**4. Discussion**

The anti-inflammatory effects of CBD isolate or CBD-rich extracts have been reported [44], but the differential effects of CBD in female pre- and postmenopausal states remains largely unexplored. The decline in ovarian E2 during perimenopause and after menopause is associated with a pro-inflammatory state which promotes several metabolic disorders, including diabetes, osteoporosis, and neurodegeneration [45]. Due to the drawbacks of HRT use, especially in older postmenopausal women [12], other strategies are needed to address chronic disease burden. CBD products are currently marketed for a variety of indications, including female menopause, but often without adequate evidence [46,47]. Building upon prior work that suggested CBD may have therapeutic application in E2-deficient females [26], in this study, the RNA-Seq analysis of colon tissues revealed that the effect of CBD on inflammatory response pathways depends on E2 status. While CBD decreased the expression of inflammatory response pathway genes in E2-deficient OVX mice (Figure 1B), CBD had the opposite effect in E2-sufficient SS mice (Figure 1C), where the expression of inflammatory response genes was increased. Indeed, the CBD-treated SS and VEH-treated OVX groups showed a similar increase in inflammatory response genes (Figure 1B,D). While the loss of ovarian E2 is known to increase the expression of inflammatory markers in murine tissues [48], the elevated expression of inflammatory response pathway genes in the CBD-treated SS group was unexpected, especially given that the prior qPCR analysis of these tissues showed decreased mRNA levels of selected inflammatory markers (*Il1b*, *Il6*, and *Tnf*) in both CBD-treated OVX and SS groups relative to VEH-treated controls [26]. Notably, compared to VEH-treatment, CBD-treated SS mice had decreased mRNA levels of *Ocln* and *Tjp1*, while the latter was increased in CBD-treated OVX mice, which suggested CBD compromised gu<sup>t</sup> barrier integrity in the E2-sufficient state but was beneficial in E2-deficiency [26]. It remains to be determined whether the induction of the inflammatory response pathway in CBD-treated SS mice is unique to colon tissue or whether this extends to other segments of the intestine or other tissues.

The loss of E2-producing ovary cells with increasing age in perimenopause through postmenopause leads to elevated oxidative stress, which induces inflammation [49]. In young reproductive adult females, ovarian cells have abundant mitochondria that require the high amounts of oxygen for oxidative phosphorylation and optimal cell survival [49]. Reactive oxygen species (ROS), such as superoxide ion and hydrogen peroxide, are created by oxidative phosphorylation and quenched by endogenous glutathione and dietary antioxidants (e.g., vitamins E, C, polyphenols) [49–51]. As aging progresses, ovarian cell membranes and mitochondria are in danger of oxidative damage due to the imperfect detoxification of oxy-radicals and reduced mitochondrial regeneration [49]. Oxidative damage leads to E2 deficiency and subsequent decline in the function and homeostasis of E2-dependent cells throughout the body [49]. CBD reduced ROS production and had a protective effect on Caco-2 monolayer integrity [17]. Together ROS and E2 deficiency may induce systemic inflammation and contribute to menopausal symptoms, such as hot flashes, an increased risk of arteriosclerosis, and decreased gu<sup>t</sup> barrier integrity [17,49].

CBD is a potent antioxidant [14]. Besides its use for patients with epilepsy [21–24], CBD is being investigated for treatment of other neurodegenerative diseases, such as Huntington's disease and schizophrenia [21,50,52,53]. CBD inhibits ROS production and modifies redox balance by activating the redox-sensitive nuclear factor erythroid 2-related factor (Nrf2) in multiple cell types [54,55]. Nrf2 transactivates several antioxidant and cytoprotective genes [55,56]. CBD was shown to reduce ROS production via the inhibition of Tnf α and iNOS [49,50], which is consistent with the CBD suppression of TL-induced inflammation in ileal organoids (Figure 3). CBD is also used for pain relief as it is a cyclooxygenase (COX)-2 inhibitor that reduces glutathione-dependent prostaglandin E2 (PGE2) signaling and subsequent inflammation [49,50]. In the present study, *Ptges*, which encodes PGE2 synthase, was downregulated in the colon tissue of CBD-treated OVX mice compared to VEH-treated OVX mice (Figure 1B and Supplementary File S1).

The presence of ROS species is sensed and monitored by the hypoxia inducible factor (HIF) pathway [57]. In hypoxic conditions, HIF1 α is stable and reduces the levels of oxidative phosphorylation and ROS [57]. HIF1 α stabilization plays an important role in activating osteoclast activity and bone resorption [58–60]. E2 destabilizes HIF1 α, even under hypoxic conditions while E2-deficient OVX mice have stabilized HIF1 α, which leads to bone loss [58]. Consistent with these published reports, *Hif1α* was upregulated in VEH-treated OVX mice compared to VEH-treated SS mice (Figure 1D and Supplementary File S1) and exhibited an osteoporotic bone phenotype [26]. The administration of a HIF1 α inhibitor was protective against bone loss in OVX mice [58]. In the present study, *Hif1α* was downregulated in CBD-treated OVX mice compared to the VEH-treated mice (Figure 1B and Supplementary File S1) and had improved bone phenotypes [26]. It remains to be determined whether CBD directly or indirectly inhibits HIF1 α.

