METHOD FOR PREVENTING OR TREATING LIVER TISSUE DAMAGE AND ASSOCIATED DISEASES

Abstract

The present invention relates to the use of plasminogen in the treatment and/or elimination of hepatic injury, thereby providing a new therapeutic strategy for treating different types of hepatic injuries.

Claims

1. A method for preventing and/or treating hepatic tissue injury and its related disorders in a subject, comprising administering an effective amount of plasminogen to the subject.

2. The method of claim 1, wherein the hepatic tissue injury and its related disorders are hepatic injury and its related disorders caused by radiation or chemical substances.

3. The method of claim 1, wherein the hepatic tissue injury and its related disorders are toxic hepatic injury and its related disorders.

4. The method of claim 1, wherein the hepatic tissue injury and its related disorders are diabetic hepatic injury and its related disorders.

5. The method according to claim 4, wherein the diabetic hepatic injury and its related disorders are caused by diabetes mellitus-induced angiopathy of large vessels, small vessels and microvessels.

6. The method of claim 1, wherein the hepatic tissue injury and its related disorders comprise hepatic dysfunction, abnormal hepatic enzymology, liver discomfort and haphalgesia, hepatomegaly, splenomegaly, hepatosplenomegaly, hepatitis, fatty liver, cholangitis, hepatic cirrhosis, hepatic necrosis and hepatic carcinoma caused by hepatic tissue injury.

7. The method of claim 1, wherein the plasminogen has at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID No. 2, 6, 8, 10 or 12, and still has the plasminogen activity.

8. The method of claim 1, wherein the plasminogen is a protein that comprises a plasminogen active fragment and still has the plasminogen activity.

9. The method of claim 1, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, ? (delta)-plasminogen or any combination thereof.

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

11. An article for preventing and/or treating hepatic tissue injury and its related disorders in a subject, comprising a container containing an effective dosage of plasminogen, and instructions for directing the administration of the article to prevent and/or treat hepatic tissue injury and its related disorders in the subject.

12. The article of claim 11, further comprising a container containing one or more other drugs.

13. The article of claim 12, wherein the instructions further indicate that the plasminogen is administered before, simultaneously with and/or after the administration of the other drugs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0091] FIG. 1 shows changes in body weight after administration of plasminogen to 24-25-week-old diabetic mice.

[0092] FIG. 2 shows the observed results of HE staining of the liver after administration of plasminogen to 24-25-week-old diabetic mice for 15 consecutive days.

[0093] FIG. 3 shows the observed results of fibrin immunostaining of the liver under a microscopy after administration of plasminogen to 24-25-week-old diabetic mice for 15 consecutive days.

[0094] FIG. 4 shows changes in body weight after administration of plasminogen to 24-25-week-old diabetic mice for 31 consecutive days.

[0095] FIG. 5 shows the observed results of HE staining of the liver after administration of plasminogen to 24-25-week-old diabetic mice for 31 consecutive days.

[0096] FIG. 6 shows the observed results of fibrin immunostaining of the liver after administration of plasminogen to 24-25-week-old diabetic mice for 31 consecutive days.

[0097] FIG. 7 shows the observed results of F4/80 immunostaining of the liver after administration of plasminogen to 24-25-week-old diabetic mice for 31 consecutive days.

[0098] FIG. 8 shows the detection results of alanine transaminase (ALT) in serum after administration of PBS or plasminogen to 24-25-week-old diabetic mice for 31 days.

[0099] FIG. 9 shows the observed results of HE staining of the liver on days 0, 2 and 7 after administration of plasminogen to mice with acute hepatic injury induced by carbon tetrachloride.

[0100] FIG. 10 shows the observed results of HE staining of the liver at hours 18, 24 and 48 after administration of plasminogen to plg.sup.?/? mice with acute hepatic injury induced by carbon tetrachloride.

[0101] FIG. 11 shows the observed results of fibrin immunostaining of the liver at hours 18, 24 and 48 after administration of plasminogen to pig.sup.?/? mice with acute hepatic injury induced by carbon tetrachloride.

[0102] FIG. 12 shows the observed results of F4/80 immunostaining of the liver 10 days after administration of plasminogen to mice irradiated with 5.0 Gy X-rays.

