DRUG FOR PREVENTING AND TREATING OSTEOPOROSIS AND USES THEREOF
20190365871 ยท 2019-12-05
Assignee
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
International classification
Abstract
The present invention provides uses of plasminogen in preventing and/or treating osteoporosis and diseases related to the osteoporosis. The present invention also provides a drug and a product for preventing and/or treating osteoporosis.
Claims
1. A method for preventing and treating osteoporosis and its related conditions, comprising administering a therapeutically effective amount of plasminogen to a subject.
2. The method of claim 1, wherein the osteoporosis comprises primary osteoporosis and secondary osteoporosis.
3. The method of claim 1, wherein the primary osteoporosis comprises postmenopausal osteoporosis and senile osteoporosis.
4. The method of claim 1, wherein the secondary osteoporosis comprises osteoporosis secondary to an endocrine disease, a rheumatic disease, and a gastrointestinal disease, and osteoporosis caused by a drug therapy.
5. The method of claim 4, wherein the secondary osteoporosis comprises osteoporosis caused by a glucocorticoid, primary hyperparathyroidism, hyperthyroidism, primary biliary cirrhosis, hypogonadism, diabetes mellitus, hypertension, atherosclerosis, a chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, osteoarthritis, a gonadal hormone therapy, an antiepileptic drug therapy, and a chemotherapeutic drug therapy.
6. The method of claim 1, wherein the osteoporosis is osteoporosis complicated with a disease, and wherein the osteoporosis complicated with a disease comprises osteoporosis complicated with a glucocorticoid therapy, primary hyperparathyroidism, hyperthyroidism, primary biliary cirrhosis, hypogonadism, diabetes mellitus, hypertension, atherosclerosis, a chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, osteoarthritis, a gonadal hormone therapy, an antiepileptic drug therapy, and a chemotherapeutic drug therapy.
7. (canceled)
8. The method of claim 7, wherein the subject comprises a subject receiving a glucocorticoid, or a subject with primary hyperparathyroidism, hyperthyroidism, primary biliary cirrhosis, hypogonadism, diabetes mellitus, hypertension, atherosclerosis, a chronic kidney disease, rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, or osteoarthritis.
9. (canceled)
10. A method for enhancing activity of osteoblasts, comprising administering an effective amount of plasminogen to a subject.
11. A method for regulation of bone mineral metabolism, comprising administering an effective amount of plasminogen to a subject.
12. The method of claim 11, wherein the regulation comprises lowering a blood calcium level, increasing a blood phosphorus level, promoting calcium deposition in a bone matrix and/or reducing calcium deposition in a blood vessel wall and an internal organ.
13. The method of claim 1, wherein the plasminogen has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 2 and still has the plasminogen activity.
14. (canceled)
15. The method of claim 1, wherein the plasminogen is a protein that comprises a plasminogen active fragment and still has the plasminogen activity.
16. The method of claim 1, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, delta-plasminogen or their variants that retain the plasminogen activity.
17. The method of claim 1, wherein the plasminogen is a natural or synthetic human plasminogen, or a variant or fragment thereof that still retains the plasminogen activity.
18-20. (canceled)
21. The method of claim 1, wherein the subject is a human.
22-29. (canceled)
30. The method of claim 1, wherein the plasminogen is administered in combination with one or more additional means or additional drugs.
31. The method of claim 30, wherein the additional drugs comprise drugs for treating osteoporosis, or drugs for treating other diseases complicated with osteoporosis.
32-35. (canceled)
36. The method of claim 1, wherein the plasminogen is administered to the subject at a dosage of 1-100 mg/kg, 1-50 mg/kg, or 1-10 mg/kg, daily, every other day, or weekly.
37. The method of claim 36, wherein the dosage of the plasminogen is repeated at least once.
38. The method of claim 36, wherein the plasminogen is administered at least daily.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0117]
[0118]
[0119]
[0120]
[0121]
[0122]
[0123]
[0124]
[0125]
[0126]
[0127]
[0128]
[0129]
[0130]
[0131]
[0132]
[0133]
[0134]
[0135]
[0136]
[0137]
[0138]
[0139]
[0140]
[0141] In summary, plasminogen can remarkably ameliorate osteoporosis, promote the increase in the bone mineral density and bone mass in various portions of the femur, and the amelioration of the trabecular bone is particularly obvious.
[0142]
[0143]
[0144]
[0145]
[0146]
[0147]
EXAMPLES
[0148] Materials and Methods:
[0149] Animals: C57 mice, and Plg.sup.+/+ and Plg.sup./ mice (Jackson Lab) were used for related experiments. The animals were fed in an experimental animal use environment that meets the national standard.
[0150] Reagents: vitamin D (Sigma Aldrich, Cat# D1530), corn oil (Sigma Aldrich, Cat# C8267), low calcium special diet (0.2% of calcium, 1% of a phosphate, and 2000 U vitamin D3/kg, from Nantong TROPHIC Feed Technology Co., Ltd., 15 kg), calcium content assay Kit (Nanjing Jiancheng Bioengineering Institute, Cat# C004-2), and human plasminogen (10 mg/ml, purified from healthy plasma donors).
[0151] Aloka Micro CT, which is designed exclusively for the observation of mouse and rat morphologies and incorporates the latest third-generation X-ray measurement, is capable of presenting high-quality tomographic images within a short time. It can be used for bone measurement (bone mineral density, bone mineral content, bone volume, bone microstructure, etc.), body fat percentage measurement, visceral and subcutaneous fat identification and measurement, synchronous photography, etc. Bone measurements were carried out on mouse femurs, craniums or lumbar vertebrae as a detection object. After the mice were sacrificed, the femurs, craniums and lumbar vertebrae were taken therefrom and fixed in 4% paraformaldehyde, and the bones were measured using Micro CT (Aloka, manufactured by HITACHI, Japan).
Example 1. Plasminogen Deficiency is Closely Related to Osteoporosis
[0152] 15-week-old wild-type and plasminogen-deficient (Plg.sup./) mice, five in each group, were used. Knee joints were taken and fixed in 4% paraformaldehyde for 24 hours, then decalcified in 10% EDTA for three weeks, and washed with a gradient sucrose solution. The above operations need to be carried out at 4 C. The materials were then embedded in paraffin, sectioned into 8 m and stained with Safranin O. The sections were observed under an optical microscope at 200.
