Use of yigong powder in treating myelodysplastic syndrome

Abstract

This invention provides the use of Yigongsan in the treatment of myelodysplastic syndrome (MDS). Specifically, this invention has found that Yigongsan improves ineffective hematopoiesis in MDS patients and significantly alleviates traditional Chinese medicine symptoms of MDS, such as fatigue, lethargy, lack of thirst, and sallow complexion. Furthermore, it has been discovered that Yigongsan regulates hematopoietic function in MDS through the S100A9-TLR4-NF-κB signaling pathway. This invention also provides a treatment method for MDS.

Description

Technical Field

[0001] This invention belongs to the technical field, and more specifically, this invention relates to the use of Yigongsan in the treatment of myelodysplastic syndrome (MDS). Background Technology

[0002] Myelodysplastic syndromes (MDS) are a group of heterogeneous clonal hematopoietic stem / progenitor cell dysfunctions, characterized by dysplastic hematopoiesis in one or more bone marrow cell lineages, ineffective hematopoiesis, and a high risk of transformation into acute myeloid leukemia (AML). The incidence of MDS is 4.5 per 100,000 people per year; the incidence is higher in men than in women (6.2 per 100,000 people per year), and the incidence increases significantly with age.

[0003] Ineffective hematopoiesis is a significant cause of pancytopenia in MDS patients, severely impacting their quality of life. Ineffective hematopoiesis leads to excessive proliferation of erythroid progenitor cells, stimulating the body to secrete growth differentiation factor 15 (GDF15) and erythrone (ERFE). The expression of ERFE and GDF15 is significantly elevated in MDS, reflecting the degree of ineffective hematopoiesis. Furthermore, high expression of ERFE in erythrocyte progenitor cells is closely associated with poor prognosis in MDS, independent of international prognostic stratification. S100A9, through TLR4 signaling and NF-κB-mediated innate immune activation, leads to the overexpression of pro-inflammatory factors, a crucial factor in MDS clonal evolution and alterations in the bone marrow inflammatory microenvironment, especially in low-risk MDS, directly causing pyroptosis and ineffective hematopoiesis.

[0004] Currently, there are limited treatment options for MDS, and the efficacy is not good. Therefore, there is an urgent need in this field to develop new strategies for treating myelodysplastic syndromes. Summary of the Invention

[0005] The purpose of this invention is to provide a method for improving the inflammatory microenvironment of bone marrow in MDS patients and reducing ineffective hematopoiesis in MDS patients.

[0006] In a first aspect of the invention, there is provided a use of Yigongsan for preparing a medicine, said medicine having uses selected from the group consisting of:

[0007] 1) Improve the inflammatory microenvironment of bone marrow in MDS patients and reduce ineffective hematopoiesis in MDS patients;

[0008] 2) Improve the TCM symptoms of MDS patients;

[0009] 3) Improve the hematopoietic function of MDS patients;

[0010] 4) Treatment of MDS.

[0011] In another preferred embodiment, the MDS patient is a low-risk or intermediate-risk MDS patient.

[0012] In another preferred embodiment, the pathological characteristics of the ineffective hematopoiesis are: bone marrow immature erythrocyte proliferation accompanied by a decrease in the peripheral blood erythropoiesis index.

[0013] In another preferred embodiment, the immature erythrocytes are erythroid precursor cells.

[0014] In another preferred embodiment, the immature red blood cells include MDS cells.

[0015] In another preferred embodiment, the MDS cells include MDS with single-lineage dysplastic hematopoietic cells (MDS-SLD), MDS with ring sideroblasts (MDS-RS), MDS with multi-lineage dysplastic hematopoietic cells (MDS-MLD), SKM-1 cell line, MUZT-1 cell line, MDS-IB1, and MDS-IB2.

[0016] In another preferred embodiment, the TCM symptoms include: fatigue, lethargy, lack of appetite, loss of thirst, sallow complexion, and edema.

[0017] In another preferred embodiment, the drug is an oral dosage form.

[0018] In another preferred embodiment, the drug is an ointment, granules, decoction, or capsule.

[0019] In another preferred embodiment, the formula of the Yigongsan includes ginseng, atractylodes macrocephala, poria cocos, prepared licorice root, and tangerine peel.

[0020] In another preferred embodiment, the proportions of ginseng, atractylodes macrocephala, poria cocos, prepared licorice root, and tangerine peel in the formula of the Yigongsan are (1±0.2):(1±0.2):(1±0.2):(1±0.2):(1±0.2), more preferably (1±0.1):(1±0.1):(1±0.1):(1±0.1):(1±0.1), and most preferably (1±0.05):(1±0.05):(1±0.05):(1±0.05):(1±0.05) or 1:1:1:1:1.

[0021] In another preferred embodiment, the administration regimen of the Yigongsan is as follows: 100g of medicinal materials per person per day (20g each of raw ginseng (sun-dried ginseng), poria cocos (peeled), stir-fried atractylodes macrocephala, tangerine peel, and prepared licorice).

[0022] In another preferred embodiment, the administration method of the Yigongsan includes: mixing and decocting the 100g of medicinal materials to concentrate the decoction into 100ml of medicinal juice.

[0023] In another preferred embodiment, the drug inhibits the S100A9-TLR4-NF-κB signaling pathway.

[0024] In another preferred embodiment, the drug inhibits the expression of GDF15 and ERFE.

[0025] In another preferred embodiment, the GDF15 and ERFE are GDF15 and ERFE in a mononuclear cell.

[0026] In another preferred embodiment, the GDF15 and ERFE are GDF15 and ERFE in serum.

[0027] In another preferred embodiment, S100A9 is linearly correlated with GDF15.

[0028] In another preferred embodiment, the S100A9 is linearly correlated with TLR4.

[0029] In another preferred embodiment, the GDF15 is linearly correlated with S100A9 and TLR4.

[0030] In another preferred embodiment, the drug is a pharmaceutical composition comprising:

[0031] 1) Extraordinary Power Powder; and

[0032] 2) Pharmaceutically acceptable carrier.

[0033] In a second aspect of the invention, a method for inhibiting the S100A9-TLR4-NF-κB signaling pathway in vitro is provided, the method comprising administering an effective amount of oxy-powder to in vitro cells.

[0034] In another preferred embodiment, the effective amount is an inhibitory effective amount, and the dosage is adjusted according to different cells.

[0035] In another preferred embodiment, the formula of the Yigongsan includes ginseng, atractylodes macrocephala, poria cocos, prepared licorice root, and tangerine peel.

[0036] In another preferred embodiment, the proportions of ginseng, atractylodes macrocephala, poria cocos, prepared licorice root, and tangerine peel in the formula of the Yigongsan are (1±0.2):(1±0.2):(1±0.2):(1±0.2):(1±0.2), more preferably (1±0.1):(1±0.1):(1±0.1):(1±0.1):(1±0.1), and most preferably (1±0.05):(1±0.05):(1±0.05):(1±0.05):(1±0.05) or 1:1:1:1:1.

