Gut Microbiota-Derived Butyric Acid Alleviates Glucocorticoid-Associated Osteonecrosis of the Femoral Head via Modulating Inflammatory Cytokines in Bone Marrow Mesenchymal Stem Cells

Abstract

Background: The role of gut microbiota and its metabolites in regulating bone metabolism has been well established, with inflammatory immune responses potentially playing a critical role. Glucocorticoid-associated osteonecrosis of the femoral head (GA-ONFH), caused by high-dose glucocorticoid use for inflammatory or immune-related diseases, is a prevalent condition of bone metabolic imbalance. However, the regulatory role and mechanisms of gut microbiota and its metabolites in the development and progression of GA-ONFH remain unclear. This study aims to investigate the intervention effects of gut microbiota and its metabolite butyric acid on GA-ONFH through a series of multi-omics in vitro and in vivo experiments. Methods: Sprague Dawley rats were randomly divided into four groups. The gut microbial composition of the groups was analyzed through 16S rDNA sequencing. Targeted metabolomics was employed to assess differences in short-chain fatty acids (SCFAs) among the groups. Butyric acid, identified as a key differential metabolite, was then selected for further exploration of its effects on bone marrow mesenchymal stem cells (BMSCs) and GA-ONFH rat models through in vitro and in vivo experiments. Results: 16S rDNA sequencing revealed alterations in gut microbiota structure in GA-ONFH rats. Micro-CT and HE staining demonstrated that depletion of gut microbiota with broad-spectrum antibiotics prior to GA-ONFH modeling exacerbated the disease's development. In contrast, fecal microbiota transplantation (FMT) was shown to alleviate GA-ONFH progression. Targeted metabolomics indicated that FMT mitigated the reduction in butyric acid levels induced by dexamethasone (DXM). Subsequent in vitro cell experiments confirmed that butyric acid promotes BMSC proliferation, migration, and osteogenic differentiation. RNA sequencing revealed that butyric acid regulates T cell-mediated inflammatory cytokine genes in BMSCs, while Western blot and immunofluorescence assays confirmed that butyric acid modulates the expression of TNF-α and IL-2/IL-4 in BMSCs. Finally, in vivo experiments demonstrated that butyric acid supplementation attenuated the progression of GA-ONFH and improved the expression of inflammation-related cytokines in femoral head tissue. Conclusions: Our study demonstrates that gut microbiota depletion exacerbates GA-ONFH, while FMT restores butyric acid levels and alleviates disease severity. Butyric acid reduced the expression of TNF-α and IL-2 while increasing the level of IL-4 in vivo and in vitro, thereby improving the local inflammatory environment of the femoral head and alleviating the progression of GA-ONFH. These findings highlight that reduction in butyric acid levels due to gut microbiota dysbiosis is a crucial factor in the progression of GA-ONFH.

Keywords: BMSCs; GA-ONFH; bone metabolism; butyrate acid; gut microbiota.

Figure 1

Difference in gut microbiota between Control group and GA-ONFH Model group (n = 10). (A) Principal component analysis. (B) Volcano plots of the gut microbiota gene profiles in Control group and Model group. (C) Histogram of the distribution of LDA effect size values. (D) Species composition abundance map of family level. (E) Species composition abundance map of genus level. GA-ONFH, glucocorticoid-associatedosteonecrosis of the femoral head.

Figure 2

FMT alleviate the progress of GA-ONFH and decrease the expression of TNF-α and IL-2. (A) Microbiota depletion experiment. (B) Schematic diagram of the experimental design for the microbiota depletion experiment and GA-ONFH modeling. (C) Representative images of micro-CT (scale bars, 500 μm), HE staining (scale bars, 100 μm) and immunohistochemistry (scale bars, 100 μm). Red arrow showed subchondral sclerosis band. Blue arrow showed microfractures in subchondral sclerosis band. Black arrow showed necrotic regions in subchondral sclerosis band. (D–G) Quantification of Tb. Th (D), empty lacuna rate (E), immunohistochemical evaluation of TNF-α (F) and IL-2(G). ⁣^∗p < 0.05. GA-ONFH, glucocorticoid-associated osteonecrosis of the femoral head; IL, interleukin; TNF-α, tumornecrosisfactor-alpha.

Figure 3

Targeted metabolomics analysis and cell proliferation assay. (A) Butyric acid concentration in feces. (B) Butyric acid concentration in blood. (C) CCK-8 analysis of BMSCs cultured with different doses of sodium butyrate. BMSCs, bone marrow mesenchymal stem cells; CCK-8, Cell Counting Kit-8. ⁣^∗p < 0.05.

Figure 4

Cell scratch test and osteogenic differentiation assay. (A) Cell scratch test (scale bars, 200 μm). (B) Quantitative analysis of migration area after BMSCs cultured with different doses of sodium butyrate. (C) Osteogenic differentiation assay and ALP staining (Scale bars, 200 μm). (D) Quantitative analysis of ALP staining. ALP, alkaline phosphatase; BMSCs, bone marrow mesenchymal stem cells. ⁣^∗p < 0.05.

Figure 5

RNA-sequencing. (A) Top 20 of functional genome enrichment in sodium butyrate-treated BMSCs. (B) Functional genome enrichment scatterplot in sodium butyrate-treated BMSCs. (C) Loop circos of KEGG functional genome enrichment analysis. (D) GSEA of positive regulation of IL-4 production. (E) GSEA of T cell receptor binding. (F) GSEA of positive regulation of T cell proliferation. BMSCs, bone marrow mesenchymal stem cells; GSEA, gene set enrichment analysis; IL, interleukin; KEGG, Kyoto Encyclopedia of Genes and Genomes.

Figure 6

The expression of TNF-α, IL-2, and IL-4 in BMSCs treated with different doses of sodium butyrate. GC -: DXM 0 mM; GC +: DXM 100 mM; SCFA -: sodium butyrate 0 mM; SCFA +: sodium butyrate 1 mM; SCFA ++: sodium butyrate 5 mM. (A–C) Immunofluorescence staining of TNF-α (A), IL-2(B) and IL-4(C) in BMSCs. (D to G) Western blot (D) and quantitative analysis of TNF-α (E), IL-2(F), and IL-4(G) in BMSCs. ⁣^∗p < 0.05. BMSCs, bone marrow mesenchymal stem cells; GC, glucocorticoids; IL, interleukin; SCFA, short-chain fatty acids; TNF-α, tumor necrosis factor-alpha.

Figure 7

Sodium butyrate alleviates the progress of GA-ONFH and regulates the expression of cytokines in vivo. (A) Representative images of HE (scale bars, 200 μm), masson staining (scale bars, 200 μm) and immunohistochemistry (scale bars, 100 μm). Black arrow showed discontinuity of the epiphyseal plate in Model group. (B to F) Quantification of empty lacuna rate (B), Subchondral trabeculae area (C), immunohistochemical evaluation of TNF-α (D), IL-2 (E), and IL-4 (F). ⁣^∗p < 0.05.GA-ONFH, glucocorticoid-associated osteonecrosis of the femoral head; IL, interleukin; TNF-α, tumor necrosis factor-alpha.