Mechanisms and therapeutic potential of glycyrrhizic acid: Insights into key signaling pathways and disease modulation (Review).

1.
Introduction
Licorice is a well-known herb that has been widely used in traditional Chinese medicine. According to the Chinese Pharmacopoeia (1), three original plants from the family Leguminosae, Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat. and Glycyrrhiza glabra L. are prescribed as licorice. Numerous studies indicate that the most pharmacologically important bioactive compounds in licorice include ~20 types of triterpenoids, 300 types of flavonoids and various polysaccharides (2,3). Among these, the triterpenoid glycyrrhizic acid (GL) is particularly notable. GL is a sweet-tasting compound extensively used as a flavoring agent in the food industry. Upon hydrolysis, GL yields two molecules of glucuronic acid and one molecule of glycyrrhetinic acid (GA). In the intestines, GL undergoes enzymatic conversion to GA-3-O-mono-β-d-glucuronide, a derivative with enhanced bioavailability and metabolic potential (4).
GL-based therapies, including compound GL tablets and compound GL injections, have been prescribed to treat a number of inflammatory and immune diseases, including viral and autoimmune hepatitis (5). The therapeutic effects of GL have also been validated in diseases such as cirrhosis, eczema, chronic urticaria, psoriasis, and ulcerative colitis (6–8). Furthermore, as an inhibitor of cellular signal transduction molecules that has been demonstrated to suppress angiogenesis and the secretion of tumor-associated cytokines, GL effectively inhibits the migration and invasion of various types of cancer cells (9). Due to its low toxicity and minimal side effects, GL exhibits a wide range of potential therapeutic applications (10).
The present review discusses the effects of GL on intracellular and extracellular inflammatory signaling pathways, aiming to provide a theoretical foundation for the clinical applications of GL and its formulations. Furthermore, the present review highlights emerging therapeutic strategies involving GL, underscoring its potential as a cornerstone for developing new treatments across a spectrum of diseases.

2.
Chemical structure and pharmacokinetics of GL
GL, also known as glycyrrhizin, is the principal bioactive component of licorice. This compound is a pentacyclic triterpenoid saponin composed of one molecule of GA conjugated with two molecules of glucuronic acid (11). The molecular formula of GL is C42H62O16, with a relative molecular weight of 822.93 kDa, whereas its derivative GA, which has a relative molecular weight of 470.69 kDa, has the molecular formula C30H46O4 (Fig. 1). The structure of GL includes five interconnected rings termed rings A-E, with functional groups present on rings A, C and E; hydroxyl and carboxyl groups are present at the 3rd and 30th carbon positions, respectively. These positions are the primary sites for chemical modification of GL, enabling the synthesis of various GA derivatives with potential therapeutic applications (12).
Following its intake, the aglycone section of GL is hydrolyzed by glucuronidase to form two isomers, 18α-GA and 18β-GA (13). The α-form has higher lipophilicity than the β-form, making it more likely to bind to receptor proteins within the body. The conformation of the D and E rings closely resemble that of prednisolone, allowing GA to easily bind to target cells of steroid hormones, which confers its antitoxic effects (14). 18β-GA, with a structure similar to cortisol, can mimic the activity of cortisol by inhibiting 11β-hydroxysteroid dehydrogenase activity (11b-HSDs) (12). This isomer also inhibits the activation of the classical complement pathway and reduces reactive oxygen species (ROS) levels, exerting anti-inflammatory and glucocorticoid hormone-like effects (6).
When administered orally, GL is broken down by β-D-glucuronidase in the intestines to produce GA. Following intravenous administration, GL is metabolized by β-D-glucuronidase in liver lysosomes to produce 3-monoglucuronic GA, which is subsequently distributed via the enterohepatic circulation. GL is metabolized to GA under the action of gut bacteria and is subsequently reabsorbed through the intestinal wall to exert its pharmacological effects (15).

3.
