CN117562899A - Application of DNJ composition in preparation of medicament for treating osteoporosis - Google Patents
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
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- 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
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- Health & Medical Sciences (AREA)
- Physical Education & Sports Medicine (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Chemical & Material Sciences (AREA)
- Rheumatology (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Organic Chemistry (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses an application of a DNJ composition in preparing a medicament for treating osteoporosis, and relates to the field of biopharmaceuticals. The active ingredients of the DNJ composition comprise DNJ and DNJ homologs; wherein DNJ homologs include, but are not limited to, alpha-1-C-alkyl-1-deoxynojirimycin, beta-1-C-alkyl-1-deoxynojirimycin. The content of effective components in DNJ composition is 6.9mg/kg-8.8mg/kg. The application of the DNJ composition in preparing the medicament for treating the osteoporosis can be widely used for preparing the medicament for treating the osteoporosis.
Description
Technical Field
The invention relates to the technical field of biological pharmacy, in particular to application of a DNJ composition in preparation of a medicament for treating osteoporosis.
Background
Osteoporosis is the most common skeletal disease affecting the health of residents in China, the early stage of the disease usually has no obvious clinical manifestation, if the disease is not emphasized, the conditions such as pain, spinal deformation, fracture and the like can be caused along with the progress of the disease, and the life quality of patients is seriously affected. Bone homeostasis is maintained by a balance between osteoblast bone formation and osteoclastic bone resorption. The existing anti-osteoporosis drugs on the market mainly comprise basic supplements, namely, drugs for inhibiting bone resorption and drugs for promoting bone formation, but more drugs for inhibiting bone resorption are actually clinically used, so that the basic problem of the senile osteoporosis is difficult to solve, and part of the anti-osteoporosis drugs have partial side effects, so that a natural product capable of promoting bone formation is searched for to develop a new anti-osteoporosis drug.
DNJ full name 1-Deoxynojirimycin, chinese name 1-Deoxynojirimycin, molecular formula C 6 H 13 NO 4 The molecular weight is 163.17, CAS number is 19130-96-2, and the mulberry extract is obtained. DNJ, a natural product, is a potent oral alpha-glucosidase (alpha-glucosidase) inhibitor. The mulberry can grow in tropical to arctic regions, the variety is various, the planting is wide, DNJ can be extracted from different parts of the mulberry, the content of the mulberry is high in the mulberry leaves and the mulberries, and the mulberry branches are the next step. The mulberry species contains various bioactive substances and is widely studied, and the natural product DNJ extracted from the mulberry is known to inhibit postprandial blood sugar, prevent diabetes mellitus and have the effects of reducing blood sugar, losing weight and resisting viruses. There are four main routes to produce DNJ: firstly, extracting from plants such as mulberry and the like; (II) extracting from silkworms; (III) chemical synthesis adopting different synthesis strategies; (IV) its adjuvant therapeutic effects on diabetes have been studied extensively before by fermentation with various Bacillus or Streptomyces, which show that DNJ plays a role in bone formation and bone content in wild-type micePromoting effect, and can be used for preparing medicine for treating osteoporosis.
Disclosure of Invention
The invention aims to provide an application of DNJ composition in preparing a medicament for treating osteoporosis, which can be widely used for preparing the medicament for treating osteoporosis.
To achieve the above object, the present invention provides an application of a DNJ composition in preparing a medicament for treating osteoporosis.
Further, the active ingredients of the DNJ composition comprise DNJ and DNJ homologs; wherein DNJ homologs include, but are not limited to, alpha-1-C-alkyl-1-deoxynojirimycin, beta-1-C-alkyl-1-deoxynojirimycin.
Further, the content of the active ingredient in the DNJ composition is 6.9mg/kg-8.8mg/kg.
Further, the medicament comprises powder, granule, tablet, capsule, pill, solution, suspension or injection.
Further, the structure of DNJ is shown as
Further, the DNJ composition may further comprise auxiliary ingredients such as carriers, excipients, flavoring agents, etc., which do not affect the effect of the active ingredient.
Further, the DNJ composition increases osteoblast differentiation, repairing damage to osteoblasts from oxidative stress.
The DNJ composition has the advantages and positive effects that:
1. the DNJ composition has the functions of increasing osteoblast differentiation and preventing osteoporosis of a high-fat mouse, and can be used for preparing medicines for treating osteoporosis.
2. The DNJ composition provided by the invention can repair the damage of oxidative stress to osteoblasts, increase the osteogenic differentiation of the cells, and can be used for preparing medicines for treating osteoporosis.
The technical scheme of the invention is further described in detail through the drawings and the embodiments.