[0037] In a third aspect of the invention, a method for treating MDS is provided, the method comprising: administering a therapeutically effective amount of Yigongsan to a subject.

[0038] In another preferred embodiment, the method further includes the administration of Yigongsan in combination with conventional Western medicine treatment.

[0039] In another preferred embodiment, the conventional Western medicine treatment is described in the Chinese Expert Consensus on the Diagnosis and Treatment of Myelodysplastic Syndromes (2019 Edition).

[0040] In another preferred embodiment, the MDS is a low-risk or intermediate-risk MDS.

[0041] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0042] Figure 1 The normal PP plot of the regression standardized residuals of serum S100A9 and GDF15 in MDS patients is shown.

[0043] Figure 2 The normal PP plot of the regression standardized residuals of serum S100A9 and TLR4 in MDS patients is shown.

[0044] Figure 3 The normal PP plot of the regression standardized residuals of serum GDF15, S100A9, and TLR4 in MDS patients is shown.

[0045] Figure 4 The LC-HRMS analysis of Yigongsan is shown: (top): Yigongsan negative control group, (bottom): Yigongsan.

[0046] Figure 5 The network pharmacology analysis of Yigongsan is shown: (A) Intersecting targets of Yigongsan and MDS disease (B) Enrichment analysis.

[0047] Figure 6 The results of the KEGG enrichment analysis are shown.

[0048] Figure 7 The results of the GO enrichment analysis are shown.

[0049] Figure 8 The target-network is shown: diamonds represent signaling pathways, and circles represent genes enriched in those pathways.

[0050] Figure 9 The NF-κB signaling pathway was shown: the red gene is a possible target for the predicted action of the drug on MDS.

[0051] Figure 10The PPI network of cytokinesin and NF-κB protein is shown.

[0052] Figure 11 The LC-HRMS analysis of Yigongsan is shown: (a): negative control group of Yigongsan, (b): Yigongsan.

[0053] Figure 12 The study shows a comparison of SKM-1 cell viability at different concentrations and time points.

[0054] Figure 13 The expression of NF-κB, ERFE, and GDF15 mRNA in SKM-1 cells was compared.

[0055] Note: Compared with the control group, *P<0.05; compared with the model group, △P<0.05; compared with the blocking agent group, ▲P<0.05;

[0056] (a) Comparison of NF-κB mRNA expression in SKM-1 cells; (b) Comparison of ERFE mRNA expression in SKM-1 cells; (c) Comparison of GDF15 mRNA expression in SKM-1 cells.

[0057] Figure 14 The comparison of TLR4 and p-p65 grayscale values ​​for each group of SKM-1 is shown.

[0058] Note: Compared with the control group, *P<0.05; compared with the model group, △P<0.05; compared with the blocking agent group, ▲P<0.05.

[0059] (a) WB results of TLR4 and p-p65 protein expression in SKM-1 cells; (b) Gray value of TLR4 in SKM-1 cells; (c) Gray value of p-p65 in SKM-1 cells.

[0060] Figure 15 The study shows a comparison of the viability of MUZT-1 cells at different concentrations and time points.

[0061] Figure 16 The mRNAs of NF-κB, ERFE, and GDF15 in MUZT-1 cells were displayed.

[0062] Note: *P<0.05 compared with the control group; △P<0.05 compared with the model group; ▲P<0.05 compared with the blocking agent group. (a) Comparison of NF-κB mRNA expression in MUZT-1 cells; (b) Comparison of ERFE mRNA expression in MUZT-1 cells; (c) Comparison of GDF15 mRNA expression in MUZT-1 cells.

[0063] Figure 17 The presence of TLR4 and p in MUZT-1 cells was shown. -Comparison of p65 protein expression.

[0064] Note: Compared with the blank group, *P < 0.05; compared with the model group, △P < 0.05; compared with the blocker group, ▲P < 0.05.

[0065] (a) WB results of TLR4 and p-p65 protein expression in MUZT-1 cells; (b) Gray value of TLR4 in MUZT-1 cells; (c) Gray value of p-p65 in MUZT-1 cells. - (a) WB results of TLR4 and p-p65 protein expression in MUZT-1 cells; (b) Gray value of TLR4 in MUZT-1 cells; (c) Gray value of p-p65 in MUZT-1 cells. Detailed implementation manners

[0066] After extensive and in-depth research, the inventor of the present invention has developed an application of Yigong Powder in the treatment of MDS. The present invention has verified through clinical trials that Yigong Powder can improve ineffective hematopoiesis (decrease in GDF15 and ERFE) in MDS patients, and significantly improve the TCM syndromes of MDS patients such as fatigue, listlessness, lack of thirst, sallow complexion, etc.; in addition, the present invention has also further verified through cell experiments and database analysis that Yigong Powder can improve the inflammatory microenvironment of MDS and relieve ineffective hematopoiesis through the S100A9-TLR4-NF-κB signaling pathway. On this basis, the present invention has been completed.

[0067] Terms

[0068] To make it easier to understand the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary.

[0069] As used herein, the terms "comprising", "including", "containing" can be used interchangeably, and not only include closed definitions, but also semi-closed and open definitions. In other words, the said terms include "consisting of...", "consisting essentially of...".

[0070] Myelodysplastic syndrome (MDS)

[0071] Myelodysplastic syndrome (MDS) is a heterogeneous myeloid clonal disease originating from hematopoietic stem cells, characterized by ineffective hematopoiesis and a high risk of transformation into acute myeloid leukemia (AML).

[0072] Myelodysplastic syndrome (MDS) is a group of clonal hematopoietic stem cell diseases, and its main characteristics include:

[0073] Ineffective hematopoiesis: Abnormal development of hematopoietic stem cells, resulting in insufficient blood cell production.

[0074] Blood cell count decreases: A drop in peripheral blood cell count. Common symptoms include anemia, increased risk of infection, and bleeding tendency.

[0075] Conversion risk: MDS patients have a higher risk of developing acute myeloid leukemia (AML).

[0076] The clinical manifestations of MDS are often nonspecific. Patients present with signs and symptoms associated with pancytopenia, such as fatigue due to anemia, infection due to neutropenia, and / or bleeding due to thrombocytopenia. Ineffective hematopoiesis is a significant cause of pancytopenia in MDS patients. Ineffective hematopoiesis leads to excessive proliferation of erythroid progenitor cells, which can stimulate the body to secrete growth differentiation factor 15 (GDF15) and erythrone (ERFE). The expression of ERFE and GDF15 is significantly elevated in MDS, reflecting the degree of ineffective hematopoiesis. Furthermore, high expression of ERFE in erythrocyte progenitor cells is closely associated with poor prognosis in MDS.

