Traditional Chinese medicine composition for treating aplastic anemia and application thereof

Abstract

The invention discloses a traditional Chinese medicine composition for treating aplastic anemia and application thereof, and belongs to the technical field of traditional Chinese medicines. The traditional Chinese medicine composition comprises active substances, and the active substances are prepared from, by weight, 6-12 parts of semen cuscutae, 6-12 parts of fructus psoraleae, 6-12 parts of fructus ligustri lucidi, 10-14 parts of radix rehmanniae recen, 4-12 parts of radix scutellariae, 4-12 parts of cortex phellodendri, 10-20 parts of poria cocos, 10-30 parts of radix codonopsis, 6-12 parts of rhizoma atractylodis macrocephalae stir-fried with bran and 2-10 parts of radix glycyrrhizae preparata. The traditional Chinese medicine composition is low in cost, high in AA treatment effect and free of toxic and side effects, and has the effects of tonifying kidney, replenishing essence, detoxifying, descending turbidity and the like; the therapeutic effect on AA can be achieved by adjusting glycolysis of macrophages.

Description

Technical Field

[0001] The present invention belongs to the technical field of traditional Chinese medicine, and in particular relates to a traditional Chinese medicine composition for treating aplastic anemia and application thereof. Background Art

[0002] Aplastic anemia (AA) is a bone marrow failure disease characterized by pancytopenia. It can occur in people of all ages, with an overall incidence of approximately 0.74 / 100,000 in my country. AA can be divided into severe (SAA) and non-severe (NSAA) according to severity. The clinical symptoms of AA are mainly anemia, infection, and bleeding caused by decreased hemoglobin (HB), neutrophils (Neutrophil, Neu), and platelets (PLT). Severe cases may develop sepsis and visceral hemorrhage. AA is a complex disease with a long-lasting course that is difficult to cure. It not only seriously affects the patient's survival, but also severely reduces the patient's quality of life, placing a heavy economic burden on the patient's family and society.

[0003] Abnormal activation of T cells, which induces apoptosis of hematopoietic stem and progenitor cells (HSPCs) through the secretion of inflammatory factors and cytotoxicity, is the direct cause of AA. Recent studies have shown that macrophages play a key role in the activation of AAT cells. Bone marrow macrophages in AA patients are often polarized toward the M1 polarization, and the secretion of TNFα induces AAT cell activation and IFNγ secretion. M1 macrophages are a pro-inflammatory phenotype. Glycolysis is the primary energy source for M1 macrophages and is also a key driver of M1 macrophage polarization.

[0004] Antithymocyte globulin (ATG / ALG) + cyclosporine A (CsA) combined with androgens and other hematopoietic drugs is currently the first-line treatment for severe aplastic anemia (SAA). Approximately 70% of patients achieve hematologic remission after six months of standard first-line treatment, but 30% to 40% of these patients relapse. Non-severe aplastic anemia (NSAA) can be treated with CsA combined with hematopoietic drugs. After six months of treatment with this regimen for NSAA, approximately 70% of patients achieve hematologic remission, but long-term follow-up reveals that only 20% to 30% achieve complete remission. Therefore, the primary therapeutic mechanisms of existing Western medicines are inhibition of T cell function (ATG / ALG, CsA) and promotion of hematopoietic cell proliferation and differentiation (androgens).

[0005] Currently, Traditional Chinese Medicine treatments primarily focus on formulas that replenish essence and nourish the marrow. These mechanisms primarily promote the proliferation and differentiation of hematopoietic cells, with limited involvement of metabolic and immune regulation. The efficacy of existing treatment options still has room for improvement.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] In view of the deficiencies in the prior art, the first object of the present invention is to provide a traditional Chinese medicine composition for treating aplastic anemia. The present invention names this traditional Chinese medicine composition Huangzi Shengxue Fang.

[0008] The second object of the present invention is to provide an application of a traditional Chinese medicine composition for treating aplastic anemia, namely, the application of Huang Zi Sheng Xue Fang.

[0009] In a first aspect, the present invention provides a traditional Chinese medicine composition for treating aplastic anemia, the traditional Chinese medicine composition comprising an active substance, which is prepared from the following raw materials in parts by weight: 6-12 parts of Cuscuta seeds, 6-12 parts of Psoralea corylifolia, 6-12 parts of Ligustrum lucidum fruits, 10-14 parts of Rehmannia root, 4-12 parts of Scutellaria baicalensis, 4-12 parts of Phellodendron amurense, 10-20 parts of Poria cocos, 10-30 parts of Codonopsis pilosula, 6-12 parts of Atractylodes macrocephala with bran flour, and 2-10 parts of roasted Licorice root.

