Huanggan decoction ameliorates cholestatic hepatic fibrosis in rats via TGF-β1/Smad3 signaling pathway

TL;DR

Huanggan Decoction helps reduce liver fibrosis in rats by affecting the TGF-β1/Smad3 pathway and reducing inflammation.

Contribution

The study reveals the mechanism by which Huanggan Decoction ameliorates cholestatic liver fibrosis through the TGF-β1/Smad3 signaling pathway.

Findings

HGD reduced serum liver fibrosis biomarkers and inflammatory cytokines in rats.

HGD decreased collagen deposition and improved liver pathology in cholestatic liver fibrosis.

HGD modulated the TGF-β1/Smad3 pathway by altering MMP1, TIMP1, and p-Smad3 levels.

Abstract

Huanggan Decoction (HGD), as a special Chinese medicine preparation, has good effects in clearing heat, removing dampness, eliminating jaundice. HGD has been used in medical institutions for more than 40 years, and shows an outstanding curative effect in jaundice and cholestatic liver diseases (CLD), makes up for the deficiency of the treatment of CLD. However, the underlying mechanisms of HGD for its therapeutic effects are still not well understood.

The hepatoprotective properties of HGD were assessed using a cholestatic liver fibrosis (CLF) rat model induced by ANIT. Serum liver function index was analyzed by automatic chemical analyzer. Serum biomarkers of liver fibrosis and inflammatory factors were detected by ELISA kits. Liver pathology and collagen fiber extent were assessed using HE and Masson’s stains. Expressions of pro-fibrotic cytokine TGF-β1 and the indicator of HSC activation α-SMA in liver were assayed by immunohistochemistry. The levels of Smad3, phosphorylated Smad3, MMP1 and TIMP1 were assayed by western blotting.

HGD dramatically decreased the serum biochemical indexes, down-regulated the serum biomarkers of liver fibrosis and inflammatory cytokines, reduced the collagen deposition, ameliorated pathological damage. At the same time, HGD notably reduced the level of α-SMA. Additionally, HGD increased MMP1 protein level while decreasing TIMP1 protein level and the p-Smad3 to Smad3 ratio.

Findings suggest that HGD demonstrated a remarkable liver-protective effect, potentially linked to halting liver fibrosis progression by maintaining the equilibrium between MMP1 and TIMP1, modulating TGF-β1/Smad signal pathway, suppressing HSC activation, and exhibiting anti-inflammatory characteristics.

Figures

HGD alleviates ANIT-induced CLF liver injury.(A) The experiment design scheme in this study. (B) Body weight of rats between different groups (n = 6 per group). (C) Survival rate of rats between different groups (n = 6 per group). (D) Serum biochemical indicators of ALT, AST, ALP, γ-GT, TBIL and TBA (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, P < 0.01, *P < 0.001, P < 0.0001. ANIT, ɑ-Naphthylisothiocyanate; HGD, Huanggan Decoction; UDCA, Ursodeoxycholic Acid; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; ALP, Alkaline Phosphatase; γ-GT, Γ-Glutamyltranspeptidase; TBIL, Total Bilirubin; TBA, Total Bile Acid.

HGD attenuates ANIT-induced liver fibrosis.(A) HE staining and masson staining of rat liver in different groups (n = 6 per group). (B) Serum biochemical indicators of liver fibrosis in Col-Ⅳ, HA, LN and PC-Ⅲ in different groups (n = 6 per group). (C) Western blot was used to detect the expression of Col-Ⅰ and α-SMA (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, P < 0.01, *P < 0.001. ANIT, ɑ-Naphthylisothiocyanate; HGD, Huanggan Decoction; UDCA, Ursodeoxycholic Acid; Col-Ⅰ, Collagen I; Col-IV, Collagen IV; HA, Hyaluronic Acid; LN, Laminin Protein; PC-III, Procollagen III; α-SMA, α-Smooth Muscle actin.

HGD ameliorates ANIT-induced liver inflammatory responses.(A) Serum pro-inflammatory cytokines of TGF-β, TNF-α, IL-1β and IL-6 in different groups (n = 6 per group). (B) Venny diagram of target gene intersection of HGD and CLF. (C-D) Immunohistochemical detection of TGF-β1 expression in the liver in different groups (n = 6 per group, 40x). (E) Serum pro-inflammatory cytokines of TGF-β, TNF-α, IL-1β and IL-6 in HSC-T6 culture supernatants (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, P < 0.01, *P < 0.001, ns, not significant. ANIT, ɑ-Naphthylisothiocyanate; HGD, Huanggan Decoction; UDCA, Ursodeoxycholic Acid.

