Hepatoprotective effects of silybin in liver fibrosis

Abstract

Chronic liver disease results in a response resembling "wound healing", also known as fibrosis, resulting in the progressive accumulation of connective tissue. Excessive fibrogenesis that results in the disruption of intercellular connections, interactions, and extracellular matrix composition are features of the fibrotic process mediated by various cell types and chemical mediators such as transforming growth factor-β. Redox-sensitive processes are major contributors to controlling this inflammatory and pro-fibrogenic cytokine's production and synthesis. Other essential hepatic fibrogenesis activities, such as the activation of stellate cells, the expression of metalloproteinases and their inhibitors can also be linked to generation of reactive oxygen species and lipid peroxidation products, which are implicated in development and progression of fibrosis. The herb Silybum marianum, also known as milk thistle, is widely studied for its potential to treat liver illnesses. Silymarin contains 50% to 70% silybin, which has the highest level of biological activity. In comparison, silybin seems to be relatively safer and the available evidence on its potential mechanisms of action is encouraging. The aim of this article is to analyze the increasing evidences linking biochemical oxidative events to excessive fibrogenesis and silybin's inhibitory mechanisms that aid in the reversal of fibrosis and fibrotic lesions.

Key Words: Fibrosis; Inflammation; Kupffer cells; Liver; Silybin

Core Tip: Liver fibrosis, a wound-healing response to chronic hepatic injury, is driven by redox-sensitive pathways, reactive oxygen species, and pro-fibrogenic cytokines like transforming growth factor-β. Silybum marianum (milk thistle), particularly its active compound silybin, exhibits promising antioxidant and anti-fibrotic properties. This study explores the biochemical oxidative mechanisms behind fibrogenesis and highlights silibin’s potential in reversing liver fibrosis through inhibition of oxidative stress and fibrogenic signaling pathways.

INTRODUCTION

Silybum marianum (L.) is an annual or biennial plant belonging to the Asteraceae family. Some of the common names for Silybum marianum include milk thistle (MT), Marian thistle, Mary thistle, St. Mary's thistle, Our Lady's thistle, Holy thistle, Sow thistle, Blessed Virgin thistle, and Holy thistle[1,2]. The plant blooms between July and August and flourishes in warm, dry soil[3]. The obliquely obovoid fruits (achenes), from which the silvery pappus is removed, make up the crude medication[4]. Each fruit has a glossy, brownish black to greyish-brown husk and measures around 5-7 mm long, up to 2-3 mm wide, and 1.5 mm thick[5]. Freshly milled fruits smell like cocoa and taste harsh and greasy[6]. Since the time of the ancient physicians and herbalists, MT has been used to treat a variety of liver and gallbladder disorders, such as cirrhosis, hepatitis, and jaundice, as well as to protect the liver from chemical and environmental toxins such as alcohol, snakebites, insect stings, and mushroom poisoning[7-9].

Silymarin and silybin the two main active ingredients in MT have been the subject of several pharmacological studies[6]. These compounds have been reported to have hepatoprotective, antioxidant, anti-inflammatory, and anti-fibrotic characteristics[10,11]. In addition, they promote protein biosynthesis and liver regeneration, boost lactation, and have immunomodulatory activity[12]. Silybin is the primary substance and naturally occurring flavonoid derived from the seeds of the Silybum marianum[13]. Silymarin and silybin demonstrated strong anti-fibrotic potential in different experimental liver intoxication models (carbon tetrachloride ethanol, acetaminophen, and phenylhydrazine)[14]. Although the effects of silybin on liver cells and functions have been widely studied but it is difficult to ascertain how it affects the immunomodulatory axis within the liver, especially the impact of Kupffer cells on liver injury[10,15]. Kupffer cells are widely accepted to play a significant part in the liver injury by releasing various mediators[16]. Therefore, the purpose of this review is to analyze how silybin affected several Kupffer cell functions in relation to liver fibrosis.

