Probiotics ameliorate alveolar bone loss by regulating gut microbiota

Abstract Objectives Oestrogen deficiency is an aetiological factor of postmenopausal osteoporosis (PMO), which not only decreases bone density in vertebrae and long bone but also aggravates inflammatory alveolar bone loss. Recent evidence has suggested the critical role of gut microbiota in osteoimmunology and its influence on bone metabolisms. The present study aimed to evaluate the therapeutic effects of probiotics on alveolar bone loss under oestrogen‐deficient condition. Materials and Methods Inflammatory alveolar bone loss was established in ovariectomized (OVX) rats, and rats were daily intragastrically administered with probiotics until sacrifice. Gut microbiota composition, intestinal permeability, systemic immune status and alveolar bone loss were assessed to reveal the underlying correlation between gut microbiota and bone metabolisms. Results We found administration of probiotics significantly prevented inflammatory alveolar bone resorption in OVX rats. By enriching butyrate‐producing genera and enhancing gut butyrate production, probiotics improved intestinal barrier and decreased gut permeability in the OVX rats. Furthermore, the oestrogen deprivation‐induced inflammatory responses were suppressed in probiotics‐treated OVX rats, as reflected by reduced serum levels of inflammatory cytokines and a balanced distribution of CD4+IL‐17A+ Th17 cells and CD4+CD25+Foxp3+ Treg cells in the bone marrow. Conclusions This study demonstrated that probiotics can effectively attenuate alveolar bone loss by modulating gut microbiota and further regulating osteoimmune response and thus represent a promising adjuvant in the treatment of alveolar bone loss under oestrogen deficiency.


| INTRODUC TI ON
Osteoporosis is a systematic bone disorder characterized by decreased bone mineral density (BMD) and compromised bone microarchitecture, leading to an increased risk of bone fracture. 1 Postmenopausal osteoporosis (PMO) is the most common type but also in the progression of inflammatory arthritis and alveolar bone loss. 4,[9][10][11] Periodontitis and periapical periodontitis are polymicrobial infectious diseases characterized by local inflammatory response within the supporting tissue of teeth, leading to alveolar bone loss. 12,13 Many studies have reported exacerbated alveolar bone resorption under oestrogen deficiency, [14][15][16][17] imposing challenges to the clinical treatment of these diseases. Conventional therapies of periodontitis and periapical periodontitis mainly rely on mechanical removal of plaque biofilm and root canal therapy. However, poor clinical treatment outcome has been noted in the elder women with PMO. [18][19][20] The traditional PMO drugs such as bisphosphonates and RANK ligand inhibitor have been demonstrated effective in inhibiting osteoclast activity, 21 but with non-negligible adverse effects such as medication-related osteonecrosis of the jaw (MRONJ), 22,23 limiting routine use of these drugs as an adjuvant treatment of periodontitis and periapical periodontitis.
Increasing evidence has indicated a close association between gut microbiota and bone metabolism, and the gut-bone axis has been proposed. [24][25][26] Aberrant gut microbiota is associated with decreased BMD and osteopenia. 27,28 In addition, the oestrogendepleted germ-free mice present decreased expression of TNFα, RANKL and IL-17 and increased BMD as compared to the SPF mice, 5,29 further underlining the critical roles of gut microbiota in skeletal homeostasis. Modulation of gut microbiota has been proposed as a potential approach to the management of skeletal disorders. 26 Probiotics, which confer a health benefits on the host mainly via modulating gut microbiota, have demonstrated effectiveness in the treatment of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), antibiotic-associated diarrhoea and necrotizing enterocolitis. 30,31 Recent studies have also demonstrated the protective effects of probiotics towards bone. 5,32,33 Although specific mechanisms are still unclear, oral administration of certain probiotic strains has shown effectiveness in suppressing alveolar bone loss in animal models. [34][35][36][37] Our previous work showed that berberine, a natural alkaloid, was able to ameliorate periodontal bone loss by regulating gut microbiota of OVX rats, 38 further underscoring the importance of gut microbiota modulation in the reversion of alveolar bone resorption. To further delineate the mechanism by which probiotics ameliorate alveolar bone loss, we hypothesize that probiotics can promote the intestinal barrier function, which subsequently alleviate osteoimmune response and consequently ameliorate periodontal and periapical bone loss under oestrogen deficiency. To validate this hypothesis, we administered probiotics to the OVX rats with either periodontitis or periapical periodontitis and investigated the effect of probiotics on the inflammatory alveolar bone loss. The effect of probiotics on gut barrier and osteoimmune response was further explored to delineate the underlying mechanisms.

| Animals and experimental design
A 10-week-old Sprague-Dawley female rats (Da Shuo, China) were housed under specific pathogen-free condition. After acclimatization for 1 week, rats were intraperitoneally anaesthetized by pentobarbital sodium (2%, 40 mg/kg) and bilaterally ovariectomized or subjected to sham surgery. At the same time, rats were administered with probiotics or vehicle until sacrificed. Rats in probiotics groups were supplemented with 1 × 10 7 CFU/day commercially available infant probiotics production by intragastric gavage with the blunt syringe inserted into the stomach. The probiotics production contained 10 39 Rats were intraperitoneally anaesthetized by pentobarbital sodium (2%, 40 mg/kg) and were ligated with a 5-0 silk suture around the bilateral maxillary first molars to establish experimental periodontitis. Porphyromonas gingivalis ATCC 33277, which was anaerobically grown and resuspended to a concentration of 1 × 10 7 CFU/mL in saline, was smeared on the silk suture every 3 days after ligation. 38 The periapical periodontitis rat model was established as reported by Brasil with minor modifications. 17 The tooth pulps of bilateral mandibular first molars were exposed to oral environment by making occlusal class I cavity near mesial margin using micro-round bur in a highspeed motor, leading to a spontaneous development of periapical periodontitis.

