Application of compound wintercreeper mixture in preparation of medicine for treating aplastic anemia

Abstract

The invention relates to the technical field of biological medicines, and discloses an application of a compound wintercreeper mixture in preparation of a medicine for treating aplastic anemia. Two aplastic anemia mouse models are created by irradiating compound cyclophosphamide and chloramphenicol and busulfan compound cyclophosphamide; it is proved that the compound wintercreeper mixture can reduce the death rate of mice with aplastic anemia, improve blood indexes of the mice with aplastic anemia and obviously recover the bone marrow hematopoietic function of the mice.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to the application of a compound fufangteng mixture in the preparation of a medicine for treating aplastic anemia. Background Art

[0002] Aplastic anemia, referred to as aplastic anemia, is a group of bone marrow hematopoietic failure syndromes caused by multiple causes. It is characterized by decreased proliferation of bone marrow hematopoietic cells and pancytopenia in peripheral blood. Clinically, it is mainly manifested by anemia, bleeding and infection. Aplastic anemia is divided into congenital aplastic anemia and acquired aplastic anemia. The annual incidence rate in my country is 0.74 / 100,000, and the high-risk groups are young people aged 15-25 and the elderly aged 65-69. At present, hematopoietic stem cell transplantation and immunosuppression are mainly used. According to the symptoms and development speed of the disease, it can be divided into acute aplastic anemia and chronic aplastic anemia. The latter accounts for the vast majority clinically. Chronic aplastic anemia has the characteristics of a long course of disease and easy recurrence of the disease. The first choice of drug for the treatment of chronic aplastic anemia is immunosuppressive drugs. Hematopoietic drugs can also be used to stimulate the proliferation and differentiation of bone marrow hematopoietic stem cells, promote the proliferation of erythroid progenitor cells, and increase the number of white blood cells. Androgen replacement therapy can also be used according to the doctor's advice, and blood transfusion can be used in emergency situations. Among the above treatment methods, hematopoietic stem cell transplantation has high cost, and immunosuppressive drug treatment has large toxic side effects and may cause new diseases.

[0003] In recent years, research on the treatment of chronic aplastic anemia with traditional Chinese medicine has made certain progress. From the form of onset and clinical manifestations, aplastic anemia can be classified into the categories of "deficiency", "marrow fatigue", "blood syndrome", "blood deficiency", "deficiency", etc. The failure of bone marrow hematopoietic function is the direct cause of aplastic anemia. The clinical manifestations are deficiency of both qi and blood. Qi and blood cannot nourish the marrow, and blood stasis is blocked in the marrow, which affects the normal hematopoietic function of the bone marrow. Therefore, "deficiency" and "stasis" are the key factors in the pathogenesis of patients with aplastic anemia. Deficiency is mainly spleen and kidney deficiency. Therefore, the treatment of aplastic anemia should be based on strengthening the spleen and kidney as the fundamental principle, combined with removing blood stasis and detoxification. The summary of famous experts' experience, clinical trials, and animal experimental studies have provided objective basis for the treatment of chronic aplastic anemia with traditional Chinese medicine. Although the current scheme has achieved certain clinical efficacy, these drugs or prescriptions are complex, composed of more than a dozen Chinese medicinal materials, and it is difficult to stably control the quality, or they are made from a single effective part of the medicinal materials, and the efficacy is limited. Clinically, there is still a lack of scientific formulations, precise efficacy, stable medicinal material sources, easy-to-control quality, and safe and controllable therapeutic drugs. Summary of the invention

[0004] In order to overcome the above-mentioned defects existing in the prior art, the present invention provides the use of compound Fuyuan truncatum mixture in the preparation of drugs for treating aplastic anemia. The compound Fuyuan truncatum mixture can significantly improve the blood indexes of animals with aplastic anemia and enhance the hematopoietic function of the bone marrow; it reflects that the compound Fuyuan truncatum mixture can be used to prepare drugs for treating aplastic anemia.

