265 BERBERINE AMELIORATES OVARIECTOMY-INDUCED ANXIETY-LIKE BEHAVIORS BY ENRICHMENT IN EQUOL GENERATING GUT MICROBIOTA

Background Decline in circulating ovarian hormone levels is associated with increment in anxiety and alteration in intestinal microbiota. As a kind of gut microbiota modulator, berberine (BBR) has shown efficacy in treating diseases such as menopausal osteoporosis and type 2 diabetes through regulating bacteria. However, the effects of BBR on ovariectomy-induced anxiety are still unclear and whether or not gut microbiota modifies the possible effect of BBR remain to be fully elucidated. Aim To demonstrate that BBR is able to alleviate anxiety through modulating intestinal microbiota under estrogen-deficient conditions. Methods Female specific pathogen-free (SPF) SD rats were ovariectomized (OVX) or shamoperated (sham). BBR (100mg/kg) or control vehicle was administered via oral gavage every day for four weeks. Open field test (OFT) and elevated plus maze test (EPM) were used to detect the anxiety-like behaviors. Moreover, the 16S rRNA sequencing and liquid chromatography/mass spectrometry (LC/MS) analysis were utilized to validate the changes in intestinal microbes and microbiota isoflavone metabolites, respectively. The concentration of serum estradiol (E2) was measured using ELISA. Further, female germ-free (GF) SD rats were operated and treated with BBR or vehicle under sterile conditions. Fecal microbiota transplantation (FMT) was conducted using fecal samples from SPF rats to colonize GF-OVX rats. Results Compared with SPF-sham rats, OFT and EPM showed increased anxiety in SPFOVX rats, and BBR treatment significantly ameliorated anxiety-like behaviors compared with SPF-OVX rats. The serum level of isoflavone metabolite equol and the ratio of equol to daidzein were decreased by ovariectomy and elevated by BBR treatment. Furthermore, BBR treatment altered the microbiota diversity and taxa richness of the gut microbiome. The alpha diversity revealed a significant reduction in richness and diversity after BBR administration compared with SPF-OVX group. The beta diversity showed dissimilarity in composition among each group. At the genus level, BBR-treated rats harbored a higher abundance of beneficial gut microbes, such as Bacteroides, Bifidobacterium, Lactobacillus, and Akkermansia, among which many species are involved in isoflavone bioconversion (Fig.1). Notably, estrogen deficiency in GF rats also resulted in anxiety-like behaviors, however gavage feeding of BBR to GF-OVX rats had no significant effects on behavior improvement and equol metabolism. FMT of SPF-BBR rats to GF-OVX rats improved anxiety-like behaviors and increased the serum concentration of isoflavone metabolite equol (Fig.2). Conclusions Gut microbiota is critical in BBR treatment of ovariectomy-aggravated anxiety, indicating a promising therapeutic strategy for postmenopausal anxiety. Download : Download high-res image (316KB) Download : Download full-size image Fig. 1 . BBR ameliorates the anxiety-like behaviors and gut dysbiosis of SPF-OVX rats. (A) Elevated plus-maze test results. (B) Open field test results. (C) Ovarian hormone levels. (D) Isoflavone levels. (E) α-diversity indices. (F) β-diversity measured by PCoA. (G) LEfSe analysis from phylum to genus. Data are expressed as the mean ± SEM. *P Download : Download high-res image (217KB) Download : Download full-size image Fig. 2 . GF condition eliminates the anti-anxiety effects of BBR, while fecal microbes from SPF-BBR rats ameliorates anxiety of GF-OVX rats. (A) Elevated plus-maze test results of GF rats. (B) Open field test results of GF rats. (C) Ovarian hormone levels of GF rats. (D) Isoflavone levels of GF rats. (E) Elevated plus-maze test results of FMT rats. (F) Open field test results of FMT rats. (G) Ovarian hormone levels of FMT rats. (H) Isoflavone levels of FMT rats. Data are expressed as the mean ± SEM. *P