HGD modulates cholestasis through the TGF-β1/Smad3 signaling pathway.(A-B) Western blot detection of protein expression and statistical plots of TGF-β1 and Smad3 in different groups (n = 6 per group). (C) The mRNA expression of Col1a1, α-SMA, TGF-β1 and Smad3 in different groups of HSC-T6 cells was detected by qPCR (n = 3). (D-E) The protein expression of Col-Ⅰ, α-SMA, TGF-β1 and Smad3 in different groups of HSC-T6 cells was detected by western blot (n = 3). Data were presented as mean ± SD. *P < 0.05, P < 0.01, *P < 0.001, ns, not significant.

HGD suppress the deposition of ECM in ANIT-induced CLF rats.(A-B) Immunohistochemical detection of α-SMA expression in different groups (n = 6 per group, 20x). (C-D) Protein expressions of MMP1 and TIMP1 in different groups (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, P < 0.01, *P < 0.001.

Full text

1. Introduction

Cholestatic liver disease (CLD) is characterized by progressive biliary injury, accumulation of bile acids, persistent inflammation, and subsequent damage to both cholangiocytes and hepatocytes, resulting from various hepatic disorders [1,2]. CLD is associated with a high incidence, complex etiology, and often unclear origin. Without appropriate intervention, it can progress to liver fibrosis and eventually lead to severe liver diseases [3,4]. The therapeutic approach to CLD primarily focuses on treating the underlying cause or alleviating symptoms. Ursodeoxycholic acid and obeticholic acid are the two most commonly used clinical agents for managing cholestasis. However, up to 40% of patients exhibit no response to UDCA, and more than 90% of patients experience multiple adverse effects during OCA treatment, limiting its continued use. For patients with end-stage primary sclerosing cholangitis, liver transplantation remains the only definitive treatment option [5–8]. Therefore, there is a need to develop drugs for the treatment of CLD.

Currently, there is growing interest in the discovery of novel therapeutics for cholestasis derived from natural plants [9]. Traditional Chinese Medicine (TCM), primarily composed of natural products and their processed forms, offers a distinctive approach based on syndrome differentiation and individualized treatment [10–13]. The role of TCM in the prevention and treatment of CLD is gaining increasing recognition and consensus. Huanggan Decoction (HGD) is a classical TCM formula, which primarily used for the treatment of jaundice and cholestatic liver diseases, with over four decades of clinical application. The prescription of HGD is recorded in the 1995 version of Ningxia Hospital preparation specification, and approved as a preparation of Chinese medicine by Drug Administration of Ningxia Hui Autonomous Region for clinical application (Approval NO: Z20200020000). HGD can clear heat and dampness, promote bilirubin excretion to eliminate jaundice, which can make up deficiency for treating of CLD. However, the systematically hepatoprotective mechanisms of HGD remain unclear.

Liver fibrosis is a pivotal pathological process in the progression of chronic liver diseases to cirrhosis and liver failure, which characterized by intrahepatic connective tissues atypical hyperplasia and excessive extracellular matrix (ECM) deposition [14–16]. Fortunately, the process of hepatic fibrosis is reversible, and early interventions could improve clinical outcomes, which has become a major strategy in cholestasis therapy [14,16]. Research indicates that the activation of hepatic stellate cells (HSCs) plays a vital role in immunomodulation during hepatic damage, resulting in heightened HSC activity and elevated extracellular matrix production [17,18]. Cytokines that promote inflammation, including IL-6, IL-1β and TNF-α participate in HSCs’ activation. Moreover, TGF-β1 is a pro-fibrotic cytokine stimulates HSCs, prevents ECM breakdown by increasing TIMP levels and decreasing MMP levels, resulting in excessive collagen fiber accumulation and advancing hepatic fibrosis [19–22].

