Use of cell globin in the preparation of a medicament for the prevention and / or treatment of diabetes

Abstract

The present application belongs to the technical field of pharmaceutical preparations, and particularly relates to application of cellular globin in preparation of a medicine for preventing and / or treating diabetes. The present application uses cellular globin for treating diabetes, especially type II diabetes. In the case of persistent hyperglycemia of diabetes, the content of cellular globin is increased to effectively eliminate the oxidative damage of various sugar metabolism byproducts to the body in various tissues and organs of the body, thereby slowing down the occurrence and development of complications caused by diabetes, which is specifically manifested in: reducing the content of blood sugar; reducing the content of total cholesterol; reducing the content of triglyceride; reducing the level of low-density lipoprotein; improving and restoring the normal morphological structure of pancreatic tissue; promoting the proliferation of islet beta cells; and promoting the secretion of insulin and glucagon of islets. In summary, the cellular globin can be used for treating diabetes.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and particularly relates to application of cellular globin in preparation of a medicine for preventing and / or treating diabetes. BACKGROUND

[0002] Diabetes mellitus (DM) is a disease caused by metabolic disorders, which leads to long-term high blood sugar levels in the body. Symptoms of high blood sugar include frequent urination, increased thirst, and increased hunger. If not treated in time, diabetes can cause a variety of complications. Among them, acute complications include diabetic ketoacidosis, hyperosmolar hyperglycemic state, or death. Severe long-term complications include cardiovascular disease, stroke, chronic kidney disease, foot ulcers, and eye damage. Elevated blood glucose usually comes from overeating, lack of exercise, or insufficient ability of the body to take up and use glucose, and the process of glucose synthesis into glycogen is affected.

[0003] Type II diabetes (T2D) is the most common type of diabetes, which is caused by the body's inability to effectively produce or use insulin. It is characterized by relative insulin deficiency, and glucose will remain in the blood. Over time, the presence of too much glucose in the blood can cause serious problems: sustained high blood sugar, increased blood viscosity, multi-organ hypoxia, abnormal glucose metabolism, and oxidative stress damage to cells. Over time, it can lead to damage to organs such as eyes, kidneys, and nerves, and cause heart disease, stroke, and even necrosis of extremities. There are many causes of type 2 diabetes, including genetics, abnormal function of downstream receptors under the action of insulin, and abnormal pancreatic beta cells. According to the latest data from the International Diabetes Federation (IDF), as of 2017, more than 425 million people worldwide have diabetes. By 2040, this number is expected to increase to 629 million, so effective prevention and treatment of diabetes can greatly improve the quality of life of people around the world, and exploring a safe and effective new treatment for diabetes is imminent! SUMMARY

[0004] The purpose of the present application is to provide application of cellular globin in preparation of a medicine for preventing and / or treating diabetes, and to use cellular globin for the treatment of diabetes, which has a significant effect and is superior to Exendin-4.

[0005] The present application provides application of cellular globin in preparation of a medicine for preventing and / or treating diabetes.

[0006] Preferably, the diabetes includes type II diabetes.

[0007] Preferably, the prevention and / or treatment comprises at least one of the following: (1) reducing blood sugar content;

[0008] (2) reducing total cholesterol content;

[0009] (3) reducing triglyceride content;

[0010] (4) reducing low-density lipoprotein level;

[0011] (5) improving and restoring normal morphology of pancreatic tissue;

[0012] (6) promoting proliferation of islet beta cells;

[0013] (7) promoting insulin and glucagon secretion of islets.

[0014] Preferably, the cell globin comprises recombinant human cell globin.

[0015] Preferably, the intake dose of the cell globin is 5 mg / kg.

[0016] Preferably, the intake method comprises injection.

[0017] Preferably, the intake is a PBS solution containing the cell globin.