High-mobility group box-1 (HMGB1)
HMGB1 is a nuclear protein that functions as a structural chromatin-binding factor, contributing to the maintenance of nucleosome architecture and the regulation of gene transcription. In response to various stimuli, HMGB1 can be actively secreted by immune and non-immune cells or passively released into the extracellular milieu (16). HMGB1 is a multi-functional regulator: Intracellularly, it acts as an architectural chromatin-binding factor. It can be passively released by damaged or virus-infected cells, or actively secreted by innate immune cells in response to exogenous bacterial products (e.g. endotoxin or CpG-DNA) or endogenous inflammatory stimuli (17). Extracellular HMGB1 mediates the inflammatory response upon binding to inflammation mediators, such as lipopolysaccharide (LPS), DNA or the cytokine interleukin-(IL-)1β. Once released, HMGB1 can bind to the receptor for advanced glycation end-products (RAGE) in soluble or cell-bound forms and to toll-like receptor-2 (TLR2) and TLR4 (16). This binding induces the production of inflammatory cytokines, chemokines, adhesion molecules and ROS, activating downstream signaling pathways such as the p38 mitogen-activated protein kinase (MAPK) kinase (MEK) (18), c-Jun N-terminal kinase (JNK) (19) and nuclear factor-κB (NF-κB) pathways. These pathways, in turn, stimulate the expression and release of other pro-inflammatory cytokines, initiating a positive feedback loop via the NF-κB signaling pathway, which leads to cellular inflammation, damage and apoptosis. HMGB1 is therefore implicated in the pathogenesis of a variety of chronic inflammatory diseases, autoimmune disorders and malignant tumors (16,20).
In recent years, research on GL has primarily focused on its role as a specific inhibitor of HMGB1 (21,22). Experimental studies have demonstrated that GL can directly bind to both high-mobility group boxes of HMGB1 with a dissociation constant of ~150 µM, thereby suppressing its chemotactic and mitogenic activities (18,23,24). Previous studies have explored the mechanisms by which GL-mediated inhibition of HMGB1 exerts protective effects in various disease models (25–27). GL effectively suppresses the pro-inflammatory cytokine activity of extracellular HMGB1 and confers protection against ischemia-reperfusion (I/R)-induced injury in the spinal cord, liver, brain and myocardium in animal models (16). For instance, in a rat liver model of I/R, GL administration was shown to reduce HMGB1 expression in Kupffer cells, demonstrating potential as a preventive treatment for I/R injury, particularly with regards to hepatobiliary surgery (28). Furthermore, a study by Zhai et al (29) reported that GL alleviated I/R injury in rat myocardium by directly inhibiting the cytokine activity of extracellular HMGB1 and blocking the phosphorylation of the JNK/apoptosis regulator BAX (Bax) pathway.
In spinal cord injury models, GA, the active metabolite of GL, was found to reduce inflammation by inhibiting HMGB1 activity via the p38/JNK signaling pathway (30). Additionally, GL has been shown to reduce the HMGB1-induced apoptosis of hepatocytes via a p38-dependent mitochondrial pathway, further supporting the therapeutic potential of GL in alleviating HMGB1-mediated liver injuries, such as viral hepatitis, liver I/R injury and sepsis-associated liver injury (31).
The HMGB1 is also stimulates the proliferation of cancer and endothelial cells, actives angiogenesis and induces inflammation formation, which has a negative impact on tumor progression and recurrence (32–34). As a HMGB1 inhibitor, GL hinders tumor regeneration in mice by blocking protein-stimulated cell proliferation and migration, inhibiting HMGB1-mediated angiogenesis, and reducing inflammatory conditions levels (16).
In addition, GL has previously been evaluated as a potential therapeutic agent targeting sepsis. Promising results indicate that GL modulates the serum level and gene expression of HMGB1 and other pro-inflammatory cytokines, thereby maintaining hemodynamic stability and protecting vital organs from LPS-induced endotoxemia in a porcine model (35).
