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杨梅素减轻去卵巢大鼠骨质疏松
甘宁1,5,6, 郁雪松2,5, 陈军3, 叶国4,5,6    
1. 401147 重庆,重庆医科大学附属口腔医院牙体牙髓科
2. 401147 重庆,重庆医科大学附属口腔医院修复科
3. 401147 重庆,重庆医科大学附属口腔医院正畸科
4. 401147 重庆,重庆医科大学附属口腔医院牙周科
5. 401147 重庆,口腔疾病与生物医学重庆市重点实验室
6. 401147 重庆,重庆市高校市级口腔生物医学工程重点实验室
摘要目的 观察杨梅素对去卵巢大鼠骨量的影响,初步探讨机制。 方法 12只雌性SD大鼠随机分为假手术组、去卵巢组(OVX)和杨梅素注射组(OVX+Myricetin)。建模后1周,OVX+Myricetin组、假手术组和OVX组分别腹腔注射杨梅素85 mg/kg和甲醇溶剂,2次/周,8周。取材前10 d 和前3 d分别皮下注射茜素红。microCT扫描股骨远心端。HE染色观察骨小梁。Elisa检测血清PINP浓度。荧光显微镜观察茜素红荧光条带。ALP染色检测BMMSCs成骨能力。荧光标记法检测BMMSCs活性氧水平。Real-time PCR检测基因表达。 结果 OVX组的骨量/组织量(BV/TV)、骨小梁数量(Tb.N)和骨小梁厚度(Tb.Th)低于假手术组;骨小梁间隙(Tb.Sp)大于假手术组,HE切片OVX组的骨小梁少于假手术组,OVX组的血清PINP、茜素红荧光条带间距低于假手术组。OVX+Myricetin组的BV/TV、Tb.N 和Tb.Th大于OVX组;Tb.Sp小于OVX组,HE切片OVX+Myricetin组的骨小梁多于OVX组,OVX+Myricetin组的血清PINP、茜素红荧光条带间距高于OVX组。OVX组BMMSCs的ROS水平高于假手术组,SOD表达降低,ALP活性和成骨基因表达降低;OVX+Myricetin组低于OVX组,SOD表达升高,ALP活性和成骨基因表达升高。H2O2处理后的BMMSCs的ROS水平升高,SOD表达降低,ALP活性和成骨基因表达降低;加杨梅素处理后的BMMSCs的ROS水平降低,SOD表达升高,ALP活性和成骨基因表达升高。 结论 杨梅素通过降低ROS水平恢复内源性BMMSCs的成骨能力,减少去卵巢大鼠骨量丢失。
关键词杨梅素     去卵巢     骨质疏松    活性氧    成骨分化    
Effect of myricetin on alleviating osteoporosis in ovariectomized rats
Gan Ning1,5,6, Yu Xuesong2,5, Chen Jun3, Ye Guo4,5,6     
1. Department of Conservation Dentistry and Endodontics, Chongqing Medical University, Chongqing, 401147;
2. Department of Prosthodontic, Chongqing Medical University, Chongqing, 401147;
3. Department of Orthodontics, Chongqing Medical University, Chongqing, 401147;
4. Department of Periodontics, Chongqing Medical University, Chongqing, 401147;
5. Chongqing Key Laboratory of Oral Diseases and Biomedicine, Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147;
6. Key Laboratory of Oral Biomedical Engineering of Chongqing Higher Education, China
Supported by the Natural Science Foundation of Chongqing(CSTC2014jcyjA10055) and the Project of Science and Technology Commission of Yubei District, Chongqing(2013-31).
Corresponding author: Ye Guo, E-mail: ygcqykd@163.com
Abstract: Objective To observe the effect of myricetin on bone volume of ovariectomized (OVX) rats and primarily explore the mechanism. Methods Twelve female SD rats were randomly divided into sham group, OVX group and OVX+myricetin group. In 1 week after modeling, the OVX+myricetin group, sham group and OVX group were intraperitoneally injected with myricetin 85 mg/kg or methanol solvent, twice per week, for 8 weeks. Subcutaneous injection of alizarin red was conducted on the 10th day and 3rd day before the rats were sacrificed. Distal femurs were analyzed by MicroCT scanning. Trabecular bone (Tb) was tested by HE staining. Serum amino-terminal propeptide of typeⅠprocollagen (PINP) was detected by ELISA. Alizarin red fluorescent strips were observed with fluorescence microscopy. Osteogenesis of BMMSCs was analyzed by ALP staining. ROS level was detected by fluorescence tagging. Gene expression was tested by real-time PCR. Results The bone volume/tissue volume (BV/TV), Tb number and Tb thickness of the OVX group were lower than those of the sham group, whereas the Tb space was higher. Tb of the OVX group was less than that of the sham group. Serum PINP and the distance between two alizarin red fluorescent strips in the OVX group were lower than those in the sham group. BV/TV, Tb number and Tb thickness of the OVX+myricetin group were higher than those of the OVX group, whereas Tb space was lower. Tb of the OVX+myricetin group was more than that of the OVX group. Serum PINP and the distance between two alizarin red fluorescent strips in the OVX+myricetin group were higher than those in the OVX group. The ROS level of endogenous BMMSCs in the OVX group was higher than that in the sham group, the expression of SOD was lower, and ALP activity and osteogenic genes were higher. ROS level of endogenous BMMSCs in the OVX+myricetin group was lower than that of the OVX group, the expression of SOD was higher, and ALP activity and osteogenic genes were lower. Furthermore, ROS level of H2O2-treated BMMSCs was higher than that of non-treated BMMSCs, the expression of SOD was lower, and ALP activity and osteogenic genes were higher. ROS level of H2O2-treated BMMSCs declined after treatment with myricetin, the expression of SOD was higher, and ALP activity and osteogenic genes were lower. Conclusion Myricetin reduces bone loss of OVX rats through lowering endogenous ROS level and restoring osteogenesis of endogenous BMMSCs.
Key words: myricetin     ovariectomization     ROS     osteogenesis    