[0153] The results showed that compared with the wild-type mice (
Example 2. Comparison in Calcium Loss Between Wild-Type Mice and Plasminogen-Deficient Mice
[0154] 15-week-old wild-type (wt) and plasminogen-deficient (ko) mice, five in each group, were used. Blood was taken from eyeballs removed from the two groups of mice to detect the blood calcium concentration. Under normal conditions, the calcium homeostasis in vivo is very precisely regulated. However, in the case of osteoporosis, calcium loss is a key marker of osteoporosis. In the study of calcium levels in wild-type and Plg.sup./ mice, we found that the Plg.sup./ (Ko) mice had significantly higher blood calcium levels at 15 weeks of age than the wild-type mice, and the statistical difference was significant (* indicates P<0.05) (
Example 3. Protective Effect of Plasminogen on Knee Tissue Structure of Plg.SUP./ Mice
[0155] Eight 20-week-old mice were randomly divided into two groups, i.e. a control group administered with vehicle PBS and a group administered with plasminogen, with 4 mice in each group. On the first day of the experiment, the groups were weighed and grouped, and administered with plasminogen or vehicle PBS. The group administered with plasminogen was injected with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the control group administered with vehicle PBS was injected with an equal volume of PBS via the tail vein. The mice were administered consecutively for 30 days and sacrificed on Day 31. The knee joints were taken and fixed in a fixative at 4 C. for 24 hours. The composition of the fixative is: 2% of paraformaldehyde, 0.075 mol/L of lysine, and 0.01 mol/L of sodium periodate. After the fixation, each material was washed with a PBS washing liquid gradient at 4 C. for 12 hours, and then placed in a decalcifying liquid at 4 C. for decalcification for 2 weeks, with the decalcifying liquid being changed every 5 days. After the decalcification was completed, the knee joints were washed with a PBS washing liquid gradient at 4 C. for 12 hours, and were subjected to alcohol gradient dehydration, permeabilization with xylene, paraffin immersion, and paraffin embedding. The sections were 5 m thick. The sections were dewaxed and rehydrated, stained with hematoxylin and eosin (H&E staining), differentiated with 1% hydrochloric acid in alcohol, and returned to blue with ammonia water. The sections were dehydrated with alcohol gradient, permeabilized with xylene, mounted with a neutral gum, and observed under an optical microscope at 200.
[0156] The results showed that in the control group (
Example 4. Plasminogen Promotes the Increase in the Activity of Osteoblasts on the Surface of Articular Cartilage of the Knee Joint of Plg.SUP./ .Mice
[0157] Eight 20-week-old mice were randomly divided into two groups, i.e. a control group administered with vehicle PBS and a group administered with plasminogen, with 4 mice in each group. On the first day of the experiment, the groups were weighed and grouped, and administered with plasminogen or vehicle PBS. The group administered with plasminogen was injected with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the control group administered with vehicle PBS was administered with an equal volume of PBS. The mice were administered consecutively for 30 days and sacrificed on Day 31. The femurs were taken and fixed in a fixative at 4 C. for 24 hours. The composition of the fixative is: 2% of paraformaldehyde, 0.075 mol/L of lysine, and 0.01 mol/L of sodium periodate. After the fixation, each material was washed with a PBS washing liquid gradient at 4 C. for 12 hours, and then placed in a decalcifying liquid at 4 C. for decalcification for 2 weeks, with the decalcifying liquid being changed every 5 days. After the decalcification was completed, the knee joints were washed with a PBS washing liquid gradient at 4 C. for 12 hours, and were subjected to alcohol gradient dehydration, permeabilization with xylene, and paraffin embedding. The materials were sectioned into 5 um, deparaffinized, rehydrated, and incubated in a magnesium chloride buffer at 4 C. overnight. The sections were incubated in an alkaline phosphatase substrate solution for 1 hour at room temperature and counterstained with hematoxylin for 2 minutes. The sections were rinsed with running water for 5 minutes, baked at 60 C. for 30 minutes, mounted with a neutral gum, and observed under an optical microscope at 200.
[0158] Alkaline phosphatase (ALP) is a marker of early differentiation of osteoblasts.sup.[33]. The results showed that there was only an extremely small amount of alkaline phosphatase staining (indicated by an arrow) on the surface of the articular cartilage in the control group administered with vehicle PBS (
Example 5. Plasminogen Promotes Increase in the Activity of Osteoblasts of the Knee Joint Growth Plate in Plg.SUP./ Mice
[0159] Eight 20-week-old mice were randomly divided into two groups, i.e. a control group administered with vehicle PBS and a group administered with plasminogen, with 4 mice in each group. On the first day of the experiment, the groups were weighed and grouped, and administered with plasminogen or vehicle PBS. The group administered with plasminogen was injected with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the control group administered with vehicle PBS was administered with an equal volume of PBS. The mice were administered consecutively for 30 days and sacrificed on Day 31. The femurs were taken and fixed in a fixative at 4 C. for 24 hours. The composition of the fixative is: 2% of paraformaldehyde, 0.075 mol/L of lysine, and 0.01 mol/L of sodium periodate. After the fixation, each material was washed with a PBS washing liquid gradient at 4 C. for 12 hours, and then placed in a decalcifying liquid at 4 C. for decalcification for 2 weeks, with the decalcifying liquid being changed every 5 days. After the decalcification was completed, the femurs were washed with a PBS washing liquid gradient at 4 C. for 12 hours, and were subjected to alcohol gradient dehydration, permeabilization with xylene, and then paraffin embedding. The materials were sectioned into 5 um, deparaffinized, rehydrated, and incubated in a magnesium chloride buffer at 4 C. overnight. The sections were incubated in an alkaline phosphatase substrate solution for 1 hour at room temperature and counterstained with hematoxylin for 2 minutes. The sections were rinsed with running water for 5 minutes, baked at 60 C. for 30 minutes, mounted with a neutral gum, and observed and photographed under a microscope at 200.
[0160] The results showed that in the control group administered with vehicle PBS (
Example 6. Plasminogen Improves the Activity of Serum Alkaline Phosphatase in Vitamin D-Induced Ageing Model Mice
[0161] Twenty-five 5- to 6-week-old male C57 mice were taken, weighed and randomly divided into three groups, a blank control group of 5 mice, a group of 10 mice administered with plasminogen, and a control group of 10 mice administered with vehicle PBS. The mice in the blank control group were intraperitoneally injected with 50 l of corn oil per day; and the mice in the group administered with plasminogen and in the group administered with vehicle PBS were intraperitoneally injected with vitamin D (Sigma Aldrich) at 0.5 g/kg/day to induce senescence.sup.[34,35]. At the same time, the mice were administered in such a manner that the mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, and the mice in the blank control group were not administered. Model establishment and administration were performed continuously for 28 days. During the period of administration, the mice in the blank control group were fed with a low-calcium diet, and the mice in the group administered with plasminogen and in the control group administered with vehicle PBS were fed with a low-calcium diet. The first day of model establishment and administration was set as Day 1. On Day 29, the blood was collected from removed eyeballs and centrifuged to obtain a supernatant to be detected for the activity of serum alkaline phosphatase (ALP).
