Melatonin alleviates titanium nanoparticles induced osteolysis via activation of butyrate/GPR109A signaling pathway

Abstract

Background: Inflammatory osteolysis after total joint replacement (TJR) may cause implant failure, periprosthetic fractures, and be a severe threat to global public health. Our previous studies demonstrated that melatonin had a therapeutic effect on wear-particles induced osteolysis. Gut microbiota is closely related to bone homeostasis, and has been proven to be affected by melatonin. However, whether melatonin could play its anti-osteolysis effects through reprogramming gut microbiota remains elusive.

Results: Here, we demonstrated that melatonin could alleviate Ti-particles induced osteolysis, while this therapeutic effect was blocked by antibiotic cocktail treatment. Interestingly, transplantation of fecal microbiota from mice treated with melatonin reappeared the same beneficial effect. Analysis of the 16S rRNA revealed that melatonin could reverse dysbacteriosis triggered by osteolysis, and elevate the relative abundance of some short chain fatty acid (SCFA) producing bacteria. Moreover, butyrate was enriched by exogenous melatonin administration, while acetate and propionate did not show an evident difference. This was consistent with the results of the metagenomic approach (PICRUSt2) analysis, which revealed a general increase in the synthetic enzymes of butyrate. More importantly, direct supplementation of butyrate could also recapitulate the anti-osteolysis effect of melatonin. Further analysis identified that butyrate alleviated osteolysis via activating its receptor GPR109A, and thus to suppress the activation of NLRP3 inflammasome triggered by Ti-particles.

Conclusions: Taken together, our results suggested that the benefits of melatonin mainly depend on the ability of modulating gut microbiota and regulating butyrate production.

Keywords: Butyrate; GPR109A; Gut microbiota; Inflammatory osteolysis; NLRP3 inflammasome.

Fig. 1

The benefits of melatonin was correlated with gut microbiota. A Representative view of calvarium in each group via Micro-CT 3D reconstruction. B–D Quantification of bone erosion parameters (BV/TV) bone volume to tissue volume ratio, (BMD) bone mineral density, and total porosity. n = 6. E Percentage of osteoclasts surface per bone surface (OCs/BS, %). n = 5. F H&E and G TRAP staining of calvarium slices from each group. H Principle coordinate analysis (PCoA) plot from sham (PBS), Ti (Ti exposure), and MT (Ti exposure and melatonin treatment) groups based on the Bray Curits distance. n = 6. I Relative abundance of SCFA producing microbes in mice feces. n = 6. Results are expressed as mean ± SEM (One-way ANOVA[post hoc:SNK] *p < 0.05, **P < 0.01, ***p < 0.001)

Fig. 2

Melatonin enriched the abundance of butyrate related enzymes and fecal butyrate concentration in mice. A The concentration of acetate, propionate, and butyrate in fecal samples. n = 6. B–D Relative abundance of acetate, propionate, and Butyrate synthesis related enzymes from PICRUSt2. n = 6. Results are expressed as mean ± SEM (One-way ANOVA[post hoc:SNK] *p < 0.05, **P < 0.01, ***p < 0.001, # significantly different from Sham, Ti and MT group)

Fig. 3

The anti-osteolysis effect of melatonin could be transmitted by fecal microbiota transplantation. A Representative view of calvarium in each group via Micro-CT 3D reconstruction. B Relative abundance of SCFA producing microbes in fecal samples n = 6. C Three major SCFA concentration in fecal samples. n = 6. D Butyrate synthesis related enzymes. n = 6 in each group. Results are expressed as mean ± SEM (Unpaired t-tests * p < 0.05, ** p < 0.01). E–G Quantification of bone erosion parameters (BV/TV) bone volume to tissue volume ratio, (BMD) bone mineral density, and total porosity. n = 6. Results are expressed as mean ± SEM (One-way ANOVA[post hoc:SNK] ***p < 0.001)

