Effects and mechanisms of 8-prenylnaringenin on osteoblast MC3T3-E1 and osteoclast-like cells RAW264.7 - PubMed (original) (raw)

Effects and mechanisms of 8-prenylnaringenin on osteoblast MC3T3-E1 and osteoclast-like cells RAW264.7

Dan Luo et al. Food Sci Nutr. 2014 Jul.

Abstract

8-Prenylnaringenin (8-PN) is a phytoestrogen with the highest estrogenic activity. The objective of the present study was to confirm the superiority of 8-PN on bone metabolisms and the estrogen receptor (ER) subtype mediating effects of 8-PN. The osteoblast MC3T3-E1 and osteoclast-like cell line RAW264.7 were treated with 17β-estradiol (10(-8) mol/L), genistein (10(-5) mol/L), daidzein (10(-5) mol/L), 8-PN (10(-5) mol/L) alone or in the presence of ERα antagonist MPP (10(-7) mol/L) and ERβ antagonist PTHPP (1.5 × 10(-7) mol/L). It has been found that 8-PN did not affect osteoblast proliferation, and that 8-PN increased alkaline phosphatase (ALP) activity, osteocalcin (OCN) concentrations, and the mineralized nodules. 8-PN inhibited RAW264.7 differentiating into osteoclasts and reduced the pit area of bone resorption. 8-PN could also inhibit the protein and mRNA expression of receptor activator of nuclear factor-κB ligand (RANKL) in osteoblasts, and conversely promote the expression of osteoprotegerin (OPG). These effects of 8-PN were mainly inhibited not by PTHPP but by MPP and they were weaker than estrogen's effects but stronger than those of genistein and daidzein. In conclusion, the effects of 8-PN on promoting osteoblastic bone formation and inhibiting osteoclastic bone resorption were mediated by ERα instead of ERβ and the efficacy was more potent than that of the two classic phytoestrogens: genistein and daidzein.

Keywords: 8-Prenylnaringenin; daidzein; estrogen receptor; genistein; osteoblast; osteoclast.

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Figures

Figure 1

Effects of 48 h treatment of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the number of MC3T3-E1 cells. Con, control group; ER, estrogen receptor.

Figure 2

Effects of 48 or 72 h treatment of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN,10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the alkaline (ALP) activity (U/L) of MC3T3-E1. *P < 0.05 and **P < 0.01 versus control group (Con), #P < 0.05 versus 8PN group. ER, estrogen receptor; ALP, alkaline phosphatase.

Figure 3

Effects of 48 or 72 h treatment of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the osteocalcin (OCN) concentration (ng/mL) of MC3T3-E1. *P < 0.05 versus control group (Con). #P < 0.05 versus 8PN group. ER, estrogen receptor.

Figure 4

(A) Effects of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on mineralized nodules of MC3T3-E1 demonstrated by alizarin red staining at day 28 (100×). (B) Different counts of calcified nodes among groups. **P < 0.01 versus control group (Con), #P < 0.05 versus 8PN group. ER, estrogen receptor.

Figure 5

Effects of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the number of osteoclast-like cells characterized via tartrate-resistant acid phosphatase (TRAP) staining from RAW264.7 induced by macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factor-κ B ligand (RANKL) for 6, 9, 12 days (×200). *P < 0.01 and **P < 0.01 vs. control group (Con), #P < 0.05 versus 8PN group. ER, estrogen receptor.

Figure 6

The characteristics of bone resorption pits on bone slices as shown by arrows (400×).

Figure 7

(A) Effects of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro- methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on bone resorption pits on bone slice observed under optical microscope (100×). (B) Different area of bone resorption pits calculated by the IPP software among groups. **P < 0.01 versus control group (Con), #P < 0.05 and ##P < 0.01 versus 8PN group. ER, estrogen receptor.

Figure 8

Effects of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the secretion of receptor activator for nuclear factor-κ B ligand (RANKL) protein in MC3T3-E1 cells. **P < 0.01 versus control group (Con), #P < 0.05 versus 8PN group. ER, estrogen receptor.

Figure 9

Effects of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the secretion of osteoprotegerin (OPG) protein in MC3T3-E1 cells. *P < 0.05 and **P < 0.01 versus control group (Con), #P < 0.05 versus 8PN group. ER, estrogen receptor.

Figure 10

Effects of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the relative value of secreting protein of receptor activator for nuclear factor-_κ_B ligand (RANKL) to osteoprotegerin (OPG) in MC3T3-E1 cells. *P < 0.05 and **P < 0.01 versus control group (Con), #P < 0.05 and ##P < 0.01 versus 8PN group. ER, estrogen receptor.

Figure 11

Effects of 17_β_-estradiol (E2, 10−8 mol/L), genistein (Gen, 10−5 mol/L), daidzein (Dai, 10−5 mol/L), and 8-prenylnaringenin (8PN, 10−5 mol/L) alone or supplemented with ER_α_ antagonist methyl-piperidino-pyrazole (MPP, 10−7 mol/L) or ER_β_ antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol (PTHPP, 1.5 × 10−7 mol/L) on the expression of receptor activator for nuclear factor-_κ_B ligand (RANKL) and osteoprotegerin (OPG) mRNA in MC3T3-E1 cells. ER, estrogen receptor.

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