Method for treating coronary atherosclerosis and complications thereof
11547746 · 2023-01-10
Assignee
Inventors
Cpc classification
A61K45/06
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61P9/10
HUMAN NECESSITIES
International classification
A61P9/10
HUMAN NECESSITIES
A61P1/16
HUMAN NECESSITIES
Abstract
The present invention relates to a method for preventing and/or treating coronary atherosclerosis and its related conditions in a subject, comprising administering a prophylactically and/or therapeutically effective amount of plasminogen to the subject, wherein the subject suffers from, is suspected of suffering from coronary atherosclerosis and its related conditions, or has a risk of suffering from coronary atherosclerosis and its related conditions. The present invention further relates to a medicament, a pharmaceutical composition, an article of manufacture, and a kit comprising plasminogen which are useful for preventing and/or treating coronary atherosclerosis and its related conditions in a subject.
Claims
1. A method for preventing and/or treating coronary atherosclerosis and its related conditions in a subject, comprising administering a prophylactically and/or therapeutically effective amount of plasminogen to the subject, wherein the subject suffers from, is suspected of suffering from coronary atherosclerosis and its related conditions, or has a risk of suffering from coronary atherosclerosis and its related conditions, and wherein plasminogen promotes serum level of the high-density lipoprotein cholesterol (HDL-C) in the subject.
2. The method of claim 1, wherein the coronary atherosclerosis-related conditions comprise coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, and heart failure caused by coronary atherosclerosis.
3. The method of claim 1, wherein the coronary atherosclerosis is coronary atherosclerosis complicated with diabetes mellitus.
4. The method of claim 1, wherein the plasminogen prevents and/or treats coronary atherosclerosis in one or more ways selected from: lowering a serum total cholesterol level in the subject, lowering a serum triglyceride level in the subject, lowering a serum low-density lipoprotein level in the subject, elevating a serum high-density lipoprotein level in the subject, reducing lipid deposition on an arterial wall of the subject, promoting fat metabolism in the liver, promoting fat transport in the liver, and reducing fat deposition in the liver of the subject.
5. The method of claim 1, wherein the coronary atherosclerosis-related conditions comprises an ischemic injury of a tissue or organ caused by coronary atherosclerosis.
6. The method of claim 1, wherein the plasminogen has at least 75% sequence identity with SEQ ID No. 2, and still has the plasminogen activity.
7. The method of claim 1, wherein the plasminogen is a protein that comprises a plasminogen active fragment and still has the plasminogen activity.
8. 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.
9. 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.
10. The method of claim 1, wherein the plasminogen is administered to the subject at a dosage of 1-100 mg/kg at a frequency of weekly to daily.
11. The method of claim 10, wherein the dosage of the plasminogen is repeated at least once.
12. The method of claim 10, wherein the plasminogen is administered at least daily.
13. A method for preventing and/or treating atherosclerosis and its related conditions in a subject, comprising administering an effective amount of plasminogen to the subject, and wherein plasminogen promotes serum level of the high-density lipoprotein cholesterol (HDL-C) in the subject.
14. The method of claim 13, wherein the atherosclerosis comprises aortic atherosclerosis, coronary atherosclerosis, cerebral atherosclerosis, renal atherosclerosis, mesenteric atherosclerosis, and lower limb atherosclerosis.
15. The method of claim 13, wherein the atherosclerosis-related conditions comprise related conditions caused by tissue and organ ischemia due to atherosclerosis, comprising coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, and heart failure caused by coronary atherosclerosis; cerebral ischemia, cerebral thrombosis, brain atrophy, cerebral hemorrhage, and cerebral embolism caused by cerebral atherosclerosis; renal insufficiency, hypertension, glomerular fibrosis, renal failure, and uremia caused by renal atherosclerosis; postprandial abdominal pain, dyspepsia, constipation, intestinal wall necrosis, and hemafecia caused by mesenteric atherosclerosis; and intermittent claudication,and gangrene caused by lower limb atherosclerosis.
16. The method of claim 13, wherein the atherosclerosis is atherosclerosis complicated with diabetes mellitus.
17. The method of claim 13, wherein the plasminogen prevents and/or treats atherosclerosis in one or more ways selected from: lowering a serum total cholesterol level in the subject, lowering a serum triglyceride level in the subject, lowering a serum low-density lipoprotein level in the subject, elevating a serum high-density lipoprotein level in the subject, reducing lipid deposition on an arterial wallof the subject, promoting fat metabolism in the liver, promoting fat transport in the liver, and reducing fat deposition in the liver of the subject.
