Application of colla corii asini bone-strengthening oral liquid in preventing and / or treating anemia

Abstract

The invention discloses application of colla corii asini bone-strengthening oral liquid to prevention and / or treatment of anemia, and belongs to the technical field of medicines, and the anemia comprises iron-deficiency anemia, renal anemia and myelosuppression anemia. The colla corii asini bone strengthening oral liquid can increase erythrocyte count, hemoglobin concentration, hematocrit, erythrocyte average hemoglobin amount, erythrocyte average hemoglobin concentration and / or erythrocyte average volume; the serum iron content is increased; a transporter divalent metal transporter and a membrane iron transporter are increased; the intestinal iron absorption is promoted. The composition specifically comprises urea nitrogen and creatinine; related indexes of renal anemia are improved; the immune index of renal anemia can be improved. The percentage of leukocytes and lymphocytes of anemia caused by myelosuppression can be recovered; the hemoglobin content is increased; the number of macrophage colony stimulating factors and / or bone marrow cells in serum is increased; the immunocompetence is improved; the proliferation of lymphoid B cells and macrophages is promoted.

Description

Technical Field

[0001] The use of donkey-hide gelatin bone strengthening oral liquid for preventing and / or treating anemia, wherein the anemia includes iron deficiency anemia, renal anemia, and bone marrow suppression anemia, belongs to the field of medical technology. Background Art

[0002] Anemia refers to a common clinical symptom in which the volume of red blood cells in human peripheral blood decreases below the lower limit of the normal range. It can be complicated by many diseases. It is defined as the hemoglobin content, red blood cell count and hematocrit per unit volume in peripheral blood are lower than the normal standard. According to WHO statistics, about 2 billion people suffer from iron deficiency or iron deficiency anemia to varying degrees, accounting for about 30% of the world's population. It is one of the four major nutritional deficiency diseases in the world. The annual incidence of anemia in infants and children in my country is 45.7%, and the incidence of anemia in the elderly over 65 years old is 52%. Therefore, the prevention and treatment of anemia is urgent.

[0003] According to the clinical incidence, anemia can be divided into iron deficiency anemia, megaloblastic anemia, aplastic anemia, hemolytic anemia, hemorrhagic anemia, pure red cell aplasia, etc.

[0004] Iron deficiency anemia is a common nutritional deficiency anemia in my country, affecting a wide range of people, with a high incidence rate, which seriously affects people's health and quality of life. Its cause is mostly insufficient intake, malabsorption or excessive consumption, which leads to the depletion of iron stored in the body. If it is not supplemented during this period, it will develop into iron deficiency anemia. Iron is one of the basic raw materials for synthesizing hemoglobin. Iron deficiency leads to the obstruction of hemoglobin synthesis in the bone marrow, causing microcytic hypochromic anemia. Iron deficiency can lead to a decrease in physical strength, a significant weakening of labor and work ability, and a significant decrease in immunity. It can also have a serious impact on women's fertility.

[0005] Iron deficiency anemia refers to anemia caused by hemoglobin (Hb) synthesis disorder due to iron deficiency and iron utilization disorder in the body. In the process of hemoglobin synthesis, erythrocyte free protoporphyrin first binds to iron under the action of heme synthetase. When the iron content is relatively deficient, free protoporphyrin in erythrocytes accumulates in free form. Therefore, the content of free protoporphyrin (FEP) in erythrocytes is one of the sensitive indicators for diagnosing iron deficiency. Serum ferritin (SF) can reflect the amount of iron stored in the body and is a specific indicator for reflecting the body's iron reserves. Serum transferrin (sTRF) receptor is a transferrin receptor fragment circulating in serum, which can accurately reflect the iron storage status in the body and is an effective indicator for diagnosing iron deficiency anemia. Erythropoietin (EPO) can stimulate the division of erythroid progenitor cells and precursor erythrocytes, promoting the production of erythrocytes.

[0006] Hepcidin is a hormone synthesized and secreted by the liver. The homeostasis of iron and bone metabolism in the body are mainly regulated by hepcidin. The main function of hepcidin is to regulate the level of endogenous circulating iron. Hepcidin reduces the iron content in the blood by binding to ferroportin 1. Ferroportin is the only pathway for iron to enter the extracellular fluid and plasma. The lack of hepcidin will lead to iron overload, which in turn upregulates the expression of hepcidin through a feedback mechanism.

[0007] Iron in the diet is mainly absorbed into the blood through the transport proteins divalent metal transporter (DMT-1) and ferroportin (FPN-1) expressed by duodenal epithelial cells. DMT1 is widely distributed in various tissues of the body, with the highest expression in the proximal small intestine. It is mainly distributed in the brush border of the duodenal villi and transports intestinal non-heme iron into cells. FPN1 is a transmembrane iron export protein. In the small intestine, FPN1 is mainly distributed in the basal end and basement membrane of the duodenal villus epithelial cells and is responsible for transporting iron in the enterocytes out of the cells and into the blood. Related studies have confirmed that hepcidin is a negative regulator of the expression of proteins related to intestinal iron absorption.

[0008] Renal anemia, as a secondary anemia, is a common complication of renal insufficiency in the terminal stage. Its incidence in patients is as high as more than 95%, which is very representative of clinical significance. Modern medical research has found that the main causes of renal anemia include relatively insufficient secretion of erythropoietin (EPO) in the body, bone marrow hematopoiesis inhibition caused by retained toxic substances, interference with the production and metabolism of red blood cells, and shortened red blood cell survival. The degree of anemia is often related to the degree of renal dysfunction, mainly normochromic and normocytic anemia. At present, the main treatment for renal anemia in clinical practice is injection of recombinant human EPO. Although it can achieve a certain therapeutic effect, it is expensive.

[0009] Anemia caused by tumors belongs to aplastic anemia. Severe bone marrow suppression anemia often leads to some serious complications, such as infection, bleeding, fever, etc., making it difficult to continue chemotherapy. Therefore, the prevention and treatment of bone marrow suppression caused by chemotherapy is particularly important. To some extent, the control of this side effect of bone marrow suppression is the key to the success of cancer chemotherapy and radiotherapy. At present, the drugs used for tumor anemia are some hematopoietic growth factors, such as granulocyte macrophage colony stimulating factor (GM-GSF), erythropoietin (EPO), thrombopoietin (TPO), etc., but they are expensive. Summary of the invention

[0010] Iron deficiency anemia refers to anemia caused by hemoglobin (Hb) synthesis disorder due to iron deficiency and iron utilization disorder in the body. The results of this study showed that the donkey-hide gelatin bone strengthening oral liquid group could significantly increase the HGB, RBC, HCT, MCH, MCHC, and MCV of the model rats; increase the serum iron content of rats; and increase the expression of DMT-1 and FPN-1 proteins in the duodenum of rats. It shows that donkey-hide gelatin bone strengthening oral liquid can improve iron deficiency anemia, promote intestinal iron absorption by upregulating the negative feedback regulation mechanism of iron absorption in rats, improve the blood picture and iron metabolism of rats with iron deficiency anemia, increase hemoglobin, and promote oxygen supply to the body. The expression of DMT1 and FPN1 proteins in the duodenum of rats in the medium and high dose groups of donkey-hide gelatin bone strengthening oral liquid was higher than that in the normal control group and the model group. It suggests that donkey-hide gelatin bone strengthening oral liquid can increase the degree of negative feedback regulation of intestinal iron absorption. This process may be related to the fact that donkey-hide gelatin bone strengthening oral liquid can inhibit the level of hepcidin in the liver. It promotes the absorption of iron in the duodenum by upregulating DMT1 and FPN1.

