Abstract

Background and purpose

   As a traditional Chinese medicine formula, Yangyinghuoxue Decoction
   (YYHXD) is used clinically for therapy of hepatic fibrosis. The
   pharmacological profile of YYHXD comprises multiple components acting
   on many targets and pathways, but the pharmacological mechanisms
   underlying its efficacy have not been thoroughly elucidated. This study
   aimed at probing the pharmacological mechanisms of YYHXD in the
   treatment of hepatic fibrosis.

Methods

   YYHXD aqueous extract was prepared and quality control using HPLC-MS
   fingerprint analysis was performed. A CCl[4]-induced rat model of
   hepatic fibrosis was established, and animals were randomly assigned to
   six groups: control, low-dose YYHXD (L-YYHXD), medium-dose YYHXD
   (M-YYHXD), high-dose YYHXD (H-YYHXD), CCl4 model, and colchicine group.
   Rats in the treatment groups received daily oral administration of
   YYHXD (5, 10, or 20 g/kg) or colchicine (0.2 mg/kg) for 6 weeks, while
   the control and model groups received distilled water. Histological
   analysis, including hematoxylin and eosin (HE) and Masson’s trichrome
   staining, was performed to evaluate hepatic fibrosis. Serum biochemical
   markers, such as AST, ALT, HA, and LN, were measured. Inflammatory
   cytokines (IL-6 and TNF-α) and oxidative stress indicators (SOD,
   GSH-Px, and MDA) in hepatic tissue were also assessed. Additionally,
   transcriptomic analysis using RNA-sequencing was conducted to identify
   differentially expressed genes (DEGs) between the control, CCl4 model,
   and H-YYHXD groups. Bioinformatics analysis, including differential
   expression analysis, protein-protein interaction analysis, and
   functional enrichment analysis, were performed to probe the
   pharmacological mechanisms of YYHXD. The regulatory effects of YYHXD on
   fatty acid metabolism and biosynthesis were further confirmed by Oil
   Red O staining, enzyme activity assays, qPCR, and Western blotting.
   Western blotting and immunofluorescence staining also validated the
   involvement of the AMPK signaling pathway in the occurrence and
   progression of hepatic fibrosis.

Results

   HE and Masson’s trichrome staining revealed reduced collagen deposition
   and improved liver architecture in YYHXD groups compared to the CCl[4]
   model group. Serum biochemical markers, including AST, ALT, HA, and LN,
   were significantly improved in the YYHXD-treated groups compared to the
   CCl[4] model group. The levels of inflammatory cytokines (IL-6 and
   TNF-α) and oxidative stress indicators (decreased SOD and GSH-Px,
   increased MDA) in hepatic tissue were significantly ameliorated by
   YYHXD treatment compared to the CCl[4] model group. Moreover, 96 genes
   implicated in YYHXD therapy of hepatic fibrosis were screened from the
   transcriptomic data, which were principally enriched in biological
   pathways such as fatty acid metabolism and biosynthesis, and the AMPK
   signaling pathway. Oil Red O staining showed reduced hepatic lipid
   accumulation by YYHXD in a dose-dependent manner, along with decreased
   serum TG, TC, and LDL-C levels. Additionally, qPCR and Western blot
   analyses demonstrated upregulated mRNA and protein expression of key
   enzymes involved in fatty acid metabolism and biosynthesis, Fasn and
   Fads2, modulated by YYHXD. YYHXD also dose-dependently enhanced
   phosphorylation of AMPK as evidenced by Western blotting and
   immunofluorescence assays.

Conclusion

   YYHXD ameliorated CCl[4]-induced hepatic fibrosis in rats through
   pharmacological mechanisms that involved manifold targets and pathways,
   including aliphatic acid synthesis and metabolism pathways and the AMPK
   signaling pathway. This study provided a reference and basis for
   further research and clinical utilization of YYHXD.

   Keywords: hepatic fibrosis, Yangyinghuoxue Decoction (YYHXD),
   transcriptomics, aliphatic acid synthesis and metabolism, AMPK
   signaling pathway

1 Introduction

   Hepatic fibrosis, a pathological process involving deposition of
   fibrous connective tissue in the liver, is a consequence of diverse
   chronic hepatic conditions, such as alcoholic hepatitis, hepatitis B
   virus infection, hereditary and metabolic liver disorders, and
   cholestasis ([33]Jangra et al., 2022). It is essentially a
   manifestation of the body’s self-repair process that occurs over the
   course of chronic liver injury ([34]Yang et al., 2022). While hepatic
   fibrosis serves as an integral part of the evolution of chronic hepatic
   diseases into cirrhosis or hepatocellular carcinoma (HCC), the abnormal
   tissue structure formed by this repair response is reversible and
   restorable ([35]Caligiuri et al., 2021). The pathological hallmark of
   hepatic fibrosis is excessive release and accretion of extracellular
   matrix (ECM). Activated hepatic stellate cells are responsible for the
   majority of the ECM release and are critical players in the occurrence
   of hepatic fibrosis. Currently, there is no FDA-approved anti-fibrotic
   drug for clinical use, emphasizing the urgent clinical need for
   anti-fibrotic therapy.

   Yangyinghuoxue Decoction (YYHXD) is a traditional Chinese medicine
   (TCM) formula that has the effects of yangyinghuoxue, promoting qi flow
   and resolving stasis ([36]Liang et al., 2023). Clinically, YYHXD can be
   used to treat hepatic fibrosis. Cirrhosis and hepatoma caused by yin
   deficiency with blood stasis, qi stagnation, and blood stasis ([37]Zhou
   and Liang, 2015; [38]Liang et al., 2023). YYHXD is a derivative of Siwu
   Tang, a classic Chinese botanical formula that has been shown to
   ameliorate hepatic fibrosis by reducing lipid accretion ([39]Chiu et
   al., 2016), regulating bile acid metabolism ([40]Xue et al., 2021), and
   modulating immune function ([41]Ma et al., 2022). YYHXD consists of 14
   botanical medicines: Danggui, Dihuang, Chuanxiong, Baishao, Chaihu,
   Chenpi, Chishao, Dahuang, Danshen, Zelan, Huangqin, Zhebeimu, Gancao,
   and Dazao. According to the principles of traditional Chinese medicine,
   the combination of these botanical drugs in YYHXD may exert synergistic
   effects in nourishing blood, promoting blood circulation, resolving
   blood stasis, regulating qi flow, and related therapeutic actions.
   Previous studies have demonstrated that YYHXD can ameliorate
   CCl4-induced hepatic fibrosis in rats ([42]Ma et al., 2018; [43]Liang
   et al., 2022). However, the pharmacological mechanisms underlying YYHXD
   therapy of hepatic fibrosis are unclear. Additionally, like other
   traditional Chinese medicines, understanding of the pharmacological
   mechanisms of YYHXD is limited by its manifold components and their
   manifold targets and pathways.

   Transcriptomics is a technique that is utilized to explore gene
   expression profiles, which helps to elucidate gene regulation,
   signaling pathways, and their interactions. Transcriptomics has
   significant value in TCM pharmacological research, especially when
   deconvoluting manifold metabolites and targets ([44]Zhang et al.,
   2021). By quantifying mRNA expression of all genes in cells or tissues,
   transcriptomics can unravel multiple target effects. Moreover,
   transcriptomics can build a TCM pharmacological network model, which
   describes the array of metabolites and targets that are characteristic
   of TCM, allowing more accurate prediction of TCM efficacy through
   systems biology and network medicine approaches ([45]Liu et al., 2021).
   Therefore, in this work, the pharmacological mechanisms underlying
   YYHXD therapy of hepatic fibrosis were first probed using a
   transcriptomic approach combined with experimental validation. The
   study workflow is illustrated in [46]Figure 1.

FIGURE 1.

   [47]FIGURE 1
   [48]Open in a new tab

   Workflow diagram summary.