Previous experimental studies have shown that HGD significantly reduces serum biochemical markers in rats with ANIT-induced cholestatic liver injury, increases bile flow, and improves hepatic histopathological alterations [23]. Therefore, the present study aimed to investigate how HGD counteracts ANIT-induced (ɑ-naphthylisothiocyanate) cholestatic liver fibrosis (CLF) in rats and to explore the underlying mechanisms that prevent the progression of liver fibrosis, thereby providing a theoretical basis for the use of HGD in the treatment of cholestasis.

2. Methods

2.1 Preparation of HGD

All herbal decoction pieces in the HGD formula were provided by Hebei Shuangning Pharmaceutical Co., Ltd (Hebei, China). Different doses of HGD were prepared by Department of Pharmaceutical Preparation, General Hospital of Ningxia Medical University. ANIT was purchased from Sigma-Aldrich (USA) and UDCA came from Losan Pharma GmbH in Germany. Nanjing Jiancheng Institute of Biotechnology (Nanjing, China) provided the assay kits. The antibodies were sourced from Abcam (Abcam Plc.). HGD was prepared using the traditional water boiling method. Specifically, the raw medicinal ingredients were boiled in water twice, first for 2 hours and then for an additional hour. The resulting decoctions were filtered, combined, and concentrated to 0.815 g/mL^-1^, corresponding to 7.70 g/kg of crude drug. Based on our earlier research [24]. HGD used the same formulation at different concentrations in both in vivo and in vitro experiments.

2.2 Animal experiments

SD rats weighing 200 ± 20g were from the Laboratory Animal Center at Ningxia Medical University (Yinchuan, China). Rats were acclimated for five days before the experiment under suitable temperature and humidity conditions, following a light-dark cycle. Subsequently, they were randomly assigned into control and ANIT group (n = 6 per group). The ratio of males to females in each group was 1:1. Rats in the ANIT group received intragastric administration of 80 mg/kg ANIT every three days for five weeks; rats in the control group received equal amounts of olive oil. CLF rats were divided into five groups, including the ANIT model group (ANIT + vehicle), the UDCA-treated group (ANIT + UDCA), the low/medium/high dose of HGD-treated groups (ANIT + HGD-L, ANIT + HGD-M, ANIT + HGD-H). Rats in HGD-treated groups were intragastrically given HGD at doses of 3.85, 7.70 or 15.40 g/kg, and in UDCA-treated (ursodeoxycholic acid) group received a continuous of UDCA at doses of 63 mg/kg by intragastric administration, twice a day for 3 weeks. UDCA was used as a positive control for the treatment of CLF rats in this study. In the meantime, the regular control group and ANIT model group received an equal volume of saline. The survival and body weight of rats were recorded weekly in the process of experiment. We first analyzed the survival rate of rats, with 8 rats assigned to each group. Following the treatment, surviving rats (n = 6 per group) were fasted for 24 hours and then humanely euthanized in accordance with the AVMA Guidelines for the Euthanasia of Animals (2020). Euthanasia was performed by deep anesthesia with 4–5% isoflurane in oxygen until complete loss of the pedal withdrawal reflex was confirmed, followed by cervical dislocation to ensure death [25,26]. This combination method was chosen to achieve a rapid and painless euthanasia. Serum and liver tissue samples were collected immediately after euthanasia for biochemical and histopathological analyses. Animals that died before the scheduled endpoint were excluded from biochemical and histopathological analyses. Prior to all surgical or invasive procedures, rats were anesthetized with 2–3% isoflurane in oxygen using an induction chamber and maintained under 1.5–2% isoflurane via a nose cone to ensure adequate sedation and minimize pain. Body temperature was maintained at 37°C using a thermostatic heating pad during the procedure. Throughout the experimental period, all efforts were made to minimize animal suffering. Animals were observed at least once daily for signs of pain, distress, or illness, including changes in body weight, posture, fur grooming, jaundice, food and water intake, and general activity. Humane endpoints were predefined, and any rat showing signs of severe distress (e.g., marked lethargy, severe jaundice, inability to access food or water) or a body weight loss exceeding 20% of baseline was humanely euthanized immediately. This study adheres to internationally accepted standards for animal research, following the 3Rs principle. The ARRIVE guidelines were employed for reporting experiments involving live animals, promoting ethical research practices. The design of drug administration scheme is shown in Fig 1A.