Drawings
FIG. 1 shows changes in cancellous bone of an OVX model mouse after DNJ treatment in the present invention, wherein A is a 3D schematic diagram of cancellous bone of the mouse, B is the bone mass/tissue mass of cancellous bone of the mouse, C is the development thickness of cancellous bone structure of the mouse, D is the number of trabeculae of bone of the mouse, and E is the separation degree of cancellous bone structure of the mouse;
FIG. 2 shows the change of cancellous bone of a mouse after DNJ treatment of a high fat diet osteoporosis mouse model according to the present invention, wherein A is a 3D schematic diagram of cancellous bone of the mouse, B is the bone mass/tissue mass of cancellous bone of the mouse, C is the development thickness of cancellous bone structure of the mouse, D is the number of trabeculae of the mouse, and E is the separation degree of cancellous bone structure of the mouse;
FIG. 3 is a graph showing the effect of DNJ on mineral nodule staining following primary osteoblast differentiation in accordance with the present invention;
FIG. 4 shows the effect of DNJ on RNA molecular signaling of HIF1α and AXIN2 in primary osteoblasts according to the present invention.
Detailed Description
The technical scheme of the invention is further described below through the attached drawings and the embodiments. Unless defined otherwise, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this invention belongs. In the examples below, biochemical reagents not specifically described are all conventional in the art and can be formulated according to conventional methods in the art or are commercially available in the laboratory pure grade format.
In the drug screening experiment adopted by the invention, the micro-CT detection of the thighbone after various mice are killed is a classical animal bone mass detection experiment, and the osteoblast mineralization nodule staining experiment is a classical experiment for detecting the osteoblast function, so that the reliability of the screening result can be ensured.
Example 1 effect of DNJ on wild type C57BL6J female mouse bone after OVX surgery.
Experimental animals: c57BL6J mice, females, supplied by si Bei Fu laboratory animal technologies limited. Mice were subjected to OVX surgery at 16 weeks of age, single cage after surgery, free water, i.e. i.p. injection of DNJ and control saline for 6 weeks starting at 17 weeks of age, then sacrificed and femur was taken for micro-CT scan to detect bone mass.
Experimental medicine: DNJ drug was purchased from selectk reagent company under the product number S3839.NaCl was purchased from biosharp reagent company.
Experimental equipment: weighing scale, scissors, forceps and injector.
The experimental steps are as follows:
OVX surgery:
after anesthetizing the mice, the mice were shaved, and no hair was required for about 1 cm of wound. The skin surface of the mice was sterilized with iodophor and then sterilized with 75% alcohol. The abdominal cavity was opened, the ovaries were found, the ovaries were removed after ligating the fallopian tubes (the control group opened the abdominal cavity, and the ovaries were found by the same procedure, but without excision). The peritoneal and skin layers were sutured separately, the wound surface was spiked with antibiotic, and mice were injected with 0.1g/kg body weight of antibiotic and 200 μl of 20% glucose.
Treatment of the abdominal cavity of the animal:
the mice were intraperitoneally injected with DNJ at 80mg/kg body weight daily, and the control group was intraperitoneally injected with physiological saline, and the body weight was measured at 10 morning daily. After 6w of intraperitoneal injection, the femur was sacrificed and collected for scanning analysis of bone mass and thickness.
Statistical analysis:
experimental results are expressed as mean ± SEM, and significance between different treatment groups is analyzed by t-test.
Analysis of results:
as shown in fig. 1, adult wild-type C57BL6J females were subjected to OVX surgery as a model of estrogen deficiency induced osteoporosis, and DNJ (80 mg/kg body weight/day) treated by intraperitoneal injection for 6 weeks showed a significant increase in bone mass in the DNJ-treated groups (animals=6, p < 0.05 per group). The results show that DNJ can significantly increase bone mass/tissue mass and cancellous bone content of the femur of OVX mice.
Example 2 effect of DNJ on bone of wild type C57BL6J male mice fed with high fat.
Experimental animals: c57BL6J mice, male, supplied by s Bei Fu laboratory animal technologies inc. Wild mice are fed with high-fat grains from 4 weeks of age, DNJ and normal saline of a control group are injected intraperitoneally after 14w for 8 weeks, and the mice are sacrificed, and femur is taken for micro-CT scanning to detect bone mass.
Experimental medicine: DNJ drug was purchased from selectk reagent company under the product number S3839.NaCl was purchased from biosharp reagent company. High fat feeds were purchased from research diets, cat No. D12493. The common feed is provided by animal houses of Chinese agricultural university.
The experimental steps are as follows:
constructing a high-fat mouse model:
wild mice are fed with high-fat grains from the age of 4 weeks, the weight of the wild mice is obviously different from that of a control group after feeding for 10 weeks, the model construction is completed, and the wild mice are continuously fed with high-fat feed in the subsequent intraperitoneal injection process.
Treatment of the abdominal cavity of the animal:
the mice were intraperitoneally injected with DNJ at 80mg/kg body weight daily, and the control group was intraperitoneally injected with physiological saline, and the body weight was measured at 10 morning daily. After 8w of intraperitoneal injection, the femur was sacrificed and collected for scanning analysis of bone mass and thickness.
Statistical analysis: experimental results are expressed as mean ± SEM, and significance between different treatment groups is analyzed by t-test.
