WO2017054337A1 - 硝酸盐在制备预防或治疗骨代谢疾病的药物中的应用 - Google Patents

硝酸盐在制备预防或治疗骨代谢疾病的药物中的应用 Download PDF

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WO2017054337A1
WO2017054337A1 PCT/CN2015/098314 CN2015098314W WO2017054337A1 WO 2017054337 A1 WO2017054337 A1 WO 2017054337A1 CN 2015098314 W CN2015098314 W CN 2015098314W WO 2017054337 A1 WO2017054337 A1 WO 2017054337A1
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nitrate
bone
medicament
preparation
preventing
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PCT/CN2015/098314
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French (fr)
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王松灵
庞宝兴
曲兴民
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首都医科大学
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Priority to US15/764,877 priority Critical patent/US20180280429A1/en
Publication of WO2017054337A1 publication Critical patent/WO2017054337A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/02—Ammonia; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/06—Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention relates to the field of medicine, and more particularly to the use of nitrate in the preparation of a medicament for preventing or treating bone metabolic diseases, and the preparation of a medicament for regulating abnormalities of the body and bone marrow mesenchymal stem cells. .
  • Nitrate is an inorganic substance widely found in nature. Plants can absorb and utilize nitrates in the soil to supply the nitrogen required in the body. Most of the nitrate in food comes from green vegetables. The traditional view is that nitrates and nitrites in the diet have a potential carcinogenic tendency, and it has been recognized that nitrites combine with amines under specific conditions to form nitrite compounds and reach a certain concentration. Oh, it will show carcinogenic effects. At present, the exact role of oral nitrate in enhancing blood flow perfusion and regulating energy metabolism has reached a consensus, which laid the foundation for its application in bone metabolic diseases.
  • Bone metabolic disease refers to a bone disease in which the body destroys or interferes with normal bone metabolism and biochemical state due to congenital or acquired factors, resulting in bone biochemical metabolic disorders.
  • Abnormal bone density disease is one of the common types.
  • Abnormal bone mineral density refers to a systemic bone disease caused by direct or indirect causes, but may also be highlighted as a bone change in a certain part of the body. For example, hyperparathyroidism can cause general decalcification of the whole body, and malignant tumors invade local bone resorption caused by bone tissue.
  • Abnormal bone mineral density is mainly characterized by disturbance or abnormality between bone formation and bone resorption.
  • Bone formation or bone resorption can be reduced or increased, bone matrix formation can be lacked or increased, and mineralization can be lacked, insufficient or deposited. Too much, the result shows that the bone can show looseness, softening, hardening or excessive calcification, and can have two or more manifestations.
  • osteoporosis is a most common degenerative bone mineral density disorder in the elderly, especially in postmenopausal women. It has systemic bone mass reduction, bone tissue microstructural degeneration, bone strength reduction, bone fragility, and easy Fractures and generalized pain are the main features of the lesion.
  • osteoporosis drugs can be divided into anti-bone resorption drugs, bone-promoting drugs and bone mineralization drugs, the most commonly used is estrogen replacement therapy (ERT), its efficacy has been Fully affirmed, but long-term use of estrogen There is an increased risk of endometrial cancer, breast cancer and cardiovascular disease.
  • ERT estrogen replacement therapy
  • the technical problem to be solved by the present invention is to provide a solution for preparing a medicament for preventing or treating a bone metabolic disease, and a nitrate containing the same, in view of the defect of the poor therapeutic effect of the therapeutic drug for the existing bone metabolic disease.
  • the technical solution adopted by the present invention to solve the technical problems thereof is to provide an application of nitrate in the preparation of a medicament for preventing or treating a bone metabolic disease.
  • This invention has for the first time discovered and provided this new use of nitrates.
  • the nitrate is used for the preparation of a medicament for the prevention or treatment of osteoporosis.
  • the osteoporosis includes primary or postmenopausal or senile osteoporosis, as well as osteoporosis secondary to multiple endocrine disorders or disuse.
  • the invention proves that the addition of nitrate to the drinking water of the ovariectomized rats can significantly alleviate the loss of bone mass in the rats through a large number of experiments, and the nitrate has no obvious adverse reaction in the preservation of the bone mass, and has no significant effect on the body weight of the rats. .
  • the medicament for preparing a nitrate of the present invention is also suitable for a disease in which bone loss is required to be alleviated. Further studies have shown that nitrate can also regulate immune abnormalities and abnormal bone marrow mesenchymal stem cells. Therefore, the first use of nitrates in the treatment of osteoporosis in the first time of the present invention may be to regulate body immunity and bone marrow through nitrate. Mesenchymal stem cells are used to achieve this.
