基于RANKL/RANK/OPG轴的绝经后骨质疏松症发病机制及健骨颗粒干预研究
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摘要
一、目的：
阐明去卵巢大鼠骨质疏松症与RANKL/RANK/OPG系统的关系,以及健骨颗粒干预对该系统及破骨细胞RANK信号通路关键节点的调节作用,从破骨细胞的分化和功能调节的角度揭示健骨颗粒防治绝经后骨质疏松症的作用机制。
二、方法：
1去卵巢骨质疏松大鼠OPG/RANKL表达的动态变化及健骨颗粒的干预作用。
1.1动物实验：6月龄雌性SD大鼠160只,随机分成假手术组、去卵巢模型组、去卵巢+雌激素组和去卵巢+健骨颗粒组各40只。各组行双侧卵巢结扎切除术,假手术组除未行卵巢结扎切除外,其余步骤同模型组。术后3天健骨颗粒组给予健骨颗粒2g·kg-1·d-,雌二醇组给予17β--雌二醇100μg·kg-1·d-1,假手术组和模型组以生理盐水灌胃。术后2、6、12、24周每组分别处死10只,取血清、股骨、胫骨和腰椎骨备用。
1.2指标检测：双能X射线检测骨密度；三点弯曲法行股骨生物力学检测；胫骨上段制作石蜡切片,MASSON染色和TRAP染色后进行形态学分析骨小梁面积和破骨细胞量;ELISA检测血清中的TRAP、BALP、OC、RANKL和OPG含量；腰椎骨分别提取组织RNA和蛋白,实时荧光定量PCR法检测RANKL/OPG的mMRNA表达,Western Blot法检测RANKL/OPG蛋白的表达。
2健骨颗粒对破骨细胞分化和RANK信号通路的影响
2.1含药血清制备：取3月龄雄性SD大鼠20只,分为中药组和对照组各10只,中药组给予健骨颗粒2g·kg-1·d-1,对照组给予生理盐水2m1·d-,连续灌胃7天,于最后一次灌胃后1h腹主动脉无菌取血,凝固后离心取血清备用。
2.2细胞培养和指标检测：破骨细胞前体细胞株RAW264.7分为阴性对照组、空白血清组和含药血清组。阴性对照组不加RANKL,空白血清组和含药血清组在50ng/ml RANKL诱导下,分别加入10%空白血清和健骨颗粒含药血清培养。干预6天后行TRAP染色,观察计算破骨细胞分化情况；干预24、48、96小时后分别提取细胞RNA蛋白,实时荧光定量PCR法和Western Blot法检测RANK信号通路受体、TRAF6、NF-κB(P50和P52亚基)、c-Fos和NFATcl的mRNA及蛋白的表达。
三、结果：
1去卵巢术后12周模型组与假手术对照组相比,骨密度和骨生物力学指标持续下降,造模成功。去卵巢术后2-12周模型组破骨细胞增加,血清TRAP和血清BALP, OC等骨重建指标均明显升高,补充雌激素可逆转去卵巢导致的骨密度和骨生物力学指标下降,以及骨重建指标变化。与假手术组相比,模型组术后2周骨RANKL表达升高而血清RANKL下降,术后6-24周与假手术组相比均无显著差别。术后2周模型组骨组织OPG明显下降,随后逐渐升高,但与同期假手术对照组相比,模型组骨OPG始终较低；血清OPG在术后6-12周明显低于假手术组。补充雌激素可缓解去卵巢后骨组织和血清RANKL/OPG的变化。相关性分析表明骨RANKL与破骨细胞骨吸收呈正相关,但与血清RANKL无相关性；骨OPG与骨重建指标负相关,与血清OPG表达水平呈正相关。
2健骨颗粒干预可明显增加去卵巢大鼠的骨密度,提高骨生物力学性能,降低去卵巢大鼠骨组织破骨细胞量和血清TRAP、BALP和0C等骨重建指标表达。健骨颗粒可降低去卵巢大鼠骨组织RANKL的表达,提高去卵巢大鼠骨组织和血清OPG的表达。
3RANKL刺激增加破骨前体细胞RANK受体及其信号通路下游转录因子NF-κB (P50)、c-Fos和NFATcl的mRNA和蛋白表达,从而诱导破骨细胞分化。健骨颗粒含药血清能抑制RANKL诱导的体外破骨细胞分化,降低RANK通路受体、c-Fos、NFATcl的mRNA和蛋白表达,但对NF-KB和TRAF6的表达无明显作用。
四、结论：
1RANKL和OPG表达水平均随大鼠去卵巢术后时间呈动态非线性变化。血清OPG水平可作为绝经后骨质疏松症预测和防治疗效观察的参考指标。
2健骨颗粒能缓解去卵巢后骨组织RANKL和OPG表达的异常变化,降低骨重建水平,达到防治绝经后骨质疏松症的目的。
3健骨颗粒可通过下调RANK受体及其信号通路下游转录因子c-Fos和NFATcl的表达,抑制破骨细胞的分化,降低骨吸收水平,从而防治骨质疏松症。
Objective
To clarify the relationships between osteoporosis of ovariectomized rats and OPG/RANK/RANKL system, and reveal the mechanism of Jiangu Granule preventing postmenopausal osteoporosis from the aspect of osteoclast differentiation and function.