MECHANISMS OF INJURY IN LIVER FIBROSIS

Oxidative stress is a common hallmark in liver fibrosis from various etiologies[12]. Indices of increased polyunsaturated membrane lipid oxidation are always present in liver fibrosis caused by prolonged ethanol consumption[17]. Alcohol metabolism, and in particular the activation of the 2E1 isoform of the cytochrome P450 (CYP2E1) family, has been shown to be essential for the pro-oxidant impact of ethanol (CYP2E1)[18-20]. Indeed, by the reduction of dioxygen to superoxide anion, which then dismutates to hydrogen peroxide, the hemoprotein isoform produced by ethanol is able to produce significantly more reactive oxygen species (ROS) than other P450 forms[21-23]. As a result, CYP2E1 activity causes a considerable amount of 1-hydroxyethyl free radicals to be generated in the liver of chronically inebriated animals and in alcoholics[12,24]. The hydroxyethyl free radical interacts with glutathione and other cellular thiols as well as macromolecules, changing their structure and function and producing antigenic epitopes[25]. By doing so, it helps ROS deplete the intracellular tripeptide pool and disrupt cellular redox equilibrium, which upsets the balance of the antioxidant enzyme system, including glutathione and superoxide dismutase (SOD)[26]. When following careful procedures, such as measuring malonaldehyde (MDA) in a protein and lipid free solution, where all interfering reactions with a given chromogen can be excluded, it has been possible to obtain reliable evidence of ethanol-induced stimulation of membrane lipid oxidation in alcoholic patients[27,28]. In keeping with aldehydic end-products of membrane lipid oxidation, immunohistochemistry has identified 4-hydroxy-2,3-nonenal (HNE), a significantly more important aldehyde in pathobiology, in the human alcoholic liver[29]. A relatively old finding of a large rise in HNE- and MDA-protein adducts in the plasma of healthy alcoholics - patients who were still showing no signs of hepatic function impairment was given new consideration by this discovery[30].

By creating a persistent state of oxidative stress, it is now widely acknowledged that primary or secondary iron overload can have pro-fibro genic and even pro-carcinogenic consequences on the liver[31]. It’s evident that multiple fibrogenesis pathways might be triggered and aided by iron overload. An effective mechanism of gradual, progressive, fibrotic liver degeneration is a tiny but prolonged neurogenic stimulation, such as that actually caused by iron in hemosiderosis patients (frequently with normal or borderline blood transaminase levels)[32,33]. In fact, it has been shown that hereditary hemochromatosis always has foci of chronic inflammation, with phagocytes both locally resident (Kupffer cells) and recruited from the blood stream (mononuclear cells) playing a significant part in this[34,35]. In particular, iron-induced liver fibrosis is strongly associated with increased expression of the pro-fibrotic cytokine transforming growth factor-β (TGF-β)1, which plays a central role in activating hepatic stellate cells (HSC) and promoting extracellular matrix (ECM) deposition[36]. Recent advances in the molecular control of the two main participants in the hepatic fibrogenesis process, activated macrophages and stellate cells, have helped researchers identify a second potential method by which iron might promote liver fibrosis that can occur without involving necrotic damage[26,37]. In other words, iron excess could trigger the direct activation of macrophages and stellate cells as shown in Figure 1.

Figure 1 Pathophysiological mechanisms of liver injury leading to fibrogenesis. This schematic illustrates the key mechanisms underlying chronic liver injury and the progression to liver fibrosis. Persistent liver damage due to viral infections, toxic insults, or metabolic disorders—induces oxidative stress, inflammation, and hepatocyte apoptosis. These insults activate Kupffer cells (liver-resident macrophages), which in turn release pro-inflammatory cytokines (e.g., tumor necrosis factor α, interleukin-6) and reactive oxygen species. These mediators stimulate hepatic stellate cells and drive extracellular matrix deposition, initiating fibrogenesis. Sustained activation of these pathways leads to scar tissue formation and, if unregulated, culminates in liver cirrhosis. TNF-α: Tumor necrosis factor α; ROS: Reactive oxygen species; TGF-β: Transforming growth factor-β.