Analysis of results:
as shown in fig. 2, wild type C57BL6J male mice were fed a high fat diet for 10 weeks from 4 weeks of age, induced osteoporosis models, and DNJ (80 mg/kg body weight/day) treated by intraperitoneal injection for 8 weeks showed a significant increase in bone mass in the DNJ-treated groups (animals=6, p < 0.05 per group). The results indicate that DNJ can significantly increase bone mass/tissue mass and cancellous bone content of the femur of high fat fed mice.
Example 3 effect of DNJ on osteogenic differentiation of primary osteoblasts in mice.
Experimental animals: the C57BL6J fetal mouse just born. Supplied by the laboratory animal technologies company, s Bei Fu.
Experimental drugs: DNJ drug was purchased from selectk reagent company under the product number S3839. Medium was purchased from Corning, and other chemicals were purchased from Sigma reagent.
Experimental instrument: cell culture plates, cell culture dishes, pipettes, scissors, forceps, centrifuges, 50ml centrifuge tubes.
The experimental steps are as follows:
1. primary cell isolation:
primary osteoblasts were isolated from the skull of a freshly born mouse, after shearing the skull into 2mm square fragments, digested with collagenase type I at 37 degrees celsius, the first two digestions were discarded, and the last three digestions were collected and centrifuged to obtain primary cells, which were cultured in a-MEM medium containing 10% fetal bovine serum.
2. Cell differentiation and drug treatment:
after cells had grown to a density of 100%, they were grown with 200mM H 2 O 2 Cells 4d were stimulated and 1. Mu.M DNJ-treated cells were differentiated throughout, and 10. Mu.g/ml of ascorbic acid and 10mM of beta-glycerophosphate were added to the cell differentiation medium in the form of 10% fetal bovine serum in alpha-MEM medium. Alizing and differentiating for 14 days, and staining with alizarin red to mineralize nodule substances in cells. The difference of mineralization degree is judged by observing the color depth after dyeing.
3. Expression of hif1α and AXIN2 molecular signals in cells:
200mM H 2 O 2 and 1 mu MDNJ to stimulate cells for 24 hours, collecting cells by using Trizol, extracting RNA, carrying out reverse transcription, and detecting the expression levels of HIF1α and AXIN2 by using an RT-PCR method.
Analysis of results: FIG. 3 is a graph showing the nodulation staining of mineral after differentiation of primary osteoblasts by DNJ, as can be seen from FIG. 3, H 2 O 2 Stimulation resulted in reduced primary osteoblast differentiation, and DNJ had a repair effect on this, indicating that DNJ had the effect of repairing hydrogen peroxide damage to primary osteoblasts.
FIG. 4 shows the effect of DNJ on the RNA molecular signals of HIF1α and AXIN2 in primary osteoblasts, H as shown in FIG. 4 2 O 2 Stimulation resulted in an increase in the level of hypoxia-inducible factor hif1α and the inhibitor of bone synthesis of AXIN2 RNA in primary osteoblasts, whereas DNJ treatment significantly reversed their changes, indicating that DNJ has the effect of repairing hydrogen peroxide on primary osteoblast injury.
Alizarin Red Staining (ARS) showed 200mM H 2 O 2 Stimulation of cells significantly reduced osteoblast mineralization nodules while osteoblast mineralization nodules were significantly increased with cells treated with 1 μm DNJ (fig. 3); 200mM H 2 O 2 After 24h of primary osteoblast stimulation, cellular RNA was extracted, wherein hif1α (hypoxia inducible factor 1α) was significantly increased, and hif1α was significantly decreased after 1 μm DNJ administration (fig. 4), indicating that DNJ can repair the damage of oxidative stress to osteoblast cells, increasing osteoblast differentiation of cells.
Therefore, the DNJ composition has the effects of increasing the differentiation of osteoblasts and preventing the osteoporosis of a high-fat mouse, can repair the damage of oxidative stress to the osteoblasts, and can increase the osteogenic differentiation of the cells, and can be used for preparing the medicament for treating the osteoporosis.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention and not for limiting it, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that: the technical scheme of the invention can be modified or replaced by the same, and the modified technical scheme cannot deviate from the spirit and scope of the technical scheme of the invention.
Claims (3)
- An application of a DNJ composition in preparing a medicament for treating osteoporosis, which is characterized in that: the content of the active ingredients in the DNJ composition is 6.9mg/kg-8.8mg/kg.
- 2. The use according to claim 1, characterized in that: the active ingredients of the DNJ composition comprise DNJ and DNJ homologs; wherein the DNJ homolog is alpha-1-C-alkyl-1-deoxynojirimycin or beta-1-C-alkyl-1-deoxynojirimycin.
- 3. The use according to claim 1, characterized in that: the medicine comprises powder, granule, tablet, capsule, pill, solution, suspension or injection.
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李晶晶等: "1-脱氧野尻霉素的结构修饰及其活性的研究进展", 现代药物与临床, vol. 38, no. 4, 30 April 2023 (2023-04-30), pages 2 - 5 * |
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