*HGD alleviates ANIT-induced CLF liver injury.(A) The experiment design scheme in this study. (B) Body weight of rats between different groups (n = 6 per group). (C) Survival rate of rats between different groups (n = 6 per group). (D) Serum biochemical indicators of ALT, AST, ALP, γ-GT, TBIL and TBA (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ***P < 0.0001. ANIT, ɑ-Naphthylisothiocyanate; HGD, Huanggan Decoction; UDCA, Ursodeoxycholic Acid; ALT, Alanine Aminotransferase; AST, Aspartate Aminotransferase; ALP, Alkaline Phosphatase; γ-GT, Γ-Glutamyltranspeptidase; TBIL, Total Bilirubin; TBA, Total Bile Acid.

2.3 Cell culture

HSC-T6 cells were purchased from Procell (Wuhan, China). The cells were cultured in DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS). All cultures were maintained at 37 °C in a humidified incubator with 5% CO₂ and 95% air. In this study, all experiments were conducted within ten passages after cell acquisition. For TGF-β1 stimulation, a concentration of 10 ng/mL was used after dilution in culture medium. HGD was applied to cell cultures at concentrations of 500 μg/mL.

2.4 Serum biochemistry

For the assessment of certain serum biochemical indicators, our institution is equipped with fully automated analytical instruments. Accordingly, 100 μL of serum was collected from each rat in all groups via orbital venous sinus sampling. An automated chemistry analyzer was adopted to detect the concentrations of serum TBA, TBIL, ALT, AST, γ-GT and ALP levels. Serum indicators of liver fibrosis including type-IV collagen (Col-IV), hyaluronic acid (HA), type-III procollagen (PC-III), laminin (LN), and inflammatory cytokines including transforming growth factor β1 (TGF-β1), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were determined with ELISA kits. In order to ensure the consistency and stability of the ELISA test results, the ELISA kits were obtained from CUSABIO (Wuhan) with the following information: Col-IV (Rat Collagen Type Ⅳ, Col Ⅳ ELISA KIT, CSB-E08883r), HA (Rat Hyaluronic acid, HA ELISA Kit, CSB-E08120r), PC-III (Rat procollagen type III (HPC Ⅲ) ELISA Kit, CSB-E15810r), LN (Rat Laminin, LN ELISA kit, CSB-E04646r), TGF-β1 (Rat Transforming Growth Factor β1(TGF-β1) ELISA kit, CSB-E04727r), TNF-α (Rat TNF-α ELISA kit, CSB-E11987r-IS), IL-1β (Rat Interleukin 1β,IL-1β ELISA Kit, CSB-E08055r-IS), IL-6(Rat Interleukin 6,IL-6 ELISA KIT, CSB-E04640r). The assay was performed according to the manufacturer’s instructions.

2.5 Histopathological assessment

Liver tissues were fixed with 4% paraformaldehyde, encased in paraffin, and cut into 5-μm slices. To assess liver damage and extent of collagen fibers, the samples were later stained using HE and Masson’s trichrome. For HE staining, Sections were deparaffinized in xylene and rehydrated through a graded ethanol series. The slides were stained with hematoxylin for 5 minutes, rinsed with water, and differentiated in acid alcohol. After bluing in running tap water, sections were counterstained with eosin for 2 minutes, dehydrated, cleared, and mounted with coverslips. For Masson’s Trichrome Staining, Paraffin sections were deparaffinized, rehydrated, and stained according to standard Masson’s Trichrome staining protocol. Briefly, tissues were stained with Weigert’s iron hematoxylin for 10 minutes, followed by Biebrich scarlet-acid fuchsin solution. Sections were then differentiated in phosphomolybdic-phosphotungstic acid solution and counterstained with aniline blue. After dehydration and clearing, slides were mounted with coverslips. Collagen fibers appeared blue, cytoplasm red, and nuclei black. Then, the tissue sections were viewed under light microscopy. Fibrotic area was defined as the percentage of blue-stained collagen relative to the total tissue area within each field, as measured using ImageJ software. For each sample, five non-overlapping fields were randomly selected from representative liver sections at 200 × magnification. All image analyses were performed in a blinded manner by two independent investigators. These details have now been added to the Methods section to enhance reproducibility and transparency.

