Protective effects of Astragaloside IV on various liver diseases: From chemistry to herbal medicines (Review)

Introduction

The liver is a vital and intricate organ within the human body, undertaking several important physiological functions, including metabolizing carbohydrates and lipids, detoxifying drugs, secreting bile, and synthesizing plasma proteins, which are irreplaceable for maintaining homeostasis in the body's internal environment and overall health (1,2). Liver disease is characterized by injury to hepatocytes, infiltration by inflammatory cells, and activation of hepatic stellate cells (HSCs). It covers an extensive range of conditions, ranging from acute liver damage and chronic liver conditions to end-stage liver disease (ESLD). In clinical settings, the prevalent types of liver disease consist of viral hepatitis, non-alcoholic fatty liver disease (NAFLD), liver injury, liver fibrosis (LF), liver cirrhosis (LC) and liver cancer (3,4).

In modern society, the global number of liver disease patients continues to rise, imposing a heavy economic burden worldwide (5-7). According to the global epidemiological statistics of liver disease in 2023, more than 2 million individuals perish annually because of liver-related conditions, representing 4% of the total number of deaths in the world, and ranking 11th among the causes of death in the world. LC and liver cancer account for the highest proportion of deaths (8). However, a large number of the early manifestations of liver disease lack specificity. The diagnosis of liver disease is highly dependent on liver biopsy. Given that an invasive approach is inappropriate for widespread screening, the disease often progresses to the ESLD stage (9). Despite the use of a range of drugs, such as antioxidant medications, including glutathione (GSH), choleretic agents, including ursodeoxycholic acid (10), and statins, including simvastatin (11), and corticosteroids (12) in current treatments, most patients ultimately and inevitably progress to ESLD. Traditional treatments, such as resection, transplantation and systemic chemotherapy, have certain limitations. For example, liver transplantation, a treatment for ESLD, faces the dual challenges of high cost and a shortage of donor organs (13-15). Transplantation therapy involving mesenchymal stem cells (MSCs) has undergone assessment in numerous clinical trials. To a certain extent, it has demonstrated safety and is considered a promising avenue for cellular therapy of liver disease (16). However, transplanted MSCs are at risk of unintended differentiation, may impair antitumor immune responses, and even have a tendency to form new arteries and thus promote tumor development and spread (17). Therefore, developing new, highly effective, and low-toxicity drugs for liver disease intervention remains an important task for the medical community in the modern healthcare system.

Various factors, including viruses, alcohol, metabolites, toxins and other pathogens, predispose individuals to liver diseases. Metabolic dysfunction, assessed using different indicators such as insulin resistance, disturbances in bile acid metabolism, imbalances in the intestinal flora, and elevated uric acid levels, can lead to metabolic-associated steatohepatitis (18,19). NAFLD is characterized by the abnormal accumulation of fat within hepatocytes, accompanied with the abnormal inactivation of adenosine monophosphate-activated protein kinase (AMPK) and abnormal activation of nuclear factor κB (NF-κB) (20,21). This phenomenon further exacerbates hepatic inflammatory responses and induces oxidative damage to the liver. The key pathogenic mechanism of LF is abnormal HSC activation. Upon activation, these cells trigger the excessive deposition of extracellular matrix (ECM) proteins such as collagen and fibronectin (FN), subsequently inducing hepatocyte apoptosis and cellular senescence and ultimately leading to LF development (22). The pathogenesis of hepatocellular carcinoma (HCC) is more complex than that of liver injury. HCC primarily manifests through the proliferation of cancer cells and the apoptosis of normal cells, while liver injury involves pathological processes such as oxidative stress, inflammatory responses and cellular apoptosis (23,24).

Chinese medicines have garnered significant attention on account of their minimal toxicity and side effects, and excellent tolerability. Radix Astragali (RA), predominantly sourced from the dried root of Astragalus membranaceous (A. membranaceous), a plant belonging to the legume family, has been used in pharmacological studies or as a dietary supplement in some countries (25). Astragaloside IV (AS-IV), a natural saponin obtained from RA, possesses low toxicity and various bioactivities, including anti-inflammatory, antioxidant, antiapoptotic and anticancer. These activities are important in treating neurological, cardiovascular and metabolic diseases, and cancer (26). These comprehensive summaries and exploratory research findings have substantially reinforced the medicinal value and attention from investigators of AS-IV. In recent years, the therapeutic potential of AS-IV in liver diseases has gradually attracted widespread attention from researchers. A number of in vivo and in vitro studies have confirmed that AS-IV can exert hepatoprotective effects in liver diseases via multiple signaling pathways.

Consequently, the present study conducts a comprehensive review of the hepatoprotective effects of AS-IV in various diseases, deeply analyzes the molecular mechanism underlying its role in liver disease, and summarizes the measures to improve its bioavailability. The aim is to provide innovative insights and rationale for the future development of AS-IV as a prospective new drug for treating liver diseases. In contrast to recently published reviews (27), the present review systematically summarizes the chemical structure, physicochemical properties and extraction methods of AS-IV. Regarding hepatoprotective mechanisms, liver injury was classified into four types, providing an updated analysis of iron overload-induced liver damage, and the synergistic antifibrotic effects of AS-IV combined with ferulic acid were reviewed. Moreover, the pharmacokinetics, toxicology and nano-delivery systems of AS-IV were comprehensively discussed, focusing on translational applications.

Overview of AS-IV

Sources and background

A. membranaceous, a common qi-tonifying herb, has had its medicinal use documented since 'Shen Nong's Herbal Classic,' written over 2,000 years ago in China (28). It is a member of the legume family and has a wide geographical distribution across the Northern Hemisphere, South America and Africa, featuring a distinctive plant morphology with erect and well-branched stems and neatly arranged leaflets on pinnately compound leaves (29). AS-IV is a natural saponin extracted from the root of A. membranaceous, also known as RA. The composition of RA varies by region and country. Its main components include astragali polysaccharides, saponins and flavonoids (25). AS-IV serves as a marker substance for one of the aspects of quality control for RA and its products in the Chinese Pharmacopoeia (2020 edition) (30).

RA is used in the field of ethnopharmacology in several countries in Asia, North America and Europe, such as China, the United States and Russia (31). In clinical practice, numerous proprietary and patented Chinese medicines and injections are formulated using or contain RA, including Astragalus injection, Astragalus polysaccharide injection, Yupingfeng Powder, Xuefu Zhuyu Decoction and Huangqi Siwu Decoction (32,33). These preparations have shown favorable application prospects in treating neurological, cardiovascular, renal, gynecological and pulmonary disorders, and organ injuries (26). RA is used as a dietary product in China and is also classified as a legal dietary supplement in the United States (34).

Chemical and physical properties

AS-IV appears as a white to yellow crystalline powder (35) and has a chemical name of 3-O-β-d-xylopyranosyl-6-O-β-glucopyranosyl-cyclostelli feryl alcohol, molecular formula of C41H68O14, and a relative molecular mass of 784.97 g/mol (36). This compound has a high melting point, ranging from 295-296°C. It is insoluble in water but displays solubility in certain organic solvents such as ethanol, methanol and acetone (37). AS-IV belongs to the category of triterpenoid saponins and pentacyclic triterpenoids, featuring a tetracyclic triterpenoid saponin in the form of lanolin ester alcohol. This structural configuration is vital because it underlies the manifestation of its anti-inflammatory and antioxidant effects and ability to improve lipid metabolism, thereby potentially exerting hepatoprotective effects (38,39). The commonly employed extraction methods include solvent extraction, ultrasound-assisted extraction, microwave-assisted extraction, enzyme-assisted extraction, supercritical fluid extraction and rapid extraction techniques (40) [Fig. 1 (37)].

Figure 1 Chemical formula of Astragaloside IV.