[0103] FIG. 13 shows the observed results of fibrin immunostaining of the liver 7 days after administration of plasminogen to model mice with injury induced by 10 mg/Kg cisplatin chemotherapy.

[0104] FIG. 14 shows the observed results of HE staining of the liver at hours 18, 24 and 48 and 7 days after administration of plasminogen to plg.sup.?/? mice with acute hepatic injury induced by carbon tetrachloride.

EXAMPLES

Example 1. Effect of Plasminogen on Body Weight of Late Diabetic Mice with Nerve Injury

[0105] Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 15 consecutive days, and the day was recorded as Day 1. 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 weighed on days 0, 4, 7, 11 and 16 after administration of plasminogen, respectively. The results showed that there was no significant difference in body weight between mice in the group administered with plasminogen and those in the control group administered with vehicle PBS on days 0, 4, 7, 11 and 16 (FIG. 1), indicating that plasminogen has little effect on animal body weight.

Example 2. Protective Effect of Plasminogen on Late Hepatic Tissue Injury of Mice with Diabetic Hepatic Injury

[0106] Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 15 consecutive days, and the day was recorded as Day 1. 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. Mice were sacrificed on day 16, and liver tissues were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. 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.

[0107] The HE staining results showed that in mice in the control group administered with vehicle PBS, the hepatocytes showed severe steatosis and lipid deposition, cell nuclei were squeezed to the edge, the cells showed mild hydropic degeneration, and the hepatic cord was disordered; and compared with mice in the control group administered with vehicle, in mice in the group administered with plasminogen, hepatocytes showed relieved steatosis, mild steatosis and mainly moderate hydropic degeneration. This indicated that plasminogen can promote the repair of diabetic hepatic injury.

Example 3. Plasminogen Reduces the Fibrin Level in Liver Tissues of Diabetic Mice

[0108] Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 15 consecutive days, and the day was recorded as Day 1. 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. Mice were sacrificed on day 16, and liver tissues were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. The sections were dewaxed and rehydrated and washed with water once. The sections were incubated with 3% hydrogen peroxide for 15 minutes and wash with water twice for 5 minutes each time. The sections were blocked with 10% normal goat serum solution (Vector laboratories, Inc., USA) for 1 hour; and after the time was up, the goat serum solution was discarded, and the tissues were circled with a PAP pen. The sections were incubated with rabbit anti-mouse fibrin (fibrinogen) 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, flushed with running water for 5 minutes, and then washed with TBS once. After gradient dehydration, permeabilization and sealing, the sections were observed under a microscope at 200?.

[0109] Fibrinogen is the precursor of fibrin, and in the presence of tissue injury, as a stress response to the body's injury, fibrinogen is hydrolyzed into fibrin.sup.[1244]. Therefore, the fibrinogen level can be used as a sign of the degree of injury.

[0110] The study found that compared with mice in the control group administered with vehicle PBS (FIG. 3A), those in the group administered with plasminogen (FIG. 3B) had a decreased fibrin level in the liver tissues, indicating that plasminogen has the function of inhibiting the fibrin deposition and the injury is repaired to a certain degree.

Example 4. Effect of Plasminogen on Body Weight of Diabetic Mice

[0111] Twenty male db/db mice aged 24-25 weeks were randomly divided into two groups, ten in the control group administered with vehicle PBS and ten in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 31 consecutive days, and the day was recorded as Day 1. 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 weighted on days 0, 4, 7, 11, 16, 21, 26 and 31.

[0112] The results showed that there was no significant difference in body weight between mice in the group administered with plasminogen and those in the control group administered with vehicle PBS on days 0, 4, 7, 11, 16, 21, 26 and 31 (FIG. 4), indicating that plasminogen has little effect on animal body weight.

Example 5. Protective Effect of Plasminogen on Late Hepatic Tissue Injury of Mice with Diabetic Hepatic Injury

[0113] Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 31 consecutive days, and the day was recorded as Day 1. 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. Mice were sacrificed on day 32, and liver tissues were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. 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.

