Method and drug for preventing and treating obesity

Abstract

The present invention relates to a method for preventing and/or treating overweight/obesity and their related conditions, comprising administering an effective amount of plasminogen to a subject susceptible to or suffering from obesity and its related conditions, to reduce an abnormal/excessive fat deposition at various sites of the body. The present invention further relates to a medicament for preventing and/or treating obesity, and its use in the preparation of a medicament.

Claims

1. A method for treating obesity in a subject, comprising administering an effective amount of plasminogen to the subject.

2. The method of claim 1, wherein the method reduces abnormal or excessive lipid deposition in a subcutis, a heart, a liver, lungs, kidneys, blood vessels, a mesentery, a peritoneum, or a body cavity, or around an organ.

3. The method of claim 1, wherein the method lowers a level of blood lipid in the subject.

4. The method of claim 3, wherein the blood lipid is triglyceride or low-density lipoprotein.

5. The method of claim 1, wherein the obesity is simple obesity or obesity secondary to a disease or condition selected from the group consisting of an endocrine disorder disease, a glucose metabolism disease, a liver disease, a kidney disease, a cardiovascular disease, an intestinal disease, a thyroid disease, a gallbladder or biliary tract disease, and excessive drinking.

6. The method of claim 1, wherein the obesity comprises obesity complicated with diabetes mellitus, obesity complicated with hypertension, obesity complicated with atherosclerosis, obesity complicated with a liver disease, or obesity complicated with osteoporosis.

7. The method of claim 1, wherein the method reduces abnormal or excessive fat deposition in the subject in one or more ways selected from: 1) reducing abnormal or excessive lipid deposition in one or more sites selected from: a subcutis, a heart, a liver, lungs, kidneys, blood vessels, a mesentery, a peritoneum, and a body cavity, and around an organ, 2) promoting clearance of hepatic fat, and 3) promoting clearance of lipid in blood to reduce the onset risk of heart disease in the subject.

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

9. The method of claim 1, wherein the plasminogen has at least 90% sequence identity with SEQ ID No. 2, and still has the plasminogen activity of proteolysis.

10. The method of claim 1, wherein the plasminogen is a protein that comprises a plasminogen active fragment of SEQ ID No. 14, and still has the plasminogen activity of proteolysis.

11. The method of claim 1, wherein the plasminogen is selected from Glu-plasminogen, Lys-plasminogen, mini-plasminogen, micro-plasminogen, and delta-plasminogen.

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

13. The method of claim 1, wherein the method reduces weight in the subject.

14. The method of claim 13, wherein the subject is human.

15. The method of claim 1, wherein the plasminogen is administered to the subject at a dosage of 1-100 mg/kg at a frequency of weekly to daily.

16. The method of claim 15, wherein the plasminogen is administered at least daily.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows calculation results of body weight changes after administration of plasminogen to high-calorie diet-induced obesity model mice for 28 days. The results are shown as the value of the weight on Day 29 minus the weight on Day 1. The results showed that there was no significant body weight change in the blank control group, the weight loss in the control group administered with vehicle PBS was remarkably lower than that in the group administered with plasminogen, and the statistical difference was significant (* indicates P<0.05). It indicates that plasminogen can promote weight loss in obesity model mice.

(2) FIG. 2 shows statistical results of the body mass index after administration of plasminogen to high-calorie diet-induced obesity model mice for 28 days. The results showed that the body mass index of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05, and ** indicates P<0.01); and compared with the control group administered with vehicle PBS, the body mass index of mice in the group administered with plasminogen was closer to that in the blank control group. It indicates that plasminogen can significantly lower the body mass index of obesity model mice, and alleviate obesity.

(3) FIG. 3 shows statistical results of the Lee's index after administration of plasminogen to high-calorie diet-induced obesity model mice for 28 days. The results showed that the Lee's index of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05); and compared with the control group administered with vehicle PBS, the Lee's index of mice in the group administered with plasminogen was closer to that in the blank control group. It indicates that plasminogen can significantly lower the Lee's index of obesity model mice, and alleviate obesity.

(4) FIG. 4 shows detection results of blood lipid in high-calorie diet-induced obesity model mice. A represents total cholesterol, B represents low-density lipoprotein, and C represents high-density lipoprotein. The results showed that there were no significant differences in the concentrations of total cholesterol, low-density lipoprotein and high-density lipoprotein among the group administered with plasminogen, the control group administered with vehicle PBS, and the blank control group. It indicates that there is no significant change in blood lipid of high-calorie diet-induced obesity model mice in this experiment.

