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. 2024 Jan 10;13(1):28-39.
doi: 10.1302/2046-3758.131.BJR-2023-0040.R3.

Sex differences of NF-κB-targeted therapy for mitigating osteoporosis associated with chronic inflammation of bone

Masakazu Toya  1 , Junichi Kushioka  1 , Huaishuang Shen  1 , Takeshi Utsunomiya  1   2 , Hirohito Hirata  1 , Masanori Tsubosaka  1 , Qi Gao  1 , Simon K-H Chow  1 , Ning Zhang  1 , Stuart B Goodman  1   3
Affiliations

Sex differences of NF-κB-targeted therapy for mitigating osteoporosis associated with chronic inflammation of bone

Masakazu Toya et al. Bone Joint Res. .

Abstract

Aims: Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice.

Methods: We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR).

Results: Local delivery of NF-κB decoy ODN in vivo increased osteogenesis in males, but not females, in the presence of chronic inflammation induced by cPE. Bone resorption activity was decreased in both sexes. In vitro osteogenic and osteoclastic differentiation assays during inflammatory conditions did not reveal differences among the groups. Receptor activator of nuclear factor kappa Β ligand (Rankl) gene expression by osteoblasts was significantly decreased only in males when treated with ODN.

Conclusion: We demonstrated that NF-κB decoy ODN increased osteogenesis in male mice and decreased bone resorption activity in both sexes in preclinical models of chronic inflammation. NF-κB signalling could be a therapeutic target for chronic inflammatory diseases involving bone, especially in males.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1
In vivo experimental design. The images of the murine model of continuous chronic inflammation. The hollow rod was press-fit into the distal femur canal. Timetable of the experimental design is shown. cPE, contaminated polyethylene particles; ODN, oligodeoxynucleotides; ROI, region of interest.
Fig. 2
Fig. 2
Immunofluorescence for macrophages in vivo. Representative images of immunofluorescence for macrophages are shown (scale bar = 100 μm). Quantitative assessments of the M1 positive (iNOS positive) cells and the M2 positive (Arg1 positive) cells were performed, on male (control group: n = 4; oligodeoxynucleotide (ODN) group: n = 5) and female mice (control group: n = 6; ODN group: n = 6). *p < 0.05, **p < 0.01, Mann-Whitney U test. Arg1, Arginase 1; cPE, contaminated polyethylene particles; DAPI, 4′,6-diamidino-2-phenylindole; iNOS, inducible nitric oxide synthase.
Fig. 3
Fig. 3
Tartrate-resistant acid phosphatase (TRAP) staining in vivo. Representative images of TRAP staining and quantitative analysis of TRAP-positive cells (white arrows) are shown (scale bar = 100 μm) for male (control group: n = 7; oligodeoxynucleotide (ODN) group: n = 9) and female mice (control group: n = 5; ODN group: n = 6). *p < 0.05, ***p < 0.001, Mann-Whitney U test. cPE, contaminated polyethylene particles.
Fig. 4
Fig. 4
Alkaline phosphatase (ALP) staining and micro-CT analysis in vivo. Representative images of ALP staining and quantitative analysis of ALP positive area proportion are shown (scale bar = 100 μm) for male (control group: n = 7, oligodeoxynucleotide (ODN) group: n = 9) and female mice (control group: n = 5, ODN group: n = 6). Quantitative assessments of bone mineral density (BMD) are also shown for male (control group: n = 8, ODN group: n = 7) and female mice (control group: n = 6, ODN group: n = 5). *p < 0.05, Mann-Whitney U test. cPE, contaminated polyethylene particles; ns, non-significant.
Fig. 5
Fig. 5
In vitro osteogenic differentiation assay pre-treated with contaminated polyethylene particles (cPE). Quantitative analysis of alkaline phosphatase (ALP) and Alizarin Red positive area proportion (%/well) and representative whole-well images of each group are shown (n = 5, each group). The comparison of ALP and Alizarin Red positive area proportion (%/well) between male and female cells is also shown (n = 5, each group). *p < 0.05, **p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons. cPE, contaminated polyethylene particles; ns, non-significant; ODN, oligodeoxynucleotides.
Fig. 6
Fig. 6
Gene expressions during the osteogenic differentiation assay pre-treated with contaminated polyethylene particles (cPE). The results of reverse transcription polymerase chain reaction (RT-PCR) on day 7 during the osteogenic differentiation assay are shown. The blue graph shows the results for males and the red for females. Values are shown as relative gene expression levels to the control group. The concentration of nuclear factor kappa B (NF-κB) decoy oligodeoxynucleotide (ODN) was 0.1 μM. n = 4, each group. *p < 0.05, Kruskal-Wallis test with Dunn’s multiple comparisons. Bglap2, bone gamma-carboxyglutamate protein 2; Col1α1, collagen type 1 alpha 1; Opn, osteopontin; Rankl, receptor activator of nuclear factor kappa-B ligand; Runx2, Runt-related transcription factor 2.
Fig. 7
Fig. 7
In vitro osteoclastic differentiation assay pre-treated with contaminated polyethylene particles (cPE). Quantitative analysis of tartrate-resistant acid phosphatase (TRAP) staining in vitro is shown. TRAP-positive cells were evaluated within the range of 1 mm2 (n = 5, each group). The comparison of TRAP-positive cells between male and female cells is also shown. Representative images of TRAP-positive cells (red arrows) are shown (scale bar = 250 μm). *p < 0.05, **p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons. ns, non-significant; ODN, oligodeoxynucleotides.
Fig. 8
Fig. 8
In vitro osteogenic and osteoclastic differentiation assay pre-treated with nuclear factor kappa B (NF-κB) decoy oligodeoxynucleotide (ODN). Quantitative analysis of alkaline phosphatase (ALP), Alizarin Red, and tartrate-resistant acid phosphatase (TRAP) staining pre-treated with NF-κB decoy ODN are shown (n = 5, each group). Quantitative analysis of TRAP staining between different pre-treatment groups is also shown (n = 5, each group). *p < 0.05; **p < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons. cPE, contaminated polyethylene particles; ns, non-significant.
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References