[0162] The results showed that the activity of serum alkaline phosphatase in mice in the group administered with plasminogen was significantly higher than that in mice in the control group administered with vehicle PBS, and the statistical difference was significant; and compared with the control group administered with vehicle PBS, the activity of serum alkaline phosphatase in mice in the group administered with plasminogen was closer to that in the blank control group (
[0163] Serum ALP is an isoenzyme glycoprotein, and serum ALP is mainly derived from liver and bone, wherein the ALP derived from bone accounts for 40% to 75%. ALP activity assays are mainly used for diagnosing hepatobiliary and skeletal system diseases. Clinically, in addition to factors such as liver diseases and pregnancy, serum ALP can also reflect the condition of osteogenesis. When bone metabolism is strong, osteoblasts are active, ALP secretion increases, and it is present around osteoblasts and on the surface thereof, is very easily released into the blood, thereby resulting in an increase in the serum ALP activity; therefore, serum ALP is a marker of changes in bone remodeling activity [36]
[0164] In this study, the activity of serum alkaline phosphatase in mice in the group administered with plasminogen was significantly higher than that in mice in the control group administered with vehicle PBS, and the statistical difference was significant. This suggests that the plasminogen group can significantly promote the increase in the activity of osteoblasts in vitamin D ageing model mice.
Example 7. Plasminogen Promotes the Increase in the Activity of Alkaline Phosphatase at the Knee Joint Growth Plate of Vitamin D-Induced Ageing Model Mice
[0165] Fifteen 5- to 6-week-old male C57 mice were taken, weighed and randomly divided into three groups, a blank control group, a group administered with plasminogen, and a control group administered with vehicle PBS, with 5 mice in each group. The mice in the blank control group were intraperitoneally injected with 50 l of corn oil per day; and the mice in the group administered with plasminogen and in the group administered with vehicle PBS were intraperitoneally injected with vitamin D (Sigma Aldrich) at 1 g/kg/day to induce senescence.sup.[34,35]. At the same time, the mice were administered in such a manner that the mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, and the mice in the blank control group were not administered. Model establishment and administration were performed continuously for 28 days. During the period of administration, all the mice were fed with a low-calcium diet (Nantong TROPHIC). The first day of model establishment and administration was set as Day 1. On Day 29, the mice were sacrificed, and knee joints were taken and fixed in a fixative for 24 hours. The composition of the fixative is: 2% of paraformaldehyde, 0.075 mol/L of lysine, and 0.01 mol/L of sodium periodate. After the fixation, each material was washed with a PBS washing liquid gradient at 4 C. for 12 hours, and then placed in a decalcifying liquid at 4 C. for decalcification for 2 weeks, with the decalcifying liquid being changed every 5 days. After the decalcification was completed, the knee joints were washed with a PBS washing liquid gradient at 4 C. for 12 hours, and were subjected to alcohol gradient dehydration, permeabilization with xylene, and paraffin embedding. The materials were sectioned into 5 um, deparaffinized, rehydrated, and incubated in a magnesium chloride buffer at 4 C. overnight. The sections were incubated in an alkaline phosphatase substrate solution for 1 hour at room temperature and counterstained with hematoxylin for 2 minutes. The sections were rinsed with running water for 5 minutes, baked at 60 C. for 30 minutes, mounted with a neutral gum, and observed under an optical microscope at 200.
[0166] The results showed that the alkaline phosphatase positive staining (indicated by an arrow) of the knee joint growth plate in the control group administered with vehicle PBS (
Example 8. Effect of Plasminogen on Cranium Bone Mineral Density
[0167] Plg.sup.+/+ mice and Plg.sup./ mice, 12-13, 20-21, and 29-30 weeks old, were taken, 5 in each group, with the body weights of the mice in these groups being substantially the same. The mice were all fed with the same food and water during the experiment. The craniums were taken and fixed in 4% paraformaldehyde for 24 hours, and subjected to Micro CT scanning to determine the bone mineral density.
[0168] The results showed that as the week of age increased, the cortical bone mineral density (
[0169] Osteoporosis is a systemic skeletal disease that is characterized by a reduced bone mass and a degenerated bone microstructure, and can lead to increased bone fragility and easy fracture. WHO recommends the use of bone mineral density (BMD) measurements to diagnose osteoporosis.sup.[37,38]. The above-mentioned experimental results indicate that plasminogen is involved in the regulation of the bone mineral metabolism and plays an important role in a certain period of time.
Example 9. Effect of Plasminogen on the Mineral Content of the Cranium
[0170] Plg.sup.+/+ mice and Plg.sup./ mice, 20-21 weeks old, were taken, 5 in each group, with the body weights of these mice being substantially the same. The mice were all fed with the same food and water during the experiment. The craniums were taken and fixed in 4% paraformaldehyde for 24 hours, and subjected to Micro CT scanning to determine the bone mineral content.
[0171] The results showed that the bone mineral contents of the cortical bone and total bone in the Plg.sup.+/+ mice at 20-21 weeks of age were remarkably higher than those in the Plg.sup./ mice, and the statistical difference was extremely significant or significant. This indicates that plasminogen plays an important role in the regulation of the bone mineral content of the cranium and is closely related to osteoporosis.
Example 10. Decease in the Femoral Bone Mineral Density in Plasminogen-Deficient Mice
[0172] Plg.sup.+/+ mice and Plg.sup./ mice, 12-13, 20-21, and 29-30 weeks old, were taken, 5 in each group, with the body weights of the mice in these groups being substantially the same. The mice were all fed with the same food and water during the experiment. The femurs were taken and fixed in 4% paraformaldehyde for 24 hours, and subjected to Micro CT scanning to determine the bone mineral density.
[0173] The results showed that the femoral bone mineral density of Plg.sup.+/+ mice increased gradually as the week of age increased during the period of 12-30 weeks of age, while the cortical bone mineral density (
Example 11. Decease in the Femoral Bone Mineral Content in Plasminogen-Deficient Mice
[0174] Plg.sup.+/+ mice and Plg.sup./ mice, 12-13, 20-21, and 29-30 weeks old, were taken, 5 in each group, with the body weights of the mice in these groups being substantially the same. The mice were all fed with the same food and water during the experiment. The femurs were taken and fixed in 4% paraformaldehyde for 24 hours, and subjected to Micro CT scanning to determine the bone mineral content.