2.6 Immunohistochemistry

Paraffin-embedded sections were deparaffinized, rehydrated, and subjected to antigen retrieval using citrate buffer (pH 6.0) at 95°C for 15–20 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 minutes. After blocking with 5% bovine serum albumin (BSA) for 30 minutes at room temperature, sections were incubated overnight at 4°C with primary antibodies (TGF-β1, 21898–1-AP, Proteintech, Wuhan; α-SMA, 14395–1-AP, Proteintech, Wuhan). The following day, slides were incubated with an appropriate HRP-conjugated secondary antibody (KFA021, Proteintech, Wuhan) for 30 minutes. Staining was visualized using diaminobenzidine (DAB) substrate and counterstained with hematoxylin. Finally, sections were dehydrated, cleared, and mounted. Images were observed by light microscopy.

2.7 Western blotting

Proteins were isolated from liver tissue using a lysis buffer, then separated via SDS-PAGE and transferred onto PVDF membranes. Following electrophoresis and membrane transfer, the blots were blocked by skim milk and then incubated with specific antibodies to MMP1 (26585–1-AP, Proteintech, Wuhan), TIMP1 (16644–1-AP, Proteintech, Wuhan), Smad3 (66516–1-Ig, Proteintech, Wuhan), p-Smad3(80427–2-RR, Proteintech, Wuhan), Col-Ⅰ (67288–1-Ig, Proteintech, Wuhan), α-SMA (14395–1-AP, Proteintech, Wuhan) and GAPDH (60004–1-Ig, Proteintech, Wuhan) overnight at 4°C. Following a 1-hour incubation at ambient temperature with the secondary antibodies (KFA021, Proteintech, Wuhan), the signals were identified and the protein band intensities were measured using Qinxiang chemical analysis software from Clinx Science Instruments, Shanghai.

2.8 Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from rat liver tissues using TRIzol reagent according to the manufacturer’s protocol. The concentration and purity of RNA were assessed using a spectrophotometer. One microgram of total RNA was reverse-transcribed into complementary DNA (cDNA) using a reverse transcription kit (Vazyme, China). Quantitative real-time PCR was performed using a SYBR Green master mix (Vazyme, China) on a real-time PCR system. Gene-specific primers were used for the detection of target genes. Relative gene expression levels were calculated using the 2^^–ΔΔCt^ method and normalized to the expression of GAPDH as the internal control. Primer details are listed in Table 1.

Table 1: The primer sequences used in this study.

2.9. Statistical analysis

The data were shown as the average value plus or minus the standard deviation (SD). The differences between the groups were assessed by one-way ANOVA test using Graphpad prism software. Difference was considered statistically significant when P-value less than 0.05.

3. Results

3.1 HGD alleviates ANIT-induced CLF liver injury

In this study, an ANIT-induced CLF rat model was established. To investigate the effects of HGD on CLF, rats were administered HGD at different concentrations via oral gavage (Fig 1A). The results showed that rats in the ANIT group exhibited significant reductions in body weight and survival rate, whereas HGD treatment effectively alleviated weight loss and improved survival in a dose-dependent manner. Notably, the high-dose HGD group (HGD-H) demonstrated therapeutic effects comparable to those of the positive control, UDCA (Fig 1B–1C). In addition, serum liver injury markers were markedly elevated in the ANIT group, while HGD administration significantly reduced the expression of these markers in a dose-dependent fashion (Fig 1D). Collectively, these findings suggest that HGD ameliorates ANIT-induced liver injury in rats.

3.2 HGD attenuates ANIT-induced liver fibrosis

To further elucidate the protective effects of HGD in the ANIT-induced liver fibrosis model, collagen accumulation was evaluated using immunohistochemical analysis. Masson’s staining revealed extensive extracellular matrix deposition (blue regions) in the livers of ANIT-treated rats, which was markedly reduced following HGD treatment (Fig 2A). These findings provide visual evidence of collagen deposition in ANIT-induced CLF rat tissues. To more comprehensively assess the antifibrotic properties of HGD, we analyzed serum markers of hepatic fibrosis, including Col-IV, LN, HA and PC-III (Fig 2B). Levels of Col-IV, LN, HA, and PC-III were significantly elevated in the ANIT group. However, compared with the ANIT group, all HGD-treated groups showed significantly reduced concentrations of HA, LN and Col-IV in a dose-dependent manner. To verify the antifibrotic effect of HGD in vivo, the expression levels of Col-I and α-SMA were examined by Western blot analysis (Fig 2C). Consistently, HGD treatment resulted in a dose-dependent decrease in the protein expression of Col-I and α-SMA. These results indicate that HGD confers robust hepatic protection and effectively attenuates ANIT-induced liver fibrosis.

