FR429 from Polygonum capitatum Demonstrates Potential as an Anti-hepatic Injury Agent by Modulating PI3K/Akt Signaling Pathway

Abstract

FR429, an ellagitannin isolated and purified from the whole herb Polygonum capitatum (P. capitatum), possesses a robust pharmacological profile, which is particularly noteworthy for its anti-inflammatory and anticancer properties. Despite these established effects, its potential in mitigating hepatic injury remains to be fully explored. The present investigation delineates the hepatoprotective efficacy of FR429 and unveils its underlying molecular mechanisms. Initially, of the tested compounds, 10 compounds (specifically, compounds 2, 4, 5, 6, 7, 8, 9, 12, 13, and 14) exhibited significant protective effects at a concentration of 10 μM, elevating HepG2 (human liver cancer cell) cell viability from 43.4 to 70% following carbon tetrachloride (CCl₄) exposure. Among them, compounds 2 (FR429, half-maximum effective concentration (EC₅₀) = 6.46 μM) and 6 (2ʺ-O-galloylquercitrin, EC₅₀ = 5.36 μM) demonstrated the highest cytoprotective activities. In the murine model, FR429 dramatically attenuated serum levels of alanine transaminase, aspartate transaminase, and alkaline phosphatase, indicative of its hepatoprotective potential. Histopathological evaluation further substantiated these findings, as FR429 noticeably mitigated CCl₄-induced hepatic lesions, involving necrosis, ballooning degeneration, and neutrophil infiltration. Transcriptomic analysis unveiled 178 differentially expressed genes in FR429-treated mice liver tissue, with significant alterations indicative of a hepatoprotective response. Mechanistic investigations revealed that FR429’s hepatoprotective effects involve modulation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, evidenced by downregulation of toll-like receptor 2, phosphorylated PI3K, phosphorylated Akt, nuclear factor-kappa-B, interleukin-1 beta, and tumor necrosis factor-alpha expression. Furthermore, FR429 modulated the gene and protein expression levels of apoptotic markers (apoptotic protein (Bax) and B-lymphoblastoma-2 gene (Bcl2)), reinforcing its anti-hepatic damage efficacy. This study represents the first report establishing FR429 as an effective hepatoprotective compound, paving the way for further investigation into its therapeutic applications.

INTRODUCTION

The liver is the largest physiological organ in the human body. Its functions in material metabolism, blood volume regulation, endocrine control, cholesterol homeostasis, and immune system support are noteworthy.¹⁾ Notably, the liver is susceptible to damage arising from chemicals, drugs, alcohol, and environmental factors. It was reported that over 2 million deaths were annually recorded owing to hepatic diseases, universally accounting for 4% of all deaths.²⁾ Acute liver injury (ALI) is a condition causing rapid damage to liver cells, accompanied by the onset of inflammation.³⁾ Long-term persistent hepatic injury may lead to liver fibrosis, cirrhosis, and even liver cancer.^4,5) The process entails intricate interactions involving hepatocyte degeneration, inflammatory responses, and reactive oxygen species (ROS), as well as necrotic and apoptotic pathways in hepatocytes.⁶⁾

Owing to a rising prevalence of liver disease, the quest for a therapeutic agent that is both efficacious and economically feasible is highly noteworthy. Herbal medicines, in particular, possess distinct advantages and potential in the prophylaxis of hepatic diseases, which may be attributable to their structural diversity, minimal toxicity, and broad accessibility.⁷⁾ Furthermore, the majority of natural medicines, including silymarin, curcumin, and baicalin,⁸⁾ have exhibited hepatoprotective properties,^9,10) reflecting the profound implications of advancing and integrating additional natural compounds in prevention and therapy of hepatic diseases.

With a longstanding historical role in traditional Miao medicine, Polygonum capitatum (P. capitatum) has been recognized in China for centuries. Predominantly distributed across regions (such as Guizhou, Jiangxi, and neighboring provinces),¹¹⁾ this botanical species is distinguished by its diverse phytochemical profile, involving a variety of bioactive compounds, notably flavonoids, phenolic acids, and organic acids.¹²⁾ Modern pharmacological studies have demonstrated that P. capitatum exhibited anti-inflammatory, antioxidant, antibacterial, anticancer, and diuretic activities.^13,14) FR429, a polyphenolic compound, was identified in P. capitatum.^15,16) Prior research documented that P. capitatum exerts notable anti-inflammatory effects, which may primarily be attributable to the bioactive compound FR429.¹⁷⁾ However, limited research has concentrated on the anti-hepatic injury activity of FR429 and its mechanism of action.

