Method for preventing and treating liver fibrosis

Abstract

The present invention relates to a method for preventing and treating hepatic fibrosis, comprising administering an effective amount of plasminogen to a subject.

Claims

1. A method for preventing or treating hepatic collagen deposition or fibrosis caused by a hepatic tissue injury in a subject, comprising administering an effective amount of plasminogen having at least 75% sequence identity to the full-length sequence of SEQ ID NO. 2 to the subject.

2. The method of claim 1, wherein the fibrosis is caused by an ischemic reperfusion injury, drugs, systemic immune disease, chemicals, inflammatory response, alcoholism, cancer, or fat deposits.

3. The method of claim 1, wherein the plasminogen is administered in combination with one or more other drugs.

4. The method of claim 3, wherein the one or more other drugs comprise: a hypolipidemic drug, an anti-platelet drug, an antihypertensive drug, a vasodilator, a hypoglycemic drug, an anticoagulant drug, a thrombolytic drug, a hepatoprotective drug, an anti-fibrosis drug, an anti-arrhythmia drug, a cardiotonic drug, a diuretic drug, an anti-tumor drug, a radiotherapeutic or chemotherapeutic drug, an inflammatory regulatory drug, an immunomodulatory drug, an antiviral drug, or an antibiotic.

5. The method of claim 1, wherein the plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the full-length sequence of SEQ ID No. 2, and still has the plasminogen activity.

6. The method of claim 1, wherein the plasminogen comprises a plasminogen active fragment and still has the plasminogen activity.

7. The method of claim 1, wherein the plasminogen is Glu-plasminogen or Lys-plasminogen.

8. The method of claim 1, wherein the plasminogen is a natural or synthetic human plasminogen.

9. 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.

10. The method of claim 9, wherein the plasminogen is administered daily.

11. The method of claim 1, wherein the plasminogen has at least 90%, 95%, or 99% sequence identity to the full-length sequence of SEQ ID No. 2.

12. The method of claim 1, wherein the plasminogen is Glu-plasminogen.

13. A method for preventing or treating hepatic collagen deposition or fibrosis in a subject, comprising administering an effective amount of plasminogen having at least 75% sequence identity to the full-length sequence of SEQ ID NO. 2 to the subject.

14. The method of claim 13, wherein the hepatic collagen deposition or fibrosis is caused or complicated by diabetes mellitus, caused or complicated by atherosclerosis, caused or complicated by hyperlipemia, caused by drugs, or caused by chronic hepatic disease.

15. The method of claim 14, wherein the hyperlipemia comprises one or more selected from: an elevated blood triglyceride level, an elevated total blood cholesterol level, increased blood low-density lipoprotein, and increased blood very low-density lipoprotein.

16. The method of claim 14, wherein the drugs are hepatotoxic drugs.

17. The method of claim 16, wherein the drugs comprise a chemotherapeutic drug, an antibiotic drug, a hypolipidemic drug, an antihypertensive drug, or a hypoglycemic drug.

18. The method of claim 14, wherein the chronic hepatic disease is viral hepatitis.

19. A method for preventing or treating alcoholic hepatic collagen deposition, alcoholic hepatic fibrosis non-alcoholic hepatic collagen deposition or non-alcoholic hepatic fibrosis in a subject, comprising administering an effective amount of plasminogen having at least 75% sequence identity to the full-length sequence of SEQ ID NO. 2 to the subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows observed results of Sirius red staining of liver after administration of plasminogen to carbon tetrachloride-induced hepatic fibrosis model mice for 28 days. A represents the blank control group, B represents the control group administered with vehicle PBS, C represents the group administered with plasminogen, and D represents the quantitative analysis results. The results showed that the collagen deposition (indicated by arrow) in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05); compared with the control group administered with vehicle PBS, the level of collagen deposition in mice in the group administered with plasminogen was closer to that in blank control mice. It indicates that plasminogen can reduce collagen deposition in liver, and ameliorate hepatic fibrosis in hepatic fibrosis model mice.

(2) FIG. 2 shows a representative image of Sirius red staining of aortic sinus after administration of plasminogen to ApoE atherosclerosis model mice for 30 days. A and C refer to the control group administered with vehicle PBS, and B and D refer to the group administered with plasminogen. The results showed that the area of collagen deposition (indicated by arrow) in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, indicating that plasminogen can reduce the level of aortic sinus fibrosis in arteriosclerosis model mice.

(3) FIG. 3 shows a representative image of Sirius red staining of liver after administration of plasminogen to carbon tetrachloride-induced hepatic fibrosis model mice for 14 days. A represents a blank control group, B represents a control group administered with vehicle PBS, and C represents a group administered with plasminogen. The results showed that in the group administered with plasminogen, the collagen deposition was remarkably less than that in the control group administered with vehicle PBS, and the level of collagen deposition in mice was close to that in blank control mice. It indicates that plasminogen can reduce collagen deposition in liver, and ameliorate hepatic fibrosis in hepatic fibrosis model mice.