18. The method of claim 13, wherein the atherosclerosis-related conditions comprise arterial thrombosis and its related conditions caused by atherosclerosis in the subject.
19. The method of claim 18, wherein the conditions comprise coronary heart disease, angina pectoris, myocardial infarction, arrhythmia, heart failure, cerebral ischemia,cerebral thrombosis, brain atrophy, cerebral hemorrhage, cerebral embolism, cerebral infarction, renal insufficiency, hypertension, glomerular fibrosis, renal failure, uremia, intestinal necrosis, intermittent claudication, and gangrene.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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EXAMPLES
Example 1
Plasminogen Ameliorates Lipid Plaque Deposition in Aorta of ApoE Atherosclerosis Mice
(23) 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 hyperlipemia model .sup.[47,48], 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 10 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. On Day 11, one mouse from each group was randomly sacrificed. The aortas were fixed in 4% paraformaldehyde for 24 to 48 hours, and dissected for general oil red O staining. The aortas were observed and photographed under a stereo microscope at 7×.
(24) Oil red O staining can show lipid deposition and reflect the severity of injury .sup.[49]. The staining results (
Example 2
Plasminogen Reduces Lipid Plaque Deposition in Aorta of ApoE Atherosclerosis Mice
(25) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 20 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. On Day 21, one mouse from each group was randomly sacrificed. The aortas were fixed in 4% paraformaldehyde for 24 to 48 hours, and dissected for general oil red O staining. The aortas were observed and photographed under a stereo microscope at 7×.
(26) The area of lipid plaques (indicated by arrow) at the aortic arch, thoracic aorta and abdominal aorta of mice was remarkably less than that in the control group administered with vehicle PBS; and the area ratios of lipid to blood vessel were 48.1% in the control group administered with vehicle PBS, and 39.4% in the group administered with plasminogen (
Example 3
Plasminogen Ameliorates Lipid Deposition in Aortic Sinus of ApoE Atherosclerosis Mice
(27) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The mice were administered for 30 days and sacrificed on Day 31. The hearts were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C. overnight, respectively, and embedded in OCT. The frozen sections were 8 μm thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 200×.
(28) The results showed that the fat deposition (indicated by arrow) in aortic sinus of mice in the group administered with plasminogen (
Example 4
Plasminogen Ameliorates Aortic Sinus Injury in ApoE Atherosclerosis Mice
(29) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. The mice were sacrificed on Day 31. The hearts were fixed in 4% paraformaldehyde for 24 to 48 hours. The fixed tissue samples were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The aortic sinus tissue sections were 3 μm thick. The sections were dewaxed and rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid in alcohol, and returned to blue with ammonia water. The sections were sealed after dehydration with alcohol gradient, and observed under an optical microscope at 40× (
(30) The staining results showed that the lipid plaque deposition (indicated by arrow) in aortic sinus of mice in the group administered with plasminogen (
Example 5
Plasminogen Reduces Lipid Deposition in aorta of ApoE Atherosclerosis Mice
(31) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. The mice were sacrificed on Day 31. The aortas were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C. overnight, respectively, and embedded in OCT. The frozen sections were 8 μm thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 200×.
(32) The staining results showed that the area of deposition stained with oil red O in aorta (indicated by arrow) of mice in the group administered with plasminogen (
Example 6
Plasminogen Elevates the High-Density Lipoprotein Cholesterol in Serum of Diabetic Mice
(33) Twenty 26-week-old male db/db mice were randomly divided into groups, 11 mice in the group administered with plasminogen, and 9 mice in the control group administered with vehicle PBS. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 2 mg/0.2 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. After continuous injection for 35 days, the whole blood was collected from removed eyeballs, and centrifuged at 3500 r/min at 4° C. for 10 min, and the supernatant was taken and detected for the high-density lipoprotein cholesterol (HDL-C). The high-density lipoprotein cholesterol was detected using a kit (Nanjing Jiancheng Bioengineering Institute, Cat# A112-1) according to the method of the kit.
(34) The detection results showed that after continuous injection of human plasminogen into db/db mice for 35 days, the content of HDL-C in serum of mice in the group administered with plasminogen was higher than that in the control group administered with vehicle PBS (
(35) Diabetes mellitus is usually complicated with cardiovascular atherosclerosis .sup.[45,46]. High-density lipoprotein is an anti-atherosclerosisplasma lipoprotein, a protective factor of coronary heart disease, commonly known as “vascular scavenger”. The detection results demonstrate that plasminogen can elevate the level of serum HDL-C, and thus contribute to the improvement of atherosclerosis in diabetic mice.
