Use of helenalin in the preparation of a medicament for alleviating and treating erythroid differentiation disorder of cells in a subject

Abstract

The present invention discloses the application of helenalin in the preparation of a medicament for alleviating and treating erythroid differentiation disorder of cells in a subject, belonging to the field of pharmaceutical preparations. The chemical structure of helenalin disclosed in the present invention is shown in Formula 1. It has been experimentally verified that helenalin can effectively correct the erythroid differentiation disorder caused by ribosomal protein gene mutations, specifically manifested as promoting the differentiation of hematopoietic stem cells with erythroid differentiation disorder into multipotent progenitor cells, promoting the differentiation of common myeloid progenitor cells with erythroid differentiation disorder into megakaryocyte-erythroid progenitor cells, alleviating problems such as the decrease in the proliferation ability of erythroid phenotype cells and the increase in apoptosis in erythroid differentiation disorder caused by ribosomal protein gene mutations, so that the erythroid phenotype cells are overall alleviated or restored. Therefore, the present invention can provide ideas for the treatment of diseases with erythroid differentiation disorder, such as DBA drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical formulations, and in particular to the use of helichrysin in the preparation of a drug for alleviating and treating erythroid differentiation disorders in a subject. Background Art

[0002] Congenital pure red cell aplasia (Diamond Blackfan anemia, DBA) is a rare congenital erythrocyte dysplasia disease with no difference in the incidence between males and females. It usually manifests in infancy and is a ribosomal disease. The common clinical features of DBA include anemia, macrocytosis, and elevated erythrocyte adenosine deaminase activity, and are usually associated with short stature, physical abnormalities, and cancer susceptibility. Erythrocyte dysplasia is the main hematological feature of DBA patients, mainly manifested as macrocytic anemia, reduced erythroid progenitor cells, and reduced reticulocytes. Studies have shown that DBA patients are significantly deficient in red blood cells, mainly due to the lack of erythroid progenitor cells in the bone marrow (BM) at the time of initial diagnosis, such as burst-forming unit-erythroid (BFU-E) cells, but all other hematopoietic lineages are minimally affected.

[0003] The genetic basis of DBA has been widely characterized, and approximately 60-70% of DBA patients carry lesions in genes encoding small 40s ribosomal protein subunits (RPS) or large 60s ribosomal protein subunits (RPL). Among the RP genes, RPS19 is the most commonly mutated gene, accounting for approximately 25% of DBA patients. The next most common mutated genes are RPL5 (approximately 7% of DBA patients), RPS26 (approximately 7% of DBA patients), and RPL11 (approximately 5% of DBA patients). In addition, mutations in a small number of non-RP genes have been found, such as the erythroid transcription factor GATA1 and the RPS26 interacting protein TSR2. Possible pathological mechanisms include abnormal ribosome biogenesis in DBA patient cells due to mutations in RPs genes, leading to the release and binding of RPL5 and RPL11 to MDM2. Since MDM2 is a negative regulator of p53, p53 activity is activated. P53 is a key cell cycle regulatory protein that plays an important role in cell stress situations such as DNA damage. Due to the high sensitivity of erythroid progenitor cells to p53 activation, cell cycle arrest and apoptosis occur, ultimately leading to erythroid hematopoietic failure. Another triggering factor is thought to be the accumulation of cytotoxic free heme due to an imbalance between globin protein synthesis and heme production caused by a global reduction in ribosome supply, which impairs the growth of red blood cells. Studies have also pointed out that in bone marrow tissue, during the process of other lineage hematopoietic stem and progenitor cells differentiating into erythrocytes, a large amount of ribosomes are required for protein synthesis due to the activation of the cell cycle at the erythroid progenitor stage. However, due to RPs mutations leading to ribosomal haploinsufficiency, erythroid-specific defects occur. Recent studies have shown that due to the excessive cell cycle speed at the BFU-E stage, replication stress is induced, activating the expression of TP53 and resulting in a large number of apoptotic cells at the BFU-E stage, affecting erythroid development. In summary, previous reports on the pathological mechanism of erythroid-specific defects in DBA mainly focused on the study of the erythroid progenitor (EP) stage, which may explain to some extent the reasons for erythroid-specific defects in DBA. However, in the context where all cell types have a general requirement for ribosome biogenesis, especially when the erythroid system (nucleated erythrocytes) in the bone marrow accounts for not the largest proportion, approximately 20% of nucleated cells, while the granulocyte system accounts for the largest proportion, approximately 40-60% of nucleated cells, the reason for erythroid production failure due to abnormal ribosome synthesis caused by RPs mutations not being able to meet the demand for a large amount of protein synthesis during erythroid development still seems to be a paradox.