Recently, transcriptomics has surged in prominence across disciplines, comprising foundational research, clinical diagnostics, and pharmacological innovation. Transcriptome sequencing, a sophisticated analytical methodology utilizing high-throughput sequencing, facilitates the sequencing of cDNA molecules that were reversely transcribed from the entirety of mRNA in cellular or tissue samples, thereby providing an exhaustive catalog of mRNA sequence data.¹⁸⁾ This technique has increasingly demonstrated its efficacy as a comprehensive tool for elucidating genome-wide transcriptional landscapes, involving the detection of differentially expressed genes (DEGs), intricate signaling cascades, and biomarkers linked to disease states in complex tissue architectures or cellular milieu.^19,20)

In this investigation, a cohort of 27 bioactive compounds extracted from P. capitatum underwent assessment for their hepatoprotective potential in a HepG2 (human liver cancer cell) cell model challenged by carbon tetrachloride (CCl₄)-induced cytotoxicity. With the assistance of transcriptomic analysis, the study further elucidated the in vivo hepatoregenerative influences of FR429, concentrating on its molecular mechanisms in countering CCl₄-induced hepatic injury in Kunming mice.

MATERIALS AND METHODS

Plant Materials

A comprehensive specimen of P. capitatum was harvested in September 2020 from Qiannan City in Guizhou Province, China, with botanical authentication conducted by the corresponding author, as documented in advance.²¹⁾ The voucher specimen (No. THL-202001) was securely obtained from the Laboratory of Molecular Pharmacognosy at the School of Pharmacy, Guizhou Medical University. Through precise isolation processes, FR429 and an additional 26 compounds were acquired from the ethyl acetate (EtOAc) extract of P. capitatum, each compound achieving a confirmed purity level exceeding 95%²¹⁾ (Table 1).

Table 1. Twenty-Seven Compounds Isolated from P. capitatum

Serial number	Name	Formula	Molecular weight
1	Kurarinol B	C24H28O14	476
2	FR429	C41H30O26	938
3	Quercetin-3-O-β-d-galactopyranoside	C22H24O12	480
4	Quercetin-3-O-β-d-glucopyranoside	C21H20O11	448
5	Quercetin-3-O-α-l-rhamnopyranoside	C22H24O11	464
6	2″-O-Galloylquercitrin	C28H24O15	600
7	3″-Galloylquercitrin	C28H24O15	600
8	Quercetin-3-O-sophoroside	C27H30O16	610
9	Quercetin-3-O-α-l-rhamnopyranoside	C20H18O13	466
10	Kaempferol-7-O-β-d-glucopyranoside	C21H20O10	432
11	Kaempferol-3-O-α-l-rhamnoside	C21H20O11	448
12	Kaempferol	C15H10O6	286
13	Quercetin	C15H10O7	302
14	1,2,6-Trigalloyl-β-d-glucose	C27H24O18	636
15	Stigmast-5-en-3-O-β-d-glucoside	C35H60O6	576
16	Catechin	C15H14O6	290
17	(−)-Epigallocatechin-3-O-gallate	C23H20O10	456
18	Silybin	C25H22O10	482
19	Apigenin	C15H10O5	270
20	Alpine isoflavones	C21H18O4	334
21	3, 3′, 4′-Trimethyl ellagic acid	C14H6O8	302
22	5,7-Dihydroxy-4H-4-chromone	C9H6O4	178
23	Gallic acid	C7H6O5	170
24	Vanillic acid	C7H6O5	168
25	Ethyl gallate	C9H10O5	198
26	β-Sitosterol	C29H50O	414
27	Methyl arachidonate	C21H34O2	318

Chemicals and Reagents

The high-glucose Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Yeasen Biotechnology Co., Ltd., headquartered in Shanghai, China, and fetal bovine serum (FBS) was supplied by Zhejiang Aerospace Biotechnology Co., Ltd., headquartered in Zhejiang, China. Cell-grade dimethyl sulfoxide, along with assay kits for quantifying aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and DEPC-treated water, were acquired from Beijing Solarbio Science & Technology Co., Ltd., headquartered in Beijing, China. The MTS assay reagent was obtained from BestBio, headquartered in Shanghai, China, while silymarin was provided by Shanghai Yuanye Bio-Technology Co., Ltd., headquartered in Shanghai, China. RNA extraction and reverse transcription kits were obtained from Tiangen Biochemical Technology Co., Ltd. (Beijing, China), with TB Green Premix supplied by Bao Bio-engineering Co., Ltd. (Yucheng, China). Antibodies for p65, phosphorylated p65 (p-p65), Bax (apoptotic protein), Bcl2 (B-lymphoblastoma-2 gene), Akt, and phosphorylated Akt (p-Akt) were supplied by Proteintech, headquartered in Wuhan, China. PI3K and phosphorylated PI3K (p-PI3K) antibodies were sourced from Abcam, headquartered in Cambridge, U.K., while antibodies for interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) were acquired from Affinity Biosciences (Jiangsu, China).