[0175] The results showed that the mineral contents in different portions of the femur of the Plg.sup.+/+ mice did not change much or increased gradually as the week of age increased during the period of 12-30 weeks of age, while the mineral contents of the cancellous bone (
Example 12. Decease in the Lumbar Vertebra Bone Mineral Density in Plasminogen-Deficient Mice
[0176] Plg.sup.+/+ mice and Plg.sup./ mice, 12-13, 20-21, and 29-30 weeks old, were taken, 5 in each group, with the body weights of the mice in these groups being substantially the same. The mice were all fed with the same food and water during the experiment. The lumbar vertebrae were taken and fixed in 4% paraformaldehyde for 24 hours, and subjected to Micro CT scanning to determine the bone mineral density.
[0177] The results showed that the lumbar vertebra bone mineral density of Plg.sup.+/+ mice increased gradually as the week of age increased during the period of 12-30 weeks of age, while the cortical bone mineral density (
Example 13. Decease in the Mineral Content of the Lumbar Vertebra in Plasminogen-Deficient Mice
[0178] Plg.sup.+/+ mice and Plg.sup./ mice, 12-13, 20-21, and 29-30 weeks old, were taken, 5 in each group, with the body weights of the mice in these groups being substantially the same. The mice were all fed with the same food and water during the experiment. The lumbar vertebrae were taken and fixed in 4% paraformaldehyde for 24 hours, and subjected to Micro CT scanning to determine the bone mineral content.
[0179] The results showed that the mineral content of the lumbar vertebra in the Plg.sup.+/+ mice did not change much as the week of age increased during the period of 12-30 weeks of age, while the mineral contents of the cortical bone (
Example 14. Effect of Plasminogen Deficiency on the Activity of Serum Alkaline Phosphatase in Mice
[0180] Plg.sup.+/+ mice and Plg.sup./ mice, 12-13, 20-21, and 29-30 weeks old, were taken, 5 in each group, with the body weights of the mice in these groups being substantially the same. The mice were all fed with the same food and water during the experiment. The blood was collected from removed eyeballs from all the mice and centrifuged to obtain a supernatant. The serum alkaline phosphatase activity was measured using an alkaline phosphatase assay kit.
[0181] The results showed that the activity of serum alkaline phosphatase in the Plg.sup.+/+ mice fluctuated but did not change significantly at 12-30 weeks of age, while the activity of serum alkaline phosphatase in the Plg.sup./ mice decreased gradually as the week of age increased; the activity of serum alkaline phosphatase in the Plg.sup.+/+ mice was significantly higher than that in the Plg.sup./ mice, and the activities of serum alkaline phosphatase in the two strains of mice showed a remarkable difference at 12 weeks of age; furthermore, as the week of age increased, the difference became more and more significant (
Example 15. Plasminogen Reduces the Blood Calcium Concentration in Atherosclerosis ApoE Mice
[0182] Thirteen 6-week-old male ApoE mice were fed with a high-fat and high-cholesterol diet for 16 weeks to induce atherosclerosis.sup.[39,40]. 50 L of blood was taken from each mouse three days before administration, and the total cholesterol concentration was detected. The mice were randomly divided into two groups based on the detection results, 7 mice in the control group administered with vehicle PBS, and 6 mice in the group administered with plasminogen. The first day of administration was set as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein. During the administration, the mice continued to be fed with the high-fat diet. On Day 30, the mice were fasted for 16 hours. On Day 31, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant to be detected for the serum calcium concentration. The blood calcium detection was carried out using a calcium detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# C004-2) according to the method in the instructions.
[0183] The results showed that the blood calcium concentration in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (
Example 16. Plasminogen Ameliorates Aortic Sinus Calcification in Atherosclerosis ApoE Mice
[0184] Thirteen 6-week-old male ApoE mice were fed with a high-fat and high-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to induce the atherosclerosis.sup.[39,40] 50 L of blood was taken from each mouse three days before administration, and the total cholesterol concentration was detected. The mice were randomly divided into two groups based on the detection results, 7 mice in the control group administered with vehicle PBS, and 6 mice in the group administered with plasminogen. The first day of administration was set as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein. During the administration, the mice continued to be fed with the high-fat diet. The mice were sacrificed on the 31st day of administration. The hearts were taken and fixed in 4% paraformaldehyde for 24 to 48 hours, dehydrated and sedimented in 15% and 30% sucrose, and embedded with OCT. They were sectioned into 8 m thick frozen sections, and stained with alizarin red S for 3 min. The sections were observed under an optical microscope at 40.
[0185] Alizarin red staining can indicate the degree of calcification. The results showed that the calcium deposition in aortic sinus of mice in the group administered with plasminogen (
Example 17. Effect of Plasminogen on Femoral Bone Mineral Density in AopE Atherosclerosis Model Mice
[0186] Nineteen 6-week-old male ApoE mice were fed with a high-fat and high-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to induce atherosclerosis.sup.[39,40] 50 L of blood was taken from each mouse three days before administration, and the total cholesterol concentration was detected. The mice were randomly divided into two groups based on the detection results, 9 mice in the control group administered with vehicle PBS, and 10 mice in the group administered with plasminogen. The first day of administration was set as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein. During the administration, the mice continued to be fed with the high-fat diet. On the 11th day of administration, 5 mice were taken from each group and sacrificed, the femurs were taken therefrom and fixed in 4% paraformaldehyde. On the 31th day of administration, the remaining mice were sacrificed, the femurs were taken therefrom and fixed in 4% paraformaldehyde. The femurs were subjected to Micro CT scanning for determining the bone mineral density.
[0187] The correlation between atherosclerosis and osteoporosis has been reported since a long time ago, and hyperlipemia is an important pathogenic factor of atherosclerosis. Recent studies have shown that apolipoprotein E (ApoE) not only affects lipid metabolism, but is also associated with bone mineral density, bone loss, and osteoporotic fractures.sup.[41,42].
[0188] The results showed that after 10 days of administration, the femoral bone mineral density in mice in the group administered with plasminogen was remarkably higher than that in the control group administered with vehicle PBS, and the statistical difference in terms of cancellous bone mineral density (
[0189] Studies have shown that the essence of vascular calcification is the phenotype transformation of vascular smooth muscle cells into osteoblasts and the transformation of vascular tissues into bone tissues. Furthermore, the formation of vascular calcification is also significantly associated with bone mineral loss.sup.[10]. From the summary of the experimental results given in Examples 16 and 17 above, it can be seen that plasminogen can enhance bone mineral density while reducing calcium deposition on an arterial wall. It is of great significance for the prevention and treatment of osteoporosis and cardiovascular diseases.