(5) FIG. 5 shows detection results of serum leptin in high-calorie diet-induced obesity model mice. The results showed that there were no significant differences in the leptin concentration among the group administered with plasminogen, the control group administered with vehicle PBS, and the blank control group. It indicates that there is no significant change in leptin of high-calorie diet-induced obesity model mice in this experiment.

(6) FIG. 6 shows detection results of serum insulin in high-calorie diet-induced obesity model mice. The results showed that there were no significant differences in the insulin concentration among the group administered with plasminogen, the control group administered with vehicle PBS, and the blank control group. It indicates that there is no significant change in insulin of high-calorie diet-induced obesity model mice in this experiment.

(7) FIG. 7 shows statistical results of the abdominal fat coefficient after administration of plasminogen to high-calorie diet-induced obesity model mice for 28 days. The results showed that the abdominal fat coefficient of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05); and compared with the control group administered with vehicle PBS, the abdominal fat content of mice in the group administered with plasminogen was closer to that in the blank control group. It indicates that plasminogen can significantly reduce abdominal fat deposition in obesity model mice.

(8) FIG. 8 shows statistical results of fat vacuolar area in abdominal fat by HE staining after administration of plasminogen to high-calorie diet-induced obesity 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 average fat vacuolar area in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, and the statistical difference was extremely significant (** indicates P<0.01); and compared with the control group administered with vehicle PBS, the fat vacuolar area of mice in the group administered with plasminogen was closer to that in the blank control group. It indicates that plasminogen can significantly reduce the size of adipose cells and abdominal fat deposition of obesity model mice.

(9) FIG. 9 shows detection results of serum leptin after administration of plasminogen to 14- to 15-week-old diabetic mice for 28 days. The results showed that the serum leptin concentration in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was extremely significant (** indicates P<0.01); and compared with the control group administered with vehicle PBS, the serum leptin level of mice in the group administered with plasminogen was closer to that of normal mice. It indicates that plasminogen can reduce the serum leptin level in mice with early-stage type 2 diabetes mellitus.

(10) FIG. 10 shows detection results of serum leptin after administration of plasminogen to 23- to 25-week-old diabetic mice for 28 days. The results showed that the serum leptin concentration of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was extremely significant (** indicates P<0.01). It indicates that plasminogen can reduce the serum leptin level in mice with late-stage type 2 diabetes mellitus.

(11) FIG. 11 shows observed results of oil red O staining of liver after administration of plasminogen to 16-week hyperlipemia model mice for 30 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 fat deposition in liver of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the quantitative analysis showed significant statistical difference (* indicates P<0.05). It indicates that plasminogen can ameliorate fat deposition in liver of hyperlipemia model mice.

(12) FIG. 12 shows observed results of oil red O staining of aortic sinus after administration of plasminogen to 16-week hyperlipemia model mice for 30 days. A and C represent the control group administered with vehicle PBS, B and D represent the group administered with plasminogen, and E represents the quantitative analysis results. The results showed that the fat deposition in aortic sinus of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05). It indicates that plasminogen can ameliorate fat deposition in aortic sinus of hyperlipemia model mice.

(13) FIG. 13 shows observed results of oil red O of kidney after administration of plasminogen to 3% cholesterol diet-induced 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 fat deposition in kidney (indicated by arrow) of mice in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, and the quantitative analysis showed significant statistical difference; in addition, the lipid deposition level in the group administered with plasminogen was similar to that in mice in the blank control group. It indicates that plasminogen can reduce the fat deposition in kidney of hyperlipemia model mice, and thus reduce renal injury caused by fat deposition.

(14) FIG. 14 shows detection results of serum low-density lipoprotein cholesterol after administration of plasminogen to 3% cholesterol diet-induced hyperlipemia model mice for 20 days. The results showed that the concentration of serum low-density lipoprotein cholesterol in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05). It indicates that plasminogen can lower the content of low-density lipoprotein cholesterol in serum of hyperlipemia model mice, and has an effect of improving hyperlipemia.