Cell Culture and Treatment

It was attempted to obtain HepG2 cell lines from the National Collection of Authenticated Cell Cultures and precisely cultivate them in DMEM, enriched with 10% FBS, 1% MEM nonessential amino acid solution (100X), and a 1% antibiotic mixture of penicillin–streptomycin, under strictly controlled conditions, particularly at 37°C in a humidified 5% CO₂ incubator. Protocols were adhered to rigorously as previously outlined.²²⁾ The hepatoprotective potential was determined through the MTS assay. In this process, seeding of cells was undertaken at a concentration of 1 × 10⁵ cells per well in 96-well plates and allowed to establish over a 12-h period. Subsequently, pretreatment of cells was undertaken utilizing experimental compounds at a concentration of 10 μM for 2-h, then exposed to a 0.35% CCl₄ solution for a duration of 6 h to induce cytotoxic stress. Thereafter, administration of 10 μL of MTS reagent was implemented, and a 4-h incubation of cells was followed particularly at 37°C to facilitate colorimetric development. Absorbance measurements were precisely acquired at 490 nm via a multifunctional enzyme marker to quantify cell viability. It was attempted to implement all assays in triplicate to ensure the reproducibility and statistical robustness of the data.

Test Animals and Treatment

The animal research committee of Guizhou Medical University has approved all experimental schemes and surgical operations (license number: SYXK (Gui) 2023-0002). Animal experiments were carried out following the recommendations of the animal experiment guidelines of Guizhou Medical University and the ARRIVE guidelines.

Six-week-old male Kunming mice were acquired from the Experimental Animal Department of Guizhou Medical University and subsequently maintained under exacting environmental parameters to preserve physiological equilibrium. Each animal had unrestricted access to filtered drinking water and a rigorously standardized, nutritionally calibrated diet, ensuring a consistent baseline across the cohort. Housing conditions were stringently monitored to provide a controlled 12-h photoperiod (light/dark cycle), with ambient temperature precisely regulated at 22 ± 2°C and relative humidity maintained in a narrow range of 55–60%, thereby minimizing any extrinsic variables that could impact study outcomes. After a necessary 3-d acclimatization period in sterile, pathogen-free quarters to stabilize baseline responses, experimental interventions were initiated.

The experimental animals were precisely stratified into 5 experimental groups (n = 6 per group): control, model, silymarin treatment (50 mg/kg/d), and 2 FR429 treatment cohorts (10 and 50 mg/kg/d). Prior to model induction, mice in the treatment groups were administered prophylactic agents daily for 7-d, while animals in the control and model groups received pure water as a baseline comparator. The FR429 and silymarin groups received their designated treatments via daily gavage over a 7-d period. Precisely 6 h following the final administration, mice in the control group were intraperitoneally administered corn oil (7 mL/kg), while all other experimental groups were subjected to modeling via intraperitoneal injection of a 2% CCl₄ solution in refined corn oil, after which they underwent a 24-h fasting regimen. It was thereafter attempted to collect blood samples, which were centrifuged (2000 × g for 15 min at 4°C), and serum isolation was implemented for further biochemical assays. Fixation of liver tissues in 10% neutral-buffered formalin was conducted, facilitating histopathological examination through hematoxylin–eosin (H&E) staining, while additional liver samples were preserved at –80°C for molecular assays.

Biochemical Analysis

For biochemical profiling, the serum samples stored at –80 °C were precisely thawed, and the enzymatic activities of AST, ALT, and ALP were accurately quantified on the based on the documents provided by the assay kit instructions.

Histological Analysis

Hepatic tissues fixed in 10% formalin solution were sent to Guangxi Zhuo Yi Biotechnology Co., Ltd. for paraffin embedding, sectioning, H&E staining, and photographing. Histopathological assay was conducted microscopically, employing a precisely controlled double-blind methodology to ensure unbiased assessment.

Transcriptomic Analysis

RNA extraction, Library Construction, and Sequencing

Informed by the outcomes obtained from blood biochemical parameters and comprehensive histological assessments, the control group, model group, and FR429 group underwent transcriptomic analysis, involving 3 samples from each experimental cohort. Total RNA extraction from hepatic tissue was executed utilizing TRIzol reagent (Magen), adhering precisely to the manufacturer’s detailed protocol. The absorbance ratio of A260/A280 for the extracted RNA was quantitatively figured out through the Nanodrop ND-2000 (Thermo Scientific), while the RNA integrity number was precisely determined via the Agilent Bioanalyzer 4150 from Agilent Technologies (U.S.A.). Upon successful validation of the RNA quality, eukaryotic mRNA was selectively enriched through the utilization of magnetic beads functionalized with oligo(dT). Thereafter, a fragmentation buffer was incorporated to induce random cleavage of the mRNA strands. Employing the mRNA as a template, the synthesis of the first strand of cDNA was accomplished via 6-base random primers (random hexamers). This was followed by the synthesis of the 2nd cDNA strand, facilitated by the addition of a specialized buffer, deoxynucleotide triphosphates, and DNA polymerase I. The resulting double-stranded cDNA was subsequently purified utilizing AMPure XP beads to ensure the removal of impurities. The purified double-stranded cDNA underwent a series of sophisticated modifications, involving repair of the ends, addition of a poly-A tail, and ligation of sequencing adapters. Fragment size selection was precisely undertaken utilizing AMPure XP beads, leading to the enrichment of the final cDNA library through a series of PCR amplification cycles. Ultimately, the sequencing data generated from the Illumina (or BGI) platform were employed for the purpose of comprehensive bioinformatics analysis, providing noticeable insights into the transcriptomic landscape. These complex procedures were executed with precision by Shanghai Applied Protein Technology.