Example 18. Protective Effect of Plasminogen on the Structure of Knee Joint Tissue of AopE Atherosclerosis Model Mice
[0190] Seven 6-week-old male ApoE mice were fed with a high-fat and high-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to induce atherosclerosis.sup.[39,40]. 50 L of blood was taken from each mouse three days before administration, and the total cholesterol concentration was detected. The mice were randomly divided into two groups based on the detection results, 3 mice in the control group administered with vehicle PBS, and 4 mice in the group administered with plasminogen. The first day of administration was set as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein. During the administration, the mice continued to be fed with the high-fat diet. On the 31th day of administration, the mice were sacrificed, the femurs were taken therefrom and fixed in 4% paraformaldehyde. The materials were then decalcified with an acidic decalcifying liquid (a decalcifying liquid of 8% of hydrochloric acid and 10% of formic acid by volume, prepared in ultrapure water) for 3.5 hours. Then, they were paraffin-embedded, and sectioned into 8 m for H&E staining, and the sections were observed under an optical microscope at 100 (A and D) and 200 (B, C, E, and F).
[0191] The results showed that in the control group administered with vehicle PBS (
Example 19. Effect of Plasminogen on Body Weight of Ovariectomy- and Dexamethasone-Induced Osteoporosis Model Mice
[0192] Seventeen 8- to 10-week-old C57 female mice were weighed for body weight, and the mice were randomly divided into two groups based on the body weight, a normal control group of 3 mice and a model group of 14 mice. The mice in the model group were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method. For the normal control mice, they were only cut open at the same position without ovariectomy. 14 days after the ovariectomy, the mice in the model group were randomly divided into two groups based on the body weight, a group administered with plasminogen and a control group administered with vehicle PBS, with 7 mice in each group. The mice in the model group were intraperitoneally injected with dexamethasone at a dose of 125 g/mouse with an injection frequency of 5 days/week for 12 days in total to induce osteoporosis.sup.[43], and the mice in the normal control group were not treated with injection. The mice were administered with drugs while injecting dexamethasone, in such a manner that the mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 16 consecutive days, and the mice in the normal control group were not injected with plasminogen or PBS. The first day of administration was set as Day 1, and on Day 17, the body weights of the mice were measured.
[0193] The results showed that the body weight of mice in the control group administered with vehicle PBS was remarkably lighter than that in the normal control group, while the body weight in the group administered with plasminogen was significantly higher than that in the control group administered with vehicle PBS, and the statistical difference was significant (P<0.05) (
Example 20. Effect of Plasminogen on the Femur of Ovariectomy- and Dexamethasone-Induced Osteoporosis Model Mice
[0194] Fourteen 8- to 10-week-old C57 female mice were weighed for body weight. All the mice were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method. 14 days after the ovariectomy, the mice were randomly divided into two groups based on the body weight, a group administered with plasminogen and a control group administered with vehicle PBS, with 7 mice in each group. The mice in the two groups were intraperitoneally injected with dexamethasone at a dose of 125 g/mouse with an injection frequency of 5 days/week for 12 days in total to induce osteoporosis.sup.[43]. The mice were administered with drugs at the same time of model establishment, in such a manner that the mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 16 consecutive days. The first day of administration was set as Day 1. On Day 17, the mice were sacrificed, and the femurs were taken therefrom and fixed in a 4% paraformaldehyde fixative. Micro CT scanning was carried out for determining various femoral indicators.
[0195] The results showed that the volumes (
Example 21. Plasminogen Improves the Femoral Structure of Ovariectomy- and Dexamethasone-Induced Osteoporosis Model Mice
[0196] Seventeen 8- to 10-week-old C57 female mice were weighed for body weight, and the mice were randomly divided into two groups based on the body weight, a normal control group of 3 mice and a model group of 14 mice. The mice in the model group were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method. For the normal control mice, they were only cut open at the same position without ovariectomy. 14 days after the ovariectomy, the mice in the model group were intraperitoneally injected with dexamethasone at a dose of 125 g/mouse with an injection frequency of 5 days/week for 12 days in total to induce osteoporosis.sup.[43], and the mice in the normal control group were not treated with injection. After the injection with dexamethasone was completed, the mice in the model group were randomly divided into two groups based on the body weight, a group administered with plasminogen and a control group administered with vehicle PBS, with 7 mice in each group. After the model was established (the 2nd day after the dexamethasone injection is completed), the mice were administered with drugs. The mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 16 consecutive days, and the mice in the normal control group were not injected with plasminogen or PBS. The first day of administration was set as Day 1. On Day 17, the mice were sacrificed, and the femurs were taken therefrom and fixed in a 4% paraformaldehyde fixative. The femurs were subjected to Micro CT scanning for determining the femoral bone mineral density.
Bone Mineral Density
[0197] The results showed that the bone mineral densities of the cortical bone, cancellous bone, trabecular bone and total bone of the femur of mice in the control group administered with vehicle PBS were all smaller than those in the normal control group, while the bone mineral density in each portion of mice in the group administered with plasminogen was greater than that in the control group administered with vehicle PBS. The trend was clear; however, due to the small number of mice, the statistical difference was only close to significant. It can be expected that a statistical difference appears in the case of increasing the number of mice (
Bone Mineral Content
[0198] The results showed that the bone mineral content in each portion of the femur of mice in the control group administered with vehicle PBS was smaller than that in the normal control group, while the bone mineral content of each portion of mice in the group administered with plasminogen was greater than that in the control group administered with vehicle PBS. The trend was clear; however, due to the small number of mice, the statistical difference was only close to significant. It can be expected that a statistical difference appears in the case of increasing the number of mice (
Bone Volume
[0199] The results showed that the trabecular bone volume of the femur of mice in the control group administered with vehicle PBS was smaller than that in the normal control group, while the trabecular bone volume of the femur of mice in the group administered with plasminogen was greater than that in the control group administered with vehicle PBS. The trend was clear; however, due to the small number of mice, the statistical difference was only close to significant. It can be expected that a statistical difference appears in the case of increasing the number of mice (
[0200] In summary, plasminogen can remarkably ameliorate osteoporosis, promote the increase in the bone mineral density and bone mass in various portions of the femur, and the amelioration of the trabecular bone is particularly obvious.
