Oridonin promotes osteogenesis and suppresses osteoclastogenesis in vitro.

doi:10.3877/cma.j.issn.2095-9605.2020.02.005

Abstract

Abstract:

Objective

To study the effect and molecular mechanism of oridonin on osteoblast differentiation and osteoclast formation.

Methods

BMSCs were isolated from the femur and tibia of SD rats and cultured in α-MEM containing 10% fetal bovine serum; after 80%~90% of the cells confluent, cells were treated with oridonin in different concentrations. CCK-8 and live/dead staining were used to detect the viability of BMSCs and BMMs. ALP straining, alizarin red S staining, ALP activity and TRAP staining were used to detect the effects on osteogenesis and osteoclast differentiation. Related gene expressions (wnt1、β-catenin、Runx2、ALP、col-1、OCN、TRAP、c-Fos、NFATc1 and CTSX) were analyzed by qRT-PCR. Related protein expressions (wnt1、β-catenin、ALP、col-1、OCN、Runx2、RANKL、OPG、GSK-3β、p-GSK-3β) were measured by Western-blot and immunofluorescence.

Results

(1) Compared with the control group, oridonin (0-2 μM) can promote ALP activity and stimulate the differentiation of BMSCs into osteoblasts. (2) Oridonin can stimulate the expression of wnt1, β-catenin, Runx2. The expressions of osteogenic differentiation gene decreased following with adding Wnt/β-catenin/TCF selective antagonist ICG-001. (3) Oridonin can promote the expression of OPG and inhibit expression of RANKL. (4) Oridonin can directly/indirectly inhibit the expression of osteoclast-related genes TRAP, NFATc1 and c-Fos.

Conclusions

Oridonin may promote BMSCs differentiate into osteoblasts through the Wnt/β-catenin signaling pathway. At the same time, it may also inhibit the formation of osteoclasts mediated by nuclear factor-κB receptor activator (NF-κB) ligand (RANKL).

Key words: Oridonin, Mesenchymal stem cells, Osteoporosis, Wnt, β-cantenin, Receptor Activator of Nuclear Factor-κB Ligand, Osteoprotegerin

Feng Kuai, Liang Zhou. Oridonin promotes osteogenesis and suppresses osteoclastogenesis in vitro.[J]. Chinese Journal of Obesity and Metabolic Diseases(Electronic Edition), 2020, 06(02): 96-106.

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References 33

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基因	序列	?
Runx2	forward	5’-GTTCCCAGGCATTTCATCCC-3’
Runx2	reverse	5’-AAGGTGGCTGGATAGTGCAT-3
ALP	forward	5′-CAAGGATGCTGGGAAGTCCG-3’
ALP	reverse	5′-CTCTGGGCGCATCTCATTGT-3’
Col-I	forward	5′-TAA AGGGTCATCGTGGCTTC-3’
Col-I	reverse	5′-ACTCTCCGCTCT TCCAGTCA-3’
OCN	forward	5’-CTTGAAGACCGCCTACAAAC-3’
OCN	reverse	5’-GCTGCTGTGACATCCATAC-3’;
CTSK	forward	5’-TAATGTGAACCATGCCGTGT-3’;
CTSK	reverse	5’-CGAGCCAAGAGAACATAGCC-3’;
TRAP	forward	5’-TCCTGGCTCAAAAAGCAGTT-3’;
TRAP	reverse	5’-ACATAGCCACACCGTTCTC-3’;
c-Fos	forward	5’-CCAGTCAAGAGCATCAGCAA-3’;
c-Fos	reverse	5’-AAGTAGTGCAGCCCGGAGTA-3’;
NFATc1	forward	5’-GGGTCAGTGTGACCGAAGAT-3’;
NFATc1	reverse	5’-GGAAGTCAGAAGTGGGTGGA-3’;
GAPDH	forward	5’-ACCCAGAAGACTGTGGATGG-3’;
GAPDH	reverse	5’-CACATTGGGGGTAGGAACAC-3’;

基因	序列	?
Runx2	forward	5’-GTTCCCAGGCATTTCATCCC-3’
Runx2	reverse	5’-AAGGTGGCTGGATAGTGCAT-3
ALP	forward	5′-CAAGGATGCTGGGAAGTCCG-3’
ALP	reverse	5′-CTCTGGGCGCATCTCATTGT-3’
Col-I	forward	5′-TAA AGGGTCATCGTGGCTTC-3’
Col-I	reverse	5′-ACTCTCCGCTCT TCCAGTCA-3’
OCN	forward	5’-CTTGAAGACCGCCTACAAAC-3’
OCN	reverse	5’-GCTGCTGTGACATCCATAC-3’;
CTSK	forward	5’-TAATGTGAACCATGCCGTGT-3’;
CTSK	reverse	5’-CGAGCCAAGAGAACATAGCC-3’;
TRAP	forward	5’-TCCTGGCTCAAAAAGCAGTT-3’;
TRAP	reverse	5’-ACATAGCCACACCGTTCTC-3’;
c-Fos	forward	5’-CCAGTCAAGAGCATCAGCAA-3’;
c-Fos	reverse	5’-AAGTAGTGCAGCCCGGAGTA-3’;
NFATc1	forward	5’-GGGTCAGTGTGACCGAAGAT-3’;
NFATc1	reverse	5’-GGAAGTCAGAAGTGGGTGGA-3’;
GAPDH	forward	5’-ACCCAGAAGACTGTGGATGG-3’;
GAPDH	reverse	5’-CACATTGGGGGTAGGAACAC-3’;

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