  • the nitrate can also be used to prepare a medicament for preventing or treating a disease of abnormal bone density.
  • abnormal bone density disorders are caused by congenital or acquired factors that disrupt or interfere with normal bone metabolism and biochemical status, leading to a breakdown of the osteogenesis and osteoclast balance, resulting in decreased bone density and loss of bone mass. It mainly includes osteolytic changes caused by malignant tumors invading bone tissue, rheumatoid arthritis invading bone loss caused by cartilage and bone tissue, and loss of bone mass caused by thyroid dysfunction.
  • the present invention proves by experiments that nitrate can significantly alleviate the loss of bone mass in rats, it can also be used to treat these bone density abnormal diseases, especially bones directly caused by abnormal body immunity and/or abnormality of bone marrow mesenchymal stem cells.
  • the density is abnormal.
  • the nitrate is a salt composed of a metal ion and a nitrate ion.
  • the active ingredient in the nitrate of the present invention is mainly a nitrate ion, and therefore any pharmaceutically acceptable salt containing a nitrate may be used, and a salt composed of a metal ion and a nitrate ion or ammonium nitrate or the like is preferable.
  • the nitrate of the present invention may be selected from the group consisting of one or more of the following: sodium nitrate, potassium nitrate and calcium nitrate, ammonium nitrate, and the like.
  • the nitrate is provided in the form of a nitrate-rich food or extract.
  • the nitrate salt in the present invention may be provided not only in the form of a compound but also in a food or extract.
  • the present invention accordingly also provides a new use of these nitrate-rich foods or extracts for the preparation of a medicament for the prevention or treatment of bone metabolic diseases.
  • the concentration of nitrate in spinach, beetroot, white radish, celery and other vegetables can reach 3000mg/Kg or more, which is several times to ten times that of other common foods.
  • the nitrate is administered in an amount of from 0.1 to 0.5 mmol/kg b.w. per day. More preferably, the daily dose of nitrate is 0.5 mmol/kg b.w.
  • the present invention also provides a medicament for preventing or treating a bone metabolic disease, which comprises a nitrate as an active ingredient. It is preferable to provide a medicament for preventing or treating a disease of abnormal bone density, which comprises nitrate as an active ingredient. More preferably, a medicament for preventing or treating osteoporosis is provided which contains nitrate as an active ingredient.
  • the nitrates can also be provided in a variety of forms and dosages as previously described.
  • the medicament for preventing or treating a bone metabolic disease is a tablet, a capsule, a pill, an injection, a syrup, an oral solution, a granule or a patch.
  • a nitrate oral solution can provide a more convenient and effective administration route, wherein the concentration of the nitrate oral solution is preferably 0.5 mmol/L to 2.5 mmol/L, more preferably 2.5 mmol/L.
  • the present invention also provides the use of nitrate in the preparation of a medicament for regulating immune abnormalities in the body, more preferably for the regulation of T lymphocyte down-regulation, IL-17 up-regulated immune abnormal disease, nitrate plays a good regulatory role .
  • the invention adopts a technique such as flow cytometry to detect the immune regulation of ovariectomized rats, and finds that nitrate can regulate the immunobiological behavior of ovariectomized rats, for example, alleviating T lymphocytes in ovariectomized rats.
  • the decline of TGF- ⁇ in ovariectomized rats was alleviated and the up-regulation of IFN- ⁇ and IL-17 was attenuated.
  • the present invention also provides a drug for regulating abnormality of the body, which comprises nitrate as an active ingredient.
  • the nitrates can also be provided in a variety of forms and dosages as previously described.
  • the present invention also provides the use of nitrate in the preparation of a medicament for regulating abnormality of bone marrow mesenchymal stem cells, and more preferably, the nitrate is well regulated for diseases in which bone marrow mesenchymal stem cells have decreased osteogenic capacity. effect.
  • the invention detects the stem cell activity of ovariectomized rats by a technique such as stem cell culture, and finds that nitrate can regulate the biological behavior of bone marrow mesenchymal stem cells in ovariectomized rats, for example, significantly reducing the proliferation ability of BMMSCs in ovariectomized rats. Significantly enhance its osteogenic differentiation ability.
  • the present invention also provides a medicament for regulating abnormality of bone marrow mesenchymal stem cells, which comprises nitrate as an active ingredient.
  • the nitrates can also be provided in a variety of forms and dosages as previously described.