Example 22. Plasminogen Improves the Condition of the Structure of Knee Joint Tissue in Ovariectomy- and Dexamethasone-Induced Osteoporosis Model Mice
[0201] Fourteen 8- to 10-week-old C57 female mice were weighed for body weight. All the mice were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method. 14 days after the ovariectomy, the mice were randomly divided into two groups based on the body weight, a group administered with plasminogen and a control group administered with vehicle PBS, with 7 mice in each group. The mice in the two groups were intraperitoneally injected with dexamethasone at a dose of 125 g/mouse with an injection frequency of 5 days/week for 12 days in total to induce osteoporosis.sup.[43]. The mice were administered with drugs at the same time of model establishment, in such a manner that the mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 16 consecutive days. The first day of administration was set as Day 1. On Day 17, the mice were sacrificed, and the knee joints were taken therefrom and fixed in a 4% paraformaldehyde fixative. The materials were then decalcified with an acidic decalcifying liquid (a decalcifying liquid of 8% of hydrochloric acid and 10% of formic acid by volume, prepared in ultrapure water) for 3.5 hours. Then, they were paraffin-embedded, and sectioned into 3 m for H&E staining (A and B) and Safrain 0 staining (C and D), and the sections were observed under an optical microscope at 100.
[0202] The results showed that in the groups administered with vehicle PBS (
Example 23. Plasminogen Improves the Activity of Osteoblasts in the Knee Joint of Ovariectomy- and Dexamethasone-Induced Osteoporosis Model Mice
[0203] Fourteen 8- to 10-week-old C57 female mice were weighed for body weight. All the mice were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method. 14 days after the ovariectomy, the mice were randomly divided into two groups based on the body weight, a group administered with plasminogen and a control group administered with vehicle PBS, with 7 mice in each group. The mice in the two groups were intraperitoneally injected with dexamethasone at a dose of 125 g/mouse with an injection frequency of 5 days/week for 12 days in total to induce osteoporosis.sup.[43]. The mice were administered with drugs at the same time of model establishment, in such a manner that the mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 16 consecutive days. The first day of administration was set as Day 1. On Day 17, the mice were sacrificed, and knee joints were taken therefrom and fixed in a fixative. The composition of the fixative is: 2% of paraformaldehyde, 0.075 mol/L of lysine, and 0.01 mol/L of sodium periodate. After the fixation, each material was washed with a PBS washing liquid gradient at 4 C. for 12 hours, and then placed in a decalcifying liquid at 4 C. for decalcification for 2 weeks, with the decalcifying liquid being changed every 5 days. After the decalcification was completed, the knee joints were washed with a PBS washing liquid gradient at 4 C. for 12 hours, and were subjected to alcohol gradient dehydration, permeabilization with xylene, and paraffin embedding. The materials were sectioned into 3 um, deparaffinized, rehydrated, and incubated in a magnesium chloride buffer at 4 C. overnight. The sections were incubated in an alkaline phosphatase substrate solution for 1 hour at room temperature and counterstained with hematoxylin for 2 minutes. The sections were rinsed with running water for 5 minutes, baked at 60 C. for 30 minutes, mounted with a neutral gum, and observed under an optical microscope at 200.
[0204] The results showed that the alkaline phosphatase staining of the knee joint cartilage tissue (indicated by thin arrow) and the growth plate (indicated by thick arrow) of mice in the control groups administered with vehicle PBS (
Example 24. Plasminogen Reduces the Blood Calcium Concentration in Ovariectomy-Induced Osteoporosis Model Mice
[0205] Eleven 8- to 10-week-old Plg.sup.+/+ female mice were used. The mice were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method.sup.[44,45]. 65 days after the ovariectomy, the mice were weighed and randomly divided into two groups based on the body weight, a group of 6 mice administered with plasminogen and a control group of 5 mice administered with vehicle PBS, and administration was started. The mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 11 consecutive days. The first day of administration was set as Day 1. On Day 12, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant to be detected for the blood calcium concentration. The blood calcium detection was carried out using a calcium detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# C004-2) according to the method in the instructions.
[0206] The results showed that the serum calcium concentration in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05) (
Example 25. Plasminogen Increases the Blood Phosphorus Concentration in Ovariectomy-Induced Osteoporosis Model Mice
[0207] Eleven 8- to 10-week-old Plg.sup.+/+ female mice were used. The mice were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method.sup.[44,45]. 65 days after the ovariectomy, the mice were weighed and randomly divided into two groups based on the body weight, a group of 6 mice administered with plasminogen and a control group of 5 mice administered with vehicle PBS, and administration was started. The mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 11 consecutive days. The first day of administration was set as Day 1. On Day 12, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant to be detected for the blood phosphorus concentration. The blood phosphorus detection was carried out using a phosphorus detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# C006-3) according to the method in the instructions.
[0208] The results showed that the serum phosphorus concentration in mice in the group administered with plasminogen was remarkably higher than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05) (
Example 26. Plasminogen Increases the Activity of Osteoblasts in the Knee Joint of 3% Cholesterol Hyperlipemia Model Mice
[0209] Sixteen 9-week-old male C57 mice were fed with a 3% cholesterol high-fat diet (Nantong TROPHIC) for 4 weeks to induce hyperlipemia.sup.[46,47]. This model was designated as the 3% cholesterol hyperlipemia model. The model mice continued to be fed with a 3% cholesterol high-fat diet. 50 L of blood was taken from each mouse three days before administration, and the total cholesterol was detected. The mice were randomly divided into two groups based on the total cholesterol concentration and the body weight, 8 mice in each group. The first day of administration was recorded as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 20 days. On Day 20, the mice were fasted for 16 hours; and on Day 21, the mice were sacrificed, and the knee joints were taken therefrom and fixed in a fixative. The composition of the fixative is: 2% of paraformaldehyde, 0.075 mol/L of lysine, and 0.01 mol/L of sodium periodate. After the fixation, each material was washed with a PBS washing liquid gradient at 4 C. for 12 hours, and then placed in a decalcifying liquid at 4 C. for decalcification for 2 weeks, with the decalcifying liquid being changed every 5 days. After the decalcification was completed, the knee joints were washed with a PBS washing liquid gradient at 4 C. for 12 hours, and were subjected to alcohol gradient dehydration, permeabilization with xylene, and paraffin embedding. The materials were sectioned into 3 um, deparaffinized, rehydrated, and incubated in a magnesium chloride buffer at 4 C. overnight. The sections were incubated in an alkaline phosphatase substrate solution for 1 hour at room temperature and counterstained with hematoxylin for 2 minutes. The sections were rinsed with running water for 5 minutes, baked at 60 C. for 30 minutes, mounted with a neutral gum, and observed under an optical microscope at 200
[0210] Hyperlipemia is a lipid metabolism disorder that can cause a series of complications. In recent years, a number of studies have found that hyperlipemia is a common cause of osteoporosis and atherosclerosis.sup.[48,49].
