneys. The fluorescent signal in the liver was strongest between 30 min and 2 h, after which it gradually diminished over time. The Cy5.5 was first detected in the kidneys at 4 h. While the fluorescent signals of Cy5.5-LBPW accumulated mainly in the liver, kidneys and gallbladder from 15 min to 12 h, with the spleen, lungs and heart showing faint fluorescence (Fig. 3b). The average fluorescence intensity‒time curves of the major organs (cholecyst, spleen, liver, lungs, heart, kidneys and brain) indicated that the liver was the main organ where Cy5.5-LBPW was distributed following i.p. administration (Supplementary Fig. 5a). Furthermore, ex vivo imaging of the gastrointestinal tract was also conducted. Only weak fluorescence of Cy5.5 was observed in the gastrointestinal tract, with Cy5.5-LBPW being distributed in the stomach, duodenum, jejunum, ileum, cecum and colon (Fig. 3c). Additionally, we evaluated the distribution of Cy5.5-LBPW after i.g. administration (Fig. 3d–f). The fluorescence signal was primarily detected in the abdominal region of the mouse, with stronger signals observed at 2 h and 6 h until the signal essentially disappeared by 12 h after administration (Fig. 3d). In contrast, the fluorescence intensity of free Cy5.5 peaked 2 h after i.g. administration and subsequently decreased [55]. A small amount of dispersed fluorescence was observed in the liver, spleen, lungs and gallbladder only at 15 min and 30 min after administration (Fig. 3e), and this distribution was almost undisturbed by free Cy5.5 [55]. The relative quantification of the fluorescence intensity in the major organs shown in Supplementary Fig. 5b suggested that the liver was the main organ in which Cy5.5-LBPW was located after i.g. administration. The intestinal distribution is shown in Fig. 3f. Strong fluorescence signals in the intestine gradually advanced from the small intestine to the colon over time. At 12 h after administration, only weak signals were detected in the cecum and colon. In contrast to the Cy5.5-LBPW group, the free Cy5.5 group displayed rapid diffusion of signals throughout the intestine within 1 h, with a subsequent decrease observed after 2 h [55]. Taken together, these collective findings indicated that Cy5.5-LBPW could be detected in organs, irrespective of the mode of administration in our study. The distribution of Cy5.5-LBPW in the liver, kidneys, gallbladder, spleen, lungs, heart and brain following i.p. injection was more pronounced than that observed after i.g. administration (Supplementary Fig. 5c–i). While after i.g. administration, Cy5.5-LBPW was primarily distributed in the intestine, with a minor amount detected in the liver (Fig. 3e, f). However, whether the fluorescent substance that enter the liver and other organs are intact or degraded remains to be determined.

Fig. 3. Real-time distribution of Cy5.5-LBPW following i.p. and i.g. administration via NIR imaging.

a In vivo imaging of the mice after i.p. administration of free Cy5.5 and Cy5.5-LBPW. b Ex vivo imaging of main organs (cholecyst, spleen, lungs, kidneys, brain, heart and liver) after i.p. administration of free Cy5.5 and Cy5.5-LBPW. c Ex vivo imaging of the excised intestine after i.p administration of Cy5.5 and Cy5.5-LBPW, respectively. d In vivo imaging of the mice after i.g. administration of Cy5.5-LBPW. e Ex vivo imaging of main organs (cholecyst, spleen, lungs, kidneys, brain, heart and liver) after i.g. administration of Cy5.5-LBPW. f Ex vivo imaging of the excised intestine after i.g. administration of Cy5.5-LBPW.

LBPW may retard the activation of HSCs by upregulating Smad7 and subsequently inhibiting the TGF-β/Smad signaling pathway

LX-2 cells treated with TGF-β1 in the presence (cHdose group) or absence (cModel group) of LBPW were subjected to RNA sequencing (RNA-Seq) to elucidate the potential molecular mechanism underlying the inhibitory effect of LBPW on HSC activation. The hierarchical clustering analysis of gene expression revealed that the cModel and cHdose groups presented distinct gene expression profiles (Fig. 4a). The TGF-β signaling pathway was downregulated in the cHdose group compared with the cModel group, as shown by the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (Fig. 4b). Indeed, Western blotting analysis confirmed the RNA-seq results, indicating that the expression of TGF-β1, TGFβR2 and p-Smad2/3 was markedly downregulated and suggesting that LBPW might inhibit TGF-β/Smad signaling (Fig. 4c, d). Notably, LBPW downregulated the expression of TGFβR2 but barely changed that of TGFβR1, which is probably due to differences in the trafficking and recycling of receptors [56]. Intriguingly, LBPW significantly upregulated the expression of Smad7 (Fig. 4c, d).