[0005] Furthermore, the aplastic anemia drug is a pharmaceutical preparation prepared with the compound fufangteng mixture as the active ingredient and pharmaceutically acceptable excipients or auxiliary ingredients.

[0006] Furthermore, the compound Fufangteng mixture can significantly increase the levels of white blood cells, red blood cells, hemoglobin, erythropoietin and stem cell factor in mice with aplastic anemia, and enhance the hematopoietic function of the bone marrow.

[0007] Furthermore, the compound Fufangteng mixture can improve the spleen and thymus damage in aplastic anemia mice and enhance immune function.

[0008] The present invention also provides the use of the compound fufangteng mixture in treating aplastic anemia.

[0009] In summary, the present invention has the following beneficial effects:

[0010] 1. The present invention constructs an aplastic anemia (AA) animal model to study the pharmacological effects of Fufangteng mixture and finds that:

[0011] (1) Fufangteng compound can significantly increase the decreased levels of white blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), and reticulocytes (Ret) in mice with aplastic anemia, and reverse other abnormal blood indicators;

[0012] (2) The compound Fufangteng mixture can improve the bone marrow hematopoietic function of mice with aplastic anemia, reduce spleen and thymus damage, and improve immune function, indicating that the compound Fufangteng mixture can be used to treat mice with aplastic anemia.

[0013] 2. The present invention provides the use of the compound Fufangteng mixture for treating aplastic anemia.

[0014] 3. Fufangteng mixture has good safety when used to treat aplastic anemia or when used as a drug to treat aplastic anemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a statistical chart of body weight, thymus and spleen index of each group of experimental mice in the embodiment of the present invention;

[0016] Figure 2 Statistical diagram of blood routine test of each group of experimental mice in the embodiment of the present invention;

[0017] Figure 3 This is a statistical diagram of the detection results of apoptosis factors and anti-inflammatory factors in the blood of each group of experimental mice in the embodiment of the present invention;

[0018] Figure 4It is a statistical chart of the blood erythropoietin and stem cell factor detection results of each group of experimental mice in the embodiment of the present invention;

[0019] Figure 5 is a Wright-Giemsa staining image of the bone marrow smear of each group of experimental mice in the examples of the present invention (×200);

[0020] Figure 6 : HE staining images of bone marrow pathological sections of experimental mice in each group in the embodiment of the present invention (×100);

[0021] Figure 7 : HE staining images of the liver of each group of experimental mice in the examples of the present invention (×40);

[0022] Figure 8 It is the HE staining picture of spleen of each group of experimental mice in the example of the present invention (×200). DETAILED DESCRIPTION

[0023] The following is combined with Figure 1-8 The present invention is described in further detail.

[0024] Example:

[0025] (1) Animals, drugs and reagents

[0026] Animals: SPF male Kunming mice, weighing 20-22 g, kept in an environment with a temperature of 18-22°C, a relative humidity of 45-75%, 12-h light-dark cycle, and free access to purified water.

[0027] Drugs and reagents: Cyclosporine A soft capsules, cyclophosphamide, busulfan, compound Fufangteng mixture, Swiss-Giemsa staining solution, interleukin-3 (IL-3) ELISA detection kit, cell apoptosis key protease Caspase-3 ELISA detection kit, stem cell factor (SCF) ELISA detection kit, erythropoietin (EPO) ELISA detection kit.

[0028] (2) Preparation of drug solution

[0029] Preparation of positive control drug cyclosporine A solution (40 mg / kg·d): cut a small hole in 100 mg (4 capsules) cyclosporine soft capsule with scissors, squeeze out the liquid, add 50 mL of normal saline and mix well for use; preparation of low-dose Eugenia group (1.5 mL / kg·d) solution: accurately measure 3 mL of compound Eugenia mixture solution into a 50 mL EP tube, add 37 mL of normal saline and mix well for use; preparation of medium-dose Eugenia group (3 mL / kg·d) solution: accurately measure 6 mL of compound Eugenia mixture solution into a 50 mL EP tube, add 34 mL of normal saline and mix well for use; preparation of high-dose Eugenia group (6 mL / kg·d) solution: accurately measure 12 mL of compound Eugenia mixture solution into a 50 mL EP tube, add 28 mL of normal saline and mix well for use.