(4) FIG. 4 shows a representative image of Sirius red staining of aortic sinus after administration of plasminogen to 16-week hyperlipemia model mice for 30 days. A and C refer to the control group administered with vehicle PBS, and B and D refer to the group administered with plasminogen. The results showed that the area of collagen deposition (indicated by arrow) on the intima of the aortic sinus wall in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, indicating that plasminogen can reduce the level of intimal fibrosis of the aortic sinus wall in hyperlipemia model mice.

(5) FIG. 5 shows a representative image of Sirius red staining of skin after administration of plasminogen to bleomycin-induced systemic sclerosis model mice for 21 days. A represents the blank control group, B represents the control group administered with vehicle PBS, C represents the group administered with plasminogen, and D represents the group with impaired PLG activity. The results showed that in the bleomycin-induced systemic sclerosis mouse model, the collagen fiber bundles in the upper dermis were remarkably increased, the collagen fibers were thick and big, and dense in arrangement, and the dermal layer was thickened in the group administered with vehicle PBS and the group with impaired PLG activity; while in the group administered with plasminogen, the fibroblasts in the dermal layer were remarkably less than those in the group administered with vehicle PBS, and the thickness of the dermal layer of the skin was close to the normal level.

(6) FIG. 6 shows a representative image of Sirius red staining of lung after administration of plasminogen to bleomycin-induced systemic sclerosis model mice for 21 days. A represents the control group administered with vehicle PBS, B represents the group administered with plasminogen, and C represents the quantitative analysis results. The results showed that in the bleomycin-induced systemic sclerosis mouse model, the degree of pulmonary fibrosis (indicated by arrow) in mice in the group administered with vehicle PBS was higher than that in the group administered with plasminogen; while in the group administered with plasminogen, the morphology of pulmonary alveolar walls in mice was close to normal, cells with the inflammatory level was remarkably reduced, the degree of fibrosis was remarkably lower than that in the group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05).

(7) FIG. 7 shows a representative image of Sirius red staining of heart after administration of plasminogen to bleomycin-induced systemic sclerosis model mice for 21 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. Studies have found that in the bleomycin-induced systemic sclerosis mouse model, the degree of collagen deposition (indicated by arrow) in heart in the control group administered with vehicle PBS was higher than that in the group administered with plasminogen. It indicates that plasminogen can effectively reduce bleomycin-induced cardiac fibrosis.

(8) FIG. 8 shows a representative image of Sirius red staining of kidney after administration of plasminogen to bleomycin-induced systemic sclerosis model mice for 21 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that in the bleomycin-induced systemic sclerosis mouse model, the degree of collagen fibrosis (indicated by arrow) in the kidney in the control group administered with vehicle PBS was higher than that in the group administered with plasminogen. It indicates that plasminogen can effectively reduce bleomycin-induced renal fibrosis.

(9) FIG. 9 shows observed results of immunostaining of type IV collagen in the kidney after administration of plasminogen to 24- to 25-week-old diabetic mice for 31 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that the positive staining (indicated by arrow) of IV collagen in the group administered with plasminogen was remarkably higher than that in the control group administered with vehicle PBS, indicating that plasminogen can ameliorate renal fibrosis in diabetic mice.

(10) FIG. 10 shows observed results of masson staining of kidney after administration of plasminogen to 26-week-old diabetic mice for 35 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that in the control group administered with vehicle PBS, glomerular mesangial hyperplasia existed, mesangial matrix increased, renal interstitial fibrosis was mild (indicated by arrow), and the hyperplastic fibrosis was blue. In the group administered with plasminogen, the glomerular mesangial cells and matrix were remarkably less than those in the control group, and renal interstitial fibrosis was remarkably reduced. It indicates that plasminogen can ameliorate fibrotic lesions in the kidneys of diabetic mice.

(11) FIG. 11 shows observed results of masson staining of heart after administration of plasminogen to 24- to 25-week-old diabetic mice for 31 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that in the control group administered with vehicle PBS, blue hyperplastic collagen fibers (indicated by arrow) could be seen between myocardial fibers, showing mild myocardial fibrosis; while in the group administered with plasminogen, a few light blue hyperplastic collagen fibers could be seen between myocardial fibers, and the myocardial fibrosis was remarkably alleviated compared with the control group. It indicates that plasminogen can ameliorate cardiac fibrosis in diabetic mice.

(12) FIG. 12 shows a representative image of Sirius red staining of heart after administration of plasminogen to 17- to 18-week-old diabetic mice for 35 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that the deposition of collagen fibers (indicated by arrow) in mice in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS. It indicates that plasminogen can reduce cardiac fibrosis in relatively young (17- to 18-week-old) diabetic mice.