[0114] The HE staining results showed that in mice in the control group administered with vehicle PBS (FIG. 5A), the liver showed severe steatosis and lipid deposition, and fusion into large fat vacuoles, cell nuclei were squeezed to the edge (?), the hepatic cord was disordered, the hepatic sinus was narrowed, and there were different numbers of inflammatory foci at the hepatic cord (?);

[0115] and in mice in the group administered with plasminogen (FIG. 5B), the liver showed mild steatosis, mainly mild hydropic degeneration at the injury, and dissolved cytoplasm (?), which were mainly distributed in the area between the portal area and the central veins; the area around the portal area and the central veins were affected mildly; and at the same time, mild inflammatory cell infiltration was seen at the hepatic cord. This indicated that hepatic injury is obviously repaired after administration of plasminogen.

Example 6. Plasminogen Reduces the Fibrin Level in Liver Tissues of Diabetic Mice

[0116] Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 31 consecutive days, and the day was recorded as Day 1. 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. Mice were sacrificed on day 32, and liver tissues were fixed in 10% neutral formalin fix solution for 24 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. The sections were dewaxed and rehydrated and washed with water once. The sections were incubated with 3% hydrogen peroxide for 15 minutes and wash with water twice for 5 minutes each time. The sections were blocked with 10% normal goat serum solution (Vector laboratories, Inc., USA) for 1 hour; and after the time was up, the goat serum solution was discarded, and the tissues were circled with a PAP pen. The sections were incubated with rabbit anti-mouse fibrin (fibrinogen) 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 and flushed with running water for 5 minutes. After gradient dehydration, permeabilization and sealing, the sections were observed under a microscope at 200?.

[0117] Fibrinogen is the precursor of fibrin, and in the presence of tissue injury, as a stress response to the body's injury, fibrinogen is hydrolyzed into fibrin.sup.[12-14]. Therefore, the fibrinogen level can be used as a sign of the degree of injury.

[0118] The study found that compared with mice in the control group administered with vehicle PBS (FIG. 6A), those in the group administered with plasminogen (FIG. 6B) had a remarkably lower fibrin level in the liver tissue, indicating that injection of plasminogen can significantly reduce the deposition of fibrin in diabetic mice, reflecting the significant repair function of plasminogen on the body's injury of diabetic mice.

Example 7. Plasminogen Reduces Inflammation of Liver Tissues of Diabetic Mice

[0119] Ten male db/db mice aged 24-25 weeks were randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 31 consecutive days, and the day was recorded as Day 1. 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. Mice were sacrificed 31 days after administration of plasminogen, and liver tissues were fixed in 10% neutral formalin fix solution for 24 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. The sections were dewaxed and rehydrated and washed with water once. The sections were incubated with 3% hydrogen peroxide for 15 minutes and wash with water twice for 5 minutes each time. The sections were blocked with 10% normal goat serum 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 a rabbit polyclonal antibody against F4/80 (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. 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 and flushed with running water for 5 minutes. After gradient dehydration, permeabilization and sealing, the sections were observed under a microscope at 400?.

[0120] F4/80 is a macrophage marker that can indicate the extent and stage of an inflammatory response. The results showed that compared with mice in the control group administered with vehicle PBS (FIG. 7A), the F4/80 positive level was significantly reduced in those in the group administered with plasminogen (FIG. 7B), indicating that inflammation of the liver tissues is reduced after administration of plasminogen. FIG. 7C shows the results of quantitative analysis of F4/80 immunohistochemical positive expression, in which the expression of F4/80 in mice in the group administered with plasminogen was significantly reduced with statistical difference, indicating that injection of plasminogen can significantly promote the repair of liver inflammation of diabetic mice.

Example 8. Plasminogen Promotes the Repair of Liver Injury of Diabetic Mice

[0121] Nine male db/db mice aged 25-28 weeks were randomly divided into two groups, three in the control group administered with vehicle PBS and six in the group administered with plasminogen, respectively. The day when the experiment began was recorded on Day 0, and the mice were weighed and grouped. From the second day of the experiment, plasminogen or PBS was administered to the mice for 31 consecutive days, and the day was recorded as Day 1. 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. Whole blood was taken from the removed eyeballs 31 days after administration of plasminogen. After the serum was precipitated, it was centrifuged at 3500 r/min for 10 minutes at 4? C., and the supernatant was taken for detection. In this experiment, the content of alanine transaminase (ALT) in serum was detected by Reitman-Frankel colorimetry using an alanine transaminase detection kit (Nanjing Jiancheng Biological Engineering Research Institute, Catalog No. C009-2).