Differential Gene Expression Analysis

Junction sequences were systematically excised from the original sequencing data, and reads exhibiting low quality, defined as those with an average base quality score falling below 20 and containing more than 5 instances of “N” (indicative of unidentifiable bases), were precisely filtered out. This process yielded clean reads that were regarded as appropriate for subsequent assays. Each gene’s expression level in the various samples was quantified using featureCounts software, employing the fragments per kilobase per million mapped reads (FPKM) metric. FPKM quantifies the number of bases per thousand of transcript length per million alignment fragments, thereby effectively controlling for the confounding influences of gene length and sequencing depth on gene expression calculations. The derived genes’ expression levels provide a robust basis for comparative assay of expression differences across diverse samples. For the purpose of identifying DEGs, differential gene expression analysis was executed through the DESeq2 R package. The criteria for screening DEGs were stringently defined, with a significance threshold of p_adj <0.05 and a log 2 (fold change) value exceeding 1 in absolute terms.

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis

Comprehensive GO and KEGG assays were conducted on the filtered significant DEGs to enable a more in-depth exploration of the markedly involved signaling pathways.

Real-Time Quantitative PCR Analysis

The initial step involved the extraction of total RNA from the hepatic tissues of each mouse, utilizing an RNA extraction reagent, followed by RNA concentration quantification via an ultra-micro-UV spectrophotometer. Subsequently, the isolated RNA was subjected to reverse transcription into cDNA via a 10 μL reverse transcription system. This system comprised 3.5 μL of DEPC-treated H₂O, 0.5 μL of cDNA, 0.5 μL of forward primer, 0.5 μL of reverse primer, and 5 μL of TB Green Premix, executed in strict accordance with the protocol delineated in the PrimeScriptTM RT kit. Amplification of the resulting cDNA was implemented through the Mx3000 Multiplex Quantitative PCR system from Stratagene, headquartered in La Jolla, CA, U.S.A. The specific primer information for the target genes under investigation is detailed in Table 2, and glyceraldehyde phosphate dehydrogenase (GAPDH) was regarded as a reference control for amplification. The computation of gene expression level was through the 2^−∆∆CT method.

Table 2. PCR Primer Sequences

Primer name	Sequence	Length (bp)
GAPDH	F: 5′-AGGTCGGTGTGAACGGATTTG-3′	20
	R: 5′-TGTAGACCATGTAGTTGAGGTCA-3′	18
TNF-α	F: 5′-GGACTAGCCAGGAGGGAGAACAG-3′	23
	R: 5′-GCCAGTGAGTGAAAGGGACAGAAC-3′	24
IL-1β	F: 5′-CACTACAGGCTCCGAGATGAACAAC-3′	25
	R: 5′-TGTCGTTGCTTGGTTCTCCTTGTAC-3′	25
TLR2	F: 5′-CTCCCAGATGCTTCGTTGTTCCC-3′	23
	R: 5′-GTTGTCGCCTGCTTCCAGAGTC-3′	22
NF-κB	F: 5′-AGACCCAGGAGTGTTCACAGACC-3′	23
	R: 5′-GTCACCAGGCGAGTTATAGCTTCAG-3′	25
Bax	F: 5′-GCTACAGGGTTTCATCCAGGATCG-3′	24
	R: 5′-TGCTGTCCAGTTCATCTCCAATTCG-3′	25
Bcl2	F: 5′-TGTAGAGAGGAGAACGCAGGTAGTG-3′	25
	R: 5′-GGCTTCTTCTTCTGTGTGGTGGTC-3′	24

Western Blotting (WB)

The expression levels of PI3K, p-PI3K, Akt, p-Akt, P65, p-P65, IL-1β, TNF-α, Bax, and Bcl2 could be quantified through a series of precisely controlled procedures. Initially, hepatic tissues were homogenized in radioimmunoprecipitation assay lysis buffer to facilitate the extraction of total proteins, which was augmented by the addition of phosphatase inhibitors and phenylmethylsulfonyl fluoride to inhibit proteolytic activity. Following homogenization, the protein samples were resolved by electrophoresis on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, after which their transfer to polyvinylidene difluoride membranes was carried out. To minimize nonspecific binding, the membranes were blocked for 2-h at room temperature with a solution of 5% skimmed milk powder. Subsequently, the incubation of membranes was executed overnight at 4°C with the appropriate primary antibody, facilitating specific binding to the target proteins. Thereafter, a horseradish peroxidase-conjugated secondary antibody, diluted to 1 : 5000, was applied for an additional 2-h, particularly at room temperature, enabling the amplification of the signal for detection. The visualization of protein expression was achieved utilizing an ECL (Enhanced Chemiluminescence) kit provided by Amersham, which facilitated the detection of the HRP activity. Finally, the grayscale values of the resultant protein bands were quantitatively accomplished through ImageJ software.