[0077] Low-risk MDS: The proportion of bone marrow blasts (usually <5%), only mild to moderate monocytopenia, slow disease progression, and low leukemia conversion rate.

[0078] Intermediate-risk MDS: The proportion of bone marrow blasts (5%–10%) is between low-risk and high-risk, the reduction of three blood cell lines is more obvious, the chromosomal abnormalities are relatively complex but do not reach the high-risk standard, there may be a risk of transformation into acute leukemia, the transformation risk is moderate (about 30%–40%), and the median survival is 2–3 years.

[0079] High-risk MDS: The proportion of blast cells in the bone marrow (>10%) is aggressive, there are complex chromosomal abnormalities, severe reduction of three blood cell lines, the disease progresses rapidly, and it is very easy to transform into acute leukemia, with a transformation rate of more than 50%.

[0080] Extraordinary Power Powder

[0081] Yigong San (YGS) is recorded in "Xiao'er Yaozheng Zhijue" (Direct Guide to Pediatric Drug Syndromes). Its function is to tonify qi, strengthen the spleen, and promote qi circulation and resolve stagnation. This formula is made by adding ingredients to Sijunzi Tang (Four Gentlemen Decoction). Sijunzi Tang contains "ginseng, atractylodes, poria, and licorice, which are sweet and warm, benefit the stomach, have the function of strengthening the spleen and promoting its function, and possess the virtue of harmony" ("Mingyi Fanglun"). It is a representative formula for strengthening the spleen. The addition of tangerine peel is intended to promote qi circulation and resolve stagnation, awaken the spleen and assist its function. It has the advantages of tonifying without causing stagnation and tonifying while promoting circulation.

[0082] The inventors have demonstrated through clinical trials that Yigongsan can effectively improve the symptoms of spleen deficiency in MDS patients according to traditional Chinese medicine, increase platelet count in low-risk MDS patients, reduce the mRNA expression of GDF15 and ERFE in peripheral blood mononuclear cells of MDS patients, and improve ineffective hematopoiesis in patients.

[0083] Furthermore, through database analysis and cell experiments, the inventors discovered that Yigongsan can reduce the levels of S100A9 and TLR4 in the peripheral blood of low-risk or intermediate-risk MDS patients, and improves hematopoietic function in MDS by regulating the S100A9-TLR4-NF-κB signaling pathway.

[0084] S100A9-TLR4-NF-κB signaling pathway

[0085] S100A9 protein (also known as calcein B or MRP14) is a member of the S100 family of calcium-binding proteins. It usually forms a heterodimer (calprotectin) with S100A8 and plays a key role in inflammation regulation, immune response, and tumorigenesis. It regulates intracellular signaling pathways by binding to calcium ions and participates in physiological and pathological processes such as neutrophil chemotaxis and antibacterial activity.

[0086] Toll-like receptor 4 (TLR4) is an important member of the Toll-like receptor family. As a pattern recognition receptor (PRR), it mainly recognizes pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), playing a key role in innate immunity and inflammatory responses.

[0087] This application screened the components of Yigongsan and MDS disease targets, identifying 268 targets. Then, it enriched 167 signaling pathways, including the NF-κB signaling pathway and TLRs signaling pathway, using KEGG and GO. Among them, 21 pathways were found to be related to the NF-κB signaling pathway. Combining the target-network diagram and PPI network diagram, the application finally focused on the TLR4-NF-κB signaling pathway.

[0088] Pharmaceutical Composition

[0089] The present invention also provides a pharmaceutical composition. In a preferred embodiment, the pharmaceutical composition is a purpuric powder and a pharmaceutically acceptable carrier / delivery carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the formulated substance and the condition to be treated.

[0090] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): oral, intravenous, or topical administration. The pharmaceutical composition is available in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably in the form of powder, ointment, tablet, capsule, granule, injection, or infusion.

[0091] The pharmaceutical carrier described herein is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents.

[0092] Preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by employing experiments not exceeding the norm. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are within the scope of a skilled physician's expertise.

[0093] Compared with the prior art, the main advantages of the present invention include:

[0094] 1. This invention has found that Yigongsan significantly improves the TCM symptoms of MDS patients, such as fatigue, lethargy, lack of thirst, sallow complexion, and edema.

[0095] 2. This invention has found that Yigongsan can enhance the hematopoietic function of MDS patients and inhibit the activity of MDS cells.

[0096] 3. This invention discovers that Yigongsan regulates hematopoietic function through the S100A-LR4-NF-κB signaling pathway.

[0097] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0098] Example 1. Clinical trials have demonstrated that Yigongsan improves ineffective hematopoiesis in MDS patients.

[0099] Experimental methods

[0100] 1. Source of MDS patients: Outpatients and inpatients of Baoshan District Integrated Traditional Chinese and Western Medicine Hospital, Shanghai Sixth People's Hospital, and Ruijin Hospital, Shanghai. The diagnostic criteria for MDS were based on the "Chinese Expert Consensus on the Diagnosis and Treatment of Myelodysplastic Syndromes (2019 Edition)".

[0101] 2. Diagnostic criteria

[0102] 2.1 Diagnostic criteria for MDS:

[0103] Referencing the "Chinese Expert Consensus on the Diagnosis and Treatment of Myelodysplastic Syndromes (2019 Edition)":

[0104] (1) Necessary conditions (both conditions must be met);

[0105] ① A persistent decrease in one or more blood cell lines for 4 months (if an increase in primitive cells or MDS-related cytogenetic abnormalities are detected, MDS can be diagnosed without waiting)

[0106] ② Exclude other hematopoietic and non-hematopoietic system diseases that can lead to decreased blood cell count and developmental abnormalities;

[0107] (2) MDS-related (major) standards (at least one must be met)

[0108] ① Developmental abnormalities: The proportion of abnormal cells in the erythroid, granulocytic, and megakaryocyte lineages in bone marrow smears is >10%;

[0109] ② The proportion of ring sideroblasts among nucleated erythrocytes is >15%, or 35%, and is accompanied by SF3B1 mutation;

[0110] ③ Primitive cells: 5%–19% of primitive cells in bone marrow smears (or 2%–19% in peripheral blood smears);

[0111] ④ Routine karyotype analysis or FISH detection of chromosomal abnormalities that are diagnostically significant for MDS;

[0112] (3) Auxiliary criteria (For patients with common MDS clinical manifestations such as macrocytic anemia that meet the necessary conditions but do not meet the main criteria, and who are transfusion-dependent, if they meet ≥2 auxiliary criteria, they are diagnosed as suspected MDS)

[0113] ① The morphological or immunohistochemical results of bone marrow biopsy sections support the diagnosis of MDS;

[0114] ② Flow cytometry analysis of bone marrow cells revealed multiple MDS-related phenotypic abnormalities, suggesting the presence of monoclonal cell populations in the erythroid and / or myeloid lines;

[0115] Gene sequencing detected mutations in MDS-related genes, suggesting the presence of a clonal population of myeloid cells.

