This study investigated the therapeutic effects of BCP on an experimental model of liver fibrosis induced by intraperitoneal administration of TAA. In order to evaluate the therapeutic rather than preventive potential of BCP, treatment was initiated after 6 weeks of TAA administration, a time point at which hepatic fibrosis is known to be established. Our findings demonstrated that BCP markedly attenuated TAA‐induced liver damage, as evidenced by improvements in liver function markers, histopathological architecture, inflammatory mediators, oxidative stress parameters, and fibrosis‐associated markers. Furthermore, the antifibrotic effects of BCP were attenuated by co‐administration of the CB2 receptor antagonist AM630, indicating that CB2 receptor activation contributes to the observed effects of BCP.

Elevated aminotransferase enzyme levels are a reflection of liver damage. Our results showed that TAA significantly elevated serum ALT and AST levels, which is consistent with the results reported by other researchers [16]. In contrast, BCP effectively mitigated the rise and restored enzyme levels toward normal values as previously documented [17]. Moreover, BCP reduced the liver index, which has been elevated following TAA administration, supported by earlier studies [18].

Activation of hepatic stellate cells (HSC) is considered the cornerstone in the induction of hepatic fibrosis [19]. TGF‐β is a regulator of fibrogenesis, acting primarily through its receptors on HSC to induce their activation. Once activated, HSCs express several markers that are typically absent in quiescent cells, such as α‐SMA [20]. In our study, immunostaining revealed an increased expression of both TGF‐β and α‐SMA in the TAA‐injected group, consistent with previous reports [21]. Activated HSC subsequently promotes excessive synthesis of extracellular matrix proteins, including collagen and fibronectin. In addition, the gene expression of both collagen and fibronectin was significantly upregulated in the TAA group. Sirius Red staining also confirmed the buildup of collagen in TAA liver sections. In agreement with earlier studies, BCP treatment ameliorated these histopathological changes induced by TAA [22, 23, 24]. Moreover, hydroxyproline, an essential amino acid in collagen, has been evaluated as a marker of collagen accumulation and fibrosis development. The levels of hydroxyproline were significantly elevated in the TAA group compared with the CON group, which corroborates the development of fibrosis in TAA‐injected rats. This is similar to what is documented earlier showing that fibrosis is correlated with an elevated hydroxyproline level [25]. However, the BCP‐treated group showed a marked reduction in hydroxyproline. The effect of BCP on hydroxyproline content has not been previously described; our findings introduce new evidence supporting its antifibrotic potential.

Chronic inflammation is a central pathogenic mechanism in liver fibrogenesis. Persistent inflammatory signaling deteriorates tissue damage and promotes wound healing response and fibrotic scar formation. TAA stimulates an inflammatory injury due to its induced oxidative damage and cellular demise [26]. Rats treated with TAA showed significantly elevated gene expression of inflammatory cytokines, IL‐1β, IL‐6, MCP‐1, and TNF‐α, in parallel with previous studies [27]. In contrast, BCP‐treated rats exhibited a remarkable decrease in their expression. Our findings are consistent with the reported anti‐inflammatory effect of BCP in experimental liver fibrosis [22, 24]. Immunohistochemical staining showed a profound increase in the infiltrated macrophages in TAA‐injected rats, indicating an active recruitment and activation of immune cells within periportal and parenchymal regions of the liver. BCP treatment was correlated with diminished expression of CD68⁺ and F4/80⁺ cells. Our data align closely with previous reports about the anti‐inflammatory effect of BCP [17], and also remain consistent with other reports for the protective effects of herbal constituents in TAA‐induced liver injury [28].