[0010] Furthermore, the active substance is prepared from the following raw materials in parts by weight: 9-12 parts of Cuscuta seeds, 9-12 parts of Psoralea corylifolia, 9-12 parts of Ligustrum lucidum fruits, 9-12 parts of Rehmannia root, 9-12 parts of Scutellaria baicalensis, 9-12 parts of Phellodendron chinense, 15-20 parts of Poria cocos, 10-20 parts of Codonopsis pilosula, 9-12 parts of Atractylodes macrocephala with bran, and 9-12 parts of roasted Licorice root.

[0011] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 10-20 parts of Codonopsis pilosula, 10 parts of stir-fried Atractylodes macrocephala, and 10 parts of roasted Licorice root.

[0012] Furthermore, the active substance is prepared from the following raw materials in parts by weight: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 10 parts of Codonopsis pilosula, 10 parts of stir-fried Atractylodes macrocephala, and 10 parts of roasted Licorice root;

[0013] Alternatively, the active substance is prepared from the following raw materials in parts by weight: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 20 parts of Codonopsis pilosula, 10 parts of stir-fried Atractylodes macrocephala, and 10 parts of roasted Licorice root.

[0014] Furthermore, the dosage form of the Chinese medicine composition is selected from any one of extracts, granules, pills, capsules and oral liquids.

[0015] Furthermore, the traditional Chinese medicine composition further comprises excipients, which include at least one of a filler, a binder, a disintegrant, a lubricant, an absorbent and a diluent.

[0016] In a second aspect, the present invention provides use of the traditional Chinese medicine composition in the preparation of a medicament for preventing and / or treating aplastic anemia.

[0017] Furthermore, the aplastic anemia includes severe aplastic anemia and non-severe aplastic anemia.

[0018] Furthermore, the drug exerts its efficacy through at least one of the following effects:

[0019] (1) Increased platelet and / or hemoglobin levels;

[0020] (2) reduce the myelodysplasia symptom score in the Guidelines for the Diagnosis and Treatment of Aplastic Anemia with Integrated Traditional Chinese and Western Medicine;

[0021] (3) inhibiting monocyte and / or macrophage glycolysis;

[0022] (4) Inhibit macrophage M1 polarization.

[0023] Furthermore, the main active ingredients of the drug that inhibit glycolysis in monocytes and / or macrophages include diterpene glycosides, 4'-O-methylglycyrrhizin, 7-acetoxy-2-methylisoflavone, 4',7-dimethoxyisoflavone, 7-hydroxy-2methyl-3-phenylpropiochromone, 7-methoxy-2-methylisoflavone, schizosporin and flavonoids;

[0024] And / or, the drug inhibits glycolysis in monocytes and / or macrophages mainly by regulating the high expression of key glycolysis enzymes, wherein the key glycolysis enzymes include HK1, PGK1 and PKM.

[0025] In the Huangzi Shengxue Recipe, a traditional Chinese medicine composition for treating aplastic anemia, Cuscuta chinensis balances yin and yang, Ligustrum lucidum nourishes kidney yin, and Psoralea corylifolia warms kidney yang, collectively tonifying essence and promoting marrow production, serving as the monarch drug. Radix Rehmanniae nourishes yin and cools blood, Scutellaria baicalensis "can reduce internal heat and promote downward movement," and Phellodendron amurense "can relieve heat and strengthen yin." These three herbs, when used together, serve as the assistant drug, clearing exogenous pathogenic toxins and suppressing internal heat, preventing heat toxicity from damaging the bone marrow and worsening deficiency. Codonopsis pilosula replenishes qi and strengthens the spleen, while Poria cocos not only strengthens the spleen and replenishes qi but also promotes dampness-removing effects. Roasted Atractylodes macrocephala dispels dampness and reduces turbidity, and its spleen-tonifying and qi-invigorating effects are even stronger than those of raw Atractylodes macrocephala. The combination of these three herbs can eliminate heat and serve as the adjuvant. Roasted Licorice root, which has a stronger effect than raw licorice in tonifying the middle and replenishing qi, serves as the guiding drug, harmonizing the other herbs. In summary, the Huangzi Shengxue Recipe can replenish the kidney and essence, detoxify and reduce turbidity, exerting a therapeutic effect on AA. What is special is that Huang Zi Shengxue Fang emphasizes both cold and heat when tonifying the kidney and replenishing essence, and replenishing the deficiency of kidney yin and yang. It follows the treatment principle of "less fire generates qi" in "The Yellow Emperor's Classic of Internal Medicine" and promotes blood metabolism with a small amount of warm medicine. At the same time, this prescription uses both attack and supplement, giving equal importance to drugs that tonify the kidney and replenish essence and drugs that clear heat and dry dampness, strengthen the spleen and replenish qi. On the one hand, it can clear away external invading evil heat and internally generated phlegm and turbidity, avoid the adhesion of phlegm and heat, generate turbidity and toxins and inhibit blood metabolism. On the other hand, it can also avoid excessive use of drugs that replenish essence and benefit marrow and block yang qi, causing yang qi to accumulate and turn into fire, resulting in the disadvantage of "strong fire and scattered qi".

