中国组织工程研究 ›› 2021, Vol. 25 ›› Issue (29): 4644-4649.doi: 10.12307/2021.163

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

电针干预去卵巢骨质疏松模型大鼠骨组织Runx2启动子甲基化水平的变化

陈  赵1,2,莫雨晴3,唐宏宇4   

  1. 1广州中医药大学,广东省广州市   510405;2肇庆市第一人民医院,广东省肇庆市   526000;3广州中医药大学第一临床医学院,广东省广州市  510405;4广州中医药大学第一附属医院,广东省广州市   510405
  • 收稿日期:2020-09-07 修回日期:2020-09-09 接受日期:2020-11-11 出版日期:2021-10-18 发布日期:2021-06-02
  • 通讯作者: 唐宏宇,硕士,医师,广州中医药大学第一附属医院,广东省广州市 510405
  • 作者简介:陈赵,1989年生,广东省河源市人,2013年广州中医药大学毕业,主管技师,主要从事针灸治病机制方面的研究。
  • 基金资助:
    国家自然科学基金项目(81774339),项目参与人:唐宏宇;广东省中医药管理局资助项目(20191099),项目负责人:唐宏宇

Runx2 promoter methylation level changes in bone tissue of rats with ovariectomized osteoporosis treated by electroacupuncture

Chen Zhao1, 2, Mo Yuqing3, Tang Hongyu4    

  1. 1Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 2Zhaoqing First People's Hospital, Zhaoqing 526000, Guangdong Province, China; 3First Clinical School of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; 4The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China
  • Received:2020-09-07 Revised:2020-09-09 Accepted:2020-11-11 Online:2021-10-18 Published:2021-06-02
  • Contact: Tang Hongyu, Master, Physician, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, Chin
  • About author:Chen Zhao, Technician in charge, Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong Province, China; Zhaoqing First People's Hospital, Zhaoqing 526000, Guangdong Province, China
  • Supported by:
    the National Natural Science Foundation of China, No. 81774339 (to THY); the grant from Guangdong Provincial Administration of Traditional Chinese Medicine, No. 20191099 (to THY)

摘要:

文题释义:
骨质疏松:是以骨强度(骨质量和骨密度)下降和骨折风险增加为特征的骨骼疾病,初期通常无明显临床表现,但随着骨量不断丢失,骨微结构破坏,患者会出现骨痛、活动受限、脊柱变形,甚至发生骨质疏松性骨折等严重并发症。
甲基化:调节基因的表达和关闭,包括DNA甲基化和蛋白质甲基化。DNA甲基化所属的表观遗传修饰是一种重要的基因表达调控机制,研究的是基因序列不变,产生可遗传的基因表达改变。DNA甲基化是在DNA甲基转移酶的催化下,将甲基基团可逆性转移到基因组鸟苷酸 (Cp G)二核苷酸上,而启动子序列DNA甲基化抑制基因表达,异常的DNA甲基化在骨质疏松症等许多疾病中存在。

背景:肾俞、足三里为针刺治疗骨质疏松症的常用穴位,但其具体机制尚不明确。
目的:基于骨组织Runt相关转录因子2启动子甲基化水平,探究电针肾俞、足三里等穴位对去卵巢骨质疏松症大鼠的疗效机制。
方法:将50只SD大鼠随机分为假手术组、模型组、电针穴位组、电针非穴位组、雌二醇组,每组10只。假手术组以外的4组大鼠采用去卵巢法建立骨质疏松症模型,电针穴位组选取肾俞、足三里穴位进行电针治疗,电针非穴位组选取非穴位处进行干预,其余3组不进行电针干预;雌二醇组给予雌二醇片(0.09 mg/kg)灌胃治疗,假手术组和模型组均灌胃10 mL/kg的生理盐水。干预12周后,测定各组大鼠右侧下肢股骨及椎骨骨密度、股骨生物力学性能、血清骨钙与Ⅰ型原胶原N端前肽表达水平、骨组织骨桥蛋白与骨唾液蛋白表达水平、骨组织Runt相关转录因子2启动子甲基化表达水平。
结果与结论:①与假手术组相比,模型组大鼠股骨、椎骨骨密度明显下降(P < 0.05);股骨最大载荷、刚度及弹性模量、血清骨钙、Ⅰ型原胶原N端前肽水平均明显下降(P < 0.01);骨组织中骨桥蛋白和骨唾液蛋白、Runt相关转录因子2启动子CpG1和CpG2、CpG3和CpG4.5的表达明显上升(P < 0.01);②与模型组相比,电针穴位组和雌二醇组对上述各指标改善明显(P < 0.01);而电针非穴位组骨组织中Runt相关转录因子2启动子CpG1、CpG2、CpG3、CpG4.5的表达下降不明显(P > 0.05),此外其余上述指标均有所改善(P < 0.05);③与雌二醇组相比,电针穴位组对血清Ⅰ型原胶原N端前肽水平的上调较为明显(P < 0.05),而其余指标差异无显著性意义(P > 0.05);④提示电针肾俞、足三里穴位可通过降低骨组织Runt相关转录因子2启动子甲基化水平,增强骨密度,改善股骨的生物力学性能等,从而有效防治骨质疏松症。
https://orcid.org/0000-0001-9502-8046 (陈赵)

