Chronic administration of a cannabis-derived mixture at an antihyperalgesic dose does not significantly enhance hepatotoxicity or the development of metabolic dysfunction-associated steatohepatitis in male mice

Abstract

Cannabis and cannabinoid mixtures have been linked to a variety of health benefits including pain mitigation, suppression of nausea produced by chemotherapeutic agents, anti-inflammatory effects, and effects on energy homeostasis, glucose, and lipid metabolism. The latter properties have led to the suggestion that these products could have therapeutic effects on the development of metabolic dysfunction-associated steatohepatitis (MASH) - a severe type of liver pathology in obese and diabetic patients. However, varying agonist and antagonistic properties of different cannabinoids on the endogenous cannabinoid system make prediction regarding hepatic effects and diet interactions difficult. The current study was designed to examine hepatic pathology following chronic administration of a cannabinoid mixture (NEPE14) at a dose equivalent to one previously demonstrating antihyperalgesic effects in rats. The effects of NEPE14 were investigated in a mouse model of MASH produced by feeding a Western diet rich in fat and simple sugars. After 24 weeks of NEPE14 administration, there was no hepatotoxicity in mice receiving the control diet and no significant exacerbation of MASH in mice receiving the Western diet. In conclusion, no chronic liver toxicity was observed, but there was also no evidence for protection against MASH by this product.

Keywords: MASH; Western diet; cannabis; liver; metabolic dysfunction-associated steatohepatitis; steatosis.

FIGURE 1

The Western diet promotes obesity. Mice were fed either a control diet or a Western diet and administered daily with corn oil or with NEPE14 for a dosage of 0.23 µg cannabinoids per Gram of body weight. (A) The total energy intake from pelleted food, drinking water with glucose and fructose, and corn oil/NEPE14 treatment in the first four weeks of the experiment was calculated. N = 5 cages per group with two mice per cage. (B) Time course of body weight; N = 10 mice per group. At sacrifice, body weight (C), liver weight (D), relative liver weight (E), gonadal fat pad weight (F) and relative gonadal fat pad weight (G) were determined. ns: not significant. *, **, ****: P < 0.05, 0.01, 0.0001 for post-hoc comparisons after two-way ANOVA with Tukey’s adjustment.

FIGURE 2

Serum parameters. Blood drawn from mice fasted overnight was used to determine serum glucose (A), insulin (B), HOMA-IR (C), triglycerides (D), cholesterol (E), alanine transaminase (ALT) (F), and aspartate aminotransferase (AST) (G). *, **, ****: P < 0.05, 0.01, 0.0001 for post-hoc comparisons after two-way ANOVA with Tukey’s adjustment.

FIGURE 3

The Western diet promotes liver steatosis. Examples of H&E stained liver sections from each of the four treatment groups (A–D). The livers were scored for microsteatosis (E), macrosteatosis (F), overall steatosis (G) and difference (Δ score) in macrosteatosis and microsteatosis scores (H). *, **, ***, ****: P < 0.05, 0.01, 0.001, 0.0001 for post-hoc comparisons after Kruskal-Wallis test with Dunn’s test. (I) Liver triglyceride content was determined. ns: not significant. *: P < 0.05 for post-hoc comparisons after two-way ANOVA with Tukey’s adjustment. Hepatic concentrations of Cd36 mRNA (J) and Fabp1 mRNA (K) were determined and scaled to a value of 1 for the mean of the control diet + corn oil group. **, ****: P < 0.01, 0.0001 for post-hoc comparisons after two-way ANOVA with Tukey’s adjustment.

FIGURE 4

The Western diet promotes liver inflammation. Examples of liver sections stained with picrosirius red from each of the four treatment groups (A–D). The sample in (D) was taken from one of the mice with a stiff, fibrotic liver. (E) Quantification of parenchymal PSR stained area. (F) Counting of inflammatory foci from H&E stained liver sections. In panels E and F, the horizontal lines indicate medians. The data points indicated by letter “F” are from stiff, fibrotic livers. ns: not significant. **: P < 0.01 for post-hoc comparisons after Kruskal-Wallis test with Dunn’s test. (G,H) An example of immunohistochemistry of a hyperplastic lymphoid follicle from a mouse fed the control diet and exposed to NEPE14 with the red-purple color demonstrating the occurrence of both CD3e-positive (G) and CD19-positive cells (H). (I) Incidence of mice with hyperplastic lymphoid follicles observed in H&E stained liver sections.

FIGURE 5

Hepatic mRNA expression. qRT-PCR was conducted for 18S rRNA as a house keeping gene, genes involved in fibrosis (Co1a1 and αSMA), genes involved in inflammation (TNFα, IL-6, IFN-γ, B220, CD3e and Cybb), and the gene encoding cannabinoid receptor 2 (Cnr2). N = 10 mice per experimental group. *, **, ****: P < 0.05, 0.01, 0.0001 for post-hoc comparisons after two-way ANOVA with Tukey’s adjustment.