[0030] (3) Modeling and grouping

[0031] Modeling method 1: After 3 days of adaptive feeding, the mice were randomly divided into a normal control group and a modeling group. The modeling group was injected with cyclophosphamide and busulfan (prepared immediately before use, ultrasound at 40°C for 1h) every other day, and 15mg / (kg·d) busulfan solution was injected intraperitoneally on days 1, 3, 5, 7, 9, and 11, and 20mg / (kg·d) cyclophosphamide was injected on days 2, 4, 6, 8, 10, and 12. On the 13th day, blood was collected from the tail tips of all mice for routine blood tests, and whether the level of red blood cells, white blood cells, and hemoglobin was significantly lower than that of the blank group was used to determine whether the modeling was successful.

[0032] Modeling method 2: mice were irradiated with 5 Gy of whole body X-rays once, and intraperitoneally injected with 25 mg / kg cyclophosphamide and 50 mg / kg chloramphenicol on the 4th, 5th and 6th days after irradiation.

[0033] Grouping: After successful modeling, the mice were randomly divided into the model group (Mod group), the positive drug cyclosporine A group, the low-dose Euonymus fortunei group, the medium-dose Euonymus fortunei group, and the high-dose Euonymus fortunei group. Normal mice were set as the normal control group (CON group). The mice in each group were gavaged with the corresponding drug solution at a volume of 100mL / kg according to the weight of the mice, twice a day, and the model group and the normal control group were gavaged with the same volume of normal saline for 25 consecutive days. During the period, the mice were observed for food and water intake, activity, appearance, body weight and number of deaths.

[0034] (4) Statistics

[0035] SPSS22 software was used for data processing and statistics, and one-way ANOVA was used to analyze the significance. The data were expressed as mean±SD, and GraphPad 8.0.2 software was used for graphing. P<0.05 indicated a significant difference, and P<0.01 indicated a very significant difference.

[0036] Experiment 1: Compound Fufangteng mixture can increase the body weight and thymus index of AA mice and reduce the spleen index.

[0037] Steps: (1) On the 26th day after administration, the total mortality rate was calculated. The surviving mice were anesthetized 40 minutes after the last oral administration, and the eyeballs were removed to collect whole blood, which was anticoagulated with heparin sodium and counted for peripheral blood cells. The remaining blood was centrifuged at 12000 rpm for 15 minutes to obtain plasma, which was packaged and prepared for testing. The thymus, spleen, and liver were dissected, the surface was washed with physiological saline, the water was absorbed with filter paper, and the weight was weighed to calculate the thymus and spleen indexes.

[0038] (2) SPSS22 software was used for data processing and statistics, one-way ANOVA was used to analyze the significance, and GraphPad 8.0.2 software was used to draw the graph. P < 0.05 indicated a significant difference, and P < 0.01 indicated a very significant difference.

[0039] Experimental results: Compared with the Con group, ## Indicates P < 0.01, compared with the Mod group, * indicates P < 0.05, ** Indicates P<0.0. All mice in the Con group survived, and different numbers of mice in the Mod group using the two modeling methods died. The compound Fufang teng mixture can significantly reduce the number of deaths induced by modeling method 1. Compared with the Con group, the body weight of mice in the Mod group decreased significantly (P<0.01), and compared with the Mod group, the body weight of mice in each dose of Fufang teng group increased to varying degrees; compared with the Con group, the thymus index of mice in the Mod group decreased significantly (P<0.01), and the thymus index of mice in each dose of Fufang teng group increased (P<0.05); compared with the Con group, the spleen index of mice in the Mod group increased, the spleen index of mice in the low-dose and medium-dose Fufang teng groups did not increase significantly, and the spleen index of mice in the high-dose Fufang teng group decreased (P<0.05, Figure 1 ). The thymus is an important immune organ. This study found that the Fufangteng mixture can increase the reduced thymus index of AA mice and protect the immune function of the thymus. The spleen is a secondary organ for hematopoiesis in adult mice. Under normal physiological conditions, the spleen's hematopoietic function will not be activated. However, under stress conditions such as myelofibrosis, anemia, pregnancy, infection and myocardial infarction, the spleen's extramedullary hematopoiesis is induced, causing splenomegaly. The Fufangteng mixture can reduce the increase in the spleen index of AA mice and reduce spleen enlargement, reflecting that the drug can protect the normal hematopoietic function of mice without activating the spleen's hematopoietic function.