TAA is a well‐established inducer of hepatic fibrosis. The reactive metabolites of TAA interact with cellular components causing the generation of highly reactive free radicals. Oxidative stress drives an acute inflammatory reaction that progresses to hepatocellular damage [29, 30]. This injury is associated with dysregulated redox balance, characterized by reduced activity of the anti‐oxidant enzymes GPX1 and GPX4, along with potentiated NOX2‐mediated ROS generation. Study outcomes showed that treating TAA‐induced rats with BCP exhibited a pronounced protection on the level of hepatic antioxidant enzymes. BCP reinstated redox balance, lowering NOX2 expression, while restoring GPX1 and GPX4 activities. This aligns with published findings showing BCP suppresses NOX2 activity and enhances GPX‐dependent antioxidant capacity [31, 32]. Oxidative stress has a role in inducing and amplifying necroptosis. Generated ROS promotes autophosphorylation and activation of RIPK1 and the subsequent formation of necrosome through binding with RIPK3. Therefore, suppressing ROS production inhibits necroptosis induction [33].

In liver fibrosis, damaged hepatocytes undergo necroptosis, a regulated form of cell death, associated with an amplified inflammation due to the release of damage‐associated molecular patterns from necroptotic cells. Necroptosis is mediated by RIPK1 and RIPK2 with the executioner protein MLKL. This type of cellular death is considered a driving mechanism of hepatocellular injury and fibrogenesis and a novel mode of cellular demise in various liver diseases such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) [34]. Inhibiting necroptotic mediators, such as blocking RIPK1 activation and suppressing RIPK3 phosphorylation and MLKL membrane translocation, attenuates hepatic fibrosis, as proven in various experimental models [35, 36, 37]. TAA exposure induces the generation of ROS, release of cytokines, and activation of death receptors, all of which converge on stimulating the necroptosis pathway. In our study, TAA administration markedly increased the expression of necroptotic markers; RIPK1, RIPK3 and p‐MLKL, indicating activated necroptotic cell death in fibrotic liver. This aligns with other studies showing RIPK1/RIPK3/p‐MLKL signaling as a key driving force of fibrosis in other hepatic disease models [38]. Notably, BCP treatment suppressed the RIPK1/RIPK3/p‐MLKL axis, evident in their downregulated expression, thereby limiting hepatocyte death.

CB2 receptor is a Gi/o‐coupled G protein coupled receptor (GPCR), a signaling molecule that can inhibit adenylate cyclase and modulate several signaling cascades intracellularly [39]. Activation of CB2 receptors is correlated to the inhibition of necroptosis through various mechanisms. CB2 receptors inhibit the upstream signaling which ultimately suppresses necroptosis induction. CB2 receptors' signaling blocks the production of the inflammatory cytokine TNF‐α, thereby abrogating TNFR1‐mediated stimulation of RIPK1–RIPK3 necrosome formation and necroptosis initiation [40]. In addition, activated CB2 receptors favor an anti‐inflammatory environment. The decreased cAMP due to inhibited adenylyl cyclase enzyme suppresses PKA, and thus inhibits the phosphorylation of the 65 subunit and prevents NF‐κB nuclear translocation [41]. This allows shifting toward pro‐survival and promotes RIPK1 ubiquitination, preventing its transition into pro‐necroptotic complexes. It is also reported that attenuated oxidative stress is linked to weakened RIPK1/RIPK3 signaling and thereby less MLKL activation and membrane permeabilization [42].

Platelet derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) are profibrogenic mediators involved in the initiation of liver fibrosis. PDGF is a potent mitogen that induces the activation, proliferation, and migration of HSC, thereby accelerating ECM deposition [43]. Similarly, VEGF promotes pathological angiogenesis, which enhances HSC activation and sustains the inflammatory response and therefore contributes to fibrogenesis. PDGF also plays a role in vascular stabilization and promotes maturation of new blood vessels [44]. Our study demonstrated that the administration of TAA markedly increased hepatic expression of both PDGF and VEGF, promoting angiogenic signaling during fibrosis development. Other studies also reported increased hepatic VEGF and PDGF following TAA exposure [45, 46]. Administered BCP reduced the upregulated level of PDGF and VEGF, suppressing angiogenic pathways. Although the data on BCP effect on PDGF and VEGF within hepatic tissue remain scarce, its regulatory effects on these mediators have been reported in several non‐hepatic models [47]. Both PDGF and VEGF have pro‐fibrogenic effects; however, hepatocyte growth factor (HGF‐α) is a key anti‐fibrotic mediator with hepatoprotective effects. HGF‐α is an endogenous mediator that promotes liver reaeration and opposes fibrogenesis by stimulating hepatocyte proliferation, suppressing stellate cells transdifferentiation, and downregulating TGF‐β. During chronic liver injury, HGF‐α levels decline, which impairs the liver's ability to repair itself [48]. Therefore, enhancing HGF‐α signaling is valuable in counteracting fibrosis. Consistent with this concept, our findings demonstrate that BCP treatment increased HGF‐α levels, reinforcing the liver's intrinsic regenerative mechanisms. Compounds with pro‐regenerative actions have been shown to counteract TAA‐induced suppression of liver regenerative capacity, supporting the therapeutic value of restoring regenerative pathways [49].