In ophthalmic applications, the topical administration of glycyrrhizinate-genistein micelle-based eye drops has been shown to markedly promote corneal epithelial and nerve regeneration in diabetic mice. This therapeutic effect is likely mediated through the inhibition of HMGB1 signaling via downregulation of HMGB1 and its receptors RAGE and TLR4, as well as the suppression of inflammatory cytokines such as IL-6 and IL-1β (36). Furthermore, as an inhibitor of HMGB1, GA has been shown to alleviate symptoms of conjunctivitis, blepharitis and dry eye disease by reducing pro-inflammatory protein levels in tear fluid (37).
Additionally, GL provides neuroprotection by inhibiting HMGB1 activity in the nervous system, thereby improving chronic stress-induced depressive behavior. This is achieved via modulation of the kynurenine pathway, which has been linked to stress-induced neuroinflammation and depressive symptoms (38).

4.
Relevant signaling pathways

NF-κB and its signaling pathway
NF-κB is an important transcription factor responsible for regulating inflammation and immune responses, and is closely associated with immune cell activation, T- and B-lymphocyte development, stress responses and apoptosis (39). Under normal conditions, NF-κB remains bound to inhibitor of κB (IκB) and remains inactive in the cytoplasm. However, when stimulated by upstream factors, IκB-α undergoes ubiquitination and degradation. This process releases the NF-κB p65 subunit from its inhibitory complex with IκB-α, allowing it to translocate to the nucleus and activate the transcription of various genes (40).
Once activated, NF-κB promotes the expression of genes that facilitate cell proliferation, inhibit apoptosis and support cancer cell proliferation. In human glioblastoma U251 cells, GL suppresses cell proliferation in a dose- and time-dependent manner. This effect is mediated through the downregulation of p65 expression and inhibition the NF-κB pathway, demonstrating GL's anti-inflammatory and antitumor properties (41). Additionally, dipotassium glycyrrhizate, a potassium salt of GL, has been shown to exhibit anti-proliferative effects in U251 and U138MG cells by inducing apoptosis and upregulating microRNA (miR)-4443 and miR-3620, which inhibit NF-κB post-transcriptionally (42). Given that overexpression of NF-κB is a hallmark of malignant glioma, this transcription factor remains a key target for GL in treating such cancers (43,44). GL has also been found to reduce the ratio of M1-like macrophages in colon. Furthermore, by inhibiting the LPS/HMGB1/NF-κB signaling pathway, it suppresses the production of C-C motif chemokine 2 and TNF-α in colonic macrophages (45). Furthermore, in hepatocellular carcinoma (HCC) cells, GL induces DNA damage and inactivates NF-κB, which collectively contribute to G1-phase arrest. This arrest is mediated through the activation of ataxia-telangiectasia mutated proteins, increased expression of cell cycle inhibitors p21 and p27, and the inhibition of NF-κB-mediated cyclin D1 expression (46).
GL has also been shown to improve bone loss and trabecular parameters in ovariectomized mice. Bone marrow stromal cells isolated from these mice have been shown to exhibit enhanced receptor activator of NF-κB-induced osteoclast formation capabilities, a characteristic that GL notably reverses. NF-κB plays an important role in osteoclastogenesis, yet glycyrrhizin inhibits the NF-κB signaling pathway in ovariectomized mice, posing GL administration as a potential adjunctive therapy for postmenopausal osteoporosis (47,48).
Furthermore, GL has been shown to alleviate acute lung injury induced by LPS by reducing the production of inflammatory factors, such as IL-1β, monocyte chemoattractant protein-1 and cyclooxygenase-2, HMGB1 and adhesion molecules. This effect is mediated by the upregulation of angiotensin-converting enzyme 2 and inhibition of the caveolin-1/NF-κB signaling pathway (49). In human bronchial epithelial cells treated with toluene diisocyanate-albumin conjugate, GL-mediated inhibition of HMGB1 has been demonstrated to lower nuclear factor erythroid 2-related factor 2 (Nrf2) expression and reduce ROS production, resulting in increased matrix metalloproteinase (MMP) levels and reduced NOD-like receptor pyrin domain-containing 3 (NLRP3) inflammasome activation. As such, GL has been shown to enhance the activation of the NLRP3 inflammasome by modulating the HMGB1-regulated ROS/NF-κB pathway (50).