[0040] Experiment 2: The compound Fufangteng mixture can improve the peripheral blood index of AA mice, reflecting that the mixture has a significant effect on hematopoiesis.

[0041] Steps: Perform routine blood tests on the whole blood collected in test one.

[0042] Experimental results: Compared with the Con group, ## Indicates P < 0.01; compared with the Mod group, * indicates P < 0.05, ** Indicates P<0.01; Compared with the Con group, the WBC, RBC, HGB, PLT, Ret, neutrophil (NEU), medium fluorescence intensity reticulocyte (MFR), and hematocrit (HCT) of the mice in the Mod group were significantly decreased (P<0.05), and the above indicators of the mice in each dose group of Fufangteng increased to varying degrees, indicating that the Fufangteng mixture can improve the peripheral blood count and hematopoietic function of AA mice ( Figure 2 ).

[0043] Experiment 3: The compound Fufangteng mixture can significantly inhibit the apoptosis of blood cells in AA mice, promote the production of anti-inflammatory factors, and improve the body's immune function.

[0044] Steps: The whole blood collected in Experiment 1 was anticoagulated with sodium heparin, and peripheral blood cell count was performed. The remaining blood was centrifuged at 12000 rpm for 15 min to obtain plasma, which was tested using IL-3 ELISA detection kit and cell apoptosis key protease Caspase-3 ELISA detection kit.

[0045] Experimental results: Compared with the Con group, the plasma Caspase-3 level of mice in the Mod group increased and IL-3 level decreased, indicating that cell apoptosis was aggravated and inflammation occurred; Fufangteng mixture could increase the plasma IL-3 level to a certain extent, and high-dose Fufangteng mixture reduced the plasma Caspase-3 level of mice ( Figure 3 ).

[0046] Experiment 4: The compound Fufangteng mixture can increase the levels of erythropoietin (EPO) and stem cell factor (SCF) in the blood of AA mice and promote bone marrow hematopoietic function.

[0047] Steps: The whole blood collected in Test 1 was anticoagulated with sodium heparin, and peripheral blood cell count was performed. The remaining blood was centrifuged at 12000 rpm for 15 min to obtain plasma, which was tested using SCF ELISA test kit and EPO ELISA test kit.

[0048] Experimental results: Compared with the Con group, the EPO and SCF levels in the plasma of the Mod group mice decreased significantly. SCF is an acidic glycoprotein produced by stromal cells in the bone marrow microenvironment, which induces the proliferation of erythroid progenitor cells. This result shows that the Mod group mice have hematopoietic dysfunction; Fufangteng mixture can increase the EPO and SCF levels of AA mice in a dose-dependent manner, indicating that Fufangteng mixture can significantly promote bone marrow hematopoietic function ( Figure 4 ).

[0049] Experiment 5: The compound Fufangteng mixture can increase the number of bone marrow nucleated cells in AA mice and promote bone marrow hematopoietic function.

