Osteoporosis (OP) is a systemic skeletal disease characterized by decreased bone mass and microarchitecture destruction, with elevated risk of fracture (Glaser and Kaplan, 1997). PMO induced by estrogen deficiency is the most common clinical form, and OP can be also secondary to medication or other systemic diseases, such as hypercortisolism and hyper-parathyroidism (Glaser and Kaplan, 1997). Osteopenia (ON) is an abnormal state with low bone density but not as severe as osteoporosis (Karaguzel and Holick, 2010). Estrogen deficiency due to gonadotropin-releasing hormone agonists induced no significant bone loss in GF mice, suggesting gut microbiota as a key determinant in PMO development (Li J.Y et al., 2016). PMO or ON patients showed distinct alterations in gut microbial taxa and metabolites compared to healthy individuals, including elevated levels of phylum Gemmatimonadetes and Chloroflexi, as well as genera Blautia, Parabacteroides, and Ruminococcaceae (Wang et al., 2017; Das et al., 2019; He et al., 2020). In addition, the genus Bacteroides and family Rikenellaceae were linked with high risk of fracture and low BMD in Japanese postmenopausal women (Ozaki et al., 2021). Glucocorticoid (GC) caused bone loss along with altered gut microbiota in mice, which can be prevented by antibiotic or probiotic treatment (Schepper et al., 2020). The mice that received fecal material from GC-treated subjects also showed pathological bone loss, supporting the involvement of gut microbiota in GC-induced osteoporosis (Schepper et al., 2020). In addition, the gut taxa SFB-dependent gut-bone crosstalk is also involved in the hyperparathyroidism-induced bone loss (Yu et al., 2020).

Osteoarthritis (OA) is the most common degenerative joint disease. The results in both Rotterdam Study and LifeLines-DEEP cohort showed an association between higher abundance of gut Streptococcus spp. and increased OA-related knee pain and joint inflammation (Boer et al., 2019). Another pilot study also demonstrated a distinct alteration in gut microbiota composition in knee OA patients compared to healthy individuals (Ramasamy et al., 2021). Additionally, animal models demonstrated the involvement of gut microbiota in OA development as well as the association between OA severity and certain gut genera, including Fusobacterium, Faecalibacterium, and Ruminococcacea (Huang et al., 2020).

Lipopolysaccharides (LPS), the important component of lipoproteins derived from Gram-negative gut microbes, is a strong stimulatory endotoxin triggering inflammatory immune responses and affecting general health. As a result of enrichment of gut LPS-containing microbes and increased gut paracellular permeability, gut-derived LPS expansion in circulation, defined as endotoxemia, or LPS translocation in distant tissues can potentially induce low-grade inflammation and promote the development of systemic disorders, such as Parkinson’s disease, obesity, insulin resistance, diabetes, NAFLD, ST-elevation myocardial infarction, and atherosclerosis (Cani et al., 2007; Cani et al., 2008; Geng et al., 2016; Perez-Pardo et al., 2019; Carnevale et al., 2020; Cho et al., 2021). In addition, GF mice model further provided strong evidence for the causative role of LPS in NAFLD development, as indicated by that only simultaneous high-fat diet administration and endotoxin-producing pathobiont mono-association induce NAFLD (Fei et al., 2020). The administration of gut commensal microbes, such as A. muciniphila and Roseburia intestinalis, and the modulation of gut microbiota via certain natural substances induced improved gut barrier function and attenuated LPS-associated systemic inflammation, consequently improving related systemic diseases (Cani et al., 2009; Jiang et al., 2016; Li J. et al., 2016; Song et al., 2016; Kasahara et al., 2018; Bian et al., 2020). In addition to paracellular pathway, LPS can translocate gut barrier in transcellular way via the phagocytosis of enterocyte mediated by toll-like receptor 4 (TLR4) (Neal et al., 2006). The obese rats administrated by natural pectin exhibited both increased TJ protein expression and reduced TLR4 expression in the ileum, leading to attenuated systemic inflammation (Jiang et al., 2016).