[0026] The Huangzi Shengxue Recipe of the present invention has been found to have at least the following effects in clinical applications: 1. increasing the levels of PLT and HB; 2. reducing the myelopathy symptom score in the "Guidelines for the Diagnosis and Treatment of Aplastic Anemia with Integrated Traditional Chinese and Western Medicine".

[0027] The present invention also conducted network pharmacology and single-cell sequencing analysis, wherein the network pharmacology analysis and single-cell sequencing results showed that Huang Zi Sheng Xue Fang may exert its therapeutic effect on AA by inhibiting glycolysis of macrophages of AA patients to inhibit their M1 polarization.

[0028] Therefore, the beneficial effects of the present invention include: the Chinese medicine composition of the present invention has low cost, high efficacy in treating AA, no toxic side effects, and has the effects of tonifying the kidney and replenishing essence, detoxifying and reducing turbidity; it can exert its therapeutic effect on AA by regulating macrophage glycolysis, and its therapeutic mechanism is innovative; clinical trials have also demonstrated that the Chinese medicine composition has good efficacy and high safety in treating AA, and is worthy of clinical application and promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 The figures show the peripheral blood cell count levels and symptom score of AA patients after 3 and 6 months of treatment with the Huangzi Shengxue Recipe in Example 1 of the present invention (A: PLT level; B: HB level; C: Neu level; D: symptom score level), where * indicates P < 0.05 compared with before treatment, and ** indicates P < 0.01 compared with before treatment.

[0031] Figure 2 These are the routine blood test results of male patient 1 during six months of treatment.

[0032] Figure 3 These are the routine blood test results of male patient 2 during six months of treatment.

[0033] Figure 4 These are the routine blood test results of female patient 1 during six months of treatment.

[0034] Figure 5 The core targets of Huang Zi Sheng Xue Fang for treating AA in Example 1 (Figure A is a Venn diagram of Huang Zi Sheng Xue Fang targets, AA disease targets and metabolic pathway targets. In the figure, the green set is AA disease (aplastic anemia)-related targets, the red is Huang Zi Sheng Xue Fang (HZSXF) regulation targets, and the blue is metabolic pathway (metabolism) targets; Figure B is a protein interaction network diagram of the metabolic targets of Huang Zi Sheng Xue Fang for treating AA. Each node in the figure represents a target, and there are links between the nodes, indicating that there is an interaction relationship between the two; Figure C is the key metabolic targets of Huang Zi Sheng Xue Fang for treating AA).

[0035] Figure 6Figure 3 shows the results of single-cell sequencing analysis of the bone marrow of AA patients (Figure A shows the clustering results of bone marrow cells of AA patients. As shown in the legend, different colors represent different cell groups; Figure B shows the marker expression dot plot of bone marrow cell clustering of AA patients. The horizontal axis represents cell grouping and the vertical axis represents cell surface markers. The dots in the figure represent the expression of surface markers in each group of cells. The size of the dots represents the expression ratio of the corresponding surface marker in each group of cells. The color of the dots represents the average expression level of the corresponding surface marker in each group of cells; Figure C shows the annotation results of bone marrow cell clustering of AA patients. In the legend, BC represents B lymphocytes, CD4T represents CD4+ T lymphocytes, CD8T represents CD8+ T lymphocytes, HSC / MPP represents hematopoietic stem / progenitor cells, Mon represents monocytes / macrophages, NK represents natural killer cells, PC represents plasma cells, preRBC represents immature red blood cells, and preT represents immature T cells; Figure D shows the distribution of core metabolic targets in the bone marrow of AA patients. The darker the color, the higher the relative quantitative expression of the gene in that type of cell).

[0036] Figure 7 The metabolic pathway of AA treated by Huang Zi Sheng Xue Fang in Example 1 (the bubble color in the figure indicates -log 10 (pvalue), the redder the bubble color, the -log 10 The larger the pvalue, the greater the number of core target genes in the metabolic pathway. The larger the bubble, the more genes there are. Enrichment fold is the enrichment fold.

[0037] Figure 8 This is the drug-active ingredient-core target network diagram of Huangzi Shengxue Recipe in Example 1 (the red triangles in the figure represent traditional Chinese medicine, the blue squares represent the medicinal ingredients in the traditional Chinese medicine, and the green circles represent the regulated targets). DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. It should be understood by those skilled in the art that the embodiments are merely to help understand the present invention and should not be regarded as specific limitations of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The process parameters for which specific conditions are not specified in the following examples are generally in accordance with conventional conditions.

[0039] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to form one or more new numerical ranges, and these numerical ranges should be considered to be specifically disclosed in the present invention.

[0040] The present invention will be further described in detail below with reference to specific examples and comparative examples.