[0116] 2.2 Spleen Deficiency Syndrome

[0117] 2.2.1 Diagnostic criteria: The "Reference Standards for Differentiation of Deficiency Syndromes in Traditional Chinese Medicine" were revised with reference to the 1986 National Conference on the Study of Deficiency Syndromes and Geriatric Diseases in Integrated Traditional Chinese and Western Medicine.

[0118] (1) Main symptoms: poor appetite, fatigue, abdominal distension after meals or in the afternoon, and abnormal bowel movements (loose, rotten, hard at first and then loose, sometimes loose and sometimes hard).

[0119] (2) Secondary symptoms: fatigue and reluctance to speak, bland taste and no thirst, persistent abdominal pain, nausea and vomiting, epigastric fullness, borborygmus, sallow complexion, edema, weak defecation, pale tongue, swollen tongue or with teeth marks, thin white coating, and weak pulse.

[0120] A diagnosis can be made if two main symptoms are present, or one main symptom plus two secondary symptoms are present.

[0121] 2.2.2 Assessment of syndrome and therapeutic effect (referring to the revised "Reference Standards for Differentiation of Deficiency Syndromes in Traditional Chinese Medicine" from the 1986 National Conference on the Study of Deficiency Syndromes and Geriatric Diseases in Integrated Traditional Chinese and Western Medicine)

[0122] (1) Effective: The clinical symptoms and signs of spleen deficiency syndrome have improved, and the syndrome score has decreased by ≥30%.

[0123] (2) Ineffective: The clinical symptoms and signs of spleen deficiency syndrome show no significant improvement, or even worsen, and the syndrome score decreases by less than 30%. Note: The calculation formula is: [(Pre-treatment score - Post-treatment score) ÷ Pre-treatment score] × 100%

[0124] The syndrome scoring table is attached at the end of this article.

[0125] 2.3 Prognostic assessment

[0126] The current prognostic assessment of MDS is based on the revised International Prognostic Scoring System (IPSS-R) standard: a prognostic score of 1.5 or less is classified as very low risk, 2-3 as low risk, 3.5-4.5 as intermediate risk, 4.5-6 as high risk, and greater than 6 as very high risk (Table 1).

[0127] Table 1. Revised International Prognostic Scoring System (IPSS-R) for Myelodysplastic Syndromes

[0128] Prognostic parameters 0 0.5 1 1.5 2 3 4 chromosome very good - good - medium Difference Range Bone marrow blast cells 2％ - ＞2％-＜5％ - 5％-10％ ＞10％ - hemoglobin ≥10 - 8-＜10 ＜8 - - platelets ≥100 50-100 ＜50 - - - - Neutrophil count ≥0.8 ＜0.8 - - - - -

[0129] 3. Selection Criteria

[0130] Patients diagnosed with spleen deficiency syndrome MDS were identified and informed consent was obtained.

[0131] 4. Exclusion Criteria

[0132] ① Patients undergoing chemotherapy, demethylation, or bone marrow stem cell transplantation;

[0133] ② Patients with concurrent non-hematologic malignancies;

[0134] ③ Blood transfusion dependence;

[0135] ④ Severe liver and kidney dysfunction;

[0136] ⑤ Recently participated in other drug clinical trials (within 2 weeks of discontinuing the previous drug);

[0137] ⑥ Those who are selected but cannot complete the prescribed observation items.

[0138] 5. Criteria for Suspension and Withdrawal of Cases

[0139] Those who experienced adverse events; those who developed other complications or whose condition worsened.

[0140] 6. Dropout cases

[0141] Any participant who is selected and enters the clinical study is considered a dropout if they withdraw at any time or for any reason, as long as they have not completed the tasks outlined in the protocol.

[0142] 7. Research Design

[0143] This study is a randomized, double-blind, placebo-controlled, parallel-group trial.

[0144] 8. Sample size calculation

[0145] Following the repeated measures study methodology, the sample size was estimated using PASS software 15.0, with the change in platelet count as the primary endpoint of clinical efficacy used for estimation. Based on the results of the preliminary experiment, the experimental group showed a 10 (×10) increase in platelet count compared to the control group. 9 / L), with a standard deviation of 15 (×10) 9 / L), α=0.05, β=0.9, PASS software calculated the sample size of each group to be 19 cases, considering a 20% loss to follow-up rate, the sample size of each group to be 24 cases, for a total of 48 cases.

[0146] 9. Dosing regimen

[0147] The administration was carried out using a randomized, double-blind method.

[0148] 9.1 Dosage of Yigongsan (Table 2): 20g each of raw ginseng (sun-dried), poria cocos (peeled), stir-fried atractylodes macrocephala, tangerine peel, and prepared licorice root. The drugs were made into granules and manufactured by Jiangyin Tianjiang Pharmaceutical Co., Ltd. under GMP conditions according to the 2015 edition of the Chinese Pharmacopoeia. The placebo mainly consisted of maltodextrin, lactose, and food coloring. It was made into granules according to modern pharmaceutical processes and placebo preparation standards. The outer packaging, weight, odor, and color were similar to those of Yigongsan granules.

[0149] The production process and quality standards for mixed packaging of traditional Chinese medicine formula granules were adopted; a quality inspection report was provided. All experimental drugs were administered twice daily, two packets each time, once after breakfast and once after dinner, dissolved in water, for a total of 12 weeks.

[0150] Table 2. Granule weight (g)

[0151] name Place of origin Weight (g) Yigongsan granules 44.8 Ginseng Jilin 6.7 Atractylodes macrocephala Anhui 14.7 Poria Zhejiang 6.7 Dried tangerine peel Zhejiang 8.2 licorice Inner Mongolia 8.5 Placebo Particles 44.8

[0152] 9.2 Conventional Western Medicine Treatment

[0153] Conventional Western medicine treatment, including EPO, cyclosporine, testosterone undecanoate, etc., is carried out according to the "Chinese Expert Consensus on Diagnosis and Treatment of Myelodysplastic Syndrome (2019 Edition)". At the same time, symptomatic and supportive treatment is given according to the patient's condition.

[0154] 10. Sample Collection

[0155] Fasting peripheral blood samples were collected from subjects before treatment and at 4, 8, and 12 weeks after treatment. Complete blood count and liver and kidney function tests were performed by the patients themselves. Of the blood samples collected, one tube of EDTA-anticoagulated blood sample was used to isolate mononuclear cells and stored at -80°C with Trizol; the other tube of procoagulant blood sample was immediately centrifuged at 3500 rpm for 20 minutes to prepare serum, which was then stored at -80°C.