[0004] In addition, some transcription factors play important roles in the process of erythroid differentiation, including GATA1, KLF1, etc. The abnormal expression of these transcription factors may be related to the occurrence of DBA. Recent studies have shown that the pathways of inflammation, immunity, oxidative phosphorylation, lipogenesis, etc. in DBA bone marrow are significantly abnormal, and the transcriptome maps caused by different RP mutations vary greatly, which may indicate that in addition to erythroid hematopoietic defects, there are also other abnormal cellular components with unknown functions in the bone marrow. It is worth pondering that through the single-cell data of DBA bone marrow, it is found that immune cells also have the activation of the cell cycle, but the clinical manifestations show that the fate of immune cells is quite different from that of erythroid cells. This indicates that there are other reasons in addition to the erythroid defect mechanism caused by ribosomal haploinsufficiency. Interestingly, in the single-cell data of normal controls, it is observed that compared with cells in the later stage of development, more primitive hematopoietic stem / progenitor cells seem to have a greater demand for ribosomal protein synthesis. Therefore, it is speculated that the cell defects caused by RP mutations in DBA may not only start at the erythroid progenitor (EP) stage, but may already appear at the more primitive hematopoietic stem / progenitor cell (HSPC) stage.

[0005] The mechanism of DBA erythroid cell failure has not been fully determined, and there are few reports on the impact of RP haploinsufficiency on HSPCs and the entire bone marrow microenvironment. The hematopoietic system can be traced back to the seed cells, hematopoietic stem cells (HSCs), which are a type of cells that can self-renew and differentiate into various blood cells, including red blood cells, white blood cells, and platelets. HSCs are mainly present in the bone marrow and placenta and are key cells necessary for maintaining the normal function of the blood system. There are rare reports that certain gene expressions are abnormal in hematopoietic stem / progenitor cells. In addition, DBA patients are prone to age-related pancytopenia, myelodysplastic syndrome, and myeloid leukemia. Although Rps19+ / - mice do not show hematopoietic abnormalities, further reducing Rps19 by expressing short hairpin RNA in vivo leads to trp53-dependent anemia and HSC failure. HSPCs from DBA patients have intrinsic quality defects and show reduced clonogenic output in long-term culture-initiating assays. Two studies examined HSPCs from DBA patients after xenotransplantation into immunodeficient mice. One of the studies showed that transducing CD34+ mutant HSPCs from DBA patients with a lentiviral vector (LV) expressing rps19 improved their engraftment, but healthy donor HSPCs were not examined as a control. In another report, HSPCs from DBA patients engrafted normally in NOD / SCID mice, but erythropoiesis was reduced. The engraftment rates of both DBA and control HSPCs were less than 1%. A recent literature report indicated that HSPCs in DBA are impaired. Editing RPS19 in HSPCs in a cell model resulted in erythroid lineage defects, indicating that HSPCs are impaired, but the internal abnormalities in hematopoietic stem / progenitor cells were not given. The current research on hematopoietic stem / progenitor cells in Diamond-Blackfan anemia is still in its preliminary stage.

[0006] Despite the gradual in - depth study of the pathogenesis, the treatment methods for DBA still mainly rely on glucocorticoids, suspension red blood cell transfusion, and allogeneic hematopoietic stem cell transplantation. All three treatment methods have side effects to varying degrees and need to be carefully considered in clinical applications. Oral glucocorticoids remain the first - line treatment for DBA currently. Although the dose of glucocorticoids is small, the patients are young when receiving treatment and need to take the drugs for a long time. Some patients even need to take the drugs for life, and the side effects of hormones cannot be ignored. Moreover, the most important current clinical problem is that different DBA patients have very different sensitivities to glucocorticoids (such as prednisone, whose mechanism of action can drive the expansion of erythroid progenitor cells). Although about 80% of DBA children respond to glucocorticoids, ultimately only 20% of the patients can achieve remission, 40% of the patients rely on drugs to maintain, and the other 40% of the patients need blood transfusion to maintain. However, the iron overload caused by long - term blood transfusion has an equally non - negligible impact on the growth and physical condition of children. Hematopoietic stem cell transplantation is the only current treatment method that can cure DBA. Some studies have shown that the 5 - year overall survival rate is about 74%, and chronic graft - versus - host disease after transplantation is the main factor affecting its overall survival rate. In addition, cyclosporine A, lenalidomide, and L - leucine have also been applied to the treatment of DBA, but their mechanisms of action are not yet clear and the curative effects are inconsistent. Therefore, finding other action pathways, further exploring the genetic abnormalities of DBA, and precise drug use are the current research focuses to alleviate clinical problems. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a drug that can correct the erythroid differentiation disorder of cells, thereby alleviating and treating erythroid differentiation disorder diseases.

[0008] In the first aspect, the present invention provides the use of a compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for alleviating and / or treating erythroid differentiation disorder of cells in a subject;

[0009] 。

[0010] For the use as described above, the compound represented by Formula 1 is named Helichrysetin.

[0011] For the use as described above, the pharmaceutically acceptable salt refers to a salt formed by the compound represented by Formula 1 and a pharmaceutically acceptable acid or base. The term "pharmaceutically acceptable" refers to a substance that is suitable for humans without excessive adverse reactions and side effects.