    1. Zhou Y, Li J, Xu F, Ji E, Wang C, Pan Z. Long noncoding RNA H19 alleviates inflammation in osteoarthritis through interactions between TP53, IL-38, and IL-36 receptor. Bone Joint Res. 2022;11(8):594–607. doi: 10.1302/2046-3758.118.BJR-2021-0188.R1. - DOI - PMC - PubMed
    1. Liu YD, Liu JF, Liu B. N,N-Dimethylformamide inhibits high glucose-induced osteoporosis via attenuating MAPK and NF-κB signalling. Bone Joint Res. 2022;11(4):200–209. doi: 10.1302/2046-3758.114.BJR-2020-0308.R2. - DOI - PMC - PubMed
    1. Kim MH, Choi LY, Chung JY, Kim E-J, Yang WM. Auraptene ameliorates osteoporosis by inhibiting RANKL/NFATc1 pathway-mediated bone resorption based on network pharmacology and experimental evaluation. Bone Joint Res. 2022;11(5):304–316. doi: 10.1302/2046-3758.115.BJR-2021-0380.R1. - DOI - PMC - PubMed
    1. Mauro D, Thomas R, Guggino G, Lories R, Brown MA, Ciccia F. Ankylosing spondylitis: an autoimmune or autoinflammatory disease? Nat Rev Rheumatol. 2021;17(7):387–404. doi: 10.1038/s41584-021-00625-y. - DOI - PubMed
    1. Ji M, Ryu HJ, Hong JH. Signalling and putative therapeutic molecules on the regulation of synoviocyte signalling in rheumatoid arthritis. Bone Joint Res. 2021;10(4):285–297. doi: 10.1302/2046-3758.104.BJR-2020-0331.R1. - DOI - PMC - PubMed