The present study demonstrated that SRD-fed rats developed hypertension, dyslipidemia, liver damage, hepatic steatosis, lipid peroxidation, and oxidative stress. This was accompanied by upregulation of liver CB1 receptor expression. CBD-rich CO, CBD:THC 1:1 ratio CO; CBD:THC 2:1 ratio CO and CBD:THC 3:1 ratio CO showed antihypertensive properties. THC-rich CO, CBD:THC 1:1 ratio CO; CBD:THC 2:1 ratio CO showed the greatest beneficial effects against hepatic steatosis and liver damage. All COs exhibited antioxidant effects in liver tissue. This was associated with normal liver CB1 receptor expression (Table 5).

Hypertension is a hallmark of MS and is frequently associated with NAFLD, contributing to an elevated risk of cardiovascular complications. In this study, systolic and diastolic blood pressure levels were significantly increased in SRD-fed rats for 3 weeks, which mirrors the hypertensive phenotype often associated with NAFLD. Interestingly, CBD-rich CO and with varying CBD:THC ratios, such as 1:1, 2:1, and 3:1, demonstrated significant antihypertensive effects, restoring blood pressure values to near reference levels. This suggests that CBD may help alleviate the hypertensive component of MS, potentially due to its vasodilatory properties, as previously documented (Degrave et al. 2023b; Karimian Azari et al. 2020; Rajesh et al. 2010). In addition to cannabinoids, terpenes present in COs, such as myrcene, d-limonene, and linalool, may also contribute to these antihypertensive effects. These terpenes can improve vascular function through mechanisms such as nitric oxide release, vascular smooth muscle relaxation, and modulation of calcium channels, all of which are crucial in blood pressure regulation. The “entourage effect” between CBD and terpenes may further enhance these cardiovascular benefits by promoting vasodilation and modulating vascular tone (Silva et al. 2019; Alves Silva et al., 2016; Russo 2011). In contrast, THC-rich CO did not confer similar cardiovascular protection, highlighting a differential role of THC in blood pressure regulation. These findings align with previous research suggesting that CBD, either alone or in combination with certain terpenes, may have a more substantial impact than THC in lowering blood pressure and improving vascular function (Vallée 2023; Sultan et al. 2020; Jadoon et al. 2017; Russo 2011).

Dyslipidemia, a characteristic of MS and NAFLD, is strongly associated with impaired lipid metabolism and hepatic steatosis. In our study, SRD-fed rats exhibited significantly higher levels of triglycerides, cholesterol, liver damage markers (AST, ALT, and AP), elevated activity of enzymes involved in de novo lipogenesis and reduced CPT-1 activity, a marker of impaired fatty acid oxidation in NAFLD. Steatogenic agents such as ethanol, high-fat diet or sucrose rich diet can upregulate the activity of CB1 receptor via increasing synthesis of endocannabinoids, 2-arachidonyl glycerol (2-AG), and anandamide. CB1 receptor activation results in upregulation of lipogenic transcription factor and its target enzymes ACC and FAS and concomitantly downregulation of CPT-1. This leads to increase de novo fatty acid synthesis as well as decreased fatty acid oxidation, culminating into the development of fatty liver (Bazwinsky-Wutschke et al. 2019; Silvestri and Di Marzo 2013; Silvestri et al. 2011; Purohit et al. 2010; Osei-Hyiaman et al. 2005). Interestingly, while the CBD-rich and the CBD: THC 3:1 ratio COs did not improve lipid profiles, the THC-rich, CBD:THC 1:1 and CBD:THC 2:1 ratio COs significantly reduced triglycerides and cholesterol levels, improved liver histology, reduced NAS scores, and restored lipid balance, indicating their therapeutic potential in NAFLD. Notably, the THC-rich, CBD:THC 1:1, and CBD:THC 2:1 ratio COs reduced the activity of enzymes involved in de novo lipogenesis and enhanced hepatic fat oxidation. The modulation of lipid metabolism through cannabinoid signaling, particularly via CB1 receptor pathways, emerged as a key mechanism. In these line, Assa Glazer et al. (2020) demonstrated that the treatment with THC-rich or similar concentrations CBD:THC extract alleviated NAFLD development. The limited metabolic efficacy observed with the CBD-rich formulation may be related to the CBD dose used in this study. Indeed, the dose–response relationship of CBD remains controversial, particularly regarding its effects on lipid metabolism and liver function. While several studies have shown that CBD can exert beneficial effects on hepatic lipid accumulation and inflammation at moderate doses [3–20 mg/kg] (Flores-Cortez et al. 2025, Jiang et al. 2021; Silvestri et al. 2015), others have reported no significant effects [5–100 mg/kg] (Zandani et al. 2020, Kutanzi et al. 2020; Wang et al. 2017) or even potential hepatotoxicity at high doses [> 100 mg/kg] (Ewing et al. 2019; Watkins et al., 2021; Lo et al. 2023). However, it is important to note that none of these studies evaluated full-spectrum CO as used in the present study, which may involve an “entourage effect” from other bioactive components such as terpenes and cannabinoids. These findings highlight the need for further investigation into optimal cannabinoid ratios and dosing strategies, particularly for CBD, to balance efficacy and safety in the context of NAFLD. Moreover, terpenes have been reported to exert beneficial effects on lipid metabolism and to reduce hepatic steatosis in animal models of NAFLD. Several preclinical studies have shown that the administration of terpene-rich CO can lead to significant improvements in liver lipid accumulation and circulating lipid profiles. These effects are associated with reductions in hepatic fat deposition and modulation of key enzymes involved in the synthesis of triglycerides in animal models (Kim et al. 2024; Ghasemi-Gojani et al. 2023; Jing et al. 2013; Victor Antony Santiago et al. 2012).

