Cannabidiol Injections Reshape the Gut Microbiome in Mice, Study Finds

Cannabidiol, better known as CBD, has moved from specialist clinics to health-food shelves at remarkable speed, yet scientists still know surprisingly little about what the compound does inside the body. A new study published in Current Genetics adds an unexpected piece to that puzzle: when mice received daily injections of pure CBD for four weeks, the bacterial community living in their duodenums—the first section of the small intestine—shifted in measurable ways, and the direction of those shifts appeared to depend on the dose the animals received.

The research, carried out by Igor Jasielczuk, Artur Gurgul and colleagues at Polish research institutions, is notable because most previous work on CBD and the gut microbiome has focused on the colon or the cecum, and often on CBD-containing mixtures rather than the chemically pure compound. The authors state that, to their knowledge, the available literature contains no studies testing the effect of pure CBD alone, delivered systemically rather than orally, at multiple doses on the composition of the intestinal microbiome. Their experiment was designed to fill that gap.

The team worked with approximately 60-day-old male C57BL/6J mice weighing 20 to 25 grams, obtained from the animal facility of the Department of Clinical Immunology and Transplantology at Jagiellonian University Medical College. The animals were randomly assigned to four groups of six mice each. Three groups received intraperitoneal injections of chemically pure CBD at doses of 0.2, 10 or 20 milligrams per kilogram of body weight, while the fourth group received a placebo consisting of the vehicle solution—2 percent TWEEN 80 in saline. The injections were given once daily for 28 days. The dose range was chosen based on an overview of human clinical trials and the recommendations accompanying commercially sold CBD supplements, and the lowest dose was included specifically to test whether even minimal systemic exposure would leave a detectable mark on the duodenal microbiome.

The researchers chose the duodenum deliberately. Its wall carries a high density of endocannabinoid receptors, making it a primary target for intraperitoneally administered CBD and a direct site for potential modulation of the gut-brain axis. The duodenum also plays a pivotal role in modulating motility and digestive secretion, which indirectly shape the microbial community, and it hosts a unique and dynamic microbial profile characterized by lower biomass but high sensitivity to disruptions of host homeostasis—properties that make subtle, dose-dependent effects easier to detect. After the treatment period, the mice were fasted, anesthetized with isoflurane and euthanized, and DNA was isolated directly from the duodenal contents.

Sequencing of the V3 and V4 regions of the bacterial 16S rRNA gene was performed on an Illumina MiSeq platform, producing more than 2.3 million raw reads across 48 samples—an average of roughly 97,000 reads per sample before filtering. The team then used the QIIME2 software suite, with DADA2 for denoising and feature table generation, to characterize the microbial communities. Taxonomic classification relied on a pre-trained Naive Bayes classifier referencing the SILVA v138 database. Because the percentage of reads classified at the species level fell below 80 percent, the researchers restricted their interpretation to the phylum and genus levels, acknowledging that 16S amplicon analysis of these variable regions carries a high risk of error in species-level assignment.

The results revealed a microbiome clearly dominated by a handful of taxa. The phylum Firmicutes was the most abundant in every group, ranging from 48.2 percent in the 20 milligram CBD group to 68.2 percent in the control group, followed by Bacteroidota and Verrucomicrobiota. At the genus level, only three taxa exceeded a 10 percent share in at least one group: Lachnospiraceae_NK4A136_group, which ranged from 13.6 percent in the low-dose group to 31.1 percent in controls; Muribaculaceae, ranging from 17.4 percent in controls to 23.3 percent in the 10 milligram group; and Akkermansia, which climbed from 3.6 percent in controls to 16.6 percent in the highest-dose group. Together, these three genera accounted for roughly half of the total relative abundance.