*HGD attenuates ANIT-induced liver fibrosis.(A) HE staining and masson staining of rat liver in different groups (n = 6 per group). (B) Serum biochemical indicators of liver fibrosis in Col-Ⅳ, HA, LN and PC-Ⅲ in different groups (n = 6 per group). (C) Western blot was used to detect the expression of Col-Ⅰ and α-SMA (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001. ANIT, ɑ-Naphthylisothiocyanate; HGD, Huanggan Decoction; UDCA, Ursodeoxycholic Acid; Col-Ⅰ, Collagen I; Col-IV, Collagen IV; HA, Hyaluronic Acid; LN, Laminin Protein; PC-III, Procollagen III; α-SMA, α-Smooth Muscle actin.

3.3 HGD ameliorates ANIT-induced liver inflammatory responses

The occurrence and progression of cholestasis disease accompany an inflammatory response, which can aggravate cholestasis. To investigate the effect of HGD on ANIT-induced inflammation, we analyzed pro-inflammatory cytokines in the serum of cholestatic rats. The results showed that the serum levels of pro-inflammatory cytokines, including TGF-β1, TNF-α, IL-1β, and IL-6, were significantly elevated in the ANIT group compared with the normal control group. In contrast, HGD treatment at various doses significantly reduced the levels of these pro-inflammatory cytokines compared to the ANIT group (Fig 3A). Furthermore, by analyzing the target genes of the herbal components in HGD via the TCMSP database and integrating them with CLF-related targets, we found that HGD may regulate cholestasis by modulating TGF-β1 and Smad3 signaling pathways (Fig 3B). Considering that TGF-β1 is a well-recognized cytokine involved in fibrosis, we assessed its hepatic protein expression using immunohistochemistry. Interestingly, HGD significantly attenuated the ANIT-induced upregulation of TGF-β1, although the low-dose HGD group (HGD-L) did not exhibit a notable reduction in TGF-β1 expression (Fig 3C–3D). Subsequently, we treated HSC-T6 cells with TGF-β1 to establish an in vitro hepatic fibrosis model. Treatment with 10 ng/mL TGF-β1 for 24 hours significantly increased the levels of TNF-α, IL-1β, and IL-6 in the cell culture supernatant, whereas HGD significantly decreased the expression of these pro-inflammatory factors (Fig 3E). Collectively, these results suggest that HGD effectively ameliorates inflammation associated with cholestasis.

*HGD ameliorates ANIT-induced liver inflammatory responses.(A) Serum pro-inflammatory cytokines of TGF-β, TNF-α, IL-1β and IL-6 in different groups (n = 6 per group). (B) Venny diagram of target gene intersection of HGD and CLF. (C-D) Immunohistochemical detection of TGF-β1 expression in the liver in different groups (n = 6 per group, 40x). (E) Serum pro-inflammatory cytokines of TGF-β, TNF-α, IL-1β and IL-6 in HSC-T6 culture supernatants (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001, ns, not significant. ANIT, ɑ-Naphthylisothiocyanate; HGD, Huanggan Decoction; UDCA, Ursodeoxycholic Acid.

3.4 HGD modulates cholestasis through the TGF-β1/Smad3 signaling pathway

The TGF-β signaling pathway plays a crucial regulatory role in liver diseases. Our preliminary analysis also indicated that HGD may modulate the TGF-β1/Smad3 signaling pathway. The results of this signaling pathway showed that HGD reduced the expression levels of ANIT-induced TGF-β1 and p-Smad3 (Fig 4A–4B). We further validated the regulatory effect of HGD using HSC-T6 cells. Consistently, HGD effectively decreased both the mRNA and protein expression levels of TGF-β1 and Smad3/p-Smad3 (Fig 4C–4D). Moreover, HGD suppresses the activation of HSC-T6 cells by reducing the expression of Col-Ⅰ and α-SMA (Fig 4C–4D). These findings suggest that HGD may exert its therapeutic effects through modulation of the TGF-β1/Smad3 signaling pathway.