Oxidative stress is a key contributor to the progression from simple hepatic steatosis to more severe forms of liver injury, such as NASH, where increased ROS production exacerbates inflammation, hepatocyte damage, and fibrosis (Arroyave-Ospina et al. 2021; Chen et al. 2020). In the present work, SRD group showed a significant increase in hepatic ROS and TBARS levels, accompanied by a marked depletion of GSH and reduced activities of key antioxidant enzymes, including CAT, GPx, and GR. These findings align with established NAFLD models, where high-sugar diets amplify oxidative stress by promoting lipid peroxidation and impairing the antioxidant defense system. Oxidative stress is a central driver in NAFLD progression, contributing to mitochondrial dysfunction, excessive ROS production, and sustained hepatocyte injury (García-Berumen et al. 2019; Jensen et al. 2018; Prasad and Dhar 2014). Conversely, the CO administration (SRD + CBD-rich CO, SRD + THC-rich CO, CBD:THC 1:1 CO, CBD:THC 2:1 CO and CBD:THC 3:1 CO) significantly reduced hepatic ROS and TBARS levels while enhancing GSH content and the activities of antioxidant enzymes (CAT, GPx, and GR). These results are consistent with previous studies highlighting the potent antioxidant properties of cannabinoids, particularly THC and CBD, in mitigating oxidative damage (Pereira et al. 2021; Rajesh et al. 2010; Hampson et al. 1998). In this line, Yang et al. (2014) demonstrated that CBD treatment effectively reversed lipid accumulation and liver damage, attenuated the increase in hepatic 4-Hydroxynonenal (4-HNE) levels induced by acute ethanol administration in mice. Similarly, Erukainure et al. (2021) showed that treatment with Cannabis sativa extracts significantly mitigated oxidative damage and enhanced both GSH levels and catalase activity in liver tissue exposed to FeSO₄ in vitro. Furthermore, terpenes, a key component of CO, have demonstrated strong antioxidant and anti-inflammatory properties (Masyita et al. 2022; Downer 2020; Nuutinen et al. 2018). In this context, several preclinical studies have demonstrated that terpenes exert antioxidant effects in experimental models of liver damage and metabolic disturbances (Ahmad et al. 2018; Bacanlı et al., 2017; Baldissera et al. 2017; Basha and Sankaranarayanan 2016; Ciftci et al. 2011). Terpene administration has been associated with increased levels of endogenous antioxidant molecules such as GSH, and enhanced activity of key antioxidant enzymes including CAT, SOD, and GPx. These changes were accompanied by a reduction in markers of oxidative stress and lipid peroxidation. Overall, these findings support the role of terpenes in mitigating oxidative damage and preserving hepatic antioxidant defenses under conditions of metabolic or toxic challenge.