(15) FIG. 15 shows detection results of serum atherosclerosis index after administration of plasminogen to 3% cholesterol diet-induced hyperlipemia model mice for 20 days. The results showed that the atherosclerosis index of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was extremely significant (* * indicates P<0.01). It indicates that plasminogen can effectively lower the risk of atherosclerosis in hyperlipemia model mice.

(16) FIG. 16 shows detection results of serum total cholesterol after administration of plasminogen to ApoE atherosclerosis model mice for 30 days. The results showed that the concentration of total cholesterol in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05). It indicates that plasminogen can lower the content of total cholesterol in serum of ApoE atherosclerosis model mice, and improve the dyslipidemia in atherosclerosis model mice.

(17) FIG. 17 shows detection results of serum triglyceride after administration of plasminogen to ApoE atherosclerosis model mice for 30 days. The results showed that the concentration of triglyceride in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05). It indicates that plasminogen can lower the content of triglyceride in serum of ApoE atherosclerosis model mice, and improve the dyslipidemia in atherosclerosis model mice.

(18) FIG. 18 shows detection results of serum low-density lipoprotein cholesterol after administration of plasminogen to ApoE atherosclerosis model mice for 30 days. The results showed that the concentration of serum low-density lipoprotein cholesterol in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05). It indicates that plasminogen can lower the content of low-density lipoprotein cholesterol in serum of ApoE atherosclerosis model mice, and improve the dyslipidemia in atherosclerosis model mice.

(19) FIG. 19 shows immunohistochemical staining results of hypothalamic leptin receptor after administration of plasminogen to obesity model mice. A and D represent the blank control group, B and E represent the control group administered with vehicle PBS, C and F represent the group administered with plasminogen, and G represents the quantitative analysis results. The results showed that the expression of hypothalamic leptin receptor in mice in the control group administered with vehicle PBS was remarkably greater than that in the blank control group; while the expression of hypothalamic leptin receptor in mice in the group administered with plasminogen was remarkably less than that in the control group administered with vehicle PBS, and was close to the blank control group in the expression level, and the statistical difference was significant (P=0.01). It indicates that plasminogen can significantly reduce expression of hypothalamic leptin receptor in obese mice.

EXAMPLES

Example 1. Effect of Plasminogen on the High-Calorie Diet-Induced Obese Mice Model

(20) Mouse Model and Grouping

(21) Fourteen 8-week-old male C57 mice were randomly divided into two groups based on the body weight, a blank control group of 4 mice and a model group of 10 mice. Mice in the blank control group were fed with a normal maintenance diet; mice in the model group were fed with a high-fat diet containing 45% fat calories (TP23000, Nantong TROPHIC Feed Technology Co., Ltd.) for model establishment for 12 weeks to establish an obesity model .sup.[30]. A high-fat diet containing 45% fat calories is herein referred to as a high-calorie diet. After 12 weeks, mice in the model group were weighed and randomly divided into two groups again based on the body weight, 5 mice in each of a group administered with plasminogen and a control group administered with vehicle PBS. Human plasminogen was dissolved in PBS. The mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein. The blank control group received no treatment. The above-mentioned experimental animals were administered for 28 consecutive days (the first day of administration was recorded as Day 1), and treated and detected as follows on Day 29.

(22) Detections and Results

(23) Detection of Body Weights

(24) The above-mentioned experimental animals were weighed on Day 1 and Day 29 to calculate the changes in body weight. The results are shown as the value of the weight on Day 29 minus the weight on Day 1.

(25) The results showed that there was no significant body weight change in the blank control group, the weight loss in the control group administered with vehicle PBS was remarkably less than that in the group administered with plasminogen, and the statistical difference was significant (* indicates P<0.05) (FIG. 1). It indicates that plasminogen can significantly lower the body weight of obesity model mice.

(26) Determination of Body Mass Index

(27) On Day 29, the above-mentioned mice were weighed and measured for body length to calculate the body mass index. Body mass index=Weight (kg)/Body length (in).

(28) Body mass index is a commonly used international standard to measure body fatness degree and health of human beings. Body mass index can also be used as an index of fatness degree in obesity model animals .sup.[43, 44]. The results showed that the body mass index of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05); and compared with the control group administered with vehicle PBS, the body mass index of mice in the group administered with plasminogen was closer to that in the blank control group (FIG. 2). It indicates that plasminogen can significantly lower the body mass index of obesity model mice, and alleviate obesity.