[0041] All the Chinese medicinal raw materials used in the present invention are in line with the description of the corresponding medicinal materials in the 2020 edition of the "Chinese Pharmacopoeia", especially the medicinal parts and the content of the effective ingredients. The raw materials used in the following examples are their corresponding medicinal material slices, which are all obtained by the preparation / processing methods in the 2020 edition of the "Chinese Pharmacopoeia". For example: Raw Rehmannia glutinosa is to slowly bake Rehmannia glutinosa to about 80% dry; Fried Atractylodes macrocephala with bran is to take bran, sprinkle it into a hot pot, and when it smokes, add Atractylodes macrocephala slices, stir quickly, and stir-fry over low heat until the surface is yellow-brown. When the aroma overflows, take it out, sieve out the bran, and let it cool; Roasted Licorice Root, take refined honey and dilute it with an appropriate amount of boiling water, add clean licorice slices, mix well, stew thoroughly, put it in a pot, stir-fry over low heat until the surface is yellow to dark yellow, take it out when it is no longer sticky, and let it cool (25 kg of refined honey is used for every 100 kg of licorice).

[0042] The Chinese medicine composition of the present invention can be prepared into the following dosage forms: granules, capsules, pills, oral liquids, etc.

[0043] When preparing different dosage forms, corresponding excipients are added, such as fillers, binders, disintegrants, lubricants, absorbents and diluents, etc. The preparation of different dosage forms can be carried out by conventional methods in the art, which will not be described in detail here.

[0044] Example 1

[0045] A traditional Chinese medicine composition for treating aplastic anemia is prepared by weighing the following raw materials according to weight ratio: 12g of Cuscuta seed, 12g of Psoralea corylifolia, 12g of Ligustrum lucidum fruit, 12g of Rehmannia root, 12g of Scutellaria baicalensis, 10g of Phellodendron chinense, 20g of Poria cocos, 10g of Codonopsis pilosula, 10g of stir-fried Atractylodes macrocephala, and 10g of roasted Licorice root.

[0046] Example 2

[0047] A traditional Chinese medicine composition for treating aplastic anemia is prepared by weighing the following raw materials according to weight ratio: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 20 parts of Codonopsis pilosula, 10 parts of stir-fried Atractylodes macrocephala, and 10 parts of roasted Licorice root.

[0048] Example 3

[0049] A traditional Chinese medicine composition for treating aplastic anemia is prepared by weighing the following raw materials according to weight ratio: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 15 parts of Codonopsis pilosula, 10 parts of stir-fried Atractylodes macrocephala, and 10 parts of roasted Licorice root.

[0050] Example 4

[0051] A traditional Chinese medicine composition for treating aplastic anemia is prepared by weighing the following raw materials according to weight ratio: 10g of Cuscuta seed, 6g of Psoralea corylifolia, 10g of Ligustrum lucidum fruit, 14g of Rehmannia root, 4g of Scutellaria baicalensis, 12g of Phellodendron chinense, 10g of Poria cocos, 30g of Codonopsis pilosula, 12g of stir-fried Atractylodes macrocephala, and 10g of roasted Licorice root.

[0052] Example 5

[0053] A traditional Chinese medicine composition for treating aplastic anemia is prepared by weighing the following raw materials according to weight ratio: 6g of Cuscuta seeds, 10g of Psoralea corylifolia, 6g of Ligustrum lucidum fruit, 10g of Rehmannia root, 10g of Scutellaria baicalensis, 4g of Phellodendron chinense, 15g of Poria cocos, 10g of Codonopsis pilosula, 6g of Atractylodes macrocephala with bran, and 2g of roasted Licorice root.

[0054] The following test examples use a decoction or concentrated solution prepared using the raw material drug (medicinal material slice) of the present invention, and the specific preparation method is as follows:

[0055] Preparation method of clinical prescription:

[0056] According to the raw material formula of Example 1, the raw materials (medicinal material slices) are all from Beijing Kangrentang Co., Ltd. and are prepared into water decoctions by the decoction room of Xiyuan Hospital of China Academy of Chinese Medical Sciences. Each dose of medicine is decocted twice with 500ml of water, that is, 500ml of water is added to each dose of medicine and decocted once, and then 500ml of water is added to each dose of medicine and decocted once. The two decoctions total 1.5h, and finally 400ml of water decoction is decocted per dose. The raw material formulas given in the above examples are all the dosages of one dose of medicine.

[0057] 1. Clinical Trials

[0058] The Chinese medicine composition of Example 1 was prepared into a decoction according to the aforementioned method and administered to patients diagnosed with AA by Western medicine. One dose was taken orally twice daily, 200 ml each time, warm and on an empty stomach. A course of treatment consisted of four weeks, and 3-6 courses were generally administered. Once clinical symptoms and signs were alleviated and laboratory tests returned normal, an additional course of treatment was administered to consolidate the therapeutic effect.