Fig. 4. LBPW hampers the activation of HSCs possibly by upregulating Smad7 and subsequently inhibiting the TGF-β/Smad signaling pathway.

a Hierarchical clustering analysis of differential genes based on the RNA sequencing of the LX-2 cells from cModel (LX-2 induced by TGF-β1) and cHdose (LX-2 treated with 1 mg/mL LBPW in the presence of TGF-β1) group (biological replicates, n = 3). b KEGG-based functional analysis of RNA sequencing data (cHdose vs cModel, results shown are downregulated pathways). c The expression of TGF-β/Smad7 signaling-related proteins in LX-2 cells was detected by Western blotting. d Quantification of the expression of proteins in (c) by ImageJ. e The expression of TGF-β/Smad7 signaling-related and fibrosis-related proteins in LX-2 cells was detected by Western blotting. f Quantification of the expression of proteins in (e) by KwikQuant Image Analyzer. g The expression of Smad7 in the liver of mice in Animal Experiment 1 was measured by Western blotting. h Quantification of the expression of Smad7 in (g) by ImageJ. i The expression of Smad7 in the liver of mice in Animal Experiment 2 was detected by Western blotting. j Quantification of the expression of Smad7 in (i) by ImageJ. Ctr, control; i.p., intraperitoneal injection; i.g., intragastric administration. Data are presented as the mean ± SD. The P values were calculated by one-way ANOVA followed by Dunnett’s multiple-comparison test (d, h, j) or Šídák’s multiple comparisons test (f).

Smad7 acts as a negative regulator of the TGF-β/Smad signaling pathway and has been shown to retard the activation of HSCs and prevent liver fibrosis [21]. Since the in vitro results revealed that LBPW upregulated the expression of Smad7 in activated LX-2 cells, we wondered whether Smad7 was required for the LBPW-mediated inactivation and inhibition of the fibrogenesis of HSCs. We addressed this question by knocking down Smad7 with a siRNA in LX-2 cells. Smad7 knockdown exacerbated fibrosis in TGF-β1-activated LX-2 cells, as evidenced by an apparent increase in the expression of FN1 and a higher level of α-SMA (Fig. 4e, f). We found that silencing Smad7 abolished the inhibitory effect of LBPW on the expression of profibrotic proteins, including α-SMA and FN1 (Fig. 4e, f). Consistently, the decreased expression level of p-Smad2/3 was notably reversed in the Smad7-knockdown group (Fig. 4e, f). Moreover, i.p. and i.g. treatment of LBPW increased Smad7 expression in CCl₄-induced liver fibrosis model mice (Fig. 4g–j). Overall, the upregulation of Smad7 by LBPW substantially impaired the TGF-β/Smad signaling pathway, suggesting that LBPW might rebalance TGF-β/Smad7 signaling.

LBPW modifies the composition of the gut microbiota and increases the abundance of Akkermansia muciniphila