(29) Determination of Lee's Index

(30) On Day 29, the above-mentioned mice were weighed and measured for body length to calculate the Lee's index.

(31) ${\mathrm{Lee}}^{\prime }s\mathrm{index}=\sqrt[3]{\mathrm{Body}\mathrm{weight}\left(g\right)}/\mathrm{Body}\mathrm{length}\left(\mathrm{cm}\right).$

(32) Body Weight(g)

(33) Lee's index is an effective index for reflecting the degree of obesity .sup.[31-32] The results showed that the Lee's index of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (* indicates P<0.05); and compared with the control group administered with vehicle PBS, the Lee's index of mice in the group administered with plasminogen was closer to that in the blank control group (FIG. 3). It indicates that plasminogen can significantly lower the Lee's index of obesity model mice, and alleviate obesity.

(34) Detection of Blood Lipid Levels

(35) On Day 29, the blood was collected from removed eyeballs in the above-mentioned model mice, and centrifuged to obtain a supernatant, which was detected for concentrations of serum total cholesterol, low-density lipoprotein, and high-density lipoprotein using the serum total cholesterol, low-density lipoprotein, and high-density lipoprotein detection kits (Nanjing Jiancheng Bioengineering Institute, Cat #A111-1, A113-1, and A112-1) according to the method of the corresponding kit.

(36) The results showed that there were no significant differences in the concentrations of total cholesterol (FIG. 4A), low-density lipoprotein (FIG. 4B) and high-density lipoprotein (FIG. 4C) among the group administered with plasminogen, the control group administered with vehicle PBS, and the blank control group. It indicates that there is no significant change in blood lipid of high-calorie diet-induced obesity model mice in this experiment.

(37) Detection of Serum Leptin Levels

(38) The leptin level in the above-mentioned serum was detected using a serum leptin detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #H174) according to the method of the detection kit.

(39) The results showed that there were no significant differences in the leptin concentration among the group administered with plasminogen, the control group administered with vehicle PBS, and the blank control group (FIG. 5). It indicates that there is no significant change in leptin of high-calorie diet-induced obesity model mice in this experiment.

(40) Leptin (LP) is a hormone secreted by an adipose tissue. Previously, it is generally believed that it will be involved in the regulation of sugar, fat and energy metabolisms after entering the blood circulation, prompting the body to reduce food intake, to increase energy release, to inhibit the synthesis of adipose cells, and thus to reduce body weight. However, some obese individuals have leptin resistance and an elevated leptin level in blood .sup.[34]. Relevant studies showed that db/db mice had leptin resistance, and serum leptin levels were significantly elevated .sup.[35-36].

(41) Detection of Serum Insulin Levels

(42) The insulin level in the above-mentioned serum was detected using a serum insulin detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #H174) according to the method of the detection kit.

(43) The results showed that there were no significant differences in the insulin concentration among the group administered with plasminogen, the control group administered with vehicle PBS, and the blank control group (FIG. 6). It indicates that there is no significant change in insulin of high-calorie diet-induced obesity model mice in this experiment.

(44) Detection of Abdominal Fat Contents

(45) On Day 29, the above-mentioned mice were weighed and sacrificed to weigh the abdominal fat. Abdominal fat coefficient (%)=(Abdominal fat mass/Body weight)*100.

(46) The results showed that the abdominal fat coefficient of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS with a significant statistical difference (* indicates P<0.05), and was close to the fat coefficient of mice in the blank control group (FIG. 7). It indicates that plasminogen can significantly reduce abdominal fat deposition in obesity model mice.

(47) Detection of Abdominal Subcutaneous Fat Vacuolar Area

(48) The above-mentioned mice were sacrificed on Day 29. The abdominal fat was fixed in 4% paraformaldehyde for 24 to 48 hours. The fixed tissue samples were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The tissue sections were 4 μm thick. The sections were dewaxed and rehydrated, stained with hematoxylin and eosin (HE staining), differentiated with 1% hydrochloric acid in alcohol, and returned to blue with ammonia water. The sections were sealed after dehydration with alcohol gradient, and observed under an optical microscope at 200×. Image-pro plus image processing software was used to analyze the fat vacuolar area.