2 Materials and methods

2.1 Preparation of YYHXD

   YYHXD is a botanical formula consisting of 14 crude drugs in a fixed
   ratio: Bai-shao, Chai-hu, Chen-pi, Chi-shao, Chuan-xiong, Da-huang,
   Da-zao, Dan-shen, Dang-gui, Huang-qin, Gan-cao, Di-huang, Ze-lan and
   Zhe-beimu in a ratio of 4:5:5:5:5:3:3:4:4:4:3:4:3:3 ([49]Table 1). The
   School of Traditional Chinese Medicine at Ningxia Medical University
   provided all of the botanical drugs used. The quality of all botanical
   drugs was corroborated by the School of Pharmacy of Ningxia Medical
   University. To prepare the medicine, the prescription was crushed and
   heated twice with eight volumes of water for 1 h each time. The
   extracts from the two extractions were filtered, combined and
   concentrated under reduced pressure at 70°C to yield a condensed
   extract with a concentration of 1.0 g crude drug per mL. This extract
   was used for quality control and animal administration.

TABLE 1.

   Composition of YYHXD.
   Chinese name English name Latin name Application area Raw botanical
   drug weight (g)
   Bai-shao White Peony Root Paeonia lactiflora Pall. Root 12
   Chai-hu Bupleurum Root Bupleurum chinense DC. Root and rhizome 15
   Chen-pi Tangerine Peel Citrus reticulata Blanco Fruit peel 15
   Chi-shao Red Peony Root Paeonia lactiflora Pall. Root 15
   Chuan-xiong Sichuan Lovage Rhizome Ligusticum sinense “Chuanxiong” Root
   15
   Da-huang Rhubarb Rheum palmatum L. Root and rhizome 9
   Da-zao Chinese Date Ziziphus jujuba Mill. Fruit 9
   Dan-shen Salvia Root Salvia miltiorrhiza Bunge. Root and rhizome 12
   Dang-gui Chinese Angelica Root Angelica sinensis (Oliv.) Diels Root 12
   Huang-qin Baikal Skullcap Root Scutellaria baicalensis Georgi Root 12
   Gan-cao Licorice Root Glycyrrhiza uralensis Fisch. Root and rhizome 9
   Di-huang Rehmannia Root Rehmannia glutinosa (Gaert.) Libosch. ex Fisch.
   et Mey. Root 12
   Ze-lan Common Bletilla Tuber Lycopus lucidus Turcz. var. hirtus Regel
   Tuber 9
   Zhe-beimu Zhejiang Fritillary Bulb Fritillaria thunbergii Miq. Bulb 9
   [50]Open in a new tab

2.2 Quality control of YYHXD

   To ensure the quality of YYHXD, a high-performance liquid
   chromatography-mass spectrometry (HPLC-MS) fingerprint of the final
   extract was determined. Separation of YYHXD extract by HPLC was
   performed on a C18 column (100 mm × 2.1 mm, 2.6 µm) (Thermo Fisher
   Scientific, Wilmington, NC, United States) with a column temperature of
   30°C and a flow rate of 1.0 mL/min. The sample injection volume was
   0.5 µL. The mobile phase consisted of acetonitrile (A) and water (B).
   The separation conditions were as follows: 0–10 min, 8% A; 10–20 min,
   8%–20% A; 20–30 min, 20%–30% A; 30–35 min, 30%–50% A; 35–40 min,
   50%–55% A; 40–50 min, 55%–60% A; and 50–60 min, 90% A. The mass
   spectrometer was equipped with an electrospray ionization source
   operating in both positive and negative modes, with spray voltages set
   at 3,500 and −3,500 V, respectively. The capillary temperature was set
   at 320°C, and the ion scanning range was 100–1,000 m/z. Pure nitrogen
   gas was used as the carrier gas at a flow rate of 1.0 L/h. Finally, the
   acquired mass spectral data were compared with those of known reference
   standards to ensure the quality and consistency of the YYHXD extract.

2.3 Animals

   Sixty male Wistar rats (200 ± 10 g) were sourced from the Experimental
   Animal Center of Ningxia Medical University. The ethical protocols for
   the study were compliant with the Animal Management Regulations
   provided by the Ministry of Health of the People’s Republic of China,
   and were ratified by the Ethics Committee of Ningxia Medical University
   (Approval No. IACUC-NYLAC-2021-009). The rats were housed under
   controlled environmental conditions (temperature: 22°C–24°C, humidity:
   45%–50%, 12-h light/dark cycle), with ad libitum access to food and
   water. Following a 1-week adaptation period, the rats were randomly
   assigned into six groups (n = 10 per group), including a control group,
   a carbon tetrachloride (CCl4) model group, a low-dose YYHXD group
   (L-YYHXD), a mid-dose YYHXD group (M-YYHXD), a high-dose YYHXD group
   (H-YYHXD), and a colchicine group.

2.4 Drug administration

   For hepatic fibrosis induction, all groups, except the control group,
   were subjected to subcutaneous injections of CCl[4] (Sigma-Aldrich,
   Shanghai, China) diluted with 40% olive oil ([51]Zhang et al., 2013;
   [52]Fan et al., 2018; [53]Kong et al., 2023). The initial dose of CCl4
   was 5 mL/kg, administered for the first week. Subsequently, the rats
   received 3 mL/kg of CCl[4] injections twice weekly for the remaining 5
   weeks. Starting from the fifth week, the rats in the L-YYHXD, M-YYHXD
   and H-YYHXD groups received daily oral administration of YYHXD
   suspension (prepared in distilled water) at doses of 5, 10 and
   20 g/10 mL/kg, respectively, for 6 weeks. Rats in the colchicine group
   received daily oral administration of a colchicine suspension mixed in
   distilled water at a dose of 0.2 mg/10 mL/kg for 6 weeks. The control
   and CCl[4] model groups were orally administered 10 mL/kg of distilled
   water daily for 6 weeks.

   All rats were euthanized 24 h after the final drug treatment using 3%
   pentobarbital sodium (Servicebio Technology). Blood samples were
   harvested from the inferior vena cava, and hepatic tissues were
   acquired.

2.5 Histological analysis

   To evaluate liver pathology, tissue was fixed in 10% formalin for 24 h,
   harvested, and then fixed again in 4% paraformaldehyde. Subsequently,
   the tissue was sliced into 5 µm sections and subjected to hematoxylin
   and eosin (HE) (Solarbio, Beijing, China) staining to observe
   histological changes. Masson’s trichrome (Sangon, Shanghai, China)
   staining was used to assess the degree of hepatic fibrosis. This
   analysis of hepatic tissue morphology and the extent of fibrosis
   enabled deeper understanding of the therapeutic effects of YYHXD on
   hepatic fibrosis.

2.6 Serum and hepatic tissue biochemical analysis

   Colorimetric assay kits from Shanghai Enzyme Research Biotechnology
   (Shanghai, China) were utilized to determine the levels of specific
   serum liver enzymes, including aspartate aminotransferase (AST) and
   alanine aminotransferase (ALT), according to the manufacturer’s
   instructions. Enzyme-linked immunosorbent assay (ELISA) kits from
   Bioswamp (Wuhan, China) were employed to determine serum levels of
   hyaluronic acid (HA) and laminin (LN) in accordance with the
   manufacturer’s instructions. Interleukin-6 (IL6) and tumor necrosis
   factor-alpha (TNF-α) concentrations in hepatic tissue were assessed
   using ELISA kits from LC-Bio Technologies (Hangzhou, China) following
   the manufacturer’s instructions. To assess the extent of oxidative
   stress in each hepatic tissue group, the activities of superoxide
   dismutase (SOD) and glutathione peroxidase (GSH-Px), together with the
   level of malondialdehyde (MDA), were determined using an assay kit from
   Nanjing Jiancheng Biological Engineering Institute (Jiangsu, China)
   following the provided instructions. Additionally, levels of
   triglycerides (TG), total cholesterol (TC), and low-density lipoprotein
   cholesterol (LDL-C) in hepatic tissue were determined using a
   commercial assay kit from the same supplier in accordance with the
   manufacturer’s instructions.