[0011] Renal anemia refers to anemia caused by various factors that cause insufficient production of erythropoietin in the kidney or some toxins in uremic plasma that interfere with the production and metabolism of red blood cells. It is a common clinical symptom of anemia. The present invention uses continuous administration of adenine to establish a rat renal anemia model, and observes the effect of donkey-hide gelatin strong bone oral liquid on rats with renal anemia by administering donkey-hide gelatin strong bone oral liquid. The present invention can cause renal fibrosis through the modeling drug adenine, resulting in insufficient EPO secretion, insufficient secretion of erythroid and megakaryocyte cells, and obtain renal failure anemia. After the modeling of this experiment, the rats' urea nitrogen, creatinine, body weight and red blood cell count and other indicators changed significantly, indicating that the experimental modeling was successful. After 6 weeks of administration, it was found that donkey-hide gelatin strong bone oral liquid had a significant regulatory effect on the renal functional indicators urea nitrogen and creatinine, indicating that it has the ability to restore the rat kidney damage caused by adenine, and has a significant restorative effect on the anemic indicators caused by it. The test kit revealed that the renal anemia-related indicators SOD, GST and EPO in the model group were significantly reduced, which was consistent with the clinical indications of renal anemia. The same dose of donkey-hide gelatin bone strengthening oral liquid in the medium-dose group had a significant improvement on these indicators, further confirming that donkey-hide gelatin bone strengthening oral liquid has a significant restorative effect on renal anemia.

[0012] Bone marrow suppression anemia caused by tumor radiotherapy and chemotherapy belongs to aplastic anemia. The present invention establishes a mouse bone marrow suppression model by combining cyclophosphamide and chloramphenicol, and observes the effect of donkey-hide gelatin fortifying bone oral liquid on mice with bone marrow suppression anemia by administering donkey-hide gelatin fortifying bone oral liquid. By comparing hematological indicators, it was found that donkey-hide gelatin fortifying bone oral liquid can restore the percentage of white blood cells and lymphocytes in model mice. Compared with positive drug colony stimulating factor, donkey-hide gelatin fortifying bone oral liquid has a more targeted effect on the proliferation of lymphocytes T, B and NK cells; at the same time, the experiment found that donkey-hide gelatin fortifying bone oral liquid can have a significant restorative effect on anemia caused by bone marrow suppression, especially in terms of hemoglobin content, and its effect is higher than positive drug colony stimulating factor and compound donkey-hide gelatin slurry. Combined with the results of macrophage colony stimulating factor and interleukin 6 in mouse serum and bone marrow cell count, it was shown that donkey-hide gelatin bone strengthening oral liquid can enhance the immune ability of immunosuppressed mice. One of its mechanisms of action is to promote the differentiation of myeloid progenitor cells into lymphocytes, especially the proliferation of lymph B cells and macrophages. At the same time, it can increase the production of hemoglobin and improve the body's energy metabolism level.

[0013] Beneficial technical effects

[0014] The Ejiao Qianggu oral liquid group can significantly increase the red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV). It increases serum iron content and increases the expression of duodenal DMT-1 and FPN-1 proteins. This shows that Ejiao Qianggu oral liquid can improve iron deficiency anemia, promote intestinal iron absorption by upregulating the negative feedback regulation mechanism of iron absorption, improve the blood picture and iron metabolism of rats with iron deficiency anemia, increase hemoglobin, and promote oxygen supply to the body.

[0015] Donkey-hide gelatin bone strengthening oral liquid has a significant regulatory effect on kidney function indicators urea nitrogen and creatinine; it has a significant recovery effect on anemia indicators caused by kidney function damage; it can increase the content of SOD, GST and EPO related indicators of renal anemia. This shows that donkey-hide gelatin bone strengthening oral liquid has the following effects: it can restore kidney damage, has a significant recovery effect on renal anemia, and has a regulatory effect on the immune indicators of rats with renal anemia.

[0016] Donkey-hide gelatin for bone strengthening oral liquid can restore the percentage of white blood cells and lymphocytes in anemia caused by bone marrow suppression; it has a significant restorative effect on anemia caused by bone marrow suppression, especially significantly increasing the hemoglobin content; it can also increase the macrophage colony stimulating factor and bone marrow cell count in serum. This shows that donkey-hide gelatin for bone strengthening oral liquid can improve immunity, and one of its mechanisms is to promote the differentiation of myeloid progenitor cells into lymphocytes, especially the proliferation of lymph B cells and macrophages; it can promote the production of hemoglobin in bone marrow suppressed mice, improve the body's energy metabolism level, and has a certain effect on the recovery of anemia caused by bone marrow suppression. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1-1 Routine blood tests of rats after modeling was completed.

[0018] Figure 1-2 Body weight changes of rats before and after modeling.

[0019] Figure 1-3 Body weight changes of each group during drug intervention.

[0020] Figure 1-4 Effects of donkey-hide gelatin bone-strengthening oral liquid on routine blood tests (HGB, HCT, MCH, MCHC, MCV) in rats with iron deficiency anemia.

[0021] Figure 1-5 Effects of donkey-hide gelatin bone strengthening oral liquid on serum iron and DMT-1 and FPN-1 proteins in rats.

[0022] Figure 2-1 Body weight changes of rats after drug administration.

[0023] Figure 2-2 SOD label song.

[0024] Figure 2-3 SOD label song.

[0025] Figure 2-4 EPO label song.

[0026] Figure 3-1 MCSF label song.

[0027] Figure 3-2 IL-6 standard curve. DETAILED DESCRIPTION

[0028] Example 1 Improvement of iron deficiency anemia by donkey-hide gelatin bone strengthening oral liquid.

[0029] 1 Materials

[0030] 1.1 Drugs

[0031] The donkey-hide gelatin bone-strengthening oral liquid used in this study was provided by Xinjiang Huashidan Pharmaceutical Co., Ltd. It is a modern oral liquid preparation made of Rehmannia glutinosa, donkey-hide gelatin, wolfberry, oyster, astragalus, and Codonopsis pilosula (batch number: 2019111802); ferrous succinate (batch numbers: 180319 190410 190610); compound donkey-hide gelatin slurry (batch numbers: 1806027 1904070); low-iron feed (Shenzhen Ruidi Biotechnology Co., Ltd., batch number: 18073112, iron content 6.7 mg / kg).

