Understanding the molecular mechanisms and pathways underlying liver fibrosis progression is crucial for developing effective therapeutic strategies, as thioacetamide (TAA) knowledge enables the identification of potential therapeutic targets and the development of novel treatments that can prevent or reverse hepatic damage and fibrotic changes.

TAA, a sulfur-containing compound widely used as an experimental model for inducing liver fibrosis and cirrhosis, exerts its hepatotoxic effects through metabolic activation by cytochrome P450 enzymes in the liver. The resulting toxic metabolite, TAA-S-oxide, triggers a complex pathological cascade primarily through the generation of reactive oxygen species (ROS) and subsequent oxidative stress in hepatic tissue (Ezhilarasan, 2023). TAA oxidative insult triggers enhanced nuclear factor erythroid 2-related factor-2 (Nrf2) translocation to the nucleus, where it binds to antioxidant response elements to regulate key antioxidant enzymes, including glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT), that work synergistically to neutralise ROS (Carmo de Carvalho e Martins et al., 2022; Bardallo et al., 2022; Jomova et al., 2024). Concurrently, TAA activates Kupffer cells to release pro-inflammatory cytokines including interleukin-1 beta (IL-1β) and tumour necrosis factor-alpha (TNF-α), initiating an inflammatory cascade (Dwivedi and Jena, 2022). The combined effects of oxidative stress and inflammatory mediators activate hepatic stellate cells, promoting their transformation into myofibroblasts that produce excessive collagen, ultimately leading to liver fibrosis (Salguero Palacios et al., 2008) and potential progression to cirrhosis if left untreated (Poynard et al., 1997).

The increased oxidative stress and inflammation in hepatic tissue promotes apoptotic cell death through multiple interconnected pathways (Li et al., 2016). Under oxidative stress conditions, the balance between pro-apoptotic Bcl-2-associated X protein (Bax) and anti-apoptotic B-cell lymphoma 2 (Bcl-2) proteins becomes disrupted, favoring increased Bax expression (Roufayel, 2016). Bax protein translocation to the mitochondria leads to the release of cytochrome c, which subsequently activates caspase cascades, particularly cysteine-aspartic acid protease 3 (caspase-3), the main executioner of apoptosis (Kirkland and Franklin, 2003).

Shikimic acid (SA) (3,4,5-trihydroxy-1-cyclohexene-1-carboxylic acid) is a crucial natural compound predominantly found in star anise (Illicium verum), with significant quantities also present in sweetgum fruit (Liquidambar styraciflua), pine needles (Pinus spp.), and various other plant species (Estevez and Estevez, 2012; Candeias et al., 2018). TAA compound plays a vital role in the biosynthesis of aromatic amino acids in plants and microorganisms through the shikimate pathway, and has gained considerable attention as a key starting material for the synthesis of various pharmaceutical compounds (Santos-Sánchez et al., 2019). Its pharmacological derivatives possess a wide range of bioactivities, including antioxidant (Sun et al., 2016), antiviral, anti-inflammatory (Gandhi et al., 2023), and antibacterial properties, combined with low toxicity, making them promising candidates for therapeutic applications across various organ systems (Quiroz et al., 2014).

Previous studies have demonstrated the therapeutic efficacy of SA in various pathological conditions, including cardiovascular diseases (Gu et al., 2024), viral disease (Xing et al., 2013), renal injuries (Lee et al., 2020), and neural disease (Bao et al., 2023). Therefore, TAA study was designed to investigate the potential protective effects of SA against TAA-induced liver fibrosis in rats by examining various biochemical, molecular, and histological parameters.

TAA and SA were purchased from Sigma-Aldrich, St. Louis, MO, United States.

The present study demonstrates the hepatoprotective potential of SA against TAA-induced liver injury through multiple interconnected mechanisms, primarily involving modulation of oxidative stress, inflammation, and apoptotic pathways. Our findings reveal a complex network of protective mechanisms that collectively contribute to the therapeutic efficacy of SA.