中国组织工程研究杂志出版内容重点:组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程

关键词: 电针, 骨质疏松, 表观遗传, 甲基化, 生物力学, 大鼠, Runx2启动子

Abstract: BACKGROUND: Shenshu and Zusanli are commonly used acupuncture acupoints in the treatment of osteoporosis; however, the specific mechanism is still unclear.
OBJECTIVE: To explore the therapeutic mechanism of electroacupuncture at Shenshu and Zusanli acupoints in ovariectomized rats with osteoporosis based on the methylation level of Runx2 promoter in bone tissue.
METHODS: Fifty Sprague-Dawley rats were randomly divided into sham operation group, model group, electroacupuncture acupoint group, electroacupuncture non-acupoint group and estradiol group (n=10 per group). The model of osteoporosis was established by ovariectomy in all the groups except for sham operation group. Shenshu and Zusanli acupoints were selected for electroacupuncture treatment in the electroacupuncture acupoint group. Non-acupoint electroacupuncture was selected for intervention in the electroacupuncture non-acupoint group, while the other three groups did not receive electroacupuncture intervention. The estradiol group was given estradiol tablets (0.09 mg/kg) by gavage, and the sham operation group and the model group were given 10 mL/kg normal saline. Femoral and vertebral bone mineral density, biomechanical properties of the femur, serum bone calcium, type I tropocollagen n-terminal propeptide (P1NP), bone tissue osteopontin and bone sialoprotein levels, and Runx2 promoter methylation level in bone tissue were determined at 12 weeks after intervention.
RESULTS AND CONCLUSION: Bone mineral density levels of the femur and vertebrae decreased significantly in the model group compared with the sham operation group (P < 0.05). The maximum load, stiffness and elastic modulus of the femur, serum calcium, and P1NP levels were significantly decreased in the model group compared with the sham operation group (P < 0.01). The osteopontin and bone sialoprotein levels and expressions of Runx2 promoters CpG1, CpG2, CpG3 and CpG4.5 in bone tissue were significantly increased in the model group compared with the sham operation group (P < 0.01). Compared with the model group, electroacupuncture at acupoints and estradiol significantly improved the above-mentioned indicators (P < 0.01), while the expression of Runx2 promoters CpG1, CpG2, CpG3 and CpG4.5 in the bone tissue of the electroacupuncture non-acupoint group showed no significant decrease (P > 0.05). In addition, the other indicators were all improved in the electroacupuncture non-acupoint group (P < 0.05). Compared with the estradiol group, the level of serum P1NP was significantly increased in the electroacupuncture acupoint group (P < 0.05), while the difference in the other indicators was not statistically significant (P > 0.05). To conclude, electroacupuncture at Shenshu and Zusanli acupoints can enhance bone mineral density and improve the biomechanical properties of the femur by reducing the methylation level of Runx2 promoter in bone tissue, thus effectively preventing and controlling osteoporosis.

Key words: electroacupuncture, osteoporosis, epigenetics, methylation, biomechanics, rat, Runx2 promoter

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