[0122] Alanine transaminase is an important index of liver health status.sup.[15,16], and the normal reference value interval of alanine transaminase is 9-50 U/L. The detection results showed that the ALT content in serum of mice in the control group administered with vehicle PBS was significantly higher than the normal physiological index, whereas the content in mice in the group administered with plasminogen had returned to normal levels in the body; and the content in mice in the group administered with plasminogen was significantly lower than that in mice in the control group administered with vehicle PBS, and there was a statistical difference (FIG. 8). This indicated that injection of plasminogen can effectively repair the liver injury in model mice with late diabetic diabetes.

Example 9. Protective Effect of Plasminogen on the Liver in Case of Acute Hepatic Poisoning

[0123] Eighteen male or female 7-8-week-old plg.sup.?/? mice were randomly divided into two groups, nine in the control group administered with vehicle PBS and nine in the group administered with plasminogen, respectively. Two groups of mice were administered with carbon tetrachloride via intraperitoneal injection at 0.5 mL/kg body weight for 2 consecutive days to establish an acute hepatic injury model.sup.[17,18]. Carbon tetrachloride should be diluted with corn oil before use, and the volume ratio of the former to the latter is 1:7. The day of model establishment was day 0, and plasminogen or PBS was administered to the mice from day 1. Mice in the group administered with plasminogen were administered with plasminogen at a dosage 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, both for 7 consecutive days. Three mice from both groups were respectively sacrificed on days 0, 2 and 7, the mice were dissected and the liver conditions were observed and recorded, and then liver tissues were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. 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 a microscope at 200?.

[0124] The HE staining results showed that on day 0, the livers of mice in the control group administered with vehicle PBS (FIGS. 9A-C) and the group administered with plasminogen (FIGS. 9D-F) mainly showed fragmented necrosis around the central veins, in which cell nuclei were fragmented and cytoplasm was stained lightly in the necrotic area, and there were moderate hydropic degeneration and cellular edema also occurring in other non-necrotic areas. On day 2, the central veins expanded, the structures of hepatocytes were disordered, and there was a little inflammatory cell infiltration. There was no significant difference between two groups. However, on day 7, mice in the control group administered with vehicle PBS still had a little hepatocyte degeneration, mild cellular edema, disordered hepatic cord and narrowed hepatic sinus, and there was mild inflammatory cell infiltration around the portal area; whereas the liver of mice in the group administered with plasminogen had basically returned to normal cytoplasm red staining, and had regular hepatic cord and clear hepatic sinus. This indicated that plasminogen can promote the repair of hepatic injury.

Example 10. Protective Effect of Plasminogen on the Liver in Case of Acute Hepatic Poisoning

[0125] Eighteen male 7-11-week-old plg.sup.?/? mice were randomly divided into two groups, nine in the control group administered with vehicle PBS and nine in the group administered with plasminogen, respectively. Two groups of mice were administered with carbon tetrachloride via intraperitoneal injection at 0.5 mL/kg body weight once to establish an acute hepatic injury model.sup.[17,18]. Carbon tetrachloride should be diluted with corn oil before use, and the volume ratio of the former to the latter is 1:7. Plasminogen or vehicle PBS was administered to the mice within half an hour after completion of model establishment. Mice in the group administered with plasminogen were injected with plasminogen at a dosage 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, both for 2 consecutive days. Three mice from both groups were respectively sacrificed at hours 18, 24 and 48 after administration, the mice were dissected and the liver conditions were observed and recorded, and then liver tissues were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. 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 a microscope at 200?.

[0126] The results showed that mice in the control group administered with vehicle PBS (FIGS. 10A-C) showed different levels of necrosis at 18 h, 24 h and 48 h, and mainly fragmented necrosis at 18 h and 24 h, and bridging necrosis, nuclear fragmentation and lightly stained cytoplasm occurring at 48 h; the injury was progressively worsening and mainly around the central veins; and there was moderate inflammatory cell infiltration in the necrotic area (?), and there was mild necrosis, mainly mild hydropic degeneration around the portal area, accompanied by mild inflammatory cell infiltration and mild bile duct hyperplasia (?); and compared with mice in the control group, the mice in the group administered with plasminogen (FIGS. 10D-F) showed no apparent necrosis at 18 h, 24 h and 48 h; the injury was mainly mild hydropic degeneration and distributed around the portal area; hepatocytes around the central veins were not affected; and these conditions were better at 24 h than those at 18 h, in which hydropic degeneration was reduced, hepatocytes around the central veins showed mild steatosis and lightly stained cytoplasm, both accompanied by mild inflammatory cell infiltration. This indicated that plasminogen can promote the repair of hepatic injury of plg model mice with acute hepatic injury.