Importantly, a physiologically beneficial level of ROS is required for pathogen resistance and cell signaling [61]. The excessive suppression of ROS was reported to induce inflammation [62], which may be the case in CBD-treated SS mice (Figure 1C). The effects of CBD on ROS in OVX and SS mice remain to be investigated.

While CBD isolate was used in this study, hemp-derived (defined as having <0.3% tetrahydrocannibinol) extract preparations contain other phytocannabinoids as well as terpenes and flavonoids [63,64]. Complex extracts are thought to have superior efficacy compared to CBD isolate preparations due to the synergistic activities of the phytochemical constituents, termed the "entourage effect" [65–67]. Whether other phytochemicals in a CBD-rich extract would temper CBD's stimulation of colonic inflammation in E2-sufficent females remains to be examined. Interestingly, when male C57BL6/J mice were orally administered a CBD-rich cannabis extract (CRCE) for 5 days per week for 2 weeks, they

showed a higher colonic expression of pro-inflammatory markers (*Il1ß*, *Cxcl1*, and *Cxcl2*) and a decreased expression of *Muc2*, suggesting an induction of intestinal inflammation [68]. Male mice have low circulating E2 levels making them more similar to OVX female mice then SS mice; therefore, the increased expression of colonic markers of inflammation may be due to sex-based difference in profile of hormones other than E2, CBD dose, and/or the presence of other phytochemicals in the CRCE.

The endocannabinoid system functions to maintain the homeostasis of central and peripheral tissues and displays cross-talk with estrogen signaling [69]. Endogenous cannabinoids (i.e., endocannabinoids) arachidonoylethanolamide (anandamide) and 2- arachidonoylglycerol (2-AG) are lipid messengers that signal to CB1 and CB2 endocannabinoid receptors present in central and peripheral tissues [70–72]. CBD activity at endocannabinoid receptors is limited but it can interact with over 65 molecular targets throughout the body [73,74]. The molecular basis of CBD bioactivity in E2-deficient vs. -sufficient states remains to be investigated.

CBD is a partial agonist for CB2, which is mainly expressed in immune cells and peripheral tissues [75]. CB2 is expressed in osteoblasts, osteoclasts, and osteocytes and is an important target for improving bone phenotypes [26,76]. In a previous study, CBD-treated OVX mice had increased femoral mRNA expression of *Cnr2*, which encodes CB2, compared to VEH-treated OVX mice and was associated with decreased bone loss [26]. In the present study, *Cnr2* was upregulated in VEH-treated OVX mice compared to the SS group and CBD treatment lead to a downregulation in the OVX mice (Figure 2B and Supplementary File S1). CBD activity at endocannabinoid receptors is limited, but it has been reported to interact with over 65 molecular targets throughout the body [73,74]. The molecular basis of CBD bioactivity in E2-deficient vs. -sufficient states remains to be investigated.

When *Corynebacterium parvum*-primed and unprimed male mice were treated with CB2 agonist WIN 55212-2 or with CB2 antagonist SR141716A, the levels of pro-inflammatory cytokines in serum was suppressed [77], suggesting that opposing effects on the CB2 receptor can result in the same outcome. Similarly, compared to VEH-treated OVX mice, CBD-treated OVX mice had increased *Cnr2* expression in bone [26] but decreased *Cnr2* expression in the colon (Figure 2B) and in both cases resulted in the reduced expression of inflammatory markers in bone and colon. Further study is needed to explain the mechanism behind these observations.

BAs are synthesized in the liver, stored in the gallbladder, and secreted into the duodenum for the digestion of lipophilic compounds [78]. 95% of Bas are reabsorbed in the ileum and return to the liver via portal circulation, while 5% enter the colon for excretion [78]. Low levels of BAs enter circulation and act as signaling molecules in diverse tissues [78]. Glycine-conjugated BAs have been correlated with increased small intestinal inflammation in rats [79]. Compared to SS groups, VEH-treated OVX mice had increased concentrations of glycine-conjugated Bas, which were reduced in CBDtreated OVX mice [26]. CBD did not alter the BA profiles in colon content (Table S3) but induced the expression of the bile secretion pathway genes in colon tissue independently of E2 status (Figure 2B,C)**.** The CBD-induced increase in bile secretion genes is likely due to it being a lipophilic compound that requires bile-mediated micelle formation for intestinal absorption [80]. An increased BA pool in the liver would be an indicator of hepatic inflammation and damage; however, CBD did not alter hepatic BA profiles in SS or OVX mice (Table S3). OVX has been associated with hepatic tissue inflammation as mice age, becoming apparent 6–7 months after the surgery [48]. There was no observation of OVX-associated increase in the expression of hepatic inflammatory markers (Figure S4), perhaps due to the mice being less than 6 months post-OVX when tissue qPCR analysis was performed.