Many studies have suggested a vital role for gut dysbiosis in the progression of liver fibrosis. We next aimed to determine whether LBPW could reverse gut dysbiosis following i.g. administration. Thus, we first performed sequencing of the full-length 16S rRNA gene extracted from the cecal contents of mice to analyze the composition and diversity of the gut microbiota. Principal coordinate analysis (PCoA) based on the Adonis and ANOSIM tests revealed distinct clustering of microbial communities among the control, model (CCl₄), and Hdose groups (CCl₄ + 300 mg ·kg^–1 ·d^–1 LBPW) (P = 0.001) (Fig. 5a, b). The community heatmap revealed that among the top 30 most abundant species, LBPW increased the abundance of A. muciniphila compared with that in the model group, reaching a level close to that in the control group (Fig. 5c). Comparisons between the two groups at the species level via the Wilcoxon rank-sum test and linear discriminant analysis effect size (LEfSe) analysis were subsequently performed to identify distinct species between the model and Hdose groups, revealing that A. muciniphila was significantly enriched after the LBPW intervention (Fig. 5d, e). Furthermore, A. muciniphila formed a co-occurrence relationship with the Hdose group (Fig. 5f). Interestingly, it seemed that the abundance of A. muciniphila was negatively correlated with the serum ALT and AST levels, at least there was a tendency that this bacterium had benefit in liver fibrosis (Fig. 5g). The above results suggest that the effect of LBPW on A. muciniphila may provide another clue to understand the molecular mechanism of action of LBPW in liver fibrosis. We thus verified these results via metagenomics sequencing. A comparison of the taxonomic annotations with the nonredundant protein sequence database revealed that 10,656 species were shared between the Hdose and model groups, whereas 603 species were specific to the Hdose group, and 531 species were unique to the model group (Supplementary Fig. 6a). PCoA revealed a remarkable separation between the model group and the Hdose group (P = 0.003) (Supplementary Fig. 6b). Heatmap analysis showed that LBPW increased the abundance of A. muciniphila compared with that in the model group (Supplementary Fig. 6c). Similarly, A. muciniphila was significantly more abundant in the Hdose group than in the model group (Supplementary Fig. 6d, e).

Fig. 5. LBPW reshapes the composition of gut microbiota and propagates A. muciniphila.

The cecal contents of mice from Animal Experiment 2 were subjected to diversity sequencing based on the full-length 16S rRNA gene. a PCoA analysis based on the bray-curtis distance (Adonis test). b PCoA analysis based on the bray-curtis distance (ANOSIM test). c Community heatmap analysis at the species level. d Comparison between model and Hdose group at the species level by Wilcoxon rank-sum test; ^*P < 0.05, ^**P < 0.01. e LEfSe analysis at the level from genus to species and ranked based on the linear discriminant analysis (LDA) score (threshold = 3). f Co-occurrence network analysis. g Spearman correlation (two-tailed Spearman’s rank test) between the bacterium abundance at the species level and serum AST and ALT levels.

As noted above, the results from 16S rRNA gene sequencing aligned with those of metagenomics sequencing and confirmed that LBPW modified the gut microbiota and that A. muciniphila was a selectively enriched species.

LBPW restores gut barrier function in mice with liver fibrosis

Since mice i.g. treated with LBPW exhibited a modified composition of the gut microbiota, especially an increase in A. muciniphila abundance, which has been demonstrated to improve intestinal barrier function [57, 58], we, therefore, evaluated the gut barrier function in these mice. Compared with those in the model group, the depth of the crypts in the LBPW-treated groups increased, and no obvious inflammatory cells were observed, indicating that LBPW ameliorated colonic lesions (Fig. 6a). Furthermore, the expression of Mucin2 (MUC2, the most important mucin that acts as the first barrier) in the colon and ileum was elevated in the LBPW-treated groups compared with that in the model group, as shown by IHC (Fig. 6b–d). Consistently, TEM revealed that CCl₄ compromised ileal barrier function, as indicated by the loss of tight junctions, which was restored by LBPW (Fig. 6e). Moreover, the expression of occludin (a tight junction protein that serves as a marker of gut barrier integrity) in the colon was increased by LBPW (Fig. 6f, g). Taken together, these results suggested that LBPW could reinforce gut barrier function in mice with CCl₄-induced liver fibrosis. These findings suggested that A. muciniphila might play a crucial role in the inhibitory effect of LBPW on liver fibrosis.

Fig. 6. LBPW improves gut barrier function in mice with liver fibrosis.

Samples examined are from Animal Experiment 2. a H&E staining against the colon sections (black arrow indicates the inflammatory cells) (scale bar = 100 μm). b The expression of MUC2 of the colon and ileum was detected by IHC staining, respectively (scale bar = 100 μm). c The expression of MUC2 of the colon was quantified by ImageJ. d The expression of MUC2 of the ileum was quantified by ImageJ. e Representative TEM images of the ileum epithelium (scale bar = 500 nm), arrows indicate tight junctions. f The expression of occludin in the colon was detected by Western blotting. g Quantification of the expression of occludin in (f) by ImageJ. Ctr, control. Data are presented as the mean ± SD. The P values were calculated by one-way ANOVA followed by Dunnett’s multiple-comparison test.