In a rat model of isoproterenol-induced myocardial ischemia, GL has been shown to dose-dependently downregulate phosphorylated-(p-)NF-κB p65 and p-IκBα levels, enhancing cardiac antioxidant capacity and reducing cardiomyocyte apoptosis (51). Similarly, a mouse model of myocardial fibrosis induced by isoproterenol has demonstrated that inflammatory responses are amplified by NF-κB-mediated TLR4 activation. However, magnesium isoglycyrrhizinate (MgIG) has been shown to protect against isoproterenol-induced myocardial fibrosis by inhibiting the TLR4/NF-κB p65 signaling pathway (52).
Additionally, GL has demonstrated renal protective effects in an insulin-resistant rat model of aluminum-induced renal toxicity by inhibiting oxidative stress as well as the TLR4/NF-κB pathway (53). Cisplatin (CP), a commonly used anti-cancer drug, often causes nephrotoxicity (54). Treatment with GL or 18β-GA has been shown to restore oxidative homeostasis and reduce inflammation in the kidneys of CP-treated mice to near-normal levels, likely via upregulation of Nrf2 and downregulation of activated NF-κB (55).
Endothelial dysfunction is an important factor in the pathogenesis of diabetes and its vascular complications. Pre-treatment with GL has been shown to markedly reduce human umbilical vein endothelial cell apoptosis induced by advanced glycation end-products, as well as exhibit protective effects against endothelial dysfunction by inhibiting the RAGE/NF-κB pathway. These anti-apoptotic, anti-inflammatory and antioxidant activities enable GL to demonstrate potential therapeutic benefits for diabetic vascular complications (56).
In a model of cerebral I/R injury, GL has been shown to inhibit the secretion of inflammatory cytokines, including IL-1β, IL-6 and TNF-α, in serum and brain tissue. Additionally, GL been shown to protect against I/R-induced cerebral ischemic disease by inhibiting the expression of the HMGB1-mediated TLR4/NF-κB pathway (57).
Furthermore, MgIG, a magnesium salt of the 18α-GA derivative of GL, has been shown to possess liver-protective, anti-inflammatory, antioxidant and antiviral properties (52). These properties underlie its clinical efficacy; for example, MgIG has been shown to markedly ameliorate liver fibrosis by preventing the nucleus translocation of NF-κB (58).

Phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway
PI3Ks are enzymes that catalyze the conversion of phosphatidylinositol 4,5-bisphosphate into phosphatidylinositol 3,4,5-trisphosphate. These enzymes play notable roles in a number of cellular processes, such as proliferation, cancer progression and inflammation. Akt is a key player in the PI3K/Akt signaling pathway, which has been implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases and neurological disorders (59–61).
The effects of GL on the PI3K/Akt pathway vary depending on cellular context, tissue type and disease model. In numerous pathological conditions, GL functions primarily as an inhibitor of PI3K activity (62). As PI3K/Akt signaling acts upstream of the NF-κB signaling pathway, experimental evidence has demonstrated that GL suppresses IL-1β-induced phosphorylation of PI3K/Akt and subsequent NF-κB activation, thereby attenuating the inflammatory response (62) and subsequently mitigating liver injury. Additionally, GL suppresses both inflammation and apoptosis via the inhibition of HMGB1 and the PI3K/mammalian target of rapamycin (mTOR) signaling pathway (63). In LPS-stimulated macrophages, both GL and 18β-GA have been shown to inhibit the activity of the p110δ and p110γ subunits of PI3K, therefore inhibiting subsequent NF-κB activation. This inhibition has also been shown to result in a dose-dependent reduction in LPS-induced TNF-α, IL-6 and IL-1β production in RAW264.7 cells (64).