  • the present invention provides a new use of nitrate in the preparation of a medicament for preventing or treating bone metabolic diseases, and proves that nitrate can significantly alleviate the loss of bone mass in rats through a large number of experiments, and the nitrate is preserved in the same amount of bone. ⁇ No obvious adverse reactions were found, which had no significant effect on the body weight of rats. In addition, it can also regulate the immunity of ovariectomized rats and the biological behavior of bone marrow mesenchymal stem cells, so that it can be prepared into drugs for the prevention and/or treatment of bone mass. Abnormal bone mineral density diseases such as loose, and diseases such as abnormal immune system and abnormal bone marrow mesenchymal stem cells.
  • 1A-1F are graphs showing the effect of nitrate on bone mineral density and trabecular bone in ovariectomized rats;
  • 2A and 2B are graphs showing the effects of nitrate on the weight and body weight of tibia in ovariectomized rats, respectively;
  • FIGS. 3A and 3B are respectively a graph showing the difference in the contents of nitrate and nitrite in serum after drinking nitrate for 3 months, respectively.
  • 4A-4D are graphs showing the results of immunomodulatory effects of nitrate on ovariectomized rats
  • 5A and 5B are graphs showing the results of defects in nitrate-improving ovariectomized rat BMMSCs.
  • Osteoporosis model 12-week female SD rats were selected and bilateral ovaries were removed.
  • the experiment was divided into three groups: sham operation group (Sham), castration group (OVX) and nitrate group (Nitrate), with 10 rats in each group.
  • the nitrate group was given sodium nitrate drinking water on the second day of Ovariectomy (ovarian removal) at a concentration of 2.5 mmol / L.
  • the daily intake of nitrate in each rat was about 0.5 mmol/Kg bw, continuous nitric acid.
  • the salt was given water for 12 weeks, and the rest of the group was drinking normally.
  • the rat tibia was completely isolated, all attached muscles and connective tissues were removed, wrapped in wet gauze soaked in physiological saline, and sealed at -20 °C.
  • the upper part of the tibia was scanned with a Skyscanl type l72 Micro-CT.
  • the thickness of the scan layer was 9 ⁇ , and the bone density and trabecular bone structure were quantitatively analyzed using the software attached.
  • FIGS. 1A-1F show the results of nitrate alleviation of bone mineral density and trabecular bone in ovariectomized rats.
  • Figure 1A is a schematic diagram of the experimental procedure of the nitrate group. Sodium nitrate was given to the siblings of the SD rats after 12 weeks of castration surgery, and the animals were sacrificed 12 weeks later.
  • Figure 1B is a microCT of the upper part of the rat tibia, which shows that the bone mass of the tibia is significantly lost in the ovariectomized group (OVX), and the bone loss of the tibia lost in the ovariectomized rats after nitrate application is significantly relieved.
  • Figure 1C-1F shows the bone mineral density (BMD) and bone volume fraction of each group of MicroCT quantitative analysis.
  • 2A and 2B are graphs showing the effect of nitrate on the weight and body weight of the tibia of ovariectomized rats, respectively (*P ⁇ 0.05).
  • NADH nicotinamide adenine dinucleotide
  • FIGS. 4A-4D are graphs showing the results of immunomodulatory effects of nitrate on ovariectomized rats (*P ⁇ 0.05).
  • Figure 4A shows the proportion of regulatory T lymphocytes in peripheral blood CD4 positive cells obtained by flow cytometry analysis of each group of rats. As shown in the figure, nitrate drinking can alleviate the decrease of regulatory T lymphocytes (Treg) in ovariectomized rats.
  • Figures 4B - 4D are graphs showing the levels of TGF-pi, IFN- ⁇ and IL-17 in each group of serum, respectively. The results showed that nitrate drinking water can alleviate the down-regulation of TGF- ⁇ in ovariectomized rats and alleviate the up-regulation of IFN- ⁇ and IL-17.
  • the skin of the hind limb was peeled off, and after exposing the femur and tibia, the ot-MEM complete medium (containing 10% of bovine serum) was aspirated from a broken end with a 1 mL syringe, and the cell clone was grown to 80%. Fusion can be passed down and subcultured to the third generation.
  • the ⁇ cell suspension was inoculated in a 96-well plate, and the plate was pre-incubated in an incubator for 24 hours (at 37 ° C, 5 % C0 2 ), and then the plate was incubated in a incubator for 72 hours. ⁇ , replace each well with a new medium ⁇ , and add 1 (Vl CCK-8 solution to each well. After 2 hours, measure the absorbance at 450 nm with a microplate reader.
  • [0070] formulated into osteoinductive culture medium containing 10% fetal calf serum ⁇ - ⁇ medium added 2 mmol / L glutamine, 100 U / ml penicillin and 10 (Vg / ml streptomycin, ⁇ ⁇ ⁇ -glycerophosphate Sodium, 10 ⁇ dexamethasone, 50mg/L vitamin C.
