CN106668001A - Application of cnidium lactone to preparation of osteoportic fracture drugs - Google Patents
Application of cnidium lactone to preparation of osteoportic fracture drugs Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to application of cnidium lactone to the preparation of osteoportic fracture drugs. According to the application, the cnidium lactone is discovered to promote osteogenesis in membranes and cartilages by regulating activities of cartilage cells and osteoblast, and promote the healing of osteoportic fracture. The mechanism that the cnidium lactone accelerates the healing of osteoportic fracture is to promote the proliferation of muscle-derived stem cells and osteoblast differentiation and migration by being applied to a beta-Catenin signal path. The cnidium lactone can be applied to the preparation of drugs for treating osteoportic fracture.
Description
Technical Field
The invention relates to application of osthole in preparing a medicine for treating osteoporotic fracture.
Background
Osteoporosis is a common metabolic, complex bone disease.
With the aging population, the incidence of osteoporosis in China increases year by year, and is better for postmenopausal women and old men. The population number of people over 40 years old in China is up to 5.68 hundred million, while the number of continental osteoporosis patients is 1.12 hundred million, accounting for 19.7 percent of the total number, wherein the number of women is up to 13.2 percent, and the number of men is up to 6.5 percent. The pathology of the fracture is characterized by reduction of bone mass and degradation of microstructure of bones, which leads to increase of brittleness of bones, and fracture can be caused under the action of slight external force, and fracture of parts such as centrum, hip, humerus, distal radius and the like is common.
The fracture caused by osteoporosis has the characteristics of high morbidity, disability causing rate, high fatality rate and high medical cost, in 2006, 68.7 million people with hip fracture are expected to happen in China over 50 years old, 163.8 and 590.8 million people with hip fracture are expected to happen in 2020 and 2050 years, 3675 and 4850 million people with vertebral body fracture are expected to happen, and in 2020 and 2050 years, the medical cost on the aspect of osteoporosis fracture is expected to rise from 103.8 million yuan to 850 and 18000 million yuan in 2006, so the osteoporosis fracture seriously threatens the health of middle-aged and elderly people. At the same time, bone metabolism disorders (osteoclast hyperfunction, osteoblast hypofunction) lead to a decrease in bone quality. The clinical treatment of osteoporotic fracture is easy to cause complications such as fracture nonunion or malformation union, or secondary operation, etc.
Therefore, there is a need to find an economical, safe and effective drug or method for promoting the healing of osteoporotic fracture, so as to solve the delayed or nonunion of fracture, reduce the physical pain and economic burden of patients.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, it is desirable to provide an economical, safe and effective drug or method for promoting osteoporotic fracture healing, which can solve delayed or nonunion of fracture, reduce physical pain and economic burden of patients.
Osthole (Osthole) also called methoxy-parsley phenol or parsley phenol methyl ether is the main effective component of Chinese herbal medicines such as cnidium monnieri (L.) DC and pubescent angelica root (L.) DC of Umbelliferae. Modern pharmacological research finds that osthole has various biological activities, such as antioxidation, anti-inflammation, anti-osteoporosis, anti-apoptosis, estrogen-like and broad-spectrum antibacterial action. The invention provides application of osthole in preparing a medicament for treating osteoporotic fracture.
The medical application of the invention is obtained by finding the effect and curative effect of osthole on an osteoporosis fracture animal model.
The subsequent in vivo experiments show that osthole can obviously promote the healing of callus of aged osteoporotic fracture mice in different periods, including promoting intramembranous osteogenesis and intrachondral osteogenesis in 0-7 days of the early stage of fracture, and a large amount of hypertrophic chondrocyte hyperplasia can be seen near the broken end; in the middle and later period of fracture, 14-28 days later, chondrocytes are apoptotic, a large amount of proliferation of osteoblasts is promoted, and the number of osteoclasts is not inhibited, so that the callus can be shaped and reconstructed, and the marrow cavity is communicated. Osthole promotes the expression of beta-catenin in broken muscle and callus of fracture. Osthole increases the number of myogenic stem cells in the fractured muscle and promotes proliferation thereof. Osthole promotes myogenic stem cells to migrate to callus, thereby stimulating osteogenesis and accelerating fracture healing.
The dose of the osthole of the invention is 5mg/kg/d when the osthole is used for treating osteoporotic fracture. Wild type C57BL/6 mice were dosed with osthole solution at a dosing concentration of 5mg/kg/d and a maximum dosing volume of 60ul/d (40ul intraperitoneal injection, 20ul injection at the broken end of fracture), and were observed for three periods of 7, 14 and 28 days, with no mice dying and no obvious toxic reaction. The maximum dose of osthole in pharmacopoeia is 9g crude drug per day, calculated by 70kg body weight, which is equivalent to 0.13g crude drug/kg/d, and the prepared concentration is 5g crude drug/ml. The maximum administration volume of each gastric lavage of the mouse is 40ml/kg, the single gastric lavage administration is carried out, the total amount is 200g of crude drugs/kg, and the maximum daily dose is 1538 times of the maximum daily dose of the human drug dictionary.
