Lycium barbarum glycopeptide reduces bone loss caused by exosomes derived from human gingival fibroblasts with radiation exposure

doi:10.12122/j.issn.1673-4254.2024.09.15

Abstract

Abstract:

Objective To explore the protective effect of Lycium barbanun glycopeptide (LbGP) against osteogenic inhibition induced by exosomes derived from human gingival fibroblasts (HGFs) exposed to radiation. Methods Cultured HGFs with or without LbGP pretreatment were exposed to 8 Gy X-ray radiation, and the changes in cell apoptosis, senescence and α-SMA level were detected using RT-qPCR, Western blotting and β-galactosidase staining. The exosomes secreted by the treated cells were extracted, and after identification by electron microscopy, particle size analysis and Western blotting, the exosomes were added into primary cultured bone mesenchymal stem cells (BMSCs), and osteoclast activity and osteogenesis in the cell cultures were detected by Trap staining, Alizarin red staining, ALP staining, RT-qPCR and Western blotting. Results In cultured HGFs, X-ray radiation significantly increased the percentage of senescent cells, which was obviously lowered by LbGP treatment. X-ray radiation significantly reduced Bcl-2/Bax ratio and increased α-SMA expression in HGFs, and these changes were significantly suppressed by LbGP pretreatment. In rat BMSCs, incubation with the exosomes derived from HGFs with radiation exposure caused a significant increase of osteoclasts, reduced calcium nodules and lowered alkaline phosphatase expression in the cells; The opposite changes were observed in the cells treated with exosomes from LbGP-pretreated HGFs, which also significantly increased the cellular expressions of the osteogenic genes (BMP2, ALP, and RUNX2) and proteins (ALP and RUNX2) as compared with the exosomes from irradiated HGFs. Conclusion LbGP can effectively inhibit osteoclast activity and promote osteogenesis by acting on exosomes secreted by irradiated HGFs, suggesting its potential value for treatment of osteoradionecrosis of the jaw.

Key words: Lycium barbarum glycopeptide, exosomes, osteoradionecrosis of the jaw, osteogenic differentiation

Siqi HE, Nan WEN, Xun CHEN, Yue WANG, Tin ZHANG, Yandong MU. Lycium barbarum glycopeptide reduces bone loss caused by exosomes derived from human gingival fibroblasts with radiation exposure[J]. Journal of Southern Medical University, 2024, 44(9): 1752-1759.

Figures/Tables 6

Tab.1 Primer sequences

Genes

Sequence primers (5'→3')

GAPDH-F

GAPDH-R

BMP2-F

BMP2-R

ALP-F

ALP-R

RUNX2-R

RUNX2-F

CCTCGTCTCATAGACAAGATGGT

GGGTAGAGTCATACTGGAACATG

CTATATGCTCGACCTGTACCG

CACTCATTTCTGAAAGTTCCTCG

CAGAGAAAGAGAAAGACCCCAG

CTGTCACTGTGGAGACGC

AAGAGGGGTAAGACTGGTCATAGG

CAGTATGAGAGTAGGTGTCCCGC

Fig.1 Identification of human gingival fibroblasts (HGFs) and rat bone mesenchymal stem cells (BMSCs). A: Morphology of HGFs under optical microscope (a) and immunocytochemical staining showing positive expression of anti-vimentin (b) and negative expression of anti-ck8 (c) in HGFs. B: Morphology of BMSCs under optical microscopy (a) and identification of BMSCs surface markers by flow cytometry (b).

Fig.2 Characterization of HGFs with X-ray radiation and Lycium barbanun glycopeptide (LbGP) treatment. A: β-galactosidase staining. B: Relative protein expression of Bcl-2 and Bax in each group. C: Relative protein expression of α-SMA in each group. *P<0.05,**P<0.01, ****P<0.0001.

Fig.3 Identification of the exosomes derived from HGFs.A: Electron microscopy for observing structure of the exosomes. B: Exosome particle size analysis. C: Exosome concentration distribution map. D: Western blotting for detecting exosomes surface markers CD81 and Tsg101. E: Exosomes under confocal microscope.

Fig.4 Osteoblast and osteogenic staining results of rat BMSCs treated with exosomes from HGFs in different groups. A: Optical microscopy of the osteoclasts and Trap staining. B: Alizarin red staining. C: ALP staining.

Fig.5 Expression of osteogenesis-related factors in BMSCs treated with exosomes from HGFs in different groups. A: Relative mRNA expression of BMP2, ALP, and RUNX2 in BMSCs in each group. B: Relative protein expressions of RUNX2 and ALP in each group. *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.

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