Fig. 4

Butyrate suppressed NLRP3 inflammasome activation in vivo and in vitro. A Representative view of calvarium in each group via Micro-CT 3D reconstruction. B–D Quantification of bone erosion parameters (BV/TV) bone volume to tissue volume ratio, (BMD) bone mineral density, and total porosity. n = 6. E Percentage of osteoclasts surface per bone surface (OCs/BS, %). n = 5. F, G BMDMs were incubated with Ti particles (0.1 mg/ml) and butyrate (1 mM) for 6 h after being primed with LPS (100 ng/ml) for 3 h, then cell lysates and supernatants were used for analysis of western blot. Cleaved Caspase-1 (P20) was detected from supernatant samples. NLRP3, Caspase-1 pro, IL-1β pro and Actin were detected in cell lysates (F). IL-1β were detected from supernatant samples (G). n = 3. H Representative immunohistochemical staining of NLRP3 and IL-1β. I Quantification of NLRP3 and IL-1β expression using integrated optical density/specimen area (IOD/Area). J Representative immunofluorescence staining of Caspase-1 in calvarial specimens. K Quantification of Caspase-1 using mean density (integrated density/specimen area) in calvarial specimens. n = 5. Results are expressed as mean ± SEM (One-way ANOVA[post hoc:SNK] **p < 0.01, ***p < 0.001)

Fig. 5

Butyrate exerted anti-osteolysis effects via its receptor GPR109A. A Western blot analysis of GPR109A in BMDMs. n = 3. B Representative immunohistochemical staining and quantification of GPR109A. n = 5. C Representative view of calvarium in each group via Micro-CT 3D reconstruction and quantification of bone erosion parameters (BV) bone volume, (BV/TV) bone volume to tissue volume ratio, (BMD) bone mineral density, and total porosity. n = 6. D Representative immunohistochemical staining of NLRP3, IL-1β and immunofluorescence staining of Caspase-1 in calvarial specimens. E Quantification of NLRP3 and IL-1β expression using integrated optical density/specimen area (IOD/Area). F Quantification of Caspase-1 using mean density (integrated density/specimen area) in calvarial specimens. n = 5. G BMDMs from wild type or GRP109A^−/− mice were incubated with Ti particles (0.1 mg/ml) and butyrate (1 mM) for 6 h after being primed with LPS (100 ng/ml) for 3 h, then cell lysates and supernatants were used for analysis of western blot. NLRP3, Caspase-1 pro, and Actin were detected in cell lysates. Cleaved Caspase-1 (P20) were detected from supernatant samples. n = 3. H The cytokine of IL-1β in supernatants was measured by ELISA. Results are expressed as mean ± SEM (One-way ANOVA[post hoc:SNK] **p < 0.01, ***p < 0.001)

Fig. 6

The ability of melatonin to inhibit NLRP3 inflammasome was evidently diminished by antibiotics. A Representative immunohistochemical staining of NLRP3 in calvarial specimens. B Representative immunofluorescence staining of Caspase-1 in calvarial specimens. C Representative immunohistochemical staining of IL-1β in calvarial specimens. D Representative immunohistochemical staining of GPR109A in calvarial specimens

Fig. 7

Metabolic pathways of three major SCFAs from carbohydrate fermentation. The gut microbiota synthetizes three major SCFAs, acetate, propionate, and butyrate through dietary fiber. Acetate is synthesized via the Wood-Ljungdahl pathway and pyruvate. Propionate can be produced from the propanediol, succinate, and acrylate pathway. Butyrate is formed through the molecule of acetyl-CoA

Fig. 8

Melatonin alleviates Ti-particles induced osteolysis through the enrichment of gut microbiota metabolite butyrate. Melatonin reverses the dysbacteriosis triggered by inflammatory osteolysis and raises the relative abundance of SCFA producing bacterium. Enrichment of butyrate following the change of microbiota activates GPR109A and suppresses osteolysis via inhibiting NLRP3 inflammasome activation