The pharmaceutical compositions provided herein can be prepared according to methods known in the art and can be administered orally, sublingually, transdermally, intramuscularly, subcutaneously, mucocutaneously, urethrally, vaginally, or intravenously. The oral preparation comprises tablet, capsule, granule, pill, chewing agent, solution, etc. The parenteral preparation can be prepared into injection, freeze-dried powder and the like. The topical administration can be made into cream, emulsion, ointment, patch or spray. But is not limited thereto.
Drawings
The effect and the curative effect of osthole on the osteoporosis fracture animal model are particularly clear in the following relevant experimental results, and the following drawings are referred: wherein,
FIG. 1 is an X-Ray observation picture of the fracture line of osthole-intervened osteoporotic fracture at 7, 14 and 28 days.
FIG. 2 is a diagram of the microbubt three-dimensional reconstruction of callus at 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 3 is a graph comparing data of callus volume, joint density and bone density analyzed by Micro-CT scanning of osthole intervention osteoporotic fracture 7, 14, 2 days 8.
FIG. 4 is a graphical representation of finite element analysis of osthole biomechanics for 28 days of osthole intervention in osteoporotic fractures.
FIG. 5 is a view showing the observation of osthole-eosin (HE) staining in 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 6 is a graph showing the staining observation of Elsinoe carinii blue/orange red (ABH/OG) on 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 7 is a photograph showing the staining observation of callus osteoclast (Trap) in 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 8 is a photograph showing immune histochemical staining of callus Osteoprotegerin (OPG) for 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 9 is an immunohistochemical staining observation picture of beta-catenin in the fracture broken end skeletal muscle of 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 10 is the immune histochemical staining observation picture of callus beta-catenin for 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 11 is the photograph of immune histochemical staining for callus Runx2 in 7, 14 and 28 days after osthole intervention in osteoporotic fracture.
FIG. 12 is a graph showing the observation of double fluorescence immunohistochemical staining of Pax7 (paired box gene 7, a unique marker protein molecule of skeletal muscle stem cells) and Sca1 (stem cell surface marker) at the fracture ends of 7, 14 and 28 days after osthole intervention in osteoporotic fractures.
FIG. 13 is a photograph of Bifluorescence immunohistochemical staining of skeletal muscle Pax7 and Brdu (5-bromodeoxyuridine-showing cell proliferation) at the fracture ends on days 7, 14, and 28 of osthole-mediated osteoporotic fracture.
FIG. 14 is a picture of observation of double-fluorescence immunohistochemical staining of callus Pax7 and Sca1 on days 7, 14 and 28 of osthole-mediated osteoporotic fracture.
FIG. 15 is a picture of observation of double-fluorescence immunohistochemical staining of callus Pax7 and Sca1 on days 7, 14 and 28 of osthole-mediated osteoporotic fracture.
Detailed Description
The invention is further illustrated with reference to the following figures and specific examples. These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.
Research on effect of osthole in promoting healing of senile (12-month-old) osteoporosis fracture mice
1 materials and methods
1.1 Experimental animals
SPF grade C57BL/6 mice at 12 months of age were purchased at the institute of Chinese college animals.
1.2 Experimental drugs
Osthole has purity of more than 98.5% and CAS NO of 484-12-8 from Shanghai Yonghengshi Co., Ltd; 10% chloral hydrate injection, batch No.: 20081027, national drug group chemical reagents, Inc.; DMSO (dimethyl sulfoxide), CODE:0231 amresco; corn Oil (Corn Oil), sigma.