Furthermore, GL has been shown to induce excessive autophagy in HCC cells both in vitro and in vivo, a process that is regulated by the concurrent inhibition of the Akt/mTOR and extracellular signal-regulated kinase (ERK)1/2 pathways. This has highlighted autophagy-mediated cell death as a potential strategy for tumor suppression (65). A study reported by Tsai et al (66) found that GL markedly inhibited tumor cell growth, invasion and the phosphorylation of ERK, Akt and epidermal growth factor receptor. In HCC cells, GL treatment inhibited anti-apoptotic and metastatic protein expression, thereby promoting caspase-8/9-mediated apoptosis in both in vitro and in vivo models. In the gastric cancer cell line MGC-803, GL has been shown to induce apoptosis. This effect is accompanied by inhibition of PI3K/Akt, downregulation of the expression of Bcl-1, survivin and p65 expression, upregulation of Bax and promotion of poly(ADP-ribose) polymerase (PARP) (67). A study reported by Niu et al (68) supported the GL-mediated inhibition of HMGB1 both in vitro and in vivo, which affected the protein brahma homolog 1 and PI3K/Akt/mTOR pathways and suppressed the epithelial-mesenchymal transition in pulmonary fibrosis in mice, ultimately slowing the progression of silicosis.
Furthermore, GL and 18β-GA have been shown to reduce mitochondrial bioenergetics and activate the PI3K/Akt pathway in PC12 cells, therefore protecting these cells from ischemic injury by modulating the intracellular antioxidant system and reducing mitochondria-induced apoptosis (69).

MAPK signaling pathway
Numerous studies have shown that ROS can activate the MAPK pathway. In mammals, there are three subgroups of MAPKs: ERKs, JNKs and p38 MAPKs. All three MAPK subgroups are involved in regulating cellular growth and apoptosis, which are important for normal cellular function (70). The Ras/Raf/MAPK/MEK/ERK pathway is activated by receptor tyrosine kinase signals from growth factors and cytokines. Once activated, Ras activates Raf, which subsequently activates MEK1/2, ultimately leading to ERK1/2 activation; continuous ERK pathway activation promotes cellular proliferation (71). Furthermore, the JNK pathway is activated by MEK7 and MEK4, leading to the translocation of activated JNK to the nucleus, where it further activates various transcription factors to regulate cellular proliferation (72).
As an inhibitor of HMGB1, GL reduces inflammation and fibrosis by inhibiting the MAPK and SMAD family member (Smad)3 signaling pathways, respectively (73); this has been shown to alleviate pulmonary toxicity induced by bleomycin, a drug used for treating various tumors (74). Additionally, GL markedly inhibits ROS production, blocking a cascade of events comprising endoplasmic reticulum calcium release, endoplasmic reticulum stress (ERS), MAPK activation and cell death (75). This GL-mediated ROS suppression provides effective photoprotection, offering potential for cosmetic or therapeutic purposes. In human skin fibroblasts (Hs68 cell line), GL protects against UV-B-induced damage by mitigating both Ca2+ imbalance, ERS, as well as by suppressing MAPK activation and subsequent apoptosis (70). Furthermore, GL has been shown to exhibit cytotoxic effects, induce apoptosis and promote G0/G1 phase cell cycle arrest in the rat pituitary adenoma-derived MMQ and GH3 cell lines. These results indicate that GL promotes cell cycle arrest and apoptosis through a ROS-dependent activation of the MAPK pathway (76).
The p38 MAPK pathway is activated by pro-inflammatory cytokines such as IL-1, IL-6 and TNF-α. Activated p38 MAPK influences downstream transcription factors, including NF-κB and transcription factor (TF)-1, −2 and −6, in order to regulate cellular proliferation, differentiation and growth processes (77). An experimental study has shown that GL may prevent colitis by reducing the expression of NF-κB p65 and p38 MAPK (40). Additionally, GL has been shown to modulate the T helper 1/T helper 2 cell balance by suppressing OX40 (CD134)-OX40 ligand signaling and p38 MAPK activity, thus reducing disease severity in ovalbumin-induced asthma models (78). GL also mitigates inflammation following spinal cord injury by regulating the p38/JNK pathway to inhibit HMGB1 expression (30). Furthermore, GL has previously been used as an anti-apoptotic agent due to its inhibition of JNK1/2 and p38 MAPK phosphorylation, as well as its suppression of CCAAT/enhancer-binding protein (C/EBP) homologous protein, resulting in the reduction of ERS (79).