2.7 RNA extraction and sequencing

   Hepatic tissue samples were harvested from nine rats: three from the
   CCl[4] model group, three from the control group, and three from the
   H-YYHXD group. TRIzol Reagent (Life Technologies, Carlsbad, CA, United
   States) was employed for total RNA extraction from hepatic tissue
   samples according to the manufacturer’s instructions. RNA purity was
   ascertained using a NanoDrop 2000 spectrophotometer (Thermo Fisher
   Scientific). The NEBNextUltra™ RNA library prep kit (New England
   Biolabs, Ipswich, MA, United States) was used to generate the cDNA
   library according to the manufacturer’s guidelines. The library quality
   was determined using an Agilent Bioanalyzer 2100 (Agilent Technologies,
   Santa Clara, CA, United States). In addition, cluster generation was
   completed on a cBot Cluster Generation System utilizing TruSeq PE
   Cluster Kit v4-cBot-HS (Illumina, San Diego, CA, United States) in
   accordance with the manufacturer’s instructions, followed by library
   sequencing on an Illumina platform. Clean reads were acquired in
   accordance with the standard protocols for high-quality sequencing
   data. Finally, gene expression was quantified using the reads per
   kilobase per million reads (RPKM) method, which is generally employed
   for transcriptomic studies to survey gene expression.

2.8 RNA sequencing (RNA-seq) data analysis

2.8.1 Gene differential expression analysis

   Differential gene expression analysis was conducted using the “limma” R
   package. Differentially expressed genes (DEGs) in control and CCl[4]
   model groups, as well as YYHXD and CCl[4] model groups, were screened
   with the threshold of |Fold change| ≥ 2 and p-value <0.01. Genes that
   were significantly elevated in the CCl[4] model group compared with the
   control group, and simultaneously significantly suppressed in the YYHXD
   group compared with the CCl[4] model group were selected, as well as
   genes with significantly lower expression in the CCl[4] model group
   compared with the control group, and simultaneously significantly
   elevated expression in the YYHXD group compared with the CCl[4] model
   group. These genes were considered to be implicated in the treatment of
   hepatic fibrosis by YYHXD (therapeutic genes).

2.8.2 Functional enrichment and protein-protein interaction (PPI) analysis

   Functional enrichment analysis of the obtained therapeutic genes was
   conducted by uploading them to the DAVID database
   ([54]https://david.ncifcrf.gov/). DAVID provides a comprehensive set of
   functional annotation tools to understand the biological meaning behind
   large gene lists. The analysis in DAVID included Gene Ontology (GO)
   term enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG)
   pathway enrichment. A p-value <0.05 was used as the threshold to
   identify significantly enriched GO terms and KEGG pathways associated
   with the therapeutic genes.

   Subsequently, Gene Set Enrichment Analysis (GSEA) was performed to
   identify differential expression of target signaling pathways among the
   control group, YYHXD group, and CCl[4] model group using GSEA v4.0.3
   software. A normalized enrichment score (NES) > 1 and p-value <0.05
   were considered significant for differential expression of the
   signaling pathway.

   To further investigate the interactions among the therapeutic genes, a
   protein-protein interaction (PPI) network analysis was conducted using
   the STRING database ([55]https://string-db.org/). The therapeutic genes
   were uploaded to the STRING database, and the resulting PPI network
   data were imported into Cytoscape software (version 3.7.1) for
   visualization and topological analysis. Cytoscape was used to identify
   hub genes within the PPI network based on their ctopological parameters
   (degree values), which may represent key regulator in the underlying
   biological processes.

2.8.3 Quantitative reverse transcription-PCR (q-PCR)

   The expression levels of fatty acid desaturase 2 (Fads2) and fatty acid
   synthase (Fasn) mRNA in hepatic tissue were determined using the q-PCR
   method. Total RNA extraction from each sample was accomplished using
   TRIzol. RNA purity and concentration were then determined using a
   NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific).
   Subsequently, the RNA extracted from the liver was reverse transcribed
   into cDNA using the PrimeScript RT Master Mix kit (Takara Bio, Kusatsu,
   Japan). Primers for fatty acid Fads2, Fasn and β-actin were designed
   using Primer Premier 5.0 software. β-actin was used as the internal
   reference gene. q-PCR was implemented on a CFX Connect machine
   (BIO-RAD, Hercules, CA, United States) using SYBR Green PCR Mix (Monad
   Biotech, Wuhan, China). The primer sequences utilized in the q-PCR are
   shown in [56]Table 2.

TABLE 2.

   Primer sequences for q-PCR.
   Gene Forward primer (5ʹ–3ʹ) Reverse primer (5ʹ–3ʹ) Product size (bp)
   Fads2 ATT​CGG​GAG​AAG​ATG​CTA​CG CCC​TGA​AGT​CCT​CGG​TGA​T 156
   Fasn TCG​CAT​GAA​CAC​TCT​GGA​GAT​G TCT​GTG​AGG​GAC​TCT​GGT​CTT​TG 146
   β-actin AGA​TTA​CTG​CCC​TGG​CTC​CTA​G CAT​CGT​ACT​CCT​GCT​TGC​TGA​T 144
   [57]Open in a new tab

2.9 Western blotting

   Western blot analysis was used to determine the protein expression
   levels of FASN, FADS2, peroxisome proliferator-activated receptor-alpha
   (PPARα), AMPK, and p-AMPK in hepatic tissue. Briefly, appropriate
   amounts of frozen hepatic tissue were ground in liquid nitrogen, and
   total protein extraction was completed using RIPA lysis buffer mixed
   with phosphate inhibitor. Protein concentrations were then determined
   using the BCA protein assay reagent (Abcam, Cambridge, United Kingdom).
   Protein samples were denatured by heating for 10 min and then separated
   by 10% sodium dodecyl-sulfate polyacrylamide gel electrophoresis
   (SDS-PAGE). Subsequently, the samples were transferred onto
   polyvinylidene difluoride (PVDF) membranes on an electrophoresis
   system. Blocking of the PVDF membranes was accomplished at room
   temperature during 1 h using 5% skim milk. Primary antibodies,
   including FASN (1:1,000), FADS2 (1:1,000), PPARα (1:1,000), AMPK
   (1:1,000), p-AMPK (1:1,000) and β-actin (1:1,000) (Elabscience
   Biotechnology, Wuhan, China) were added and incubated overnight at 4°C.
   The membranes were then incubated with a fluorescent secondary antibody
   (1:5,000) (Elabscience Biotechnology) for 1 h at room temperature. The
   membranes were developed by enhanced chemiluminescence (ECL) and
   exposed in a gel imager. The relative gray value of the target protein
   was assessed using Image J software. β-Actin acted as a loading
   control.

2.10 Immunofluorescence staining

   Immunofluorescence staining was used to analyze the phosphorylation
   level of AMPK in hepatic tissues from CCl[4] model rats treated with
   YYHXD. Specifically, paraffin-embedded hepatic tissues were sliced into
   4 µm sections, and then dewaxed and rehydrated. Endogenous peroxidase
   activity was quenched by incubating the sections in 3% hydrogen
   peroxide for 10 min. Section blocking was achieved using 5% bovine
   serum albumin (BSA) solution for 30 min, which was followed by
   incubation with primary antibody against p-AMPK (1:200) (Abcam) at 4°C
   overnight. After washing with phosphate-buffered saline (PBS), sections
   were incubated with secondary antibody (1:1,000) (Elabscience
   Biotechnology) at room temperature for 30 min, followed by
   counterstaining with 4,6-diamidino-2-phenylindole (DAPI) (Thermo Fisher
   Scientific). Fluorescence images were acquired using a fluorescence
   microscope (Nikon Corporation, Tokyo, Japan).

3 Results

3.1 Quality control of YYHXD

   The chemical composition of YYHXD was assayed by HPLC-MS and a
   characteristic fingerprint was constructed. The fingerprint contained
   24 metabolites (shown in [58]Figure 2), including 17 metabolites
   identified in positive ion mode: paeoniflorin, isoferulic acid,
   isoliquiritigenin, naringenin chalcone, senkyunolide H, baicalin,
   apigenin 7-O-glucuronide, 6-O-methylscutellarin, emodin, oroxindin,
   aloe-emodin, tangeritin, nobiletin, wogonin,
   5,2′-dihydroxy-6,7,8,6′-tetramethoxyflavone, cryptotanshinone, and
   tanshinone IIA; and 7 metabolites detected in negative ion mode:
   catechin, albiflorin, rutin, neohesperidin, chrysin, luteolin, and
   apigenin. The characteristic chemical composition of YYHXD is shown in
   [59]Table 3.