[0018] Beneficial effects: the present application provides application of cell globin in preparation of a drug for prevention and / or treatment of diabetes, and embodiments use cell globin for treatment of diabetes, especially type II diabetes. In the case of persistent hyperglycemia in diabetes, the content of cell globin can effectively remove oxidative damage of various sugar metabolism byproducts to the body in various tissues and organs of the body, thereby slowing down occurrence and development of complications caused by diabetes, which is specifically manifested in: reducing blood sugar content; reducing total cholesterol content; reducing triglyceride content; reducing low-density lipoprotein level; improving and restoring normal morphology of pancreatic tissue; promoting proliferation of islet beta cells; and promoting insulin and glucagon secretion of islets. In summary, the cell globin of the present application can be used for treatment of diabetes. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0020] Figure 1The blood glucose change graph of the modeling group and the blank control group during the modeling of C57BL / 6J mice, in which *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; the same below

[0021] Figure 2 The body weight change graph of the modeling group and the blank control group during the modeling of C57BL / 6J mice;

[0022] Figure 3 The blood glucose change graph of the randomly selected model group and the ordinary group of mice before treatment;

[0023] Figure 4 The body weight change graph of the randomly selected model group and the ordinary group of mice before treatment;

[0024] Figure 5 The comparison graph of the area under the curve of oral glucose tolerance of the blank control group and the diabetic model group of mice;

[0025] Figure 6 The blood glucose value comparison graph of the placebo group, the rhCygb treatment group and the EX-4 treatment group of mice; in the graph, NS (no significant) means no significant difference;

[0026] Figure 7 The blood glucose value comparison graph of the blank group, the placebo group, the rhCygb treatment group and the EX-4 treatment group of mice; the p value marked in the graph is the comparison of the blood glucose value of the PBS group and the rhCygb group every week;

[0027] Figure 8 The T-CHO kit detection result graph of the treatment effect of different treatment groups on the total cholesterol of C57BL / 6J mice;

[0028] Figure 9 The TG kit detection result graph of the treatment effect of different treatment groups on the triglyceride of C57BL / 6J mice;

[0029] Figure 10 The HDL-C kit detection result graph of the treatment effect of different treatment groups on the high-density lipoprotein of C57BL / 6J mice;

[0030] Figure 11 The LDL-C kit detection result graph of the treatment effect of different treatment groups on the low-density lipoprotein of C57BL / 6J mice;

[0031] Figure 12 The HE staining result graph of the pancreatic tissue sections of the mice in each group; in the graph, A, B, C and D are the NC group, the PBS group, the rhCygb group and the EX-4 group, respectively;

[0032] Figure 13 Figure for statistical analysis result of HE staining result of pancreas tissue section of each group of mice;

[0033] Figure 14 Figure for statistical analysis result of PCNA positive cell rate of different treatment groups;

[0034] Figure 15 Figure for PCNA group chemical staining result (200X magnification); A, B, C and D in the figure are NC group, PBS group, rhCygb group and EX-4 group respectively;

[0035] Figure 16 Figure for statistical analysis result of Glucagon average optical density value of different treatment groups;

[0036] Figure 17 Figure for statistical analysis result of Insulin average optical density value of different treatment groups;

[0037] Figure 18 Figure for Glucagon group chemical staining result (200X magnification); A, B, C and D in the figure are NC group, PBS group, rhCygb group and EX-4 group respectively;

[0038] Figure 19 Figure for Insulin group chemical staining result (200X magnification); A, B, C and D in the figure are NC group, PBS group, rhCygb group and EX-4 group respectively. DETAILED DESCRIPTION

[0039] The application provides application of a cell globin in preparation of a medicine for preventing and / or treating diabetes.

[0040] The diabetes preferably includes type II diabetes. The application establishes a C57BL / 6J diabetes model mouse fed with high-fat feed for a long time, uses a method of subcutaneous injection of rhCygb, and comprehensively evaluates the effectiveness of rhCygb in treating diabetes of the mouse, and the effectiveness is specifically shown in the following aspects: (1) reducing blood sugar content;

[0041] (2) reducing total cholesterol content;

[0042] (3) reducing triglyceride content;

[0043] (4) reducing low-density lipoprotein level;

[0044] (5) improving and restoring normal morphological structure of pancreas tissue;

[0045] (6) promoting proliferation of islet beta cells;

[0046] (7) promoting insulin and glucagon secretion of islets.