(13) FIG. 13 shows a representative image of Sirius red staining of heart after administration of plasminogen to 26- to 27-week-old diabetic mice for 35 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that the collagen deposition (indicated by arrow) in mice in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS. It indicates that plasminogen can attenuate cardiac fibrosis in relatively old (26- to 27-week-old) diabetic mice.

(14) FIG. 14 shows observed results of immunostaining of type IV collagen in the kidney after administration of plasminogen to cisplatin-induced renal fibrosis model mice for 7 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that the positive expression (indicated by arrow) of type IV collagen in the kidney in the control group administered with vehicle PBS was remarkably higher than that in the group administered with plasminogen. It indicates that plasminogen can ameliorate renal fibrosis in cisplatin-induced renal fibrosis model mice.

(15) FIG. 15 shows a representative image of Sirius red staining of heart after administration of plasminogen to ApoE atherosclerosis model mice for 30 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that the collagen deposition (indicated by arrow) in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, indicating that plasminogen can alleviate cardiac fibrosis in ApoE atherosclerosis model mice.

(16) FIG. 16 shows a representative image of Sirius red staining of heart after administration of plasminogen to C57 hyperlipemia model mice for 30 days. A represents the control group administered with vehicle PBS, and B represents the group administered with plasminogen. The results showed that the collagen deposition (indicated by arrow) in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, indicating that plasminogen can alleviate cardiac fibrosis in hyperlipemia model mice.

(17) FIG. 17 shows observed results of Sirius red staining of kidney after administration of plasminogen to purine-induced chronic renal injury model mice for 10 days. A represents the control group administered with vehicle PBS, B represents the group administered with plasminogen, C represents the group with impaired PLG activity, and D represents the quantitative analysis results. The collagen deposition (indicated by arrow) in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS and the group with impaired PLG activity, and the quantitative analysis showed a significant statistical difference between the group administered with plasminogen and the group with impaired PLG activity (* indicates P<0.05). It indicates that plasminogen can alleviate renal fibrosis induced by chronic renal injury, and promote the repair of renal injury.

(18) FIG. 18 shows the observed results of Sirius red-staining for pancreatic islets after administration of plasminogen to 24- to 25-week-old diabetic mice for 31 days. A represents the control group administered with vehicle PBS, B represents the group administered with plasminogen, and C represents the quantitative analysis results. The results showed that the collagen deposition (indicated by arrow) in the pancreatic islet of mice in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05). It indicates that plasminogen can ameliorate injury and fibrosis of the pancreatic islet caused by diabetes mellitus.

(19) FIG. 19 shows observed results of Sirius red staining of kidney after administration of plasminogen to 3% cholesterol hyperlipemia model mice for 30 days. A represents the blank control group, B represents the control group administered with vehicle PBS, C represents the group administered with plasminogen, and D represents the quantitative analysis results. The results showed that the collagen deposition in kidney (indicated by arrow) in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, and the statistical difference was significant; and in the group administered with plasminogen, fibrosis was substantially restored to a normal level. It indicates that plasminogen can effectively reduce renal fibrosis in 3% cholesterol hyperlipemia model mice.

EXAMPLES

Example 1. Plasminogen Reduces Collagen Deposition in Liver During Induction of Hepatic Fibrosis by Carbon Tetrachloride

(20) Twenty 7- to 8-week-old female C57 mice were randomly divided into three groups, 5 mice in the blank control group, 7 mice the control group administered with vehicle PBS, and 8 mice in the group administered with plasminogen. Mice in the control group administered with vehicle PBS and the group administered with plasminogen were injected with carbon tetrachloride intraperitoneally at a dose of 1 mL/kg body weight, three times a week for four consecutive weeks, to establish the hepatic fibrosis model.sup.[36,37]; while the blank control mice were injected with a corresponding volume of corn oil intraperitoneally. Carbon tetrachloride required to be diluted with corn oil, and the dilution ratio of carbon tetrachloride to corn oil was 1:3. Administration began on the day of model establishment, i.e., 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 28 days. The blank control group was not treated with injection. The mice were sacrificed on Day 29. The livers were fixed in 4% paraformaldehyde for 24 hours. The fixed livers 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 for 60 min, the sections were flushed with running water. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol and returned to blue with ammonia water, flushed with running water, dried and sealed. The sections were observed under an optical microscope at 200×.

(21) The results showed that the collagen deposition in the group administered with plasminogen (FIG. 1C) was remarkably less than that in the control group administered with vehicle PBS (FIG. 1B), and the statistical difference was significant (FIG. 1D); compared with the control group administered with vehicle PBS, the level of collagen deposition (indicated by arrow) in mice in the group administered with plasminogen was closer to that in blank control mice (FIG. 1A). It indicates that plasminogen can reduce collagen deposition in liver, and ameliorate hepatic fibrosis in hepatic fibrosis model mice.