In gut-bone axis, leaky gut-induced LPS translocation and systemic inflammation potentially link to bone pathologies. Transplantation of fecal materials from metabolic syndrome patients in GF mice enhanced gut permeability by downregulating TJ proteins ZO-1 and occludin, causing elevated plasma level of LPS and subsequently exacerbated OA severity (Huang et al., 2020). Antibiotic treatment altered gut microbiota and decreased serum LPS level, alleviating circulating inflammation and preventing OA progression (Guan et al., 2020). Estrogen-deficient mice exhibited pathological bone loss and impaired bone microarchitecture in femur, with downregulated gut epithelial TJ protein expression and increased serum endotoxin levels, and these pathologies could be prevented by probiotics administration (Li J. Y. et al., 2016). Consistently, our previous studies observed enhanced gut permeability, elevated serum LPS, and systemic inflammation in OVX mice, consequently resulting in Th17-related immune responses in alveolar bone and aggravated periodontitis (Jia et al., 2019; Jia et al., 2021). In addition, administration of probiotics or berberine increased gut butyrate-producing bacteria, enhanced gut barrier function with reduced circulating LPS, and thus ameliorated estrogen deficiency-induced alveolar bone loss (Jia et al., 2019; Jia et al., 2021). Estrogen is potentially an important modulator of gut barrier. 17β-estradiol (E2), the predominant form of estrogen, can directly enhance gut barrier function by upregulating the expression of gel-forming mucin 2 (MUC2) and TJ proteins (ZO-1, occludin, and claudin 4) in colonic mucosa upon estrogen β signaling (Song et al., 2018). Additionally, estrogen can indirectly affect gut barrier by modulating gut microbiota. Estrogen deficiency induces reduction in gut butyrate-producing bacteria (Jia et al., 2019; Jia et al., 2021). Butyrate is a beneficial bacterial metabolite that reinforces gut barrier (Geirnaert et al., 2017). As a result, increased gut permeability due to estrogen deficiency induces gut pathogens translocation into circulation, causing inflammatory responses and promoting alveolar bone loss. Thus, gut barrier can be a potential therapeutic target for periodontitis under estrogen deficiency. Probiotics, such as Lactobacilli and Bifidobacteria, are live bacteria that benefit the host if provided as dietary or medical supplements in adequate quantities. Berberine is an alkaloid present in some medicinal plants, such as Berberis vulgaris, and Hydrastis canadens. With selective enrichment of gut butyrate-producing bacteria, probiotics and berberine supplements enhance gut barrier function and exert protective roles on alveolar bone under estrogen deficiency, representing a promising adjuvant treatment against periodontitis in postmenopausal women.

Short-chain fatty acids (SCFAs) are end-products deriving from gut microbial fermentation of dietary indigestible fibers, which escape from host digestion and absorption, mainly including butyrate, propionate, and acetate (Koh et al., 2016; Morrison and Preston, 2016). The type and amount of SCFAs generated in the gut depend on gut microbial composition, substrate types, and intestinal transit time (Macfarlane and Macfarlane, 2003). SCFA concentration varies among different segments of human intestinal tract, with high level in the cecum and proximal colon (Cummings et al., 1987). SCFAs are absorbed in the cecum and colon via active transport mediated by Na⁺- and H⁺-coupled monocarboxylate transporters, or via protonation-dependent non-ionic diffusion, or possibly via special exchange with intracellular bicarbonate or other SCFAs (Titus and Ahearn, 1988; Canfora et al., 2015; Koh et al., 2016). In addition to being partially metabolized by colonocytes as energy source, other absorbed SCFAs translocate into circulation. Although distinctly lower than the concentration in colonic contents, three major SCFAs are detected in human portal, hepatic, and peripheral venous blood with a descending trend among the levels of acetate, propionate, and butyrate (Cummings et al., 1987). Consistently, propionic and butyric acid levels in cecal contents are also correlated with that in aortic serum of the rats fed with dietary fibers (Jakobsdottir et al., 2013).