Methods
1Dynamic changes of OPG/RANKL expression in ovariectomy-induced osteoporotic rats and effects of Jiangu Granule treatment.
1.1Animal experiments:160female SD rats of6months old were randomly divided into4groups:sham operated, Ovariectomized, ovariectomized+estrogen and OVX+Jiangu Granule,40each group. Each group underwent bilateral ovariectomy exepted for the sham group, which were subjeted to sham surgery.3days after operation, OVX+Jiangu Granule group were given Jiangu Granule2g·kg-1·d-1, estradiol17p-estradiol group received100μg·kg-1·d-1, sham operation group and model group were given saline gavage. After2,6,12and24weeks,10rats of each group were sacrificed, the serum, femur, tibia and lumbar spine bone were harvested.
1.2Detection of indexes:bone density was measured by dual-energy X-rays; three-point bending method were used in femur biomechanical testing; tibia were made paraffin setions, trabecular area and the amount of osteoclasts were measured after MAS SON staining and TRAP staining; serum TRAP, BALP, OC, RANKL and OPG levels were detected by ELISA; lumbar were extracted mRNA and protein, RANKL/OPG mRNA expression was detected by real-time PCR, and the protein was detectd by Western Blot assay.
2The effect of Jiangu Granule on osteoclast differentiation and RANK signaling. pathway
2.1Preparation of serum-containing drugs:20male SD rats of3months old were divided into Jiangu Granule group and control group,10each. Jiangu Granule group received medicine of2g·kg-1·d-1, the control group received saline2ml·d-1for7days.1h after the last gavage, abdominal aortic blood was harvested and centrifugated after solidification to obtain the serum.
2.2Cell culture and index detection:osteoclast precursor cell line RAW264.7were cultured and separated into3groups:negative control group, blank serum and drug serum group. Negative control group without RANKL, blank serum and drug serum group containing with50ng/ml RANKL, and10%blank serum or drug serum. After6days culture, the cells were TRAP stained to calculate differentiated osteoclasts;24,48and96hours after the treatment, cells were extracted RNA and proteins, real-time quantitative PCR and Western Blot assay were applied to detect mRNA and protein expression of RANK and TRAF6, NF-κB (P50and P52subunits), c-Fos and NFATc1.
Results
1Compared with the sham-operated group, bone mass and biomechanical indicators of model group continued to decline, which indicates the success of osteoporosis model.2-12weeks post operation, bone osteoclasts, indicators of bone remodeling including serum TRAP, serum BALP and OC of the model group were significantly increased, and estrogen replacement therapy can reverse the decline of bone density and the increase of bone biomechanical indicators induced by ovariectomy. Compared with the sham operated group, bone RANKL in model group increased at2week post OVX, after which they fell to near the level of the sham group. OPG of bone model group decreased sharply at2week post OVX, and subsequently increased, but compared with the same period of sham control group, bone OPG in model group consistently lower; Serum OPG in OVX group was significantly lower than that in the sham group during6-12week post OVX. Estrogen can relieve bone and serum RANKL/OPG changes after ovariectomy. Correlation analysis showed a positive correlation between bone RANKL and osteoclast resorption indicators, but no correlation in RANKL levels between bone and serum. There was a positive correlation in OPG levels between bone and serum, while a negative correlation between bone OPG level and bone resorption markers.
2Jiangu granule treatment can significantly increase bone density and bone strength in ovariectomized rats, and reduce osteoclasts volume and indicators of bone remodeling including serum TRAP, BALP and OC. Jiangu Granule can reduce bone RANKL expression, and increase OPG expression in bone and serum of ovariectomized rats.
3RANKL stimulation increased mRNA and protein expression of NF-κB (P50), c-Fos and NFATc1, thus induced osteoclast differentiation; Jiangu Granule containing serum can reduce the rate of osteoclast differentiation induced by RANKL, and reduce mRNA and protein expression of RANK, c-Fos and NFATc1.
Conclusions
1RANKL and OPG showed nonlinear variations with time post ovariectomy in rats. Serum OPG level can be used as an indicator of postmenopausal osteoporosis disease.
2Jiangu Granule may regulate the expression of RANKL and OPG to affect bone remodeling, so as to prevent and treat osteoporosis induced by ovariectomy.
