冬凌甲草素在体外促进成骨分化和抑制破骨形成及其机制研究

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

目的

研究冬凌甲草素对成骨细胞分化和破骨细胞形成的作用及分子机制。

方法

从SD大鼠股骨和胫骨中分离出骨髓间充质干细胞和骨髓单核细胞，培养于含10%胎牛血清的α-MEM培养基，细胞80%~90%汇合后采用不同浓度的冬凌甲草素（0、1、2、3、4 μM）进行分组干预，CCK-8法及活死染色检测不同浓度的冬凌甲草素对间充质干细胞及骨髓单核细胞活力的影响；通过碱性磷酸酶（ALP）染色、茜素红染色、ALP活性的测定以及TRAP染色检测冬凌甲草素对成骨和破骨分化的影响；qRT-PCR检测wnt1、β-catenin、Runx2、ALP、collage-1(col-1)、骨钙蛋白(OCN)、TRAP、c-Fos、NFATc1和CTSK的表达；Western-blot及免疫荧光检测wnt1、β-catenin、ALP、col-1、OCN、Runx2、核因子κB受体活化因子配体（RANKL）、骨保护素（OPG）、GSK-3β、p-GSK-3β的表达。

结果

（1）与对照组比较，冬凌甲草素（2 μM）具有促进间充质干细胞向成骨细胞分化和提高ALP活性的作用；（2）冬凌甲草素可以促进wnt1、β-catenin、Runx2的表达，在加入Wnt/β-catenin/TCF通路选择性拮抗剂ICG-001后，成骨相关标志物表达下降。（3）冬凌甲草素可以促进OPG而抑制RANKL的表达。（4）冬凌甲草素可以直接或间接抑制破骨相关基因TRAP，NFATc1和c-Fos的表达。

结论

冬凌甲草素可能通过Wnt/β-catenin信号通路促进骨髓间充质干向成骨细胞分化，同时，它还可能抑制RANKL介导的破骨细胞的形成。

关键词: 冬凌甲草素, 间充质干细胞, 骨质疏松症, Wnt, β-连环蛋白, 核因子κB受体活化因子配体, 骨保护素

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

表1 qRT-PCR引物

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’;

表1 qRT-PCR引物

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’;

图1 高浓度的ORI抑制骨髓间充质干细胞的增殖。在测量细胞活力之前，用ORI干预原代骨髓间充质干细胞。CCK-8分析（1B）和活/死染色（1C）表明，在浓度大于2 μM的ORI处理72 h后，死亡的BMSC数量显着减少。细胞毒性（活/死）测定图像显示活细胞（绿色）和死细胞（红色）。

图2 ORI对BMSCs成骨分化的影响。以高糖培养基（HSM）作为对照组，成骨培养基（OM）和ORI培养骨髓间充质干细胞。7 d后使用碱性磷酸酶染色法进行测定。2B：在用HSM，OM和ORI培养7天的BMSC上测试了ALP活性。^*P<0.05和^**P<0.01 vs.对照组（n=3）。2C、2D：将骨髓间充质干细胞与HSM（对照）一起，在有或没有ORI的情况下培养14 d。通过茜素红S染色定量。^*P<0.05和#P<0.01 vs.对照组（n=3）。2E、2F、2G：在第2 d收获用OM（对照），ORI（0.5μM、1μM、2μM）培养的骨髓间充质干细胞。通过qRT-PCR评估ALP、col-1和OCN的mRNA表达水平并定量。^*P<0.05和^#P<0.01 vs.对照组。

图3 ORI通过Wnt/β-catenin信号通路促进骨髓间充质干细胞的成骨分化。3A：在第3 d收集分别用OM（对照），ORI（0.5μM、1μM、2μM）培养的骨髓间充质干细胞。通过Westernblot评估Wnt1、β-catenin、GSK-3β和p-GSK-3β的蛋白表达水平。^*P<0.05和^#P<0.01 vs.对照组。3B：测定Wnt1和β-连环蛋白的蛋白质表达水平。^*P<0.05和^#P<0.01 vs.对照组。3C：在48 h收集含OM（对照），有或没有ORI（2 μM）或ICG-001的骨髓间充质干细胞，测定ALP，col-1和OCN的蛋白表达。^*P<0.05和^#P<0.01 vs.对照组。3D：在48 h收集经OM培养的骨髓间充质干细胞（对照）、有或没有ORI或ICG-001组，测定ALP染色和茜素红染色。3E：在48 h收集用OM（对照），有或没有ORI培养的骨髓间充质干细胞。通过qRT-PCR评估并定量β-catenin和Runx2的mRNA表达水平。^*P<0.05和^#P<0.01 vs.对照组。3F：免疫荧光检测β-catenin蛋白的易位。用OM（对照）和ORI（2μM）干预骨髓间充质干细胞。β-catenin在细胞质和细胞核中均表达，并且荧光密度和强度在第5 d呈剂量依赖性增加。细胞核用DAPI染色并显示为蓝色荧光。比例尺=100 μM。3G、3H：在48 h收集用OM（对照），ORI处理的骨髓间充质干细胞。Westernblot评估RANKL和OPG的蛋白表达水平，^*P<0.05和^#P<0.01 vs.对照组。

图4 ORI直接抑制骨髓单核细胞的增殖和分化。4A、4B：ORI在48 h或96 h对骨髓单核细胞生存能力的影响。4C：活/死染色。4D、4E、4H：用M-CSF和RANKL以及不同浓度的ORI干预5 d产生的的TRAP阳性骨髓单核细胞。TRAP阳性多核细胞的定量和破骨细胞面积。^*P<0.05和^#P<0.01 vs.对照组。4F、4G：用ORI处理5 d的骨髓单核细胞中的NFATc1，c-Fos和TRAP表达并定量。^*P<0.05和^#P<0.01 vs.对照组。4I：ORI干预12 h、24 h或48 h的骨髓单核细胞中NFATc1和c-Fos表达水平。^*P<0.05和^#P<0.01 vs.对照组。

图5 ORI间接抑制骨髓单核细胞的分化。5A：用不同浓度的上清液干预5 d的骨髓单核细胞中NFATc1，c-Fos和TRAP的表达并定量。^*P<0.05和^#P<0.01 vs.对照组。5B、5C、5D：TRAP阳性骨髓单核细胞用不同浓度的上清液干预，然后用M-CSF和RANKL孵育5 d。定量TRAP阳性多核细胞和破骨细胞面积。

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Oridonin promotes osteogenesis and suppresses osteoclastogenesis in vitro.

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Oridonin promotes osteogenesis and suppresses osteoclastogenesis in vitro.