| Specimens collection
Four weeks after ligation or pulp exposure, samples were harvested for analyses. Faeces were collected in 1.5-mL sterile Eppendorf tubes and immediately stored at −80℃. Blood samples were collected from the abdominal aorta under intraperitoneal anaesthesia. Rats were then immediately sacrificed by cervical dislocation. 2-cm segments of ileum from all rats were immediately excised and submerged into 1 mL of TriZol reagent for RNA isolation. Bilateral maxilla from rats representing periodontitis, bilateral mandibles from rats representing periapical periodontitis and 2-cm segments ileum from all rats were removed and fixed in 4% paraformaldehyde for 24 hours. Femurs were dissected thoroughly free from soft tissue. The tips of the femurs were removed and bone marrow (BM) was harvested by inserting a syringe needle into one end of the bone and flushing with phosphate-buffered saline (PBS).

| Micro-CT scanning and analysis
To evaluate the bone destruction and microarchitecture of alveolar bone and femur bone, micro-CT was performed as previously de-

| Analysis of serum proinflammatory cytokine
Serum was isolated by centrifuging the blood after clotting at 1500 g for 10 minutes. The serum levels of TNFα and IL-17A were assayed by ELISA kits (Invitrogen) according to the manufacturer's instructions.

| Histologic analyses of alveolar bone and ileum tissue
Decalcified for 28 days, paraformaldehyde-fixed and paraffinembedded 5μm thickness bone tissue sections were prepared for histologic analyses, which were performed with the method previ- Intestinal barrier integrity was also examined by H&E staining.
Images captured at ×100 magnification were randomly selected,

| RNA isolation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Intestinal RNA was isolated and purified from an ileum segment with TriZol Reagent (Invitrogen). Reverse transcription of RNA into cDNA was performed with the Primer-Script RT Reagent Kit with gDNA Eraser (RR047A; Takara Bio). The expression of genes encoding intestinal tight junction (TJ) proteins, including occludin, claudin1, claudin 3, zo-1 and Jam3 (encoding genes were named as ocln, cldn 1, cldn 3, TJP1 and jam 3, respectively) was quantified with the β-actin as internal control. The primer sequences are presented in Table 1. Relative quantitative analysis was performed with the 2 −ΔΔCT method.  Table 3.
Relative quantitative analysis was performed with the 2 −ΔΔCT method.

| Butyrate treatment
To further validate the role of intestinal butyrate in maintaining gut permeability and preventing skewed Th17/Treg-induced bone resorption, additional OVX/sham rats were gavage-fed with sodium butyrate (400 mg/kg) every day for 8 wk until sacrifice. Serum, ileum and bone marrow cells were collected for further analyses with the same methods as described above.

| Statistical analysis
All data were statistically analysed by SPSS v25.0 (Statistical

| Probiotics restore the gut permeability of OVX rats by enriching butyrate-generating bacteria
Principal components analysis (PCA) and principal coordinate analysis (PCoA) based on Bray-Curtis distance showed significant alteration of gut microbiota on operational taxonomic unit (OTU) level in OVX rats. The altered microbial community structure was reversed by the administration of probiotics ( Figure 1A). More importantly, intragastric administration of probiotics significantly elevated the levels of butyrate-producing genera that were deprived in OVX rats, including Clostridium leptum subgroup, Clostridium coccoides subgroup, Fecalibacterium prausnitzii and Roseburia/E rectale cluste ( Figure 1B). Consistently, we found that transcripts encoding butyrate synthesis-associated enzymes, including But and Buk were downregulated in the OVX rats and the administration of probiotics significantly upregulated But and Buk expression ( Figure 1C).
In parallel, the scarcity in butyrate as observed in the intestine of OVX rats was reversed by the intragastric administration of probiotics ( Figure 1D). Furthermore, we observed an increased abundance of gut segmented filamentous bacteria (SFB) in OVX rats, and administration of probiotics significantly reduced the intestinal level of SFB ( Figure 1E).

| Probiotics ameliorate periodontal bone loss in OVX rats
The effects of oestrogen-deficiency and probiotics treatment on the

| Probiotics ameliorate periapical bone loss in OVX rats
The periapical periodontitis rat model was further established to investigate the effect of oestrogen deficiency and probiotics on the inflammatory alveolar bone loss. Oestrogen deficiency aggra- Of note, the current study showed that intragastric administration of probiotics exerted no positive effects on the alveolar bone destruction in rats free of OVX or inflammatory state. This is consistent with our previous findings of berberine or probiotics treatment outcome reported by others. 38,84 This may suggest the unnecessity

ACK N OWLED G EM ENTS
This study was supported by the National Natural Science

CO N FLI C T O F I NTE R E S T
All authors state that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this article.