Example 2. Plasminogen Ameliorates Aortic Sinus Fibrosis in ApoE Atherosclerosis Mice

(22) 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× (FIGS. 2A and 2B) and 200× (FIGS. 2C and 2D).

(23) The results showed that the area of collagen deposition (indicated by arrow) on the intima of the aortic sinus wall in the group administered with plasminogen (FIGS. 2B and D) was remarkably less than that in the control group administered with vehicle PBS (FIGS. 2A and C), indicating that plasminogen can reduce the level of aortic sinus fibrosis in arteriosclerosis model mice.

Example 3. Plasminogen Ameliorates Carbon Tetrachloride-Induced Hepatic Fibrosis

(24) Fifteen 9-week-old female C57 mice were randomly divided into three groups, a blank control group, a control group administered with vehicle PBS, and a group administered with plasminogen, 5 mice in each group. Mice in the control group administered with vehicle PBS and the group administered with plasminogen were injected with carbon tetrachloride intraperitoneally at a dose of 1 mL/kg body weight, three times a week for two consecutive weeks, to establish the hepatic fibrosis model.sup.[36,37]; while the blank control mice were injected with a corresponding volume of corn oil according to the injection method of model mice.

(25) Carbon tetrachloride required to be diluted with corn oil, and the dilution ratio of carbon tetrachloride to corn oil was 1:3. Administration began after model establishment. 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 mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 14 consecutive days. The blank control group was not treated with injection. The mice were sacrificed on Day 15. The livers were fixed in 4% paraformaldehyde for 24 hours. The fixed livers 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 for 60 min, the sections were flushed with running water. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol and returned to blue with ammonia water, flushed with running water, dried and sealed. The sections were observed under an optical microscope at 200×.

(26) The results showed that the collagen deposition in the group administered with plasminogen (FIG. 3C) was remarkably less than that in the control group administered with vehicle PBS (FIG. 3B), and compared to the group administered with PBS, the level of collagen deposition in mice in the group administered with plasminogen was closer to that in blank control mice (FIG. 3A). It indicates that plasminogen can reduce collagen deposition in liver, and ameliorate hepatic fibrosis in hepatic fibrosis model mice.

Example 4. Plasminogen Reduces Aortic Sinus Fibrosis in 16-Week Hyperlipemia Model Mice

(27) Eleven 6-week-old male C57 mice were fed with a high-fat and high-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to induce the hyperlipemia model.sup.[30,31]. This model was designated as the 16-week hyperlipemia model. The model mice continued to be fed with a high-cholesterol diet. 50 μL of blood was taken from each 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, 6 mice in the control group administered with vehicle PBS, and 5 mice in the group administered with plasminogen. 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. The mice were administered for 30 days and sacrificed on Day 31. The heart materials were taken and 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 aortic sinus 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 40× (FIGS. 4A and 4B) and 200× (FIGS. 4C and 4D).

(28) The results showed that the area of collagen deposition (indicated by arrow) on the intima of the aortic sinus wall in the group administered with plasminogen (FIGS. 4B and 4D) was remarkably less than that in the control group administered with vehicle PBS (FIGS. 4A and 4C), indicating that plasminogen can alleviate the level of aortic sinus fibrosis in hyperlipemia model mice.

Example 5. Plasminogen Lowers Skin Fibrosis in Systemic Sclerosis Mice

(29) Fifteen 12-week-old male C57 mice were randomly divided into three groups, a blank control group, a control group administered with vehicle PBS (PBS refers to Phosphate Buffer Saline, as a vehicle of plasminogen herein), and a group administered with plasminogen, 5 mice in each group, and five 13-week-old mice with impaired PLG activity were taken. The mice were weighed and grouped on the day when the experiment began, i.e., Day 0. Model establishment and administration began from the next day, wherein mice in the control group administered with vehicle PBS and the group administered with plasminogen as well as mice with impaired PLG activity were injected with bleomycin subcutaneously at a dose of 0.1 mg/0.1 mL/mouse/day to induce systemic sclerosis.sup.[26]. Mice in the blank control group were injected with normal saline subcutaneously at a dose of 0.1 mL/mouse/day; meanwhile, on Day 1, plasminogen or PBS was administered for 21 consecutive days for model establishment. Mice in the group administered with plasminogen were injected with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, an equal volume of PBS was administered to mice in the control group administered with vehicle PBS, and the normal mouse group and mice with impaired PLG activity were not treated. The mice were sacrificed on Day 22. The back skin tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed skin 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 100×.

