Gut Microbiota Influence Bone Health In Menopausal Women Via Bacteroides Vulgatus And Valeric Acid Pathway

[Background and Objective]Osteoporosis(OP)is a human complex disease with unclear mechanism.Strong association was found between gut microbiota(GM)and bone mineral density(BMD)in experimental animal models.However,this relationship and the potential underlying mechanism in humans are unknown.Thus human studies will be investigated here comprehensively.[Methods]We randomly recruited a cohort composed of 517 Chinese peri-and post-menopausal women and performed analyses in metagenomics,targeted metabolomics,and whole-genome sequencing to identify BMD-associated bacterial species,along with significant functional short chain fatty acids(SCFAs)derived from gut microbiome.Subsequent validations in vivo in mice,in vitro in osteoblasts/osteoclast-like cells,and an in vivo in an independent human population were performed for the results we identified.Specifically,first,we randomly recruited eligible volunteers to collect blood and fecal samples,and measured BMD in different sites using dual energy X-ray absorptiometry(DXA).Second,we performed metagenomic analysis to evaluate the association between GM biodiversity/function and human BMD,and identified specific BMD-associated species.Third,we used targeted metabolome analysis to measure concentration of SCFAs,then found BMD-associated SCFAs.Fourth,we performed whole genome sequencing,genome-wide association study,plus Mendelian randomization(MR)to identify potential causal relationship between GM and SCFA.Fifth,we performed the same analysis methods in a US Caucasian females cohort for validation of the results.Meanwhile,C57BL/6 mice(specific-pathogen free[SPF]grade)underwent bilateral ovariectomy were gavaged with Bacteroides vulgatus(B.vulgatus)/normal saline(NS)for eight weeks.Then we measured BMD and serum biomarkers of bone metabolism.We also observed structure of trabecular bone by using MicroCT and hematoxylin-eosin staining.RAW264.7 cells and MC3T3-E1 cells were used as osteoclast/osteoblast precursors,respectively,cultured with various concentrations of valeric acid to observe differentiation of osteoclast-like cells and osteoblasts.Total protein and RNA were extracted from the osteoclast-like cells/osteoblasts for western blot analysis and real-time PCR analysis to obtain expressions of NF-κB p65,IκB-α and IL-10.[Results]1.Both GM biodiversity and functional modules were negatively associated with BMD(p-values<0.05).In the Chinese cohort,seven species were found to be associated with BMD of various skeletal sites(e.g.,B.vulgatus,regression coefficient[β]=-0.027,p-value=0.032).Meanwhile,in the US Caucasian females cohort,the B.vulgatus was also negatively associated with BMD(p=-0.018,p-value=0.029).One of the microbiota-derived SCFAs,valeric acid,was positively associated with spine BMD(p=0.046,p-value=0.013).Meanwhile,the results of MR analysis indicated that the B.vulgatus may causally down regulate valeric acid(β’s<0,p-values<0.05).2.In mice in vivo,gavaging with B.vulgatus can cause significantly lower BMD of the 5th lumbar vertebral body(p-value<0.05)and osteocalcin concentration(p-value<0.05),while a higher concentration of C-telopeptide of type I collagen(p-value<0.05),compared with the NS treated group.MicroCT and hematoxylin-eosin staining results demonstrated increased disconnection and separation of trabecular bone network,as well as reduction of trabecular bone mass in the B.vulgatus treated group.3.In osteoclast-like cells/osteoblasts in vitro,after adding valeric aicd into mediums,the proportion of tartrate-resistant acid phosphatase-positive multinucleated(≥3 nuclei)osteoclast-like cells significantly decreased;while the differentiation of osteoblasts,as well as mineralization of extracellular matrix were significantly increased.The expression of IL-10 was significantly increased,while the expressions of NF-κB p65 and IκB-α were decreased in the valeric acid treated group.[Conclusion]This study supports the correlation between GM and BMD in human.We also considered the possibility that the GM might affect human BMD through its SCFA byproducts(e.g.,valeric aicd).IL-10 may also play a role in the mechanism.Hence,based on our findings,B.vulgatus,valeric acid and the involved pathway may represent novel treatment/intervention targets for OP prevention and treatment.