The CB1 receptor plays a crucial role in regulating lipid metabolism, and its activation has been shown to promote lipogenesis and impair fatty acid oxidation, leading to the accumulation of fat in the liver. Over the last few years, the ECS has attracted significant attention due to the connection between endocannabinoids, their lipid analogues, and various redox-dependent processes (Jorgačević et al. 2021; Lipina and Hundal 2016). Activation of the ECS via CB1 modulation likely underpins these metabolic effects, consistent with previous reports linking CB1 signaling to impaired energy balance, oxidative stress, and disrupted lipid metabolism (Silvestri and Di Marzo 2013; Rajesh et al. 2012). Furthermore, oxidative stress and the development of liver fibrosis are intricately related to CB1 receptor activation. By increasing the production of ROS and diminishing antioxidant defenses, CB1 receptor activation exacerbates oxidative stress, contributing to hepatocyte damage and the progression of fibrosis (Tan et al. 2020; Rivera et al. 2020; Bazwinsky-Wutschke et al. 2019; Mallat et al. 2013). Our results further support these findings, as we observed a significant overexpression of CB1 receptor protein mass levels in the liver of SRD-fed rats, consistent with previous studies linking high-fat/high-sugar diets to increased CB1 receptor expression. This overexpression was associated with heightened oxidative stress, lipid peroxidation, and markers of fibrosis, reinforcing the role of CB1 receptor in the progression of hepatic steatosis and related complications (Mallat et al. 2011; Osei-Hyiaman et al. 2005). These observations suggest that the pathological upregulation of CB1 receptor contributes to the metabolic and redox imbalances underlying NAFLD, highlighting its potential as a therapeutic target.

In the present study, the administration of COs (CBD-rich CO, THC-rich CO, CBD:THC 1:1 ratio CO, CBD:THC 2:1 ratio CO and CBD:THC 3:1 ratio CO) significantly reduced liver CB1 receptor protein mass levels. This reduction was accompanied by marked improvements in hepatic steatosis, oxidative stress, and fibrosis, except for lipid metabolism (lipogenesis and fatty acid oxidation) in the SRD + CBD group. The less pronounced effect observed in this group may be attributed to the specific properties of the CBD-rich formulation (SRD + CBD), which, while exhibiting potent antioxidant and anti-inflammatory effects, it may not influence the key pathways of lipid metabolism as effectively as other formulations. The decrease in CB1 receptor expression likely contributed to the observed restoration of lipid metabolism and redox balance, as previous studies have demonstrated that downregulation or inhibition of CB1 receptor can reverse steatosis, reduce oxidative damage, and mitigate fibrotic processes (Jorgačević et al. 2021; Gruden et al. 2016; Silvestri and Di Marzo 2013). Some authors suggest that the loss of CB1 receptor in mouse liver or the administration of CB1 receptor antagonists can reduce hepatic steatosis, as CB1 can be blocked by THC or CBD from cannabis (Chen and Kim 2024; Dibba et al. 2018). Although THC theoretically acts as an agonist of CB1 receptor, repetitive THC use may induce tolerance and downregulate CB1 receptor expression, potentially explaining the dose-dependent inverse relationship between marijuana use and NAFLD occurrence reported in other studies (Hirvonen et al. 2012; Le Strat and Le Foll 2011; Pertwee 2008). These mechanisms align with our findings, as the modulation of the endocannabinoid system, particularly the reduction in CB1 receptor density, likely contributed to the restoration of lipid metabolism, reduction of oxidative damage, and mitigation of fibrotic processes observed in our experimental model.

The role of terpenes in modulating the endocannabinoid system (ECS) has gained attention in recent years. Although the terpene content was broadly similar across all CO formulations, the presence of β-caryophyllene, humulene, guaiol, and bisabolol—all with reported anti-inflammatory and antioxidant properties—may have contributed to the observed effects through synergistic interactions with cannabinoids. This concept, known as the “entourage effect,” suggests that terpenes can enhance or modulate the actions of cannabinoids, improving therapeutic efficacy (André et al. 2024; Yao and Liu, 2023; LaVigne et al. 2021; Russo 2011). For instance, β-caryophyllene is a selective CB2 agonist with known hepatoprotective effects, which could complement the reduction in CB1 receptor expression observed in our study (Gertsch et al. 2008). However, due to the lack of significant differences in terpene composition among the oils, it is not possible to attribute specific outcomes to individual terpenes. Our findings instead support the hypothesis that a consistent profile of bioactive terpenes, acting in concert with THC and CBD, may contribute to the overall efficacy of cannabis-based treatments in NAFLD.

One of the main limitations of this study lies in the use of full-spectrum cannabis oils, which, although they have a well-characterized profile of cannabinoids and terpenes, do not allow us to discriminate the specific effect of each of their components on the ECS. Future studies should focus on the evaluation of isolated cannabinoids or terpene-rich essential oils to directly determine their impact on hypertension, lipid metabolism, oxidative stress, and CB1 receptor expression in an experimental model of NAFLD. It would also be beneficial to conduct further analysis of other related key components of the ECS, such as endocannabinoids, additional receptors or specific enzymes. These studies would be fundamental for a more comprehensive understanding of the mechanisms involved in the proposed experimental model.