The decreased abundance of Akkermansia muciniphila caused by antibiotics counteracts the beneficial effects of LBPW

To explore the role of A. muciniphila in the beneficial effects of LBPW on liver fibrosis progression, antibiotics were administered to decrease the abundance of A. muciniphila to detect whether LBPW still exerts inhibitory effects on hepatic injury and fibrosis. First, we screened the susceptibility of A. muciniphila to antibiotics encompassing ampicillin, vancomycin, neomycin and spectinomycin. A. muciniphila was more susceptible to ampicillin (minimum inhibitory concentration (MIC) = 0.5 mg/L), vancomycin (MIC = 64 mg/L) and spectinomycin (MIC = 64 mg/L) but was resistant to neomycin (MIC > 256 mg/L) (Supplementary Fig. 7a). We thus selected ampicillin, vancomycin and spectinomycin as an antibiotics cocktail to suppress A. muciniphila in mice. A schematic diagram of Animal Experiment 3 is shown in Fig. 7a. The cecal contents were collected from model mice treated with LBPW alone or in combination with Abs to evaluate the abundance of A. muciniphila via sequencing of the 16S rRNA gene. PCoA analysis demonstrated distinct clustering patterns in the microbiota composition between the two groups (Supplementary Fig. 7b, c). The community heatmap indicated that the antibiotic cocktail decreased the abundance of A. muciniphila (Supplementary Fig. 7d). Apparently, the decline in the abundance of A. muciniphila following the administration of the antibiotic cocktail was significant (Fig. 7b, Supplementary Fig. 7e, f).

A striking decrease in ALT level, but not AST level, was detected after an antibiotic cocktail was administered to CCl₄-treated mice (Fig. 7c, d). The antibiotic cocktail led to more severe liver pathological damage in the model mice, as indicated by vacuoles within the hepatocytes and more disordered liver lobules (Fig. 7e). LBPW significantly decreased the serum ALT and AST levels, accompanied by a notable amelioration of liver pathology. However, these protective effects of LBPW on liver damage were largely negated by the administration of antibiotics (Fig. 7c–e).

In addition, the ability of LBPW to reduce collagen fiber deposition was vitiated upon the administration of antibiotics (Fig. 7f, g). Moreover, the inhibition of α-SMA expression by LBPW was abolished by the administration of antibiotics (Fig. 7h, i). These results suggested that A. muciniphila might be responsible for the efficacy of LBPW since the decrease in the abundance of A. muciniphila caused by antibiotics offset the beneficial effects of LBPW on liver injury and fibrogenesis.

Next, we explored the effect of a reduced abundance of A. muciniphila on the improvement in colonic barrier function caused by LBPW. As shown in Supplementary Fig. 8a, the colonic lesions of CCl₄-induced hepatic fibrosis model mice treated with antibiotics were deeper, and the effect of LBPW on ameliorating colonic lesions disappeared with the administration of antibiotics. Furthermore, antibiotic treatment markedly reduced the expression of MUC2 in hepatic fibrosis model mice, and the reduced abundance of A. muciniphila offset the ability of LBPW to induce MUC2 expression in the colon (Supplementary Fig. 8b, c). Moreover, the expression of occludin in the colon was notably decreased in fibrosis model mice treated with antibiotics, and the ability of LBPW to increase occludin expression was reversed after the abundance of A. muciniphila was decreased by antibiotics (Supplementary Fig. 8d, e). These results suggested that the reduced abundance of A. muciniphila caused by antibiotics counteracted the beneficial effect of LBPW on restoring colonic barrier function.