[0156] The health check center obtained peripheral blood from 22 healthy individuals, and the serum was separated and stored at -80°C.

[0157] 11. Detection Indicators

[0158] 1) Isolate lymphocytes from peripheral blood for testing;

[0159] 2) RT-PCR was used to detect the expression levels of GDF15 and ERFE in peripheral blood;

[0160] 3) Detection of serum IL-6, IL-1β, TNF-α, GDF15, S100A9, and TLR4 by enzyme-linked immunosorbent assay (ELISA);

[0161] 4) Before and after medication, peripheral blood samples were collected from the subjects, and liver and kidney function were tested to evaluate the safety.

[0162] Clinical trial conclusions

[0163] 1. Patient's condition

[0164] This trial collected 48 cases. Three patients withdrew from the trial on a voluntary basis, and two patients discontinued the trial due to disease progression. A total of 43 patients were finally included in the statistical analysis, including 22 patients in the Yigongsan group and 21 patients in the placebo group.

[0165] The male-to-female ratio was roughly equal in both groups: 54.50% male and 45.50% female in the Yigongsan group, and 42.9% male and 57.10% female in the placebo group. Both groups were predominantly composed of patients over 60 years of age, at 59.10% and 57.10% respectively. The predominant disease type in both groups was MDS-MLD, at 63.60% and 76.20% respectively. 40.90% of patients in the Yigongsan group had gene abnormalities, compared to 61.90% in the placebo group. 36.60% of patients in the Yigongsan group had chromosomal abnormalities, compared to 28.40% in the placebo group. According to the R-IPSS score, both groups were predominantly low-risk, at 54.50% and 71.40% respectively. The baseline difference between the two groups was minimal (P > 0.05) as determined by chi-square test (Table 3).

[0166] Table 3 Comparison of general information between the two groups of patients [n(%)]

[0167]

[0168] 2. The effect of Yigongsan on the TCM syndromes of MDS patients

[0169] 2.1 Comparison of TCM syndrome differentiation and efficacy between the two groups of MDS patients

[0170] The effective rates of spleen deficiency syndrome in the Yigongsan group at 4, 8, and 12 weeks after treatment were 54.50%, 75.00%, and 68.20%, respectively; the effective rates in the placebo group were 42.90%, 33.30%, and 33.30%, respectively. Chi-square test analysis showed that compared with the placebo, the Yigongsan group showed significant improvement in symptoms at 12 weeks of treatment (P < 0.05), while there was a trend of improvement in spleen deficiency at weeks 4 and 8, but the differences were not statistically significant (P > 0.05). (Table 4)

[0171] Table 4. Treatment efficacy for spleen deficiency syndrome in two groups of MDS patients before and after treatment [cases (%)]

[0172]

[0173]

[0174] 2.2 Comparison of TCM syndrome score stratification between the two groups of MDS patients

[0175] The TCM syndrome scoring standard is a standardized tool for quantitatively assessing the severity of patients' symptoms and the effectiveness of treatment. It scores patients based on dimensions such as symptom frequency, intensity, and duration, helping clinicians to standardize syndrome differentiation and objectify treatment outcomes.

[0176] Repeated measures ANOVA showed that, compared with pre-treatment levels, the syndrome scores of MDS patients in the Yigongsan group and low-risk MDS patients at 4, 8, and 12 weeks after treatment, as well as those of intermediate-risk patients at 8 and 12 weeks after treatment, were significantly lower than those in the placebo group (P < 0.05). Compared with 4 weeks after treatment, the total syndrome scores of MDS patients and low-risk MDS patients at 8 and 12 weeks after treatment were significantly lower than those in the placebo group (P < 0.05). (Table 5-7)

[0177] Table 5. Improvement of TCM syndrome scores in MDS patients ( point)

[0178]

[0179] Note: Compared with before treatment, * P < 0.05; compared with 4 weeks, # P < 0.05

[0180] Table 6. Improvement of TCM syndrome scores in low-risk MDS patients ( point)

[0181]

[0182] Note: Compared with before treatment, * P < 0.05; compared with 4 weeks, # P < 0.05

[0183] Table 7 Improvement of TCM syndrome scores in patients with intermediate-risk MDS ( point)

[0184]

[0185] Note: Compared with before treatment, * P < 0.05

[0186] 2.3 Comparison of TCM syndrome scores between the two groups of MDS patients

[0187] Repeated measures ANOVA showed that, compared with before treatment, the scores for fatigue, lethargy, and lack of speech in the Yigongsan group were significantly lower than those in the placebo group at 4 weeks, 8 weeks, 12 weeks, and 13 weeks after treatment. Compared with 4 weeks after treatment, the scores for lack of thirst and lack of speech at 8 weeks and 12 weeks after treatment were also significantly lower than those in the placebo group, with statistically significant differences (P < 0.05). (Table 8-12)

[0188] Table 8. Improvement of patients' fatigue and weakness syndrome scores ( point)

[0189]

[0190] Note: Compared with before treatment, * P < 0.05

[0191] Table 9. Improvement of patients' fatigue and lethargy syndrome scores ( point)

[0192]

[0193] Note: Compared with before treatment, * P < 0.05; compared with 4 weeks, # P < 0.05

[0194] Table 10 Improvement of Patients' Symptoms of Bland Taste and Lack of Thirst ( point)

[0195]

[0196] Note: Compared with before treatment, * P < 0.05; compared with 4 weeks, # P < 0.05

[0197] Table 11 Improvement of Patients' Sallow Complexion Syndrome Scores ( point)

[0198]

[0199] Note: Compared with before treatment, * P < 0.05; compared with 4 weeks, # P < 0.05

[0200] Table 12 Improvement of edema syndrome scores in patients ( point)

[0201]

[0202] Note: Compared with before treatment, * P < 0.05

[0203] 3. Effects of Yigongsan on peripheral blood GDF15 and ERFE levels

[0204] ERFE is a 340-amino acid soluble protein-6, and it is part of the C1q tumor necrosis factor α-associated protein subtype 15 (CTRP15); GDF15 is a member of the transforming growth factor (TGF)-β superfamily. Both are secreted by erythroid precursors and reflect ineffective erythroid hematopoiesis.

[0205] 3.1 Comparison of serum GDF15 levels between healthy individuals and MDS patients

[0206] The rank-sum test results showed that, compared with healthy individuals, MDS patients had significantly higher serum GDF15 levels, with a statistically significant difference (P < 0.05). (Table 13)

[0207] Table 13 Comparison of serum GDF15 levels between healthy individuals and MDS patients ( pg / ml)

[0208]

[0209] 3.2 Comparison of GDF15 and EREF mRNA in peripheral blood mononuclear cells of two groups of MDS patients

[0210] Multivariate analysis of variance showed that, compared with the placebo group, the relative expression level of GDF15 mRNA in peripheral blood mononuclear cells was significantly decreased at 4 and 8 weeks after drug administration in the Yigongsan group, and the ERFE mRNA expression level was significantly decreased at 12 weeks, with statistically significant differences (P < 0.05). (Table 14-15)

[0211] Table 14 Comparison of relative GDF15 mRNA expression in mononuclear cells of MDS patients

[0212]

[0213] Compared with the placebo group, △ P < 0.05.