(30) Sirius red staining allows for long-lasting staining of collagen. As a special staining method for pathological sections, Sirius red staining can show the collagen tissue specifically.

(31) The results showed that in the bleomycin-induced systemic sclerosis mouse model, it was observed under a microscope that the collagen fiber bundles in the upper dermis were remarkably increased, the collagen fibers were thick and big, and dense in arrangement, and the dermal layer was thickened in mice in the group administered with vehicle PBS (FIG. 5B) and the group with impaired PLG activity (FIG. 5D); while in the group administered with plasminogen (FIG. 5C), the fibroblasts in the dermal layer were remarkably less than those in the control group administered with vehicle PBS, and the thickness of the dermal layer of the skin was substantially close to the normal level (FIG. 5A). It indicates that plasminogen can effectively reduce bleomycin-induced skin fibrosis.

Example 6. Plasminogen Lowers Pulmonary Fibrosis in Systemic Sclerosis Mice

(32) Seventeen 12-week-old male C57 mice were randomly divided into two groups, 11 mice in the control group administered with vehicle PBS, and 6 mice in the group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e., Day 0. Model establishment and administration began from Day 1, wherein mice in both groups were injected with bleomycin subcutaneously at a dose of 0.1 mg/0.1 mL/mouse/day to induce systemic sclerosis.sup.[26], and plasminogen or PBS was administered for 21 consecutive days for model establishment. Mice in the group administered with plasminogen were injected with 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 in the same manner. The mice were sacrificed on Day 22. The lung tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed lung 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×.

(33) Studies have found that in the bleomycin-induced systemic sclerosis mouse model, it was observed under a microscope that the degree of collagen fibrosis (indicated by arrow) in the group administered with vehicle PBS (FIG. 6A) was higher than that in the group administered with plasminogen (FIG. 6B); while in the group administered with plasminogen, the morphology of pulmonary alveolar walls in mice was close to the normal level, the inflammatory cells were remarkably reduced, the degree of fibrosis was remarkably lower than that in the group administered with vehicle PBS, and the statistical difference was significant (FIG. 6C). It indicates that plasminogen can effectively reduce lung tissue fibrosis in bleomycin-induced systemic sclerosis mice.

Example 7. Plasminogen Lowers Cardiac Fibrosis in Systemic Sclerosis Mice

(34) Ten 12-week-old male C57 mice were randomly divided into two groups, 5 mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e., Day 0. Model establishment and administration began from Day 1, wherein mice were injected with bleomycin subcutaneously at a dose of 0.1 mg/0.1 mL/mouse/day to induce systemic sclerosis.sup.[26], and plasminogen or PBS was administered for 21 consecutive days. Mice in the group administered with plasminogen were injected with 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 sacrificed on Day 22. The hearts were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed hearts 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×.

(35) Studies have found that in the bleomycin-induced systemic sclerosis mouse model, it was observed under a microscope that the collagen deposition in heart in the control group administered with vehicle PBS (FIG. 7A) was higher than that in the group administered with plasminogen (FIG. 7B). It indicates that plasminogen can effectively reduce bleomycin-induced cardiac fibrosis.

Example 8. Plasminogen Lowers Renal Fibrosis in Systemic Sclerosis Mice

(36) Ten 12-week-old male C57 mice were randomly divided into two groups, 5 mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e., Day 0. Model establishment and administration began from Day 1, wherein all mice were injected with bleomycin subcutaneously at a dose of 0.1 mg/0.1 mL/mouse/day to induce systemic sclerosis, and plasminogen or PBS was administered for 21 consecutive days for model establishment. Mice in the group administered with plasminogen were injected with 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. The mice were sacrificed on Day 22. The kidneys were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidneys 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×.

(37) The results showed that in the bleomycin-induced systemic sclerosis mouse model, the degree of collagen fibrosis (indicated by arrow) in the kidney in the control group administered with vehicle PBS (FIG. 8A) was higher than that in the group administered with plasminogen (FIG. 8B). It indicates that plasminogen can effectively reduce bleomycin-induced renal fibrosis.

Example 9. Plasminogen Lowers Collagen Deposition in Kidney of Diabetic Mice

(38) Ten 24- to 25-week-old male db/db mice were randomly divided into two groups, five mice in each of a control group administered with vehicle PBS and a group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS 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. The mice were sacrificed after administration of plasminogen for 31 days. The kidney tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 μm. The sections were dewaxed and rehydrated and washed with water once. The sections were 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 10% normal goat serum (Vector laboratories, Inc., USA) for 1 hour, and after the time was up, the serum was thrown away, and the tissues were circled with a PAP pen. The sections were incubated with rabbit anti-mouse polyclonal antibody (Abcam) against IV collagen overnight at 4° C. and washed with TBS twice for 5 minutes each time. The sections were incubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperature and washed with TBS twice. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washed with water three times, the sections were counterstained with hematoxylin for 30 seconds and flushed with running water for 5 minutes. After dehydration with alcohol gradient, permeabilization with xylene, and sealing with a neutral gum, the sections were observed under an optical microscope at 200×.