研究表明,内源性骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMMSCs)成骨活性的减弱可以打破骨稳态,引发骨质疏松[1, 2]。黄酮类小分子化合物与雌激素具有类似的化学结构,能有效恢复骨质疏松动物的骨量[3],是骨质疏松研究领域的焦点。杨梅素作为一种黄酮类化合物,不仅具有优良的抗氧化活性[4, 5, 6, 7],在体外还能可以促进细胞成骨活性[8, 9],抑制破骨活性[10, 11],而目前尚少见其治疗骨质疏松的报道。本研究拟观察腹腔注射杨梅素能否减少去势大鼠的骨量丢失,探讨其对内源性BMMSCs的影响及其明确机制,以期为临床应用提供研究基础。

1 材料与方法 1.1 实验动物与试剂

SPF级雌性SD大鼠12只,体质量约180 g[合格证号SCXK(渝2007-0001)]。杨梅素标准品(上海永叶生物,UV≥98%),茜素红(上海谱振生物),血清PINP Elisa试剂盒(上海研辉生物),ALP染色试剂盒、活性氧检测试剂盒(碧云天),Trizol reagent、RNA反转录试剂盒及大鼠β-actin、ALP和Osterix引物(TaKaRa公司)。

1.2 方法 1.2.1 模型建立及腹腔注射

实验分为假手术组、OVX组和OVX+Myricetin组。术后1周,OVX+Myricetin组以85 mg/kg、2次/周腹腔注射杨梅素溶液,注射8周;假手术和OVX组以相同的频率注射等体积的甲醇。

1.2.2 皮下注射茜素红

取材前10 d和前3 d,以30 mg/kg 的剂量分别皮下注射。

1.2.3 取材

8周后取材,常规麻醉,心脏采血并分离血清。分离股骨,用于细胞学与组织学检测。

1.2.4 microCT扫描及茜素红荧光带检测

取左侧股骨远心端2 cm,扫描重建;左侧股骨近心端树脂包埋,50 μm厚硬组织切片,荧光显微镜观察荧光条带。

1.2.5 HE染色

常规脱钙,石蜡包埋,染色。

1.2.6 血清Ⅰ型前胶原氨基端前肽(Amino-terminal propeptideof typeⅠprocollagen,PINP)检测

按操作说明,取10 μL血清检测,检测浓度×5获得终浓度。

1.2.7 BMMSCs成骨能力检测

BMMSCs以1×105接种于12孔板,成骨诱导7 d后,ALP染色。同组复孔用于提取总RNA,检测成骨基因表达。

1.2.8 BMMSCs活性氧水平检测

BMMSCs以4×104接种于24孔板,贴壁后,加入800 μmol/L的H2O2处理6 h,再加入10 nmol/L的杨梅素预处理18 h。按活性氧试剂盒操作说明染色,荧光显微镜下拍照。同组复孔用于提取总RNA,检测SOD基因表达。