Statistical Analysis

It was attempted to express data in the form of the mean ± standard deviation that was obtained from 3 separate experiments. Implementation of statistical analysis was through one-way ANOVA, and post hoc tests were employed to compare the mean values. Statistical significance was recognized by p falling below 0.05, and this threshold below 0.01 was representative of extremely significant differences.

RESULTS

Screening of Hepatoprotective 27 Compounds from P. capitatum against CCl₄-Induced Injury in HepG2 Cells

To elucidate the in vitro hepatoprotective influences, the EtOAc extract of P. capitatum and its 27 constituent compounds were accurately assessed by culturing HepG2 cells in conjunction with the administration of CCl₄. CCl₄ is a highly toxic chemical agent recognized for its ability to induce lipid peroxidation in HepG2 and LO2 cell lines, resulting in substantial cellular damage, and it is broadly utilized as a model for determining the hepatoprotective influences of natural products.²³⁾

After 6-h incubation with 0.35% CCl₄, HepG2 cell viability was notably diminished to 48.5 ± 3.2%, thereby demonstrating the potent hepatotoxicity associated with this agent. Conversely, treatment with silymarin, which served as the positive control, at a concentration of 10 μM remarkably elevated cell viability to 79.6 ± 3.2% relative to the CCl₄-treated cohort. These findings reveal that the EtOAc extract of P. capitatum confers substantial protective influences against CCl₄-induced hepatic injury, achieving a notable viability rate of 78% at a concentration of 50 μg/mL (Fig. 1). Moreover, 10 of the evaluated compounds (specifically, 2, 4, 5, 6, 7, 8, 9, 12, 13, and 14) demonstrated a remarkable enhancement in cell viability, surpassing 70% at a concentration of 10 μM. Particularly noteworthy were compounds 2 (FR429) and 6 (2″-O-galloylquercitrin), which exhibited the most noticeable protective activities, with half-maximum effective concentration (EC₅₀) values of 6.04 and 5.28 μM, respectively. It is also noteworthy that compounds 2, 6, 7, 8, 9, 12, and 14 were reported for the first time in this investigation.

Fig. 1. Effect of Ethyl Acetate Site Extract of P. capitatum and Compounds 1–27 on the Activity of CCl₄-Injured HepG2 Cells

*p < 0.05, **p < 0.01, ***p < 0.001 vs. the model group; ^###p < 0.001 vs. the control group.

Protective Influences of FR429 against CCl₄-Induced Hepatic Injury in Mice

To further elucidate the hepatoprotective influences of FR429, the compound was administered orally to CCl₄-treated mice. Under normal physiological conditions, enzymes such as AST, ALT, and ALP are predominantly localized in the nucleus of hepatocytes. However, upon hepatocellular damage, these enzymes are released into the cytoplasm in significant quantities, rendering serum levels of AST, ALT, and ALP useful for determining out hepatic function.²⁴⁾ As depicted in Fig. 2, the model group displayed a noticeable elevation in serum levels of ALT, AST, and ALP, escalating from baseline values of 2106, 56, and 519 U/L in the control group to levels of 8285, 655, and 1867 U/L following CCl₄ administration, respectively. In contrast, pretreatment with the positive control silymarin at a dosage of 50 mg/kg, along with FR429 at doses of 50 and 10 mg/kg, resulted in a notable reduction in serum levels of ALT, AST, and ALP to ranges of 3197–6475 (p < 0.001), 142–427 (p < 0.001), and 779–1343 (p < 0.001) U/L, respectively (Fig. 2A). Moreover, the high-dose groups exhibited noticeably diminished serum enzyme levels relative to their corresponding low-dose counterparts, emphasizing the efficacy of FR429. In alignment with the serum analyses, histopathological assessments provided further confirmation that FR429 markedly mitigated liver lesions precipitated by CCl₄ exposure. This comprised reductions in necrosis, ballooning degeneration, and neutrophil infiltration in liver tissues. Consequently, these outcomes robustly support the conclusion that FR429 possesses remarkable anti-hepatic injury effects in vivo (Fig. 2B).

Fig. 2. Protective Effect of FR429 on Acute Liver Injury Caused by CCl₄

(A) Blood biochemical indicators ALT, AST, and ALP values, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the model group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the control group. (B) H&E staining of mouse liver induced by CCl₄-induced liver injury under ×20 magnification microscope. Arrows point to vacuoles of lipid droplets in the plasma of hepatocytes.

