Effect of Rhizoma Drynariae on osteoclast differentiation is related to the concentration of drug-containing serum

doi:10.3969/j.issn.2095-4344.2777

Abstract

Abstract:

BACKGROUND: Total flavonoids of Rhizoma Drynariae can be used to prevent and treat osteoporosis, but its mechanism of action is mostly focused on osteoblasts rather than osteoclast differentiation.

OBJECTIVE: To investigate the effect of Rhizoma Drynariae on osteoclast differentiation through T-cell nuclear factor 1 (NFATc1) signaling pathway.

METHODS: Forty Sprague-Dawley rats were randomly divided into four groups. Total flavonoids of Rhizoma Drynariae were intragastrically administered at 0, 11.6, 34.8 and 104.4 g/kg, respectively. Control serum and low-, middle-, high-concentration drug-containing sera were obtained. Bone marrow macrophages were isolated from 5-week-old rats and the effects of different drug-containing sera on macrophage activity were detected by cell counting kit-8 method. The macrophages were divided into low-, middle- and high-concentration groups, negative control group and blank group, with 5 multiple holes in each well. The low, middle and high concentration of drug-containing serum, negative control serum culture medium and normal culture medium were added respectively. All culture media were used to induce osteoclast differentiation with 20 μg/L macrophage colony-stimulating factor and 100 μg/L receptor activator of nuclear factor kappa B ligand. The number of osteoclasts and fusion index were detected by tartrate-resistant acid phosphatase staining 7 days after induction of differentiation. The expressions of c-Fos, Fra-1, Fra-2, NFATc1 and cathepsin K were detected by real-time fluorescence quantitative PCR and western blot at 7 days after differentiation. The number of bone resorption lacunae and the proportion of lacunae area in osteoclasts after 14 days of differentiation were detected by bone fragment absorption lacunae test. The study protocol was approved by the Animal Experiment Ethics Committee of Anyang District Hospital of Puyang City with approval No. PYSAYDQ-2019096.

RESULTS AND CONCLUSION: Cell counting kit-8 results showed that there was no significant change in macrophage activity after intervention with different concentration of sera containing Rhizoma Drynariae. The morphology of macrophages was regular before differentiation. After differentiation, osteoclasts were identified by tartrate-resistant acid phosphatase staining. Compared with the blank group and negative control group, the number of osteoclasts, fusion index, number of bone resorption lacunae and proportion of lacunae area and the relative expressions of c-Fos, Fra-1, Fra-2, NFATc1, cathepsin K mRNA and protein in the low-, middle- and high-concentration groups decreased significantly (P < 0.05). The above indicators showed a decreasing trend with the increase of serum concentration of Rhizoma Drynariae: low-concentration group > middle-concentration group > high-concentration group, and there were significant differences between different concentration groups (P < 0.05). In conclusion, Rhizoma Drynariae may inhibit the differentiation of rat macrophages into osteoclasts through NFATc1 signaling pathway, and the degree of differentiation inhibition is related to the serum concentration of Rhizoma Drynariae.

Key words: Rhizoma Drynariae, nuclear factor-activated T cell 1, osteoclast, differentiation, inhibition

CLC Number:

Xu Jinsong, Deng Na, Zhang Xiao. Effect of Rhizoma Drynariae on osteoclast differentiation is related to the concentration of drug-containing serum[J]. Chinese Journal of Tissue Engineering Research, 2020, 24(29): 4620-4625.

Figures/Tables 9

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