  • the 3-4rd generation cells were seeded in a 6-well plate at a concentration of 2xl03/cm2. After the cells were grown to 80% confluence, they were replaced with osteogenic induction medium. After 2 days of fluid change, the formation of calcium nodules was observed under light microscope. After 2 weeks of induction, alizarin red staining was performed.
  • FIGS. 5A and 5B Please refer to FIGS. 5A and 5B for the results of defect improvement of nitrate-induced BMMSCs in ovariectomized rats (*P ⁇ 0.05).
  • Fig. 5A the primary cultures of the BMMSCs of each group were taken according to the above method, and the OD450 values of the third generation cells were detected by CCK8. The results showed that nitrate drinking significantly reduced the proliferation of BMMSCs in ovariectomized rats.
  • Fig. 5B is a graph showing the osteogenic induction of BMSSC in each group for 21 days according to the above method, and the results of parallel alizarin red staining and quantitative results. The results showed that nitrate drinking significantly enhanced the osteogenic differentiation ability of ovariectomized rat BMMSCs.

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Abstract

一种预防或治疗骨代谢疾病的含有硝酸盐的药物，以及硝酸盐在制备预防或治疗骨代谢疾病的药物、制备调节机体免疫异常和骨髓间充质干细胞异常的药物中的应用。

Description

硝酸盐在制备预防或治疗骨代谢疾病的药物中的应用 技术领域
[0001] 本发明涉及医药领域， 更具体地说， 涉及硝酸盐在制备预防或治疗骨代谢疾病 的药物中的应用， 以及在制备调节机体免疫异常和骨髓间充质干细胞异常的药 物中的应用。
背景技术
[0002] 硝酸盐是自然界广泛存在的一种无机物， 植物能够吸收和利用土壤中的硝酸盐 以供应体内所需的氮元素。 食物中的硝酸盐大部分来源于绿色蔬菜中。 传统的 观点认为， 饮食中的硝酸盐和亚硝酸盐类具有潜在的致癌倾向， 随后人们认识 到， 亚硝酸盐只有特定条件下跟胺类物质结合形成亚硝酸胺类化合物， 且达到 一定的浓度吋， 才会显现出致癌作用。 目前， 口服硝酸盐在增强机体血流灌注 ， 调节能量代谢等方面的确切作用已经达到共识， 这为其在骨代谢疾病的应用 奠定了基础。