Free iron's redox cycling potential most likely sets off ROS-driven reactions, which now directly affect Kupffer cells and perhaps ECM-producing cells even in the absence of necrosis[12,38]. Viral infection, chronic cholestasis, or congestive conditions all result in irreversible damage to parenchymal cells, which invariably sets off an inflammatory response in the liver that is first driven by neutrophils, which build up in the portal spaces and eventually infiltrate the lobule itself[39,40]. Reticuloendothelial cells quickly get involved when they are stimulated by the creation of ROS by activated neutrophils as well as by a number of degradation products that emerge from the injured area[41]. Kupffer cells make up roughly 80%-90% of the sessile macrophages in the reticuloendothelial system, making them the primary site for the removal of this debris. Twenty percentage of the liver's cells are Kupffer cells, which are found near the portal vein[42]. Several signals in the liver's microenvironment influence how Kupffer cells behave. Direct activation of Kupffer cells, however, seems extremely likely in cases of liver damage brought on by alcoholism or iron overload, where, incidentally, irreversible damage is noticeably less severe than in other disease processes[43,44]. An increase in the formation of oxidants occurs in Kupffer cells, parenchymal cells submerged in an ECM, and the hepatic lobule through the catalysis of ethanol and/or iron[45]. In theory, it is anticipated that such an oxidative burst will have a varied zonal distribution[31,46]. Both times, significant amounts of oxygen-derived reactive species, such as lipid peroxidation products, are produced. The NADPH oxidase at the plasma membrane level, which is strongly up-regulated by the increased phagocytic activity, is undoubtedly involved in the activation of Kupffer cells, as is the inducible NO synthase, which results in a harmful reaction between ROS and NO, the pathophysiology of which is still largely unknown[47,48]. These findings point to the potential for additional research into the function of lipid peroxidation and oxidative damage at the very earliest stages of Kupffer cell activation[49]. On the other hand, in inflammatory situations, HSC and Kupffer cells cause fibrosis of the liver tissue and lose their function by creating various ECM components and differentiating into myofibroblasts[50]. This interaction can worsen tissue damage, boost the generation of ROS, and harm liver tissue[12] as shown in Table 1.

Table 1 Different phases of inflammatory conditions in liver that progress towards the fibrosis of the liver tissues.

Extent of damage	Possible cellular mechanisms
Stage 1: Cell recruitment	Neutrophills and macrophages activation, hence, activation of reticuloendothelial cells
Stage 2: Inflammatory cytokines	Increase in the levels of TNF-α, TGF-β, PGE2, IL-1 along with decrease in the levels of SOD, GSH
Stage 3: Tissue damage	Increase in extracellular matrix production, deposition and degradation along with decrease in Tissue inhibitors of matrix metalloproteinases
Stage 4: Fibrinogenesis	Persistant activation of inflammatory cascade is associated with the destruction or liver parenchyma which eventually leads to fibrosis

PGE₂: Prostaglandin E₂; TNF-α: Tumor necrosis factor α; IL: Interleukin; SOD: Superoxidedismutase; TGF-β: Transforming growth factor-β.