(39) Diabetic nephropathy is a chronic complication of diabetes mellitus, and glomerular sclerosis and renal interstitial fibrosis are typical pathological changes.sup.[27].

(40) The results showed that the positive staining of IV collagen in the group administered with plasminogen (FIG. 9B) was remarkably more than that in the control group administered with vehicle PBS (FIG. 9A), indicating that plasminogen can reduce collagen deposition (indicated by arrow) in the kidney tissue, and suggesting that plasminogen is expected to prevent renal tissue fibrosis caused by diabetes mellitus by reducing collagen deposition in the kidney tissue.

Example 10. Plasminogen Ameliorates Renal Fibrosis in Diabetic Mice

(41) Ten 26-week-old male db/db mice were randomly divided into two groups, 5 mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS was administered from day 1 for 35 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. The mice were sacrificed on Day 36. The kidney tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 μm. The sections were dewaxed and rehydrated and then put into a potassium dichromate solution overnight. The sections were stained with iron hematocylin for 3 to 5 minutes, and flushed slightly with running water. The sections were differentiated with 1% hydrochloric acid in alcohol, treated with ammonia water for 1 second, and rinsed with water. The sections were stained in ponceau acid fuchsin fluid for 8 minutes, and rinsed rapidly in water. The sections were treated with 1% phosphomolybdic acid aqueous solution for about 2 minutes, and counterstained with aniline blue solution for 6 minutes. The sections were rinsed with 1% glacial acetic acid for about 1 minute. The sections were sealed after dehydration with absolute ethanol, and permeabilization with xylene, and were observed under an optical microscope at 200×.

(42) Masson staining can reveal tissue fibrosis. The results showed that in the control group administered with vehicle PBS (FIG. 10A), glomerular mesangial hyperplasia existed, mesangial matrix increased, renal interstitial fibrosis was mild (indicated by arrow), and the hyperplastic fibrosis was blue. In the group administered with plasminogen (FIG. 10B), the glomerular mesangial cells and matrix were remarkably less than those in the control group, and renal interstitial fibrosis was remarkably reduced. It indicates that plasminogen can ameliorate renal fibrosis in diabetic mice.

Example 11. Plasminogen Ameliorates Cardiac Fibrosis in 24- to 25-Week-Old Diabetic Mice

(43) Ten 24- to 25-week-old male db/db mice were randomly divided into two groups, five mice in each of a control group administered with vehicle PBS and a group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS 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. The mice were sacrificed after administration of plasminogen for 31 days. The heart tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed heart tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 μm. The sections were dewaxed and rehydrated and then put into a potassium dichromate solution overnight. The sections were stained with iron hematocylin for 3 to 5 minutes, and flushed slightly with running water. The sections were differentiated with 1% hydrochloric acid in alcohol, treated with ammonia water for 1 second, and rinsed with water. The sections were stained in ponceau acid fuchsin fluid for 8 minutes, and rinsed rapidly in water. The sections were treated with 1% phosphomolybdic acid aqueous solution for about 2 minutes, and counterstained with aniline blue solution for 6 minutes. The sections were rinsed with 1% glacial acetic acid for about 1 minute. The sections were sealed after dehydration with absolute ethanol, and permeabilization with xylene, and were observed under an optical microscope at 200×.

(44) The most common complication of diabetes mellitus is excessive accumulation of connective tissues (pathological fibrosis). Myocardial interstitial fibrosis may be the characteristic pathological change of diabetic cardiomyopathy.sup.[28-29].

(45) Masson staining can reveal tissue fibrosis. The results showed that in the control group administered with vehicle PBS (FIG. 11A), blue hyperplastic collagen fibers (indicated by arrow) could be seen between myocardial fibers, showing mild myocardial fibrosis; while in the group administered with plasminogen (FIG. 11B), a few light blue hyperplastic collagen fibers could be seen between myocardial fibers, and the myocardial fibrosis was remarkably alleviated compared with the control group. It indicates that plasminogen can ameliorate cardiac fibrosis in diabetic mice.

Example 12. Plasminogen Lowers Collagen Deposition in Heart of 17- to 18-Week-Old Diabetic Mice

(46) Eight 17- to 18-week-old male db/db mice were randomly divided into two groups, four mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS was administered from day 1 for 35 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. The mice were sacrificed after administration of plasminogen for 35 days. The heart tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed hearts 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×.