1.2.9 real-time PCR检测

TRIzol法提取BMMSCs总RNA,反转录,real-time PCR扩增。β-actin上游引物5′-CCCATCTATGAGGGTTACGC-3′(20 bp),下游引物5′-TTTAATGTCACGCACGATTTC-3′(21 bp)。 ALP上游引物5′-GCACAACATCAAGGACATCG-3′(20 bp),下游引物5′-AGGGAAGGGTCAGTCAGG-3′(18 bp)。Osterix 上游引物5′-GAGTAGGATTGTAGGATTGG-3′(20 bp),下游引物5′-TGCTTGAGGAAGTTCACTATGGC-3′(23 bp)。SOD上游引物5′-TGACCTGCCTTACGACTA-3′(18 bp),下游引物5′-CGACCTTGCTCCTTATTG-3′(18 bp)。

1.3 统计学分析

采用SPSS17.0统计软件,数据以x±s表示,3组间比较采用单因素方差分析。

2 结果 2.1 成功建立骨质疏松模型

OVX组的BV/TV、Tb.N和Tb.Th低于假手术组,而Tb.Sp高于假手术组(图 1表 1);HE切片中,OVX组的骨小梁少于假手术组(图 2)。OVX组的血清PINP浓度低于假手术组(表 1)。OVX组的成骨速率低于假手术组(图 3表 1)。

A:假手术组;B:去卵巢组;C:去卵巢+杨梅素组 图 1 各组大鼠股骨远心端microCT扫描观察
表 1 各组大鼠股骨远心端骨小梁参数、血清浓度及成骨速率
(n=4, x±s)
组别BV/TVTb.N(/mm)Tb.Th(mm)Tb.Sp(mm)PINP(ng/mL)成骨速率(μm/d)
假手术组0.42±0.023.54±0.130.072±0.0030.047±0.0135.818±0.9743.319±0.346
OVX组0.22±0.03a1.56±0.12a0.040±0.006a0.161±0.015a2.088±0.489a1.553±0.189*
OVX+Myricetin组0.31±0.02ab2.34±0.09ab0.059±0.002ab0.117±0.010ab3.565±0.527ab2.195±0.163ab
a: P<0.05,与假手术组比较;b: P<0.05,与OVX组比较
图 2 各组大鼠股骨远心端HE染色观察 (×100)
A:假手术组;B:去卵巢组;C:去卵巢+杨梅素组
A:假手术组;B:去卵巢组;C:去卵巢+杨梅素组 图 3 茜素红荧光标记各组大鼠新生骨带 (×200)
2.2 杨梅素可以有效缓解骨质疏松

OVX+Myricetin组的BV/TV、Tb.N参数高于OVX组,而Tb.Sp低于OVX组(图 1表 1);HE切片中,OVX+Myricetin组的骨小梁多于OVX组(图 2)。 OVX+Myricetin组的血清PINP浓度高于OVX组(表 1)。 OVX+Myricetin组的成骨速率高于OVX组(图 3表 1)。

2.3 杨梅素可以降低内源性ROS水平,并恢复内源性BMMSCs的成骨活性

与假手术组相比,OVX组内源性BMMSCs的ROS水平升高,SOD表达降低。而与OVX组相比,OVX+Myricetin组的内源性ROS水平降低,SOD表达升高。OVX组内源性BMMSCs的ALP活性、成骨基因ALP和Osterix的表达量低于假手术组,而OVX+Myricetin组则高于OVX组。见图 4图 5表 2

A:假手术组;B:去卵巢组;C:去卵巢+杨梅素组 图 4 各组大鼠内源性BMMSCs的ROS水平观察 (×40)
A:假手术组;B:去卵巢组;C:去卵巢+杨梅素组 i 图 5 各组大鼠内源性BMMSCs的ALP活性观察
表 2 各组大鼠内源性BMMSCs基因的表达
(n=4, x±s)
组别SODALPOsterix
假手术组9.25±0.6311.12±1.1410.73±1.20
OVX组3.03±0.80a2.90±0.80a4.27±0.92a
OVX+Myricetin组6.07±0.47ab5.92±0.63ab8.05±0.48ab
a: P<0.05,与假手术组比较; b: P<0.05,与OVX组比较
2.4 杨梅素通过降低活性氧水平恢复BMMSCs的成骨活性