[0032] 1.2 Experimental animals

[0033] Healthy male SD rats (SPF grade 90, purchased from the Experimental Animal Research Center of Xinjiang Medical University, Experimental Animal Production License No.: SCXK (New) 2018-0002, experimental animal breeding environment: temperature 24-26 ° C, humidity 60%-70%, 12h / 12h interval lighting, free access to food and water, adaptive feeding after 1 week of weighing, 180-220g.

[0034] 1.3 Instruments and reagents

[0035] FA1004 analytical balance (Shanghai Hengping Scientific Instrument Co., Ltd.); 5417R desktop refrigerated high-speed centrifuge (Eppendorf, Germany); 750 fully automatic hematology analyzer (Backman Coulter); Olympus 5400 fully automatic biochemical analyzer (OLYMPUS Optical Co., Ltd., Japan); Model 680 microplate reader (Bio-Rad, USA); mouse divalent metal transporter 1 (DMT-1) enzyme-linked immunosorbent assay kit (Shanghai ELISA Biotechnology Co., Ltd.); mouse membrane transporter (FPN-1) enzyme-linked immunosorbent assay kit (Shanghai ELISA Biotechnology Co., Ltd.).

[0036] 2 Methods

[0037] 2.1 Animal modeling and grouping

[0038] Replication of IDA rat model: After 5 days of adaptive feeding, 60 male SD rats were randomly divided into 5 normal control groups and 55 modeling groups according to body weight using SPSS software. According to the modeling method reported in the literature, the modeling group was given a low-iron feed of about 20g per day and drank deionized water. And blood was drawn from the ear vein of the rat every 3 days, with 20 drops of blood drawn each time. The normal control group was given a normal basic feed and drank tap water. The body weight was weighed and recorded weekly. After 28 days of modeling, blood was drawn from the inner canthus and blood routine analysis was performed using anticoagulant tubes. If the results show the characteristics that meet the requirements, the average hemoglobin (Hb) of the experimental rats is ≤90g / L, and the modeling is successful.

[0039] Animal grouping: Select rats with successful modeling according to the degree of anemia using SPSS software, randomly select 5 rats as the model group, and the rest are randomly divided into 5 groups, each with 9 rats. Therefore, all rats were divided into 7 experimental groups: A normal control group, B model control group, C ferrous succinate group, D compound donkey-hide gelatin syrup group, E low-dose donkey-hide gelatin bone strengthening oral liquid group, F medium-dose donkey-hide gelatin bone strengthening oral liquid group, and G high-dose donkey-hide gelatin bone strengthening oral liquid group.

[0040] 2.2 Drug solution and administration

[0041] The dosages of the ferrous sulfate group (5 ml / kg of ferrous sulfate suspension and 10 ml / kg of deionized water were administered orally per day for each mouse), the compound donkey-hide gelatin slurry group (6.3 ml / kg), the high-dose donkey-hide gelatin bone-strengthening oral liquid group (12.6 ml / kg), the medium-dose donkey-hide gelatin bone-strengthening oral liquid group (6.3 ml / kg), and the low-dose donkey-hide gelatin bone-strengthening oral liquid group (3.15 ml / kg) were all converted to clinical dosages. The mice were gavaged continuously for 28 days, and their body weights were measured every 3 days and their conditions were observed by taking photos.

[0042] 2.3 Index detection

[0043] 2.3.1 Routine blood test

[0044] 28 days after modeling, blood was collected from the orbits of all rats and placed in EP tubes containing anticoagulant EDTA-2K for routine blood analysis. After the last administration of each group of animals, fast for 12 hours. After chloral hydrate anesthesia, blood was collected from the carotid artery and 1 mL was placed in EP tubes containing anticoagulant EDTA-2K for routine blood analysis.

[0045] 2.3.2 Blood biochemical test

[0046] The collected blood was injected into a 2mL EP tube (without adding anticoagulant), incubated at 37°C for 1h to allow it to fully coagulate, then placed in a 4°C refrigerator overnight, centrifuged at 3000r / min for 20min, and the supernatant was carefully aspirated with a 1mL pipette and sent to the laboratory of XX Hospital for determination of serum iron SI.

[0047] 2.3.3 Detection of proteins related to iron metabolism

[0048] The detection of divalent metal transporter DMT-1 and ferroportin FPN-1 in rat duodenal epithelial cells was detected by ELISA. The same part of the duodenum of rats in each group was taken, and the specimens were cut and weighed. After adding a certain amount of PBS, the specimens were homogenized with a homogenizer, and the supernatant was collected after centrifugation for about 20 minutes (2000-3000 rpm). Standard wells and sample wells were set up, and 50 μL of standards of different concentrations were added to each standard well. Blank wells and test sample wells were set up respectively. 40 μl of sample diluent was first added to the test sample well on the enzyme-labeled plate, and then 10 μl of the test sample was added. The sample was added to the bottom of the enzyme-labeled plate well, and 100 μl of the enzyme-labeled reagent was added to each well, except for the blank well. The plate was sealed with a sealing film and incubated at 37°C for 60 minutes. Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard it, repeat this 5 times, and pat dry. First add 50u1 of colorimetric reagent A to each well, then add 50u1 of colorimetric reagent B, gently shake and mix, and color at 37℃ in the dark for 15 minutes. Add 50u1 of stop solution to each well to stop the reaction (the blue color immediately turns yellow). Zero the blank well and measure the absorbance (OD value) of each well in sequence at a wavelength of 450nm. Draw a standard curve based on the concentration and OD value of the standard, and use the ELISA Calc regression / fitting calculation program to reversely calculate the protein content of each administration group.

[0049] 2.4 Statistical methods

[0050] All experimental data were statistically analyzed using SPSS 21.0 statistical software.

[0051] 3. Results and Analysis

[0052] 3.1 Results of blood routine tests after successful modeling of rats

[0053] 28 days after modeling, the following six indicators were detected by routine blood test, namely, red blood cell count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV). Red blood cell count refers to the number of red blood cells contained in a unit volume of blood, which is of great significance for indicating diseases involving the red blood cell system. Hemoglobin, also known as hemoglobin, is the main component of red blood cells and can bind to oxygen to transport oxygen and carbon dioxide. The clinical significance of increased and decreased hemoglobin is basically similar to that of red blood cell count, and hemoglobin can better reflect the degree of anemia. The results of the blood routine test of rat modeling are shown in Table 1. The hemoglobin concentration (HGB) of rats in the model group decreased from (179.00±8.60) to (78.29±8.60), P<0.001. According to the clinical identification standard, hemoglobin (HGB) is lower than 90g·L -1, which met the evaluation criteria of mild iron deficiency anemia model. At the same time, compared with the blank group, there were extremely significant differences in red blood cell count (RBC), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV), P<0.001.