(47) The results showed that the deposition of collagen fibers (indicated by arrow) in mice in the group administered with plasminogen (FIG. 12B) was remarkably less than that in the control group administered with vehicle PBS (FIG. 12A). It indicates that plasminogen can reduce collagen deposition in the heart tissue, and suggests that plasminogen is expected to reduce heart tissue fibrosis in relatively young (17- to 18-week-old) diabetic mice by lowering collagen deposition in the heart tissue.

Example 13. Plasminogen Lowers Collagen Deposition in Heart of 26- to 27-Week-Old Diabetic Mice

(48) Nine 26- to 27-week-old male db/db mice were randomly divided into two groups, 5 mice in the control group administered with vehicle PBS, and 4 mice in the group administered with plasminogen. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS was administered from day 1 for 35 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. The mice were sacrificed after administration of plasminogen for 35 days. The heart tissues were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed hearts 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 for 60 min, the sections were flushed with running water. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol and returned to blue with ammonia water, flushed with running water, dried and sealed. The sections were observed under an optical microscope at 200×.

(49) The results showed that the deposition of collagen fibers (indicated by arrow) in mice in the group administered with plasminogen (FIG. 13B) was remarkably less than that in the control group administered with vehicle PBS (FIG. 13A). It indicates that plasminogen can reduce collagen deposition in the heart tissue, and suggests that plasminogen is expected to reduce heart tissue fibrosis in relatively old (26- to 27-week-old) diabetic mice by lowering collagen deposition in the heart tissue.

Example 14. Plasminogen Lowers Renal Fibrosis in Cisplatin-Induced Renal Fibrosis Model Mice

(50) Ten healthy 8-9-week-old male C57 mice were randomly divided into two groups, five mice in each of the control group administered with vehicle PBS and the group administered with plasminogen. After the completion of grouping, single intraperitoneal injection of cisplatin was performed at 10 mg/Kg body weight to establish the renal fibrosis model.sup.[30]. After the model was established, mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day via tail vein injection, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS. The mice were weighed and grouped on the day when the experiment began, i.e. day 0; the mice received intraperitoneal injection of cisplatin for modelling on day 1, and were administered with plasminogen or vehicle PBS 3 hours after the modelling, for an administration period of 7 days. The mice were sacrificed on Day 8. The kidneys were fixed in 4% paraformaldehyde fixative for 24 hours. The fixed kidney tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 μm. The sections were dewaxed and rehydrated and washed with water once. The sections were repaired with citric acid for 30 minutes, and gently rinsed with water after cooling at room temperature for 10 minutes. The sections were incubated with 3% hydrogen peroxide for 15 minutes, and the tissues were circled with a PAP pen. The sections were blocked with 10% goat serum (Vector laboratories, Inc., USA) for 1 hour, and after the time was up, the goat serum liquid was discarded. The sections were incubated with rabbit anti-mouse IV collagen antibody (Abcam) overnight at 4° C. and washed with TBS twice for 5 minutes each time. The sections were incubated with a secondary antibody, goat anti-rabbit IgG (HRP) antibody (Abcam), for 1 hour at room temperature and washed with TBS twice for 5 minutes each time. The sections were developed with a DAB kit (Vector laboratories, Inc., USA). After washing with water three times, the sections were counterstained with hematoxylin for 30 seconds, returned to blue with running water for 5 minutes, and washed with TBS once. After dehydration with a gradient, permeabilization and sealing, the sections were observed under an optical microscope at 200×.

(51) Cisplatin is a broad-spectrum anti-tumor drug with extensive clinical application and reliable efficacy. However, it has severe nephrotoxicity, mainly results in renal tubular and renal interstitial injuries which eventually develop into renal fibrosis.sup.[30]. The experimental results showed that the positive expression (indicated by arrow) of type IV collagen in the kidney in the control group administered with vehicle PBS (FIG. 14A) was remarkably higher than that in the group administered with plasminogen (FIG. 14B). It indicates that plasminogen can ameliorate renal fibrosis in cisplatin-induced renal fibrosis model mice.

Example 15. Plasminogen Ameliorates the Level of Cardiac Fibrosis in ApoE Atherosclerosis Mice

(52) 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.[31,32]. 50 μL 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 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 which 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×.

(53) The results showed that the collagen deposition (indicated by arrow) in the group administered with plasminogen (FIG. 15B) was remarkably less than that in the control group administered with vehicle PBS (FIG. 15A), suggesting that plasminogen can prevent and lower cardiac fibrosis induced by atherosclerosis by reducing collagen deposition in the heart tissue of ApoE atherosclerosis model mice.

Example 16. Plasminogen Lowers Cardiac Fibrosis in Hyperlipemia Model Mice

(54) Eleven 6-week-old male C57 mice were fed with a high-fat and high-cholesterol diet (Nantong TROPHIC, TP2031) for 16 weeks to induce hyperlipemia.sup.[33,34]. 50 μL 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, 6 mice in the control group administered with vehicle PBS, and 5 mice in the group administered with plasminogen. 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. The administration lasted for 30 days, during which mice continued to be fed with a high-fat and high-cholesterol diet. The mice were sacrificed on Day 31. The heart tissues 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×.