The ERK and JNK signaling pathways are both associated with learning and memory functions (80). A study has demonstrated that GL improves short-term memory by reducing the phosphorylation of ERK and JNK, both of which are important for regulating neuroplasticity and inflammatory responses (81). In vascular endothelial cells, GL has demonstrated the potential to inhibit angiogenesis by suppressing the ROS/ERK signaling axis, potentially slowing the progression of angiogenesis-dependent diseases such as various types of cancer (82).
In a chicken model of Mycoplasma gallisepticum infection, GL has been found to suppress the infection-induced expression of MMP2, MMP9 and inflammatory cytokines via the p38 and JNK signaling pathways. In vivo histopathological analysis has revealed that GL treatment markedly alleviates tracheal and lung injuries resulting from M. gallisepticum infection (83). In a rat model of sepsis-induced kidney injury, GL has been shown to markedly suppress LPS-induced oxidative stress by activating the ERK pathway (84). Furthermore, the C/EBP family plays a core role in regulating adipogenesis within the transcriptional network that controls this process. GL has been shown to inhibit early-stage adipogenesis in the 3T3-L1 cell line by inhibiting the MEK/ERK-mediated expression of C/EBPβ and C/EBPδ (85).

Janus kinase (JAK)/signal transducer and activator of transcription (STAT)3 signaling pathway
JAKs, such as JAK1, JAK2, JAK3 and non-receptor tyrosine-protein kinase TYK2, are transmembrane tyrosine kinases that activate STAT proteins (86). Upon cytokine receptor binding, JAKs become activated and phosphorylate tyrosine residues on the receptor tail. This phosphorylation facilitates the binding of STAT3. The phosphorylated STAT3 then undergoes dimerization and translocates from the cytoplasm to the nucleus, where it regulates target gene expression, including NF-κB, cyclin D1, survivin, apoptosis regulator Bcl-2 and vascular endothelial growth factor, in order to increase cell proliferation. JAK1 in particular plays a notable role in cytokine signaling pathways that regulate inflammatory cytokine expression (87).
Several studies have shown that phosphorylated non-receptor tyrosine kinases, such as Akt and mTOR, can activate STAT3 proteins (88–90). GL has been shown to suppress the Akt/mTOR/STAT3 signaling pathway, downregulate cyclin D1 and survivin, promote the cleavage of caspase-3 and PARP, and inhibit TF-1 cell proliferation in vitro; this inhibition of cell proliferation has resulted in reductions in TF-1 tumor volume in vivo (91). A previous study on the non-small cell lung cancer cell line HCC827 has shown that GL inhibits cancer cell migration and invasion by targeting the JAK/STAT/HMGB1 pathway (92).
Additionally, a study reported by Tian et al (93) demonstrated that GL ammonium salt reduced hepatocyte apoptosis by suppressing the JAK1/STAT1/interferon regulatory factor 1 signaling pathway, inhibiting oxidative stress, downregulating p-JNK expression and modulating apoptosis-related protein expression, therefore alleviating liver injury and restoring T helper cell balance in the liver. Another study reported by Guo et al (94) observed that GL promoted CYG-binding protein 1-mediated activation of the interferon-γ (IFN-γ)/STAT1/Smad7 signaling pathway, which reduced liver fibrosis and suppressed hepatic stellate cell (HSC) activation.

Pyroptosis signaling pathway
Pyroptosis is a regulated form of programmed cell death that is characterized by inflammatory responses. Pyroptotic signaling pathways are primarily categorized into canonical pathways and non-canonical pathways, which are mediated by caspase-1 and caspase-11, respectively. Pyroptotic cell death is marked by the activation of inflammatory caspases, predominantly caspase-1, −4, −5 and −11, and the cleavage of gasdermin family proteins. Cleaved gasdermin proteins subsequently form membrane pores, resulting in cell membrane rupture, the release of inflammatory mediators and cell death (95). The NLRP3 inflammasome is a key regulator of pyroptosis. The inflammasome recruits apoptosis-associated speck-like protein to form an active inflammasome complex that responds to various exogenous and endogenous stressors by secreting inflammatory factors such as IL-1β or IL-18, leading to inflammation (96).