[0054] Table 1-1 Blood routine indicators of rats after modeling

[0055]

[0056] Note: Compared with the control group, *p<0.05, **P<0.001, the same as the notes in the following table.

[0057] 3.2 Body weight changes of rats before and after modeling

[0058] During the induction of the iron deficiency anemia model, the changes in rat body weight are shown in Table 2. Starting from the second week of modeling, the weight of the rats gradually decreased and the amount of food consumed decreased. From the third week, there was a significant difference in body weight, indicating that iron deficiency can cause weight loss and reduced food intake. In the fourth week, the rats in the model group generally showed whitening of the auricle, eyeballs, and toes, lighter color of feces, messy and dull hair, and a preference for chewing on the mouse cage. This meets the morphological evaluation criteria for the iron deficiency anemia model.

[0059] Table 1-2 Changes in body weight of rats before and after modeling

[0060]

[0061] Note: Compared with the control group, *P<0.05, **P<0.001

[0062] 3.3 Effects of donkey-hide gelatin bone strengthening oral liquid on body weight and organ indexes in rats with iron deficiency anemia

[0063] The body weight and organ indexes of rats after 28 days of drug intervention are shown in Table 1. The body weight of each drug-treated group tends to the normal group weight, and there is no significant difference between the groups, P>0.05. This shows that the weight of rats rebounded after iron supplementation and tended to normal weight. The liver is the body's most important iron storage organ. Iron in the body mainly exists in the liver in the form of ferritin and hemosiderin. From Table 3, it can be found that there are significant differences between each group and the normal control group after iron supplementation (P<0.05). The reason may be that the weight of rats in the control group is slightly heavier than that of the drug-treated group, or that the iron source has a certain effect on the growth, development and renewal of rat liver tissue. There is no difference between the spleen index and heart index among the groups, p>0.05.

[0064] Table 1-3. Effects of donkey-hide gelatin bone strengthening oral liquid on body weight and organ indexes of rats with iron deficiency anemia ( n=8)

[0065]

[0066] Note: Compared with the normal control group A, #p<0.05

[0067] 3.4 Effect of donkey-hide gelatin bone strengthening oral liquid on blood routine in rats

[0068] The following blood routine indicators of each group of rats were tested, namely, hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and mean corpuscular volume (MCV). Hemoglobin, also known as hemoglobin, is the main component of red blood cells and can bind to oxygen to transport oxygen and carbon dioxide. The clinical significance of increased or decreased hemoglobin is basically similar to that of red blood cell count, and hemoglobin can better reflect the degree of anemia. As shown in Table 4, after 28 days of drug intervention, the hemoglobin (HGB) values ​​of each group increased to varying degrees, and the HGB of groups E, F, and G (i.e., low-, medium-, and high-dose groups of donkey-hide gelatin bone strengthening oral liquid) increased from (91.0±6.98) in the model group to (127.8±7.33), (131.0±9.67), and (131.4±8.33), respectively, with increases of 40.4%, 43.95%, and 44.4%, respectively; the hematocrit (HCT) of the model group (26.05±1.64) increased from (32.78±1.60), (35.2±3.73), and (33.5±2.02), respectively, with increases of 25.8%, 35.12%, and 28.6%, respectively. The decrease in the mean hemoglobin content and mean volume of red blood cells is manifested in microcytic hypochromic anemia, which is mostly normocytic anemia, and is more common in clinical iron deficiency anemia. Normal or high hemoglobin content indicates that the body is rich in iron and no iron supplementation is needed. Low hemoglobin content indicates iron deficiency in the body, which in turn affects the synthesis and oxygen-carrying capacity of hemoglobin, causing anemia and hindering the normal progress of various physiological activities of the body. As shown in Table 4, during the administration period, the MCH values ​​and MCV values ​​of rats in all experimental groups were significantly greater than those in the model group B, and groups E, F, and G were close to normal group A. In summary, donkey-hide gelatin bone strengthening oral liquid has a significant promoting effect on the six indicators of blood routine. After 28 days of administration, there is a trend of gradually approaching the indicators of normal rats.

[0069] Table 1-4. Effects of donkey-hide gelatin bone strengthening oral liquid on blood routine tests (RBC, HGB, HCT, MCH, MCHC, MCV) in rats with iron deficiency anemia ( n=8)

[0070]

[0071] (1) Note: #Compared with the normal control group, P<0.05; *Compared with the model group, P<0.05, the same as the notes in the following table 3.5 Effects of donkey-hide gelatin bone strengthening oral liquid on serum iron and DMT-1 and FPN-1 proteins in rats

[0072] Two-thirds of the iron in the human body is present in the hemoglobin of red blood cells, and the remaining one-third is stored in the liver, spleen and bone marrow. All serum iron ions are bound to transferrin, which is the transport form of iron ions. The serum iron content of each group in this experiment is shown in Table 5. The serum iron content of E, F, and G groups of gavage for 28 days was significantly higher than that of model group B, P < 0.05; and it was linearly increasing with the dosage; and compared with the normal group A, there were significant differences in groups F and G, p < 0.05, indicating that Ejiao bone strengthening oral liquid can increase the serum iron content of normal rats. The iron in the diet is mainly absorbed into the blood through DMT1 and FPN1 expressed by duodenal epithelial cells. DMT1 is widely distributed in various tissues of the body, with the highest expression in the proximal small intestine, mainly distributed in the brush border of the duodenal villi, and transports intestinal non-heme iron into cells. FPN1 is a transmembrane iron export protein. In the small intestine, FPN1 is mainly distributed in the basal end and basement membrane of the duodenal villus epithelial cells, responsible for transporting iron in the intestinal cells out of the cells and into the blood. The results of the experimental study on the determination of DMT1 and FPN1 protein content in the duodenum of rats are shown in Table 5. In the determination of DMT1 protein, there were extremely significant differences between groups F and G compared with the normal group A and the model group B, P<0.001; indicating that the medium and high doses of donkey-hide gelatin strong bone oral liquid significantly increased the expression of DMT1 protein in the duodenum of rats with iron deficiency anemia. In the determination of FPN1 protein, there was a difference between group G and blank group A, P<0.05; there was an extremely significant difference compared with model group B, P<0.001. It indicates that the high-dose group of donkey-hide gelatin strong bone can significantly increase the expression of FPN1 protein in the duodenum of rats with iron deficiency anemia.

[0073] Table 1-5. Effect of donkey-hide gelatin bone strengthening oral liquid on serum iron in rats with iron deficiency anemia

[0074]

[0075] Note: #Compared with blank control group, P<0.05; *Compared with model group, P<0.05.