[0214] Table 15 Comparison of relative ERFE mRNA expression in mononuclear cells of MDS patients

[0215]

[0216] Compared with the placebo group, ΔP < 0.05.

[0217] 4. The effect of the dissipation of variability on the S100A9-TLR4 shaft

[0218] 4.1 Comparison of S100A9 levels between the two groups of MDS patients

[0219] Repeated measures ANOVA showed that, compared with 4 weeks after treatment, the serum S100A9 level in the Yigongsan group was significantly lower than that in the placebo group after 8 weeks of treatment, with a statistically significant difference (P < 0.05). (Table 16).

[0220] Table 16 Comparison of changes in serum S100A9 levels in MDS patients ( pg / ml)

[0221]

[0222] Note: Compared to 4 weeks,# P < 0.05

[0223] 5. Correlation between peripheral blood S100A9 and GDF15

[0224] Multiple linear regression analysis showed that serum S100A9 and GDF15 in MDS patients before treatment were linearly correlated, with a statistically significant difference (P < 0.05). (Table 17) Figure 1 )

[0225] Table 17. Correlation analysis between S100A9 and GDF15

[0226]

[0227] 6. Correlation between peripheral blood S100A9 and TLR4

[0228] Multiple linear regression analysis showed that serum S100A9 and TLR4 were linearly correlated before treatment, with a statistically significant difference (P < 0.05). (Table 18) Figure 2 )

[0229] Table 18 Correlation Analysis between S100A9 and TLR4

[0230]

[0231] 7. Correlation between peripheral blood GDF15 and S100A9-TLR4 axis-related proteins

[0232] Multiple linear regression analysis showed that serum GDF15, S100A9, and TLR4 in MDS patients before treatment were linearly correlated with each other, with statistically significant differences (P < 0.05). (Table 19) Figure 3 )

[0233] Table 19 Correlation analysis between GDF15 and S100A9, TLR4

[0234]

[0235] 8. Safety Evaluation

[0236] No obvious toxic side effects were observed in any of the patients. Two patients experienced mild liver dysfunction (alanine aminotransferase levels increased by less than 1 time), and four patients experienced mild kidney dysfunction (serum creatinine levels increased by less than 1 time). Since these patients were all taking Western medicines that can cause liver and kidney damage, such as cyclosporine, testosterone undecanoate, and eltrombopag, and with the addition of liver and kidney protection treatments, and continued use of traditional Chinese medicine, their liver and kidney function did not worsen. Therefore, liver and kidney dysfunction caused by traditional Chinese medicine is not considered.

[0237] Conclusion: Based on the inclusion criteria, this trial collected 48 cases. Three patients withdrew from the study, and two were excluded, leaving 43 patients for this unblinding and statistical analysis. Of these, 22 were in the Yigongsan group and 21 in the placebo group. Clinical data and peripheral blood samples were collected before and after medication. Efficacy was analyzed by observing complete blood counts and traditional Chinese medicine (TCM) syndromes. The results showed that Yigongsan can improve the TCM syndrome of spleen deficiency in MDS patients. Further analysis of S100A9, TLR4, and GDF15, and detection of GDF15 and ERFE mRNA in peripheral blood mononuclear cells of some patients, indicated that compared with healthy individuals, MDS patients had significantly higher levels of S100A9 in peripheral blood and a trend towards higher levels of TLR4. A linear correlation exists between peripheral blood S100A9 and TLR4 levels in MDS patients, suggesting that the S100A9-TLR4 axis has been activated in MDS patients. Yigongsan reduces peripheral blood S100A9 and TLR4 levels in low-risk or intermediate-risk MDS patients, indicating that Yigongsan can downregulate the S100A9-TLR4 axis. Peripheral blood S100A9 and GDF15 expression are linearly correlated, and Yigongsan can reduce GDF15 and ERFE mRNA expression in peripheral blood mononuclear cells of MDS patients, suggesting that Yigongsan may intervene in hematopoietic function of MDS by regulating the S100A9-TLR4 axis.

[0238] Example 2. Target Analysis of Yigongsan in the Treatment of MDS

[0239] Experimental methods

[0240] Preparation of Yigongsan dry powder

[0241] The formula for Yigong Powder is as follows: Sun-dried ginseng (origin: Jilin Province, Fusong County Baicaowang Organic Plant Professional Cooperative, batch number: 20062711); stir-fried Atractylodes macrocephala (origin: Anhui Province, Hebei Jincao Pharmaceutical Co., Ltd., batch number: 191101001); Poria cocos (origin: Anhui Province, Yuexi County Baoya Traditional Chinese Medicine Professional Cooperative, batch number: 20062811); dried tangerine peel (origin: Suizhou City, Hubei Province, Suizhou City Hongsheng Traditional Chinese Medicine Planting Farmers Professional Cooperative, batch number: 20062811); prepared licorice root (origin: Inner Mongolia Province, Hebei Jincao Pharmaceutical Co., Ltd., batch number: 191115001). These five ingredients are decocted twice with 12 times the amount of water, 2.5 hours each time. The decoctions are combined, filtered, and the filtrate is concentrated under reduced pressure (60-80℃) to a relative density of 1.10-1.20 (60℃). The filtrate is then spray-dried into a dry extract powder. Provided by Shenwei Pharmaceutical Co., Ltd.

[0242] The compounds in Yigongsan dry powder were analyzed by LC-HRMS, and the target genes that interact with the compounds were screened. The target-channel network was drawn to predict the possible signaling pathways of Yigongsan's effect on MDS and to conduct subsequent experimental verification.

[0243] result

[0244] 1. LC-HRMS analysis of the components of the dry extract of Yigongsan

[0245] The LC-HRMS results are as follows. Based on comparison with previous test results (Evidence-Based Complementary and Alternative Medicine, Volume 2016, Article ID 2696480), the YGS used in this study was determined to contain ginseng, atractylodes macrocephala, poria cocos, licorice, and tangerine peel as the main components. Specific components and compounds can be found in [link to specific list]. Figure 4 Table 20.

[0246] Table 20 Composition of Yigongsan Dry Extract Powder

[0247]

[0248]

[0249]

[0250] Note: RS: Ginseng; BZ: Atractylodes macrocephala; FL: Poria cocos; GC: Licorice; CP: Tangerine peel

[0251] The GeneCards database was used to search for the targets of 65 compounds, and after screening based on a "relevance score ≥ 2", 587 target genes were obtained. Using "Myelodysplastic syndrome" as the keyword, the GeneCards database was used to search for genes related to MDS, and after screening based on a "relevance score ≥ 2", 1506 genes were obtained. Figure 5 A). Taking the intersection of the two yielded 268 intersection target sites, and network pharmacology analysis was performed on these 268 genes. Figure 5 B).

