Source / Quelle: https://link.springer.com/article/10.1007/s10787-026-02358-4

Abstract

Cannabidiolic acid (CBDA) is a phytocannabinoid found in the Cannabis plant. Understanding the effects of CBDA is essential to uncover its full potential and possible health benefits. The study was conducted on rats receiving standard rat chow (control) and a high-fat diet (HFD). Half of the animals in each group were administered CBDA intragastrically. The total lipid fractions and arachidonic acid (AA) contents were measured in the frontal and posterior cortex, hippocampus, and subcortical nuclei using gas-liquid chromatography. The expression of proteins involved in neurodegenerative diseases and insulin signaling pathway proteins in the frontal and posterior cortex was measured using Immunoblotting. RT-PCR was used to assess the expression of pro-inflammatory pathway proteins in the same regions. Additionally, untargeted and targeted metabolomic analyses were performed on cerebrospinal fluid (CSF). The results showed that a decrease in arachidonic acid levels and pro-inflammatory precursor proteins after CBDA treatment in high-fat-fed rats was simultaneous with improved insulin signaling, particularly in the posterior cortex. Inactivation of glycogen synthase kinase 3 (GSK-3β) in this region was concomitant with changes in neurodegenerative biomarkers in the cortex and CSF. Metabolomic studies revealed a significant diminishment in creatinine, phenylalanine, and sarcosine levels in the HFD+CBDA group, suggesting it plays an important role in neurological disorders. The results suggest that CBDA has anti-inflammatory properties by reducing the synthesis of lipid inflammatory mediators, which are concomitant with improved insulin signaling and probably reduced neurodegeneration. Thus, CBDA could be considered as a part of future clinical treatment for many inflammatory conditions.

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Introduction

In today’s modern society, characterized by unlimited access to high-calorie diets, sedentary lifestyles, and chronic stress, there is an increased prevalence of obesity and its comorbidities(Celletti et al. 2026). One of them is inflammation, characterized by excessive production and release of pro-inflammatory cytokines, which in the central nervous system may lead to impairment in many signaling pathways (Van Greevenbroek et al. 2013; Arruda et al. 2011). Chronic, excessive availability of fatty acids in the diet leads to increased deposition of lipids not only in adipose tissue or the liver but also in the brain. As proven in studies on other tissues, not only is excessive accumulation associated with impairment in tissue function, but also the type of deposited lipids is vital, as some of them, like palmitic acid or arachidonic acid (AA), may be lipid precursors of inflammation development. Studies conducted in vitro on isolated rat astrocytes showed that incubation with saturated fatty acids, namely palmitic acid, induced increased release of tumor necrosis factor α (TNFα) and interleukin-6 (IL-6)(Gupta et al. 2012) - two of the most important indicators of neuroinflammation occurrence. Moreover, studies on immortalized embryonic rat and mouse hypothalamic cell lines indicated that exposure to TNFα induced insulin resistance (IR) development through inhibited phosphorylation of protein kinase B (Akt), which has a similar effect to that observed in peripheral tissues (Clemenzi et al. 2019). Evidence has shown that changes in the signaling pathway of Akt that lead to glycogen synthase kinase 3 (GSK-3α/β) dysregulation and tau hyperphosphorylation have been associated with the development of Alzheimer’s disease (AD)(Lauretti et al. 2020; Razani et al. 2021). Thus, there is a strong link between obesity, inflammation, and the development of neurodegenerative diseases. Despite constant progress in pharmacology, new safe treatment methods targeting all elements in the etiopathogenesis of neurodegenerative diseases are still desired. The substance with great potential to become a new remedy for neurodegenerative diseases is cannabidiolic acid (CBDA). CBDA is a cannabinoid isolated from Cannabis sativa, as a precursor of cannabidiol (CBD). Apart from structural similarity to CBD, CBDA also shares similar beneficial effects, such as anti-inflammatory, anti-nociceptive, and anti-convulsant properties(Takeda et al. 2008; Vigli et al. 2021; Ben-Cnaan et al. 2022). The biological effects of CBDA are attributed to its pleiotropic action, characterized by the modulation of key enzymatic pathways involved in immune responses, among which the most important is the inhibition of cyclooxygenase-2 (COX-2) activity (Takeda et al. 2008, 2014; Singh et al. 2026a). Although CBDA is an acidic precursor to CBD, it does not interact with cannabinoid receptors (CB1 and CB2). However, CBDA exerts a remarkably high affinity to the transient receptor potential vanilloid 1 (TRPV1) and the 5-hydroxytryptamine receptor 1 A (HTR1A), alongside the peroxisome proliferator-activated receptors (PPARs) (Muller et al. 2019; D’Aniello et al. 2019; Rock et al. 2021; Hirao-Suzuki et al. 2022; Singh et al. 2026a). COX-2 emerges as of particular significance due to its pivotal role in inflammation development. It has been demonstrated that pharmacological inhibition of COX-2 diminishes the synthesis of prostaglandin E2 (PGE2), which suppresses high-fat diet (HFD)-induced neuroinflammation and ameliorates neuronal insulin signaling in the rat brain (Zeng et al. 2025; Hassan et al. 2026).

