Lactobacillus Reuteri 6475 Prevents Bone Loss in a Clinically Relevant Oral Model of Glucocorticoid-Induced Osteoporosis in Male CD-1 Mice

Abstract

Glucocorticoids (GCs) are commonly used anti-inflammatory medications with significant side effects, including glucocorticoid-induced osteoporosis (GIO). We have previously demonstrated that chronic subcutaneous GC treatment in mice leads to gut barrier dysfunction and trabecular bone loss. We further showed that treating with probiotics or barrier enhancers improves gut barrier function and prevents GIO. The overall goal of this study was to test if probiotics could prevent GC-induced gut barrier dysfunction and bone loss in a clinically relevant oral-GC model of GIO. Eight-week-old male CD-1 mice were treated with vehicle or corticosterone in the drinking water for 4 weeks and administered probiotics Lactobacillus reuteri ATCC 6475 (LR 6475) or VSL#3 thrice weekly via oral gavage. As expected, GC treatment led to significant gut barrier dysfunction (assessed by measuring serum endotoxin levels) and bone loss after 4 weeks. Serum endotoxin levels significantly and negatively correlated with bone volume. Importantly, LR 6475 treatment effectively prevented both GC-induced increase in serum endotoxin and trabecular bone loss. VSL#3 had intermediate results, not differing from either control or GC-treated animals. GC-induced reductions in femur length, cortical thickness, and cortical area were not affected by probiotic treatment. Taken together, these results are the first to demonstrate that LR 6475 effectively prevents the detrimental effects of GC treatment on gut barrier, which correlates with enhanced trabecular bone health in an oral mouse model of GIO. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Keywords: BARRIER; GLUCOCORTICOID; GUT‐BONE INTERACTIONS; OSTEOPOROSIS; PROBIOTIC.

Fig. 1

Glucocorticoid and probiotic treatment does not cause differences in body weight. (A) Average percent body weight change after 4 weeks of treatment. Bar graph is mean + SEM. (B) Final body weights after 4 weeks of treatment. Violin plot represents minimum to maximum values with lines at the median and quartiles. No significant differences were observed. Statistical analyses via one‐way ANOVA with Tukey's post‐test. n = 8/group. GC = glucocorticoid; LR 6475 = Lactobacillus reuteri ATCC 6475.

Fig. 2

Lactobacillus reuteri ATCC 6475 prevents glucocorticoid‐induced distal femoral trabecular bone loss. Eight‐week‐old male CD‐1 mice were treated +/− GC and probiotics LR 6475 or VSL#3 for 4 weeks. (A) Representative iso‐surface images of distal femur trabecular bone. (B, C) μCT analysis of distal femur trabecular bone expressed as (B) bone volume fraction and (C) bone volume fraction corrected for body weight. (D) Distal femur trabecular bone mineral density and bone mineral content. (E) Distal femur trabecular bone microarchitectural analyses. n = 7–8 per group. Violin plots represent minimum to maximum values with lines at the median and quartiles. Statistical analysis performed via one‐way ANOVA with Tukey's post‐test. BMC = bone mineral content; BMD = bone mineral density; BV/TV = bone volume/total volume; BV/TV/BW = bone volume/total volume/body weight; GC = glucocorticoid; LR 6475 = Lactobacillus reuteri ATCC 6475; Tb.N = trabecular number; Tb.Sp = trabecular spacing; Tb.Th = trabecular thickness.

Fig. 3

Lactobacillus reuteri ATCC 6475 prevents glucocorticoid‐induced vertebral body trabecular bone loss. (A, B) μCT analysis of vertebral body trabecular bone expressed as (A) bone volume fraction and (B) bone volume fraction corrected for body weight. (C) Vertebral body trabecular bone mineral density and bone mineral content. (D) Vertebral body trabecular bone microarchitectural analyses. n = 7–8 per group. Violin plots represent minimum to maximum values with lines at the median and quartiles. Statistical analysis performed via one‐way ANOVA with Tukey's post‐test. BMC = bone mineral content; BMD = bone mineral density; BV/TV = bone volume/total volume; BV/TV/BW = bone volume/total volume/body weight; GC = glucocorticoid; LR 6475 = Lactobacillus reuteri ATCC 6475; Tb.N = trabecular number; Tb.Sp = trabecular spacing; Tb.Th = trabecular thickness.

Fig. 4

Probiotics do not prevent reduced femur strength caused by glucocorticoid treatment. Data points represent ultimate load and fail load along with respective displacements via three‐point bending to determine structural‐level biomechanical properties. n = 6–8. Values are mean ± SEM. Error bars correspond to respective axis. GC = glucocorticoid; LR 6475 = Lactobacillus reuteri ATCC 6475.

Fig. 5

Lactobacillus reuteri ATCC 6475 improves gut barrier function. (A) Serum endotoxin unit quantification normalized to control levels. (B) Correlation analysis between normalized serum endotoxin units and distal femur trabecular BV/TV%. n = 7–8 per group. Violin plots represent minimum to maximum values with lines at the median and quartiles. Statistical analysis performed via one‐way ANOVA with Tukey's post‐test and Pearson's correlation analysis. BV/TV = bone volume/total volume; GC = glucocorticoid; LR 6475 = Lactobacillus reuteri ATCC 6475.