1.3 Experimental Equipment
uCT80 MicroCT (SCANCO Medical, CHE), DISCOVERY dual-energy X-ray bone densitometer, CMISA-99B image analysis management system, LE-80K ultracentrifuge, Leica TP1020 tissue hydroextractor, Leica EG1160 type full-automatic paraffin embedding machine, Leica RM2135 rotary microtome, Leica Histobarth type HI1210 sheet spreader, Leica Histoplast type HI1220 drying machine, Leica LBS EMLR microscope and photographic system, Olympus full-tissue scanner, Olympus VS120-SL, Eppendorf 5415D high speed centrifuge
1.4 animal grouping and processing methods
SPF grade C57BL/6 mice of 12 months of age were selected, 16 mice per time point on days 7, 14, 28, and randomized into osthole and placebo groups of 8 mice each. After a mouse is anesthetized by chloral hydrate (0.3ml/100g) through intraperitoneal injection, the mouse is taken out of the supine position, the left tibia is cleaned, the mouse hair is cleaned, iodophor is sterilized, the skin is cut through the front edge of the tibia in an aseptic operation, fascia and muscle on the inner side and the outer side of the upper part 1/3 (above the tibial spine) of the tibia are separated in a blunt manner, an intramedullary needle head is inserted into the upper 1/3 part (the upper part of the broken end) of the tibia from the tibial platform in advance, the deep muscle on the inner side of the tibia is avoided at the 1/3 part by surgical scissors, the tibia is completely transected, the needle head is inserted into the bone cavity on the lower part of the broken end, the length of about 3/4 is cut, and then. On the next day after operation, 40ul and 20ul of osthole are injected into the fracture part of the tibia on the abdominal cavity and the left side respectively by the observation group, mice are killed and materials are taken after 7 days, 14 days and 28 days after the fracture operation, after all tissues are fixed by 10% neutral formaldehyde for 48 hours, 75% ethanol is replaced for long-term fixation, and meanwhile, relevant index detection is carried out.
1.5 detection index
1.5.1 imaging examination:
and (4) carrying out X-ray examination on the broken end of the left tibia fracture, and carrying out Micro-CT scanning analysis on the left tibia callus.
1.5.2 bone histomorphometry analysis:
after the imaging detection, the left tibia is decalcified by 14% EDTA, after the calcium is completely decalcified for about 20 days, a Leica TP1020 tissue dehydrator is used for gradient dehydration, wax dipping and embedding, a slicer is used for cutting into sections with the thickness of 5mm along the sagittal plane of the tibia, airing and baking the sections, and later pathological tissue morphological analysis (such as HE, ABH/OG, Trap staining, immunohistochemistry, immunofluorescence staining and the like) is carried out.
1.5.3 muscle tissue section analysis:
taking out fixed soleus muscle, washing with PBS, soaking for 1-2 hr, performing gradient dehydration, wax soaking, embedding with Leica TP1020 tissue dehydrator, slicing into 5mm thick slices along the coronal plane of muscle with a slicing machine, air drying, baking, and performing later stage pathological histomorphometry (such as HE and immunofluorescence staining).
1.5.4 biomechanical index detection:
and (3) carrying out mechanical analysis on the 28d callus reconstructed in the Micro-CT three-dimensional mode through finite element analysis software, wherein the measured parameters comprise structural rigidity, breaking load, cross-sectional area and elastic modulus, and calculating by the software.
1.6 statistical analysis:
the counting index is mean number plus or minus standard deviationOne-Way ANOVA analysis was performed between multiple samples with the SPSS18.0 software package, and q-test was performed between two samples, with double side α being 0.05.
2 results
2.1 action of osthole in promoting formation and development of tibial callus of senile osteoporosis fracture mouse in different time periods
FIG. 1X-Ray shows that the tibial fracture line of mice in 7 days and 14 days of osthole group is fuzzy compared with that of blank group, the tibial fracture line disappears in 28 days, the blank group can still see the existence of the tibial fracture line, and no obvious healing trend is seen at the fracture broken end at three time points.
Fig. 2 Micro-CT shows that the morphology and density of the callus at 7, 14 and 28 days of the osthole group are better than those of the blank group, and fig. 3 data analysis further shows that the osthole group promotes the formation of the callus at the fractured ends of the mice at the early stage (7 and 14 days), and indexes such as trabecular bone volume to callus volume ratio (BV/TV), connection density (Conn-Dens) and callus bone density (BMD) are better than those of the blank group (P is less than 0.01), although the data at 28 days has no obvious difference from the blank group, the fractured ends of the osthole group are considered to be basically healed at 21 days, and the white group in 28 sky is considered to be basically healed, so that no obvious difference is seen between the two groups. However, the biomechanics-finite element analysis result in fig. 4 shows that the structural rigidity, the breaking load and the elastic modulus of the osthole in the osthole group are higher than those of the blank group, and the combination of the results shows that the healing time and the quality of the osthole in the osthole group are better than those of the blank group.
2.2 Effect of osthole in regulating and controlling the activity of cartilage and osteoblast of mice with senile osteoporosis fracture
FIG. 5 HE staining and FIG. 6ABH/OG staining revealed that the cartilage at the fractured end of the fracture was greatly proliferated at 7 days, the chondrocytes were apoptotic and the osteoblasts began to increase at 14 days in the osthole group, while the blank group formed soft callus at 14 days, which was delayed in healing by one week compared to the osthole group. The osthole can remarkably regulate the activities of cartilage and osteoblast in different time periods, and promote intramembranous osteogenesis and intrachondral osteogenesis of the osteoporotic fracture so as to improve delayed healing and nonhealing conditions of the osteoporotic fracture.