[0059] 1. Patients

[0060] The selected cases were 30 AA patients (including 6 SAA patients and 24 NSAA patients) who were diagnosed and treated in the outpatient department of the hematology department of our hospital from May to December 2024. There were 14 males and 16 females. The median age of the patients was 39 (31.5, 47) years, and the median disease course was 60 (12, 114) months. All patients took Huang Zi Sheng Xue Fang for at least 3 courses of treatment, of which 18 patients (including 4 SAA patients and 14 NSAA patients) took Huang Zi Sheng Xue Fang for 6 courses of treatment.

[0061] 2. Methods

[0062] (1) Treatment plan

[0063] Of the 30 patients, 8 had discontinued immunosuppressants, androgen-based medications for at least 3 months, and 22 had not achieved clinical efficacy after 6 to 1 year of androgen-based androgen-based treatment. All patients took the Chinese herbal decoction obtained in Example 1 of the present invention twice daily, without taking other medications for the disease while taking the decoction. Follow-up was conducted every 3 months, including symptoms such as fatigue, anorexia, fever, and bleeding, and blood tests and liver and kidney function indicators.

[0064] (2) The efficacy standard refers to the "Chinese Guidelines for the Diagnosis and Treatment of Aplastic Anemia (2022 Edition)"

[0065] SAA:

[0066] Complete remission (CR): HB>100g / L; Neu>1.5×10 9 / L;PLT>100×10 9 / L.

[0067] Partial remission (PR): Independence from blood transfusions and no longer meeting the diagnostic criteria for SAA.

[0068] Not Relevant (NR): The diagnostic criteria for SAA are still met.

[0069] NSAA:

[0070] Complete remission (CR): Same as SAA efficacy criteria.

[0071] Partial remission (PR): independence from blood transfusion (if previously transfusion dependent), or at least one lineage cell count doubles or reaches normal, or any lineage cell baseline level increases: HB>30g / L (e.g. <60g / L before treatment, increase after treatment>30g / L), Neu>0.5×10 9 / L (if <0.5×10 9 / L, the increase after treatment>0.5×10 9 / L)、PLT>20×10 9 / L (if <20×10 9 / L, the increase after treatment>20×10 9 / L).

[0072] No effect (NR): disease progression or failure to reach the above-mentioned efficacy indicators.

[0073] (3) Symptom score (determined with reference to the myelopathy symptom score in the Guidelines for the Diagnosis and Treatment of Aplastic Anemia with Integrated Traditional Chinese and Western Medicine (2022 Edition)).

[0074]

[0075]

[0076] Refer to this table to evaluate the efficacy based on the changes in the patient's TCM symptom scores before and after treatment:

[0077] 1) Effective symptom improvement: a group or single symptom score is reduced by ≥60%;

[0078] 2) No improvement in symptoms: the score of a group or single symptom is reduced by <40%;

[0079] (4) Statistical analysis

[0080] Statistical analysis was performed using paired t-test, with P < 0.05 indicating significant significance.

[0081] 3. Results

[0082] (1) Clinical efficacy

[0083] After 3 months of treatment, 14 patients (approximately 46.67%) achieved hematological remission among all patients, of which approximately 66.67% (4 cases) of SAA patients were free from blood transfusion and achieved partial remission; approximately 41.67% (10 cases) of NSAA patients achieved partial remission, of which 3 of 4 transfusion-dependent NSAA patients were free from blood transfusion and 1 patient's Neu returned to normal; among 6 non-transfusion-dependent NSAA patients, 2 had an increase in or normalization of Neu baseline levels, 3 had an increase in PLT baseline levels, and 2 had normalization of HB; no patient achieved complete remission. According to statistics, the peripheral blood PLT of 30 patients (P<0.01) was higher than before, and the difference was statistically significant ( Figure 1 A); at the same time, peripheral blood HB and Neu also increased, but the difference did not show statistical significance ( Figure 1 B, 1C).

[0084] After 6 months of treatment, 12 of the 18 patients (approximately 66.67%) achieved hematological remission, of which 75% (3 cases) of SAA patients were free from blood transfusion and achieved partial remission; 64.29% (9 cases) of NSAA patients achieved partial remission, of which 3 of the 4 transfusion-dependent NSAA patients were free from blood transfusion and 1 patient's Neu returned to normal; of the 5 non-transfusion-dependent NSAA patients, 3 had an increase in or normalization of Neu baseline levels, 3 had an increase in or normalization of HB baseline levels, and 2 had an increase in or a 2-fold increase in PLT baseline levels. No patient achieved complete remission. According to statistics, the peripheral blood PLT (P<0.01) and HB (P<0.05) of the 18 patients were higher than before, and the difference was statistically significant ( Figure 1 A, 1B), and peripheral blood Neu also increased, but the difference did not show statistical significance ( Figure 1 D). Attached are the blood test results of some patients before and after taking the medicine: The blood test results of male patient 1 during the six-month treatment are as follows: Figure 2 As shown; the blood test results of male patient 2 during the six-month treatment are as follows Figure 3 The blood test results of female patient 1 during the six-month treatment period are as follows: Figure 4 shown.

