This intervention study characterized the gut microbiota in the Depression (CES-D scores ≥ 21, untreated) group compared with those in the flavonoid-rich versus placebo treatment groups. Furthermore, the associations between gut microbiota and depression were investigated. In this study, the 8-week flavonoid intervention changed the gut microbiota taxonomic composition and diversity in the Depression group. The gut microbiota composition in the Depression group was dominantly characterized by Firmicutes. However, a clinical report on major depressive disorder (MDD) has shown that low counts of Firmicutes may lead to a reduction in short-chain fatty acids, which can serve as a physiological basis for low levels of inflammation against depression [33]. However, no consensus results have emerged from clinical studies on depression and gut microbiota taxonomic composition that are relevant to depression [34].
We found that the generally dominant bacteria in the Orange group were unclassified Lachnospiraceae, unclassified Bacteroides, Eubacterium_g4, Clostridium_g24, unclassified Bifidobacterium, Eubacterium ventriosum, unclassified Roseburia, Coprococcus_g2, and unclassified Agathobacter. The genera that most significantly increased in number in the Orange group were unclassified Lachnospiraceae, unclassified Roseburia, and unclassified Bifidobacterium (p < 0.001). Intestinal microbial groups are affected by environmental factors, such as food and the presence of parasites, and genetic factors may also cause such differences [35]. Despite the vast diversity of gut bacteria, only 13 identified core bacterial genera, including unclassified Lachnospiraceae, were common among 95% of individuals [36].
Naseribafrouei et al. (2014) reported that the abundance of higher-order Bacteroidales and the Oscillibacter and Alistipes genera and lower abundance of the family Lachnospiraceae were associated with depression in the comparison of the gut microbiota between 37 patients with depression and 18 healthy controls [37]. Moreover, Jiang et al. (2015) reported that the abundance of Lachnospiraceae and Ruminococcaceae decreased in patients with MDD compared with that in the control group [38]. In our study, after the intervention, the relative taxonomic abundance of Lachnospiraceae increased in both groups (Orange and Flavored) compared with that in the Depression group. Furthermore, the abundance of genus Lachnospiraceae significantly increased (p < 0.0001) in the Orange group compared with that in the Flavored group (p = 0.0002) (Supplementary Fig. 1A and 2A). Moreover, significantly increased expression of Lachnospiraceae_uc and Roseburia_uc were observed in the Orange group compared with that in the Flavored group as shown by real-time PCR results of stool samples (Fig. 5). Thus, we found that Lachnospiraceae responds to flavonoid treatment in patients with depression.
In the Orange group, the relative taxonomic abundance of Bifidobacterium, Roseburia, Ruminococcus, and Akkermansia significantly increased compared to that in the Flavored group (Supplement Figs. 1 and 2). The relative expression of Bifidobacterium_uc in the Orange group was also significantly increased compared to that in the Flavored group (Fig. 5C and F). Bifidobacterium is a genus that influences intestinal function in infants who have received healthy breast milk, while low but relatively stable Bifidobacterium counts are observed in adulthood [39]. The abundance of Bifidobacterium increases in the late stages of pregnancy in both women and mice, indicating the causative role of progesterone [40]. Bifidobacterium bifidum colonization increases interleukin 6 (IL-6) and IL-8 cytokine levels through NF-κB activation in mice [41]. Supplementation of Bifidobacterium has also been shown to increase the fecal levels of immunoglobulin A in young women [42] and to lead to changes in the levels of human immune cells [43]. Furthermore, exogenous probiotic microbes, such as Bifidobacterium and Lactobacillus, have been shown to reduce anxiety in human participants [44].
elative abundances of bacterial genera, including Akkermansia spp., were significantly reduced in socially defeated animals, which was positively correlated with both anxiety and depression [45]. Moreover, the administration of prebiotics increased the relative abundance of Akkermansia during exposure to stressors [46, 47]. The abundance of Akkermansia has been suggested as a biomarker for healthy intestines and has an inverse correlation with several intestinal disorders [48]. Generally, Akkermansia and Bifidobacterium are well-known health-associated genera that protect against inflammation, promote immunomodulation, and promote healthy metabolic homeostasis [49]. These findings corroborate our study on flavonoid intervention, and the relative abundance of Akkermansia significantly increased in the Orange group compared to that in the Flavored group.