FIGURE 2.

   [60]FIGURE 2
   [61]Open in a new tab

   Total ion chromatogram of YYHXD based on HPLC-MS analysis. (A) Positive
   ion mode. (B) Negative ion mode.

TABLE 3.

   Identification of characteristic components in YYHXD.
   ID Name Formula RT [min] Calc. MW m/z Reference ion mz/mz Structure
   1 Catechin C[15]H[14]O[6] 4.935 290.07952 289.07205 [M-H]^−
   335.07767([M+FA-H]-), 245.08189, 221.08162, 205.05019, 123.04431,
   109.02861 graphic file with name FPHAR_fphar-2024-1364023_wc_tfx1.jpg
   2 Paeoniflorin C[23]H[28]O[11] 14.622 497.19109 498.19836 [M+H]^+
   197.08177, 179.07076, 151.07571, 133.06520, 105.07053 graphic file with
   name FPHAR_fphar-2024-1364023_wc_tfx2.jpg
   3 Albiflorin C[23]H[28]O[11] 14.645 480.16325 525.16144 [M+FA-H]^−
   362.26999, 327.10812, 280.79984, 220.24370, 121.02087 graphic file with
   name FPHAR_fphar-2024-1364023_wc_tfx3.jpg
   4 Isoferulic acid C[10]H[10]O[4] 16.676 194.05852 195.0658 [M+H]^+
   177.05501, 145.02875, 117.03398 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx4.jpg
   5 Isoliquiritigenin C[15]H[12]O[4] 19.213 256.07417 257.08145 [M+H]^+
   239.07040, 211.07571, 147.04439, 137.02367, 119.04963 graphic file with
   name FPHAR_fphar-2024-1364023_wc_tfx5.jpg
   6 Rutin C[27]H[30]O[16] 19.751 564.14818 609.14636 [M+FA-H]^−
   300.02774, 271.02499, 255.02986, 227.03465, 151.00288, 108.02105
   graphic file with name FPHAR_fphar-2024-1364023_wc_tfx6.jpg
   7 Naringeninchalcone C[15]H[12]O[5] 21.554 272.06913 273.07645 [M+H]^+
   171.02922, 153.01859, 147.04442, 119.01889, 107.04961 graphic file with
   name FPHAR_fphar-2024-1364023_wc_tfx7.jpg
   8 Neohesperidin C[28]H[34]O[15] 22.754 610.19037 609.18262 [M-H]^−
   343.08179, 325.07324, 301.07199, 286.04849, 196.00092, 174.03175,
   125.02368 graphic file with name FPHAR_fphar-2024-1364023_wc_tfx8.jpg
   9 Senkyunolide H C[12]H[16]O[4] 23.029 206.09503 207.10231 [M+H]^+
   189.09155, 165.05524, 119.08614, 105.07050 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx9.jpg
   10 Baicalin C[21] H[18]O[11] 25.064 446.0856 447.09308 [M+H]^+
   469.07315 ([M+Na]), 423.79083, 271.06061, 225.05571, 105.03415 graphic
   file with name FPHAR_fphar-2024-1364023_wc_tfx10.jpg
   11 Apigenin 7-O-glucuronide C[21]H[18]O[11] 26.979 446.08562 447.09323
   [M+H]^+ 395.81219, 370.43393, 271.06073, 237.06723, 225.05511,
   180.41391, 169.01369, 105.03422 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx11.jpg
   12 6-O-Methylscutellarin C[22]H[20]O[12] 27.448 476.09655 477.10425
   [M+H]^+ 499.08658([M+Na]), 396.56082, 301.07132, 286.04779, 202.01146,
   184.00076, 156.00566, 115.08618 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx12.jpg
   13 Chrysin C[15]H[10]O[4] 28.189 254.05781 253.05045 [M-H]^− 225.05571,
   210.03284, 181.06470, 115.70124 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx13.jpg
   14 Emodin C[15]H[10]O[5] 29.032 270.05325 271.06076 [M+H]^+ 229.04991,
   225.05487, 201.05585, 197.06047, 169.06615 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx14.jpg
   15 Oroxindin C[22]H[20]O[11] 29.508 460.10118 461.10867 [M+H]^+
   483.09113 ([M+Na]), 285.07642, 270.05286, 144.05705 graphic file with
   name FPHAR_fphar-2024-1364023_wc_tfx15.jpg
   16 Luteolin C[15]H[10]O[6] 31.788 286.04817 285.04089 [M-H]^−
   255.03049, 227.03520, 213.05630, 143.04944, 109.02866 graphic file with
   name FPHAR_fphar-2024-1364023_wc_tfx16.jpg
   17 Aloe-emodin C[15]H[10]O[5] 31.985 270.05364 271.06091 [M+H]^+
   253.05025, 241.05038, 225.05519, 197.05988, 169.06531, 141.06972
   graphic file with name FPHAR_fphar-2024-1364023_wc_tfx17.jpg
   18 Tangeritin C[20]H[20]O[7] 34.856 372.12175 373.12903 [M+H]^+
   343.082, 329.10251, 315.08722, 296.06866, 254.05923, 181.01445,
   151.03897 graphic file with name FPHAR_fphar-2024-1364023_wc_tfx18.jpg
   19 Nobiletin C[21]H[22]O[8] 35.584 402.13278 403.14005 [M+H]^+
   425.12191([M+Na]+), 369.09665, 355.08261, 345.06381, 284.06857 graphic
   file with name FPHAR_fphar-2024-1364023_wc_tfx19.jpg
   20 Wogonin C[16]H[12]O[5] 35.952 284.06892 285.07635 [M+H]^+ 269.04425,
   242.05856, 197.05891, 116.06232 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx20.jpg
   21 5,2′-Dihydroxy-6,7,8,6′-tetramethoxyflavone C[19]H[18]O[8] 36.374
   374.10105 375.10852 [M+H]^+ 327.05078, 302.04294, 271.06088, 227.05565,
   215.01918, 149.06023, 121.06543 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx21.jpg
   22 Apigenin C[15]H[10]O[5] 39.244 270.05314 269.04587 [M-H]^−
   241.05096, 225.05565, 181.06563, 135.66243, 120.49317 graphic file with
   name FPHAR_fphar-2024-1364023_wc_tfx22.jpg
   23 Cryptotanshinone C[19]H[20]O[3] 43.396 296.14206 297.14932 [M+H]^+
   319.13132([M+Na]+), 268.11017, 261.12799, 254.09451, 221.09726,
   207.08147, 181.10123, 116.64880 graphic file with name
   FPHAR_fphar-2024-1364023_wc_tfx23.jpg
   24 Tanshinone IIA C[19]H[18]O[3] 50.732 294.12669 295.13394 [M+H]^+
   277.12308, 252.07915, 235.07915, 207.08168, 192.09383, 111.03707
   graphic file with name FPHAR_fphar-2024-1364023_wc_tfx24.jpg
   [62]Open in a new tab

   The bold font (ID) numbers correspond to the IDs of each feature peak
   in [63]Figure 2.

3.2 YYHXD alleviated histopathological changes in hepatic fibrosis rats

   HE and Masson staining of rat hepatic tissues were used to probe the
   effects of YYHXD on pathological changes in rats with hepatic fibrosis.
   The results are shown in [64]Figures 3, [65]4. Hepatic tissues in the
   control group exhibited normal lobule and cellular structures, with no
   inflammatory cell infiltration or fibrous tissue proliferation between
   the hepatic lobules. In the CCl[4] model group, however, the hepatic
   tissue showed obvious steatosis and vacuolization, disorganized hepatic
   lobule structure, disorganized arrangement of hepatocyte cords, massive
   deposition of collagen fibers, and obvious infiltration of inflammatory
   cells. Compared with the CCl[4] model group, the severity of
   CCl[4]-induced hepatic pathological changes was remarkably attenuated
   in the YYHXD and positive control groups. Only a small amount of
   collagen fiber deposition was apparent in the YYHXD high dose group.
   The results suggested that YYHXD ameliorated CCl[4]-induced hepatic
   injury and fibrosis in rats.