Pharmacological effects

AS-IV, an emerging natural compound in the medical realm, is a highly promising nutritional therapeutic agent. It displays an array of advantageous biological functions, such as anti-inflammatory, antioxidant (41), antiapoptotic (42), anticancer (43) and antitumor (44). These properties hold promise for its applications in neurological disorders such as stroke, dementia, epilepsy and Alzheimer's disease (29). AS-IV is also of great significance in cardioprotection. It may play a role, either directly or indirectly, in the treatment of cardiac disorders. It controls myocardial hypertrophy and fibrosis processes and effectively reduces cardiomyocyte apoptosis (45-47). In addition, AS-IV shows remarkable efficacy in combating diabetes and its complications (48), along with certain viruses (49). In the area of intestinal health, it modulates intestinal function, alleviates intestinal inflammation, and effectively restores damaged intestinal barrier function (50,51). In the treatment of kidney and gynecological diseases, AS-IV can ameliorate chronic glomerulonephritis and triple-negative breast cancer by participating in autophagy inhibition (52,53). Owing to its powerful antioxidant and anti-inflammatory characteristics, AS-IV exhibits significant protective effects against various lung diseases (54,55). These properties provide a strong rationale for the evaluation of AS-IV as a potential therapeutic option for various liver ailments.

Hepatoprotective effect of AS-IV

NAFLD

NAFLD belongs to the category of metabolic syndromes. It is triggered by well-defined liver-damaging factors. A prominent feature of NAFLD is the over-accumulation of fat in hepatocytes, which is evident in conditions such as non-alcoholic fatty liver, non-alcoholic steatohepatitis (NASH) and associated LC (56). In general, non-alcoholic fatty liver may progress to NASH, which in turn may eventually progress to LC and liver cancer (57). NAFLD development is associated with a variety of factors. The currently widely recognized 'multiple strike' hypothesis takes into account the synergistic effects of fat build-up, lipotoxicity, inflammation, oxidative stress, mitochondrial impairment, gut microbiota and genetic elements (58). As shown in NAFLD models, a diet rich in fat can trigger hepatic steatosis and inflammation, which subsequently lead to significant oxidative damage in the liver (59). Palmitic acid, the most prevalent free fatty acid (FFA) in dietary sources and the serum, contributes to hepatic steatosis and exacerbates insulin resistance (60).

Zhai et al (61) successfully established a cellular model of hepatic steatosis using FFA. They discovered that FFA-induced lipid accumulation led to endoplasmic reticulum (ER) stress, and triggered autophagy and inflammation. This ER stress was significantly alleviated by AS-IV treatment. It reduced the elevated expression of ER stress markers, such as glucose-regulated protein 78, protein kinase R-like ER kinase, and C/EBP homologous protein, and mitigated the increased nuclear translocation of these markers (21,62). Liu et al (63) discovered that FFA induced the blockage of autophagic fluxes, attenuating lipophagy. AS-IV triggered autophagy within HepG2 cells by inhibiting the activation of protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway, effectively attenuating FFA-induced lipid accumulation in HepG2 cells and degrading excess intracellular lipids. Moreover, AS-IV alleviates oxidative stress in hepatocytes. It reduces the elevated levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and mitigates the significant decrease in GSH peroxidase (Px) activity. And, AS-IV plays a role in hepatocyte apoptosis inhibition by downregulating the mRNA and protein expression levels of B-cell lymphoma/leukemia-2-associated X protein (Bax) and upregulating the mRNA and protein expression levels of B-cell lymphoma 2 (Bcl-2) (64). NAFLD is marked by the deposition of hepatic fat within the liver, and prolonged deposition can give rise to hepatic inflammation. AS-IV can mitigate the inflammatory response by inhibiting the generation of inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). Toll-like receptor 4 (TLR4) can initiate a series of injury-related immune responses. However, the overexpression of the TLR4 signaling pathway leads to the occurrence of immune-inflammatory responses, and AS-IV can inhibit the overexpression of this pathway (64-67). AMPK functions as a crucial regulator in hepatic lipogenesis, and acetyl-CoA carboxylase phosphorylation is a direct target of AMPK. AS-IV dose-dependently increased the phosphorylation of acetyl-CoA carboxylase (ACC) by AMPK activation. The presence of adipogenic genes such as acc1, scd1 and fas in sterol regulatory element-binding protein-1c (SREBP-1c) reduces the accumulation of mature SREBP-1c in HepG2 cells exposed to FFA. It also inhibits the enhanced nuclear translocation of SREBP-1c and downregulates adipogenic gene mRNA. Co-processing with 5-amino-4-imidazolecarboxamide ribonucleotide can further enhance this effect (68). The intestinal flora is essential for maintaining physiological balance in the human body (69). When the population of bile salt hydrolase flora decreases, AS-IV can inhibit diet-induced metabolic damage, especially hepatic steatosis. AS-IV can also inhibit intestinal farnesoid receptor (FXR) signaling, and this inhibition is accompanied with a reduction in intestinal fibroblast growth factor expression. Subsequently, hepatic FXR is activated. AS-IV increases glucagon-like peptide-1 and promotes ceramide production. All these effects collaborate to suppress the progression of hepatic steatosis (61). In a mouse model study of NAFLD, AS-IV reduced body weight gain in NAFLD mice without reducing food intake. It also decreased the levels of NAFLD markers aspartate aminotransferase (AST) and alanine aminotransferase (ALT), eliminated the tendency of elevated triglyceride (TG) and total cholesterol, and significantly reduced the release of aminotransferases due to hepatocellular injury (64,70).

All the aforementioned molecular effects of AS-IV have been validated across heterogeneous experimental systems, including in vitro fatty liver cell models, HepG2 cell lines, primary mouse hepatocytes, and in vivo NAFLD mouse and rat models. Comprehensive analysis of current evidence demonstrates that, despite variations in experimental conditions, these regulatory effects maintain an overall consistent trend. AS-IV directly reduces fat accumulation, facilitates excess fat degradation, and inhibits steatosis by regulating autophagy, promoting AMPK activation, and synergizing with the gut microbiota. Furthermore, it suppresses oxidative stress and hepatic inflammation in hepatocytes, thereby alleviating NAFLD.

LF

LF is the outcome of a continuous wound-healing response to chronic liver injury, and its main causes include chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infections, alcohol abuse that results in alcoholic liver disease (ALD), NASH, and autoimmune diseases (71). The distinguishing feature of LF is HSC activation, which subsequently triggers excessive ECM accumulation, leading to fibrous scarring. If the disease progresses further, fibrous scarring can transform into LC and ESLD. LC is defined as the presence of regenerating hepatocellular nodules in the liver, accompanied with a reduced hepatic blood supply. Oxidative stress and inflammation are as important factors in LF development (72,73).

Chen et al (74) revealed the protective effects of AS-IV on LF, it downregulated collagen type I alpha 1 chain, α-smooth muscle actin (α-SMA) and FN, thereby reducing the excessive secretion and accumulation of ECM. AS-IV can also inhibit the excessive pathological activation of the NF-κB signaling pathway, thereby exerting antifibrotic effects. It can enhance the expression levels of p65, p52 and p50, thus promoting the senescence and apoptosis of activated HSC-T6 cells. Moreover, AS-IV reduces the protein and mRNA expression of α-SMA, collagen synthesis, and FN through mediated p38 MAPK. In turn, this process inhibits HSC activation. AS-IV exhibits antioxidant properties and could decrease the levels of ROS and lipid peroxide (75). Hydroxyproline content, one of the indicators of LF, initially showed a decreasing trend after AS-IV treatment. In vitro studies showed that the platelet-derived growth factor-bb stimulated proliferation of HSC was significantly inhibited by AS-IV (76). In the bile duct ligation-induced LF model, the combined treatment of AS-IV and ferulic acid (FA) effectively alleviated LF, significantly reduced collagen deposition, and greatly improved the pathological changes in the liver. Compared with AS-IV or FA alone, the combination therapy was more effective in inhibiting HSC activation and significantly enhanced the body's capacity to resist oxidative stress. The reduction in MDA levels, elevation in GSH-Px, and enhancement of superoxide dismutase (SOD) enzyme activity are all attributed to AS-IV rather than FA. In addition, AS-IV activates antioxidant defense mechanisms via the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and promotes the phosphorylation of glycogen synthase kinase 3β (GSK-3β) at the Ser9 site (77).