[0050] Steps: (1) Bone marrow smear

[0051] Take the left femur of the mouse, remove the muscle tissue, etc., cut the femur short, suck out the bone marrow, dilute it with PBS solution 1:1, quickly smear and stain, dry it at room temperature, use a dropper to absorb methanol and evenly spread it on the slide for fixation, and dry it at room temperature; add 0.8mL of Wright Giemsa A solution to cover the specimen on the smear, stain for 30s, then add 2mL of Wright Giemsa B solution to the A solution, blow it repeatedly to mix the two solutions thoroughly, and stain for 10min. Rinse the residual dye on the slide with running water, put it on a drying rack, dry it at room temperature, and examine it under an inverted microscope after drying, and observe the number of nucleated cells at different magnifications.

[0052] (2) Bone marrow pathology section

[0053] Decalcification and fixation: The right femur of the mouse was peeled off, the muscle tissue was removed, and the femur was fixed in 4% paraformaldehyde fixative, and then placed in EDTA decalcification solution (pH 7.2) for decalcification for 15 days.

[0054] Dehydration and transparency treatment: After 14 days, the femoral bone marrow of mice was taken out from the decalcification solution, rinsed with distilled water, and then subjected to gradient dehydration (in order: 80% ethanol solution, 95% ethanol solution, anhydrous ethanol type I, anhydrous ethanol type II); after dehydration, the femoral bone marrow was placed in xylene to make the femoral bone marrow transparent; wax immersion and embedding: the treated femoral bone marrow was placed in melted paraffin and kept warm in a constant temperature box; it was taken out after cooling and solidification.

[0055] Slicing and baking: Slice the processed wax block into approximately 6 μm thin slices, flatten the slices with 60°C distilled water and spread them on a clean glass slide, then place them in a 45°C constant temperature box and store them at a constant temperature for 20 minutes.

[0056] Dewaxing and staining: Dewax the processed sections with xylene, then dewax with xylene, ethanol, and distilled water in sequence; stain the sections with hematoxylin in sequence, then rinse the sections with distilled water, differentiate with differentiation solution, soak in distilled water, then stain with eosin stain, and finally soak in distilled water again;

[0057] Washing: Wash the sections with 95% ethanol to remove excess color, dehydrate with anhydrous ethanol, absorb excess ethanol, and then treat with xylene.

[0058] Microscopic examination.

[0059] Experimental results: Compared with the Con group, the number of nucleated cells in the bone marrow of the mice in the Mod group was greatly reduced, and the bone marrow cavity was relatively empty. The number of nucleated cells in the bone marrow of the mice in the Fufangteng mixture groups at each dose was more than that in the Mod group, and the bone marrow cavity was full, reflecting that the bone marrow hematopoietic function was significantly improved ( Figure 5 , 6 ).

[0060] Experiment 6: Compound Fufang Teng mixture reduces liver damage in AA mice.

[0061] (1) Tissues from the same part of the left lobe of the liver of mice in each group were taken, washed, placed in 4% paraformaldehyde, dehydrated, embedded in paraffin, cut into 5 μm thick sections with a microtome, and stained with conventional HE. After transparent sealing, the pathological condition of the liver tissue in each section was observed under an optical microscope.

[0062] Experimental results: The liver tissue cells of mice in the Con group had normal morphology, clear boundaries and neat arrangement. The liver tissue cells of mice in the Mod group had abnormal morphology, chaotic arrangement and enlarged intercellular crypts. Compared with the Mod group, the intercellular crypts of mice in the Fufangteng mixture groups were reduced and the liver cell morphology was relatively complete ( Figure 7 ).

[0063] Experiment 7: Fufangteng mixture can reduce the spleen extramedullary hematopoiesis and abnormal hematopoiesis of AA mice.