[0003] 骨代谢疾病是指机体因先天或后天性因素破坏或干扰了正常骨代谢和生化状态 ， 导致骨生化代谢障碍而发生的骨疾患。 骨密度异常疾病是其中一种常见的类 型。 骨密度异常疾病， 是指由直接或间接原因引起的全身性骨疾病， 但也可能 突出反映为身体某一部位的骨改变。 如甲状旁腺功能亢进能引起全身普遍脱钙 ， 恶性肿瘤侵犯骨组织引起的局部骨吸收等。 骨密度异常疾病主要表现为骨形 成和骨吸收两者之间的转换紊乱或异常， 骨形成或骨吸收可减少或增加， 骨基 质形成可缺乏或增加， 其矿化也可缺乏、 不足或沉积过多， 结果骨可显示疏松 、 软化、 硬化或过度钙化， 可兼具两种或两种以上的表现。 其中， 骨质疏松症 是老年人， 尤其是绝经妇女最常见的一种退行性骨密度异常疾病， 以全身性骨 量减少、 骨组织显微结构退变、 骨强度降低、 骨脆性增加、 易骨折和全身疼痛 为主要病变特点。 目前， 临床上治疗骨质疏松的药物可分为抗骨吸收药物、 促 进骨形成药物和促进骨矿化药物等， 其中最常用的是雌激素替代疗法 （estrogen replacement therapy , ERT) ， 其疗效已经得到充分肯定， 然而长期使用雌激素 有增加子宫内膜癌、 乳腺癌和心血管疾病的风险。
[0004] 目前， 尚未有研究或专利报道硝酸盐在预防和治疗骨代谢疾病尤其是骨密度异 常方面的应用。
技术问题
[0005] 本发明要解决的技术问题在于， 针对现有骨代谢疾病的治疗药物疗效不佳的缺 陷， 提供硝酸盐在制备预防或治疗骨代谢疾病的药物中的应用， 以及含有该硝 酸盐的预防或治疗骨代谢疾病的药物。
问题的解决方案
技术解决方案
[0006] 本发明解决其技术问题所采用的技术方案是： 提供硝酸盐在制备预防或治疗骨 代谢疾病的药物中的应用。 本发明首次发现并提供了硝酸盐的这种新用途。
[0007] 在根据本发明硝酸盐应用的优选实施例中， 所述硝酸盐用于制备预防或治疗骨 质疏松症的药物。 该骨质疏松症包括原发性者如经绝后或老年性骨质疏松症， 以及继发于多种内分泌疾患的或废用引起的骨质疏松症。 本发明通过大量实验 证明给予去势大鼠的饮水中添加硝酸盐可以明显缓解大鼠骨量的丢失， 而且硝 酸盐在保存骨量的同吋未发现明显不良反应， 对大鼠体重没有明显影响。 基于 同样的原理， 本发明硝酸盐制备的药物， 也适用于需要缓解骨量丢失的疾病。 进一步的研究表明， 硝酸盐同吋还可调节机体免疫异常和骨髓间充质干细胞异 常， 因此本发明首次发现的硝酸盐治疗骨质疏松症的新用途可能是通过硝酸盐 调节机体免疫及骨髓间充质干细胞来实现的。
[0008] 在根据本发明硝酸盐应用的优选实施例中， 所述硝酸盐还可以用于制备预防或 治疗骨密度异常疾病的药物。 这些骨密度异常疾病是因先天或后天性因素破坏 或干扰了正常骨代谢和生化状态， 导致成骨和破骨的平衡被打破， 致使出现骨 密度降低乃至骨量丢失。 其主要包括恶性肿瘤侵犯骨组织引起的溶骨性改变， 类风湿关节炎侵犯软骨及骨组织引起的骨量丢失， 甲状腺功能紊乱引起的骨量 丢失等。 由于本发明通过实验证明了硝酸盐可以明显缓解大鼠骨量的丢失， 因 此也可以用于治疗这些骨密度异常疾病， 尤其是直接由机体免疫异常和 /或骨髓 间充质干细胞异常导致的骨密度异常。 [0009] 在根据本发明硝酸盐应用的优选实施例中， 所述硝酸盐为金属离子与硝酸根离 子组成的盐类。 本发明的硝酸盐中活性成分主要为硝酸根离子， 因此可以采用 含有硝酸根的任何药学上可接受的盐类， 优选为金属离子与硝酸根离子组成的 盐类或者硝酸铵等。 因此， 本发明的硝酸盐可以选自下组中一种或者多种物质 的组合： 硝酸钠、 硝酸钾和硝酸钙及硝酸铵等。
[0010] 在根据本发明硝酸盐应用的优选实施例中， 所述硝酸盐以富含硝酸盐的食物或 提取物形式提供。 本发明中的硝酸盐不仅可以化合物形式提供， 还可以存在于 食物或提取物中。 因此本发明也相应提供了这些富含硝酸盐的食物或提取物用 于制备预防或治疗骨代谢疾病的药物中的新用途。 例如， 菠菜、 甜菜根、 白萝 卜、 芹菜等蔬菜中存在的硝酸盐浓度可以达到 3000mg/Kg以上， 是其他普通食物 中硝酸盐浓度的数倍至十几倍。
[0011] 在根据本发明硝酸盐应用的优选实施例中， 所述硝酸盐每日的给药量为 0.1-0.5 mmol/Kg b.w.。 更优选地， 硝酸盐每日的给药量为 0.5 mmol/Kg b.w.。
[0012] 本发明还提供了一种预防或治疗骨代谢疾病的药物， 其中含有硝酸盐作为活性 成分。 优选提供一种预防或治疗骨密度异常疾病的药物， 其中含有硝酸盐作为 活性成分。 更优选地， 提供一种预防或治疗骨质疏松症的药物， 其中含有硝酸 盐作为活性成分。 所述硝酸盐也可以采用如前所述的各种形式和剂量提供。