[0047] The cytoglobin according to the present application preferably comprises recombinant human cytoglobin, which is the fourth member of the globin superfamily in mammals, is a six-coordinated heme globin, has a protein molecular weight of 21.4 kDa, is composed of 190 amino acids, and is located at the chromosome fragment 17q25.3. In the embodiments of the present application, the content of the cytoglobin in the body is preferably increased by injection, and the injection amount is preferably 5 mg / kg. The injection according to the present application preferably comprises injection of a PBS solution of the cytoglobin.

[0048] In order to further illustrate the present application, the application of the cytoglobin provided by the present application in the preparation of a drug for preventing and / or treating diabetes is described in detail below in combination with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present application.

[0049] In the embodiments of the present application, the materials and methods used are all conventional for those skilled in the art, unless otherwise specified.

[0050] 1. Experimental animals

[0051] 50 three-week-old C57BL / 6J male mice, SPF level, weighing about 15-20 g, were purchased from Guangzhou Saibainuo Biological Technology Co., Ltd.

[0052] 2. Main reagents and instruments

[0053] Table 1 reagents used in the embodiments

[0054]

[0055]

[0056] Table 2 instruments used in the embodiments

[0057]

[0058]

[0059] 3. Statistical processing

[0060] Statistical analysis was performed using Prism 7 / SPSS 13.0 software. All quantitative data were replicated at least three times, and are expressed as mean ± standard deviation (mean ± SE). One-way ANOVA was used for comparisons among multiple groups. Within-group pairwise comparisons were performed using the LSD test if variances were homogeneous, and Dunnet's T3 test if variances were unequal. Independent-samples t-tests were used for comparisons between two samples. A p-value < 0.05 was considered statistically significant.

[0061] Example 1

[0062] 1. Modeling methods

[0063] Fifty SPF-grade C57BL / 6J male mice were purchased from Guangzhou CyberNuo Biotechnology Co., Ltd. Their health status and sex were checked. All male mice were divided into ten cages of five each, and each cage was labeled with a number. Cages 1-8 were designated as the high-fat diet model group, fed with high-fat food and water freely. Cages 9 and 10 were designated as the normal diet model group, fed with normal food and water freely. All mice were marked with lines on their tails. During the modeling period, the weight and fasting blood glucose levels of all mice were measured weekly, and the tail markings were reinforced. Blood glucose testing was conducted every Thursday evening at 9 PM by removing all food from the cage lids and changing the bedding to ensure no food residue remained. Mice were allowed free access to water. Every Friday morning at 9:00 AM, the mice were weighed and their tails were clipped. A drop of about 1-2 μl was taken and placed on a Roche Vitality blood glucose test strip or a blood glucose meter to measure the fasting blood glucose value. The changes in blood glucose and weight were observed until the difference in fasting blood glucose between the high-fat diet model group and the normal diet model group was statistically significant. The total modeling time was 58 weeks. The mice were housed in an SPF environment with a room temperature of 22 degrees Celsius and 12 hours of light per day.