FIGURE 3.

   [66]FIGURE 3
   [67]Open in a new tab

   HE staining. HE staining in control (A), CCl[4] model (B), L-YYHXD (C),
   M-YYHXD (D), H-YYHXD (E), and colchicine (F) groups.

FIGURE 4.

   [68]FIGURE 4
   [69]Open in a new tab

   Masson staining. Masson staining in control (A), CCl[4] model (B),
   L-YYHXD (C), M-YYHXD (D), H-YYHXD (E) and colchicine (F) groups.

3.3 YYHXD ameliorated serum and hepatic tissue indicators in hepatic fibrosis
rats

   In this work, serum ALT and AST concentrations were determined to
   assess hepatic function. After CCl[4] administration, serum levels of
   ALT and AST were significantly elevated, implying that CCl[4] induced
   liver dysfunction in rats. YYHXD significantly reduced the elevated
   serum ALT and AST induced by CCl[4] in a dose-dependent manner,
   indicating that YYHXD restored liver function in rats with hepatic
   fibrosis ([70]Figures 5A, B).

FIGURE 5.

   [71]FIGURE 5
   [72]Open in a new tab

   Effect of YYHXD on serum and hepatic tissue indicators in hepatic
   fibrosis rats. (A) ALT; (B) AST; (C) HA; (D) LN; (E) IL6; (F) TNF-α;
   (G) GSH-Px; (H) SOD; (I) MDA. ^## p-value <0.01, compared with the
   control group. **p-value <0.01, compared with the CCl[4] model group.

   To further assess the anti-fibrotic effect of YYHXD, serum
   concentrations of HA and LN were determined by ELISA. The results
   showed that, compared with the control group, the CCl[4] model group
   displayed significantly elevated serum levels of HA and LN. However,
   concentrations of these indicators were notably lower in the YYHXD and
   positive control groups compared with the CCl[4] model group,
   indicating that YYHXD and colchicine have anti-fibrotic effects. It is
   worth noting that the serum concentrations of HA and LN were lower in
   the high-dose YYHXD group compared with those in the colchicine group
   ([73]Figures 5C, D).

   To assess the effects of YYHXD on immune damage in hepatic fibrosis,
   levels of inflammatory factors IL6 and TNF-α in hepatic tissue were
   also determined. The results, as shown in [74]Figures 3E, F,
   demonstrated that both YYHXD and colchicine significantly reduced the
   elevated concentrations of IL6 and TNF-α in the hepatic tissue of
   CCl[4]model rats. The antiphlogistic effect of YYHXD increased at
   higher doses.

   Analysis of oxidative stress indicators revealed that, compared with
   the control group, concentrations of GSH-Px and SOD in hepatic tissue
   fell significantly, while the concentration of MDA increased in the
   CCl[4] model group. YYHXD significantly and dose-dependently
   ameliorated oxidative stress damage in hepatic tissue of rats with
   hepatic fibrosis by upregulating levels of GSH-Px and SOD, and
   downregulating the concentration of MDA in hepatic tissue. The
   antioxidant effect of high-dose YYHXD (20 g/10 mL/kg) was superior to
   that of colchicine (0.2 mg/10 mL/kg) ([75]Figures 5G–I).

3.4 Analysis of RNA-seq data

3.4.1 Screening of genes implicated in YYHXD therapy of hepatic fibrosis

   Using transcriptome sequencing technology, gene expression in rat
   hepatic tissues from the control, YYHXD, and CCl[4] model groups was
   used to probe the pharmacological mechanism of YYHXD in the treatment
   of hepatic fibrosis. A threshold of |fold-change| ≥ 1.5 and adjusted
   p-value <0.01 was set. A total of 318 differentially expressed genes
   were identified between the control and CCl[4] model groups, including
   179 that were upregulated and 139 that were downregulated ([76]Figure
   6A). Moreover, 140 differentially expressed genes were discovered
   between the CCl[4] model and YYHXD groups, including 95 that were
   upregulated and 45 that were downregulated ([77]Figure 6B). By
   comparing these differentially expressed genes, 96 genes implicated in
   the treatment of hepatic fibrosis by YYHXD (therapeutic genes) were
   identified. Expression of these therapeutic genes was significantly
   altered by CCl[4] treatment, and the changes were significantly
   reversed by YYHXD. The changes included 23 genes that were
   significantly elevated in the CCl[4] model group compared with the
   control group, but significantly reduced in the YYHXD group compared
   with the CCl[4] model group ([78]Figure 6C), as well as 35 genes that
   were significantly suppressed in the CCl[4] model group compared with
   the control group, but significantly elevated in the YYHXD group
   compared with the CCl[4] model group ([79]Figure 6D). The expression
   heat map of these therapeutic genes is shown in [80]Figure 5E. Of note,
   Fasn and Fads2 were two genes that exhibited the largest differences
   among the control, YYHXD, and CCl[4] model groups (CCl[4] model group
   vs. control group: Fold change >2, p-value <0.05; YYHXD group vs.
   CCl[4]model group: Fold change >2, p-value <0.05), suggesting that Fasn
   and Fads2 may be key players in hepatic fibrosis and in therapy with
   YYHXD.

FIGURE 6.

   [81]FIGURE 6
   [82]Open in a new tab

   Screening of genes implicated in YYHXD therapy for hepatic fibrosis.
   (A,B) Volcano plots for differential analyses between the CCl[4] model
   and control groups, and between the YYHXD and CCl[4] model groups; (C)
   Therapeutic genes identified by comparing gene expression differences
   between the CCl[4] model and control groups, as well as between the
   CCl[4] model and YYHXD groups. Certain genes were observably
   upregulated in the CCl[4] model group but significantly downregulated
   in the YYHXD group. (D) Therapeutic gene screening. These genes were
   notably downregulated in the CCl[4] model group but significantly
   upregulated in the YYHXD group. (E) Expression heat map of therapeutic
   genes.

3.4.2 Functional enrichment and PPI analysis

   The genes implicated in YYHXD therapy of hepatic fibrosis were uploaded
   onto the DAVID database to conduct GO and KEGG pathway enrichment
   analysis to probe the possible biological pathways involved in hepatic
   fibrosis treatment by YYHXD. The GO annotation results revealed that
   the therapeutic genes were significantly enriched in 30 biological
   processes (BP), 20 cellular components (CC), and 17 molecular functions
   (MF) (p-value <0.05). The BP included response to drug, response to
   xenobiotic stimulus, innate immune response, lipid metabolism and cell
   adhesion. The CC terms included cytoplasm, plasma membrane, cytosol,
   mitochondrion, and integral component of membrane. In terms of MF, the
   therapeutic genes were enriched in protein binding, identical protein
   binding, receptor binding, integrin binding, and heme binding
   predominantly. The top 10 significantly enriched GO terms in BP, CC,
   and MF based on the count ranking are shown in [83]Figure 7A. Moreover,
   a total of eight KEGG pathways were identified after removing signaling
   pathways that were implicated in the disease ([84]Figures 7B, C). The
   obtained KEGG pathways were metabolic pathways, aliphatic acid
   metabolism, aliphatic acid biosynthesis, terpenoid backbone
   biosynthesis, cell adhesion molecules, AMPK signaling pathway, pyruvate
   metabolism and platelet activation.

FIGURE 7.

   [85]FIGURE 7
   [86]Open in a new tab

   Functional enrichment and PPI analysis of therapeutic genes. (A) GO
   annotation. (B) KEGG pathway enrichment analysis. (C) Interaction of
   KEGG signaling pathways and their associated therapeutic genes. (D) PPI
   analysis.