3Jiangu Granule may supress osteoclast differentiation in vitro by regulate RANK, c-Fos and NFATc1expression.
阐明去卵巢大鼠骨质疏松症与RANKL/RANK/OPG系统的关系,以及健骨颗粒干预对该系统及破骨细胞RANK信号通路关键节点的调节作用,从破骨细胞的分化和功能调节的角度揭示健骨颗粒防治绝经后骨质疏松症的作用机制。
二、方法：
1去卵巢骨质疏松大鼠OPG/RANKL表达的动态变化及健骨颗粒的干预作用。
1.1动物实验：6月龄雌性SD大鼠160只,随机分成假手术组、去卵巢模型组、去卵巢+雌激素组和去卵巢+健骨颗粒组各40只。各组行双侧卵巢结扎切除术,假手术组除未行卵巢结扎切除外,其余步骤同模型组。术后3天健骨颗粒组给予健骨颗粒2g·kg-1·d-,雌二醇组给予17β--雌二醇100μg·kg-1·d-1,假手术组和模型组以生理盐水灌胃。术后2、6、12、24周每组分别处死10只,取血清、股骨、胫骨和腰椎骨备用。
1.2指标检测：双能X射线检测骨密度；三点弯曲法行股骨生物力学检测；胫骨上段制作石蜡切片,MASSON染色和TRAP染色后进行形态学分析骨小梁面积和破骨细胞量;ELISA检测血清中的TRAP、BALP、OC、RANKL和OPG含量；腰椎骨分别提取组织RNA和蛋白,实时荧光定量PCR法检测RANKL/OPG的mMRNA表达,Western Blot法检测RANKL/OPG蛋白的表达。
2健骨颗粒对破骨细胞分化和RANK信号通路的影响
2.1含药血清制备：取3月龄雄性SD大鼠20只,分为中药组和对照组各10只,中药组给予健骨颗粒2g·kg-1·d-1,对照组给予生理盐水2m1·d-,连续灌胃7天,于最后一次灌胃后1h腹主动脉无菌取血,凝固后离心取血清备用。
2.2细胞培养和指标检测：破骨细胞前体细胞株RAW264.7分为阴性对照组、空白血清组和含药血清组。阴性对照组不加RANKL,空白血清组和含药血清组在50ng/ml RANKL诱导下,分别加入10%空白血清和健骨颗粒含药血清培养。干预6天后行TRAP染色,观察计算破骨细胞分化情况；干预24、48、96小时后分别提取细胞RNA蛋白,实时荧光定量PCR法和Western Blot法检测RANK信号通路受体、TRAF6、NF-κB(P50和P52亚基)、c-Fos和NFATcl的mRNA及蛋白的表达。
三、结果：
1去卵巢术后12周模型组与假手术对照组相比,骨密度和骨生物力学指标持续下降,造模成功。去卵巢术后2-12周模型组破骨细胞增加,血清TRAP和血清BALP, OC等骨重建指标均明显升高,补充雌激素可逆转去卵巢导致的骨密度和骨生物力学指标下降,以及骨重建指标变化。与假手术组相比,模型组术后2周骨RANKL表达升高而血清RANKL下降,术后6-24周与假手术组相比均无显著差别。术后2周模型组骨组织OPG明显下降,随后逐渐升高,但与同期假手术对照组相比,模型组骨OPG始终较低；血清OPG在术后6-12周明显低于假手术组。补充雌激素可缓解去卵巢后骨组织和血清RANKL/OPG的变化。相关性分析表明骨RANKL与破骨细胞骨吸收呈正相关,但与血清RANKL无相关性；骨OPG与骨重建指标负相关,与血清OPG表达水平呈正相关。
2健骨颗粒干预可明显增加去卵巢大鼠的骨密度,提高骨生物力学性能,降低去卵巢大鼠骨组织破骨细胞量和血清TRAP、BALP和0C等骨重建指标表达。健骨颗粒可降低去卵巢大鼠骨组织RANKL的表达,提高去卵巢大鼠骨组织和血清OPG的表达。
3RANKL刺激增加破骨前体细胞RANK受体及其信号通路下游转录因子NF-κB (P50)、c-Fos和NFATcl的mRNA和蛋白表达,从而诱导破骨细胞分化。健骨颗粒含药血清能抑制RANKL诱导的体外破骨细胞分化,降低RANK通路受体、c-Fos、NFATcl的mRNA和蛋白表达,但对NF-KB和TRAF6的表达无明显作用。
四、结论：
1RANKL和OPG表达水平均随大鼠去卵巢术后时间呈动态非线性变化。血清OPG水平可作为绝经后骨质疏松症预测和防治疗效观察的参考指标。
2健骨颗粒能缓解去卵巢后骨组织RANKL和OPG表达的异常变化,降低骨重建水平,达到防治绝经后骨质疏松症的目的。
3健骨颗粒可通过下调RANK受体及其信号通路下游转录因子c-Fos和NFATcl的表达,抑制破骨细胞的分化,降低骨吸收水平,从而防治骨质疏松症。
Objective
To clarify the relationships between osteoporosis of ovariectomized rats and OPG/RANK/RANKL system, and reveal the mechanism of Jiangu Granule preventing postmenopausal osteoporosis from the aspect of osteoclast differentiation and function.