The significant elevation in liver enzymes (ALP by 65%, AST by 114%, and ALT by 61%) following TAA administration indicates severe hepatocellular damage, consistent with findings by Shareef et al. (2023) and El-Demerdash et al. (2024), who reported similar enzymatic disruptions in chemical-induced liver injury. TAA elevation reflects compromised hepatocyte membrane integrity, leading to enzyme leakage into circulation. Notably, our results showed more severe AST elevation (90 ± 10) compared to El-Gendy et al. (2021), who reported a 51.93 ± 0.95 increase, suggesting potentially greater mitochondrial involvement in our model. Moreover, the pattern of enzyme elevation provides crucial insights into the nature and extent of hepatocellular damage (Kaur et al., 2019; El-Hameed et al., 2024). The pronounced elevation in AST/ALT ratio suggests significant mitochondrial involvement in the injury process, a finding that correlates with the observed oxidative stress parameters. TAA mitochondrial dysfunction likely contributes to the amplification of oxidative damage through increased electron leak and compromised ATP production (Chen et al., 2024). The ability of SA to normalize these enzyme levels suggests protection of both cellular and mitochondrial membranes, possibly through stabilization of membrane phospholipids and preservation of membrane-bound enzyme systems.

The oxidative stress parameters revealed a comprehensive disruption of cellular redox homeostasis by TAA. The severe depletion of GSH (67% reduction) represents a critical compromise of cellular antioxidant defense, exceeding the 40.6% reduction reported by Salam et al. (2013). TAA enhanced GSH depletion suggests a more severe oxidative challenge in our model. The concurrent elevation in NO (52%) and MDA (109%) levels indicates extensive oxidative damage and nitrosative stress, creating a self-perpetuating cycle of cellular injury.

SA demonstrated potent antioxidant activity by mitigating oxidative stress and restoring GSH levels. These results align with Manna et al. (2014), who reported that coconut water concentrate and its active constituent, SA, reversed H₂O₂-induced oxidative damage in hepatocytes by inhibiting NF-κB, activating the PI3K/Akt/Nrf2 pathway, and suppressing apoptosis via the SAPK/JNK/Bax axis. Furthermore, the interplay between oxidative stress markers reveals a complex cascade of cellular damage (Iqbal et al., 2024). The depletion of GSH not only compromises direct antioxidant defense but also affects multiple GSH-dependent detoxification pathways. The observed elevation in MDA levels indicates extensive lipid peroxidation, which can trigger a self-perpetuating cycle of membrane damage and cellular dysfunction. Notably, the ratio between GSH depletion and MDA elevation suggests a severe imbalance in cellular redox status that exceeds the compensatory capacity of endogenous antioxidant systems. SA ability to restore TAA balance indicates a comprehensive antioxidant mechanism that likely involves both direct radical scavenging and enhancement of cellular antioxidant capacity.

The comprehensive impairment of antioxidant enzymes by TAA (CAT ↓63%, GPx ↓58%, GR ↓50%, SOD ↓45%) reveals a systematic breakdown of cellular antioxidant defenses. These findings align with those reported by Jorgačević et al. (2022), who observed that TAA-induced nephrotoxicity increases SOD and CAT activity, as well as GSH levels. The dose-dependent restoration of these enzymes by SA, particularly at 100 mg, demonstrates a coordinated enhancement of antioxidant defense systems.

The differential effects on various antioxidant enzymes provide insights into the hierarchy of oxidative damage. The more severe reduction in CAT activity compared to other enzymes suggests particular vulnerability of peroxide detoxification pathways. The coordinated restoration of enzyme activities by SA indicates a systematic enhancement of antioxidant defense that may involve both transcriptional activation and post-translational stabilization of enzyme systems. TAA comprehensive enzyme modulation distinguishes SA from other antioxidant compounds that typically show more selective effects on specific enzymes.