All the aforementioned molecular effects of AS-IV have been validated across heterogeneous experimental systems, including in vitro HSC-T6 cells and HCC cell lines, in vivo models of the PS-induced LF rat model, the bile duct ligation rat model, and the PSmad3C and Nrf2 mouse models. Comprehensive analysis of current evidence demonstrates that, despite variations in experimental conditions, these regulatory effects maintain an overall consistent trend. AS-IV exerts antifibrotic effects in vivo by inhibiting oxidative stress and inflammation via the NF-κB and Nrf2 pathways. It also suppresses the proliferation of HSC in vitro, and its combined treatment with FA synergistically enhances the anti-fibrotic effect. Compared with other antiviral drugs, corticosteroids, immunosuppressive and antifibrotic agents, AS-IV exhibits significant advantages, including a broad application spectrum, high safety, multi-target regulation and low cost (78,79) (Fig. 2).

Figure 2 Mechanisms by which AS-Ⅳ ameliorates non-alcoholic fatty liver disease and liver fibrosis. BSH, bile salt hydrolase; IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-α; IL-6, interleukin-6; TLR4, toll-like receptor 4; FXR, farnesoid x receptor; GLP-1, glucagon-like peptide-1; ACC, acetyl-CoA carboxylase; AMPK, adenosine monophosphate-activated protein kinase; GSH-Px, glutathione peroxidase; Akt, protein kinase B; mTOR, mammalian target of rapamycin; MDA, malondialdehyde; Bax, b-cell lymphoma/leukemia-2-associated X protein; Bax, B cell lymphoma 2; ROS, reactive oxygen species; ER, endoplasmic reticulum; SREBP-1c, sterol regulatory element-binding protein-1c; GRP78, glucose-regulated protein 78; CHOP, c/edp homologous protein; PERK, phosphorylated protein kinase-like ER kinase; MAPK, mitogen-activated protein kinase; AS-IV, Astragaloside IV; Nrf2, nuclear factor erythroid 2-related factor 2; α-SMA, α-smooth muscle actin; COLα1 (I), collagen type I alpha 1 chain; FN, fibronectin; NF-κB, nuclear factor κB; GSK-3β, glycogen synthase kinase 3β; SOD, superoxide dismutase; ECM, extracellular matrix.

HCC

Liver cancer, a common solid malignancy arising from the liver, is the sixth most common type of cancer and the fourth leading cause of cancer-related deaths worldwide (80). It encompasses HCC, intrahepatic cholangiocarcinoma and fibrous laminar carcinoma. Among them, HCC is the most common type of primary liver cancer, accounting for 90% of all liver cancer cases (81-83). Numerous factors, including HBV and HCV infections (84), NAFLD (85), alcohol abuse (86) and exposure to aflatoxin (87,88), elevate the risk of developing HCC. The common symptoms of HCC are abdominal pain, abdominal distension, weight loss, fatigue, jaundice and vomiting (89). Its pathogenesis is intricate and may involve alterations in the tumor microenvironment, inflammatory response, oxidative stress, tissue hypoxia, hepatocyte necrosis, regeneration related to fibrosis, and numerous molecular-level changes (90,91). At present, surgical resection remains the preferred treatment for HCC (92). AS-IV, a typical natural antioxidant, exerts potential pharmacological effects against various malignancies. It influences the development of HCC via four principal mechanisms: (i) metabolic regulation, (ii) immunomodulatory mechanism, (iii) apoptosis induction, and (iv) inhibition of epithelial-mesenchymal transition (EMT). The specific mechanisms are described below:

First, AS-IV can inhibit the malignant proliferation of hepatoma cells by regulating glycolipid metabolism, thereby breaking the abnormal metabolic homeostasis of tumor cells. A recent study showed that AS-IV decreased glucose consumption, lactate production and extracellular acidification rate (93). It also inhibits phosphoglycerate mutase 1 (PGAM1) succinylation levels and lysine acetyltransferase 2A (KAT2A) protein levels in SNU182 and Huh7 cell lines. Overexpressing KAT2A counteracted the reduction in cell viability and glycolysis brought about by AS-IV treatment in the SNU182 and Huh-7 cell lines. Meanwhile, KAT2A increases the succinylation of PGAM1, and AS-IV appeared to inhibit tumor growth by blocking the KAT2A-mediated succinylation of PGAM1 (93). Fang Gong et al (94) and Li et al (95) revealed that AS-IV significantly reduces the proliferation, migration and invasion in Huh-7 cells and suppresses ROS production. These effects were achieved via multiple pathways, including the TGF β1/mothers against decapentaplegic homolog (Smad) and Nrf2/heme oxygenase 1 (HO-1) pathway; upregulated expression of pSmad3C, pNrf2, HO-1 and DT-diaphorase (NQO1); and decreased expression of pSmad2C, pSmad2L, pSmad3L, plasminogen activator inhibitor-1 and α-SMA. The inhibitory impact of AS-IV on HCC in vivo was further corroborated in a mouse subcutaneous tumor model, in which AS-IV suppressed the proliferation, migration and invasive capacity of Huh-7 cells by inhibiting macrophage M2 polarization and reducing the expression of M2 macrophage-stimulated phosphorylated signal transducer and activator of signal transducer and activator of transcription (STAT) 3, p-NF-κB and TLR4 expression (94,96). In a mouse model of HCC, Li et al (95) analyses further verified the reversibility and antagonism of AS-IV in regulating pSmad3C/3L and the phosphorylated expression of the Nrf2/HO-1 pathway in TGF β1-activated HSC-T6 and HepG2 cells. All these results indicated that AS-IV can effectively inhibit HCC progression by suppressing the proliferation, migration and invasion of liver cancer cells.

AS-IV exerts indirect anti-hepatoma effects by regulating the tumor immune microenvironment and enhancing antitumor immune responses. Yang et al (97) and Cui et al (98) uncovered the underlying mechanism: AS-IV could target the miR-135b-5p signaling pathway of carboxypeptidase n domain-containing protein 1, alleviating the immunosuppression caused by programmed death-ligand 1 (PD-L1) and accelerating apoptosis. In addition, AS-IV exhibited a dose-dependent inhibitory impact on the proliferation of Huh-7 and SMMC-7721 cell lines. It suppressed the interferon-γ-induced expression of PD-L1, thereby weakening PD-L1-mediated immunosuppression and immune cell cytotoxicity, and strengthening the antitumor immune response (96). Guo et al (99) demonstrated that AS-IV facilitated dendritic cell maturation and enhanced antigen presentation. It also increased the release of the functional cytokine interleukin-12 (IL-12) by upregulating various surface markers, namely, cluster of differentiation (CD)14, CD40, CD80, CD83, CD86 and human leukocyte antigen-DR. As a consequence, dendritic cell-induced specific cytotoxic T lymphocyte responses against the HCC cell line SMMC-7721 were enhanced. IL-12 is regarded as a natural immune booster in dendritic cell-mediated anti-HCC therapy. In a study by Qi et al (100), AS-IV exhibited anticancer activity by downregulating oncogenes such as vav guanine nucleotide exchange factor 3.1 and inhibiting the non-adherent-dependent growth of HepG2 cells. Another study showed that AS-IV inhibited the phosphorylation of c-Jun-terminal kinase and p-c-Jun and downregulated multidrug resistance gene 1 in Bel-7402 and Bel-7402/FU cells in a dose-dependent manner (101). It achieved this feature by decreasing the expression levels of multidrug resistance gene 1 mRNA and p-glycoprotein, thus reversing the resistance of Bel-7402/FU cells to 5-FU. Therefore, AS-IV can reverse multidrug resistance in a mouse model of HCC (101). Meanwhile, Qu et al (102) discovered that AS-IV enhanced the antitumor effect of cisplatin by inhibiting the expression of certain factors in HepG2 cells and multidrug resistance-associated protein 2. It also enhanced the responsiveness of HCC to cisplatin-based chemotherapy.