[0013] 在根据本发明的优选实施例中， 该预防或治疗骨代谢疾病的药物的剂型为片剂 、 胶囊、 丸剂、 注射剂、 糖浆、 口服液、 颗粒剂或贴剂。 其中， 以硝酸盐口服 液的方式可以提供一种更为便捷有效的给药途径， 其中硝酸盐口服液的浓度优 选为 0.5mmol/L-2.5mmol/L， 更优选为 2.5 mmol/ L。
[0014] 本发明还提供了硝酸盐在制备调节机体免疫异常的药物中的应用， 更优选地针 对调节性 T淋巴细胞下调、 白介素 17上调的免疫异常疾病， 硝酸盐起到很好的调 节作用。 本发明采用流式细胞术等技术方法对去势大鼠的免疫调控进行检测， 发现硝酸盐可以调控去势大鼠的免疫生物学行为， 例如缓解去势大鼠调节性 T淋 巴细胞 （Treg) 的下降， 缓解去势大鼠 TGF-βΙ的下调， 并缓解 IFN-γ和 IL-17的上 调。 同吋， 本发明也提供了一种调节机体免疫异常的药物， 其中含有硝酸盐作 为活性成分。 该硝酸盐也可以采用如前所述的各种形式和剂量提供。 [0015] 本发明还提供了硝酸盐在制备调节骨髓间充质干细胞异常的药物中的应用， 更 优选地， 针对骨髓间充质干细胞成骨能力下降的疾病， 硝酸盐起到很好的调节 作用。 本发明通过干细胞培养等技术方法对去势大鼠的干细胞活性进行检测， 发现硝酸盐可以调控去势大鼠的骨髓间充质干细胞生物学行为， 例如显著降低 去势大鼠 BMMSC的增殖能力， 显著增强其成骨分化能力。 同吋， 本发明也提供 了一种调节骨髓间充质干细胞异常的药物， 其中含有硝酸盐作为活性成分。 该 硝酸盐也可以采用如前所述的各种形式和剂量提供。
发明的有益效果
有益效果
[0016] 本发明提供了硝酸盐在制备预防或治疗骨代谢疾病的药物中的新用途， 并通过 大量实验证明硝酸盐能够明显缓解大鼠骨量的丢失， 而且硝酸盐在保存骨量的 同吋未发现明显不良反应， 对大鼠体重没有明显影响， 此外还可以调控去势大 鼠的免疫及骨髓间充质干细胞生物学行为， 因而可以制备成药物， 应用于预防 和 /或治疗骨质疏松等骨密度异常疾病， 以及机体免疫异常和骨髓间充质干细胞 异常等疾病。
对附图的简要说明
附图说明
[0017] 图 1A-1F为硝酸盐缓解去势大鼠骨密度和骨小梁的影响结果图；
[0018] 图 2A和 2B分别为硝酸盐对去势大鼠胫骨重量和体重的影响结果图；
[0019] 图 3A和 3B分别为硝酸盐饮水 3个月后血清中的硝酸盐和亚硝酸盐含量的差异图
[0020] 图 4A-4D为硝酸盐对去势大鼠的免疫调节作用结果图；
[0021] 图 5A和 5B为硝酸盐改善去势大鼠 BMMSC的缺陷结果图。
本发明的实施方式
[0022] 为了使本发明的目的、 技术方案及优点更加清楚明白， 以下结合附图及实施例 ， 对本发明进行进一步详细说明。 [0023] 1、 骨质疏松模型的建立及硝酸盐给药
[0024] 骨质疏松模型选择 12周齢雌性 SD大鼠， 摘除双侧卵巢。 实验分为 3组： 假手术 组 （Sham) 、 去势组 (OVX) 及硝酸盐组 （Nitrate) ， 每组 10只。 其中硝酸盐 组在去势 （Ovariectomy , 卵巢摘除术） 第二天给予硝酸钠饮水， 浓度为 2.5mmol /L， 每只大鼠每日摄取的硝酸盐含量约为 0.5mmol/Kg b.w.， 连续硝酸盐饮水 12周 ， 其余组正常饮水。
[0025] 2、 骨密度检测
[0026] 完整分离大鼠胫骨， 剔除所有附着的肌肉和结缔组织， 用生理盐水浸泡的湿纱 布包裹， -20°C密封保存。 应用 Skyscanl l72型 Micro-CT对胫骨上部进行扫描， 扫 描层厚为 9μηι， 使用其所附带的软件对骨密度及骨小梁结构进行定量分析。
[0027] 请参阅图 1A- 1F, 为硝酸盐缓解去势大鼠骨密度和骨小梁的影响结果图。 其中 图 1A为硝酸盐组的实验流程示意图， 对 12周齢 SD大鼠去势手术的同吋给予硝酸 钠饮水， 12周后处死动物收集标本。 图 1B为大鼠胫骨上部 MicroCT图， 其中显示 去势组 （OVX) 胫骨骨量明显丢失， 应用硝酸盐后去势大鼠胫骨丢失的骨量明 显缓解。 图 1C-1F分别为各组 MicroCT定量分析的骨密度 （BMD) 、 骨体积分数