Via hematogenous way, gut-derived SCFAs are potentially transmitted to distant organs and involved in physiological processes or diseases. Transported through portal circulation to liver, SCFAs are consumed by hepatocytes in energy metabolism as well as the biosynthesis of cholesterol, glucose, and fatty acids (Dalile et al., 2019). Following hepatic utilization, small amounts of SCFAs travel to other organs via systemic circulation. SCFAs translocate to brain across blood-brain barrier and affect brain physiology and pathology, such as central appetite regulation, and post-stroke improvement (Frost et al., 2014; Lee et al., 2020). SCFAs generated by maternal gut microbiota are reflected in embryo tissues or organs, including sympathetic nerves, gut epithelium, and pancreas, participating in the development of postnatal nerve and metabolic systems via GPR41 and GPR43 signaling (Kimura et al., 2020).

Gut-derived SCFAs, especially butyrate and propionate, are the regulators of bone homeostasis, traveling to bone marrow and directly affecting bone metabolism. Fructo-oligosaccharide administration significantly increases serum level of butyrate and promotes bone formation in rats, ameliorating the pathological bone loss due to estrogen deficiency (Porwal et al., 2020). Study by Lucas and colleagues directly demonstrated the translocation of SCFAs to bone tissue, as evidenced by augmented butyrate or propionate concentrations in bone marrow of the mice with exogenous supplement of corresponding SCFA in drinking water (Lucas et al., 2018). Previous studies proved the direct regulation of SCFAs on the differentiation of osteoclasts and osteoblasts (Iwami and Moriyama, 1993; Rahman et al., 2003; Chen et al., 2007; Morozumi, 2011; Chang et al., 2016; Lucas et al., 2018). Butyrate, the strong histone deacetylase inhibitor, have been reported as a strong inhibitor of osteoclastogenesis. Early study showed the inhibitory role of sodium butyrate on murine osteoclast differentiation in bone marrow, possibly depending on the cytotoxicity of sodium butyrate (Iwami and Moriyama, 1993). Subsequent researches further updated related specific mechanisms. The in vitro study on murine macrophage cell line RAW264 showed that sodium butyrate inhibited osteoclast formation via regulating nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways (Rahman et al., 2003). Furthermore, butyrate and propionate can inhibit osteoclast differentiation via enhancing glycolysis in osteoclast precursors and subsequently downregulating the expression of osteoclast marker genes TRAF6 and NFATc1 (Lucas et al., 2018). The role of SCFAs on osteoblast formation remains uncertain. Sodium butyrate can promote osteogenic differentiation of mesenchymal stem cells via activating ERK pathway (Chen et al., 2007). However, osteoblastic differentiation of ROS17/2.8 cells was halted in the presence of high-level butyrate (10^-3M) (Morozumi, 2011). MG-63 osteoblasts treated by butyrate demonstrated suppressed cell proliferation with increased cell cycle arrest and reactive oxygen species (ROS) production (Chang et al., 2016). However, the in vivo study by Lucas showed no effects of SCFAs on osteoblasts and bone formation (Lucas et al., 2018).

Our previous studies indicated that estrogen deficiency led to reduction of gut butyrate-producing bacteria, such as Clostridium leptum, Clostridium coccoides, Fecalibacterium prausnitzii, and Roseburia, as well as decreased butyrate production, leading to more severe periodontitis (Jia et al., 2019; Jia et al., 2021). Rescue of gut butyrate-producing bacteria and butyrate production by berberine or probiotics prevented the alveolar bone loss induced by estrogen deficiency, suggesting the pivotal roles of gut-derived butyrate in periodontitis (Jia et al., 2019; Jia et al., 2021). The indirect regulation of gut-derived butyrate on periodontitis possibly depends on gut barrier. In addition, butyrate may potentially translocate to alveolar bone via circulation and directly affects periodontitis progression.