At the molecular level, TAA administration led to a suppression of Nrf2 mRNA expression. In contrast, our results demonstrated that SA potently activated the Nrf2 pathway (1.7 ± 0.2 fold-change). TAA finding aligns with Manna et al. (2014), who reported significant activation of the transcription factor Nrf2 when treating hydroperoxide-induced oxidative damage in hepatocytes with coconut water concentrate containing SA as its active component. The enhanced Nrf2 activation represents a crucial molecular mechanism underlying the observed antioxidant effects, as Nrf2 regulates the transcription of multiple antioxidant enzymes. The temporal relationship between Nrf2 activation and subsequent antioxidant enzyme upregulation suggests a coordinated cellular stress response. The enhanced Nrf2 nuclear translocation observed in our study indicates efficient sensing of oxidative stress and rapid activation of protective pathways. TAA heightened Nrf2 response may explain the superior antioxidant enzyme modulation observed with SA treatment. Moreover, the sustained activation of Nrf2 suggests long-term adaptation of cellular antioxidant capacity rather than merely acute stress response.

The hierarchical organization of protective pathways reveals sophisticated regulation of cellular defense mechanisms. The primary activation of Nrf2 initiates a cascade of protective events that appear to be temporally coordinated. The rapid induction of antioxidant enzymes is followed by sustained modulation of inflammatory and fibrogenic pathways, suggesting that the antioxidant effects of SA serve as a master regulator of downstream protective mechanisms.

The inflammatory response to TAA administration was characterized by significant elevations in key pro-inflammatory mediators: TNF-α (76%), IL-1β (67%), and NF-κB (95%). The increase in IL-1β was confirmed at the transcriptional level through elevated mRNA expression, demonstrating regulation at both protein and gene levels. Zhao et al. (2021) similarly showed that dihydromyricetin inhibited NF-κB activation and reduced both TNF-α and IL-1β levels in TAA-treated mice, underscoring the central role of NF-κB–mediated inflammation in fibrogenesis, supporting the role of inflammatory cascade activation in the pathogenesis of hepatic injury. However, SA ability to normalize these inflammatory markers, particularly at 100 mg, demonstrates anti-inflammatory properties that complement its antioxidant effects.

The relationship between oxidative stress and inflammation in our model demonstrates a complex bidirectional interaction. The elevation in pro-inflammatory cytokines coincides with peaks in oxidative stress markers, suggesting that these pathways amplify each other’s effects (Kumar et al., 2012). The ability of SA to suppress both pathways simultaneously indicates interference with TAA self-perpetuating cycle. Notably, the reduction in NF-κB levels (95% to near baseline) suggests that SA may directly modulate inflammatory signaling pathways in addition to its antioxidant effects. TAA dual action potentially explains the superior anti-inflammatory effects observed in our study compared to conventional antioxidants. The temporal pattern of inflammatory marker reduction correlates strongly with the restoration of antioxidant capacity, suggesting that the anti-inflammatory effects may be secondary to oxidative stress reduction.

We observed that TAA-induced liver fibrosis was associated with increased pro-apoptotic protein expression (Bax and caspase-3), in addition to a decrease in anti-apoptotic protein expression (Bcl-2). This mirrors Eissa et al. (2018), who observed a 2.2-fold increase in caspase-3 mRNA after TAA exposure, and (Mousa et al., 2019), who showed that Moringa oleifera extract downregulated caspase-3 protein in TAA-treated rats. Importantly, our 100 mg/kg SA treatment brought Bax, caspase-3, and Bcl-2 levels back toward control values—providing precise protein-level confirmation that correcting apoptotic dysregulation is a central mechanism of SA’s protective effect.

The ability of SA to normalize these parameters suggests a comprehensive cytoprotective effect. The modulation of apoptotic pathways by SA reveals sophisticated regulation of cell death mechanisms. The observed changes in Bax/B-cell lymphoma-2 (Bcl2) ratio suggest that oxidative stress-induced apoptosis is a major mechanism of cell death in our model (Warren et al., 2000). The ability of SA to normalize TAA ratio while simultaneously reducing caspase-3 levels indicates protection at multiple points in the apoptotic cascade. The correlation between reduced oxidative stress markers and normalized apoptotic proteins suggests that SA anti-apoptotic effects are mediated primarily through its antioxidant properties. TAA protection extends to mitochondrial pathway regulation, as evidenced by the preservation of mitochondrial membrane potential and reduced cytochrome c release, effects not previously reported with similar compounds (Rodrigues et al., 1999; Petrosillo et al., 2004).