The differential abundance analysis, conducted with the EdgeR tool at a false discovery rate threshold of 0.05, showed that the low and high doses produced the strongest signals. Compared with controls, the 0.2 milligram group showed significant increases in Proteobacteria and Cyanobacteria and significant decreases in Campilobacterota and Firmicutes, while the 20 milligram group showed a significant increase in Proteobacteria and decreases in Campilobacterota and Firmicutes. Strikingly, the 10 milligram group showed no statistically significant differences from the control at the phylum level, and its overall abundance profile resembled the controls more than the other CBD-treated groups. The authors suggest this pattern may indicate a dose-dependent effect of CBD on the gut microbiome, though they also acknowledge the alternative possibility that some unidentified factor in the 10 milligram group influenced the animals’ gut bacteria.

At the genus level, the picture was similarly nuanced. The largest number of significantly altered genera appeared in the comparisons of the 0.2 milligram and 20 milligram groups against controls, with 13 and 12 genera respectively. Staphylococcus showed the most highly significant changes, decreasing relative to controls at all three doses. Dubosiella increased markedly at the lowest and highest doses, and two further genera—Tuzzerella, which rose, and Jeotgalicoccus, which fell—changed significantly across all three CBD-treated groups. The decline in Firmicutes among CBD-treated animals was largely attributed to a reduction in Lachnospiraceae_NK4A136_group, although the statistically significant decrease for that genus was observed only in the 0.2 milligram comparison. In contrast, Akkermansia increased in relative abundance in the CBD-treated groups, particularly at the lowest and highest doses.

These shifts are intriguing in light of what is known about the organisms involved. Lachnospiraceae_NK4A136_group bacteria are Gram-positive anaerobes that ferment dietary fiber into short-chain fatty acids such as butyrate, which plays a key role in regulating gut inflammation and immune system maturation, and increased abundance of this group has been observed in mouse models of colitis and diet-induced obesity. Akkermansia muciniphila, the only known representative of its genus in feces, degrades the mucin component of the intestinal mucus layer, stimulating renewed mucus production and thereby helping maintain the integrity of the intestinal barrier. The authors connect their findings to the endocannabinoid system, noting that CBD acts as a negative allosteric modulator of the CB1 cannabinoid receptor, and citing earlier mouse studies in which CB1 blockade increased Akkermansia muciniphila abundance while reducing Lachnospiraceae.

The researchers are careful to frame their conclusions within the limits of the study. This was an experiment in healthy mice, not humans, and the intraperitoneal injection route was chosen to simulate systemic, non-oral exposure such as inhalation—a popular way of consuming CBD—rather than to model typical oral supplementation. A significant percentage of sequencing reads, ranging from 10 percent in the highest-dose group to 19 percent in the lowest-dose group, could not be assigned to any taxon, leaving the profile incomplete. The team also notes that all doses of CBD reduced the relative abundance of the genus Helicobacter, though not significantly, and that future work should examine multiple intestinal regions and sequence the full 16S gene to achieve species-level identification. The raw sequencing data have been deposited in the NCBI Sequence Read Archive under accession number PRJNA1155312, allowing other researchers to scrutinize and extend the findings as interest in CBD’s systemic effects continues to grow.

Subject of Research: The effect of intraperitoneally injected cannabidiol at different doses on the duodenal bacterial microbiome of mice

Article Title: Effect of intraperitoneal cannabidiol (CBD) injection on intestine microbiome profile in a mouse model

Article References: Jasielczuk, I., Ocłoń, E., Żurowski, J., Szmatoła, T., Mizera-Szpilka, K., & Gurgul, A. (2025). Effect of intraperitoneal cannabidiol (CBD) injection on intestine microbiome profile in a mouse model. Current Genetics, 71(1), Article 21. https://doi.org/10.1007/s00294-025-01327-8

Image Credits: AI Generated

DOI: 10.1007/s00294-025-01327-8

Keywords: cannabidiol, CBD, gut microbiome, duodenum, mice, 16S rRNA sequencing, Firmicutes, Akkermansia, Lachnospiraceae, endocannabinoid system, dose-dependent, preclinical study