Interestingly, CBDA has higher oral bioavailability in the bloodstream compared to CBD. Moreover, while CBD crosses the blood-brain barrier highly efficiently due to its neutral, highly lipophilic structure, CBDA exhibits lower brain penetration in mice (Anderson et al. 2019; Singh et al. 2026b). CBDA is still understudied, and its possible clinically relevant properties have become less significant due to the popularity of its derivative. A study by Formato et al. in 2020 showed that searching for “cannabidiol” in the PubMed library returned 2997 results, but only 104 matches were found when searching for information on CBDA (Formato et al. 2020). Today, a search for ‘cannabidiol’ returns 8639 results, compared to 338 outcomes for CBDA. Over the past 6 years, researchers have not shifted their focus to cannabidiolic acid, even though it has clearly exhibited potentially useful effects in rodent models. That is why, in our research, we focused on CBDA and deeply studied its effects.

Encouraged by literature data about CBDA’s properties, the present study aimed to evaluate the effect of short-term CBDA treatment on lipid deposition, inflammatory and insulin signaling pathways, as well as neurodegenerative markers in the frontal and posterior parts of the brain cortex of high-fat diet-induced obese rats. Moreover, to investigate the impact of CBDA on the metabolic state, an untargeted and targeted metabolomic analysis of cerebrospinal fluid (CSF) was performed. The main assumption of this study was that CBDA, through a decrease in lipid deposition, may diminish inflammation development, resulting in increased insulin sensitivity of the brain cortex and improved levels of neuroinflammatory markers assessed in brain tissue and CSF. If so, CBDA may in the future become a supportive drug in the treatment of comorbidities associated with obesity.

Materials and methods

Animals and experimental design

All the experiments were performed in accordance with the ARRIVE guidelines, and the Polish Animal Ethics Committee in Olsztyn under license number 35/2023 approved all procedures. Six-week-old male Wistar rats (initial body weight 70–100 g) obtained from the Centre for Experimental Medicine of the Medical University of Bialystok (Poland) were used in this study. The rats were given 7 days to acclimatize to their housing environment. Throughout the acclimatization period and the experimental period, the rats were maintained in a temperature- and humidity-controlled facility (22 ± 2 °C, 55 ± 5% humidity) on a reverse 12-hour light/dark cycle. Animals were housed in acrylic cages with two rats per cage, with ad libitum access to provided food and water.

Animals were selected and separated into four experimental groups using a random number generator’s randomization method:

  1. (1)
    Control group—was fed a basal rodent diet consisting of 12.4 kcal% fat, 57.1 kcal% carbohydrates, and 30.5 kcal% protein purchased from Animal Feed Manufacturer “Morawski” (Labofeed B, Kcynia, Poland) for 8 weeks. The nutritional and fatty acid formulations are available in the literature (Pastuszewska et al. 2000; Nowacki et al. 2017).
  2. (2)
    CBDA group—was fed the basal rodent diet (the same as the control group) for 8 weeks and during the last 14 days of the diet also treated once a day with freshly prepared just before use synthetic CBDA (0.1 mg/kg body mass, purity ≥ 99%; THC Pharm GmbH, Frankfurt, Germany) in a single intragastric dose at the same time of the day.
  3. (3)
    HFD group—was fed a rodent diet rich in fatty acids consisting of 60% fat, 20 kcal% carbohydrates, and 20 kcal% protein, purchased from Research Diets Inc. (cat. no.: D12492, New Brunswick, NJ, USA) for 8 weeks. The nutritional and fatty acid formulations are available in the literature (Research Diets 2026; Zalewska et al. 2019).
  4. (4)
    HFD+CBDA group—was fed a rodent diet rich in fatty acids (the same as the HFD group) for 8 weeks and during the last 14 days of the diet also treated intragastrically once a day with freshly prepared just before use synthetic CBDA (0.1 mg/kg body mass, purity ≥ 99%; THC Pharm GmbH, Frankfurt, Germany) in a single dose at the same time of the day.

Sesame oil was used as a vehicle for CBDA; it was given to animals from every group with or without CBDA in a volume of 1 ml/kg body mass through a gastric tube designed for adult rats. The CBDA dose was selected based on our preliminary studies and available literature as the most effective when administered intragastrically (Nadal et al. 2017; Rock et al. 2018). The total number of animals used in the study was 40, with 10 animals in each experimental group. Body weight and the amount of food consumed were measured every day until euthanasia. At the end of the experimental period, which was 24 h after the last dose of CBDA or its solvent, animals were anesthetized intraperitoneally with ketamine: xylazine (80 mg/kg:5 mg/kg body mass). From deeply anesthetized animals, large volumes of blood were collected, and the animals were killed by heart removal. The blood was collected in centrifuge tubes containing heparin as an anticoagulant and centrifuged for 15 min at 1000 x g at 4℃ within 30 min of collection. Next, the plasma was collected and aliquoted at -80℃ for further analysis. Immediately after sacrifice, samples of four brain regions, namely the frontal and posterior cerebral cortex, hippocampus, and subcortical nuclei (amygdala and nucleus basalis of Meynert), were anatomically identified with the use of a brain matrix and tissue punch needles (Palkovits punch technique), excised, frozen in liquid nitrogen with precooled aluminium tongs, and stored at -80℃ for further analysis (Herman and Watson 1987; Jaszczyk et al. 2022).

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