Discussion

The hepatoprotective potential of the fruit of L. barbarum has been well documented for thousands of years. However, the bioactive material basis and pharmacological mechanisms of this fruit remain incompletely understood. In this study, we reported that LBPW, which was isolated from the fruit of L. barbarum, is a class of neutral O-linked peptidoglycan (Supplementary Fig. 1) that can be detected both in the liver and intestine and has potent activity against liver damage and fibrosis induced by CCl₄ in vivo and HSC activation in vivo and in vitro. The mechanisms of action of LBPW might be associated with the upregulation of Smad7 to impede the TGF-β/Smad cascade in HSCs and the increase in the abundance of A. muciniphila to refine the intestinal microenvironment (Fig. 8). Our findings increase our understanding of the active macromolecules in the fruit of L. barbarum responsible for hepatoprotection and lay the foundation for the development of health products and innovative drugs against liver fibrosis based on the fruit of L. barbarum.

Fig. 8. Graphical abstract.

LBPW might exert an anti-fibrogenesis effect on CCl₄-induced liver fibrosis in mice via rebalancing TGF-β/Smad7 signaling in HSCs and propagating gut commensal A. muciniphila.

Since some polysaccharide-type biomolecules are ineffective when administered i.g. but effective when administered i.p. [59], we first evaluated the therapeutic effects of LBPW on a CCl₄-induced liver fibrosis mouse model following i.p. injection. Our results indicated that i.p. administration of LBPW at daily doses of 50 mg/kg, 100 mg/kg and 200 mg/kg reversed the liver injury and liver fibrosis phenotypes. The mice were subsequently i.g. administered LBPW, as L. barbarum is taken orally in the clinic. Taking into account the first-pass effect, the maximum dose is 300 mg ·kg^–1 ·d^–1 for i.g. administration. Our data revealed that the i.g. treatment of mice with daily doses of 100 mg/kg, 200 mg/kg and 300 mg/kg LBPW also counteracted liver injury and fibrogenesis. However, the dose dependence was weak, possibly due to the slight variation in the concentration gradient used in the experiments. The reductions of AST and ALT levels following i.p. injection of LBPW is slightly higher than that of i.g. treatment. (Supplementary Tables 5 and 6). Both methods of administration resulted in an increase in the body weight of the mice (Supplementary Fig. 9a, b). However, i.p. administration increased the liver index, whereas i.g. administration decreased it (Supplementary Fig. 9c, d), suggesting a potential side effect of i.p. administration. Combined with the pharmacokinetic studies, we might conclude that i.g. administration was superior to i.p. administration, as i.g. administration of LBPW resulted in a more focused targeting of the target organs.

In particular, we observed that the knockdown of Smad7 in activated LX-2 cells significantly increased the expression of FN1, although the expression of α-SMA was not significantly altered. Xu et al. reported that the knockdown of Smad7 in activated LX-2 cells significantly increased the expression of α-SMA and COL1A1 [28], while a study by Yang et al. showed that this manipulation could not markedly promote fibrosis; however, a trend toward worsening fibrogenesis was observed [26]. Different knockdown efficiencies might be the cause of these discrepancies.

Some complex polysaccharides and protein/peptide-linked glycans remain intact after transit through the stomach and small intestine since the enzymes required for their degradation are absent in humans [60–62]. Hence, they have the chance to reach the lower digestive tract, where the majority of intestinal bacteria live, to regulate the intestinal microenvironment. Here, through 16S rRNA gene sequencing and metagenomics sequencing, we showed that LBPW modulated the composition of the gut microbiota, notably enriching the gut bacterium A. muciniphila. Moreover, the impaired gut barrier was reinforced by LBPW, which might be ascribed to the increased abundance of A. muciniphila, a promising ‘next-generation probiotic’ with the capacity to maintain a healthy gut barrier [47]. Furthermore, the antibiotic cocktail that decreased the abundance of A. muciniphila might abrogate the effects of LBPW on ameliorating liver damage and fibrosis, highlighting that A. muciniphila might play a favorable role in the protective effects of LBPW. Notably, the antibiotic cocktail exacerbated liver injury in the model group, and the marked decrease in ALT level might be attributed to increased hepatocyte damage that resulted in the exhaustion of ALT, which is mainly distributed in the cytoplasm, leading to a reduction in ALT level. However, A. muciniphila did not grow in LBPW-enriched media (Supplementary Fig. 10a, b), and the mechanisms underlying the bloom of A. muciniphila caused by LBPW administration are not clear. Similar findings were reported for oligofructose and Si Miao Formula [63], both of which favor the growth of A. muciniphila in vivo but cannot directly promote the growth of this bacterium in vitro. First, one explanation for this result might be that LBPW increases mucus production by stimulating goblet cells, providing A. muciniphila with its main energy source. Second, we cannot exclude the possibility that LBPW is metabolized by certain bacteria and triggers cross-feeding, benefiting the growth of A. muciniphila.