GL enhances the expression of the tumor suppressor protein p53 and upregulates the levels of caspase-9 and cleaved caspase-3 (97). These results indicated that GL treatment induces apoptosis, which is consistent with previous reports (98–100). Additionally, caspase-11 acts as an LPS receptor, mediating ferroptosis, coagulopathy and lethality in endotoxemia and bacterial sepsis. GL treatment has been shown to markedly suppress caspase-11-dependent immune responses in endotoxemia and experimental sepsis models, resulting in reduced coagulopathy, organ damage and mortality (101). Furthermore, GL treatment has been shown to inhibit liver I/R injury and promote pyroptosis in Kupffer cells through gasdermin D-mediated cell death (102).

Other related signaling pathways
In HSCs, GL exerts anti-fibrotic effects by inhibiting the expression of Smad2, Smad3 and Smad7, all of which are activated by transforming growth factor-β (TGF-β)1-actived signaling pathway (103). A previous study in a rat model of liver fibrosis found that combined administration of GL and aspartate aminotransferase notably reduced Smad3 mRNA levels and the protein levels of p-Smad2/3, Smad3 and TGF-β1. These findings further support the inhibitory effect of GL on the TGF-β1/Smad signaling pathway (104). GL has also been shown to alleviate gefitinib-induced liver injury by inhibiting the p53/p21 pathway, thereby promoting cell-cycle progression (105).
Furthermore, GL has been shown to regulate the Hippo/yes-associated protein (YAP) pathway, a key modulator of cell proliferation and apoptosis, by inhibiting YAP nuclear translocation, thus preventing myocardial I/R injury (106). Additionally, GL alleviate steroid-induced femoral head necrosis in both in vivo and in vitro models by activating the Wnt/β-catenin pathway. The activation of this pathway reduces oxidative stress, enhances osteogenic differentiation and suppresses the adipogenic differentiation of mesenchymal stem cells. These combined effects restore osteogenic homeostasis in the femoral head, thereby mitigating necrosis (107). A study reported by Lai et al (108) demonstrated that GL protected against myocardial I/R injury by mitigating inflammation and cell death, potentially through the inhibition of ERS. Furthermore, GL administration has been demonstrated to alleviate fibrosis and inflammation caused by high glucose levels in glomerular podocytes by upregulating the AMP-activated protein kinase pathway and its associated regulatory factors (109). In both in vitro and in vivo models of acute liver failure, GL treatment has been shown to markedly inhibit ferroptosis by reducing oxidative stress (110). Co-administration of glycyrrhizin mitigates triptolide (TPL)-induced nephrotoxicity. This protection is achieved though the repair of TPL-damaged tight junction structures in renal tubules, mediated via the RhoA/Rho-associated kinase-1/myosin light chain signaling pathway (111) (Fig. 2).

5.
Discussion
GL, a natural triterpenoid compound, exhibits a range of biological activities, including anti-inflammatory, antioxidant and antitumor effects. These pharmacological properties are mediated through multiple signaling pathways, including the NF-κB, PI3K/Akt, MAPK, JAK/STAT3 and pyroptotic pathways (Table I).