SILYBININ LIVER FIBROSIS

Silybin as free radicals scavenger

Mira et al[51,52] tested silybin's capacity to interact with biologic oxidants in vitro. They discovered no physiologically significant interactions with hydrogen peroxide (H₂O₂) or the superoxide anion radical at values ranging from 0.1 mmol/L to 5.0 mmol/L. Hypochlorous acid, in contrast, had a low IC₅₀ value of 5 mmol/L. It was discovered that silybin interacts with the hydroxyl radical (OH) at a rate close to that of diffusion[53,54]. The capacity of silybin to prevent deoxyribose degradation in reaction mixtures comprising Fe (III) and H₂O₂ with or without ethylene diamine tetra-acetic acid was discovered, and the authors hypothesized that this indicated that metals might have chelating characteristics[55]. Using the thiobarbituric acid test for MDA, silybin was also found to reduce lipid peroxidation in a dose-dependent manner[52]. Varga et al[53] developed a series of in vitro tests to examine the impact of different silymarin and silybin forms on polymorphonuclear leukocytes' production of superoxide anion (O₂^-) and H₂O₂ (PMNLs). The most lipophilic compound, a trimethyl derivative, showed the highest suppression of O₂^- generation and H₂O₂ production. Additionally, there was a dose-dependent reduction of O₂^- release by PMA-stimulated PMNLs as compared to controls by flavonolignans with increasing concentration and incubation period of up to 30 minutes[53]. In an in vitro experiment using rat Kupffer cells, silybin (25, 50, 75, 100, and 200 mmol/L) inhibited the production of O₂^-, NO, tumor necrosis factor α (TNF-α), prostaglandin E₂ (PGE₂), and leukotriene B₄ (LTB₄) in a dose-dependent manner, but not PGE₂ or TNF-α at doses up to 100 mmol/L. On the other hand, at a dose of 15 mmol/L, suppression of LTB4, an inflammatory leukotriene from the 5-lipoxygenase route of arachidonic acid metabolism, was found. Human Kupffer cells showed an even stronger manifestation of this action[56,57].

Effects of silybin on the level of antioxidant enzyme and inflammatory markers

When silymarin (10 mg/mL) was incubated with erythrocytes and lymphocytes from patients with chronic alcoholic liver disease and healthy controls for 18 hours, SOD levels were compared to those at baseline. Patients' flow cytometry results showed that treatment dramatically boosted SOD activity in both cell types, but not in healthy individuals. Similar amounts of silymarin, particularly silybin, may be attained in vivo, according to the scientists. Human erythrocytes were subjected to silymarin (10 mg/mL) in a different investigation. MDA, glutathione peroxidase activity, SOD, and the amount of time needed for full acid hemolysis were measured in erythrocytes. The authors also noted a trend toward increases in glutathione peroxidase activity, MDA, GSH, and time to hemolysis, as well as a significant increase in SOD activity compared to control (P < 0.05). Cell viability (measured by lactate dehydrogenase release and eosin exclusion), lipid peroxidation, and GSH depletion caused by allyl alcohol (AA) tert-butyl hydroperoxide (t-BuOOH) were used to test silymarin's potential to protect the liver. Lactate dehydrogenase leakage and eosin uptake were blocked by a 30-minute preincubation with 0.01 mmol/L silymarin before exposure to AA and similar but less pronounced responses were observed after exposure to t-BuOOH[4,58]. Preincubation with silymarin led to a dose-dependent decrease in MDA generation compared to controls, complete inhibition of AA-induced MDA synthesis at 0.025 mmol/L, and total protection against intracellular GSH depletion brought on by t-BuOOH and partial protection from AA-induced GSH depletion[59]. Silymarin has been shown in controlled in vitro tests to prevent NF-kB activation in a number of cell lines. Human histiocytic lymphoma U-937 cells, Jurkat T cells, HeLa epithelial cells, and ML-1a myeloid cells were investigated by Manna et al[60]. The viability of U-937 cells was unaffected by a 2-hour exposure to 50 mmol/L silymarin, but TNF-mediated NF-kB activation was suppressed in a dose-dependent manner. Additionally, silymarin was found to partially reduce NF-kB induction by H₂O₂ and to limit NF-kB activation in all cell types examined. It also appeared to prevent the activation of NF-kB by phorbol ester, LPS, okadaic acid, and ceramide[61,62] summarized in Table 2.

Table 2 Protection mechanisms to liver cells against various toxicities by silybin.