Inducing the apoptosis of hepatoma cells is one of the core antitumor mechanisms of AS-IV. Cui et al (98) revealed that AS-IV negatively regulated CTNNB1 levels through the binding of miR-150-5p to the untranslated region of CTNNB1. In addition, CTNNB1 overexpression partially reversed the tumor growth inhibition and apoptosis effects induced by miR-150-5p upregulation. Meanwhile, miR-150-5p upregulation suppressed β-catenin expression, thereby promoting apoptosis and inhibiting tumor growth. Furthermore, Su et al (103) showed that AS-IV could induce SK-Hep1 and Hep3B cells to halt the cell cycle in the G1 phase and activate the caspase-8-dependent exogenous and caspase-9-dependent endogenous apoptotic pathways. As a result, it suppressed the expression of apoptotic signal proteins such as myeloid cell leukemia-1 protein, x-linked inhibitor of apoptosis protein, cellular fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein, and survivin in HCC cells.

AS-IV significantly inhibits EMT in hepatoma cells. EMT is a key process for the migration, invasion and distant metastasis of hepatoma cells. Qin et al (104) reported that AS-IV could mitigate the inhibitory effect on GSK-3β by reducing Akt phosphorylation. As a consequence, the expression of the wingless-type MMTV integration site family (Wnt)/β-catenin signaling pathway is suppressed, and the EMT signaling pathway is regulated, resulting in the downregulation of N-catenin, vimentin and α-SMA. In addition, AS-IV reduces the expression of zinc finger protein transcription factor and promotes the transcription of E-cadherin to some extent, thereby weakening the invasion and migration abilities of HCC cells. Li et al (15) demonstrated that AS-IV inhibited the EMT and migration of HCC cells by downregulating long non-coding RNA-activated transcript in breast cancer. Jiang and Mao (105) demonstrated that AS-IV reduced the expression of N-calmodulin, E-calmodulin and vimentin in HepG2 and Hep3B cell lines by influencing their EMT. As a consequence, the proliferative capacity of these HCC cell lines HepG2 and Hep3B was diminished. Moreover, the enhanced apoptotic capacity was manifested by a substantial elevation in the protein amounts of caspase-3 and caspase-9, effectively inhibited tumor invasion and migration. In a mouse model of HCC, AS-IV induced the apoptosis of tumor cells by blocking the activation of the Wnt/β catenin/T cell factor-4 signaling pathway and reducing the protein expression of vascular endothelial growth factor.

All the aforementioned molecular effects of AS-IV have been validated across heterogeneous experimental systems, including in vitro HCC cell lines, in vivo nude mouse xenograft models, H22 tumor-bearing mouse models, HepG2 xenograft tumor models, and HCC rat models. Comprehensive analysis of current evidence demonstrates that, despite variations in experimental conditions, these regulatory effects maintain an overall consistent trend. AS-IV exerts a pivotal function in HCC development. It curbs the proliferation, migration, and invasion of cancer cells; restrains tumor growth; promotes apoptosis; and improves the treatment outcome by enhancing the antitumor immune response (Fig. 3).

Figure 3 Mechanisms by which Astragaloside Ⅳ ameliorate hepatocellular carcinoma. MRP2, multidrug resistance-associated protein 2; HO-1, heme oxygenase 1; NQO1, DT-diaphorase; PAI-1, plasminogen activator inhibitor-1; Smad, mothers against decapentaplegic homolog; STAT, signal transducer and activator of transcription; p-gp, p-glycoproterin; IL-12, interleukin-12; PD-L1, programmed death-ligand 1; Wnt, wingless-type mmtv integration site family; TGF4, T cell factor-4; TGF, transforming growth factor; EMT, epithelial-mesenchymal transition; ZFP, zinc finger protein transcription; KAT2A, lysine acetyltransferase 2A; CNDP1, carboxypeptidase n domain containing protein 1; miR, microRNA.

Liver injury

Liver injury refers to a series of pathophysiological alterations induced by diverse internal and external factors, with significant manifestations of liver dysfunction (106). If this condition remains inadequately controlled, then it has a high probability of further progressing to LF or LC, and may even deteriorate into acute liver failure (107). Liver injury has numerous causes, including toxins, inflammation, metabolic disorders and ischemia-reperfusion. In the present treatment of liver injury, synthetic drugs such as corticosteroids and IFN are commonly employed to manage the progression of this condition (108). When liver injury is exacerbated and progresses to liver failure, liver transplantation emerges as one of the limited therapeutic alternatives. This approach faces numerous challenges, with the low graft success rate being a prominent issue (109). Natural compounds with anti-inflammatory and antioxidant properties offer significant protection against liver injury (110,111). Among them, AS-IV, a typical natural compound, possesses strong anti-inflammatory and antioxidant activity and shows great potential for use in liver injury prevention (112).

Toxin-induced liver injury

Iron serves as a vital biological catalyst for cellular redox reactions and is essential for life. However, excessive iron absorption in the gut can lead to abnormal iron deposition within the parenchymal cells of various organs, including the liver, heart and pancreas, resulting in cytotoxicity, tissue damage and organ fibrosis. The liver is the primary site of iron storage and plays a central role in regulating iron homeostasis. Iron-induced cellular damage is caused by the oxygen-free radicals generated by iron and lipid membrane peroxidation. In the liver, particularly within the mitochondria and lysosomes, this condition is considered a factor in hepatocyte necrosis and apoptosis, ultimately leading to LF and liver damage (113-115). In a related study, Lo2 cells were continuously cultured in a medium containing iron dextrose to construct an iron overload model (116). The results revealed decreased Lo2 cell viability and increased autophagy and apoptosis, which were reversed by AS-IV. Excessive autophagy damages the cell structure, which was significantly alleviated by AS-IV. This effect manifested as a marked elevation in p62 expression and a significant decline in the microtubule-associated protein light chain 3 (LC3) Ⅱ/Ⅰ ratio in the cells treated with AS-IV. Meanwhile, the cell morphology and structure gradually returned to the normal state. Hepcidin is a well-recognized iron modulator that plays a crucial part in regulating plasma iron concentration by modulating iron release from the circulatory system or from the cells that store iron. The same study found that AS-IV dose-dependently increased the expression level of hepcidin, thus effectively inhibiting iron-induced apoptosis and autophagy in hepatocytes.