我们在体外利用H2O2处理外源性BMMSCs模拟OVX大鼠内源性BMMSCs的状态。外源性BMMSCs用H2O2处理后,ROS水平高于未处理的BMMSCs,SOD表达降低;而加入杨梅素后ROS水平降低,SOD表达升高(图 6图 7表 3)。外源性BMMSCs用H2O2处理后,ALP活性、成骨基因ALP和osterix表达低于未处理的BMMSCs;加入杨梅素后ALP活性、成骨基因ALP和osterix表达升高。

A:BMMSCs组;B:BMMSCs+H2O2组;C:BMMSCs+H2O2+杨梅素组 图 6 各组大鼠外源性BMMSCs的ROS水平观察 (×40)
A:BMMSCs组;B:BMMSCs+H2O2组;C:BMMSCs+H2O2+杨梅素组 图 7 各组大鼠外源性BMMSCs的ALP活性观察
表 3 各组大鼠外源性BMMSCs基因的表达
(n=4, x±s)
组别SODALPOsterix
BMMSCs6.73±0.539.74±0.5412.18±0.79
BMMSCs+H2O22.29±0.33a3.42±0.93a4.79±0.81a
BMMSCs+H2O2+Myr6.40±0.36b8.43±1.01b7.68±0.69ab
a: P<0.05,与假手术组比较;b: P<0.05,与OVX组比较
3 讨论

目前临床主流药物虽然能够在一定程度上治疗骨质疏松,但是仍然存在一些副作用或者应用缺陷[12, 13, 14],因此急需寻找到一种疗效与安全俱佳的药物。杨梅素不仅具有黄酮类化学结构,毒性也较其他黄酮类化合物低,并且有较好的抗氧化作用,具有成为骨质疏松治疗药物的潜质。

本实验成功建立了骨质疏松模型。通过microCT扫描及参数分析,明确腹腔注射杨梅素后,可以有效减少OVX大鼠的骨量丢失,改善骨小梁微结构;HE染色结果与microCT检测结果一致,从组织学上确认了杨梅素的疗效。血清PINP是较为公认的成骨活性指标,杨梅素注射后,血清PINP升高,说明杨梅素促进了OVX大鼠机体的成骨活性。茜素红荧光条带间距离反映两次注射间隔期间内的新骨形成情况,杨梅素注射后,OVX大鼠的荧光条带间距明显加宽,也同样说明杨梅素促进OVX大鼠的内源性成骨活性。杨梅素的抗氧化作用[5, 6, 7]和促进成骨作用已经明确,但是两者之间的上下游关系尚不明确。本研究发现,杨梅素降低ROS的同时,内源性BMMSCs的成骨活性增强。为了进一步确认这一现象,我们在体外利用H2O2处理外源性BMMSCs模拟OVX大鼠内源性BMMSCs的状态,也观察到了类似的现象。所以我们有理由推测杨梅素很可能是通过降低内源性BMMSCs的氧化应激水平,从而促进成骨,最后达到缓解骨质疏松的目的。但是,从结果中也可以看出虽然杨梅素可以有效恢复OVX大鼠的骨量,但是仍无法恢复到正常状态,提示需要对治疗策略进行优化,比如进一步探索给药剂量、频率及时间等,或者联合其他小分子化合物协同治疗,力求达到更好的疗效。

本研究初步表明,杨梅素减少骨量丢失,机制之一是通过降低内源性BMMSCs的ROS水平,促进内源性成骨活性,从而治疗骨质疏松。那么明确影响的靶细胞以及分子机制将是我们进一步研究的重点。机体的骨稳态是在成骨和破骨的动态平衡中维持的,成骨和破骨任何一方的改变都将影响到骨骼。已有研究表明,杨梅素在体外可以有效促进成骨细胞活性[8, 9],抑制破骨细胞活性[10, 11]

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http://dx.doi.org/10.16016/j.1000-5404.201507137
中国人民解放军总政治部、国家科技部及国家新闻出版署批准,
由第三军医大学主管、主办

文章信息

甘宁,郁雪松,陈军,叶国
Gan Ning, Yu Xuesong, Chen Jun, Ye Guo
杨梅素减轻去卵巢大鼠骨质疏松
Effect of myricetin on alleviating osteoporosis in ovariectomized rats
第三军医大学学报, 2016, 38(6): 614-618
J Third Mil Med Univ, 2016, 38(6): 614-618.
http://dx.doi.org/10.16016/j.1000-5404.201507137

文章历史

收稿:2015-07-23
修回:2015-09-07

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