[0076] 4. Conclusion

[0077] The results of this study showed that the expression of DMT1 and FPN1 proteins in the duodenum of rats in the medium and high dose groups of donkey-hide gelatin for strengthening bones were higher than those in the normal control group and the model group. This suggests that donkey-hide gelatin for strengthening bones oral liquid can increase the degree of negative feedback regulation of intestinal iron absorption. This process may be related to the fact that donkey-hide gelatin for strengthening bones oral liquid can inhibit the level of hepcidin in the liver. It promotes the absorption of iron in the duodenum by upregulating DMT1 and FPN1.

[0078] In summary, after 28 days of administration, the donkey-hide gelatin bone strengthening oral liquid group can significantly increase the HGB, RBC, HCT, RDW, MCH, MCHC, and MCV of the model rats; increase the serum iron content of rats; and increase the expression of DMT-1 and FPN-1 proteins in the duodenum of rats. This shows that donkey-hide gelatin bone strengthening oral liquid can improve iron deficiency anemia, promote intestinal iron absorption by upregulating the negative feedback regulation mechanism of iron absorption in rats, improve the blood picture and iron metabolism of rats with iron deficiency anemia, increase hemoglobin, and promote oxygen supply to the body.

[0079] Example 2 Effect of donkey-hide gelatin bone strengthening oral liquid on improvement of renal anemia.

[0080] 1. Materials

[0081] 1.1 Animals

[0082] Clean-grade SD rats, weighing about 180-200 g, half male and half female, 100 rats. Provided by the Animal Experiment Center of Xinjiang Medical University, license number: SCXK(Xin)2018-0001.

[0083] 1.2 Reagents

[0084] Adenine (Jiangxi Alpha Hi-Tech Pharmaceutical Co., Ltd., 20200501, 1 kg / bag);

[0085] Recombinant human erythropoietin EPO injection (Baxter Oncology GmbH, 9J341A, specification 10,000 U / vial);

[0086] Donkey-hide gelatin bone strengthening oral solution (Huashidan Pharmaceutical Co., Ltd., 19021201, specification 10ml / tube);

[0087] Mouse SOD ELISA kit, catalog number JL22893, Shanghai Future Industrial Co., Ltd.;

[0088] Mouse GST ELISA kit, catalog number JL11340, Shanghai Future Industrial Co., Ltd.;

[0089] Mouse EPO ELISA kit, catalog number JL11719, Shanghai Future Industrial Co., Ltd.

[0090] 1.3 Instruments

[0091] Disposable syringes, gavage needles, electronic balance (1 / 10,000, FA1004, Shanghai Hengping Scientific Instrument Co., Ltd.), electronic balance (1 / 100, DT500, Changshu Jinyang Weight Instrument Co., Ltd.); microplate reader: Bio-Rad 680, Bio-Institute, USA.

[0092] 2. Methods

[0093] 2.1 Modeling method

[0094] After 80 rats (half male and half female) were adapted to the environment for 3 days, they were randomly divided into 20 normal control groups and 60 modeling groups (half male and half female) according to their body weight. The modeling group was given an aqueous solution of adenine by gavage at a dose of 20 mg / ml and 250 mg / kg.d, and the normal control group was given the same volume of purified water once a day by gavage for a total of 27 consecutive days. The body weight was weighed and recorded weekly. After modeling, 20 rats (10 normal group and 10 control group) were randomly selected from the inner canthus of the animals for blood routine analysis and determination of renal function indicators serum creatinine and blood urea nitrogen. The results showed that the characteristics met the requirements and the conditions for successful modeling were as follows: the blood routine indicators RBC count, Hb and Hct of the model group rats were significantly lower than those of the normal control group (p < 0.05), and the renal function indicators BUN and Scr content were significantly increased (p < 0.05).

[0095] 2.2 Experimental methods

[0096] 2.2.1 Animal grouping

[0097] Fifty rats with successful modeling were selected and randomly divided into five groups: model control group (same volume of purified water), EPO group (200U / kg injected once every 3 days), low-dose donkey-hide gelatin strong bone oral liquid group, medium-dose donkey-hide gelatin strong bone oral liquid group, and high-dose donkey-hide gelatin strong bone oral liquid group. The drug was administered continuously for 21 days, and the body weight was weighed and recorded every week, and the general signs of the animals were observed. Animal treatment and sample collection. Specific grouping is shown in Table 1.

[0098] Table 2-1 Treatment methods and grouping of BALB / c mice with donkey-hide gelatin bone strengthening oral liquid

[0099]

[0100] All mice were divided into 6 groups: blank control group, model control group, EPO group (200U / kg), high-dose donkey-hide gelatin bone-strengthening oral liquid group (12.8ml / kg), medium-dose donkey-hide gelatin bone-strengthening oral liquid group (6.4ml / kg), and low-dose donkey-hide gelatin bone-strengthening oral liquid group (3.2ml / kg). After grouping, the drugs were continuously administered for 21 days, the body weight was weighed and recorded every day, and the general signs of the animals were observed.

[0101] 2.2.2 Animal handling and sample collection

[0102] After 41 days of gavage administration, the animals in each group were fasted for 12 hours, and their weights were weighed and recorded. Blood was collected from the inner canthus of the eye, and 1 ml of blood was placed in a tube containing an anticoagulant. The anticoagulated blood was used for routine blood analysis. About 2 ml of blood was placed in a clean EP tube, kept warm in a 37-degree water bath for 1 hour, and then centrifuged at 3500 r / min for 20 minutes to extract serum for other index detection.

[0103] After the animals were killed by cervical dislocation, the kidneys on both sides were quickly peeled off, their fascia were removed, and they were rinsed twice in saline. One kidney was fixed in 4% formaldehyde solution for making pathological sections and subsequent staining; the other kidney was quickly frozen in liquid nitrogen and stored in a refrigerator for subsequent testing.

[0104] Bone marrow nucleated cell sample collection: Aseptically take the left femur of the rat for the preparation of bone marrow image, drop 15ul of blood on one end of the slide, hold the slide with the left hand, take the long side of the pusher with the right hand and touch it with the top of the slide and pull it from the front of the blood drop to the back, so that the blood drop forms a line, and then push the pusher steadily at an angle of 45 degrees to form a blood film. After the blood smear is made, blow it dry next to the fan to make the blood film dry quickly to prevent the blood cells from shrinking. After drying, add 2-3 drops of Wright's stain to the blood film to cover the entire blood film. After 1 minute, add an equal amount of purified water to the stain and mix for 5-10 minutes. Finally, wash off the stain, dry, and examine under a microscope.

[0105] 2.3 Index detection

[0106] Renal function indicators: blood urea nitrogen (BUN), blood creatinine (Scr) levels; serum erythropoietin (EPO), superoxide dismutase (SOD), and glutathione transferase (GSH) levels.

[0107] Anemia indicators: hemoglobin (Hb) content, peripheral red blood cells (RBC), platelet count (PLT), mean corpuscular volume (MCV), mean corpuscular hemoglobin content (MCH), and bone marrow nucleated cell count.