[0064] Steps: Mouse spleen was separated and washed with sterile PBS buffer. Part of the spleen was fixed in paraformaldehyde solution for 24 hours. The paraformaldehyde fixative was rinsed with PBS, dehydrated with ethanol, and embedded in paraffin. Paraffin tissue blocks were sliced ​​continuously with a thickness of 2 μm and placed on slides. They were incubated in a 37°C incubator overnight, then kept at 65°C for 2 hours, and paraffin sections were dewaxed with water. The sections were placed in hematoxylin stain for 3 to 5 minutes, acetic acid and ammonia were used for coloring for a few seconds each, and then rinsed with running water for 1 hour and placed in distilled water for a few seconds. The sections were placed in 85% and 95% gradient alcohol for dehydration for 5 minutes each, and then stained in eosin stain for 5 minutes. Dehydrated with anhydrous ethanol, the sections were made transparent with xylene, and sealed with neutral gum. The sections were observed under an optical microscope, and images were collected for analysis.

[0065] Experimental results: The red pulp area is the main area where the spleen performs hematopoietic function. Compared with the mice in the Con group, the red pulp and white pulp of the spleen in the Mod group were clearly demarcated, and the proportion of red pulp increased. Compared with the mice in the Mod group, the proportion of red pulp in the spleen of the mice in the Fufangteng mixture administration groups was significantly reduced ( Figure 8 ).

[0066] In the above experiment, the activity and food intake of AA mice in the Mod group decreased, the lip, auricle, and limbs turned white, the body temperature decreased, the number of nucleated cells in the bone marrow decreased significantly, the blood indicators were significantly lower than those in the normal group, the bone marrow cavity was empty, the number of hematopoietic cells decreased, and the stasis, bleeding, and interstitial edema were significant. The mice given Fufangteng mixture had increased activity, normal food intake, normal color of lip, auricle, and limbs, normal body temperature, and significantly higher blood cell counts than the model group, significantly increased bone marrow nucleated cells, significantly filled bone marrow cavity, increased hematopoietic cells, and reduced stasis, bleeding, and interstitial edema.

[0067] From the above experiments 1 and 2, it can be seen that the compound Fufangteng mixture can increase the decreased thymus index of AA mice, reduce the increased spleen index, improve their immune function, and significantly restore the abnormal blood routine of AA mice;

[0068] From the above experiments three and four, it can be seen that the compound Fufang vine mixture can significantly reduce the elevated Caspase-3 level in the plasma of AA mice and reduce cell apoptosis; it can increase the plasma IL-3 of AA mice. IL-3 is a polyclonal stimulating factor that promotes the growth and differentiation of pluripotent stem cells, enables them to produce various lineages of bone marrow cells, and can increase EPO and SCF levels. These results indicate that the compound Fufang vine mixture can significantly promote bone marrow hematopoietic function.

[0069] It can be seen from the above experiment 5 that the compound Fufangteng mixture can increase the number of nucleated cells in the bone marrow of AA mice and promote bone marrow hematopoietic function, which proves that the compound Fufangteng mixture has the effect of treating aplastic anemia.

[0070] From the above experiments six and seven, it can be seen that the compound Fufangteng mixture has a protective effect on the liver of AA mice, which proves that the compound Fufangteng mixture is safe and effective when calculated according to the experimental dosage to the clinical dosage.

[0071] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. Application of Fufangteng mixture in the preparation of drugs for the treatment of aplastic anemia.

2. The use of the compound Fufangteng mixture according to claim 1 in the preparation of a drug for treating aplastic anemia, characterized in that: The aplastic anemia drug is a drug preparation prepared by taking the compound fufangteng mixture as an effective ingredient and adding pharmaceutically acceptable excipients or auxiliary ingredients.

3. The use of the compound Fufangteng mixture according to claim 1 in the preparation of a drug for treating aplastic anemia, characterized in that: The compound fufangteng mixture can significantly increase the levels of white blood cells, red blood cells, hemoglobin, erythropoietin and stem cell factor in mice with aplastic anemia, and promote bone marrow hematopoietic function.

4. The use of the compound Fufangteng mixture according to claim 1 in the preparation of a drug for treating aplastic anemia, characterized in that: The compound fufangteng mixture can improve the spleen and thymus damage of aplastic anemia mice and enhance the immune function.

5. Application of Fufangteng mixture in preventing aplastic anemia.

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