Cisplatin, a highly potent chemotherapeutic agent widely employed in the treatment of numerous cancers, exhibits remarkable anticancer effects. However, it is accompanied with severe issues of nephrotoxicity and hepatotoxicity (117,118). Cisplatin nephrotoxicity at standard clinical doses is one of the most common side effects. Although the incidence of hepatotoxicity is relatively low, it may still occur following exposure to high cisplatin doses. Hepatotoxicity occurs primarily because the liver is the main organ responsible for metabolizing a wide range of drugs and chemicals. After the kidneys, the liver is the organ in which cisplatin accumulates to the greatest extent; this is the key reason high-dose cisplatin induces hepatotoxicity. Prolonged or high-dose exposure to cisplatin may lead to hepatic necrosis, and apoptotic lesions may be observed in liver tissue. At present, research on the hepatotoxicity of cisplatin remains limited, and the potential mechanisms have not been fully elucidated (119,120). Several studies investigated cisplatin-induced toxicity and corresponding coping strategies. For instance, an experimental study using rats discovered that the hepatotoxic injury caused by cisplatin was significantly mitigated when the rats treated with cisplatin were administered AS-IV (121). In particular, the hepatic/body mass index of the rats returned to normal. The levels of ALT and AST, which had been elevated by cisplatin, showed a downward trend. This finding demonstrated that AS-IV could effectively alleviate cisplatin-induced hepatic injury in a dose-dependent manner (121). In another study, cisplatin induced an inflammatory response and autophagy in hepatocytes. AS-IV reversed this process, thereby offering protection against cisplatin-mediated liver injury in rats (122). Cisplatin-induced abnormal cellular function frequently triggers ferroptosis, a crucial type of programmed cell death in cisplatin-induced acute liver injury (123-125). Guo et al (122) distinctly demonstrated that AS-IV could inhibit excessive lipid peroxidation and iron induced cell death by targeting the peroxisome proliferator-activated receptor α (PPARα) pathway. It also restored the expression of GSH-Px 4 and ferroptosis suppressor protein 1, ultimately ameliorating cisplatin-induced liver injury.

Acetaminophen (APAP) is a well-known antipyretic and analgesic widely used in daily life. If overdosed, excessive APAP promotes high ROS generation, which induces oxidative damage and ultimately leads to liver injury (126,127). AS-IV demonstrates a positive intervention in this process. It can effectively reduce the inflammatory response and inhibit APAP-induced oxidative stress. Its mechanism of action is mainly based on the regulation of the Nrf2 pathway. AS-IV can activate Nrf2, resulting in a significant enhancement of nuclear Nrf2 expression. As a crucial transcription factor, Nrf2 governs the expression of downstream genes HO-1 and NQO1, both of which encode important antioxidant enzymes. Meanwhile, AS-IV inhibits the expression of Kelch-like ECH-associated protein 1, which is involved in the ubiquitination and degradation of Nrf2 within cells and exerts a negative regulatory influence on Nrf2 activity. These findings suggest that AS-IV alleviates liver damage caused by excess APAP (128,129).

Atorvastatin (ATO) is commonly utilized in clinical practice for its effects of lowering blood lipids and preventing atherosclerosis. However, its excessive use can give rise to hepatotoxicity (130). From a pathological perspective, an ATO overdose can trigger a series of pathological alterations in liver tissues, including inflammatory cell infiltration and apoptosis. Qin et al (131) reported that AS-IV could remarkably mitigate these adverse pathological changes. In particular, AS-IV alleviates the LF and inflammatory responses induced by ATO and activates the phosphorylation of AMPK. It also inhibits hepatocyte apoptosis by upregulating sirtuin 1 in the liver tissues of rats. As a result, AS-IV significantly reduces the hepatotoxicity caused by ATO.

Chronic excessive alcohol intake is a primary factor contributing to the development of ALD, a condition that can severely damage the liver. Regarding its pathogenesis, the metabolic process of ethanol in the body disrupts normal FA oxidation, leading to a substantial buildup of fat within the liver (132). In clinical settings, this phenomenon is demonstrated by a significant elevation in ALT and AST concentrations. In addition, the liver tissue exhibits notable pathological changes, such as cellular degeneration, hepatocellular necrosis and nuclear condensation. Treatment with AS-IV can effectively reverse these adverse conditions. It ameliorates pathological damage in ALD rats, reduces lipid accumulation in hepatocytes, and plays a positive role in repairing the damaged liver (133). Oxidative stress is a crucial indicator for evaluating the severity of alcoholic liver injury (134). AS-IV treatment effectively reverses the inhibition of SOD activity and GSH-Px content and blocks the increase in 4-hydroxy-2-nonenal and MDA levels, thereby suppressing oxidative stress. Acute alcohol stimulation prompts an upsurge in the production of macrophages and neutrophils in the liver, leading to systemic and hepatic inflammation. In this regard, AS-IV has demonstrated excellent anti-inflammatory properties. It inhibits the upregulation of F4/80 protein, effectively reduces the levels of pro-inflammatory factors, including TNF-α, IL-1β, IL-6 and myeloperoxidase; and prevents the activation of nod-like receptor protein 3 inflammatory vesicles (135). In addition, alcohol stimulation impairs the intestinal barrier function, resulting in a leaky gut and endotoxemia (136). AS-IV can significantly reduce the elevated levels of LPS, LPS-binding protein and diamine oxidase. The changes in the levels of these three indicators can effectively reflect the severity of the leaky gut. Moreover, AS-IV prevents the decrease in the expression of tight junction proteins, such as occludin and claudin 4, within the small intestine (133), thereby preserving the integrity of the intestinal barrier and lessening the harm inflicted on organs such as the liver when endotoxins infiltrate the circulatory system. Furthermore, Hao et al (137) indicated that AS-IV could improve the metabolic disorders of linoleic acid, sphingolipid and glycerophospholipid in rats with ALD through the core targets of phosphorylated receptor-interacting protein kinase 3, phosphorylated mixed lineage kinase domain-like protein, cytochrome p450 family 2 subfamily c member 19, cytochrome p450 family 1 subfamily a member 2, PPARα and proprotein convertase subtilisin/kexin type 9. It alleviates liver injury in rats with ALD at the metabolic level. Their study provided an in-depth theoretical basis for the treatment of ALD with AS-IV.

Inflammatory liver injury

In the pathology of sepsis/septic shock, lipopolysaccharide (LPS), an immunostimulatory molecule located in the outer membrane of Gram-negative bacteria, is a major causative factor for inducing organ failure, including liver injury (138). When LPS enters the liver, it induces an immune response, triggering the release of pro-inflammatory cytokines (such as TNF-α, IL-6 and IL-1β), ROS and nitrosamines, thereby significantly increasing inflammation (139). Hepatic injury is a crucial component of this inflammatory response. LPS significantly increases the mRNA expression levels of inflammatory cytokines, including IL-1β, TNF-α and IL-6, triggering a series of pathological reactions. AS-IV has demonstrated remarkable efficacy in countering LPS-induced hepatic injury. It can directly decrease ALT and AST levels in LPS-induced liver injury, a reduction in these levels directly reflects a decrease in hepatic cell damage degree. Moreover, AS-IV improves the pathological changes in liver tissue and repairs the damaged liver at the histological level. Regarding apoptosis regulation, AS-IV reduces the expression of pro-apoptotic proteins via downregulating the mRNA expression of Bax and augments the expression of anti-apoptotic proteins by upregulating the mRNA expression of Bcl-2. As a consequence, the LPS-induced apoptosis of hepatic tissues is inhibited, thereby maintaining the normal survival and function of liver cells. Oxidative stress has a crucial impact on liver injury induced by LPS. AS-IV protects liver cells from oxidative damage by reducing the MDA content and lipid peroxidation products in LPS-stimulated mice. It also elevates the levels of SOD and catalase, which bolster the antioxidant ability of liver cells. As a result, it effectively inhibits oxidative stress and ROS within liver cells. In addition, LPS induces a robust inflammatory response. AS-IV can reverse this outcome by significantly decreasing the mRNA expression levels of these inflammatory factors, thus effectively alleviating the generation of the inflammatory response and further mitigating inflammatory injury in the liver (140).