[0085] (2) TCM symptom efficacy

[0086] As shown in Table 1, among the 30 patients, 7 (male 5, male 7, male 13, male 14, female 5, female 13, female 15) did not exhibit any abnormal symptoms. Three months after treatment, 30.43% of the 23 patients with abnormal symptoms showed significant improvement. Four patients had a reduction in their total symptom score of more than 60%, and three had a reduction in their fatigue and dizziness scores of more than 60%. The main symptoms that improved were palpitations, fatigue, dizziness, and bleeding. Six months after treatment, 56.25% of the 16 patients with abnormal symptoms showed significant improvement. Eight patients had a reduction in their total symptom score of more than 60%, and one patient had a reduction in their fatigue score of more than 60%. The main symptoms that improved were palpitations, fatigue, dizziness, bleeding, and pale complexion, lips, and nails. After 3 and 6 months of treatment, the symptom scores of patients were lower than before (P<0.01), and the difference was statistically significant ( Figure 1 D).

[0087] Table 1 TCM symptom score table

[0088]

[0089]

[0090] Note: NA in the table means that the patient did not take the medication for 6 courses, but only took the medication for 3 courses.

[0091] (3) Adverse reactions

[0092] Three patients experienced mild abdominal distension during medication, and the symptoms were alleviated by increasing the dose of Codonopsis pilosula to 20g. No patients were found to have abnormal heart, liver, or kidney function.

[0093] 2. Analysis of Network Pharmacology and Single-Cell Sequencing Results

[0094] 1. Active ingredients and corresponding targets of Huangzi Shengxue Recipe

[0095] In this study, the Traditional Chinese medicine systems pharmacology database and analysis platform (TCMSP) was used to search for drugs in HZSXF to identify active ingredients, and those with oral availability (OB) greater than 20% and drug-like index (DL) greater than or equal to 0.1 were screened.

[0096] Because the raw rehmannia root and psoralea corylifolia root in the drug combination were not included in the TCMSP database, we identified all the drug's major chemical components from the SymMap database. Based on the two-dimensional structures of the chemical components, human pharmacokinetic data and drug-like properties were predicted and screened using the Swiss ADME platform. Swiss Target Prediction was used to predict the active targets of the screened active ingredients. Targets with a probability greater than 0 were screened and summarized. Ultimately, 1,081 targets regulated by the Huangzi Shengxue Recipe were identified.

[0097] 2. AA and metabolic pathway targets

[0098] Databases were queried to identify target genes and metabolic pathway targets for AA. The targets of Huang Zi Sheng Xue Fang, metabolic pathway targets, and AA targets were intersected. AA disease-related targets were retrieved from the GeneCards database using the search term "Aplastic Anemia." After summarizing the results using Excel, duplicate targets were removed, and targets with correlation scores greater than the median were selected. A total of 1513 targets associated with AA were identified. 1752 metabolic pathway targets were obtained using the KEGG platform.

[0099] 3. Metabolic targets of Huangzi Shengxue Fang in treating AA

[0100] Take the intersection of Huang Zi Sheng Xue Fang’s targets, metabolic pathway targets, and AA targets, and draw a Venn diagram at the same time, as shown in the following example: Figure 5 As shown in Figure A: the green set represents AA disease-related targets (i.e., the 1513 targets related to the pathogenesis of AA obtained in 2. AA and metabolic pathway targets), the red set represents the regulatory targets of Huangzi Shengxue Recipe (i.e., the 1083 targets obtained in 1. Active ingredients and corresponding targets of Huangzi Shengxue Recipe), and the blue set represents metabolic pathway targets (i.e., the 1752 metabolic pathway targets obtained in 2. AA and metabolic pathway targets). The intersection of the three results in a total of 48 metabolic targets for the treatment of AA by Huangzi Shengxue Recipe.

[0101] 4. Construct protein interaction networks and screen key targets

[0102] The 48 metabolic targets of Huangzi Shengxue Fang in treating AA were imported into the STRING database, and the obtained protein interactions were downloaded in the form of TSV and imported into the analysis platform to draw the PPI network diagram, as shown in Figure 5 As shown in Figure B, each node represents a target. The existence of links between nodes indicates that there is an interaction between the two. MCODE was used to screen key targets, with degree cutoff set to 2, node score cutoff set to 0.2, K-score set to 2, and Max depth set to 100. The targets in the modules with the highest scores were selected as key targets, and a total of 15 key targets were screened. Figure 5 As shown in C, the key targets are XDH, NOS3, G6PD, NOS2, HK1, ARG1, IDH1, MPO, PGK1, HMOX1, TKT, NQO1, PGD, PTGS2, and PKM.

