The liver, the largest internal organ in the human body, performs numerous critical metabolic functions, including the synthesis and metabolism of proteins, regulation of glucose homeostasis, production of bile, detoxification of harmful substances, and modulation of immune responses [1]. Liver diseases encompass a wide spectrum of conditions, such as hepatitis, cirrhosis, alcoholic and non-alcoholic fatty liver disease, and obstructive jaundice (cholestasis). Among these, cholestatic liver disease is of significant clinical concern, resulting from impaired bile formation and/or flow. This impairment leads to the accumulation of toxic hydrophobic bile salts, which can damage liver cells by generating free radicals, triggering inflammation, inducing apoptosis, and contributing to fibrosis and, eventually, cirrhosis [2]. Elevated bile acids play a key pathological role by inducing inflammation, promoting hepatocyte destruction, producing reactive oxygen species (ROS), and generating oxidative stress. Inflammation, in turn, drives fibrogenesis, transforming hepatic stellate cells (HSCs) into myofibroblasts, which produce fibrogenic cytokines and extracellular matrix (ECM) [3]. The stagnation of bile salts in the liver triggers a ductular reaction marked by the proliferation of biliary epithelial cells and intrahepatic bile ducts, accompanied by the activation of Kupffer cells and the infiltration of inflammatory cells, which release pro-inflammatory mediators [4,5].

Research has shown that HSCs undergo a specific activation process in response to liver injury, involving proliferation and transformation into myofibroblasts [6]. Once activated, these cells are major contributors to the production of fibrillar collagen, particularly types I and III, becoming central players in the matrix buildup seen in injured liver tissue [7]. During this activation phase, HSCs display pronounced immunoreactivity for alpha-smooth muscle actin (α-SMA), a marker of smooth muscle cell differentiation. Therefore, α-SMA expression is commonly used to identify activated HSCs with a myofibroblastic phenotype, which correlates with the extent of fibrotic tissue accumulation in chronic liver disease. In diseased livers, the presence of α-SMA-positive HSCs is markedly elevated compared to healthy tissue [8]. Furthermore, transforming growth factor-β (TGF-β), a multifunctional cytokine, is a critical regulator of liver fibrogenesis, promoting the excessive formation and deposition of connective tissue matrix components [9]. The activation of HSCs during liver injury is associated with increased proliferation, contractile activity, fibrogenesis, altered protease activity, loss of intracellular retinoid stores, cytokine production, and their transformation into myofibroblast-like cells [10].

Cholangiocytes, the epithelial cells that line the intrahepatic bile ducts, play a crucial role in the development of ductal hyperplasia observed in cholestatic liver diseases, such as bile duct ligation (BDL) [11]. Under normal conditions, these cells maintain bile duct homeostasis. However, in response to biliary injury, they become activated and undergo proliferation, resulting in bile duct hyperplasia. This response is mediated by signaling pathways, including cAMP/PKA and ERK1/2, which are triggered by bile acids, growth factors, and cytokines, ultimately promoting cholangiocyte expansion and the secretion of proinflammatory mediators. Furthermore, cholangiocytes interact with adjacent cells to support liver repair and fibrosis [12]. In the BDL model, the proliferation of larger cholangiocytes is associated with their vascular connections and unique receptor expression, both of which contribute to the histological features of ductal hyperplasia [13].

Ruta graveolens, commonly known as rue, is a dicot herb belonging to the Rutaceae family. Native to the Mediterranean region, it has since spread to tropical regions worldwide [14]. The genus Ruta consists of 14 species, with Ruta graveolens containing more than 100 identified compounds, including coumarins, flavonoids, lignans, quinoline alkaloids, essential oils, and fluoroquinolones. Ruta graveolens is valued for its medicinal properties, particularly its anti-inflammatory, antispasmodic, and analgesic effects [15,16]. It is known to alleviate conditions such as arthritis, muscle spasms, and menstrual cramps. The plant also possesses significant antioxidant activity, which enhances its therapeutic potential by protecting against oxidative stress [17].

Several studies have highlighted that Ruta graveolens has considerable anti-inflammatory and antioxidant effects in various experimental models. For example, Mirghazanfari et al. (2016) demonstrated that Ruta graveolens possesses both acute and chronic anti-inflammatory properties using a formalin-induced hind paw edema model in rats [18]. In hypercholesterolemic mice, it effectively reduced the atherogenic index and improved glutathione (GSH) activity [19]. Moreover, the extract significantly decreased total cholesterol and LDL-C levels in diabetic rats [20]. Panday et al. (2011) further indicated that Ruta graveolens exhibits antidiabetic activity through the inhibition of α-amylase, suggesting its leaf extract is a valuable antioxidant source with potentially protective effects against lipid oxidation [21]. In diabetic rats induced by STZ, Ruta graveolens extract has revealed antihyperglycemic and antihyperlipidemic effects, providing renal protection and outperforming synthetic drugs such as Atorvastatin, Allopurinol, and Metformin [22]. Chawale et al. (2023) highlighted the hepatoprotective effects of Ruta graveolens, exhibiting its combined antioxidant capacity with Angelica sinensis metabolic extract to protect HepG2 cells from CCl4-induced liver damage [23]. Furthermore, treatment with various doses of Ruta graveolens aqueous extracts has been demonstrated to improve liver function and ameliorate liver injury by reducing liver enzyme levels and modifying lipid profiles, facilitating hepatocyte recovery and preventing inflammatory responses caused by CCl4 induction in rats [23].

Despite these findings, no research has yet investigated the hepatoprotective and anti-fibrotic effects of hydroalcoholic extracts of Ruta graveolens on TGF-β and α-SMA gene expression in cholestatic liver tissue induced by bile duct obstruction in male Wistar rats. Therefore, the present study aims to explore the hepatoprotective effects of Ruta graveolens in cholestasis induced by bile duct obstruction in male Wistar rats.