(BV/TV) 、 骨小梁厚度 （Tb.Th) 和骨小梁数目 （Tb.N) 的结果图 (*P<0.05)。 结果显示去势组 （OVX) 的上述参数显著降低， 而硝酸盐 （Nitrate) 组能够部分 恢复以上指标。
[0028] 图 2A和 2B分别为硝酸盐对去势大鼠胫骨重量和体重的影响结果图 (*P<0.05)。
结果显示硝酸盐饮水可缓解去势大鼠胫骨重量的下降， 同吋对去势大鼠体重的 增加没有明显影响。
[0029] 3、 血清中硝酸盐及亚硝酸盐的测量
[0030] (1)配制标准品： 倍比稀释原标准品为 200μΜ-3.125μΜ
[0031] (2)亚硝酸盐浓度测定：
[0032] 1)加 50μ1反应缓冲液于空白孔中；
[0033] 2)向反应缓冲液中加入 50μ1样品或标准品；
[0034] 3)各孔中依次加入 50μ1反应缓冲液；
[0035] 4)依次加入 50μ1格里斯试剂 （Griess Reagent) I于各孔中； [0036] 5)依次加入 50μ1格里斯试剂 （Griess Reagent) II于各孔中， 室温孵育 10分钟；
[0037] 6)酶标仪 540nm读取 OD值， 690nm作为校正。
[0038] (3)硝酸盐浓度测定：
[0039] 1)加 50μ1反应缓冲液于空白孔中；
[0040] 2)向反应缓冲液中加入 50μ1样品或标准品；
[0041] 3)各孔中依次加入 25μ1烟酰胺腺嘌呤二核苷酸 （NADH) ；
[0042] 4)各孔中依次加入 25μ1硝酸盐还原酶；
[0043] 5)依次加入 50μ1 Griess Reagent I于各孔中；
[0044] 6)依次加入 50μ1 Griess Reagent II于各孔中， 室温孵育 10分钟；
[0045] 7)酶标仪 540nm读取 OD值， 690nm作为校正。
[0046] 请参阅图 3A和 3B， 分别为硝酸盐饮水 3个月后血清中的硝酸盐和亚硝酸盐含量 的差异图 (*P<0.05)。 其中， 硝酸盐饮水显著增加了血清中硝酸盐及亚硝酸盐的 含量。
[0047] 4、 免疫指标的检测
[0048] (1) 大鼠外周血调节 T细胞比率检测
[0049] 1)各组大鼠腹主动脉取血， 离心取上清备用；
[0050] 2) 10(V1 0.1<¾ BSA重悬， 加入 Anti-CD4， Anti-CD25， 冰上孵育 30分钟， 避光
[0051] 3) 1500rpm离心 10分钟， 去上清， 0.1<¾ BSA重悬；
[0052] 4)加入破膜液， 4°C， 10分钟；
[0053] 5) 1500rpm离心 10分钟， 去上清， 0.1<¾ BSA重悬；
[0054] 6)加入 Anti-Foxp3， 冰上孵育 30分钟， 避光；
[0055] 7)离心， 去上清， 50(V1 0.1<¾BSA重悬；
[0056] 8)流式细胞仪检测。
[0057] (2) TGF-β IFN-γ及 IL-17的检测
[0058] 1)将血清稀释 10倍， 取 0.1 ml置入试剂盒 96孔板中， 同吋取试剂盒内标准品按 比例稀释后置入 96孔板中， 按说明书要求室温下 /37°C孵育 30分钟 /1小吋， 使用 洗液洗涤 3次， 每次 3分钟； [0059] 2)加入酶标抗体 100μ1， 室温下 /37°C孵育 15分钟 /30分钟， 洗涤 3次， 每次 3分 钟；
[0060] 3)加底物液 ΙΟΟμΙ显色， 室温下 /37°C孵育 15分钟；
[0061] 4)加 ΙΟΟμΙ终止液终止反应 5分钟；
[0062] 5)酶标仪 450nm下读数。
[0063] 图 4A-4D为硝酸盐对去势大鼠的免疫调节作用结果图 (*P<0.05)。 其中图 4A为取 各组大鼠外周血做流式分析得到的外周血 CD4阳性细胞中调节性 T淋巴细胞比例 。 如图所示硝酸盐饮水可缓解去势大鼠调节性 T淋巴细胞 （Treg) 的下降。 图 4B -4D分别为各组血清中 TGF-pi、 IFN-γ和 IL-17的含量图。 结果显示， 硝酸盐饮水 可缓解去势大鼠 TGF-βΙ的下调， 并缓解 IFN-γ和 IL-17的上调。
[0064] 5、 干细胞的增殖及成骨分化