Disease/liver toxicity	Silybin proposed mechanisms	Ref.
Acetaminophen, methotrexate, ethanol, carbon tetrachloride toxicity	Decreased cytotoxicity, increased antioxidant enzymes; i.e., SOD, GSH, decreased cytokines production; i.e., IL-1, IL-6, TNF production	[68-71]
Free radicals toxicity	Increased antioxidant enzymes, i.e., SOD, glutathione; decreased lipoxygenase and leukotrienes	[70,72]
Iron toxicity	Decreased inflammatory cytokines; i.e., TGF-β and interleukins, decreased lipid peroxidation and oxidative damage	[12]
Viruses & natural toxins	Decreased alanine transaminase, aspartate transaminase and γ-glutamyl transpeptidase	[72]

PGE₂: Prostaglandin E₂; TNF-α: Tumor necrosis factor α; IL: Interleukin; SOD: Superoxide dismutase; TGF-β: Transforming growth factor-β.

Silybin’s potential on iron loading in fibrosis

In a carefully controlled trial, gerbils were given weekly injections of iron-dextran (1 mg/g body weight) for 8 weeks. The study looked at the impact of silybin (100 mg/kg body weight) on the advancement of fibrosis[63,64]. When silybin was administered along with iron, there was no discernible decrease in the gerbils' total hepatic iron level as measured by atomic absorption spectroscopy. However, compared to controls and iron-fed gerbils, there was a substantial decrease in the amount of mitochondrial deferoxamine cheatable iron (P < 0.01)[65]. In contrast to treatment with iron alone, silybin + iron treatment considerably (P < 0.05) reduced the level of MDA in mitochondrial membranes. Treatment with silybin appeared to considerably reduce collagen buildup and fibrosis[66,67]. Silymarin (30 mg/kg per day for 21 days) dosed intragastrically was found to be associated with a reduction in aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase levels as compared to control animals in a study looking at chronic liver injury (caused by CCl₄) ending in fibrosis or cirrhosis in rats (P < 0.001). Additionally, silymarin therapy was linked to a considerable decline in cirrhosis and necrosis of grades III and IV (P < 0.05)[68,69]. However, because the study was focused on prevention rather than the treatment of a condition that had already developed, and because the sample size was limited, comparing these results with published clinical studies is challenging and problematic task. Properties of silybin are shown in Figure 2.

Figure 2 Key biological and pharmacological properties of silybin. This figure highlights the major therapeutic properties of silybin, the principal active constituent of silymarin extracted from Silybum marianum (milk thistle). Silybin exerts strong antioxidant and anti-inflammatory effects, primarily through the modulation of oxidative stress, suppression of pro-inflammatory cytokines, regulation of apoptotic pathways, and inhibition of fibrogenic signaling. These multifaceted actions contribute to its hepatoprotective and antifibrotic potential. ROS: Reactive oxygen species; SOD: Superoxidedismutase.

Silybin in liver regeneration

Silymarin (100 mg/kg) boosted weight for treated rats in comparison to controls, demonstrating that it improved liver regeneration in hepatectomy-affected rats (P < 0.01). The autokinetic test, which counts the number of mitotic cells in prepared slides of liver tissue from hepatectomy-affected rats, showed that treatment increases proliferative activity as compared to control animals (P < 0.01). Silybin (i.p. 27 mg/mL silibinin-2',3-dihydrogensuccinate) enhanced the rate of DNA synthesis in rats after partial hepatectomy by 23% to 35% compared to controls. Normal livers showed no variation in DNA synthesis[4,70].

Silybin as anti-cancer and cell cycle regulator

According to laboratory research, silybin has cytoprotective effects on cancer cells exposed to carcinogenic chemicals[71]. Pre-inoculation of cancer cells with silybin before exposure to Adriamycin boosted the drug’s effectiveness in stopping cell proliferation[72]. It should be mentioned that because Silybum marianum has potent antioxidant properties, there is fear that this plant may interact with chemotherapy drugs that work through biochemical peroxidative pathways to fix lymphocytotoxicity[67]. There is no indication that silybin interacts with the cytotoxic effects of doxorubicin and cisplatin, although it can strengthen their synergistic effects.