Of note, some pathogenic bacteria in periodontitis, such as Porphyromonas gingivalis and Fusobacterium nucleatum, can produce SCFAs, including butyrate and propionate. Interestingly, butyrate has cytotoxic effects in patients with periodontitis. The concentration of butyrate in gingival crevices positively correlates to the severity of periodontitis. Butyric acid could reach to millimolar concentration in the gingival crevicular fluid of periodontitis patients, whereas the maximum plasma concentration of butyrate of adult leukemia patients after intravenous infusion only reached to 0.05 millimolar (Miller et al., 1987; Niederman et al., 1997; Lu et al., 2014). Oral-derived butyrate can promote periodontitis development (Guan et al., 2021), likely due to its cytotoxic effects on gingival epithelial cells and fibroblasts. Butyrate at millimolar concentrations can induce apoptosis, autophagy, and pyroptosis of gingival epithelial cells and impair epithelial TJ, leading to the destruction of gingival epithelial barrier and periodontitis initiation (Tsuda et al., 2010; Evans et al., 2017; Liu et al., 2019; Magrin et al., 2020). Butyric acid can negatively affect cell growth and cell cycle progression of gingival fibroblasts by inducing reactive oxygen species production (Kurita-Ochiai et al., 2008; Chang et al., 2013), and lead to mitochondria- and caspase-dependent apoptosis in inflamed gingival fibroblasts in a dose-dependent manner (Kurita-Ochiai et al., 2008). Long-term exposure to butyrate can also lead to cytostasis and apoptosis of healthy gingival fibroblasts via intrinsic and extrinsic pathways, promoting periodontitis progression (Shirasugi et al., 2017; Shirasugi et al., 2018). In addition, butyrate can dose-dependently induce the production of proinflammatory cytokines in gingival fibroblasts, including IL-6 and IL-11, which rescue T-cell from apoptosis and contributed to the development of periodontitis (Kurita-Ochiai et al., 2002). Thus, unlike circulating butyrate derived from gut microbiota, local accumulation of concentrated butyrate due to periodontal pathogens has deleterious effects on alveolar bone, which should be taken into consideration in future investigation.

The bone homeostasis is the dynamic balance between the coupled processes of osteoblast-mediated bone formation and osteoclast-mediated bone resorption. The immune system has long been recognized as an essential regulator of bone metabolism. The crosstalk between immune and skeletal systems, termed as “osteoimmunology,” underlies bone physiological and pathological processes (Rho et al., 2004). The components of immune systems, such as immune cells and cytokines, are involved in bone remodeling and metabolic or inflammatory bone diseases, including osteoporosis, arthritis, and periodontitis (Weitzmann and Pacifici, 2006; Hajishengallis, 2015; Kuhn and Morrison, 2020). Periodontitis is an inflammation induced by exogenous pathogens. Microbial pathogens are recognized and presented by innate immune system to activate adaptive immune responses, thereby inducing the production of effector cells and molecules and driving the initiation of periodontal destruction.