*HGD modulates cholestasis through the TGF-β1/Smad3 signaling pathway.(A-B) Western blot detection of protein expression and statistical plots of TGF-β1 and Smad3 in different groups (n = 6 per group). (C) The mRNA expression of Col1a1, α-SMA, TGF-β1 and Smad3 in different groups of HSC-T6 cells was detected by qPCR (n = 3). (D-E) The protein expression of Col-Ⅰ, α-SMA, TGF-β1 and Smad3 in different groups of HSC-T6 cells was detected by western blot (n = 3). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001, ns, not significant.

3.5 HGD suppress the deposition of ECM in ANIT-induced CLF rats

When the liver encounters external triggers, dormant hematopoietic stem cells are activated by a significant elevation of TGF-β1, transforming into myofibroblasts that highly express α-SMA and promote the secretion and accumulation of ECM. Our immunohistochemical results showed that α-SMA expression was extremely low in the conventional control group. α-SMA positive expression was evident in the portal area and central vein of the ANIT group. α-SMA protein expression in the liver was greatly reduced by HGD, which manifested itself as lighter staining and a decrease in the number of positive cells (Figs 5A-5B). The balance between ECM synthesis and degradation is tightly regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), with MMPs facilitating ECM degradation and TIMPs preventing ECM breakdown by inhibiting MMP activity. Therefore, maintaining the equilibrium between MMPs and TIMPs is critical for ECM turnover. Western blot analysis showed that, compared with the normal control group, MMP1 expression was significantly decreased in the ANIT group. However, this suppression of MMP1 expression was partially reversed by HGD treatment in a dose-dependent manner. Concurrently, TIMP1 expression was significantly upregulated in the ANIT group compared with controls (Fig 5C–5D), while HGD administration markedly reduced TIMP1 levels across all doses. Taken together, these findings suggest that HGD inhibits ECM accumulation by modulating the balance between MMPs and TIMPs.

*HGD suppress the deposition of ECM in ANIT-induced CLF rats.(A-B) Immunohistochemical detection of α-SMA expression in different groups (n = 6 per group, 20x). (C-D) Protein expressions of MMP1 and TIMP1 in different groups (n = 6 per group). Data were presented as mean ± SD. *P < 0.05, **P < 0.01, **P < 0.001.

4. Discussion

Cholestasis is a medical condition resulting from disruptions in bile acid balance, leading to accumulating harmful bile components in liver and blood, thereby worsening the progression of the disease [27–29]. Reversing the progression of liver fibrosis has become a new therapy for cholestatic diseases. As an alternative and complementary medicine, TCM provides a new opportunity for cholestasis treatment [30,31]. Modern pharmacological studies have shown that TCM compound intervention has excellent potential for delaying or reversing liver fibrosis in cholestatic liver diseases [32,33]. Interestingly, different Chinese herbal medicines exhibit varying indications for different stages of cholestatic liver disease. Yin Chen is suitable for cholestasis’ [34], Chi Shao for cholestatic hepatitis [35], Fu Ling for liver fibrosis to cirrhosis [36], and Huang Qi Decoction for liver fibrosis [37]. HGD is a classic prescription for treating liver diseases more than 40 years. HGD is composed of Artemisia capillaris Thunb (Yinchen, YC), Angelica sinensis (Oliv.), Diels (Danggui, DG), Scutellaria baicalensis Georgi (Huangqin, HQ), Gentiana scabra Bunge (Longdan, LD), Phellodendron chinense C.K.Schneid (Huangbo, HB), Curcuma longa L. (Yujin,YJ), Melia toosendan Siebold & Zucc (Chuanlianzi, CLZ), Poria cocos (Schw.) Wolf (Fuling, FL), Plantago asiatica L. (Cheqianzi, CQZ), Dioscorea opposita Thunb (Shanyao, SY), Rheum palmatum L. (Dahuang, DH), Hordeum vulgare L. (Maiya, MY), Crataegus pinnatifida Bunge (Shanzha, SZ), Massa Medicata Fermentata (Liushenqu, LSQ). The names of the plants have been verified using the ‘World Flora Online’ database (https://www.worldfloraonline.org/). Despite the fact that HGD has a foundation of more than 40 years of clinical use, little is known about its mechanism of action in the treatment of jaundice and cholestatic liver diseases. This research utilized an ANIT-induced CLF rat model to examine HGD’s liver-protective properties and delve into the potential mechanisms involved in slowing liver fibrosis progression.Our preliminary experimental research showed that HGD could still effectively ameliorate liver function and histopathological changes on liver injury in rats.