The significant upregulation of fibrotic markers (TGF-β↑125%, COL1A1↑225%) by TAA demonstrates robust activation of pro-fibrotic pathways. These results are consistent with Zeweil et al. (2020), who demonstrated that TAA administration (200 mg/kg) for 15 weeks induced liver fibrosis in rats, characterized by increased hepatic mRNA expression of TGF-β and COL1A1 compared to control rats. The superior anti-fibrotic effects of SA, particularly at 100 mg, suggest effective interruption of fibrogenic signaling cascades.

The relationship between oxidative stress and fibrogenesis in our model provides new insights into the mechanisms of hepatic fibrosis. The marked elevation in TGF-β and COL1A1 expression correlates strongly with oxidative stress markers, suggesting that redox disturbance is a key driver of fibrogenic activation. SA ability to suppress these fibrotic markers while restoring antioxidant capacity indicates that its anti-fibrotic effects may be primarily mediated through oxidative stress reduction. The observed decrease in myofibroblast activation and reduced stellate cell proliferation suggest additional mechanisms beyond direct antioxidant effects. The temporal sequence of these changes indicates that early oxidative stress reduction may prevent the initiation of fibrogenic pathways rather than merely suppressing established fibrosis.

The histological findings correlate well with the molecular and biochemical parameters, showing severe tissue architecture disruption by TAA and significant protection by SA. These observations provide structural validation of the biochemical findings and demonstrate the translation of molecular effects to tissue-level protection.

The histological findings provide crucial validation of the molecular and biochemical observations. The preservation of hepatic architecture in SA-treated groups correlates strongly with reduced oxidative stress markers and improved antioxidant capacity. The reduced inflammatory infiltration and decreased collagen deposition observed microscopically align with the molecular markers of inflammation and fibrosis. Particularly noteworthy is the preservation of sinusoidal structure and reduced stellate cell activation in treated groups, suggesting that SA antioxidant effects extend to maintaining microvascular integrity. The dose-dependent improvement in histological parameters provides visual confirmation of the biochemical findings and supports the therapeutic potential of SA in liver injury.

SA exerts its hepatoprotective and antifibrotic effects against TAA-induced liver fibrosis through multiple interconnected mechanisms. Primarily, it activates the Nrf2 signaling pathway, leading to the upregulation of antioxidant enzymes such as SOD, CAT, GPx, and GR, which collectively reduce oxidative stress. In parallel, it suppresses the NF-κB pathway, resulting in the downregulation of pro-inflammatory cytokines like TNF-α and IL-1β. Furthermore, SA modulates the expression of apoptotic markers (e.g., Bax, Bcl-2, and caspase-3), thereby preventing hepatocyte apoptosis. Additionally, it inhibits the expression of fibrogenic genes such as TGF-β1 and COL1A1, which are critical drivers of extracellular matrix deposition and fibrosis. These combined effects contribute to the overall protective role of SA in TAA-induced hepatic injury.

In this study, we demonstrate that SA significantly attenuates TAA-induced hepatic fibrosis in rats by activating the Nrf2 antioxidant pathway and suppressing NF-κB–mediated inflammatory signaling. Over the 6-week treatment period (50 and 100 mg/kg, i. p., daily), SA was well tolerated, with no observable signs of toxicity or behavioral changes at either dose. SA treatment reduced oxidative-stress markers, downregulated pro-inflammatory cytokines, inhibited hepatocyte apoptosis, and decreased expression of fibrogenic genes, collectively leading to reduced collagen deposition and improved liver histology. Although no adverse effects were noted at the doses tested, further studies are warranted to define the safety profile of higher doses and longer treatment durations. These findings highlight the therapeutic potential of SA as an effective antifibrotic agent, while underscoring the need for additional work on optimal dosing and treatment length.