[0103] 5. Distribution of core targets in the bone marrow of AA patients

[0104] The scRNA-seq of bone marrow from two AA patients in the Gene Expression Omnibus (GEO) database GSE181989 was analyzed using the R package Seurat (version 4.1.2). The two sets of data were integrated using the merge function, and the harmony function was used to remove batch effects. Cells that were considered to be of low quality based on criteria such as the minimum expression level greater than 3, the gene count less than 200, and the proportion of mitochondrial genes greater than 10% were excluded from subsequent analysis. The remaining cells were used for subsequent bioinformatics analysis, which included principal component analysis (PCA) using the top 2000 variably expressed genes. After this, the principal components were subjected to dimensionality reduction and cluster analysis using the UMAP method, as shown in Figure 5. Figure 6 As shown in A, the bone marrow cells of AA patients were clustered into 23 groups. The marker genes in CellMarker were used to annotate the cells in each group, as shown in Figure 6 As shown in Figure B, the horizontal axis represents cell grouping, the vertical axis represents cell surface markers, the dots in the figure represent the expression of surface markers in each group of cells, the size of the dots represents the expression ratio of the corresponding surface marker in each group of cells (the larger the ratio, the higher the ratio), and the color of the dots represents the average expression level of the corresponding surface marker in each group of cells. Finally, the cells were annotated into 9 categories, such as Figure 6 As shown in Figure C, BC represents B lymphocytes, CD4T represents CD4+ T lymphocytes, CD8T represents CD8+ T lymphocytes, HSC / MPP represents hematopoietic stem / progenitor cells, Mon represents monocytes / macrophages, NK represents natural killer cells, PC represents plasma cells, preRBC represents immature red blood cells, and preT represents immature T cells. Finally, the R function Featureplot is used to draw a distribution map of the core targets, and further screen the targets highly expressed in macrophages as core targets, such as Figure 6 As shown in D, the color depth in the figure represents the relative expression level of the gene in various types of cells. The darker the color, the higher the relative quantitative expression of the gene in this type of cell.

[0105] pass Figure 6 D shows that the core targets HK1, PGK1, PKM, PGD, G6PD, and TKT are highly expressed in bone marrow macrophages from AA patients. HK1, PGK1, and PKM are key enzymes in the glycolysis pathway. HK1 (Hexokinase 1) catalyzes glucose into glucose-6-phosphate in the first step of glycolysis. By phosphorylating glucose, HK1 prevents glucose from escaping through the cell membrane, ensuring that the metabolic substrate remains intracellular. This process is one of the major rate-limiting steps in glycolysis. PGK1 (Phosphoglycerate kinase 1) catalyzes the conversion of 1,3-bisphosphoglycerate to 3-phosphoglycerate in the seventh step of glycolysis, simultaneously generating one ATP molecule. This ATP generation occurs directly through the transfer of high-energy phosphate groups from the substrate, independent of mitochondrial oxidative phosphorylation. PKM (Pyruvate kinase isozyme type M1 / 2) catalyzes the conversion of phosphoenolpyruvate to pyruvate in the final step, generating two molecules of ATP. This process is the final rate-limiting step and determines the overall rate of glycolysis.

[0106] 6. Pathway Enrichment Analysis

[0107] KEGG pathway enrichment analysis was performed on the 48 metabolic targets of Huangzi Shengxue Fang in the treatment of AA, and a bubble chart of the KEGG results was drawn. P-value < 0.05 indicates that the target is significantly enriched in a specific pathway. Metabolic pathways with P-value < 0.05 and involving ≥ 5 genes were ranked according to the enrichment score [enrichment fold (enrichment fold = gene ratio / background ratio)]. The top 10 metabolic pathways are as follows ( Figure 7 ), and the top 10 pathways with the highest enrichment scores were drug metabolism other enzymes, carbon metabolism, central carbon metabolism in cancer, glutathione metabolism, biosynthesis of amino acids, type II diabetes mellitus, VEGF signaling pathway, glycolysis / gluconeogenesis, and chemical carcinogenesis-DNA adducts.

[0108] 7. Drug-Active Ingredient-Target Network

[0109] Import the active ingredients and corresponding core targets into Cytoscape 3.9.1, draw a drug-active ingredient-target network diagram, and obtain the active ingredients corresponding to the targets, such as Figure 8As shown in the figure, red triangles represent traditional Chinese medicines, blue squares represent medicinal ingredients in traditional Chinese medicines, and green circles represent regulatory targets. The above results indicate that kingiside_qt, 4'-O-methylglabridin, 7-Acetoxy-2-methylisoflavone, 4',7-dimethoxyisoflavone, 7-hydroxy-2-methyl-3-phenyl-chromone, 7-methoxy-2-methylisoflavone, menisporphine, and palmatine may be the main active ingredients of Huangzi Shengxue Recipe that regulate glycolysis in mononuclear macrophages.