Example 11. Plasminogen Reduces Fibrin Deposition in the Liver Tissues of Model Mice with Acute Hepatic Injury

[0127] Eighteen male 7-11-week-old plg.sup.?/? mice were randomly divided into two groups, nine in the control group administered with vehicle PBS and nine in the group administered with plasminogen, respectively. Two groups of mice were administered with carbon tetrachloride via intraperitoneal injection at 0.5 mL/kg body weight once to establish an acute hepatic injury model.sup.[17,18]. Carbon tetrachloride should be diluted with corn oil before use, and the volume ratio of the former to the latter is 1:7. Plasminogen or vehicle PBS was administered to the mice within half an hour after completion of model establishment. Mice in the group administered with plasminogen were injected with plasminogen at a dosage 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, both for 2 consecutive days. Three mice from both groups were respectively sacrificed at hours 18, 24 and 48 after administration, the mice were dissected and the liver conditions were observed and recorded, and then liver tissues were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. The sections were dewaxed and rehydrated and washed with water once. The sections were incubated with 3% hydrogen peroxide for 15 minutes and wash with water twice for 5 minutes each time. The sections were blocked with 10% normal goat serum solution (Vector laboratories, Inc., USA) for 1 hour; and after the time was up, the goat serum solution was discarded, and the tissues were circled with a PAP pen. The sections were incubated with rabbit anti-mouse fibrin (fibrinogen) 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 and flushed with running water for 5 minutes. After gradient dehydration, permeabilization and sealing, the sections were observed under a microscope at 200?.

[0128] Fibrinogen is the precursor of fibrin, and in the presence of tissue injury, as a stress response to the body's injury, fibrinogen is hydrolyzed into fibrin.sup.[12-14]. Therefore, the fibrinogen level can be used as a sign of the degree of injury.

[0129] The results showed that at three time points, 18 h, 24 h and 48 h, mice in the group administered with plasminogen (FIGS. 11D-F) showed significantly lighter fibrin-positive staining than that in mice in the control group administered with vehicle PBS (FIGS. 11A-C), and the fibrin staining also tended to become lighter gradually with the extension of time. This indicated that injection of plasminogen can reduce fibrin deposition and promote the repair of hepatic injury.

Example 12. Plasminogen Promotes the Repair of Liver Inflammation of Mice Irradiated with 5.0 Gy X-Rays

[0130] In this experiment, ten healthy 6-8-week-old male C57 mice were used and randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. After the grouping was completed, a radiation-induced injury model was established by uniformly irradiating the mice with 6 MV X-rays from a linear accelerator at 5.0 Gy once systemically, in which the absorbed dosage rate was 2.0 Gy/min and the absorbed dosage was 5.0 Gy (irradiation for 2.5 minutes). Plasminogen was administered to the mice within 3 hours after the model was established. The day when the experiment began was Day 0, and the mice were weighed and grouped. The mice were treated with radiation and administered with plasminogen or vehicle PBS from day 1. The administration period was 10 days. After the completion of the administration, the medication of animals was discontinued and they were observed for 11 days. The entire experimental period was 21 days. Mice in the group administered with plasminogen were injected with plasminogen at a dosage 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. Mice were sacrificed and dissected on day 21, and livers were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. The sections were dewaxed and rehydrated and washed with water once. The sections were repaired with Tris-EDTA for 30 minutes, and gently rinsed with water after cooling at room temperature for 20 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% normal goat serum (Vector laboratories, Inc., USA) for 1 hour; and after the time was up, the goat serum solution was discarded. The sections were incubated with rabbit anti-mouse F4/80 antibody (Abcam) at 4? C. overnight 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 and flushed with running water for 5 minutes. After gradient dehydration, permeabilization and sealing, the sections were observed under a microscope at 200?.