(49) When the energy intake of an obese body exceeds the energy consumption, a large amount of lipid accumulates in adipose cells, leading to expansion of adipose tissues, i.e. enlargement of adipose cells and increase of the fat vacuolar area .sup.[33].

(50) The results showed that the fat vacuolar area of mice in the group administered with plasminogen (FIG. 8C) was remarkably less than that in the control group administered with vehicle PBS (FIG. 8B), and the statistical difference was extremely significant (* * indicates P<0.01) (FIG. 8D); and compared with the control group administered with vehicle PBS, the fat vacuolar area of mice in the group administered with plasminogen was closer to that in the blank control group (FIG. 8A). It indicates that plasminogen can significantly reduce the size of adipose cells and abdominal fat deposition of obesity model mice.

Example 2. Plasminogen Lowers the Concentration of Serum Leptin in Mice with Early-Stage Diabetes Mellitus

(51) Twelve 14- to 15-week-old male db/db mice and three db/m mice were taken. db/db mice were weighed and then randomly divided into two groups based on the body weight, 6 mice in each of the group administered with plasminogen and the control group administered with vehicle PBS. The first day of administration was recorded as the Day 1. Starting from the 1st day, plasminogen or PBS was administered. The group administered with plasminogen was injected with human plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and the control group administered with vehicle PBS was injected with an equal volume of PBS via the tail vein, both lasting for 28 consecutive days. As the normal control mice, db/m mice were not administered. On Day 28, the mice were fasted for 16 hours, and on Day 29, the blood was taken from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for the concentration of serum leptin. The leptin level in the above-mentioned serum was detected using a serum leptin detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #H174) according to the method of the detection kit.

(52) The results showed that the serum leptin concentration in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (** indicates P<0.01); and compared with the control group administered with vehicle PBS, the serum leptin level of mice in the group administered with plasminogen was closer to that of normal mice (FIG. 9). It indicates that plasminogen can significantly reduce the serum leptin level in mice with early-stage type 2 diabetes mellitus.

Example 3. Plasminogen Lowers the Concentration of Serum Leptin in Mice with Late-Stage Diabetes Mellitus

(53) Thirteen 23- to 25-week-old male db/db mice were weighed and then randomly divided into two groups based on the body weight, 7 mice in the group administered with plasminogen, and 6 mice in the control group administered with vehicle PBS. Starting from the 1st day, plasminogen or PBS was administered. The group administered with plasminogen was injected with human plasminogen at a dose of 2 mg/0.2 mL/mouse/day via the tail vein, and the control group administered with vehicle PBS was injected with an equal volume of PBS via the tail vein, both lasting for 28 consecutive days. On Day 28, the mice were fasted for 16 hours, and on Day 29, the blood was taken from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for the concentration of serum leptin. The leptin level in the above-mentioned serum was detected using a serum leptin detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #H174) according to the method of the detection kit.

(54) The results showed that the serum leptin concentration of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was extremely significant (** indicates P<0.01) (FIG. 10). It indicates that plasminogen can reduce the serum leptin level in mice with late-stage type 2 diabetes mellitus.

Example 4. Plasminogen Reduces the Fat Deposition in Liver of 16-Week Hyperlipemia Model Mice

(55) 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.[37, 38]. 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 livers were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C. overnight, respectively, and embedded in OCT. The frozen sections were 8 μm thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 400×.

(56) Oil red O staining can show lipid deposition and reflect the extent of lipid deposition .sup.[39]. The results showed that the fat deposition in liver of mice in the group administered with plasminogen (FIG. 11B) was remarkably lower than that in the control group administered with vehicle PBS (FIG. 11A), and the quantitative analysis showed significant statistical difference (FIG. 11C). It indicates that plasminogen can reduce fat deposition in liver of hyperlipemia model mice.

Example 5. Plasminogen Reduces Lipid Deposition in Aortic Sinus of 16-Week Hyperlipemia Model Mice

(57) 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.[37, 38]. 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 tissues 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 of aortic sinus were 8 μm thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 40× (FIGS. 11A and 11B) and 200× (FIGS. 11C and 11D).

(58) The results showed that the fat deposition in aortic sinus of mice in the group administered with plasminogen (FIGS. 12B and 12D) was remarkably lower than that in the control group administered with vehicle PBS (FIGS. 12A and 12C), and the statistical difference was significant (FIG. 12E). It indicates that plasminogen can reduce lipid deposition in aortic sinus of hyperlipemia model mice.