Data from animal research highlight the ability of silymarin to stop carcinogenesis in a variety of cancers in rat models. For instance, feeding rats silymarin could shield them against cancer-causing chemicals and UVB rays[73]. Silybin stimulates liver DNA, which affects non-cancerous cells. In a study using rats with hepatoma, silymarin did not promote the formation of tumors[74]. It is possible to hypothesize that silymarin's stimulation of DNA polymerase causes an increase in the production of ribosomal RNA and the regeneration of liver cells by regulating cell cycle. SOD and glutathione peroxidase are stabilized by an increase in the quantity of glutamine in cells[75]. By blocking the 5-lipoxygenase cycle and preventing the liver's Kupffer cells from producing leukotrienes and free radicals, silymarin reduces the size of the enlarging liver[67]. Additionally, silybin prevents lipid peroxidation and cellular damage in the hepatocyte cells of mice.

Role of silybin in modulating key molecular targets in hepatic fibrosis

Recent studies have begun to elucidate the specific molecular pathways through which silybin exerts its antifibrotic effects. One of the central targets is the TGF-β1/Smad signaling pathway, a key mediator in HSC activation and ECM deposition. Silybin has been shown to inhibit TGF-β1 expression and suppress downstream phosphorylation of Smad2/3, thereby reducing fibrogenic gene transcription in activated HSCs[76]. In addition, silybin attenuates nuclear factor kappa-B (NF-κB) signaling, a pivotal pathway driving inflammation and fibrogenesis in the liver[77]. By inhibiting IκB degradation and preventing NF-κB nuclear translocation, silybin effectively reduces pro-inflammatory cytokine production such as TNF-α and interleukin-6[78]. Silybin has also been reported to modulate the MAPK pathway, particularly by downregulating ERK1/2 phosphorylation, thereby disrupting the fibrotic signaling cascade[79]. On a molecular level, proteomic and transcriptomic analyses suggest that silybin influences the expression of genes related to oxidative stress, apoptosis, and immune regulation in hepatic cells[79]. These findings collectively point toward a multifaceted mechanism involving antioxidant, anti-inflammatory, and anti-fibrotic actions at the molecular level, warranting further exploration through advanced genomic and proteomic platforms.

Potential drug-drug interactions of silybin in hepatic therapeutics

Although silybin has demonstrated promising hepatoprotective and antifibrotic effects, its potential for drug-drug interactions remains an underexplored yet clinically significant concern, especially in the context of liver disease, where polypharmacy is common. Silybin is known to modulate the activity of several cytochrome P450 (CYP) enzymes, particularly CYP3A4, CYP2C9, and CYP2D6, which are responsible for the metabolism of many drugs used in hepatic and systemic conditions[80]. For instance, CYP3A4 is involved in the metabolism of direct-acting antivirals used in hepatitis C treatment and statins used for managing hyperlipidemia in cirrhotic patients. Silybin has been shown to inhibit CYP3A4, which may increase plasma concentrations of these drugs and elevate the risk of adverse effects such as myopathy or hepatotoxicity. Moreover, silybin may affect drug transporters, particularly P-glycoprotein (P-gp), which influences drug absorption and excretion. Inhibition of P-gp by silybin could alter the pharmacokinetics of co-administered drugs like cyclosporine, rifampin, or certain chemotherapeutics, leading to unpredictable therapeutic responses[81]. These interactions could be especially relevant in patients with chronic liver diseases who are already on complex therapeutic regimens. As such, further in vivo pharmacokinetic and pharmacodynamic studies are needed to characterize these interactions comprehensively and to provide guidance for the rational co-administration of silybin with conventional hepatotherapeutics. Additionally, future clinical trials should include pharmacovigilance components to monitor for potential adverse interactions in real-world settings.