IGF-1 belongs to the IGF family, which plays an important role in childhood growth and has anabolic effects in adults. IGF-1 in serum, existing as a complex containing IGF-1 molecule, IGF binding protein 3 (IGFBP-3), and acid labile subunit (ALS), is mainly generated by hepatocytes in response to growth hormone and travels to distant organs via circulation (Daughaday et al., 1972). In addition, IGF-1 is also locally produced via autocrine or paracrine fashion in non-hepatic organs/tissues, including bone. Accumulating evidences have suggested IGF-1 as an important regulator of skeletal development and bone remolding (Yakar et al., 2010; Tahimic et al., 2013; Wang et al., 2013). With the absence of ALS, reduction of circulating IGF-1 results in disrupted linear growth and decreased bone mineral density (Yakar et al., 2002). Circulating IGF-1 also affects skeletal integrity and mechanical properties via regulating lateral growth of long bone and deposition of mineralized cortical bone tissue (Yakar et al., 2009; Courtland et al., 2011). Subcutaneous administration of IGF-1 improves bone density and cortical thickness in osteopenia mice by upregulating osteogenic-related proteins and reducing protein expression in osteoclastic activity (Guerra-Menéndez et al., 2013). Of note, reduced circulating IGF-1 during early lifetime can be compensated by local IGF-1 signaling, as evidenced by increased bone mass with early-life loss of circulating IGF-1 in female mice (Ashpole et al., 2016). IGF-1 binds to IGF-1 receptor (a tyrosine kinase receptor) and elicits IGF-1 signaling, which regulates the growth and differentiation of osteoclasts and osteoblasts. IGF-1 can promote osteoclast differentiation through maintaining the normal interplay between osteoblasts and osteoclast precursors, depending on regulating the production of RANKL and M-CSF (Wang et al., 2006). Furthermore, IGF/IGF-IR signaling mediates the proliferation and differentiation of osteoprogenitor regulated by PTH, influencing periosteal bone formation (Wang et al., 2007).

Mounting evidences have suggested that gut microbiota affects host development and general health via regulating systemic IGF-1 levels (Yan and Charles, 2018). Commensal microbe Acetobacter pomorum regulates insulin/IGF signaling in Drosophila depending on the activity of pyrroloquinoline quinone-dependent alcohol dehydrogenase, contributing to host development and metabolic homeostasis (Shin et al., 2011). During intestinal or pneumonic infection, gut bacterium Escherichia coli O21:H⁺ maintains systemic IGF-1 level, which links to increased IGF-1 level and E.coli O21:H⁺ colonization in white adipose tissue, thus preventing muscle wasting and promoting disease tolerance (Schieber et al., 2015). The catabolic and anabolic effects of commensal gut microbiota on bone remolding are also partially mediated by regulation of systemic and local IGF-1 signaling (Yan et al., 2016; Novince et al., 2017). Gut microbiota colonization in GF mice promotes bone turnover with elevated IGF-1 levels in serum and bone marrow, while antibiotic treatment in SPF mice reduces serum IGF-1 level and increases bone mass, which can be rescued by SCFA supplement (Yan et al., 2016).

Sex hormones, such as follicle-stimulating hormone (FSH), estrogen, and androgen, have been considered as important regulators of bone metabolism (Leder, 2007; Khosla, 2020). Both estrogen and androgen prevent bone resorption and sustain bone formation (Syed and Khosla, 2005). A clinical randomized study demonstrated that hormone/estrogen replacement therapy could ameliorate postcranial bone density and alveolar bone mass in postmenopausal women (Civitelli et al., 2002). Furthermore, estrogen deficient rats had lower periodontal bone density than control rats, while estradiol treatment protects against alveolar bone loss induced by estrogen deficiency (Duarte et al., 2006). Additionally, estrogen deficiency has adverse effects on the bone healing after bone extraction (Bezerra et al., 2013). Estrogen doesn’t directly regulate osteoclast differentiation, instead it inhibits mature osteoclast function by downregulating genes relevant to bone resorption, and controls mature osteoclast lifespan by promoting apoptosis in binding with estrogen receptor α (ERα) (Imai et al., 2009). Consistently, estrogen-deficient rats presented increased osteoclasts in alveolar bone, and exogenous estrogen supplement reduces alveolar bone osteoclasts partially due to osteoclast apoptosis (Faloni et al., 2007; Bezerra et al., 2013). Of note, instead of femur and vertebrae, estrogen affects maxillary bone phenotype via IL-33/ST2 signaling in a site-specific manner (Macari et al., 2018). In addition, without direct action on bone cells, high FSH activity promotes bone formation depending on ovary pathway (Allan et al., 2010).