Example 6. Plasminogen Lowers Fat Deposition in Kidney of 3% Cholesterol Diet-Induced Hyperlipemia Model Mice

(59) 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.[37-38]. 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, both lasting for 30 days. The mice were sacrificed on Day 31. The kidneys were fixed in 4% paraformaldehyde for 24 to 48 hours, then sedimented in 15% and 30% sucrose at 4° C. overnight, respectively, and embedded in OCT. The frozen sections were 8 μm thick, stained with oil red O for 15 min, differentiated with 75% ethanol for 5 s, followed by nuclear staining with hematoxylin for 30 s, and sealing with glycerine and gelatin. The sections were observed under an optical microscope at 400×.

(60) The results showed that the fat deposition in kidney (indicated by arrow) of mice in the group administered with plasminogen (FIG. 13C) was remarkably less than that in the control group administered with vehicle PBS (FIG. 13 B), and the quantitative analysis showed significant statistical difference (FIG. 13D); in addition, the lipid deposition level in the group administered with plasminogen was similar to that in mice in the blank control group (FIG. 13A). It indicates that plasminogen can reduce the fat deposition in kidney of 3% cholesterol hyperlipemia model mice, and thus reduce renal injury caused by fat deposition.

Example 7. Plasminogen Lowers the Serum Low-Density Lipoprotein Cholesterol Level in 3% Cholesterol Diet-Induced Hyperlipemia Model Mice

(61) 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.[37-38]. This model was designated as the 3% cholesterol hyperlipemia model. The model mice continued to be fed with a 3% cholesterol high-fat diet. 50 μL of blood was taken from each mouse three days before administration, and the total cholesterol was detected. The mice were randomly divided into two groups based on the total cholesterol concentration and the body weight, 8 mice in each group. The first day of administration was recorded as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 20 days. On Day 20, the mice fasted for 16 hours, and on Day 21, 50 μL of blood was collected from orbital venous plexus, and centrifuged to obtain a supernatant. The low-density lipoprotein cholesterol (LDL-C) was detected using a low-density lipoprotein cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #A113-1).

(62) The results showed that the concentration of LDL-C in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (FIG. 14). It indicates that plasminogen can lower the content of low-density lipoprotein cholesterol in serum of hyperlipemia model mice.

Example 8. Plasminogen Lowers Risk of Atherosclerosis Formation in 3% Cholesterol Diet-Induced Hyperlipemia Model Mice

(63) 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.[37-38]. This model was designated as the 3% cholesterol hyperlipemia model. The model mice continued to be fed with a 3% cholesterol high-fat diet. 50 μL of blood was taken from each mouse three days before administration, and the total cholesterol (T-CHO) was detected. The mice were randomly divided into two groups based on the total cholesterol concentration and the body weight, 8 mice in each group. The first day of administration was recorded as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein. After administration on Day 20, the mice began to fast for 16 hours, and on Day 21, 50 μL of blood was collected from orbital venous plexus, and centrifuged to obtain a supernatant. The total cholesterol content was detected by using a total cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #A111-1); and the high-density lipoprotein cholesterol (HDL-C) content was detected using a high-density lipoprotein cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #A112-1).

(64) Atherosclerosis index is a comprehensive index to predict atherosclerosis clinically. It is considered to be of greater clinical importance as an estimate of the risk of coronary heart disease than total cholesterol, triglyceride, high-density lipoprotein, and low-density lipoprotein alone .sup.[40]. Atherosclerosis index=(T-CHO-HDL-C)/HDL-C.

(65) The calculation results showed that the atherosclerosis index of mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (FIG. 15). It indicates that plasminogen can lower the risk of atherosclerosis in hyperlipemia model mice.

Example 9. Plasminogen Lowers the Content of Serum Total Cholesterol in ApoE Atherosclerosis Mice

(66) 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.[41-42] The model mice continued to be fed with a high-fat and 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, 7 mice in the control group administered with vehicle PBS, and 6 mice in the group administered with plasminogen. The first day of administration was set as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 30 days. On Day 30, the mice fasted for 16 hours, and on Day 31, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for the total cholesterol using a total cholesterol detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #A111-1).