Methods
1Dynamic changes of OPG/RANKL expression in ovariectomy-induced osteoporotic rats and effects of Jiangu Granule treatment.
1.1Animal experiments:160female SD rats of6months old were randomly divided into4groups:sham operated, Ovariectomized, ovariectomized+estrogen and OVX+Jiangu Granule,40each group. Each group underwent bilateral ovariectomy exepted for the sham group, which were subjeted to sham surgery.3days after operation, OVX+Jiangu Granule group were given Jiangu Granule2g·kg-1·d-1, estradiol17p-estradiol group received100μg·kg-1·d-1, sham operation group and model group were given saline gavage. After2,6,12and24weeks,10rats of each group were sacrificed, the serum, femur, tibia and lumbar spine bone were harvested.
1.2Detection of indexes:bone density was measured by dual-energy X-rays; three-point bending method were used in femur biomechanical testing; tibia were made paraffin setions, trabecular area and the amount of osteoclasts were measured after MAS SON staining and TRAP staining; serum TRAP, BALP, OC, RANKL and OPG levels were detected by ELISA; lumbar were extracted mRNA and protein, RANKL/OPG mRNA expression was detected by real-time PCR, and the protein was detectd by Western Blot assay.
2The effect of Jiangu Granule on osteoclast differentiation and RANK signaling. pathway
2.1Preparation of serum-containing drugs:20male SD rats of3months old were divided into Jiangu Granule group and control group,10each. Jiangu Granule group received medicine of2g·kg-1·d-1, the control group received saline2ml·d-1for7days.1h after the last gavage, abdominal aortic blood was harvested and centrifugated after solidification to obtain the serum.
2.2Cell culture and index detection:osteoclast precursor cell line RAW264.7were cultured and separated into3groups:negative control group, blank serum and drug serum group. Negative control group without RANKL, blank serum and drug serum group containing with50ng/ml RANKL, and10%blank serum or drug serum. After6days culture, the cells were TRAP stained to calculate differentiated osteoclasts;24,48and96hours after the treatment, cells were extracted RNA and proteins, real-time quantitative PCR and Western Blot assay were applied to detect mRNA and protein expression of RANK and TRAF6, NF-κB (P50and P52subunits), c-Fos and NFATc1.
Results
1Compared with the sham-operated group, bone mass and biomechanical indicators of model group continued to decline, which indicates the success of osteoporosis model.2-12weeks post operation, bone osteoclasts, indicators of bone remodeling including serum TRAP, serum BALP and OC of the model group were significantly increased, and estrogen replacement therapy can reverse the decline of bone density and the increase of bone biomechanical indicators induced by ovariectomy. Compared with the sham operated group, bone RANKL in model group increased at2week post OVX, after which they fell to near the level of the sham group. OPG of bone model group decreased sharply at2week post OVX, and subsequently increased, but compared with the same period of sham control group, bone OPG in model group consistently lower; Serum OPG in OVX group was significantly lower than that in the sham group during6-12week post OVX. Estrogen can relieve bone and serum RANKL/OPG changes after ovariectomy. Correlation analysis showed a positive correlation between bone RANKL and osteoclast resorption indicators, but no correlation in RANKL levels between bone and serum. There was a positive correlation in OPG levels between bone and serum, while a negative correlation between bone OPG level and bone resorption markers.
2Jiangu granule treatment can significantly increase bone density and bone strength in ovariectomized rats, and reduce osteoclasts volume and indicators of bone remodeling including serum TRAP, BALP and OC. Jiangu Granule can reduce bone RANKL expression, and increase OPG expression in bone and serum of ovariectomized rats.
3RANKL stimulation increased mRNA and protein expression of NF-κB (P50), c-Fos and NFATc1, thus induced osteoclast differentiation; Jiangu Granule containing serum can reduce the rate of osteoclast differentiation induced by RANKL, and reduce mRNA and protein expression of RANK, c-Fos and NFATc1.
Conclusions
1RANKL and OPG showed nonlinear variations with time post ovariectomy in rats. Serum OPG level can be used as an indicator of postmenopausal osteoporosis disease.
2Jiangu Granule may regulate the expression of RANKL and OPG to affect bone remodeling, so as to prevent and treat osteoporosis induced by ovariectomy.
3Jiangu Granule may supress osteoclast differentiation in vitro by regulate RANK, c-Fos and NFATc1expression.
引文
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