Metabolic liver injury

Type 2 diabetes mellitus (T2DM) is a serious lifelong metabolic disease. Among the numerous complications associated with T2DM, liver injury is one of the major ones and is triggered by multiple factors (141,142). Long-term chronic hyperglycemia leads to glycotoxicity. The insulin resistance associated with T2DM impairs insulin signaling in the liver, disrupting gluconeogenesis and glycogen synthesis, and directly damaging the normal metabolism and structure of liver cells. A large influx of FFAs into the liver leads to abnormal lipid accumulation, which activates inflammatory signaling pathways, induces oxidative stress and ER stress, and exacerbates inflammation, apoptosis and damage to liver cells. Liver damage further exacerbates hepatic insulin resistance, leading to an increase in the conversion of glucose into triglycerides and worsening of hepatic steatosis; in turn, the accumulation of hepatic fat further exacerbates insulin resistance and hyperglycemia, creating a vicious cycle that accelerates the progression of T2DM and liver damage (143-145). In the synergistic treatment with AS-IV and metformin, the abnormally elevated liver indices (which are important indicators for evaluating liver function) in patients with diabetes were significantly suppressed. As shown in the T2DM rat model, T2DM is often accompanied with dyslipidemia, characterized by significantly elevated levels of TG and IL and markedly reduced levels of high-density lipoprotein cholesterol. These manifestations can be mitigated by AS-IV treatment. Moreover, AS-IV and Met inhibited the conversion of LC3I to LC3II (a marker of autophagy) and the reduction of beclin 1 (an Essen autophagy protein at the initial stage of autophagy) accumulation. They also reduced the expression level of P62 and facilitated the generation of autophagosomes, thereby enhancing hepatic autophagy. The mechanism further involves enhancing autophagy by activating the AMPK/mTOR pathway, which helps ameliorate IR, dyslipidemia, oxidative stress and inflammation (146).

Ischemic liver injury

Ischemia-reperfusion injury mainly occurs during liver transplantation, an effective treatment for ESLD (147). According to Cheng et al (148), AS-IV pretreatment could significantly ameliorate hepatic parenchymal cell injury. It improves hepatocyte survival and liver function by downregulating TNF-α levels and NF-κB expression and transcriptional activity, and upregulating glucocorticoid receptor.

All the aforementioned molecular effects of AS-IV have been validated across heterogeneous experimental systems, including in vitro models of iron overload-induced liver injury, HepG2 cells, Bel-7402 cells, and others, in vivo models such as cisplatin-induced liver injury in rats, paracetamol-induced liver injury in mice, atorvastatin-induced liver injury in rats, and the ALD rat model. Comprehensive analysis of current evidence demonstrates that, despite variations in experimental conditions, these regulatory effects maintain an overall consistent trend.

Overall, AS-IV frequently exhibits pronounced therapeutic efficacy in oxidative stress-related liver injury. Specifically, its protective effects have been consistently demonstrated across multiple studies on toxin-induced and metabolic liver injury, where it exerts hepatoprotection mainly by activating the Nrf2 signaling pathway to exert antioxidant effects and modulating the AMPK/PPARα signaling pathway to maintain metabolic homeostasis. In inflammatory liver injury, AS-IV exerts a marked anti-inflammatory effect by inhibiting the NF κB signaling pathway. In ischemic liver injury, AS-IV confers protection by ameliorating hepatocellular damage via modulation of the AMPK/mTOR signaling pathway. However, its mechanism of action in ischemic liver injury is relatively limited (Fig. 4).

Figure 4 Mechanism of AS-Ⅳ amelioration of liver injury induced by various hepatotoxin substances. PPARα, peroxisome proliferator-activated receptor α; FSP1, ferroptosis suppressor protein 1; APAP, acetaminophen; SIRT1, sirtuin 1; ATO, atorvastatin; LPS, lipopolysaccharide; 4-HNE, 4-hydroxy-2-nonena; LBP, lipopolysaccharide-binding protein; DAO, diamine oxidase; NLRP 3, nod-like receptor protein 3; MPO, myeloperoxidase; IRI, ischemia-reperfusion injury; GCR, glucocorticoid receptor; AS-IV, Astragaloside IV; MDA, malondialdehyde; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; ROS, reactive oxygen species.

Pharmacokinetics, toxicity and nanoscale application of AS-Ⅳ

Pharmacokinetics

As a discipline focusing on drug absorption, distribution and metabolism excreted in living organisms, pharmacokinetics is crucial for understanding drug action and efficacy. AS-IV presents a unique pharmacokinetic profile because of its own characteristics, such as low intestinal permeability, a relatively high molecular weight, and limited lipophilicity, which is associated with paracellular transport.

The oral bioavailability of AS-IV remains low across different species. When orally given to rats and beagles at 10 mg/kg dose, its in vivo bioavailability was only 3.66 and 7.4%, respectively. The low oral bioavailability substantially limits systemic drug exposure, making it challenging to reach therapeutically effective concentrations in target tissues. Therefore, intravenous injection is generally adopted for AS-IV administration in clinical settings. However, when the drug concentration in various tissues was measured subsequent to the intravenous injection of AS-IV to rats, the liver and kidneys exhibited the highest levels of AS-IV, followed by the lungs, heart and spleen. By contrast, the limited distribution of AS-IV to the brain indicates that it might encounter challenges in crossing the blood-brain barrier. Within the concentration range of 250-1,000 ng/ml, ~90% of AS-IV was bound to plasma proteins. In plasma, AS-IV primarily exists in a bound state, with relatively low free drug concentrations (149,150).

The elimination of AS-IV exhibits certain variations across different species yet displays overall linear kinetic characteristics. For instance, following the intravenous injection of 0.5, 1 and 2 mg/kg of AS-IV in beagles, the plasma t1/2 was 177.18, 196.58 and 241.59 min, respectively, and the area under the curve (AUC) was 126.24, 276.28 and 724.51 µg h/ml respectively (149). By contrast, when AS-IV was administered intravenously only to healthy volunteers, the mean maximum plasma concentration (Cmax) values were 2.12, 3.59, 3.71 and 5.17 g/ml at single doses of 200, 300, 400 and 500 ml (contained 27, 36, 45 and 54 mg AS-IV, respectively), respectively. The corresponding mean values of AUC were 4.38, 9.75, 13.59 and 18.22 µg h/ml, respectively, and the mean values of elimination t1/2 were 2.14, 2.59, 2.62 and 2.69 h, respectively (151).

In terms of excretion, AS-IV is primarily eliminated via feces and bile, with urinary excretion accounting for a relatively minor proportion. For male rats, the excretion rates of the substance in feces, urine and bile within 24 h were 31.41, 13.43 and 31.92%, respectively. For female rats, the corresponding excretion rates in feces, urine and bile over the same 24-h period were 31.84, 21.77 and 36.20%, respectively (150). In healthy volunteers, the cumulative urinary excretion of AS-IV over 24 h after administration of 500 ml was 3.91% (151). However, in rat models, only 50% of AS-IV was metabolized in vivo via urine and feces, 30% was recovered in bile, and metabolism was virtually non-existent in the liver, suggesting that first-pass elimination does not occur (47,152).

Toxicity

Understanding the toxic impacts of AS-IV is essential for the development of AS-IV-related drugs. For therapeutic applications, AS-IV has a high safety profile, with low levels of toxicity at standard doses that do not induce significant adverse effects. Oral administration of AS-IV at a daily dose of 1.0 mg/kg for 14 weeks did not cause adverse effects on hepatic and renal function (153). Animal studies also confirmed that AS-IV treatment does not lead to toxicity or side effects in adult animals. The safe doses for rats and beagles are equivalent to 70 or 35-fold the human dose, respectively, such as 0.57 g/kg (154). When AS-IV was administered intravenously at a daily dose of 1.0 mg/kg, it caused maternal toxicity in rats, and doses >0.5 mg/kg led to fetal toxicity. By contrast, when AS-IV was administered intravenously daily from 4 weeks before mating until day 6 of pregnancy in rats, no maternal toxicity was observed at doses ranging from 0.25-1.0 mg/kg. However, at the highest dose of 1.0 mg/kg, suppression of female fertility and delayed hair growth and neurological development in the offspring were observed. These findings indicate that while the toxic effects of AS-IV vary markedly with the stage of pregnancy, the compound consistently lacks teratogenic effects, underscoring the need for caution in its clinical use during pregnancy (155,156). Therefore, caution should be exercised when applying AS-IV treatment in the pregnant population.