[0064] The results are as follows Figures 1-4 As shown, statistically significant differences in blood glucose levels between the NC group and the model group began to appear at week 4 of modeling, but the changes in blood glucose levels were not yet stable. In the subsequent fifth and sixth weeks, there were no statistically significant differences in blood glucose levels between the NC group and the C57BL / 6J mouse model group. Starting at week 7, the blood glucose levels in the NC group and the C57BL / 6J mouse model group began to show a stable difference, indicating that the high-fat diet method for inducing diabetes is meaningful and requires long-term feeding, tending to stabilize over time. However, in the later stages, when the mice developed diabetic complications such as skin and eye diseases, the blood glucose levels in the C57BL / 6J mouse model group began to decline. Figure 1). Statistics of body weight, the body weight of the mice in the high-fat diet feeding group was significantly higher than that of the mice in the normal diet feeding group, and the difference was stable and obvious. After a long time of high-fat diet feeding, the body weight of the mice in the diabetes model group fluctuated greatly within the group due to different individual differences in the severity of complications, but the body weight of the mice without disease was at a very high level Figure 2 ). The blood glucose of the C57BL / 6J mice fed with high-fat diet alone fluctuated greatly, which was not as high as the sustained hyperglycemia of the chemical directly damaging the islet β cells or the genetic defect model. However, it can be seen that the red part (diabetes mellitus, DM) as a whole showed a significant upward trend compared with the black part (normal control group) Figure 3 ). The body weight of the C57BL / 6J mice fed with high-fat diet alone was significantly higher than that of the mice in the normal diet model group, and the body weight was greater than 55g. However, it can be seen that after the modeling time was continuously prolonged, the C57BL / 6J mice in the diabetes group began to have individuals with suspected severe complications of diabetes, and with the appearance of symptoms such as skin damage that could not heal, vitreous opacity, cataract, stroke hemiplegia, and the like, the body weight of some mouse individuals began to drop sharply Figure 4 ).

[0065] 2. Grouping and treatment

[0066] 1) After 40 mice in the model group were successfully modeled at 58 weeks, oral glucose tolerance test (OGTT) was performed to verify that the decrease in glucose tolerance was statistically significant, and statistical analysis was performed to determine that the mean blood glucose of the fasting blood glucose of the model group and the blood glucose of the normal group was statistically significant.

[0067] The area under the curve AUC drawn by the blood glucose change curve within two hours after intragastric administration of glucose between the two groups is shown in Figure 5 , indicating that the glucose tolerance of the DM group mice is impaired.

[0068] 2) The successfully modeled mice were divided into three groups according to no statistical difference in blood glucose within the group and statistical difference in blood glucose between the groups, namely the rhCygb treatment group (5mg / kg subcutaneous injection), the exentin-4 treatment group (50μg / kg subcutaneous injection), and the placebo group (0.1mL PBS subcutaneous injection) (grouping without cage, continue to feed according to the original cage of each mouse).

[0069] Since the type 2 diabetes obtained by high-fat diet feeding is similar to the onset of normal body, there is individual difference, so the blood glucose difference between different mice cannot be used for random grouping experiment, and the statistical grouping result is shown in Figure 6 , indicating that there is no statistical difference in the fasting blood glucose value between the placebo group, the rhCygb treatment group and the EX-4 treatment group mice for two weeks, which can be used for subsequent experiments.

[0070] In summary, compared with the normal control group, the model group mice appeared serious diabetic complications, with skin damage unable to heal, vitreous opacity, cataract, stroke hemiplegia and other symptoms, and the body weight of some mouse individuals began to drop sharply. Compared with the normal control group, the glucose tolerance of the model group mice was impaired.

[0071] The placebo group, rhCygb treatment group and EX-4 treatment group mice were subcutaneously injected with PBS, rhCygb and EX-4 respectively every day for 2 months (daily injection or injection frequency was considered according to the survival status of the mice), and the blood glucose level and body weight of the mice were detected every week during the period, as shown in Figure 7 As shown in the table, rhCygb treatment for 6 weeks can significantly reduce the blood glucose of mice, and the treatment effect is stable and similar to that of the ex-4 group.

[0072] 3) During the experiment, observe the fur, diet, stool of C57BL / 6J mice, whether the limbs move normally, whether there are deaths and causes of death, etc.

[0073] 4) After 28 days of treatment, the eyeballs of the mice were bled for four blood lipid indicators, and the mice were immediately executed by cervical dislocation after the blood was collected, and the tissues were immediately dissected. The mouse liver tissue, pancreas, kidney, heart, lung, stomach, and spleen were collected in turn for biochemical and pathological examination. The specific method is as follows.