[0211] The results showed that the alkaline phosphatase staining (indicated by an arrow) of the knee joint of mice in the groups administered with plasminogen (
Example 27. Plasminogen Improves the Condition of the Structure of Knee Joint Tissue in Ovariectomy- and Dexamethasone-Induced Osteoporosis Model Mice
[0212] Fourteen 8- to 10-week-old C57 female mice were weighed for body weight. The mice were anesthetized by means of intraperitoneal injection with pentobarbital sodium at a dose of 50 mg/kg body weight. The hair on both sides of the back of the mice was removed, followed by disinfection with 70% alcohol and iodine. The skin, back muscles and peritoneum were cut open, then the white shiny cellulite was gently pulled out of the incision by means of small forceps, and after the cellulite was separated, the ovaries can be revealed. The fallopian tube at the lower end of an ovary was first ligated with a silk thread, and then the ovary was removed. The incision was sutured, followed by the external application of anti-inflammatory powder. The ovary on the other side was removed by means of the same method. 14 days after the ovariectomy, the mice in the model group were intraperitoneally injected with dexamethasone at a dose of 125 g/mouse with an injection frequency of 5 days/week for 12 days in total to induce osteoporosis.sup.[43]. After the injection with dexamethasone was completed, the mice were randomly divided into two groups based on the body weight, a group administered with plasminogen and a control group administered with vehicle PBS, with 7 mice in each group. After the model was established (the 2nd day after the dexamethasone injection is completed), the mice were administered with drugs. The mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 16 consecutive days. The first day of administration was set as Day 1. On Day 17, the mice were sacrificed, and the knee joints were taken therefrom and fixed in a 4% paraformaldehyde fixative. The materials were then decalcified with an acidic decalcifying liquid (a decalcifying liquid of 8% of hydrochloric acid and 10% of formic acid by volume, prepared in ultrapure water) for 3.5 hours. Then, they were paraffin-embedded, and sectioned into 3 m for H&E staining (A and B) and Safrain 0 staining (C and D), and the sections were observed under an optical microscope at 100
[0213] The results showed that in the groups administered with vehicle PBS (
REFERENCES
[0214] [1] Long F, Ornitz D M. Development of the endochondral skeleton [J]. Cold Spring Harb perspect biol, 2013, 5: 008334. [0215] [2] Ryan J W, R einke D, Kogawa M, et al. Novel targets of vitamin D activity in bone: action of the vitamin D receptor in osteoblasts, osteocytes and osteoclasts [J]. Curr Drug Targets, 2013, 14: 1683-1688. [0216] [3] Liu E Y, Wactawski W J, Donahue R P, et al. Does low bone mineral density start in postteenage years in women with type 1 diabetes [J]. Diabetes Care, 2003, 26(8): 2365-2369. [0217] [4] Dennison E M, Syddall H E, Aihie Sayer A, et al. Type 2 diabetes mellitus is associated with increased axial bone density in men and women from the Hertfordshire Cohort Study: evidence for an indirect effect of insulin resistance [J]. Diabetologia, 2004, 47 (11):1963-1968. [0218] [5] Schwartz A V, Sellmeyer D E, Strotmeyer E S, et al. Diabetes and bone loss at the hip in older black and white adults [J]. J Bone Miner Res, 2005, 20(4): 596-603. [0219] [6] YANG, Nailong, WANG, Jun, Q U, Ning. The Investigation and evaluation of bone mineral density in type 2 diabetic women [J]. Chinese Journal of Diabetes, 2008, 16(1): 26-28. [0220] [7] Doherty T M, Fitzpatrick L A, Inoue D, et al. Molecular, endocrine, and genetic mechanisms of arterial calcification [J]. Endocr Rev, 2004, 25: 629-672. [0221] [8] Marcovitz P A, Tran H H, Franklin B A, et al. Usefulness of bone mineral density to predict significant coronary artery disease [J]. Am J Cardiol, 2005, 96: 1 059-063. [0222] [9] Schulz E, Arfai K, Liu X, et al. Aortic calcification and the risk of osteoporosis and fractures [J]. J Clin Endocrinol Metab, 2004, 89: 4 246-253. [0223] [10] Doctoral thesis of Third Military Medical University, ZHOU, Rui, Title: Molecular perspectives of vitamin D in osteoblast and association between osteoporosis and artery calcification, May 2015. [0224] [11] Lanske, B. and Razzaque, M. S. (2007). Vitamin D and aging: old concepts and new insights. J. Nutr. Biochem. 18, 771-777. [J]. [0225] [12] Alexander C M and Werb, Z. (1991). Extracellular matrix degradation. In Cell Biology of Extracellular Matrix, Hay E D, ed. (New York: Plenum Press), pp. 255-302. [0226] [13] Werb, Z., Mainardi, C. L., Vater, C. A., and Harris, E. D., Jr. (1977). Endogenous activation of latent collagenase by rheumatoid synovial cells. Evidence for a role of plasminogen activator. N. Engl. J. Med. 296, 1017-1023. [0227] [14] He, C. S., Wilhelm, S. M., Pentland, A. P., Marmer, B. L., Grant, G. A., Eisen, A. Z., and Goldberg, G. I. (1989). Tissue cooperation in a proteolytic cascade activating human interstitial collagenase. Proc. Natl. Acad. Sci. U. S. A 86, 2632-2636. [0228] [15] Stoppelli, M. P., Corti, A., Soffientini, A., Cassani, G., Blasi, F., and Assoian, R. K. (1985). Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc. Natl. Acad. Sci. U. S. A 82, 4939-4943. [0229] [16] Vassalli, J. D., Baccino, D., and Belin, D. (1985). A cellular binding site for the Mr 55, 000 form of the human plasminogen activator, urokinase. J. Cell Biol. 100, 86-92. [0230] [17] Wiman, B. and Wallen, P. (1975). Structural relationship between glutamic acid and lysine forms of human plasminogen and their interaction with the NH2-terminal activation peptide as studied by affinity chromatography. Eur. J. Biochem. 