(55) The results showed that the collagen deposition (indicated by arrow) in the group administered with plasminogen (FIG. 16B) was remarkably less than that in the control group administered with vehicle PBS (FIG. 16A), suggesting that plasminogen can prevent and lower cardiac fibrosis induced by hyperlipemia by reducing collagen deposition in the heart tissue of hyperlipemia model mice.

Example 17. Plasminogen Repairs Renal Fibrosis in Chronic Renal Failure Model

(56) Twelve 8- to 9-week-old male mice with normal PLG activity and six male mice with impaired PLG activity were taken. The mice with normal PLG activity were randomly divided into two groups, 6 mice in each of the group administered with plasminogen and the control group administered with vehicle PBS. Three groups of mice were fed with a 0.25% purine diet (Nantong TROPHIC) every day to establish the chronic renal failure model.sup.[35]. The day of model establishment was recorded as Day 1, and administration began at the same time. Mice in the group administered with plasminogen were administered with plasminogen at a dose of 1 mg/0.1 mL/mouse/day, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS in the same manner, both lasting for 10 consecutive days for model establishment. The mice with impaired PLG activity were not treated. The mice were sacrificed on Day 11. The kidneys were fixed in 4% paraformaldehyde for 24 hours. The fixed kidneys 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 for 60 min, the sections were flushed with running water. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol and returned to blue with ammonia water, flushed with running water, dried and sealed. The sections were observed under an optical microscope at 200×.

(57) The results showed that the collagen deposition (indicated by arrow) in the group administered with plasminogen (FIG. 17B) was remarkably less than that in the control group administered with vehicle PBS (FIG. 17A) and the group with impaired PLG activity (FIG. 17C), and the statistical difference between the group administered with plasminogen and the group with impaired PLG activity was significant (P=0.018) (FIG. 17D). It indicates that plasminogen can significantly alleviate collagen deposition in kidney tissues of animals with chronic renal injury, thus preventing and alleviating renal fibrosis induced by chronic renal injury.

Example 18. Plasminogen Reduces Collagen Deposition in the Pancreatic Islet of Diabetic Mice

(58) Sixteen 24- to 25-week-old male db/db mice were randomly divided into two groups, 10 mice in the group administered with plasminogen, and 6 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. The mice were weighed and grouped on the day when the experiment began, i.e. Day 0. Plasminogen or PBS was administered from day 1 for 31 consecutive days. On day 32, the mice were sacrificed, and the pancreas was taken and fixed in 4% paraformaldehyde. The fixed pancreas 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 for 60 min, the sections were flushed with running water. After stained with hematoxylin for 1 min, the sections were flushed with running water, differentiated with 1% hydrochloric acid in alcohol and returned to blue with ammonia water, flushed with running water, dried and sealed. The sections were observed under an optical microscope at 200×.

(59) The results showed that the collagen deposition (indicated by arrow) in the pancreatic islet of the mice in the group administered with plasminogen (FIG. 18B) was remarkably lower than that in the control group administered with vehicle PBS (FIG. 18A), and the statistical difference was significant (FIG. 18C). It indicates that plasminogen can significantly alleviate collagen deposition in pancreatic tissues of diabetic mice, thus preventing and alleviating pancreatic injury and fibrosis.

Example 19. Plasminogen Lowers Renal Fibrosis in 3% Cholesterol Hyperlipemia Model Mice

(60) 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.[30,31]. This model was designated as the 3% cholesterol hyperlipemia model. The model mice continued to be fed with the 3% cholesterol high-fat diet. Another five male C57 mice of the same week age were taken as the blank control group, and were fed with a normal maintenance diet during the experiment. 50 μL of blood was taken from each mouse three days before administration, and the total cholesterol was detected. The model mice were randomly divided into two groups based on the total cholesterol concentration and the body weight, i.e., the group administered with plasminogen, and the control group administered with vehicle PBS, 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. The mice were administered for 30 days. After the mice were administered on day 30, the mice were sacrificed on Day 31. The kidney materials were taken and 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 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×.

(61) The results showed that the collagen deposition in kidney (indicated by arrow) in the group administered with plasminogen (FIG. 19C) was remarkably less than that in the control group administered with vehicle PBS (FIG. 19B), and the statistical difference was significant (FIG. 19D); while in the group administered with plasminogen, fibrosis was substantially restored to a normal level (FIG. 19A). It indicates that plasminogen can effectively reduce renal fibrosis in 3% cholesterol hyperlipemia model mice.

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