   Additionally, we confirmed the expression differences of these eight
   KEGG signaling pathways in the CCl[4] model, YYHXD, and control groups
   by Gene Set Enrichment Analysis (GSEA). The results are presented in
   [87]Table 4. Among the eight pathways, seven were found to be regulated
   by YYHXD through GSEA. Notably, two pathways, cell adhesion molecules
   and platelet activation, were markedly downregulated (NES <0, p-value
   <0.05) in the CCl[4] model group compared with the control group, but
   were upregulated (NES >0, p-value <0.05) in the YYHXD group compared
   with the CCl[4] model group. On the other hand, five pathways,
   including aliphatic acid biosynthesis, aliphatic acid metabolism,
   terpenoid backbone biosynthesis, AMPK signaling pathway, and pyruvate
   metabolism, were prominently upregulated (NES >0, p-value <0.05) in the
   CCl[4] model group compared with the control group, but displayed
   significant downregulation (NES <0, p-value <0.05) in the YYHXD group
   compared with the CCl[4] model group. The GSEA results for aliphatic
   acid biosynthesis, aliphatic acid metabolism, and the AMPK signaling
   pathway are shown in [88]Figure 8. Overall, these discoveries provide
   valuable insight into the mechanisms underlying the therapeutic effects
   of YYHXD on hepatic fibrosis, suggesting regulation of aliphatic acid
   metabolism through modulation of the AMPK signaling pathway.

TABLE 4.

   Seven KEGG signaling pathways identified by GSEA.
   ID Description CCl[4] model group vs. Control group YYHXD group vs.
   CCl[4] model group
   NES p-value NES p-value
   ko04611 Platelet activation −1.902 0.001107 1.765 0.001236
   ko04514 Cell adhesion molecules −1.876 0.001104 1.844 0.001233
   ko04152 AMPK signaling pathway 1.352 0.027778 −1.192 0.034021
   ko01212 Aliphatic acid metabolism 1.553 0.011428 −1.518 0.017921
   ko00900 Terpenoid backbone biosynthesis 1.613 0.011952 −1.804 0.008876
   ko00620 Pyruvate metabolism 1.445 0.044554 −1.489 0.026756
   ko00061 Aliphatic acid biosynthesis 1.878 0.002392 −1.446 0.034656
   [89]Open in a new tab

FIGURE 8.

   [90]FIGURE 8
   [91]Open in a new tab

   GSEA results for aliphatic acid biosynthesis, aliphatic acid
   metabolism, and the AMPK signaling pathway.

   The interactions between these 96 therapeutic genes are shown in the
   PPI network diagram ([92]Figure 7D). Fasn and Fads2 were two of the
   most significant genes in the PPI network in terms of topological
   parameters (degree values). The results suggested that FASN and FADS2
   may function as pivotal players in the treatment of hepatic fibrosis by
   YYHXD.

3.5 YYHXD regulated aliphatic acid synthesis and metabolism in hepatic
fibrosis rats

   To probe the effect of YYHXD on aliphatic acid synthesis and
   metabolism, we used Oil Red O staining to assess the degree of lipid
   accretion in hepatic tissue. Additionally, q-PCR and Western blot
   methods were employed to evaluate the expression of genes implicated in
   aliphatic acid synthesis and metabolism in hepatic tissue. ELISA was
   used to quantify TG, TC, and LDL-C in serum. Results from Oil Red O
   staining indicated no lipid accretion in the liver in the control
   group. In contrast, the CCl[4] model group exhibited significant
   amounts of lipid accretion and noticeable fatty degeneration in hepatic
   tissue. Compared with the CCl[4] model group, the YYHXD and positive
   control groups displayed a significant reduction of lipid droplets and
   less severe fat denaturation in the liver. In particular, the ability
   of YYHXD (20 g/10 mL/kg) to ameliorate hepatic lipid accretion induced
   by CCl[4] was superior to that of colchicine (0.2 mg/10 mL/kg)
   ([93]Figure 9). Serum biochemistry analysis showed that CCl[4]
   increased levels of TG, TC, and LDL-C, all of which were reduced by
   varying degrees dose-dependently by YYHXD ([94]Figure 10). Fasn, Fads2,
   and Ppara are key genes regulating aliphatic acid synthesis and
   metabolism ([95]Hayashi et al., 2021; [96]Li et al., 2020;
   [97]O'Farrell et al., 2022; [98]Pawlak et al., 2015; [99]Yamane et al.,
   2022). Among them, Fasn and Fads2 were the two most differentially
   expressed genes among the three groups according to the results of
   transcriptome sequencing. The q-PCR results indicated that expression
   of Fasn and Fads2 mRNA in hepatic tissue of CCl[4]-induced hepatic
   fibrosis rats was notably higher than that of normal rats, while YYHXD
   significantly inhibited this increase in a dose-dependent manner
   ([100]Figure 11). Additionally, the Western blot data indicated that
   expression of FASN, FADS2, and PPARα proteins in hepatic tissue of
   CCl[4]-induced hepatic fibrosis rats was notably higher than that in
   normal rats, while YYHXD dose-dependently inhibited the increases in
   these aliphatic acid metabolism-related proteins. In particular, the
   ability of high-dose YYHXD (20 g/10 mL/kg) to reduce aliphatic acid
   accretion was superior to that of colchicine (0.2 mg/10 mL/kg)
   ([101]Figure 12).

FIGURE 9.

   [102]FIGURE 9
   [103]Open in a new tab

   Oil Red O staining. Oil Red O staining in control (A), CCl[4] model
   (B). L-YYHXD (C), M-YYHXD (D), H-YYHXD (E) and colchicine (F) groups.

FIGURE 10.

   [104]FIGURE 10
   [105]Open in a new tab

   Effect of YYHXD on serum indicators of aliphatic acid synthesis and
   metabolism in hepatic fibrosis rats. (A) TG. (B) TC. (C) LDL-C. ^##
   p-value <0.01, compared with the control group. **p-value <0.01,
   compared with the CCl[4] model group.

FIGURE 11.

   [106]FIGURE 11
   [107]Open in a new tab

   Effect of YYHXD on expression of Fasn and Fads2 mRNA in hepatic
   fibrosis rats. ^## p-value <0.01, compared with the control group. ^**
   p-value <0.01, compared with the CCl[4] model group.

FIGURE 12.

   [108]FIGURE 12
   [109]Open in a new tab

   Effect of YYHXD on expression of FASN, FADS2, and PPARα proteins in
   hepatic fibrosis rats. ^## p-value <0.01, compared with the control
   group. **p-value <0.01, compared with the CCl[4] model group.

3.6 YYHXD activated the AMPK signaling pathway in hepatic fibrosis rats

   To further elucidate the effect of YYHXD on AMPK signaling, Western
   blotting analysis was used to assess expression of AMPK and p-AMPK
   proteins in rat hepatic tissues from each group. Our results
   demonstrated that AMPK protein expression was not statistically
   different in each group. Compared with the control group, the levels of
   p-AMPK were notably lower in the hepatic fibrosis CCl[4] model group,
   indicating repression of the AMPK signaling pathway. However, YYHXD
   therapy upregulated expression of p-AMPK, reversing the downregulation
   of AMPK signaling that was induced by CCl[4] ([110]Figure 13). In
   addition, immunofluorescence staining was used to confirm the levels of
   p-AMPK in hepatic tissue. The results of immunofluorescence staining
   ([111]Figure 14) were consistent with those of the Western blot
   analysis. Expression of p-AMPK was notably lower in hepatic tissue of
   rats with hepatic fibrosis compared with that in normal rat hepatic
   tissue. YYHXD treatment (5, 10, and 20 g/10 mL/kg) dose-dependently
   attenuated the significant reduction of p-AMPK expression induced by
   CCl[4] in rat hepatic tissue. Furthermore, the ability of high-dose
   YYHXD (20 g/10 mL/kg) to upregulate p-AMPK expression was superior to
   that of the positive control drug colchicine (0.2 mg/10 mL/kg).

FIGURE 13.