50, 489-494. [0231] [18] Saksela, O. and Rifkin, D. B. (1988). Cell-associated plasminogen activation: regulation and physiological functions. Annu. Rev. Cell Biol. 4, 93-126. [0232] [19] Raum, D., Marcus, D., Alper, C. A., Levey, R., Taylor, P. D., and Starzl, T. E. (1980). Synthesis of human plasminogen by the liver. Science 208, 1036-1037. [0233] [20] Wallen P (1980). Biochemistry of plasminogen. In Fibrinolysis, Kline D L and Reddy K K N, eds. (Florida: CRC) [0234] [21] Sottrup-Jensen, L., Zajdel, M., Claeys, H., Petersen, T. E., and Magnusson, S. (1975). Amino-acid sequence of activation cleavage site in plasminogen: homology with pro part of prothrombin. Proc. Natl. Acad. Sci. U. S. A 72, 2577-2581. [0235] [22] Collen, D. and Lijnen, H. R. (1991). Basic and clinical aspects of fibrinolysis and thrombolysis. Blood 78, 3114-3124. [0236] [23] Alexander, C. M. and Werb, Z. (1989). Proteinases and extracellular matrix remodeling. Curr. Opin. Cell Biol. 1, 974-982. [0237] [24] Mignatti, P. and Rifkin, D. B. (1993). Biology and biochemistry of proteinases in tumor invasion. Physiol Rev. 73, 161-195. [0238] [25] Collen, D. (2001). Ham-Wasserman lecture: role of the plasminogen system in fibrin-homeostasis and tissue remodeling. Hematology. (Am. Soc. Hematol. Educ. Program.) 1-9. [0239] [26] Rifkin, D. B., Moscatelli, D., Bizik, J., Quarto, N., Blei, F., Dennis, P., Flaumenhaft, R., and Mignatti, P. (1990). Growth factor control of extracellular proteolysis. Cell Differ. Dev. 32, 313-318. [0240] [27] Andreasen, P. A., Kjoller, L., Christensen, L., and Duffy, M. J. (1997). The urokinase-type plasminogen activator system in cancer metastasis: a review. Int. J. Cancer 72, 1-22. [0241] [28] Rifkin, D. B., Mazzieri, R., Munger, J. S., Noguera, I., and Sung, J. (1999). Proteolytic control of growth factor availability. APMIS 107, 80-85. [0242] [29] Marder V J, Novokhatny V. Direct fibrinolytic agents: biochemical attributes, preclinical foundation and clinical potential [J]. Journal of Thrombosis and Haemostasis, 2010, 8(3): 433-444. [0243] [30] Hunt J A, Petteway Jr S R, Scuderi P, et al. Simplified recombinant plasmin: production and fu-nctional comparison of a novel thrombolytic molecule with plasma-derived plasmin [J]. Thromb Haemost, 2008, 100(3): 413-419. [0244] [31] Sottrup-Jensen L, Claeys H, Zajdel M, et al. The primary structure of human plasminogen: Isolation of two lysine-binding fragments and one mini-plasminogen (MW, 38, 000) by elastase-catalyzed-specific limited proteolysis [J]. Progress in chemical fibrinolysis and thrombolysis, 1978, 3: 191-209. [0245] [32] Nagai N, Demarsin E, Van Hoef B, et al. Recombinant human microplasmin: production and potential therapeutic properties [J]. Journal of Thrombosis and Haemostasis, 2003, 1(2): 307-313. [0246] [33]. Weinreb M, Shinar D, Rodan G. Different pattern of alkaline phosphatase, osteopontin, and osteocalcin expression in developing rat bone visualized by in situ hybridization J. J Bone Miner Res, 1990, 5(8):831-842. [0247] [34]. E. DACI, A. VERSTUYF, K. MOERMANS et al. Bone Resorption Induced by 1a,25 Dihydroxyvitamin D3 In Vivo Is Not Altered by Inactivation of the Plasminogen Activator Inhibitor 1. Bone Vol. 27, No. 1 Jul. 2000:97-102. [0248] [35]. Mohammed S. Razzaque, Despina Sitara, Takashi Taguchi et al. Premature aging-like phenotype in fibroblast growth factor 23 null mice is a vitamin D-mediated process. FASEB J. 2006 April; 20(6): 720-722. [0249] [36] LIU, Yuyu, W U, Tie, CUI, Liao et al. The correlation between bone histomorphometry and serum alkaline phosphatase in ovariectomized rats. Chinese Journal of Gerontology, 2004, 1(24): 49-50). [0250] [37] Kanis J A, Melton L J, Christiansen C, et al. Perspective: The diagnosis of osteoporosis. [0251] Journal of Bone and Mineral Research, 1994, 9(11): 37-41. [0252] [38] Yu J, Yu X F. The application of the bone metabolic markers and bone mineral density in osteoporosis. J Chin Intern Med, 2009, 26(3): 155-157. [0253] [39] Yutaka Nakashima, Andrew S. Plump, Elaine W. Raines et al. Arterioscler Thromb. 1994 January; 14(1): 133-40. [0254] [40] Yvonne Nitschke, Gabriele Weissen-Plenz, Robert Terkeltaub et al. Npp1 promotes atherosclerosis in ApoE knockout mice. J. Cell. Mol. Med. Vol 15, No 11, 2011 pp. 2273-2283 [0255] [41] Plu A S, Smith J D, Hayek T et al. Severe hyercholestrolemia and antherosclerosis in apolipoprotein E-deficient mice created by homologous recombination in ES cells. cell. 1992; 71:343-353. [0256] [42] Zhang S H, Reddick R L, Piedrahita J A et al. Spontaneous hypeicholeserolemia and arterial leision in mice lacking apolipoprotrin E [J]. Science. 1992; 258:468-471. [0257] [43]. Louise Grahnemo, Caroline Jochems, Annica Andersson, Possible role of lymphocytes in glucocorticoid-induced increase in trabecular bone mineral density, Journal of Endocrinology (2015) 224, 97-108. [0258] [44]. Gustavo Duque, Dao Chao Huang, Natalie Dion et al. Interferon-g Plays a Role in Bone Formation In Vivo and Rescues Osteoporosis in Ovariectomized Mice. Journal of Bone and Mineral Research, Vol. 26, No. 7, July 2011, pp. 1472-1483. [0259] [45]. Min Hee Park, Namoh Kim, Hee Kyung Jinl et al. Neuropeptide Y-based recombinant peptides ameliorate bone loss in mice by regulating hematopoietic stem/progenitor cell mobilization. BMB Rep. 2017; 50(3): 138-143. [0260] [46] Dominika Nackiewicz, Paromita Dey, Barbara Szczerba et al. Inhibitor of differentiation 3, a transcription factor regulates hyperlipidemia associated kidney disease. Nephron Exp Nephrol. 2014; 126(3): 141-147. [0261] [47] Ming Gul, Yu Zhang., Shengjie Fan et al. Extracts of Rhizoma Polygonati Odorati Prevent High-Fat Diet-Induced Metabolic Disorders in C57BL/6 Mice. PLoS ONE 8(11): e81724. [0262] [48] Ihsane Hmamouchi, Fadoua Allali, Hamza Khazzani et al. Low bone mineral density is related to atherosclerosis in postmenopausal Moroccan women. BMC Public Health. 2009; 9: 388. [0263] [49] Hui Yang, Ahmed Salah Salem Mohamed, Sheng-hua Zhou. Oxidized low density lipoprotein, stem cells, and atherosclerosis. Lipids Health Dis. 2012; 11: 85.