Example 7
Plasminogen Lowers Low-Density Lipoprotein Cholesterol in Serum of Diabetic Mice
(36) Ten 24- to 25-week-old male db/db mice were randomly divided into groups, 5 mice in each of the group administered with plasminogen and the control group administered with vehicle PBS. Three db/m mice were taken as the normal control group. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, an equal volume of PBS was administered to mice in the PBS control group via the tail vein, and mice in the normal control group received no treatment. The first day of administration was set as Day 0. After continuous injection for 31 days, the whole blood was collected from removed eyeballs in mice, and centrifuged at 3500 r/min at 4° C. for 10 min, and the supernatant was taken and detected for the low-density lipoprotein cholesterol (LDL-C). The low-density lipoprotein cholesterol was detected using a kit (Nanjing Jiancheng Bioengineering Institute, Cat# A113-1) according to the method of the kit.
(37) The detection results showed that after continuous injection of human plasminogen into db/db mice for 31 days, the content of LDL-C in serum of mice in the group administered with plasminogen was lower than that in the control group administered with vehicle PBS (
(38) Low-density lipoprotein is a lipoprotein particle that carries cholesterol into peripheral tissue cells and can be oxidized into oxidized low-density lipoprotein. When low-density lipoprotein, particularly oxidized low-density lipoprotein (OX-LDL) is in excess, the cholesterol it carries accumulates on the arterial wall, causing arteriosclerosis. Therefore, low-density lipoprotein cholesterol is called “bad cholesterol” .sup.[52]. The experiment results demonstrate that plasminogen can reduce the content of low-density lipoprotein cholesterol in serum, and thus contribute to the control of atherosclerosis.
Example 8
Effect of Plasminogen on Body Weight of ApoE Atherosclerosis Mice
(39) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. Mice were weighed on Day 1 and Day 31 of administration.
(40) The results showed that there was no significant change in body weight of mice after administration of plasminogen for 30 days (
Example 9
Plasminogen Lowers the Content of Blood Lipid in ApoE Atherosclerosis Mice
(41) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. On Day 30, the mice fasted for 16 hours, and on Day 31, the blood was collected from removed eyeballs, and centrifuged to obtained a supernatant, which was used in detecting the content of serum total cholesterol (T-CHO), serum triglyceride (TG), and serum low-density lipoprotein cholesterol (LDL-C).
(42) 1. Content of Serum Total Cholesterol
(43) The content of serum total cholesterol was detected using a detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A111-1) according to the method of the detection kit.
(44) The detection results showed that the concentration of total cholesterol 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 (
(45) 2. Content of Serum Triglyceride
(46) The content of serum TG was detected using a TG detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A110-1) according to the COD-PAP method based on the instructions of the kit. The detection results showed that the concentration of TG 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 (
(47) 3. Content of Serum Low-Density Lipoprotein Cholesterol
(48) The content of serum low-density lipoprotein cholesterol was detected using a low-density lipoprotein cholesterol (LDL-C) detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A113-1) according to the method of the detection kit.
(49) The detection results showed that the concentration of LDL-C 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 (
(50) The above-mentioned results demonstrate that plasminogen can remarkably lower the content of serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol in atherosclerosis model mice, and improve atherosclerosis. Meanwhile, the risk of atherosclerotic complications, such as atherosclerotic cardiovascular disease, is reduced by lowering the content of serum total cholesterol, triglyceride, and low-density lipoprotein cholesterol.
Example 10
Plasminogen Ameliorates Compensatory Cardiac Hypertrophy in ApoE Atherosclerosis Mice
(51) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. After weighed on Day 31 of administration, the mice were sacrificed, their hearts were weighed, and cardiac coefficients were calculated. Cardiac coefficient (%)=heart weight/body weight×100.
(52) The results showed that the cardiac coefficient of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS (
Example 11
Plasminogen Ameliorates Lipid Deposition in Liver of ApoE Atherosclerosis Mice
(53) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. The mice were sacrificed on Day 31. The livers were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C. overnight, respectively, and embedded in OCT. The frozen sections were 8 μm thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 400×.