[0110] These results suggest that some of the active ingredients in Huangzi Shengxue Recipe regulate glycolytic metabolism primarily by modulating key glycolytic enzymes highly expressed in monocytes and macrophages. M1 macrophages are predominant in the bone marrow of AA patients. M1 macrophages secrete inflammatory factors such as TNF-α to activate T cells, playing a crucial role in the pathogenesis of AA. Glycolysis is the primary energy source for M1 macrophages and a key driver of M1 macrophage polarization. Inhibiting glycolysis in AA macrophages, thereby suppressing M1 polarization, is a key mechanism of action of Huangzi Shengxue Recipe in treating AA.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Chinese medicine composition for treating aplastic anemia, characterized in that: The traditional Chinese medicine composition includes active substances, which are prepared from the following raw materials in parts by weight: 6-12 parts of Cuscuta seeds, 6-12 parts of Psoralea corylifolia, 6-12 parts of Ligustrum lucidum fruits, 10-14 parts of Rehmannia root, 4-12 parts of Scutellaria baicalensis, 4-12 parts of Phellodendron chinense, 10-20 parts of Poria cocos, 10-30 parts of Codonopsis pilosula, 6-12 parts of stir-fried Atractylodes macrocephala, and 2-10 parts of roasted Licorice root.

2. The Chinese medicine composition according to claim 1, wherein: The active substance is prepared from the following raw materials in parts by weight: 9-12 parts of Cuscuta seeds, 9-12 parts of Psoralea corylifolia, 9-12 parts of Ligustrum lucidum fruits, 9-12 parts of Rehmannia root, 9-12 parts of Scutellaria baicalensis, 9-12 parts of Phellodendron chinense, 15-20 parts of Poria cocos, 10-20 parts of Codonopsis pilosula, 9-12 parts of stir-fried Atractylodes macrocephala, and 9-12 parts of roasted Licorice root.

3. The Chinese medicine composition according to claim 1, wherein: The traditional Chinese medicine composition is prepared from the following raw materials in parts by weight: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 10-20 parts of Codonopsis pilosula, 10 parts of stir-fried Atractylodes macrocephala, and 10 parts of roasted Licorice root.

4. The Chinese medicine composition according to claim 1, wherein: The active substance is prepared from the following raw materials in parts by weight: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 10 parts of Codonopsis pilosula, 10 parts of Atractylodes macrocephala stir-fried with bran, and 10 parts of roasted Licorice root; Alternatively, the active substance is prepared from the following raw materials in parts by weight: 12 parts of Cuscuta seeds, 12 parts of Psoralea corylifolia, 12 parts of Ligustrum lucidum fruits, 12 parts of Rehmannia root, 12 parts of Scutellaria baicalensis, 10 parts of Phellodendron chinense, 20 parts of Poria cocos, 20 parts of Codonopsis pilosula, 10 parts of stir-fried Atractylodes macrocephala, and 10 parts of roasted Licorice root.

5. The Chinese medicine composition according to claim 1, wherein: The dosage form of the Chinese medicine composition is selected from any one of extracts, granules, pills, capsules and oral liquids.

6. The Chinese medicine composition according to claim 1, wherein: The traditional Chinese medicine composition further comprises excipients, which include at least one of a filler, a binder, a disintegrant, a lubricant, an absorbent and a diluent.

7. Use of the traditional Chinese medicine composition according to any one of claims 1 to 6 in the preparation of a medicament for preventing and / or treating aplastic anemia.

8. The use according to claim 7, characterized in that: The aplastic anemia includes severe aplastic anemia and non-severe aplastic anemia.

9. The use according to claim 7, characterized in that: The drug exerts its efficacy through at least one of the following effects: (1) Increased platelet and / or hemoglobin levels; (2) reduce the myelodysplasia symptom score in the Guidelines for the Diagnosis and Treatment of Aplastic Anemia with Integrated Traditional Chinese and Western Medicine; (3) inhibiting monocyte and / or macrophage glycolysis; (4) Inhibit macrophage M1 polarization.

10. The use according to claim 9, characterized in that: The main active ingredients of the drug that inhibit glycolysis in monocytes and / or macrophages include diterpene glycosides, 4'-O-methylglycyrrhizin, 7-acetoxy-2-methylisoflavone, 4',7-dimethoxyisoflavone, 7-hydroxy-2methyl-3-phenylpropiochromone, 7-methoxy-2-methylisoflavone, schizosporin and tauropeptide; And / or, the drug inhibits glycolysis in monocytes and / or macrophages mainly by regulating the high expression of key glycolysis enzymes, wherein the key glycolysis enzymes include HK1, PGK1 and PKM.

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