Pharmacokinetic and pharmacodynamic (PK/PD) characterization of multi-herb decoctions remains challenging; however, integrated PK-based strategies have been applied to identify putative effective components of related anti-fibrotic decoctions, illustrating that multiple constituents with distinct PK profiles may act together to produce the therapeutic effect. These studies support a component-centric approach (profiling major bioactives and metabolites) when interpreting in vivo efficacy and when planning translational development [38]. Regarding safety, acute and subacute toxicity studies of related decoctions and standardized extracts generally report acceptable tolerability in rodent models at therapeutic-equivalent doses, whereas the combined chronic toxicity and systemic human safety data for HGD have been clinically validated for more than four decades.

ANIT is frequently utilized to construct models of human intrahepatic cholestatic disease by damaging biliary epithelial cells and liver cells [39]. Our research showed that HGD notably decreased collagen buildup in liver tissue, which was verified through HE and Masson staining. At the same time, HGD reduced the concentrations of serum γ-GT, ALP, ALT, AST, TBA and TBIL levels in CLF rats. Additionally, the levels of hepatic fibrosis serum indicators such as HA, PC-III, LN, and Col-IV were notably reduced following HGD therapy.

The stimulation of HSCs is crucial in the progression of liver fibrosis. In cholestasis, hepatocyte damage triggers inflammation and the release of pro-inflammatory cytokines, which promotes the excessive synthesis and deposition of ECM by activating quiescent HSCs, subsequently lead to liver fibrosis [40]. Additional research showed that varying amounts of HGD therapy notably reduced the blood concentrations of pro-inflammatory cytokines including TGF-β1, TNF-α, IL-1β and IL-6 in comparison to the ANIT-induced model group. Furthermore, administering various doses of HGD to CLF rats can notably reduce the levels of α-SMA protein and the pro-fibrogenic agent TGF-β1 in liver tissue. Additionally, western blot analysis confirmed a noticeable decline in the p-Smad3 to Smad3 ratio within the TGF-β1/Smad signal pathway. Although Smad3 mRNA expression was significantly downregulated, total Smad3 protein levels remained stable, while phosphorylated Smad3 levels were notably reduced. This discrepancy suggests that Smad3 protein may be maintained through post-transcriptional mechanisms or enhanced protein stability. The selective reduction in p-Smad3 indicates that the inhibitory effect of HGD may primarily target Smad3 activation rather than its overall expression, highlighting a regulatory checkpoint at the level of phosphorylation.

The synthesis and breakdown of ECM are regulated by MMPs and TIMPs activity. During liver fibrosis, the equilibrate of MMPs and TIMPs will be disturbed, keeping MMPs and TIMPs in balance is a key potential in liver fibrosis treatment. Our research revealed a notable reduction in MMP1 protein level and a significant rise in TIMP1 expression in CLF rats. Following HGD therapy, the irregular levels of MMP1 and TIMP1 proteins caused by ANIT were normalized, demonstrating that HGD can decrease ECM buildup in the liver.

However, this study also has certain limitations. Macrophages are the primary producers of TGF-β1 in the liver. We did not further investigate whether the reduction in TGF-β1 caused by HGD might result from decreased macrophage accumulation. Furthermore, although hematopoietic stem cells play a role in ECM deposition, duct cells also play an important role in cholestatic diseases. We did not effectively evaluate duct cell markers or proliferation. Canalicular cell profiling represents an important avenue for future research to further elucidate the mechanisms of HGD in cholestatic liver injury.

5. Conclusion

To sum up, this research shows that HGD offers notable liver protection against ANIT-induced damage and fibrosis, as indicated by lowered liver injury and cholestasis markers, reduced serum indicators of liver fibrosis and inflammatory cytokines, improved inflammation caused by ANIT, and decreased ECM accumulation. The liver-protective effects of HGD might be linked to halting liver fibrosis advancement by breaking TGF-β1/Smad pathways, reducing inflammation and HSCs activation, and maintaining the equilibrium between MMP1 and TIMP1. Nonetheless, further detailed studies on the molecular mechanisms of HGD are required in upcoming research.

Supporting information

S1 FileRaw data.(XLSX)

S2 FileWestern blots.(ZIP)