Lactulose Suppresses Osteoclastogenesis and Ameliorates Estrogen Deficiency-Induced Bone Loss in Mice
Chen Xiao1, Zhang Zheng2, Hu Yan1, Cui Jin1, Zhi Xin2, Li Xiaoqun2, Jiang Hao1, Wang Yao1, Gu Zhengrong3, Qiu Zili4, Dong Xin5, Li Yuhong1, Su Jiacan1,2,*
1Department of Orthopedics Trauma, Shanghai Changhai Hospital, Second Military Medical University, Shanghai, China. 2College of Basic Medicine, Second Military Medical University, Shanghai, China. 3Department of Orthopedics, Jing’ An District Centre Hospital of Shanghai Huashan Hospital, Fudan University, Shanghai, China. 4Jinling high school, Nanjing, Jiangsu, China. 5School of Pharmacology, Second Military Medical University, Shanghai, China.
Postmenopausal osteoporosis is characterized by excess osteoclastogenesis which leads to net bone loss and brittle fractures. Studies have demonstrated that estrogen deficiency-associated bone loss is microbiota-dependent and could be prevented by probiotics and prebiotics. In this study, we report that orally administered lactulose (20 g/kg, 6 weeks) orally administered significantly inhibited osteoclastogenesis, bone resorption, and prevented ovariectomy (OVX)-induced bone loss in mice. Lactulose increased intestinal Claudin 2, 3 and 15, compared to the OVX group, and lowered pro-osteoclastogenic cytokines levels including tumor necrosis factor-α, interleukin(IL)-6, receptor activator of nuclear factor kappa-Β ligand (RANKL), and IL-17 as well as increased the anti-inflammatory cytokine IL-10 in the intestine, peripheral blood, and bone marrow. Lactulose significantly preserved the number of Foxp3+ Treg cells in the intestines compared with that in OVX mice. Lactulose altered the composition of intestinal microbiota measured by 16s rDNA sequencing and increased intestinal and serum short-chain fatty acids (SCFAs) levels including acetate, propionate and butyrate which were decreased in OVX mice as measured by gas chromatography. Oral administration of lactulose for 2 weeks significantly lowered the level of bone resorption marker C-telopeptide of type 1 collagen-1 in healthy male young volunteers (aging 20-25 years). In conclusion, lactulose inhibited osteoclastogenesis and bone resorption by altering the intestinal microbiota and increasing SCFAs. Lactulose could serve as an ideal therapeutic agent for postmenopausal osteoporosis.
Figure 1. Lactulose prevented bone loss induced by ovariectomy in mice. (A) Representative μCT analysis of the distal femur. (B) Representative H&E staining of distal femoral sections and quantification of the trabecular area from each group 6 weeks after the operation of H&E staining. Scale bar: 200 μm. (C) Calculations of bone value / total value (BV/TV), bone surface area /total value (BS/TV), bone mineral density (BMD), trabecular number (Tb.N), bone surface area / bone value (BS/BV), trabecular space (Tb.Sp). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding group.
Figure 2. Lactulose suppressed osteoclastogenesis. (A) Tartrate-resistant acid phosphatase-stained sections of the distal femur. Scale bar: 20 μm. (B) Oc.N / B.Area. and Oc.N / B.pm. (C) Serum levels of TRAcp-5b. (D) Osteocalcin stained sections of the distal femur. Original magnification ×40. Scale bar: 20 μm. (E) Ob.N / B.Area. and Ob.N / B.pm. (F) Serum levels of osteocalcin. n= 5 mice per group in all panels. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding group.
Figure 3. Lactulose reduced intestine permeability after OVX. (A) Transcript levels of the tight junction proteins claudin 2, claudin 3 and claudin 15 of Sham, OVX and OVX mice treated with lactulose in the small intestine. (B) Immunohistological analysis of claudin 2 expressions in small intestine of Sham, OVX and OVX mice treated with lactulose. (Scale bar: 200 or 20 μm). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding group.
Figure 4. Lactulose inhibited OVX-induced pro-inflammatory cytokines. (A) Levels of the inflammatory cytokines TNF-α, IL-6, RANKL, and IL-17 and anti-inflammatory cytokine IL-10 in the small intestine of Sham, OVX and OVX mice treated with lactulose. (B) Immunofluorescent analysis of Foxp3+ cells in the intestine from Sham, OVX and OVX mice treated with lactulose small. Scale bar: 20 μm. (C) Calculation of Foxp3+ cells/Area. n = 5 mice per group in all panels. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding group.
Figure 5. Lactulose altered gut microbiota and increased SCFA production. (A) PCoA analysis. (B) Observed Species Number in different sequence number. (C) Ternary plot of different groups. (D) Taxonomy distribution at the genus level in different samples. (E) Top 10 enriched species in all groups. (F-I) Significantly differed species in the different groups; (J-L) The fecal SCFA levels of each group. (M) The serum SCFA levels of each group. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the corresponding group.
Figure 6. Graphical abstract. Estrogen withdrawal results in reduced fecal SCFA, increased systemic pro-inflammatory cytokines levels, excessive osteoclastogenesis, and significant bone loss. Lactulose increases SCFA production, lowers osteoclastogenic cytokines levels, inhibits osteoclastogenesis and ameliorates bone loss.
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