   [112]FIGURE 13
   [113]Open in a new tab

   Effect of YYHXD on protein expression of AMPK and p-AMPK in hepatic
   fibrosis rats by Western blotting analysis. ^## p-value <0.01, compared
   with the control group. **p-value <0.01, compared with the CCl4 model
   group.

FIGURE 14.

   [114]FIGURE 14
   [115]Open in a new tab

   Effect of YYHXD on expression of p-AMPK in hepatic tissue of hepatic
   fibrosis rats by immunofluorescence staining. The panels show DAPI
   nuclear staining in blue (B), staining with the corresponding p-AMPK
   antibody in red (A), and merged images (C). Scale: 200 µm.

4 Discussion

   Hepatic fibrosis is a hepatopathy that is characterized by chronic
   inflammation and incremental accretion of collagen, which can advance
   to hepatic cirrhosis and even hepatoma. The pathogenesis of hepatic
   fibrosis is complex, often involving liver dysfunction, immune injury,
   and oxidative stress damage ([116]Rodríguez et al., 2021). YYHXD is a
   traditional Chinese medicine comprising 14 botanical drugs, developed
   based on traditional Chinese medicine theory. Danggui and Dihuang are
   the principal botanical drugs, with functions that include nourishing
   blood, promoting blood circulation, replenishing yin, and tonifying
   essence. They have been proven to activate the immune regulation system
   ([117]Wu et al., 2011), reduce hepatocyte death ([118]Liu et al.,
   2012), maintain ECM balance, and boost regeneration of hepatocytes and
   blood ([119]Wang and Liang, 2010). Baishao, Chishao, Chuanxiong,
   Danshen, Zhebeimu, and Zelan are regarded as the ministerial botanical
   drugs in the formula, which function to nourish yin, cool blood, and
   promote liver circulation to dispel blood stasis. Among them, Chishao
   can stop bleeding and disperse stasis, and is particularly suitable for
   treating yin deficiency with heat stasis. Danshen, Zhebeimu, and Zelan
   boost blood circulation and resolve blood stasis, and have been shown
   to play an important role in regulating the immune system ([120]Peng et
   al., 2018), protecting the liver from damage ([121]Nagappan et al.,
   2019; [122]Wu et al., 2022), and impeding the advancement of liver
   cancer ([123]Zhang Q. et al., 2022). Chuanxiong boosts blood
   circulation and relieves stagnation, while Baishao nourishes blood and
   liver. Both Chuanxiong and Baishao have been shown to impede the
   activation of hepatic stellate cells and the inflammatory response in
   liver ([124]Kong et al., 2013; [125]Wu et al., 2015). Moreover, Chaihu,
   Chenpi, Dahuang, Huangqin, Gancao, and Dazao are the auxiliary
   botanical drugs in the formula. Traditional Chinese medicine theory
   holds that Chaihu modulates liver function and relieves depression,
   Chenpi regulates qi and relieves pain, Dahuang clears heat and
   detoxifies, Huangqin purges fire and detoxifies, and Gancao harmonizes
   the properties of the other botanical drugs. Modern pharmacological
   studies have demonstrated that Chaihu and Chenpi ameliorated hepatic
   fibrosis through anti-inflammatory, antioxidant, and anti-apoptotic
   mechanisms ([126]Zou et al., 2018; [127]Wu et al., 2021). Dahuang
   extract emodin has been discovered to reduce liver oxidative stress
   injury by activating the AMP-activated protein kinase (AMPK) signaling
   pathway ([128]Lee D. et al., 2020), while Huangqin extract baicalin
   alleviates hepatic fibrosis by inducing hepatic stellate cell
   ferroptosis (Liu et al., 2022). Dazao has been shown to have
   liver-protective, anti-cancer, antioxidant, and anti-inflammatory
   activities ([129]Sun et al., 2022), and Gancao alleviates hepatic
   tissue inflammation and fibrosis damage ([130]Guo et al., 2023). The
   combination of these botanical drugs can exert synergistic effects
   across many traditional therapeutic actions and pharmacological
   mechanisms. Therefore, YYHXD exerts yongyinghuoxue, promoting qi flow
   and resolving stasis effects according to TCM theory. And YYHXD has
   been broadly applied in China to treat various kinds of hepatopathy
   caused by yin deficiency with blood stasis, qi stagnation, and blood
   stasis, such as hepatic fibrosis, and has shown promising therapeutic
   effects. However, the pharmacological mechanism of YYHXD in the
   treatment of hepatic fibrosis is complex and not well understood, due
   to its multi-component nature, having effects on multiple targets and
   pathways. Transcriptomic techniques, with their high-throughput
   capabilities, have pivotal roles in elucidating the pathogenesis of
   hepatic fibrosis and the pharmacological mechanisms of traditional
   Chinese medicines with such complex therapeutic characteristics.

   The CCl[4]-induced rat hepatic fibrosis model used in this study is a
   widely accepted and well-established animal model that recapitulates
   many pathological features of human hepatic fibrosis, including
   excessive deposition of extracellular matrix, activation of hepatic
   stellate cells, and development of cirrhosis ([131]O’Farrell et al.,
   2022; [132]Guo et al., 2023). While there are some differences between
   the rat model and human disease, such as the etiology and the time
   course of fibrosis development, the fundamental pathogenic mechanisms
   are highly similar. Previous studies have shown that the therapeutic
   effects of traditional Chinese medicines, including YYHXD, observed in
   the CCl4-induced rat model often translate well to human hepatic
   fibrosis patients ([133]Ma et al., 2018; [134]Liang et al., 2022).
   Therefore, the findings from the present study using the CCl4-induced
   rat model provide valuable insights into the potential pharmacological
   mechanisms of YYHXD in the treatment of human hepatic fibrosis.

   In this work, transcriptomic technology was utilized to identify
   potential target genes in YYHXD therapy of hepatic fibrosis. Using
   bioinformatics analysis, the biological functions and signaling
   pathways associated with these target genes were identified. Altogether
   96 genes implicated in YYHXD therapy for hepatic fibrosis were
   identified from RNA sequencing data, with Fasn and Fads2 being
   potential key genes affected by treatment. Functional enrichment
   analysis revealed that YYHXD may exert its therapeutic effects on
   hepatic fibrosis through regulation of important pathways such as
   aliphatic acid biosynthesis and metabolism, as well as the AMPK
   signaling pathway.

   Aliphatic acid synthesis and metabolic pathways are crucial players in
   the occurrence and advancement of hepatic fibrosis. As a major organ
   for aliphatic acid synthesis and metabolism, the liver has the ability
   to synthesize, break down, and metabolize aliphatic acids ([135]Heeren
   and Scheja, 2021). In certain pathological states, such as obesity,
   hyperlipidemia, and hepatic fibrosis, the liver is susceptible to
   excessive lipid accretion ([136]Ran et al., 2023). Adipocyte
   proliferation, white blood cell infiltration, and cytokine release can
   induce the deposition and accretion of lipids in hepatocytes,
   exacerbating the occurrence and progression of hepatic fibrosis
   ([137]Suganami et al., 2012; [138]van der Windt et al., 2018;
   [139]Heeren and Scheja, 2021). Transforming growth factor-beta (TGF-β),
   a critical mediator in hepatic fibrosis, is able to stimulate hepatic
   stellate cell diffusion as well as collagen deposition. Overexpression
   of TGF-β impedes the expression of genes implicated in aliphatic acid
   metabolism and the activity of enzymes, leading to abnormal aliphatic
   acid metabolism and excessive lipid accretion, which exacerbates the
   occurrence and progression of hepatic fibrosis ([140]Kwapisz et al.,
   2021). Abnormalities in aliphatic acid synthesis and metabolism also
   cause oxidative stress and inflammatory responses, accelerating the
   development of hepatic fibrosis ([141]Zhang et al., 2023). FASN, FADS2
   and PPARα are important regulators of aliphatic acid synthesis and
   metabolism pathways. FASN boosts aliphatic acid synthesis and is
   involved in various metabolic processes in hepatocytes, and its
   expression is positively correlated with the severity of hepatic
   fibrosis ([142]O'Farrell et al., 2022). FADS2 is a pivotal enzyme
   during aliphatic acid desaturation, which is essential for biosynthesis
   of long-chain polyunsaturated aliphatic acids (PUFAs) ([143]Yamane et
   al., 2022). Increased expression of FADS2 significantly augments the
   development of hepatic fibrosis, causing accelerated synthesis of
   unsaturated aliphatic acids in hepatocytes and an imbalance of
   intracellular lipid metabolism ([144]Li et al., 2020; [145]Hayashi et
   al., 2021). PPARα is another essential regulator of aliphatic acid
   metabolism. Inhibition of PPARα can lead to lipid metabolism disorder
   and promote the progression of hepatic fibrosis. Activation of PPARα
   can accelerate the breakdown of aliphatic acids and inhibit activation
   of hepatic stellate cells, reducing the severity of hepatic fibrosis
   ([146]Pawlak et al., 2015).