AM630 is widely used as a pharmacological tool to investigate CB2‐mediated actions. The use of AM630 as a selective antagonist has been reported in various liver‐disease models [22, 50] and has also been co‐administered with BCP in non‐hepatic models to confirm CB2‐dependent actions [51, 52]. In our study, the co‐administration of AM630 completely shifted the picture of BCP protective effects. In our results, AM630 abolished the beneficial effects of BCP on all evaluated parameters, including liver function, fibrosis, inflammation, oxidative stress, and necroptosis. While BCP alone markedly improved serum functional markers and liver index, decreased pro‐inflammatory mediators, restored antioxidant defense, mitigated fibrotic and angiogenic markers, and suppressed the necroptosis cascade, these effects were lost when BCP was administered with AM630. Oxidative stress indices, fibrotic markers, and necroptotic proteins reverted toward TAA‐group values, indicating that AM630 effectively counteracted BCP hepatoprotection. This reversal of BCP actions suggests that BCP's effects are critically dependent on CB2 receptors activation. Given that CB2 receptors are expressed on hepatic stellate cells, Kupffer cells, and other non‐parenchymal cells involved in inflammation and fibrogenesis, the blockade of these receptors by AM630 inhibits BCP modulatory effects. Taken together, our findings strongly support that the hepatoprotective effects of BCP in TAA‐induced fibrosis are CB2 receptors‐dependent. Although an AM630‐alone group was not included in the present study, this experimental condition was evaluated in our previous work [24] using the same model and treatment protocol, where AM630 administration alone did not significantly affect the assessed hepatic parameters. Consequently, the current study focused on investigating the ability of AM630 to modulate the effects of BCP and thereby clarify the involvement of CB2 receptor signaling. Furthermore, the omission of an additional AM630‐only group helped avoid unnecessary repetition of experiments and reduced animal use in accordance with established ethical principles.

As explained in our previous publication [24], this study was exclusively conducted on male rats to minimize the hormonal variability associated with the estrous cycle in female rats, which is associated with significant fluctuations in estrogen, a hormone known to play a role in liver fibrosis development and progression. The exclusion of female rats was intended to provide a consistent and established baseline model for liver fibrosis that excludes confounding factors, such as the hormonal variability observed in females. Given the fact that hormonal influence is temporary and eliminated with menopause, at which the risk of liver fibrosis becomes equivalent to the risk for males [53].

The findings of the present study should be interpreted in the context of our previous publication [24] investigating the preventive effects of β‐caryophyllene (BCP) in TAA‐induced liver fibrosis. In the earlier study, BCP administration was initiated concurrently with TAA exposure, thereby evaluating its ability to prevent or attenuate the development of hepatic injury and fibrosis. In contrast, the present study employed a therapeutic intervention design, in which BCP treatment was initiated after 6 weeks of TAA administration, when liver injury and fibrotic changes had already been established. Consequently, the current investigation addresses a distinct and more clinically relevant question regarding the capacity of BCP to mitigate ongoing hepatic damage rather than prevent disease initiation. Therefore, the present work extends our earlier findings by providing evidence that BCP may exert beneficial effects not only during fibrosis development but also after disease establishment. Collectively, these findings provide preclinical evidence supporting the potential of BCP as an experimental antifibrotic intervention; however, further pharmacokinetic, toxicological, and clinical studies are required before its therapeutic applicability can be established.