The effect of AG490 on the crosstalk between osteoblasts and osteoclasts in vitro

doi:10.3877/cma.j.issn.1674-1366.2017.02.002

Abstract

Abstract:

Objective

To establish a co-culture system of MC3T3-E1 cells and RAW 264.7 cells in vitro, and then explore the effect of AG490 on osteoclastogenesis in this system.

Methods

The mineralized MC3T3-E1 cells and RAW 264.7 cells were co-cultured after the addition of different concentrations of AG490 (1 μmol/L, 5 μmol/L, 10 μmol/L) . The cell proliferation was analyzed by CCK-8 assay, ALP activity was analyzed by a simultaneous-coupling azo dye method, and osteoblast-related genes OPG, COL1, ALP, OCN were analyzed by RT-PCR in mineralized MC3T3-E1 cells. TRAP and hematoxylin staining were performed to observe the osteoclastogenesis, and Western blot was adopted to analyze the RANK expression in osteoclasts. Data were evaluated by One-Way ANOVA and Two-Way ANOVA.

Results

AG490 inhibited the ALP activity and the expression of osteoblast-related genes OPG, COL1, ALP, OCN in mineralized MC3T3-E1 cells (F_OPG= 30.120, P_{OPG, AG490 1 μmol/L}= 0.021, P_{OPG, AG490 5 μmol/L}= 0.221, P_{OPG, AG490 10 μmol/L}= 0.003; F_ALP= 67.352, P_{ALP, AG490 1 μmol/L}= 0.034, P_{ALP, AG490 5 μmol/L}= 0.001, P_{ALP, AG490 10 μmol/L}= 0.156; F_COL1= 28.158, P_{COL1, AG490 1 μmol/L}= 0.054, P_{COL1, AG490 5 μmol/L}= 0.002, P_{COL1, AG490 10 μmol/L}= 0.4; F_OCN= 125.375, P_{OCN, AG490 1 μmol/L}<0.001, P_{OCN, AG490 5 μmol/L}<0.001, P_{OCN, AG490 10 μmol/L}<0.001) . Compared with RAW264.7 cells alone group (control group) , the indirect co-culture of MC3T3-E1 cells and RAW264.7 cells successfully promoted osteoclast formation, which could be significantly inhibited by AG490 (F= 85.391, P_{co-culture-control}<0.001, P_{co-culture+AG490-co-culture}= 0.035) , meanwhile the addition of AG490 down-regulated the RANK in the osteoclasts in protein level (F_RANK= 376.25, P_{RANK, AG490 1 μmol/L}= 0.468, P_{RANK, AG490 5 μmol/L}=0.003, P_{RANK, AG490 10 μmol/L}<0.001) .

Conclusions

AG490 can inhibit osteoblastic differentiation in MC3T3-E1 cells, and might play an important role in osteoclast formation inhibition in co-culture of MC3T3-E1 cells and RAW 264.7 cells by blockade of OPG/RANKL/RANK signal axis.

Key words: Culture techniques, Receptor activator of nuclear factor-kappa B, AG490, Osteoblastic differentiation, Osteoclastic differentiation

Xin Cheng, Qilong Wan, Zubing Li. The effect of AG490 on the crosstalk between osteoblasts and osteoclasts in vitro[J]. Chinese Journal of Stomatological Research(Electronic Edition), 2017, 11(02): 73-80.

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URL: https://zhkqyxyjzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-1366.2017.02.002

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Figures/Tables 8

References 16

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基因	引物序列
OPG	F:5′-CTGGAGTGCACGATGCCAGCTA-3′
?	R:5′-TCCGTGCTCGGCGATGGACGTC-3′
RANKL	F:5′-GAGACAGACATCGGGAGGGAT-3′
?	R:5′-GTGGGATGTGAACTCGGAAGCT-3′
ALP	F:5′-ACGGAGGCATTGACTCTGAACG-3′
?	R:5′-GGAAGCACCAACGAGAGGAGCT-3′
COL1	F:5′-GAGATCGTGCTGAACGGTAATT-3′
?	R:5′-GATTGACATGCATGTTGAATGA-3′
OCN	F:5′-AGCGAAGACCTAGGAGACAAGT-3′
?	R:5′-TGAGGATCGTAACTCGCGAGAG-3′
GAPDH	F:5′-AAGGTCGGTGTGAACGGATTTA-3′
?	R:5′-TGTAGACCATGTAGTTGAGGTA-3′

基因	引物序列
OPG	F:5′-CTGGAGTGCACGATGCCAGCTA-3′
?	R:5′-TCCGTGCTCGGCGATGGACGTC-3′
RANKL	F:5′-GAGACAGACATCGGGAGGGAT-3′
?	R:5′-GTGGGATGTGAACTCGGAAGCT-3′
ALP	F:5′-ACGGAGGCATTGACTCTGAACG-3′
?	R:5′-GGAAGCACCAACGAGAGGAGCT-3′
COL1	F:5′-GAGATCGTGCTGAACGGTAATT-3′
?	R:5′-GATTGACATGCATGTTGAATGA-3′
OCN	F:5′-AGCGAAGACCTAGGAGACAAGT-3′
?	R:5′-TGAGGATCGTAACTCGCGAGAG-3′
GAPDH	F:5′-AAGGTCGGTGTGAACGGATTTA-3′
?	R:5′-TGTAGACCATGTAGTTGAGGTA-3′

AG490浓度(μmol/L)	基因相对表达量					F值					P值
AG490浓度(μmol/L)	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL
0	2.35	1.67	5.62	1.02	1.43	30.120	67.352	28.158	125.375	8.164	-	-	-	-	-
1	1.21	1.17	3.75	0.51	1.51	30.120	67.352	28.158	125.375	8.164	0.021	0.034	0.054	0.000	0.557
5	1.01	0.78	1.47	0.19	1.39	30.120	67.352	28.158	125.375	8.164	0.221	0.001	0.002	0.000	0.924
10	0.58	0.63	0.93	0.08	1.31	30.120	67.352	28.158	125.375	8.164	0.003	0.156	0.400	0.000	0.376

AG490浓度(μmol/L)	基因相对表达量					F值					P值
AG490浓度(μmol/L)	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL	OPG	ALP	COL1	OCN	RANKL
0	2.35	1.67	5.62	1.02	1.43	30.120	67.352	28.158	125.375	8.164	-	-	-	-	-
1	1.21	1.17	3.75	0.51	1.51	30.120	67.352	28.158	125.375	8.164	0.021	0.034	0.054	0.000	0.557
5	1.01	0.78	1.47	0.19	1.39	30.120	67.352	28.158	125.375	8.164	0.221	0.001	0.002	0.000	0.924
10	0.58	0.63	0.93	0.08	1.31	30.120	67.352	28.158	125.375	8.164	0.003	0.156	0.400	0.000	0.376

AG490浓度(μmol/L)	基因相对表达量		F值		P值
AG490浓度(μmol/L)	对照组	共培养组	对照组	共培养组	对照组	共培养组
0	3.27	3.18	376.25	376.25	-	0.723
1	3.25	2.94	376.25	376.25	-	0.468
5	3.39	2.16	376.25	376.25	-	0.003
10	3.13	0.12	376.25	376.25	-	0.000

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