   AMPK is an important intracellular metabolic regulator that can affect
   aliphatic acid synthesis and metabolism by modulating various signaling
   pathways. This makes it a key player in the occurrence and progression
   of hepatic fibrosis. Activation of AMPK can inhibit aliphatic acid
   synthesis and boost aliphatic acid oxidation, resulting in reduced
   accretion of liver fat and progression of hepatic fibrosis ([147]Zhao
   and Saltiel, 2020; [148]Hu et al., 2021). Activated AMPK (p-AMPK) can
   reduce the synthesis of acyl-CoA, which has a key role in aliphatic
   acid synthesis, and inhibits aliphatic acid synthesis, leading to
   reduced fat deposition ([149]Ruderman et al., 2003). Moreover,
   [150]Viollet et al. (2009) suggested that activation of AMPK can also
   promote β-oxidation of aliphatic acids in the liver, increase the rate
   of aliphatic acid metabolism, and alleviate lipid deposition. In
   addition to directly regulating aliphatic acid metabolism, AMPK
   activation can also exert regulatory effects through related genes and
   transcription factors. For example, AMPK activation can inhibit
   expression of sterol regulatory element-binding protein 1c (SREBP-1c),
   reducing the expression of genes implicated in aliphatic acid synthesis
   ([151]Yap et al., 2011). AMPK activation can also regulate aliphatic
   acid metabolism by modulating the expression of cytochrome P450 enzyme
   2E1 (CYP2E1) ([152]Ki et al., 2007; [153]Kim et al., 2016). According
   to relevant research, p-AMPK can also alleviate hepatic fibrosis by
   suppressing the release of inflammatory mediators and collagen
   synthesis ([154]Shi et al., 2017; [155]Huang et al., 2021). AMPK can
   downregulate expression of FASN, impede hepatic intracellular aliphatic
   acid synthesis and saturation, and ameliorate fatty liver disease
   ([156]Lee E. et al., 2020; [157]Irungbam et al., 2020). Furthermore,
   AMPK activation is able to enhance aliphatic acid β-oxidation by
   activating PPARα, suppressing cell diffusion, inflammation, apoptosis,
   and other cellular processes ([158]Sozio et al., 2011; [159]Mansour et
   al., 2022).

   In this work, transcriptomic sequencing and experimental verification
   were used to deconvolute the pharmacological mechanisms underlying the
   efficacy of YYHXD in therapy of hepatic fibrosis injury in rats. It was
   found that efficacy correlated with the modulation of aliphatic acid
   synthesis and metabolism pathways, as well as the AMPK signaling
   pathway. The observed dose-dependent therapeutic effects of YYHXD
   provide valuable insights into its pharmacodynamics. The superior
   efficacy of the high-dose YYHXD suggests that a more comprehensive
   modulation of the multiple pathways involved in hepatic fibrosis, such
   as aliphatic acid metabolism and the AMPK signaling pathway, may be
   achieved with higher concentrations of the decoction’s phytochemical
   constituents. This dose-dependent effect highlights the importance of
   optimizing the dosage regimen to maximize the therapeutic potential of
   YYHXD in the clinical management of hepatic fibrosis. This finding was
   novel, although some botanical drugs used in YYHXD have been found to
   regulate aliphatic acid synthesis and metabolism or activate the AMPK
   signaling pathway. For example, Qiang Zhang et al. found that the
   active metabolite of Radix Bupleuri, β-sitosterol, reduced lipid
   accretion in hepatocytes processing free aliphatic acids by activating
   the PPARγ-UCP-1 pathway, thus exerting anti-hepatic fibrosis effects
   ([160]Zhang Y. et al., 2022). [161]Lei et al. (2022) found that the
   traditional Chinese medicine formula Chaihu Shugan powder (containing
   Radix Bupleuri as the main metabolite) ameliorated lipid accretion in
   the livers of mice with non-alcoholic fatty liver disease, inhibited
   secretion of inflammatory cytokines in hepatic tissues, and inhibited
   synthesis of aliphatic acids in the liver. The active metabolite of
   Salvia miltiorrhiza, cryptotanshinone, activates the AMPK signaling
   pathway, inhibits fat formation, aliphatic acid oxidation, oxidative
   stress, and inflammatory reactions, thus ameliorating ethanol-induced
   liver damage ([162]Nagappan et al., 2019).

   However, several methodological limitations and uncertainties should be
   acknowledged. First, the small sample size for the RNA-seq analysis may
   limit the statistical power, and the use of whole liver tissue
   precludes the identification of cell-type specific effects. Second, the
   CCl4-induced rat model may not fully recapitulate the complex etiology
   and pathogenesis of human liver fibrosis, and the findings need to be
   validated in additional animal models. Lastly, this study focused on
   transcriptional changes, and further exploration of
   post-transcriptional and post-translational regulation would provide a
   more comprehensive understanding of the mechanisms. Despite these
   limitations, the results suggest that YYHXD exerts anti-fibrotic
   effects through the modulation of aliphatic acid synthesis/metabolism
   and the AMPK signaling pathway, which have potential clinical relevance
   for the management of liver fibrosis and related liver diseases. Future
   research directions include evaluating YYHXD in additional animal
   models, exploring its combination with other therapies, conducting
   human clinical trials, and deeper mechanistic investigations to
   optimize its therapeutic applications.

5 Conclusion

   In this work, the pharmacological mechanisms of YYHXD in vivo were
   investigated for the first time. Our results demonstrated that YYHXD
   effectively reversed CCl[4]-induced hepatic fibrosis in rats, with
   corresponding restoration of liver function, reduced oxidative stress,
   and alleviated inflammatory damage. The anti-hepatic fibrosis effect of
   YYHXD was characterized by modulation of aliphatic acid synthesis and
   metabolism, combined with activation of the AMPK signaling pathway.
   This work provided a reference and foundation for further research and
   clinical utilization of YYHXD.

Funding Statement

   The authors declare that financial support was received for the
   research, authorship, and/or publication of this article. This study
   was supported by the Ningxia Key Research and Development Plan project
   (2021BEG03112) and Ningxia Key Research and Development Plan project
   (2022BEG03144).

Data availability statement

   The datasets presented in this study can be found in online
   repositories. The names of the repository and accession number(s) can
   be found below: NCBI Sequence Read Archive (SRA), accession number
   PRJNA1059506.

Ethics statement

   The animal study was approved by the Ethics Committee of Ningxia
   Medical University approved the study (Approval No.
   IACUC-NYLAC-2021-009). The study was conducted in accordance with the
   local legislation and institutional requirements.

Author contributions

   YB: Writing–original draft, Writing–review and editing. SL:
   Conceptualization, Data curation, Investigation, Writing–review and
   editing. YZ: Data curation, Methodology, Writing–review and editing.
   BZ: Writing–original draft, Writing– review and editing

Conflict of interest

   The authors declare that the research was conducted in the absence of
   any commercial or financial relationships that could be construed as a
   potential conflict of interest.

Publisher’s note

   All claims expressed in this article are solely those of the authors
   and do not necessarily represent those of their affiliated
   organizations, or those of the publisher, the editors and the
   reviewers. Any product that may be evaluated in this article, or claim
   that may be made by its manufacturer, is not guaranteed or endorsed by
   the publisher.

References