Transcriptomic Analysis of FR429’s Protective Mechanisms

To elucidate the mechanism by which FR429 combats hepatic injury, transcriptomic analysis was implemented through RNA-seq on liver tissues after FR429 treatment. DEGs between the control-model and model-FR429 groups are depicted in Fig. 3A, where identification of DEGs was based on p < 0.05 and |log 2 FC| > 1. The model-FR429 group exhibited 3788 upregulated and 3085 downregulated DEGs relative to the control-model group, as displayed in the heatmap in Figs. 3B, 3C. A total of 178 target proteins were recognized to determine FR429’s regulation of barrier dysfunction, and the outcomes of cluster analysis of these significant DEGs are displayed in Fig. 3C. Subsequently, 178 DEGs underwent protein-protein interaction network analysis, as well as GO and KEGG pathway enrichment analyses (Figs. 3D, 3E).

Fig. 3. Transcriptomics Analysis

(A) Number of up/downregulated unigenes in model vs. control and FR429 vs. model comparison. (B) Venn diagram of the number of differentially expressed genes in control, model, and FR429 groups. (C) Heatmap showing the expression of the 178 DEGs in response to FR429. (D) GO terms enrichment of DEGs in model vs. control. (E) GO terms enrichment of DEGs in FR429 vs. model. (F) KEGG pathway enrichment of DEGs in model vs. control. (G) KEGG pathway enrichment of DEGs in FR429 vs. model. The x-axis represents the rich factor, while the Y-axis represents KEGG pathways enriched. The size of the bubble represents the gene number involved in the KEGG pathway. The color represents the Q-value of enrichment.

The top biological process entries were immune response, immune system process, and immune system regulation. The cellular component entries highlighted the membrane’s external side, plasma membrane, and immunoglobulin complex, while the molecular function analysis primarily enriched antigen binding. FR429 also modulated multiple pathways, including metabolic and PI3K/Akt pathways, as well as hypertrophic cardiomyopathy, dilated cardiomyopathy, and primary bile acid biosynthesis. Overall, transcriptomic analysis suggested that FR429’s hepatoprotective effects engage multiple signaling pathways, emphasizing the necessity of further molecular investigation into the PI3K/Akt pathway.

FR429 Could Protect Hepatotoxicity via the PI3K–Akt Pathway

Prior investigations have elucidated the notable function of the PI3K–Akt pathway in both the prevention and therapeutic intervention of hepatic diseases. Expanding upon the transcriptomic analysis referenced, RT-qPCR and WB were employed to authenticate critical genes and proteins within the PI3K/Akt signaling cascade at both transcriptional and translational levels. As depicted in Fig. 4A, the mRNA expression levels of toll-like receptor 2 (TLR2), nuclear factor-kappa-B (NF-кB), IL-1β, and TNF-α in the model group exhibited a noticeable elevation relative to the control group (p < 0.05). Notably, treatment with FR429 dramatically attenuated the CCl₄-induced upregulation of mRNA expression for TLR2, NF-кB, IL-1β, and TNF-α in the murine model. Furthermore, the levels of p-PI3K and p-Akt proteins were noticeably elevated in liver tissues subjected to CCl₄ injury, whereas these levels were markedly diminished following administration of silymarin and FR429 (p < 0.05) (Fig. 4B). Consequently, FR429 exerted a profound influence by dramatically reducing the expression levels of p-P65, IL-1β, and TNF-α in the groups treated with silymarin and FR429.

Fig. 4. Expression Levels of Genes and Proteins Related to PI3K/Akt Signaling Pathway

(A) Expression levels of mRNA for TLR2, NF-κB, IL-1β, and TNF-α. (B) Expression levels of proteins for p-PI3K, PI3K, p-Akt, Akt, p-P65, P65, IL-1β, and TNF-α. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the model group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the control group.

Moreover, the downstream effectors of the PI3K/Akt pathway, Bax and Bcl2, are widely distributed across various cell types and are integral to the regulation of apoptotic processes. In the model group, a notable escalation in Bax’s mRNA expression level was observed (p < 0.05) relative to the control group, while a concomitant reduction in Bcl2’s mRNA expression was noted (p < 0.01). Conversely, the FR429 group demonstrated a remarkable reduction in Bax’s mRNA level (p < 0.05) and a notable increase in Bcl2’s mRNA level (p < 0.05) in a dose-dependent manner relative to the model group (Fig. 5A). Aligning with the mRNA data, WB confirmed these outcomes, revealing a parallel trend in Bax’s and Bcl2’s protein levels (Fig. 5B). Consequently, FR429 exhibited hepatoprotective properties through modulating the PI3K/Akt signaling pathway.

Fig. 5. Expression Levels of Genes and Proteins Related to Apoptosis Signaling Pathway

(A) Expression levels of mRNA for Bax and Bcl2. (B) Expression levels of proteins for Bax and Bcl2. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the model group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. the control group.