[0108] Immune indicators: white blood cell count (WBC), neutrophil count (NEUT), lymphocyte count (LY), monocyte count (MONO) and their total proportion.

[0109] 3. Experimental results

[0110] 3.1 Modeling

[0111] After modeling, the fur color of the animals in the modeling group was dark, and the body weight was significantly lower than that of the normal group (p < 0.01). A few animals died. Among the 60 modeling rats, 8 died. 27 days after modeling, 10 rats in the normal group and 10 rats in the modeling group were randomly selected for testing. The results showed that the hemoglobin and red blood cells in the blood of the animals in the modeling group decreased significantly, and the urea nitrogen and creatinine test values ​​increased significantly. This shows that the modeling was successful. 52 models were established, with a mortality rate of 13.3%. See Table 2 and Table 3 for specific weight changes.

[0112] Table 2-2 Body weight changes of model animals (n=10)

[0113]

[0114] Note: "**" compared with the normal group p < 0.01

[0115] Table 2-3 Effects of modeling on animal blood indicators (n=10)

[0116]

[0117] Note: "**" compared with the normal group, p < 0.01.

[0118] 3.2 Changes in animal body weight after administration

[0119] After 30 days of administration, the body weight of the model group animals was significantly lower than that of the normal group, indicating that the body weight of the model animals had not recovered. The body weight of each drug group was significantly lower than that of the normal group, indicating that the body weight of the animals had not recovered, but compared with the model group, the body weight increased slightly, indicating that there was a certain recovery effect on the body weight of the model animals. See Table 2-4 and Appendix for details. Figure 2-1 .

[0120] Table 2-4 Animal body weight changes after administration (x±SD)

[0121]

[0122] Note: “*” p<0.5 compared with the normal group, “**” p<0.01 compared with the normal group; “#” p<0.5 compared with the model group, “##” p<0.01 compared with the model group.

[0123] 3.3 Effects on renal function

[0124] 3.3.1 Effects on serum urea nitrogen and creatinine

[0125] The experimental results showed that 41 days after the drug treatment, the urea nitrogen and creatinine of the model group rats were still significantly higher than those of the normal group (p < 0.01), indicating that their kidney damage was irreversible; and compared with the model group, the urea nitrogen and creatinine content of the rats in the high-dose group of donkey-hide gelatin for strengthening bones was significantly reduced, indicating that it has a restorative effect on kidney damage. Specific data are shown in Table 2-5.

[0126] Table 2-5 Effects on urea nitrogen and creatinine

[0127]

[0128] 'Note: "**" compared with the normal group P < 0.01, "*" compared with the normal group P < 0.05, "##" compared with the model group P < 0.01, "#" compared with the model group P < 0.05.

[0129] 3.3.2 Effects on superoxide dismutase (SOD) and glutathione transferase (GST)

[0130] The degree of consumption of superoxide dismutase SOD and glutathione transferase GST is directly correlated with the degree of renal damage, and they will decrease sharply when the clinical manifestation is acute renal damage. The SOD test results showed that the SOD value of the model group was significantly lower than that of the normal group, indicating that the animal model had severe renal damage, while the medium dose and optimized group of donkey-hide gelatin bone strengthening oral liquid could significantly increase the SOD value of rats; the GST test results showed that the GST value of the model group was significantly lower than that of the normal group, indicating that the animal model had severe renal damage, while the medium dose and optimized group of donkey-hide gelatin bone strengthening oral liquid could significantly increase the GST value of rats. The results showed that donkey-hide gelatin bone strengthening oral liquid had a significant restorative effect on the reduction of superoxide dismutase SOD and glutathione transferase GST caused by renal damage, indicating that it has a good renal damage recovery effect. See Table 2-6, Table 2-7, for details. Figure 2-2 , Figure 2-3 .

[0131] Table 2-6 Effects on serum superoxide dismutase (SOD) in rats (n=5)

[0132]

[0133] 'Note: "*" compared with the normal group P < 0.05, "#" compared with the model group P < 0.05, "##" compared with the model group P < 0.01.

[0134] Table 2-7 Effects on rat serum glutathione transferase GST (n=5)

[0135]

[0136] 'Note: "*" compared with the normal group P < 0.05, "##" compared with the model group P < 0.01.

[0137] 3.3.3 Effect on erythropoietin (EPO)

[0138] Erythropoietin (EPO) is a hormone-like substance secreted by the kidneys and liver that can promote the production of red blood cells. Taking erythropoietin can increase the blood flow ratio of patients with renal anemia (that is, increase the percentage of red blood cells in the blood). It is used for anemia, tissue separation, premature infants, oncology and hematology. The results of this experiment showed that the EPO value of the model group was significantly lower than that of the normal group, indicating that the animal model had severe kidney damage and renal anemia, while the donkey-hide gelatin strong bone oral liquid group was able to significantly increase the EPO value of rats, indicating that donkey-hide gelatin strong bone oral liquid has a certain recovery effect on renal damage anemia. See details. Figure 2-4 , Table 8.

[0139] Table 2-8 Effects on serum erythropoietin (EPO) in rats (n=5)

[0140]

[0141] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0142] 3.3.4 Effect on rat kidney coefficient

[0143] Anatomical observation showed that the kidneys of the rats in the model group and the drug-treated group were significantly larger in size than those in the normal group, and there were large areas of necrotic hemorrhagic white spots. The renal coefficient of the rats in the model group was significantly higher than that in the normal group (P < 0.01). The renal coefficients of the donkey-hide gelatin bone strengthening optimization group and the EPO group were significantly lower than those in the model group. The morphological results showed that the renal morphology of the high-dose donkey-hide gelatin bone strengthening oral liquid group had recovered, and a large number of white spots disappeared. The results showed that donkey-hide gelatin bone strengthening oral liquid had a significant restorative effect on renal injury. See Table 9 for specific data.

[0144] Table 2-9 Effects on rat kidney coefficient

[0145]

[0146] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0147] 3.4 Effects on anemia indexes in rats with renal anemia

[0148] The experimental results show that high doses of donkey-hide gelatin for strengthening bones oral liquid can significantly restore the number of red blood cells and hemoglobin content in rats with renal injury, and can significantly increase the number of bone marrow cells. This shows that donkey-hide gelatin for strengthening bones oral liquid has a certain restorative effect on anemia caused by renal injury. See Table 10 for specific data.

[0149] Table 2-10 Effects on anemia indicators in rats

[0150]

[0151] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0152] 3.5 Effects on immune parameters in rats with renal anemia

[0153] The experimental results show that donkey-hide gelatin bone strengthening oral liquid can increase the number of white blood cells in rats, and the high-dose group can increase the number of lymphocytes. This shows that donkey-hide gelatin bone strengthening oral liquid has a certain regulatory effect on the immune indicators of rats with renal anemia. Specific data are shown in Table 11.