Consistently, gut microbiota has been suggested as an important regulator of hormone homeostasis (Baker et al., 2017; Rizzetto et al., 2018). As early as about 40 years ago, it was reported that gut microbiota intervention by antibiotic treatment resulted in increased fecal estrogen levels with reduced urinary estrogen excretion, especially estradiol and estriol, which potentially related to the suppressed gut reductive estrogen metabolism (Adlercreutz et al., 1984). More evidences have shown the association between gut microbial composition and urinary or systemic levels of estrogen and estrogen metabolites (Flores et al., 2012; Fuhrman et al., 2014). As for testosterone, early-life colonization of gut commensal microbes or gut microbial transfer alters serum level of testosterone, affecting the development of autoimmune diseases (Markle et al., 2013). However, the mechanism whereby gut microbiota affects hormone remains uncertain. Recently, it has been reported that gut microbiota affects hormone homeostasis depending on “endobolome,” a novel term that refers to the aggregate of gut microbial genes involved in the production and metabolism of sex steroid hormones including estrogen (Aguilera et al., 2020). Gut microbiota metabolizes hormones via distinct enzymes, such as β-glucuronidases, β-glucuronides, and hydroxysteroid dehydrogenase, affecting circulating and local hormone levels (Plottel and Blaser, 2011; García-Gómez et al., 2013). Enzyme β-glucuronidases derived from gut microbiota promote deconjugated estrogens production, which travel into circulation and carry out physiological functions (Rizzetto et al., 2018). Additionally, gut microbe Clostridium scindens could transfer glucocorticoids to androgens via side-chain cleavage (Ridlon et al., 2013). Some gut bacteria, mostly belonging to the family Coriobacteriaceae, are capable of metabolizing soy-derived isoflavone-glycosides and producing metabolites, including equol, a compound with strong estrogenic activity (Guadamuro et al., 2017; Chen and Chen, 2021). Although direct evidence is still lacking, interplays in “gut-hormone-bone” axis provide another pathway whereby gut microbiota regulates periodontal diseases.

Serotonin, namely, 5-hydroxytrypatamine (5-HT), is a neurotransmitter with hormone-like activity, which can affect bone homeostasis via circulation. Circulating serotonin derived from duodenum inhibits bone formation and regulates bone mass (Yadav et al., 2008). Serotonin transporter is a plasma membrane transporter expressing on bone cells, which is responsible for the uptake of extracellular serotonin and subsequent intracellular serotonin storage and degradation. Extracellular serotonin signaling enhancement via serotonin transporter inhibition exerts detrimental effects on skeletal growth and bone homeostasis, as indicated by reduced bone mass, altered microarchitecture, as well as impaired mechanical properties, relating to increased risk of osteoporotic fracture (Warden et al., 2005; Warden et al., 2008; Eom et al., 2012; Wu et al., 2012; Kerbage et al., 2014; Gorgas et al., 2021). Serotonin can promote osteoclast proliferation and differentiation via NF-κB signaling and influence osteoblast proliferation in a dose-dependent manner (Battaglino et al., 2004; Gustafsson et al., 2006). Serotonin directly prevents osteoblast differentiation via Htr1b and CREB signaling (Yadav et al., 2008).

Serotonin is mainly synthesized by enterochromaffin cells and exerts biological effects via the circulation. Accumulating evidences indicate that gut microbiota affects the biosynthesis and availability of serotonin. SCFAs derived from gut microbiota, such as acetate and butyrate, promote serotonin secretion via upregulating the expression of tryptophan hydroxylase 1 (Tph1), which is the rate-limiting enzyme responsible for serotonin biosynthesis in enterochromaffin cells (Reigstad et al., 2015; Vincent et al., 2018). Gut microbe Clostridium ramosum or its components are also reported to increase serotonin secretion and availability from enterochromaffin cells (Mandić et al., 2019). Hence, gut microbiota also potentially regulates alveolar bone homeostasis via serotonin.