DISCUSSION

Chronic liver conditions, including hepatic injury, liver fibrosis, nonalcoholic fatty liver disease, hepatic cirrhosis, and hepatocellular carcinoma (HCC), constitute some of the most prevalent and escalating global health concerns. The scarcity of effective therapeutic interventions in current clinical practice reflects a pressing challenge in the discovery and development of hepatoprotective agents with multifaceted benefits. ALI, triggered by exposure to diverse chemical agents, biological compounds, traditional Chinese medicines, and their metabolites, serves as a notable precursor in the progression of acute hepatic dysfunction toward chronic pathologies involving fibrosis, cirrhosis, hepatic failure, and malignant transformation. Given the absence of potent anti-hepatic injury medications, there remains a critical demand for novel, efficacious hepatoprotective drugs. The underlying pathological mechanisms of hepatic injury are notably complex, primarily involving inflammation, oxidative stress mitigation, apoptosis inhibition, and other complex biochemical processes, further accentuating the need for targeted therapeutic advancements.²⁵⁾

P. capitatum, a vital constituent of the Hmong medicinal flora, is extensively distributed throughout the southwestern regions of China, where it serves as a traditional remedy for an array of urological diseases, including urinary tract infections, pyelonephritis, and the formation of urinary calculi. The phytochemical profile of P. capitatum is characterized predominantly by flavonoids and phenolic compounds integral to its medicinal efficacy.²⁶⁾ The bioactive compounds isolated from P. capitatum exhibit a remarkable range of pharmacological activities involving anti-inflammatory, antioxidant, anticancer, analgesic, and diuretic properties, thereby marking its therapeutic potential across various medical domains.

FR429, as a polyphenolic natural compound from P. capitatum, possesses a number of pharmacological activities. For instance, FR429 can directly downregulate the level of EZH2 protein and enhance proteasome-dependent degradation of EZH2 in HCC to inhibit growth and exert anticancer effects against HCC.²⁷⁾ FR429 also demonstrates antibacterial and cytotoxic effects on human oral squamous cells.²⁸⁾ However, the anti-hepatic injury activity and the mechanism of action of FR429 have not yet been reported.

Thus, this research elucidated the hepatoprotective pharmacodynamics of FR429, specifically targeting its molecular pathways and underlying mechanisms to pave a scientific basis for future anti-hepatic injury drug development.

In the preliminary phase, 27 phenolic compounds were extracted from P. capitatum, with 2 compounds exhibiting remarkable liver-protective efficacy. Notably, FR429 (compound 2) demonstrated substantial bioactivity with an EC₅₀ of 6.04 μM. Further in vivo assessments uncovered that FR429 significantly ameliorated CCl₄-induced acute hepatic injury in mice at dosages of 10 and 50 mg/kg over a 7-d intragastric (ig) regimen. Transcriptomic analysis subsequently identified 178 DEGs, revealing the PI3K/Akt signaling pathway as a potential target of FR429’s action. Mechanistic insights suggest that FR429 could mediate hepatoprotective effects by modulating expression levels of critical genes and proteins, specifically targeting TLR2, IL-1β, NF-κB, TNF-α, Bax, and Bcl2, as well as phosphorylation ratios such as p-PI3K/PI3K, p-Akt/Akt, and p-P65/P65. Through this modulation, FR429 potentially mitigates CCl₄-induced hepatic damage, unveiling insights into its therapeutic mechanism.

The PI3K/Akt signaling pathway, a quintessential regulatory mechanism, is extensively studied for its integral role in modulating inflammatory responses, apoptosis, and oxidative stress, with implications across a spectrum of pathophysiological conditions involving neurodegenerative disorders, glucose homeostasis, and oncogenesis.^29–32) Serving as a principal effector in this pathway, Akt is acknowledged as the central mediator of PI3K-dependent signaling. Upon PI3K activation, phosphatidylinositol triphosphate (PIP3) is synthesized at the plasma membrane, prompting the translocation and phosphorylation of Akt. This phosphorylation event initiates a downstream signaling cascade, transmitting extracellular stimuli to intracellular targets and facilitating crucial biological processes such as cellular metabolism, the cell cycle, and survival.³³⁾ Notably, NF-κB functions as a downstream effector of p-Akt, and the PI3K/Akt pathway is capable of stimulating NF-κB activation via transcriptional enhancement of the P65 subunit.^34,35) Furthermore, apoptosis regulation in many cell types is mediated by the Bcl2 protein family and X-linked apoptosis inhibitor protein.³⁶⁾ Upon PI3K/Akt pathway activation, there is an upregulation of antiapoptotic gene expression (e.g., Bcl2), accompanied by suppression of proapoptotic genes (e.g., Bax), resulting in diminished apoptotic activity and bolstering cell survival.³⁷⁾

In addition, FR429 may exert its hepatoprotective effect by inhibiting the PI3K/Akt signaling pathway. Under normal physiological conditions, this pathway maintains a relative equilibrium in the liver. However, when the liver is stimulated by damage-causing factors such as viral infection, drug toxicity, chemical damage, or alcohol intake, the PI3K/Akt pathway is abnormally activated. For instance, in the case of chemical damage, TLR2/4 and other related receptors are activated. This activation then triggers a series of events through adaptor proteins that ultimately activate PI3K. PI3K converts PIP2 to PIP3, which in turn recruits and activates Akt. Once activated, Akt activates NF-κB; it enters the nucleus and initiates the transcription of a series of inflammation-related genes. Simultaneously, the balance between proapoptotic proteins like Bax and antiapoptotic proteins such as Bcl2 is disrupted, leading to an increase in hepatocyte apoptosis and consequently resulting in liver damage.