[0074] 3. Four blood lipid determinations

[0075] 1) Total cholesterol (T-CHO) determination: The prepared serum was diluted ten times with normal saline at a ratio of 1:9. In each well of the 96-well plate, 2.5 μL of deionized water, 2.5 μL of standard, and 2.5 μL of sample were added in turn. Add 250 μL of working solution to each well, mix well, and incubate in a 37°C electric heating incubator for 10 minutes. Measure the absorbance of each well at a wavelength of 510 nm in an enzyme marker. Calculate the cholesterol content according to the formula:

[0076] Total cholesterol content (mmol / L) = (sample OD value - blank OD value) / (standard OD value - blank OD value) x standard concentration x 10

[0077] The serum of each C57BL / 6J mouse after treatment was determined using a total cholesterol determination kit, and the results are shown in Figure 8As shown, the total cholesterol levels of the rhCygb treatment group and the exendin-4 treatment group after treatment were statistically significantly lower than those of the placebo treatment group, and the total cholesterol levels of the rhCygb treatment group and the exendin-4 treatment group after treatment were not statistically significantly different from those of the blank control group, indicating good treatment effect.

[0078] 2) Triglyceride (TG assay): The prepared serum was diluted ten times with normal saline at a ratio of 1:9. In each well of a 96-well plate, 2.5 μL of deionized water, 2.5 μL of standard, and 2.5 μL of sample were sequentially added. 250 μL of working solution was added to each well, mixed well, and incubated in a 37°C electric heating incubator for 10 minutes. The absorbance of each well at a wavelength of 510 nm was measured in a microplate reader. The triglyceride content was calculated according to the formula:

[0079] Triglyceride content (mmol / L) = (sample OD value-blank OD value) / (calibrator OD value-blank OD value) x calibrator concentration x 10

[0080] The serum of each C57BL / 6J mouse after treatment was measured using a triglyceride level TG assay kit, and the results are shown in Table 3. Figure 9 As shown, the triglyceride levels of the rhCygb treatment group and the exendin-4 treatment group after treatment were not statistically significantly different from those of the NC group. The triglyceride level of the rhCygb group was statistically significantly lower than that of the placebo treatment group, indicating good treatment effect.

[0081] 3) High-density lipoprotein cholesterol (HDL-C) assay: The prepared serum was diluted ten times with normal saline at a ratio of 1:9. In each well of a 96-well plate, 2.5 μL of deionized water, 2.5 μL of calibrator, and 2.5 μL of sample were sequentially added. 180 μL of working solution R1 was added to each well, mixed well, and incubated in a 37°C electric heating incubator for 5 minutes. The absorbance of each well at a wavelength of 546 nm was measured in a microplate reader A1. 60 μL of working solution R2 was added to each well, mixed well, and incubated in a 37°C electric heating incubator for 5 minutes. The absorbance of each well at a wavelength of 546 nm was measured in a microplate reader A2. The high-density lipoprotein cholesterol content was calculated according to the formula:

[0082] High-density lipoprotein cholesterol content (mmol / L) = ((sample A2-sample A1) - (blank A2-blank A1)) / ((standard A2-standard A1) - (blank A2-blank A1)) x calibrator concentration x 10

[0083] The serum of each C57BL / 6J mouse after treatment was measured using a high-density lipoprotein level assay kit, and the results are shown in Table 4. Figure 10As shown, the high-density lipoprotein levels of each group after treatment had no statistically significant difference compared with the NC group, and the high-density lipoprotein level of the rhCygb group was higher than that of each group on average, but it was not statistically significant enough to prove that the rhCygb treatment could improve the high-density lipoprotein level of the diabetic mice.

[0084] 4) Low-density lipoprotein cholesterol (LDL-C) determination: The prepared serum was diluted ten times with normal saline at a ratio of 1:9. In each well of a 96-well plate, 2.5 microliters of deionized water, 2.5 microliters of calibration, and 2.5 microliters of sample were added in turn. 180 microliters of working solution R1 was added to each well, mixed well, and incubated in a 37°C electric heating incubator for 5 minutes. The absorbance value A1 of each well at a wavelength of 546 nm was measured in a microplate reader. 60 microliters of working solution R2 was added to each well, mixed well, and incubated in a 37°C electric heating incubator for 5 minutes. The absorbance value A2 of each well at a wavelength of 546 nm was measured in a microplate reader. The low-density lipoprotein cholesterol content was calculated according to the formula:

[0085] Low-density lipoprotein cholesterol content (mmol / L) = ((sample A2 - sample A1) - (blank A2 - blank A1)) / ((standard A2 - standard A1) - (blank A2 - blank A1)) x calibration concentration x 10

[0086] The serum of each C57BL / 6J mouse after treatment was determined using a low-density lipoprotein level determination kit, and the results are shown in Figure 11 As shown, the low-density lipoprotein levels of the rhCygb treatment group and the exendin-4 treatment group after treatment were statistically significantly lower than those of the placebo treatment group, and there was no statistically significant difference between the low-density lipoprotein levels of the rhCygb treatment group and the exendin-4 treatment group after treatment and those of the blank control group, indicating that the treatment effect was good.

[0087] In summary, rhCygb treatment for 2 months can significantly reduce blood glucose in mice, and the treatment effect is stable and similar to that of the ex-4 group. Moreover, rhCygb treatment can reduce the levels of total cholesterol, triglycerides, and low-density lipoprotein in the blood.

[0088] 6. HE staining

[0089] Take the tissue wax block, use xylene solution to dissolve the paraffin component in the section, and sequentially soak and wash in anhydrous ethanol, 90% alcohol, 70% alcohol, 50% alcohol, 25% alcohol, and distilled water. Soak and wash the section in the distilled water for several minutes, and then sequentially place in acid water and ammonia water for several seconds. After the section is colored, place it in running water for 1 hour, and then soak in distilled water. Take the section out of the distilled water, and sequentially perform dehydration steps in 70% and 90% alcohol for 10 minutes. Soak the dehydrated section in an alcohol-erythrosin staining solution for 2-3 minutes. Soak the colored section in anhydrous ethanol for dehydration. Soak and wash the section in a xylene solution until the section becomes transparent. Add gum to the transparent section, cover it with a cover glass, and then seal the section. Place it for about 30 minutes, label it on the glass slide, and then observe and describe it under an optical microscope. Take a photo of the main description part.

[0090] The results are shown in Table 1. Figure 12 As shown in Table 1, a large number of acinar cells are fatty degenerated in the pancreas tissue of the PBS group, and round vacuoles of different sizes are observed in the cytoplasm, which is considered to be beta cell vacuolar degeneration. The islet morphology of the rhCygb treatment group is normal, and no obvious inflammation is observed. A small amount of acinar cell degeneration is still observed, but the difference is not large compared with the NC group. It can be seen that rhCygb treatment can effectively improve and restore the normal morphology of the pancreas tissue. The Ex-4 group is the same as the PBS group, and a large number of acinar cells are fatty degenerated, which indicates that Ex-4 has no recovery effect on the fatty degeneration of the mouse pancreas.

[0091] According to the severity of the lesions observed in the HE staining results of each mouse pathological section, the grade data are subjected to non-parametric test of K independent samples by using SPSS software, and the test result is that there is a statistical difference between groups. Since SPSS cannot perform pairwise comparison between groups of multiple independent samples, the present application also ranks all the data, converts them into measurement data, and then performs one-way ANOVA analysis. The results are shown in Table 2. Figure 13 As shown in Table 2, there is no statistical difference between the rhCygb group and the NC group, and there is a statistical difference between the PBS group and the NC group and the rhCygb group. RhCygb has a significant improvement effect on the fatty degeneration of the pancreas.

[0092] 7. Immunohistochemistry

[0093] The pancreas is cut into 4 μm tissue sections, and the sections are baked in an oven at 60°C for 2 hours until the wax is melted; stained with hematoxylin-eosin;

[0094] Slices were immersed in methanol containing 3% hydrogen peroxide (blocker of endogenous peroxidase activity) for 15 minutes; after rinsing with PBS (10 mM, pH 7.0), the slices were incubated with an antibody mixture (rabbit anti-glucagon monoclonal antibody: anti-somatostatin antibody = 1:1) at 25°C for 1 hour or anti-mouse insulin antigen monoclonal antibody, anti-proliferating cell number antigen (PCNA) monoclonal antibody, or rabbit Ki67 monoclonal antibody, or the slices were incubated with guinea pig anti-PDX-1 monoclonal antibody at 4°C for 14 hours; for counterstaining, the cell nuclei were dyed blue by hematoxylin, and the slices were subjected to morphometric analysis;