2.3 Effect of osthole in regulating and controlling osteoclast activity of mice with senile osteoporosis fracture
FIG. 7 Trap staining shows that there was a large amount of osteoclast formation in the callus at the fractured end of the fracture on days 7, 14 and 28 in the blank groups, whereas osthole was formed only in a small amount at 7 days and osteoclast was increased at 14 and 28 days.
FIG. 8 shows that the deep brown positive staining of early (7 days) osthole OPG immunohistochemistry is obviously higher than that of blank group, while the positive staining of the two groups is not obviously different at 14 and 28 days.
FIGS. 7 and 8 show that osthole inhibits osteoclast proliferation in early stage, and only has a small amount to absorb necrotic bone tissue; chondrocytes started to apoptosis at 14 days, osteoblasts increased greatly, and a certain amount of osteoclasts were required to absorb apoptotic chondrocytes and to begin callus remodelling; the callus remodeling stage is reached at 28 days, requiring the resorption of excess callus by osteoclasts to recanalize the marrow cavity to form normal bone. Therefore, the traditional Chinese medicine (osthole) has the function of balancing the normal functions of cells, tissues and organs of the organism, namely the balance of yin and yang.
2.4 molecular mechanism study of osthole in promoting healing of senile osteoporosis fracture
FIG. 10 shows that beta-catenin immunohistochemical staining of the fractured muscle shows that compared with the blank group, the osthole group has more beta-catenin positive expression cells in the fractured muscle at 7, 14 and 28 days of treatment, which indicates that the osthole can activate the beta-catenin signal channel of the fractured muscle.
FIG. 11 shows that compared with the blank group, the osthole group has more beta-catenin positive expression cells in the callus at 7, 14 and 28 days of treatment, which indicates that osthole can activate callus beta-catenin signal channels.
FIG. 12 Runx2 immunohistochemical staining of callus shows that compared with blank group, osthole group has more Runx2 positive expression cells in callus at 7, 14 and 28 days of treatment, indicating that osthole can promote osteogenesis.
FIG. 13 double fluorescent immunohistochemical staining of the fractured muscles Pax7 and Sca1 shows that the osthole group has more cells with double expression of Pax7 and Sca1 in the fractured muscles at 7, 14 and 28 days of treatment compared with the blank group, and indicates that the osthole can increase the number of myogenic stem cells in the fractured muscles.
FIG. 14 double fluorescent immunohistochemical staining of the fractured muscles Pax7 and Brdu shows that the osthole group has more Pax7 and Brdu double-expressed cells in the fractured muscles at 7, 14 and 28 days of treatment compared with the blank group, and indicates that the osthole can promote the proliferation of myogenic stem cells in the fractured muscles.
FIG. 15 shows that the Pax7 and Sca1 double-fluorescence immunohistochemical staining of callus shows that compared with the blank group, the osthole group has more cells with double expression of Pax7 and Sca1 in callus at 7, 14 and 28 days of treatment, and the osthole can promote myogenic stem cells to migrate to the fracture end and stimulate osteogenic differentiation to form callus.
The results in fig. 10-15 show that the molecular mechanism of osthole for promoting the healing of senile osteoporosis fracture is to increase the number of myogenic stem cells by regulating the beta-catenin signal pathway, promote the proliferation and migration of myogenic stem cells and stimulate the differentiation of myogenic stem cells to osteogenesis.
By combining the experimental results, the osthole can promote intramembranous osteogenesis and intracartilaginous osteogenesis of osteoporotic fracture through a beta-catenin signal pathway, and accelerate fracture healing; and the beta-catenin signal channel is acted to promote the proliferation and differentiation of myogenic stem cells in skeletal muscle near the fracture end, promote the myogenic stem cells to migrate to the callus part, induce the myogenic stem cells to differentiate specifically into osteogenesis, and participate in the repair of fracture.
Claims (4)
1. Application of osthole in preparing medicine for treating osteoporosis fracture is provided.
2. Use according to claim 1, characterized in that the osthole has a purity > 98.5%.
3. Use according to claim 2, characterized in that the administration concentration is 5 mg/kg/d.
4. Use according to any one of claims 1 to 3, wherein the osthole is administered orally, sublingually, transdermally, intramuscularly, subcutaneously, mucocutaneously, urethrally, vaginally or intravenously.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114344258A (en) * | 2022-02-15 | 2022-04-15 | 上海中医药大学附属龙华医院 | A special preparation of osthole for improving sarcopenia and muscular atrophy after fracture |
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| CN114344258A (en) * | 2022-02-15 | 2022-04-15 | 上海中医药大学附属龙华医院 | A special preparation of osthole for improving sarcopenia and muscular atrophy after fracture |
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