FR429 may interact with the catalytic or regulatory subunit of PI3K, interfering with its normal function. For example, it could prevent PI3K from binding to upstream activation signals, such as those from TLR2/4 activation, thereby inhibiting the phosphorylation of PIP2 to PIP3. Without sufficient generation of PIP3, Akt cannot be effectively recruited to the cell membrane for subsequent activation steps, cutting off the “supply line” of signal transduction and preventing excessive activation of the signaling pathway from the source. Even if some PI3K is partially activated and generates a certain amount of PIP3, FR429 may also inhibit Akt. It might bind to the active site or regulatory domain of Akt, preventing its phosphorylation by kinases such as PDK1. As a result, the numerous downstream functions of Akt cannot be realized.

Moreover, FR429 may also play an inhibitory role in the activation of NF-κB. It could prevent Akt from phosphorylating IKK, thereby inhibiting the phosphorylation and degradation of IκB. As a result, NF-κB remains bound to IκB in the cytoplasm and cannot enter the nucleus to initiate the transcription of inflammation and apoptosis-related genes. For example, after inhibiting NF-κB, the production of inflammatory factors such as TNF-α and IL-1β is reduced, alleviating the damage to liver tissue caused by inflammation. At the same time, it can also maintain the normal balance of apoptosis-related proteins such as Bax and Bcl2, reducing hepatocyte apoptosis and ultimately playing a role in protecting the liver.

Unlike the positive drug, silymarin, which has a broad but less specific hepatoprotective mechanism, FR429 provides a highly targeted effect against inflammation-induced liver damage and requires a lower effective dosage.

It should be noted that after transcriptomic analysis, this study was divided into 2 verification stages. Initially, the vital genes and proteins in the PI3K/Akt pathway were verified through the significant KEGG pathway enriched by DEGs, and the mechanism of FR429 against hepatic injury was preliminarily explored and verified. Secondly, the most significant DEGs identified by transcriptomic analysis may be the target of FR429 against hepatic injury. This outcome will be combined with the follow-up study to experimentally determine the specific target of FR429 for further research.

Consequently, the present investigation unveiled that FR429 had a potent anti-hepatic injury effect, and its mechanism of action could inhibit CCl₄-induced hepatic damage in mice by regulating the PI3K/Akt signaling pathway (Fig. 6).

Fig. 6. FR429 Exerts Anti-hepatic Injury Effects through PI3K/Akt Signaling Pathways

CONCLUSION

In this investigation, 27 distinct components were totally isolated and characterized from the EtOAc extract of P. capitatum. Among them, 10 compounds exhibited significant cytoprotective effects against CCl₄-induced hepatic injury in HepG2 cells. Notably, compounds 2 (FR429, EC₅₀ = 6.46 μM) and 6 (2″-O-galloylquercitrin, EC₅₀ = 5.36 μM) demonstrated exceptional potency. Concurrently, FR429, administered at a dose of 50 mg/kg (via the ig route for 7 consecutive days), markedly mitigated hepatocellular damage in a CCl₄-induced ALI murine model. Further mechanistic studies revealed that FR429’s hepatoprotective effects are potentially mediated through modulating the PI3K/Akt signaling pathway. These outcomes unveil that FR429 represents a promising natural agent for hepatoprotection.

Acknowledgements

This research was supported by Guizhou Provincial Science and Technology (No. ZK [2022]-362; No. [2022]4028; No. ZK [2024]-047; No. ZK [2023]-378; No. gzwkj [2023]-026; and No. [2023] ZK01), the Innovation and Entrepreneurship Training Program for Undergraduates from China (No. 202310660082), the Science Foundation of Guizhou Education Technology (No.2022-064), and the University Engineering Research Center for the Prevention and Treatment of Chronic Diseases by Authentic Medicinal Materials in Guizhou Province (No. [2023]035).

Author Contributions

Yaru Yang: methodology, data curation, and writing—original draft. Lei He: software, validation, and writing—review and editing. Minghui He: data curation, methodology, and writing—review and editing. Xu Zhang: validation, methodology, and writing—review and editing. Shanggao Liao: software, validation, and writing—review and editing. Zhu Zeng: visualization, project administration, and writing—review and editing. Yan Lin: conceptualization, funding acquisition, supervision, and writing—review and editing. Bo Tu: formal analysis, methodology, supervision, and writing—review and editing.

Conflict of Interest

The authors declare no conflict of interest.

Data Availability

The authors are unable or have chosen not to specify which data has been used.

Supplementary Materials

This article contains supplementary materials.

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