Greater microbial diversity was found in the Depression group than that in the Orange and Flavored groups. To date, four studies on MDD have investigated and performed a microbial diversity analysis. While three studies reported no significant differences in microbial diversity [37, 50, 51], Jiang et al. (2015) reported greater microbial diversity in patients with MDD than in healthy individuals [38]. High microbial diversity could be easily affected by age, eating habits, and other factors [52]. Although greater diversity of bacteria is potentially beneficial to human health, the precise consequences of increased bacterial diversity for depression are still unclear [38].
In our study, the abundances of Ruminococcaceae and Lachnospiraceae were positively correlated with BDNF levels in the Depression group. After the 8-week intervention, the serum levels of BDNF in the Orange group were higher than those in the Flavored group. The abundances of Ruminococcaceae OCTT_g (p < 0.0016), Ruminococcus_g2 (p < 0.007), Ruminicoccus (p < 0.0248), and Lachnospiraceae_uc (p < 0.039) were positively correlated with serum BDNF levels. BDNF is a neurotrophin that performs multiple functions in the central nervous system and participates in the therapeutic mechanisms of antidepressants [53]. BDNF levels were significantly lower in patients with MDD than the control group, and recovery from depression after antidepressant treatment was associated with normal serum levels of BDNF in patients with MDD [54]. Similarly, there were more changes in the taxonomic composition of the microbiota in the Orange group than in the Flavored group. In the Orange group, 10 taxons, including Lachnospiraceae, Bacteroides, and Bifidobacterium, were more abundant, but only one taxon (Blautia_uc) increased in abundance in the Flavored group compared to that in the Depression_F group. Besides, the taxonomic abundance of Bifidobacterium was decreased in the Flavored group (Fig. 3F and Supplement Fig. 2B). Moreover, the relative abundance of Clostridium decreased further in the Orange group (Supplement Fig. 1F and 2F). The prevalence of Clostridium was negatively associated with serum BDNF levels [38]. Therefore, we found that the 8-week intervention changed the gut microbiota composition and increased serum BDNF levels in the Orange group compared to those in the Flavored group.
Members of the Lachnospiraceae family, such as the Roseburia, Blautia, and Coprococcus genera, are known to break down carbohydrates into short-chain fatty acids (SCFAs) [55]. Reduction in these fermentation-related bacteria precipitates a decline in SCFA production, causing intestinal barrier dysfunction [55, 56]. Moreover, SCFAs promote the differentiation of T cells and can function as a histone diacetylase inhibitor [57]; therefore, SCFAs can act as a regulator of immune homeostasis. Additionally, SCFAs are involved in neurotransmitter production [58] and neuroprotection and can penetrate the blood–brain barrier [59]; therefore, they have been proposed as potential novel antidepressants [60]. The Ruminococcaceae family was highly abundant in healthy controls compared to that in patients with MDD [38]. Moreover, the Ruminococcaceae family is characterized by anti-inflammatory activity and associated with a chronic low-grade inflammatory response [49, 61]. Therefore, the relative abundance of these genera in the gut microbiome mediated the low degree of inflammation and higher intestinal barrier function. Inflammation is associated with major depression [62]. Furthermore, flavonoids exhibit a neuroprotective effect by falsifying inflammatory reactions and have potential therapeutic effects in terms of neuroprotection [63].
In this study, the use of antibiotics, probiotics, and prebiotics was not allowed for assessing the microbial community. Additionally, we served fresh, 100%-pure, orange juice daily as the flavonoid-rich drink and investigated the associations between the relative abundance of the gut microbiome and depression in young adults. Although the particular association between flavonoids and depression is still unclear, we found that high intake of flavonoids changed the relative abundance of the gut microbiome and increased serum BDNF levels. An unidentified genus of Lachnospiraceae, Bacteroides, and Bifidobacterium was correlated with flavonoid-rich orange juice treatment.