[0095] PCNA analysis method, the percentage analysis method was used for the histological results by using the analysis software Image-pro plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA): at least 3 200x fields of view were randomly selected for each slice in each group for photographing. When photographing, the tissue was filled in the entire field of view as much as possible to ensure the uniformity of the background light of each photo. Image-Pro Plus 6.0 software was used to select the same brownish yellow cell nuclei as the uniform standard for judging the positive cells of all photos, and the same blue cell nuclei were selected as the total cells. The number of positive cells and the total number of cells were obtained by analyzing each photo, and the percentage of positive cells (positive cell number / total cell number*100) was obtained, that is, the positive rate (%).

[0096] PCNA positive staining, a marker of cell proliferation, was analyzed by using the analysis software Image-pro plus 6.0 to analyze the cell positive rate. It was found by statistical analysis that there was no statistical difference in the PCNA positive cell rate between the rhCygb treatment group and the ex-4 treatment group, and there was a statistical difference in the PCNA positive cell rate between the rhCygb treatment group and the PBS group and the NC group Figure 14 and 15 ), indicating that rhCygb treatment for 4 weeks significantly increased the PCNA staining intensity of the rhCygb treatment group C57BL / 6J mice and was similar to the positive drug treatment effect. It is indicated that the treatment of rhCygb can promote the proliferation of pancreatic beta cells.

[0097] Glucagon Insulin analysis method, using analysis software Image-pro plus 6.0 (MediaCybernetics, Inc., Rockville, MD, USA) to carry out immunohistochemical average density value analysis method on the groupization result: in each group, at least 3 200 times fields of view are selected on each slice to take a photo. When taking a photo, try to make the tissue fill the entire field of view, and ensure that the background light of each photo is consistent. Image-Pro Plus 6.0 software is applied to select the same brownish yellow on the islet as the unified standard for judging the positivity of all photos, and each photo is analyzed to obtain the cumulative optical density value (IOD) of the positivity on each photo and the pixel area (AREA) of the islet. And the average optical density value (average optical, AO value) is calculated, AO = IOD / AREA, the larger the AO value, the higher the positive expression level.

[0098] The results are shown in Figures 16-19 As shown in the results, the analysis software Image-pro plus 6.0 is used to analyze the cell positive rate, and it is found through statistical analysis that the expression levels of insulin and glucagon in the rhCygb treatment group have no statistical difference with the EX-4 group, and the expression levels of insulin and glucagon in the rhCygb treatment group have statistical differences with the PBS group and the NC group. It is proved that the treatment of rhCygb can promote the secretion of insulin and glucagon of islets.

[0099] In summary, rhCygb plays a role in reducing blood sugar by promoting islet cell proliferation and increasing insulin expression. It is shown that the cytoglobin of the application can be used for the treatment of diabetes.

[0100] Although the above embodiment makes a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, and other embodiments can be obtained according to the present embodiment without creativity, which all belong to the protection scope of the present application.

Claims

1. Use of a cellular globin in the manufacture of a medicament for the treatment of type II diabetes, characterized in that, The treatment includes at least one of (1) reducing blood sugar content; (2) reducing total cholesterol content; (3) reducing triglyceride content; (4) reducing low-density lipoprotein level; (5) improving and restoring normal morphological structure of pancreatic tissue; (6) promoting proliferation of islet beta cells; (7) promoting insulin and glucagon secretion of islets.

2. Use according to claim 1, characterized in that, The cell globin includes recombinant human cell globin.

3. Use according to claim 1 or 2, characterized in that, The intake dose of the cell globin is 5 mg / kg.

4. Use according to claim 3, characterized in that, The intake method includes injecting a PBS solution of the cell globin.

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