[0252] KEGG analysis predicted 167 signaling pathways in the MDS affected by heterogeneous dispersions, including the PI3K-Akt signaling pathway, NF-κB signaling pathway, TLRs signaling pathway, and VEGF signaling pathway. (See...) Figure 6 )

[0253] GO enrichment analysis was performed on 268 overlapping targets using the Omicshare bioinformatics platform. The results showed 3272 biological processes, 101 cellular components, and 384 molecular functions. Biological processes mainly include responses to cellular physiological processes, metabolic processes, and stimuli; cellular components mainly include cells, cellular components, and tissues; and molecular functions mainly include inhibitory effects, catalytic effects, and molecular functional regulation. (See...) Figure 7 )

[0254] Target-network diagrams were constructed using Cytoscape 3.6.1. A total of 21 genes were enriched in the NF-κB signaling pathway among the intersecting target sites, including TLR4, IL-1β, and TNF-Q (see...). Figure 8-9 The results of clinical trials suggest that the TLR4-NF-κB signaling pathway may be a target for the intervention of dysplasia in MDS.

[0255] 268 intersecting target sites were imported into the STRING database to construct a PPI protein network interaction map (see...). Figure 10 Using a minimum required interaction score ≥ 0.400 as the screening criterion, PPI network analysis revealed that proteins closely related to the interaction between Yigongsan and NF-κB include TNF, TLR4, TLR2, and STAT3. This analysis result corroborates the findings of the target-network diagram analysis.

[0256] Conclusion: Based on the intersection of the components of Yigongsan and genes related to MDS, 268 genes were obtained. Further enrichment of these genes using KEGG and GO yielded 167 signaling pathways, primarily including the PI3K-Akt, NF-κB, TLRs, and VEGF pathways. Combining target-network diagrams and PPI network diagrams, the focus was ultimately on the TLR4-NF-κB signaling pathway. Based on clinical research results, it is predicted that the target of Yigongsan may be the S100A9-activated TLR4-NF-κB signaling pathway.

[0257] Example 3. Cellular experiments verify the mechanism by which Yigongsan improves ineffective hematopoiesis through the inflammatory pathway.

[0258] Experimental subjects: human MDS cell lines SKM-1 and MUZT-1. All cells were sent to Shanghai Yihe Applied Biotechnology Co., Ltd. for cytogenetic quality assessment.

[0259] Preparation of drug-containing serum

[0260] Seven female SD rats (200-250g each) were purchased from Shanghai Bikai Experimental Animal Company and housed at the Animal Experiment Center of Shanghai University of Traditional Chinese Medicine. The animals were housed in an SPF-grade standard animal room at 24℃-26℃ with automatic cyclic lighting and a 12-hour light-off system, with free access to water and food. They were divided into two groups: a blank serum group (n=3) and a drug-containing serum group (n=4). The drug-containing serum group was administered 7.713g crude drug / kg / day, 1ml / 100g, by gavage twice a day for three consecutive days. Blood was collected one hour after the last gavage to prepare the drug-containing serum.

[0261] Modeling and Experimental Grouping

[0262] Modeling: SKM-1 cells were incubated with YGS for 1 h, and MUZT-1 cells were incubated in groups for 48 h, and then stimulated with S100A9 (4 ug / mL) for 24 h.

[0263] Blocker: NF-κB inhibitor BAY-11-7082 (BAY) to reduce NF-κB gene expression: Before drug intervention, after modeling, 25 uM NF-κB inhibitor (BAY) was added to stimulate for 1 hour.

[0264] Grouping and processing are shown in Table 21:

[0265] Table 21 Grouping and Processing Methods

[0266]

[0267] result:

[0268] 1. LC-HRMS analysis of serum components containing Yigongsan drug

[0269] A blank serum sample was used as a negative control. The test results are as follows. Based on the comparison with previous test results (Evidence-Based Complementary and Alternative Medicine, Volume 2016, Article ID 2696480), the serum components containing Yigongsan used in this study were determined to be licorice and tangerine peel. (See...) Figure 11 ab)

[0270] 2. Results of SKM-1 cell experiments

[0271] In this experiment, a high-risk MDS cell line (SKM-1 cells) was used.

[0272] 2.1 Cell viability

[0273] One-way ANOVA results showed that, compared with 10% FBS, cell viability was significantly reduced after co-incubation for 24h and 48h with blank serum (2.5%, 5%, and 7.5%) and serum containing Yigongsan (2.5%, 5%, and 7.5%), respectively. Cell viability increased after co-incubation for 72h with 2.5% NC (2.5% YGS), with statistically significant differences (P < 0.05). Based on CCK-8 and preliminary experimental results, 2.5% YGS was selected for subsequent experiments after 72h. (Table 22) Figure 12 )

[0274] Table 22 Comparison of SKM-1 cell viability at different concentrations and time points ( %)

[0275]

[0276] Note: Compared to 10% fetal bovine serum...* P < 0.05

[0277] 2.2 Comparison of intracellular NF-κB, ERFE, and GDF15 mRNA levels in each group

[0278] One-way ANOVA and rank-sum test results showed that, compared with the 2.5% NC group, the expression of NF-κB, GDF15, and ERFE mRNA in the model group was significantly increased (P < 0.05); compared with the model group, the expression of NF-κB and ERFE mRNA in the Yigongsan group was significantly decreased (P < 0.05); GDF15 mRNA showed a significant decreasing trend, but no statistical difference (P > 0.05). In the blocker group, NF-κB mRNA was significantly decreased, GDF15 mRNA expression was significantly increased (P < 0.05), and ERFE mRNA showed an increasing trend, but no statistical difference (P > 0.05); compared with the blocker group, the expression of GDF15 mRNA in the Yigongsan + blocker group was significantly decreased (P < 0.05), while there was no statistical difference in NF-κB mRNA and ERFE mRNA (P > 0.05). (See Table 23) Figure 13 ac)

[0279] Table 23 Comparison of NF-κB, ERFE, and GDF15 mRNA expression in SKM-1 cells

[0280]

[0281] Note: Compared with the blank group, * P < 0.05; compared with the model group, △ P < 0.05; compared with the blocking agent group, ▲ P < 0.05

[0282] 2.3 Intracellular expression of TLR4 and p-p65 proteins in each group

[0283] One-way ANOVA and rank-sum test results showed that, compared with the control group, the expression of TLR4 and p-p65 proteins in the model group was significantly increased (P < 0.05); compared with the model group, the expression of p-p65 protein in the Yigongsan group was significantly decreased (P < 0.05), while the expression of TLR4 protein showed no significant change (P > 0.05); the expression of TLR4 and p-p65 proteins in the inhibitor group was significantly decreased (P < 0.05); compared with the inhibitor group, the expression of p-p65 protein in the Yigongsan + inhibitor group was significantly decreased (P < 0.05), while the expression of TLR4 protein showed no significant change (P > 0.05). (See Table 24) Figure 14 ac)

[0284] Table 24 Comparison of TLR4 and p-p65 grayscale values ​​of SKM-1 groups

[0285]

[0286] Note: Compared with the blank group, * P < 0.05; compared with the model group, △ P < 0.05; compared with the blocking agent group, ▲ P < 0.05.