(54) The staining results showed that the fat deposition (indicated by arrow) in liver of mice in the group administered with plasminogen (
Example 12
Plasminogen Ameliorates Cardiac Injury in ApoE Atherosclerosis Mice
(55) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. The mice were sacrificed on Day 31. The hearts were fixed in 4% paraformaldehyde for 24 to 48 hours. The fixed tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 3 μm. The sections were dewaxed and rehydrated and washed with water once. The tissues were circled with a PAP pen, incubated with 3% hydrogen peroxide for 15 minutes, and washed with 0.01M PBS twice for 5 minutes each time. The sections were blocked with 5% normal goat serum (Vector laboratories, Inc., USA) for 30 minutes, and after the time was up, the goat serum liquid was discarded. Goat anti-mouse IgM (HRP) antibody (Abcam) was added to the sections dropwise, incubated for 1 hour at room temperature and washed with 0.01M PBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with alcohol gradient, permeabilization with xylenehe, and sealing with a neutral gum, the sections were observed under an optical microscope at 200×. IgM antibodies play an important role during the clearance of apoptotic and necrotic cells, and the local level of IgM antibodies at the injury site in tissues and organs are positively correlated with the degree of injury .sup.[50,51]. Therefore, detection of local level of IgM antibodies in tissues and organs can reflect the injury of the tissues and organs. The experiment showed that the positive expression of IgM in the heart of mice in the group administered with plasminogen (
Example 13
Plasminogen Lowers the Level of Cardiac Fibrosis in ApoE Atherosclerosis Mice
(56) 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 hyperlipemia model .sup.[47,48]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The administration lasted for 30 days. During the administration, mice continued to be fed with a high-fat and high-cholesterol diet. The mice were sacrificed on Day 31. The hearts were fixed in 4% paraformaldehyde for 24 to 48 hours. The fixed tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The tissue sections was 3 μm thick. The sections were dewaxed and rehydrated and washed with water once. After stained with 0.1% Sirius red in saturated picric acid for 30 min, the sections were flushed with running water for 2 min After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol, returned to blue with ammonia water, flushed with running water, dried and sealed with a neutral gum. The sections were observed under an optical microscope at 200×.
(57) Sirius red staining allows for long-lasting staining of collagen, and is a special staining method for collagen tissue in pathological sections to show collagen tissue specifically.
(58) The staining results showed that the collagen deposition (indicated by arrow) in the group administered with plasminogen (
Example 14
Plasminogen Lowers Lipid Deposition in Ventricle of Diabetic Mice
(59) Diabetes mellitus is usually complicated with cardiovascular atherosclerosis .sup.[45,46]. Cardiovascular atherosclerosis can lead to ischemic injury of cardiac myocytes. Oil red O staining can show lipid deposition and reflect the severity of injury .sup.[49].
(60) Nine 26-week-old male db/db mice were randomly divided into two groups, 4 mice in the group administered with plasminogen, and 5 mice in the control group administered with vehicle PBS. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 2 mg/0.2 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 35 days. The mice were sacrificed on Day 36. The hearts were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C. overnight, respectively, and embedded in OCT. The frozen sections were 8 μm thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 400×.
(61) The results showed that the lipid deposition (indicated by arrow) in ventricle of mice in the group administered with plasminogen (
Example 15
Plasminogen Ameliorates Aortic Sinus Fibrosis in ApoE Atherosclerosis Mice
(62) 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 model .sup.[31,32]. 50 μL of blood was taken from each model mouse three days before administration, and the total cholesterol (T-CHO) content was detected. The mice were randomly divided into two groups based on the T-CHO content, 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 an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. The mice were administered for 30 days and sacrificed on Day 31. The hearts were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C. overnight, respectively, and embedded in OCT. The frozen sections were 8 μm thick. After stained with 0.1% Sirius red in saturated picric acid for 30 min, the sections were flushed with running water for 2 min After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol, returned to blue with ammonia water, flushed with running water, dried and sealed with a neutral gum. The sections were observed under an optical microscope at 40×.
(63) The results showed that the area of collagen deposition (indicated by arrow) in the group administered with plasminogen (
Example 16
Protective Effect of Plasminogen on the Aortic Inner Wall Injury in Diabetic Mice
(64) Ten 24- to 25-week-old male db/db mice were weighed on the day the experiment started, i.e. Day 0, and were randomly divided into two groups based on the body weight, 5 mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. Plasminogen or PBS (PBS refers to Phosphate Buffer Saline, as the vehicle of plasminogen herein) was administered from Day 1 for 31 consecutive days. Mice in the group administered with plasminogen were injected with plasminogen at a dose of 2 mg/0.2 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 in the same manner. Mice were sacrificed on Day 32, and the aortas were fixed in 10% neutral formalin fixative for 24 hours. The fixed tissue samples were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The tissue sections were 5 μm thick. The sections were dewaxed and rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid in alcohol, and returned to blue with ammonia water. The sections were sealed after dehydration with alcohol gradient, and observed under an optical microscope at 400×.