[0131] The F4/80 immunohistochemical results showed that after model establishment with 5.0 Gy X-ray irradiation, the expression level of the macrophage marker in mice in the control group administered with vehicle PBS (FIG. 12A) was higher than that of the macrophage marker in mice in the group administered with plasminogen (FIG. 12B), indicating that the inflammation of the liver tissues of the animals was significantly reduced after administration of plasminogen.

Example 13. Plasminogen Reduces Fibrin Deposition in the Liver Tissues of Model Mice with Injury Induced by Cisplatin Chemotherapy

[0132] Ten healthy 8-9-week-old male C57 mice were used and randomly divided into two groups, five in the control group administered with vehicle PBS and five in the group administered with plasminogen, respectively. After the grouping was completed, a chemotherapy-induced injury model was established by single intraperitoneal injection of cisplatin at 10 mg/Kg body weight. After the model was established, mice in the group administered with plasminogen were administered with plasminogen at a dosage of 1 mg/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 day when the experiment began was Day 0, and the mice were weighed and grouped. The mice were injected with cisplatin intraperitoneally from day 1 for model establishment. Plasminogen or vehicle PBS was administered to the mice within 3 hours after completion of model establishment, and the administration period was 7 days. Mice were sacrificed on day 8, and livers were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. 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% normal goat serum (Vector laboratories, Inc., USA) for 1 hour; and after the time was up, the goat serum solution was discarded. The sections were incubated with rabbit anti-mouse fibrin 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 and flushed with running water for 5 minutes. After gradient dehydration, permeabilization and sealing, the sections were observed under a microscope at 200?.

[0133] Fibrinogen is the precursor of fibrin, and in the presence of tissue injury, as a stress response to the body's injury, fibrinogen is hydrolyzed into fibrin.sup.[12-14]. Therefore, the fibrinogen level can be used as a sign of the degree of injury.

[0134] The results showed that fibrin-positive staining in the liver tissues of mice in the control group administered with vehicle PBS (FIG. 13A) was significantly darker than that in the liver tissues of mice in the group administered with plasminogen (FIG. 13B). This indicated that plasminogen can significantly reduce fibrin deposited in injured liver tissues, indicating that plasminogen can promote the repair of hepatic injury caused by the chemotherapy drug cisplatin.

Example 14. Protective Effect of Plasminogen on the Liver in Case of Acute Hepatic Poisoning

[0135] Six male 7-11-week-old plg.sup.?/? mice were randomly divided into two groups, three in the control group administered with vehicle PBS and three in the group administered with plasminogen, respectively. Two groups of mice were administered with carbon tetrachloride via intraperitoneal injection at 0.5 mL/kg body weight once to establish an acute hepatic injury model.sup.[17,18]. Carbon tetrachloride should be diluted with corn oil before use, and the volume ratio of the former to the latter is 1:7. Plasminogen or vehicle PBS was administered to the mice within half an hour after completion of model establishment. Mice in the group administered with plasminogen were administered with plasminogen at a dosage 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, both for 7 consecutive days. Mice were sacrificed and dissected on day 8, the liver conditions were observed and recorded, and then liver tissues were fixed in 10% neutral formalin fix solution for 24-48 hours. The fixed liver tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 5 ?m. 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 a microscope at 200?.

[0136] The results showed that in the livers of mice in the control group administered with vehicle PBS (FIG. 14A), the central veins expanded, the endothelial cells were necrotic, hepatocytes around the central veins all had large areas of focal necrosis with fragmented and darkly stained nuclei, and there were mild hydropic degeneration, cellular edema and clear cytoplasm in other non-necrotic areas, accompanied by mild inflammatory cell infiltration in the necrotic area; and in the livers of mice in the group administered with plasminogen (FIG. 14B), there was no apparent necrosis, injury was mainly mild hydropic degeneration, and there were enhanced acidophily and red staining in the cytoplasm of a small amount of hepatocytes. Injury of mice in the group administered with plasminogen was obviously milder than that of mice in the control group administered with vehicle PBS, indicating that plasminogen can promote the repair of hepatic injury of plg.sup.?/? model mice with acute hepatic injury.

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