LIMITATION ANDFUTURE PROSPECTS

Treatment with silybin significantly decreased the number of liver-related fatalities in clinical trials including cirrhotic patients. Silybin antioxidant activity is thought to be the mechanism of action that causes these therapeutic outcomes. It is apparent that it exerts its anti-oxidative potentials by scavenging the free radicals that cause lipid peroxidation and interfering with the enzyme systems linked to the cellular damage that causes fibrosis and cirrhosis. Silybin preserves healthy liver cells or cells that have not yet sustained irreparable damage by lowering oxidative stress and the cytotoxicity that results. Through interference with the expression of cell cycle regulators and proteins involved in apoptosis, silymarin and its active component silybin alter the imbalance between cell survival and apoptosis. The hepatoprotective effects of silibinin investigated in a variety of in vitro and in vivo cancer models, including liver cancer, suggest that they prevent or reduce chemotherapy- and radiotherapy-induced toxicity. The molecular mechanisms of silybin-mediated anti-proliferative actions involve the primary receptor tyrosine kinase, androgen receptor, NF-kB, cell cycle regulation and apoptotic signaling pathways in various cancer cells.

Although the hepatoprotective effects of silybin are relatively well established but its scientific validation in clinical trials remain under reported. Further studies are also required to draw its precise mechanism of action especially as a hepatoprotective agent. Lack of toxicity studies is another area of concern where further research can be useful.

Silybin holds significant promise as a therapeutic agent for liver fibrosis; however, several critical gaps must be addressed before it can be adopted in routine clinical practice. While numerous in vitro and preclinical animal studies have demonstrated the hepatoprotective and antifibrotic properties of silybin, these findings require rigorous validation through well-designed clinical trials before silybin can be integrated into evidence-based therapeutic regimens for liver fibrosis. Current human studies are limited, small-scale, and often heterogeneous in terms of patient populations, dosages, and outcome measures, making it difficult to draw definitive conclusions about efficacy and safety[82]. To bridge this translational gap, randomized controlled trials (RCTs) - particularly Phase I and Phase II studies - are urgently needed to assess the safety, tolerability, pharmacokinetics, and dose-response relationships of silybin in patients with chronic liver disease. Furthermore, Phase III trials should evaluate long-term outcomes such as fibrosis regression, liver function improvement, and quality of life. Clinical trials should also explore potential subgroup effects, such as efficacy in patients with metabolic-associated fatty liver disease, viral hepatitis, or alcohol-related liver disease. In addition, the lack of robust biomarker data and limited mechanistic insights in human subjects highlight the importance of incorporating biomarker endpoints and mechanistic sub-studies using liver biopsies or non-invasive imaging. Equally important is the need for comprehensive toxicity studies, especially considering the long-term use of herbal compounds in chronic conditions. Future research should include dose-escalation studies and safety monitoring protocols to ensure the absence of hepatotoxic or off-target effects. Addressing these gaps will be critical to establishing silybin as a clinically viable therapeutic agent in the management of liver fibrosis. Collectively, these clinical investigations will be essential to confirm silybin’s therapeutic viability and to support its translation from bench to bedside in the management of liver fibrosis.

CONCLUSION

Future research on silybin’s therapeutic potential in liver fibrosis would greatly benefit from the application of modern biotechnological tools. Gene editing technologies, such as CRISPR/Cas9, can be employed to knock out or overexpress specific genes involved in fibrogenic signaling, enabling a more precise understanding of silybin’s molecular targets and mechanisms of action. Additionally, proteomic analyses could uncover global changes in protein expression and post-translational modifications induced by silybin, offering insights into the broader signaling networks it affects during hepatic fibrogenesis. These approaches can facilitate the identification of novel biomarkers, clarify pathway-specific effects, and potentially guide the development of combination therapies. Integrating such high-throughput and mechanistic techniques will strengthen the translational relevance of preclinical findings and accelerate the rational design of future clinical studies.