However, current research on the adverse reactions and toxic side effects of AS-IV remains limited. Most findings are based on preliminary observations, and the underlying mechanisms of its potential toxicity have not been fully elucidated. Notably, long-term human clinical data are still lacking, rendering it difficult to confirm its long-term safety profile in chronic conditions such as viral liver disease. Therefore, clinical studies on the toxicity of AS-IV in liver disease are urgently needed to facilitate its development and application in the therapeutic field of liver disease.

Nanoscale application

AS-IV suffers from poor permeability and low oral bioavailability. Nanocapsules and nanocarriers have been effective in enhancing drug bioavailability (157). In terms of brain-targeted delivery, the use of the ion cross-linking method for β-asarone-modified asi-loaded chitosan nanoparticles with 120 nm size and favorable uniformity led to ~20% cumulative release in vitro over 48 h, demonstrating sustained-release properties. Compared with unmodified nanoparticles, DiR-βCS-NP exhibited 1.52-fold enhanced uptake in 16HBE cells and 2.49-fold increased fluorescence intensity in the brain, significantly improving nasal brain targeting efficiency. ASI-βCS-NP reduced clinical scores in EAE mice by ~60%, inhibited glial cell activation, mitigated myelin loss and promoted myelin regeneration, demonstrating superior nasal-brain delivery and neuroprotective effects (158). In terms of stability and hydrophilicity, encapsulation in liposomes and loading them onto PVA nanofibers yielded particles with an average diameter of 143.23±3.25 nm and a polydispersity index of 0.11±0.048. The zeta potential was -11.2±1.35 mV, indicating uniform particle distribution without significant aggregation. The ASL/APS/PVA nanofiber surface exhibited smoothness and structural stability, protecting AS-IV from degradation while enhancing its storage and in vivo transport stability. Furthermore, the liposomes encapsulated the hydrophobic AS-IV, enabling its stable dispersion in the aqueous phase. The PVA nanofibers demonstrated excellent hydrophilicity, adsorbing bodily fluids to promote AS-IV dispersion and sustained release within aqueous environments, and significantly improving its hydrophilic properties (159). In tumor-targeted drug delivery, nanomicelles efficiently encapsulate AS-IV with a 90.92% encapsulation efficiency and 12.48% loading capacity, significantly enhancing its water solubility and dispersibility. This nanomedicine exhibited stable particle size in serum and at 4°C storage conditions, demonstrating favorable biosafety. Following PDL1 modification, the tumor-targeting capability was enhanced, enabling the precise delivery of AS-IV and inhibition of the STAT3/NF-κB pathway, substantially boosting antitumor efficacy (160). In addition, AS-IV can enhance ocular delivery. AS-IV-loaded lipid nanocapsules (LNCs) with uniform particle sizes of 19.88, 49.39 and 92.89 nm showed an encapsulation efficiency of over 90%. AS-IV-LNCs-20 also markedly reduced retinal apoptosis rate from 5.12 to 0.533% and alleviated ROS accumulation, while showing no ocular irritation and excellent biocompatibility (161). Nanoparticles, nanomicelles and LNCs are excellent in tissue-targeted delivery; and the liposome-PVA nanofiber system has advantages in improving stability and water solubility. These results demonstrate that nanotechnology greatly enhances the solubility, stability, ocular penetration and retinal targeting efficiency of AS-IV, providing new ideas and ways to improve the pharmacokinetic properties of AS-IV.

Conclusions and future perspectives

Liver disease has become one of the major perils to global public health, significantly influencing patient morbidity and mortality worldwide. At present, the issue of diverse side-effects associated with existing therapeutic drugs is prominent. Traditional Chinese medicine has garnered substantial attention mainly because of its features such as low toxicity, few side effects and favorable tolerability. AS-IV is the key active component derived from the root of A. membranaceous, a traditional Chinese herb renowned for qi-tonifying. In the present study, a comprehensive review of the hepatoprotective effects of AS-IV in NAFLD, liver injury, LF and liver cancer was conducted. Its potential hepatoprotective mechanisms encompass a wide spectrum, including attenuation of fat accumulation, modulation of apoptosis, activation of autophagy, inhibition of fibrosis, regulation of the inflammatory response, alleviation of oxidative stress, anticancer effects, and combined therapeutic effects related to intestinal microbiota. These effects are mediated through signaling pathways such as Akt/mTOR, TLR4, NF-κB, PPARα, TGF β/Smad, Wnt/β catenin and Nrf2/HO-1. Notwithstanding the extensive potential of AS-IV in liver disease treatment, current research exhibits notable limitations. Most studies remained confined to in vitro cell and animal experiments, lacking high-quality, large-scale human clinical trial evidence. As a consequence, the systematic elucidation of its overall regulatory network and underlying interactive mechanisms remains insufficient. The inherent issues with AS-IV, including its low water solubility, poor oral bioavailability, and insufficient hepatic targeting, remain key bottlenecks constraining its druggability and clinical translation. Furthermore, the process scale-up, long-term stability, and in vivo long-term safety of existing nano-delivery systems require further systematic validation. Novel drug delivery systems such as liposomes, nanoparticles and micelles have significantly enhanced the solubility and tissue distribution of AS-IV, offering viable strategies to overcome its translational barriers. Therefore, large-scale randomized clinical trials and comprehensive and in-depth scientific research are urgently required to accurately assess the safety and efficacy of AS-IV (Table I).

Table I Summary of the protective effect of Astragaloside IV in various liver diseases.

Table I

Summary of the protective effect of Astragaloside IV in various liver diseases.

