**5. Conclusions**

In conclusion, the RNA-Seq analysis of colon tissues allowed a comprehensive investigation of CBD- and OVX-induced transcriptome changes. CBD had a potent anti-

inflammatory effect in colon tissues of E2-deficient OVX mice but may contribute to inflammation in intact, E2-sufficient females. To better delineate the CBD mechanisms of action in E2-deficient and -sufficient states, additional experiments are needed to follow up on the extensive gene expression changes. The gu<sup>t</sup> organoid data sugges<sup>t</sup> that CBD may have a direct anti-inflammatory effect on the intestinal epithelium. Future gu<sup>t</sup> organoid studies that test CBD in the absence and presence of E2 treatment would contribute to understanding CBD actions in E2-deficient and -sufficient states. In both the SS and OVX groups, CBD induced similar changes to genes related to bile secretion, indicating changes that were independent of E2 status. OVX or CBD treatment did not alter BA levels in liver or colon content, suggesting that previously observed CBD-induced changes in ileal and serum BAs [26] are more relevant. There is currently widespread use but inadequate investigation of CBD and CBD-rich extracts/products in the menopause and postmenopause [25]. Whether CBD and/or other phytocannabinoids have differential effects in women based on E2 status warrants further study. Given that HRT is not recommended for the prevention of chronic conditions in postmenopausal women, CBD may offer a therapeutic option; however, more research is needed to assist women in making better-informed judgements about individualized CBD risks and benefits.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/biomedicines11010074/s1, Figure S1: Two-dimensional PCA score plots comparing difference/similarity between collection of DEGs within and between indicated biological samples; Figure S2: Top 20 enriched GOBP pathways generated in ShinyGO; Figure S3: Top 20 enriched KEGG pathways generated in ShinyGO; Figure S4: qPCR of inflammatory markers in liver tissue; Figure S5: Cell viability of ileal organoids. Table S1: Bile acid information, cannabidiol, and HPLC processing method parameters; Table S2: Calibration curves, limit of detection and quantification, and coefficient of variance for bile acid and cannabidiol analysis; Table S3: Bile acid concentrations in colon content and liver. Supplementary File: Gene annotations of DEGs in inflammatory response (S1) and bile secretion (S2) pathways.

**Author Contributions:** Conceptualization, D.E.R.; methodology, K.M.A.B. and K.M.T.; software, K.M.A.B. and K.M.T.; validation, all authors; formal analysis, K.M.A.B., K.M.T. and H.P.; investigation, all authors; resources, D.E.R. startup funds; data curation, all authors; preparation of figures, tables, and manuscript draft, all authors; writing D.E.R. and K.M.A.B.; editing of manuscript D.E.R. and K.M.A.B.; visualization, K.M.A.B. and K.M.T.; supervision, D.E.R.; project administration, D.E.R.; funding acquisition, D.E.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Rutgers University startup funds to D.E.R.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The accession number for deposited RNA sequences is PRJNA908843 and can be found at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA908843. The data can be accessed on 1 January 2023.

**Acknowledgments:** The authors thank Hung Skyler Hoang for guidance with ShinyGO.

**Conflicts of Interest:** The authors declare no conflict of interest.