(67) The detection results showed that the concentration of total cholesterol in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (P=0.014) (FIG. 16). It indicates that plasminogen can lower the content of total cholesterol in serum of ApoE atherosclerosis model mice, and improve the dyslipidemia of atherosclerosis.

Example 10. Plasminogen Lowers the Content of Serum Triglyceride in ApoE Atherosclerosis Mice

(68) 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.[41-42] The model mice continued to be fed with a high-fat and 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, 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 recorded as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 30 days. On Day 30, the mice fasted for 16 hours, and on Day 31, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for triglyceride using a triglyceride detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #A110-1).

(69) The detection results showed that the concentration of triglyceride in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (P=0.013) (FIG. 17). It indicates that plasminogen can lower the content of triglyceride in serum of ApoE atherosclerosis model mice, and improve the dyslipidemia of atherosclerosis.

Example 11. Plasminogen Lowers the Content of Serum Low-Density Lipoprotein Cholesterol in ApoE Atherosclerosis Mice

(70) 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.[41-42] The model mice continued to be fed with a high-fat and 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, 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 recorded as Day 1. Mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and an equal volume of PBS was administered to mice in the control group administered with vehicle PBS via the tail vein, both lasting for 30 days. On Day 30, the mice fasted for 16 hours, and on Day 31, the blood was collected from removed eyeballs, and centrifuged to obtain a supernatant, which was detected for LDL-C using a low-density lipoprotein cholesterol (LDL-C) detection kit (Nanjing Jiancheng Bioengineering Institute, Cat #A113-1).

(71) The results showed that the concentration of LDL-C in mice in the group administered with plasminogen was remarkably lower than that in the control group administered with vehicle PBS, and the statistical difference was significant (P=0.017) (FIG. 18). It indicates that plasminogen can lower the content of low-density lipoprotein cholesterol in serum of ApoE atherosclerosis model mice, and improve the dyslipidemia in atherosclerosis model mice.

Example 12. Plasminogen Improves Expression of Hypothalamic Leptin Receptor in Obesity Model Mice

(72) Fourteen 8-week-old male C57 mice were randomly divided into two groups based on the body weight, a blank control group of 4 mice and a model group of 10 mice. Mice in the blank control group were fed with a normal maintenance diet; mice in the model group were fed with a high-fat diet containing 45% fat calories (TP23000, Nantong TROPHIC Feed Technology Co., Ltd.) for model establishment for 12 weeks to establish an obesity model .sup.[1]. After 12 weeks, mice in the model group were weighed and randomly divided into two groups again based on the body weight, 5 mice in each of a group administered with plasminogen and a control group administered with vehicle PBS. The mice in the group administered with plasminogen were injected with human plasminogen at a dose of 1 mg/0.1 mL/mouse/day via the tail vein, and the mice in the control group administered with vehicle PBS were injected with an equal volume of PBS via the tail vein, both lasting for 28 consecutive days. The blank control group was not injected with any liquid. During the administration, mice continued to be fed with a model establishment diet. The mice were sacrificed on Day 29. The hypothalami were fixed in 4% paraformaldehyde for 24 to 48 hours. The fixed tissues were paraffin-embedded after dehydration with alcohol gradient and permeabilization with xylene. The thickness of the tissue sections was 4 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 anti-leptin receptor antibody (Abcam) overnight at 4□ and washed with PBS 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 PBS 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 PBS once. After dehydration with a gradient, permeabilization and sealing, the sections were observed under an optical microscope at 40× (Figures A-C) and 200× (Figures E and F).

(73) The leptin receptor has a main physiological function of binding with leptin, facilitates the physiological role of leptin in regulating energy balance, fat storage, reproductive activities and the like in the body, and also participates in the autocrine regulation of leptin. Different types of leptin receptors are selectively expressed in central and peripheral tissues .sup.[45-47].

(74) The results showed that the expression of hypothalamic leptin receptor in mice in the control group administered with vehicle PBS (FIGS. 19B and E) was remarkably greater than that in the blank control group (FIGS. 19A and D); while the expression of hypothalamic leptin receptor in mice in the group administered with plasminogen (FIGS. 19C and F) was remarkably less than that in the control group administered with vehicle PBS, and was close to the blank control group in the expression level, and the statistical difference was significant (P=0.01) (FIG. 19G). It indicates that plasminogen can significantly reduce expression of hypothalamic leptin receptor in obese mice.

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