[0154] Table 2-11 Effects on rat immune indicators

[0155]

[0156]

[0157] 'Note: "**" compared with the normal group P < 0.01, "*" compared with the normal group P < 0.05, "##" compared with the model group P < 0.01, "#" compared with the model group P < 0.05.

[0158] 4 Summary

[0159] The modeling drug adenine can cause renal fibrosis, insufficient EPO secretion, insufficient secretion of erythroid and megakaryocyte cells, and renal failure anemia. After the modeling of this experiment, the rats' urea nitrogen, creatinine, body weight, and red blood cell count changed significantly, indicating that the experimental modeling was successful. After 6 weeks of administration, it was found that donkey-hide gelatin strong bone oral liquid had a significant regulatory effect on the kidney functional indicators urea nitrogen and creatinine, indicating that it can restore the kidney damage caused by adenine in rats, and has a significant restorative effect on the anemia indicators caused by it. The test kit found that the renal anemia-related indicators SOD, GST and EPO in the model group were significantly reduced, which was in line with the clinical indications of renal anemia, and the same dose of donkey-hide gelatin strong bone oral liquid in the middle dose group had a significant improvement effect on these indicators, further confirming that donkey-hide gelatin strong bone oral liquid has a significant restorative effect on renal anemia.

[0160] Example 3 Improvement of bone marrow suppression anemia by donkey-hide gelatin bone strengthening oral liquid.

[0161] 1. Materials

[0162] 1.1 Animals

[0163] BALB / c mice, weighing about 18-20 g, half male and half female, 100 mice. Provided by the Animal Experiment Center of Xinjiang Medical University, license number: SCXK(Xin)2018-0001.

[0164] 1.2 Reagents

[0165] Chloramphenicol tablets (Chongqing Dikang Changjiang Pharmaceutical Co., Ltd., 200202, 0.25g / tablet)

[0166] Cyclophosphamide for injection (Baxter Oncology GmbH, 9J341A, specification 0.25 g / vial)

[0167] Recombinant human colony stimulating factor injection (Shuanglu Pharmaceutical, 20191010, specification 150ug / vial)

[0168] Donkey-hide gelatin bone strengthening oral liquid (Huashidan Pharmaceutical Co., Ltd., 19021201, specification 10ml / tube)

[0169] Compound donkey-hide gelatin syrup group (Dong'e donkey-hide gelatin, 1905026, 10mL / vial)

[0170] Mouse interleukin-6 ELISA kit, catalog number JL20268, Shanghai Future Industrial Co., Ltd.;

[0171] Mouse macrophage colony stimulating factor ELISA kit, catalog number JL11340, Shanghai Future Industrial Co., Ltd.;

[0172] 1.3 Instruments

[0173] Disposable syringes, gavage needles, electronic balance (1 / 10,000, FA1004, Shanghai Hengping Scientific Instrument Co., Ltd.), electronic balance (1 / 100, DT500, Changshu Jinyang Weight Instrument Co., Ltd.); microplate reader: Bio-Rad 680, Bio-Institute, USA.

[0174] 2. Methods

[0175] 2.1 Solution preparation

[0176] Chloramphenicol tablets: Dilute the chloramphenicol tablets with propylene glycol to a final concentration of 8 mg / ml, filter through a 0.22 um filter membrane for sterilization, and store for later use.

[0177] Cyclophosphamide injection: Dissolve cyclophosphamide with normal saline for injection to a final concentration of 10 mg / ml and set aside.

[0178] 2.2 Model making

[0179] After 100 normal mice (half male and half female) were adapted to the environment for 3 days, they were randomly divided into a blank control group (14 mice) and a modeling group (half male and half female) according to their body weight. The mice in the modeling group were intraperitoneally injected with cyclophosphamide 50 mg / kg.d and chloramphenicol 400 mg / kg.d every other day for 4 times.

[0180] 2.3 Animal grouping and drug administration

[0181] 60 mice with successful modeling were randomly divided into 6 groups, with 10 mice in each group. The specific groups are shown in Table 1.

[0182] Table 3-1 Treatment methods and grouping of BALB / c mice with donkey-hide gelatin bone strengthening oral liquid

[0183]

[0184] All mice were divided into 7 groups: blank control group, model control group, recombinant human colony stimulating factor group (0.125ml / kg), high-dose donkey-hide gelatin bone strengthening oral liquid group (16ml / kg), medium-dose donkey-hide gelatin bone strengthening oral liquid group (8ml / kg), low-dose donkey-hide gelatin bone strengthening oral liquid group (4ml / kg), and compound donkey-hide gelatin slurry group (9ml / kg). After grouping, the drugs were continuously administered for 10 days, the body weight was weighed and recorded every day, and the general signs of the animals were observed.

[0185] 2.4 Animal handling and sample collection

[0186] After 10 days of oral administration, the animals in each group were fasted for 2 hours, and their body weights were weighed and recorded. Blood was collected from the inner canthus, and 0.5 ml of blood was placed in a tube containing an anticoagulant. The anticoagulated blood was used for routine blood analysis. About 1.5 ml of blood was collected from 2 animals in each group and placed in a clean EP tube. The tube was placed in a refrigerator at 4 degrees overnight, and the serum was centrifuged at 3500 r / min for 15 minutes to absorb the serum. The serum was tested for GM-GSF and IL-6 using the ELISA method.

[0187] (1) Internal organs

[0188] After the mice were killed by cervical dislocation, their thymus and spleen were quickly removed, rinsed with physiological saline, dried with filter paper, and weighed and recorded.

[0189] (2) Preparation of bone marrow cells

[0190] After the animals were killed, the left femur was quickly separated, and its two ends were cut with scissors in a sterile clean bench. The bone marrow cells were flushed out with PBS and repeatedly blown. The bone marrow nucleated cells were extracted according to the instructions of the mouse lymphocyte separation solution for bone marrow nucleated cell counting.

[0191] (3) Index detection

[0192] Routine blood tests; thymus and spleen coefficients; bone marrow nucleated cell counts; serum levels of hematopoiesis-related cytokines GM-GSF and IL-6.

[0193] 3 Experimental Results

[0194] 3.1 Modeling results

[0195] In this experiment, 90 normal mice were selected, 14 in the blank control group and 76 in the modeling group (half male and half female). The modeling was established by 4 intraperitoneal injections of cyclophosphamide 50 mg / kg.d and chloramphenicol 40 mg / kg.d. As a result, the modeling rate was 82.89%, 13 mice died during the period, and the mortality rate was 17.1%.

[0196] 3.2 Effect on body weight

[0197] After modeling, the modeled mice showed significant weight loss, rough fur color, and reduced activity. After administration, the weight slowly increased. See Table 2 for details of weight changes. The weight of the model group mice decreased significantly 8 days after modeling, indicating that the modeling was successful. After 10 days of administration, the weight recovery of the low-dose group of donkey-hide gelatin bone strengthening oral liquid was the highest.