[0065] (1) 大鼠 BMMSC的培养
[0066] 脱颈致死后剥去后肢的皮肤， 暴露股骨和胫骨后用 1 mL注射器吸取 ot-MEM完 全培养基 （含牛血清 10%) 从断端冲出骨髓， 待细胞克隆生长至 80%融合可传代 ， 传代培养至第三代。
[0067] (2) CCK8法
[0068] 在 96孔板中接种 ΙΟΟμΙ细胞悬液， 将培养板在培养箱预培养 24小吋 （在 37°C， 5 % C0 2的条件下） ， 随后将培养板在培养箱孵育 72小吋， 每孔换新的培养基 ΙΟΟμΙ, 并向每孔加入 l(Vl CCK-8溶液。 2小吋后用酶标仪测定在 450 nm处的吸光 度。
[0069] (3) 成骨诱导分化
[0070] 配制成骨诱导培养液： 含 10%胎牛血清 α-ΜΕΜ培养基中加入 2 mmol/L谷氨酰 胺， 100U/ml青霉素和 10(Vg/ml链霉素， ΙΟ ηιΜβ-甘油磷酸钠， 10ηΜ地塞米松 ， 50mg/L维生素 C。 第 3-4代细胞以 2xl03/cm2的浓度接种于 6孔板中， 等细胞生 长至 80%融合后， 更换为成骨诱导培养液， 每 2天换液 1次， 光镜下观察钙结节 形成情况。 于诱导 2周后， 进行茜素红染色。
[0071] (4) 茜素红染色
[0072] 1)去培养基， PBS洗 2次； [0073] 2) 70%乙醇固定， 4°C， lh;
[0074] 3)双蒸水洗 2次；
[0075] 4) 40mM茜素红溶液 （pH 4.2) 室温染色 1-10分钟， 肉眼观察着色情况； [0076] 5)双蒸水洗 5次， 轻轻吹打；
[0077] 6)镜下观察并采集图像；
[0078] 请参阅图 5A和 5B， 为硝酸盐改善去势大鼠 BMMSC的缺陷结果图 (*P<0.05)。 其 中图 5A中为按照上述方法取各组大鼠 BMMSC原代培养， 取第三代细胞行 CCK8 检测的 OD450值。 结果显示硝酸盐饮水显著降低去势大鼠 BMMSC的增殖能力。 图 5B为按照上述方法取各组大鼠 BMSSC行成骨诱导 21天， 并行茜素红染色及定 量结果图。 结果显示硝酸盐饮水显著增强去势大鼠 BMMSC的成骨分化能力。
[0079] 6、 统计学方法
[0080] 采用 SPSS 17.0统计学软件进行统计学分析， 多组计量资料比较采用 ANOVA 分析， P<0.05有统计学意义。
[0081] 本发明是根据特定实施例进行描述的， 但本领域的技术人员应明白在不脱离本 发明范围吋， 可进行各种变化和等同替换。 此外， 为适应本发明技术的特定场 合或材料， 可对本发明进行诸多修改而不脱离其保护范围。 因此， 本发明并不 限于在此公幵的特定实施例， 而包括所有落入到权利要求保护范围的实施例。

Claims

权利要求书
[权利要求 1] 硝酸盐在制备预防或治疗骨代谢疾病的药物中的应用。
[权利要求 2] 根据权利要求 1所述的应用， 其特征在于， 所述硝酸盐用于制备预防 或治疗骨密度异常疾病的药物。
[权利要求 3] 根据权利要求 1所述的应用， 其特征在于， 所述硝酸盐用于制备预防 或治疗骨质疏松症的药物。
[权利要求 4] 根据权利要求 1-3中任一项所述的应用， 其特征在于， 所述硝酸盐选 自下组中一种或者多种物质的组合： 硝酸钠、 硝酸钾和硝酸钙及硝酸
[权利要求 5] 根据权利要求 4所述的应用， 其特征在于， 所述硝酸盐以富含硝酸盐 的食物或提取物形式提供。
[权利要求 6] 根据权利要求 1或 2所述的应用， 其特征在于， 所述硝酸盐每日的给药 量为 0.1-0.5mmol/Kg b.w.。
[权利要求 7] 一种预防或治疗骨代谢疾病的药物， 其特征在于， 其中含有硝酸盐作 为活性成分。
[权利要求 8] 根据权利要求 7所述的预防或治疗骨代谢疾病的药物， 其特征在于， 所述预防或治疗骨密度异常药物的剂型为片剂、 胶囊、 丸剂、 注射剂
、 糖浆、 口服液、 颗粒剂或贴剂。
[权利要求 9] 硝酸盐在制备调节机体免疫异常的药物中的应用。
[权利要求 10] 硝酸盐在制备调节骨髓间充质干细胞异常疾病的药物中的应用。
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