We found that both i.p. and i.g. treatments of LBPW increased Smad7 expression in CCl₄-induced liver fibrosis model mice, suggesting that the mechanism of these two routes of administration might involve an increase in Smad7 expression by LBPW. Our pharmacokinetic studies revealed that Cy5.5-LBPW could be detected in the liver following i.p. and i.g. administration, which supported this possible mechanism. On the other hand, the distribution of i.g. administered Cy5.5-LBPW, which mainly accumulated in the intestine, ensures the physical premise of LBPW interacts with the intestinal microenvironment. Although Cy5.5-LBPW was also observed in the intestine via near-infrared imaging after i.p. administration, this result might be attributed to the absorption of Cy5.5-LBPW through the peritoneum into the bloodstream and subsequent circulation to the intestinal vessels.

Previous studies have reported that the hepatoprotective effects of L. barbarum fruit are attributed mainly to LBPs, which relieve liver diseases in several models, including alcoholic liver injury [64], high-fat diet-induced MASH [65–67], methionine-choline-deficient-induced hepatic injury [68] and CCl₄-induced fibrosis [48]. The molecular mechanisms underlying these beneficial effects are multifaceted and involve restoring the gut microbiota [67], arresting the TGF-β/Smad pathway [68], and blocking the TLRs/NF-κB signaling pathway [48], among other processes. This study presents the first evidence that the peptidoglycan derived from L. barbarum fruit can ameliorate hepatic fibrosis through the upregulation of Smad7 expression and the propagation of A. muciniphila.

In addition, our study has some limitations. This study presents the general pharmacological effects of LBPW, yet the pharmacodynamic basis remains unknown. By isolating and characterizing the glycan and peptide components of LBPW, their respective roles in liver fibrosis can be elucidated. We also cannot exclude the possibility that LBPW is metabolized by the gut microbiota to produce active metabolites that exert an inhibitory effect on hepatic fibrosis. Finally, the direct target of LBPW related to the TGF-β/Smad7 cascade still needs to be explored.

In summary, the present study is the first to demonstrate that biomacromolecules other than polysaccharides, specifically a peptidoglycan named LBPW, in the fruit of L. barbarum has the potential to attenuate CCl₄-induced hepatic fibrosis in mice. Moreover, these findings suggest that the increased expression of Smad7 and the increase in A. muciniphila abundance contribute to its overall pharmacological effects.

Author contributions

KD and MXL conceived the investigation. KPW conceived the pharmacokinetic experiments. YMN and WQZ designed the experiments. YMN performed most of the activity evaluation and explored the mechanisms. WQZ performed the extraction and isolation of the peptidoglycan. TN performed Western blotting in Animal Experiment 3. MFM performed the chemical composition analysis. YXZ performed fluorescence imaging experiments. YL detected serum ALT and AST levels in Animal Experiment 3. YMN and WQZ performed all data analysis, drafted and revised the manuscript. KD, MXL revised the manuscript.

Funding

The work was supported by the National Key R&D Program of China [2022YFA1303802], the National Natural Science Foundation of China [32271332, 82341097] and Fund of State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences [SIMM0120232001]. This work was partially supported by the High-level Innovative Research Institute [2021B0909050003] from the Department of Science and Technology of Guangdong Province and the Ninth Batch of Innovative Scientific Research Team Projects from Zhongshan Science and Technology Bureau.

Data availability

The data that support the findings of this study are available in the Sequence Read Archive database (accession number PRJNA1097756 for RNA sequencing, PRJNA1106790 for 16S rRNA gene sequencing in Animal Experiment 2, PRJNA1110731 for 16S rRNA gene sequencing in Animal Experiment 3 and PRJNA1108440 for metagenomics sequencing).

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-024-01454-x.