Authors, year	Dosage	In vivo, in vitro	Model	Observed effects	(Refs.)
NAFLD
Zhai et al, 2022	12.5, 25, 50 mg/kg	In vivo	Mouse model of NAFLD	AS-IV inhibits intestinal FXR through intestinal microbiota remodeling and improves dietinduced hepatic steatosis in obese mice	(61)
Liu et al, 2024	0.25, 0.5, 1, 5, 10, 20, 40 µg/ml	In vitro	Fatty liver cell model	AS-IV promotes autophagy and improves cellular lipid deposition through the Akt/mTOR pathway	(63)
Liang et al, 2021	0, 20, 40, 80 mg/kg and 20, 60, 100 µg/ml	In vivo, in vitro	Mouse model of NAFLD and fatty liver cell model	AS-IV reduces liver inflammation, oxidative stress, and apoptosis	(64)
Liu et al, 2020	0, 20, 40, 80 mg/kg	In vivo	NAFLD rat model	AS-IV regulates inflammatory factor levels via the TLR4/NF-κB signaling pathway	(67)
Zhou et al, 2017	0, 50, 100, 200, 400 µg/ml	In vitro	HepG2 cells and Primary mouse hepatocytes	AS-IV alleviates free fatty acid-induced ER stress and lipid accumulation in hepatocytes through AMPK activation	(68)
Chen et al, 2019	0, 20, 40 µg/ml	In vitro	HSC-T6 cells	AS-IV regulates NF-κB-mediated hepatic stellate cell senescence and apoptosis to inhibit PDGF BB-induced activation	(74)
Liver fibrosis
Li et al, 2013	0, 1, 3, 10, 30, 100 µM	In vitro	HSC-T6 cells	AS-IV inhibits collagen production in activated hepatic stellate cells via the p38 MAPK pathway	(75)
Liu et al, 2009	0, 1.5, 3, 6, 12, 24 mg/kg	In vivo	PS-induced LF rat model	AS-IV delayed the formation of liver fibrosis	(76)
Zhao et al, 2020	0, 4.8, 10.8 mg/kg	In vivo	BDL rat model	AS-IV in collaboration with FA induced hepatic stellate cell inactivation by activating the Nrf2 pathway	(77)
Zhu and Lu, 2024	0, 10, 20, 40 µg/ml	In vitro	HCC cell lines (SNU-18 and Huh-7)	AS-IV inhibits HCC by regulating KAT2A-mediated PGAM1 succinylation	(93)
Fang Gong et al, 2023	40 mg/kg	In vivo, in vitro	PSmad3C and Nrf2 mice and HepG2 cells	The ameliorative effect of AS-IV on HCC through Nrf2-mediated pSmad3C/3L transformation	(94)
Li et al, 2022	5, 10, 20 µM	In vitro	HCC cell lines (Huh-7)	The AS-IV by regulating Nrf2/HO-1 and TGF beta inhibition of migration and invasion of cancer of the liver	(95)
HCC
Min et al, 2022	0, 40, 80, 100 mg/kg and 0, 50, 80, 100, 120, 150 µM	In vivo, in vitro	Mouse model of subcutaneous tumor and HCC cell lines (Huh-7), THP-1	AS-IV regulates macrophage polarization through the TLR4/ NF-κB/STAT3 signaling pathway	(96)
Yang et al, 2023	0, 50, 100, 150 mg/kg and 0, 10, 20, 40, 80 µg/ml	In vivo, in vitro	HCC rat model and HCC cell lines (Huh-7 and SMMC-7721), THP-1	AS-IV alleviates PD-L1-induced immunosuppression by modulating the miR-135b-5p/CNDP1 pathway	(97)
Cui et al, 2020	0, 5, 10, 20, 40, 80, 100, 120, 160, 200 µg/ml	In vivo, in vitro	Xenotransplantation model of nude mouse and HCC cell lines (Huh-7 and SMMC-7721)	AS-IV-induced up-regulation of miR-150-5p inhibits HCC progression by inhibiting β catenin	(98)
Qi et al, 2010	0, 150, 200 µg/ml	In vitro	HepG2 cells	As-iv induces anticancer activity by downregulating the expression of oncogenes such as Vav3.1	(100)
Wang et al, 2014	0.04, 0.08 mg/ml	In vitro	Bel-7402 cells, Bel-7402/FU cells	AS-IV reversed drug resistance in Bel-7402/FU cells by downregulating mdr1 expression	(101)
Qu et al, 2020	40, 60, 80 µM and 0.4, 4, 40 µM	In vivo, in vitro	H22 tumor-bearing mice and HepG2 cells	AS-IV enhances cisplatin chemotherapy sensitivity in HCC by inhibiting MRP2	(102)
Su et al, 2020	0, 100, 150, 200, 300, 400 µM	In vitro	HCC cell lines (SK-Hep1 and Hep3)	AS-IV induced G1 block and apoptosis of HCC cells	(103)
Qin et al, 2017	0, 0.5, 1, 5, 10, 50, 100 µg/ml	In vitro	HCC cell lines (Huh-7 and MHCC97-H)	AS-IV inhibits EMT by targeting the Akt/GSK-3β/β catenin pathway, thereby reducing the invasion and migration of HCC cells	(104)
Jiang and Mao, 2019	25, 50, 100 nmol/l	In vivo, in vitro	HepG2 xenograft tumor model and HCC cell lines (HepG2 and Hep3B)	AS-IV alleviates HCC via the Wnt/β catenin signaling pathway	(105)
Xie et al, 2019	2.5, 5, 10, 20, 40, 80, 160 µM	In vitro	Liver injury induced by iron loading	AS-IV protects the liver by inhibiting excessive autophagy and apoptosis	(116)
Liver injury
Qu et al, 2019	40, 80 mg/kg	In vivo	Cisplatin-induced liver injury in rats	AS-IV protects the liver by inhibiting NLRP3 through autophagy	(121)
Guo et al, 2024	40,80 mg/kg	In vivo	Cisplatin induced liver injury in rats Cisplatin induced liver injury in rats	AS-IV protects the liver by inhibiting PPARα/FSP1 signaling	(122)
Li et al, 2018	20,40 mg/kg	In vivo	Acetaminophen in mice	AS-IV alleviates liver damage by activating the Nrf2 signaling pathway	(129)
Qin et al, 2023	0,60 mg/kg	In vivo	Atorvastatin in rats	AS-IV alleviates liver damage through the AMPK/SIRT1 pathway	(131)
Wu et al, 2023	50, 150, 500 mg/kg	In vivo	Rat model of ALD	AS-IV protects the liver by regulating intestinal flora imbalance and regulating the NLRP3/Caspase-1 signaling pathway	(133)
Han et al, 2014	0.1, 1, 10, 50 mg/l	In vitro	Alcoholic and non-alcoholic oxidative damage models	AS-IV had protective effects on both alcoholic and non-alcoholic oxidative damage of Chang's liver cells	(135)
Hao et al, 2024	50, 100, 200 mg/kg	In vivo	Rat model of ALD	AS-IV reduces liver damage by regulating oxidative stress and inflammation	(137)
Sun et al, 2023	0, 80 mg/kg	In vivo	Lipopolysaccharide-induced liver injury	AS-IV reduces liver damage by regulating oxidative stress and inflammation	(140)
Zhu et al, 2021	0, 80 mg/kg	In vivo	Type 2 diabetes mellitus rat model	AS-IV alleviates liver damage by promoting AMPK/mTOR-mediated autophagy	(146)
Cheng et al, 2011	100 g/ml	In vivo	Mouse orthotopic liver transplantation model	AST-IV alleviates hepatic ischemia-reperfusion injury by inhibiting NF-κB transcriptional activity	(148)

[i] NAFLD, non-alcoholic fatty liver disease; HCC, hepatocellular carcinoma; ALD, alcoholic liver disease; AS-IV, astragaloside IV.

In summary, AS-IV demonstrates clear anti-liver disease activity and holds potential for clinical application. Nevertheless, rigorous, standardized clinical trials are required for further validation. Future research should focus on optimizing liver-targeted nano-delivery systems to enhance drug accumulation efficiency and potency within the liver. High-quality clinical studies should be conducted to systematically elucidate its efficacy, safety profile and optimal dosing regimen, thereby advancing AS-IV from basic research toward clinical application. The present review may serve as a theoretical reference for the pharmacological research and subsequent development of AS-IV in liver diseases.

Availability of data and materials

Not applicable.

Authors' contributions

JS conceptualized the study, curated data and wrote the original draft. XZ validated and curated data. JW conducted investigation and data curation. LZ conducted investigation. XW curated data. WL supervised the study. XH acquired funding, conducted project administration and supervised the study. XD supervised the study and conducted project administration. WW acquired funding and conducted project administration. XZ, LZ, XW, WL, XH, XD and WW wrote, reviewed and edited the manuscript. All authors read and approved the final version of the manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Use of artificial intelligence tools

During the preparation of this work, artificial intelligence tools were used to improve the readability and language of the manuscript or to generate images, and subsequently, the authors revised and edited the content produced by the artificial intelligence tools as necessary, taking full responsibility for the ultimate content of the present manuscript.

Abbreviations:

Acknowledgments

Not applicable.

Funding

The present study was supported by the Outstanding Youth Project of Department of Education of Hunan (grant nos. 24B1079 and 24B1077).

References