GL has also been shown to attenuate oxidative stress and mediate the activation of inflammatory signaling cascades. For example, GL inhibits the ERK, JNK and p38 MAPK pathways, resulting in the reduced secretion of pro-inflammatory cytokines. Furthermore, GL negatively regulates the JAK/STAT3 pathway by suppressing STAT3 phosphorylation (112), leading to reduced cellular proliferation and inflammatory responses. Through inhibition of the PI3K/Akt/mTOR pathway, GL modulates cell survival and apoptosis, downregulates the expression of cell cycle-related proteins, such as cyclin D1 and survivin, and promotes tumor cell apoptosis (9). In fibrosis-associated disorders, GL has been shown to exert anti-fibrotic effects primarily through inhibition of the TGF-β/Smad signaling axis, resulting in reduced HSC activation. Additionally, GL has been shown to modulate the Hippo/YAP pathway, thus mitigating myocardial I/R injury, and to activate the Wnt/β-catenin pathway, which contributes to the maintenance of osteogenic homeostasis and ameliorates steroid-induced femoral head necrosis. The antioxidative and anti-inflammatory actions of GL have also been demonstrated in models of high glucose-induced podocyte injury, acute liver failure and neuroinflammation. Notably, GL has been shown to suppress NLRP3 inflammasome-mediated pyroptosis by inhibiting the HMGB1/TLR4/NF-κB signaling pathway, reducing inflammatory damage and demonstrating notable neuroprotective and anticonvulsant effects in kainic acid-induced status epilepticus mouse model (113). In models of endotoxemia and sepsis, GL has been shown to alleviate immune dysregulation by suppressing caspase-11-dependent coagulation abnormalities and mitigating organ damage, demonstrating notable systemic protective effects.
Recent studies support the expanding pharmacological relevance of GL and related triterpenoids. Network pharmacology and molecular docking analyses have revealed that GA exerts its effects in diabetic nephropathy by targeting multiple key pathways involved in inflammation and fibrosis, providing additional mechanistic evidence for the renoprotective potential of licorice-derived compounds (114). A recent review highlighted the therapeutic potential of glycyrrhizin in neuroinflammatory and neurodegenerative diseases, emphasizing its ability to modulate oxidative stress, apoptosis and HMGB1-related signaling within the central nervous system (115). Furthermore, a recent study has examined the efficacy of traditional and nanotechnology-based delivery approaches for GL, underscoring the importance of advanced formulations, such as improved nanotechnology, for enhancing drug bioavailability and clinical utility (116). These findings have complemented the discussion of the present review by demonstrating that GL-associated compounds modulate broader pathological process (such as inflammation) across organ systems, and that the clinical administration may benefit from innovative delivery strategies.
The present review systematically summarized the signaling networks involved in the pharmacological actions of glycyrrhizin across diverse disease models (Table II) (30,40,41,42,44–53,55–57,58,63,65–70,73,79,82–85,94,95,97,103–107). A thorough understanding of the complex crosstalk among signaling pathways modulated by GL is important for informing the design of rigorous, multicenter, large-scale randomized controlled trials.
Notably, the biological activity of GL exhibits context-dependent variability, which is influenced by cell type, tissue environment and disease state. Elucidating the primary signaling mechanisms regulated by GL in each pathological context is important for optimizing targeted therapeutic strategies. Given the limited oral bioavailability of GL, previous research have focused on the development of novel strategies, such as combination therapies and nanocarrier-based delivery systems, to enhance its pharmacokinetic properties and reduce adverse effects in patients (117–119). Mechanistic insights into GL-mediated signaling regulation will facilitate the rational design of synergistic drug combinations and advanced delivery systems for GL-based therapies, therefore maximizing their therapeutic efficacy.
The present review extended the existing literature regarding GL activity by adopting a pathway-oriented perspective that integrated molecular mechanisms with disease relevance. While a previous review by Semwal et al (120) comprehensively summarized the pharmacological activities of glycyrrhizin, the present review placed greater emphasis on the organization of available evidence according to key signaling axes. HMGB1 inhibition was highlighted as a central upstream event linking inflammatory, oncological and fibrotic processes. Additionally, downstream pathways, such as the NF-κB, PI3K/Akt, MAPK, JAK/STAT3 and pyroptotic pathways, were discussed in a unified framework. To facilitate the interpretation of these complex interactions, concise mechanistic summary tables have been provided with the aim of improving accessibility for readers from diverse scientific backgrounds (Tables I and II).
In conclusion, GL exerts multifaceted biological effects in various pathologies through the modulation of diverse signaling pathways, highlighting its potential as a promising therapeutic agent for inflammatory diseases, oxidative stress-related disorders, tumor progression and fibrosis. Continued mechanistic exploration and pathway-specific studies are required to provide a solid theoretical foundation for the clinical translation of GL.