[0198] Table 3-2 Effect on mouse body weight (x±SD)

[0199]

[0200] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0201] 3.3 Effects on mouse organ coefficients

[0202] After 10 days of administration, the positive drug colony stimulating factor G-CSF significantly increased the spleen coefficient compared with the model group. Compared with the model group, the thymus coefficient of the donkey-hide gelatin bone strengthening optimization group and the compound donkey-hide gelatin syrup group increased significantly. The data are shown in Table 3.

[0203] Table 3-3 Effects on mouse organ coefficients

[0204]

[0205] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0206] 3.4 Effects on mouse hematology

[0207] The results showed that the leukocyte count in the model group was lower than that in the normal group, while the positive drug and donkey-hide gelatin bone strengthening group both had a significant effect on increasing the number of leukocytes, and the positive drug colony stimulating factor had the strongest effect on increasing leukocytes; in terms of lymphocyte percentage, the lymphocyte percentage in the positive drug colony stimulating factor group was significantly reduced, indicating that its effect mainly increased neutrophils, eosinophils and basophils among leukocytes, and did not have an effect on lymphocyte B, T and NK cells, resulting in a decrease in lymphocyte percentage, while the donkey-hide gelatin bone strengthening oral liquid group had an effect on increasing lymphocyte percentage compared with colony stimulating factors, and its target was B, T and NK cells; in terms of red blood cells, hemoglobin and average hemoglobin content, the donkey-hide gelatin bone strengthening oral liquid group had a significant recovery effect on anemia caused by bone marrow suppression, especially in terms of hemoglobin content, its effect was higher than that of positive drug colony stimulating factors and compound donkey-hide gelatin slurry. The specific data are shown in Table 4.

[0208] Table 3-4 Effects on mouse hematology

[0209]

[0210] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0211] 3.5 Effect on macrophage colony stimulating factor (M-CSF)

[0212] Colony stimulating factor mainly acts selectively on granulocyte hematopoietic progenitor cells, promoting their proliferation and differentiation, and can increase the function of terminally differentiated granulocyte cells, increasing the number of granulocytes. The experimental results show that the high-dose group of donkey-hide gelatin strong bone oral liquid has a significant effect on increasing macrophage colony stimulating factor, indicating that it can promote the generation of macrophages in mice.

[0213] Table 3-5 Effects of M-CSF on myelosuppression mouse model

[0214] Group M-CSF content (pg / ml) Normal group 195.92±12.34 Model Group 194.75±9.03 Positive control group 210.35±10.19 Low-dose donkey-hide gelatin bone strengthening group 207.49±15.32 Middle dose group of donkey-hide gelatin for bone strengthening 211.64±7.62 High-dose donkey-hide gelatin bone strengthening group 217.12±10.76*# Compound donkey-hide gelatin group 208.92±19.09

[0215] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0216] 3.6 Effect on interleukin-6 (IL-6)

[0217] Interleukin 6 is a cytokine, a protein produced by immune cells acting on other cells, which helps regulate and promote immune responses. The experimental results show that the high-dose group of donkey-hide gelatin oral liquid and the compound donkey-hide gelatin syrup group have the effect of promoting IL-6 secretion, but the effect is not significant.

[0218] Table 3-6 Effects of IL-6 on myelosuppression mouse model

[0219] Group IL-6 content (mg / ml) Normal group 18.34±3.42 Model Group 15.53±2.03 Positive control group 16.66±1.19 Low-dose donkey-hide gelatin bone strengthening group 15.83±1.31 Middle dose group of donkey-hide gelatin for bone strengthening 16.72±0.96 High-dose donkey-hide gelatin bone strengthening group 17.02±1.37 Compound donkey-hide gelatin group 16.21±1.93

[0220] 3.7 Effect on the number of mouse bone marrow cells

[0221] The results showed that the medium and high dose groups of donkey-hide gelatin bone strengthening oral liquid could significantly increase the number of nucleated cells in the femoral bone marrow of bone marrow-suppressed mice, indicating that donkey-hide gelatin bone strengthening oral liquid has the effect of promoting the recovery of anemia.

[0222] Table 3-7 Effects on bone marrow nucleated cell counts in myelosuppression mouse model

[0223] Group <![CDATA[The number of nucleated bone marrow cells (10 6 )]]> Normal group 3.18±1.30 Model Group 3.14±1.49 Positive control group 3.60±1.08 Low-dose donkey-hide gelatin bone strengthening group 5.57±5.41 Middle dose group of donkey-hide gelatin for bone strengthening 4.15±1.92 High-dose donkey-hide gelatin bone strengthening group 7.53±4.17* Compound donkey-hide gelatin group 8.38±7.79**

[0224] Note: “**” compared with the normal group, P<0.01; “*” compared with the normal group, P<0.05; “##” compared with the model group, P<0.01; “#” compared with the model group, P<0.05.

[0225] 4. Conclusion

[0226] The immunosuppressive mouse model is formed by the bone marrow suppression effect of cyclophosphamide and chloramphenicol. This animal model is simple to make, but it also has the disadvantage of fast recovery of drug suppression. After modeling in this experiment, the weight of the animals in the model group decreased significantly, and symptoms such as death and mental depression appeared, indicating that the modeling was successful. After 10 days of administration, the weight and state of the mice in the model group recovered, but the degree of recovery was lower than that of the drug group. By comparing the hematological indicators, it was found that donkey-hide gelatin strong bone oral liquid can restore the percentage of white blood cells and lymphocytes in the model mice. Compared with the positive drug colony stimulating factor, donkey-hide gelatin strong bone oral liquid has a more targeted effect on the proliferation of lymphocytes T, B and NK cells; at the same time, the experiment found that donkey-hide gelatin strong bone oral liquid can have a significant recovery effect on anemia caused by bone marrow suppression, especially in terms of hemoglobin content, and its effect is higher than that of positive drug colony stimulating factor and compound donkey-hide gelatin slurry. Combined with the results of macrophage colony stimulating factor and interleukin 6 in mouse serum and bone marrow cell count, it was shown that donkey-hide gelatin bone strengthening oral liquid can enhance the immune ability of immunosuppressed mice. One of its mechanisms of action is to promote the differentiation of myeloid progenitor cells into lymphocytes, especially the proliferation of lymph B cells and macrophages. At the same time, it can increase the production of hemoglobin and improve the body's energy metabolism level.

Claims

1. Application of donkey-hide gelatin bone strengthening oral liquid in the preparation of medicines for preventing and / or treating renal anemia.

2. Application of donkey-hide gelatin bone strengthening oral liquid in the preparation of drugs for improving indicators related to renal anemia.

3. The use according to claim 2, characterized in that The renal anemia related indexes are selected from SOD, GST and EPO.

4. Application of donkey-hide gelatin bone strengthening oral liquid in the preparation of drugs for improving immune indicators of renal anemia.

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