(65) The HE staining results showed that in the control group administered with vehicle PBS, there was a foam cell deposition (indicated by arrow) on the vascular wall, the middle elastic membrane was arranged in disorder, and the vascular wall was accidented (
Example 17
Protective Effect of Plasminogen on the Myocardial Injury in Diabetic Mice
(66) Diabetes mellitus is usually complicated with cardiovascular atherosclerosis .sup.[45,46]. Cardiovascular atherosclerosis can lead to ischemic injury of cardiac myocytes. Cardiac troponin I (CTNI) is an important marker of myocardial injury, and its serum concentration can reflect the extent of myocardial injury .sup.[44]. In this experiment, the repair effect of plasminogen on myocardial injury was observed by detecting cardiac troponin I.
(67) Twenty-eight 24- to 25-week-old male db/db mice were weighed on the day the experiment started, i.e. Day 0, and were randomly divided into two groups based on the body weight, 12 mice in the control group administered with vehicle PBS, and 16 mice in the group administered with plasminogen. From the second day after grouping, i.e. Day 1, plasminogen or PBS was administered for 31 consecutive days. Mice in the group administered with plasminogen were injected with plasminogen at a dose of 2 mg/0.2 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. On day 32, blood was taken from the removed eyeballs and centrifuged at 3500 r/min for 15-20 minutes, and the supernatant was used for detection for determining cardiac troponin I concentration. The results showed that the concentration of cardiac troponin I in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was extremely significant (
Example 18
Plasminogen Increases the Concentration of Serum High-Density Lipoprotein Cholesterol in 3% Cholesterol Hyperlipemia Model Mice
(68) 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.[52,53]. 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 μL 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 10 and Day 20, the mice fasted for 16 hours, and on Day 11 and Day 21, 50 μL of blood was collected from orbital venous plexus, and centrifuged to obtain a supernatant, which was used in detecting the serum high-density lipoprotein cholesterol (HDL-C). The content of high-density lipoprotein cholesterol herein was detected by the method as described in a detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A112-1).
(69) The detection results showed that the concentration of HDL-C in serum of mice in the group administered with plasminogen was remarkably higher than that in the control group administered with vehicle PBS, and the HDL-C concentrations of the two groups were statistically different after administration for 10 or 20 days (
Example 19
Plasminogen Lowers Risk of Atherosclerosis Formation in 3% Cholesterol Hyperlipemia Model Mice
(70) 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.[52,53]. 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 μL of blood was taken from each mouse three days before administration, and the total cholesterol (T-CHO) 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. After administration on Day 20, the mice began to fast for 16 hours, and on Day 21, 50 μL of blood was collected from orbital venous plexus, and centrifuged to obtain a supernatant. The total cholesterol content was detected by using a total cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A111-1); and the high-density lipoprotein cholesterol (HDL-C) content was detected using a high-density lipoprotein cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A112-1).
(71) Atherosclerosis index is a comprehensive index to predict atherosclerosis clinically. It is considered to be of greater clinical importance as an estimate of the risk of coronary heart disease than total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein alone .sup.[54]. Atherosclerosis index=(T-CHO-HDL-C)/HDL-C.
(72) The calculation results showed that the atherosclerosis index of 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 20
Plasminogen Lowers Risk of Onset of Heart Disease in 3% Cholesterol Hyperlipemia Model Mice
(73) 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.[52,53]. 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 μL of blood was taken from each mouse three days before administration, and the total cholesterol (T-CHO) was detected. The mice were randomly divided into two groups based on the total cholesterol concentration, 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. After administration on Day 20, the mice began to fast for 16 hours, and on Day 21, 50 μL of blood was collected from orbital venous plexus, and centrifuged to obtain a supernatant. The total cholesterol content was detected by using a total cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A111-1); and the high-density lipoprotein cholesterol (HDL-C) content was detected using a high-density lipoprotein cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat# A112-1). Cardiac risk index=T-CHO/HDL.
(74) Cardiac risk index (CRI) is used to assess the risk of heart disease induced by dyslipidemia.sup.[54].
(75) The results showed that CRI in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was extremely significant (
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