Effects of five cannabis oils with different CBD: THC ratios and terpenes on hypertension, dyslipidemia, hepatic steatosis, oxidative stress, and CB1 receptor in an experimental model

Abstract

Background: Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder caused by oxidative stress and dysregulation of lipid metabolism. The endocannabinoid system (ECS), particularly the type 1 cannabinoid (CB1) receptor, plays a crucial role in NAFLD progression. Cannabinoids, such as cannabidiol (CBD) and tetrahydrocannabinol (THC), along with terpenes, such as beta-myrcene and d-limonene, have shown potential therapeutic effects on liver health, particularly in reducing oxidative stress and modulating lipid metabolism. This study aimed to analyse the effects of five cannabis oils (COs), each with different CBD:THC ratios and terpenes content, on hypertension, dyslipidemia, hepatic steatosis, oxidative stress, and CB1 receptor expression in an experimental model of NAFLD induced by a sucrose-rich diet (SRD) in Wistar rats for 3 weeks.

Methods: Male Wistar rats were fed either a: (1) reference diet (RD; standard commercial laboratory diet) or a: (2) sucrose-rich diet (SRD) for 3 weeks. 3 to 7 SRD + CO as following: (3) SRD + THC; (4) SRD + CBD; (5) SRD + CBD:THC 1:1; (6) SRD + CBD:THC 2:1; and (7) SRD + CBD:THC 3:1. The COs were administered orally at a dose of 1.5 mg total cannabinoids/kg body weight daily. The cannabinoid and terpenes content of all COs used in the study was determined. The terpenes found in COs were beta-myrcene, d-limonene, terpinolene, linalool, beta-caryophyllene, alpha-humulene, (-)-guaiol, (-)-alpha-bisabolol. During the experimental period, body weight, food intake and blood pressure were measured. Serum glucose, triglyceride, total cholesterol, uric acid, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (AP) levels were evaluated. Liver tissue histology, NAFLD activity score (NAS), triglyceride and cholesterol content, lipogenic enzyme activities, enzyme related to mitochondrial fatty acid oxidation, reactive oxygen species (ROS), thiobarbituric acid reactive substance (TBARS), and antioxidant enzyme activities were also evaluated. The CB1 receptor expression was also determined.

Results: The results showed 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.

Conclusions: This study demonstrated that COs, particularly THC-rich CO, CBD:THC ratio 1:1 CO, CBD:THC ratio 2:1 CO and terpenes, can effectively reduce dyslipidemia, liver damage and hepatic steatosis in SRD-induced NAFLD. COs with a higher proportion of CBD in their composition showed antihypertensive properties. All the COs exhibited antioxidant properties. These findings suggest that COs, especially those with CBD:THC ratios of 1:1 and 2:1 and terpenes, may represent a promising therapeutic approach for managing NAFLD and preventing its progression to more severe liver disease.

Keywords: Cannabinoid; Cannabis oil; Hepatic steatosis; Oxidative stress; Terpenes.

Fig. 1

Cannabinoids oils profiles. (A) Cannabinoid identification solution (CIS): CBDVA (cannabidivarinic acid), CBDV (Cannabidivarin), CBDA (Cannabidiolic acid), CBGA (Cannabigerolic acid), CBG (Cannabigerol), CBD (Cannabidiol), THCV (Tetrahydrocannabivarin), THCVA (Tetrahydrocannabivarinic acid), CBN (Cannabinol), Δ-9-THC (Delta-9-Tetrahydrocannabinol), Δ-8-THC (Delta-8-Tetrahydrocannabinol), THCA (Tetrahydrocannabinolic acid), and CBC (Cannabichromene). (B-F) Chromatographic profiles of CBD-rich CO, CBD:THC 1:1 ratio CO; CBD:THC 2:1 ratio CO and CBD:THC 3:1 ratio CO

Fig. 2

Terpenes oils profiles. Terpenes contents of CBD-rich CO, CBD:THC 1:1 ratio CO; CBD:THC 2:1 ratio CO and CBD:THC 3:1 ratio CO

Fig. 3

Histological analysis, NAS score and lipid content in liver tissue. A) Representative photomicrograph of histological abnormalities observed in liver sections. The accumulation of lipid droplets (black arrow), ballooning cells (black arrowhead) and inflammatory foci (red arrow) are observed in the liver section H&E-stained. 400× magnification. An inset is used to show, at high magnification, lipid droplets and inflammation foci in the SRD group, and hepatocellular ballooning in the SRD + CO₂ group. Table insert. Histologic scoring system for activity grade of nonalcoholic fatty liver disease (NAS) in liver sections. B) Triglycerides content. C) Cholesterol content. Values are expressed as mean ± SEM, n = 6. Bars that do not share the same letter are significantly different, (P < 0.05), when one variable at a time was compared by one-way ANOVA followed by Newman-Keuls´ test. Triglycerides content: F (DFn, DFd): 1.191 (4, 29), P value: 0.0356; Cholesterol content: F (DFn, DFd): 1.313 (6, 33), P value: 0.0279

Fig. 4

Enzymes involved in hepatic steatosis. A) Acetyl CoA carboxylase (ACC). B) Fatty acid synthase (FAS). C) malic enzyme (ME). D) glucose-6-phosphate dehydrogenase (G-6-PDH). E) carnitine palmitoyltransferase-1 (CPT-1). Values are expressed as mean ± SEM, n = 6. Bars that do not share the same letter are significantly different, (P < 0.05), when one variable at a time was compared by one-way ANOVA followed by Newman-Keuls´ test. ACC: F (DFn, DFd): 0.5965 (6, 30), P value: 0.0307; FAS: F (DFn, DFd): 0.3750 (4, 23), P value: 0.0241; ME: F (DFn, DFd): 1.308 (6, 32), P value: 0.0221; G-6-PDH: F (DFn, DFd):1.139 (6, 32), P value: 0.0325; CPT-1: F (DFn, DFd): 1.099 (6, 30), P value: 0.0386

Fig. 5

Liver oxidative stress biomarkers. A) reactive oxygen species (ROS). B) Thiobarbituric acid reactive substance (TBARS). C) glutathione (GSH). D) catalase (CAT). E) glutathione peroxidase (GPx) and F) glutathione reductase (GR). Values are expressed as mean ± SEM, n = 6. Bars that do not share the same letter are significantly different, (P < 0.05), when one variable at a time was compared by one-way ANOVA followed by Newman-Keuls´ test. ROS: F (DFn, DFd): 1.377 (6, 31), P value: 0.0254. TBARS: F (DFn, DFd):1.872 (6, 36), P value: 0.1127; GSH: F (DFn, DFd): 1.778 (6, 36), P value: 0.0131; CAT: F (DFn, DFd): 1.407 (6, 37), P value: 0.0237; GPx: F (DFn, DFd): 1.199 (6, 32), P value: 0.0332; GR: F (DFn, DFd): 1.633 (6, 32), P value: 0.0170

Fig. 6

Liver CB1 receptor protein mass. Each gel contained an equal number of samples from rats fed a RD, SRD, SRD + THC, SRD + CBD, SRD + CBD:THC 1:1, SRD + CBD:THC 2:1 and SRD + CBD:THC 3:1. A. Representatives immunoblot of liver CB1 receptor. B. Densitometric immunoblot analysis of the CB1 receptor protein mass levels. Values are mean ± SEM (n = 6). Bars that do not share the same letter are significantly different, (P < 0.05) when one variable at a time was compared by one-way ANOVA followed by a Newman–Keuls test. CB1: F (DFn, DFd): 5.065 (6, 30), P value: 0.0011