[0287] 3. Results of MUZT-1 cell experiments

[0288] In this experiment, a low-risk MDS cell line (MUZT-1 cells) was used.

[0289] 3.1 Cell viability

[0290] One-way ANOVA results showed that, compared with 10% FBS, cell viability was significantly reduced after co-incubation for 48h and 72h with 2.5%, 5%, and 7.5% blank serum and 2.5%, 5%, and 7.5% drug-containing serum, respectively. Cell viability decreased after co-incubation for 24h with 7.5% blank serum and 2.5% drug-containing serum, with statistically significant differences (P < 0.05). Based on CCK-8 and preliminary experimental results, 2.5% drug-containing serum was selected for subsequent experiments after 24h. (See Table 25) Figure 15 )

[0291] Table 25 Comparison of MUZT-1 cell viability ( %)

[0292]

[0293] Note: Compared to 10% fetal bovine serum... * P < 0.05

[0294] 3.2 Intracellular NF-κB, ERFE, and GDF15 mRNA levels in MUZT-1 cells

[0295] One-way ANOVA and rank-sum test results showed that, compared with the control group, the expression of ERFE mRNA in the model group was significantly increased (P < 0.05); the expression of NF-κB and GDF15 mRNA showed no significant change (P > 0.05). Compared with the model group, the expression of NF-κB, GDF15, and ERFE mRNA in the Yigongsan group was significantly decreased, while the expression of GDF15 mRNA was significantly increased (P < 0.05). The expression of NF-κB, GDF15, and ERFE mRNA in the blocking agent group was significantly decreased (P < 0.05). Compared with the blocking agent group, the expression of NF-κB, GDF15, and ERFE mRNA in the Yigongsan + blocking agent group showed no significant change (P > 0.05). (See Table 33) Figure 16 ac)

[0296] 3.3 Intracellular expression of TLR4 and p-p65 proteins in each group

[0297] One-way ANOVA and rank-sum test results showed that, compared with the control group, the expression of p-p65 protein in the model group was significantly increased (P < 0.05), while the TLR4 protein content showed no significant change (P > 0.05). Compared with the model group, the expression of p-p65 protein in the Yigongsan group was significantly decreased (P < 0.05), while the expression of TLR4 protein showed no significant change (P > 0.05). The expression of TLR4 protein in the inhibitor group was significantly increased. - p65 protein expression was significantly decreased (P < 0.05); compared with the inhibitor group, intracellular TLR4 protein was significantly decreased in the Yigongsan + inhibitor group (P < 0.05), while p-p65 protein showed no significant change (P > 0.05). (See Table 26) Figure 17 ac)

[0298] Table 26 Comparison of gray values ​​of TLR4 and p-p65 in MUZT-1 cells of different groups

[0299]

[0300] Note: Compared with the blank group, * P < 0.05; compared with the model group, △ P < 0.05; compared with the blocking agent group, ▲ P < 0.05.

[0301] Conclusion: This application selected two MDS cell lines, SKM-1 and MUZT-1, and activated the TLR4-NF-κB signaling pathway with S100A9, respectively, and added the NF-κB inhibitor BAY. The effects of Yigongsan on the S100A9-TLR4-NF-κB signaling pathway were observed. The results showed that Yigongsan could downregulate the NF-κB gene or protein in both SKM-1 and MUZT-1 MDS cell lines through the S100A9-TLR4 pathway, thereby regulating hematopoietic function (GDF15, EFER). The effect on MUZT cells was significantly reduced after NF-κB inhibition, while the effect remained in SKM-1 cells, suggesting that the S100A9-TLR4-NF-κB signaling pathway is the key pathway for Yigongsan's effect on MUZT cells, while other pathways may also be involved in the action on SKM-1 cells.

[0302] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0303] Spleen Qi Deficiency Syndrome Scoring Table

[0304]

Claims

1. The use of a special powder, characterized in that, For the preparation of a drug, said drug having uses selected from the group consisting of: 1) Improve the inflammatory microenvironment of bone marrow in MDS patients and reduce ineffective hematopoiesis in MDS patients; 2) Improve the TCM symptoms of MDS patients; 3) Improve the hematopoietic function of MDS patients; 4) Treatment of MDS.

2. The use as described in claim 1, characterized in that, The TCM symptoms include: fatigue, lethargy, lack of appetite, bland taste in the mouth, sallow complexion, and edema.

3. The use as described in claim 1, characterized in that, The MDS cells include MDS with single-lineage dysplastic hematopoietic cells (MDS-SLD), MDS with ring sideroblasts (MDS-RS), MDS with multi-lineage dysplastic hematopoietic cells (MDS-MLD), SKM-1 cell line, MUZT-1 cell line, MDS-IB1, and MDS-IB2.

4. The use as described in claim 1, characterized in that, The drug is an oral dosage form.

5. The use as described in claim 1, characterized in that, In the formula of Yigongsan, the ratio of ginseng, atractylodes macrocephala, poria cocos, prepared licorice root, and tangerine peel is (1±0.2):(1±0.2):(1±0.2):(1±0.2):(1±0.2), preferably (1±0.1):(1±0.1):(1±0.1):(1±0.1):(1±0.1), and optimally (1±0.05):(1±0.05):(1±0.05):(1±0.05):(1±0.05) or 1:1:1:1:

1.

6. The use as described in claim 1, characterized in that, The dosage regimen of the Yigongsan is as follows: 100g of medicinal materials per person per day, wherein the 100g of medicinal materials includes 20g each of ginseng, poria cocos, stir-fried atractylodes macrocephala, tangerine peel and prepared licorice root.

7. The use as described in claim 1, characterized in that, The drug inhibits the S100A9-TLR4-NF-κB signaling pathway.

8. The use as described in claim 1, characterized in that, The drug inhibits the expression of GDF15 and ERFE.

9. The use as described in claim 1, characterized in that, The MDS patients mentioned are low-risk or intermediate-risk MDS patients.

10. A method for in vitro inhibition of the S100A9-TLR4-NF-κB signaling pathway, characterized in that, The method includes applying an effective amount of Yigongsan to in vitro cells.

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