[0012] In the applications described above, the subject can be a mammal, which can be selected from Bovidae, Equidae, Felidae, Canidae, Leporidae, Suidae, Camelidae, rodents, and primates, etc., including but not limited to cows, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, orangutans), and humans. Preferably, the mammals are cows, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys, and humans.

[0013] In the applications described above, the erythroid differentiation disorder of the cells includes erythroid differentiation disorder of the cells caused by mutations in ribosomal protein genes of the cells and / or erythroid differentiation disorder of the cells caused by mutations in non-ribosomal protein genes. Further, the erythroid differentiation disorder of the cells is caused by mutations in ribosomal protein genes of the cells. Still further, the mutations in ribosomal protein genes of the cells include at least one of RPS19 mutation, RPL5 mutation, RPS26 mutation, and RPL11 mutation. Still further, the mutations in ribosomal protein genes include RPS19 mutation, and the RPS19 mutation is a mutation that results in a decrease in the expression level of the RPS19 gene.

[0014] In the applications described above, the subject has congenital pure red cell aplasia.

[0015] In the applications described above, the alleviation and / or treatment of the erythroid differentiation disorder of the subject is manifested as an increase in the number and / or proportion of erythroid cells of the subject. Further, the erythroid differentiation disorder of the cells at least includes erythroid differentiation disorder of hematopoietic stem progenitor cells (HSPCs) that can differentiate into hematopoietic lineages and produce all blood cell types. Still further, the alleviation and / or treatment of the erythroid differentiation disorder of the subject is specifically manifested as at least one of the following:

[0016] 1) Promoting the differentiation of hematopoietic stem cells with erythroid differentiation disorder into multipotent progenitor cells;

[0017] 2) Promoting the differentiation of common myeloid progenitor cells with erythroid differentiation disorder into megakaryocyte-erythroid progenitor cells;

[0018] 3) Increasing the number and / or proportion of erythroid cells that are positive for CD71 and CD235a.

[0019] For the application as described above, the dosage form of the drug can be determined according to actual needs, and can be selected from, for example, injections, tablets, capsules, buccal tablets, oral liquids, granules, instant decoctions, pills, powders, ointments, syrups, mixtures, floral waters, effervescent agents, pastes, emulsions, tea preparations, elixirs, suspensions, powders, implants, ointments, plasters, creams, sprays, drops, patches, etc. Further, the dosage form of the drug is selected from one of injections, tablets, capsules, buccal tablets, oral liquids, suspensions, implants, patches.

[0020] Furthermore, the dosage form of the drug is an injection, such as liquid injections, powder injections, tablets for injection, etc. The injection forms include but are not limited to the form of the original drug, drug-loaded nanoparticles, etc.; the injection sites include but are not limited to intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and intraspinal injection, etc.

[0021] Furthermore, the dosage form of the drug is an implant, and the implant is selected from one of cavity-controlled implants, matrix implants, and osmotic pressure-driven implants.

[0022] For the application as described above, the drug further comprises a pharmaceutically acceptable carrier. Further, the pharmaceutically acceptable carrier includes at least one of excipients, lubricants, pH regulators, stabilizers, encapsulants, sprays, binders, fillers, disintegrants, wetting agents, transdermal absorbers, absorption promoters, surfactants, flavoring agents, coloring agents, preservatives, sweeteners, osmotic pressure regulators, adsorption carriers.

[0023] In a second aspect, the present invention provides the use of the compound shown in formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for correcting and / or alleviating erythroid differentiation disorder of isolated cells;

[0024] 。

[0025] In a third aspect, the present invention provides a method for correcting and / or alleviating erythroid differentiation disorder of isolated cells, comprising contacting the isolated cells with the compound shown in formula 1 or a pharmaceutically acceptable salt thereof to correct and / or alleviate the erythroid differentiation disorder of the isolated cells;

[0026] 。

[0027] For the method as described above, the compound shown in formula 1 can be contacted with cells having erythroid differentiation disorder in a cell culture system, or can also be contacted in other forms of carriers.

[0028] For the method as described above, the concentration of the compound shown in formula 1 in the cell culture system is 5 - 20 μM, and the culture time is 24 - 72 h.

[0029] Fourth aspect, the present invention provides a method for alleviating and / or treating erythroid differentiation disorder of a subject's cells, the method comprising administering to the subject a compound of formula 1 or a pharmaceutically acceptable salt thereof in an amount effective for alleviating and / or treating, or a medicament comprising the compound of formula 1 or a pharmaceutically acceptable salt thereof as described above.

[0030] The method as described above, wherein the subject suffers from congenital pure red cell aplasia.

[0031] For the method as described above, the dosage of the medicament for alleviating and / or treating erythroid differentiation disorder of a subject depends on the mode of administration, the route of administration, the age and / or weight of the individual, and the condition of the treated individual, and is variable and ultimately determined by the attending physician. The dosage administered to an individual, in the context of the present invention, should be for a period sufficient to elicit a beneficial response in the individual.

[0032] The chemical structure of helichrysin disclosed in the present invention is shown in formula 1. It has been experimentally verified that helichrysin can effectively correct the erythroid differentiation disorder caused by ribosomal protein gene mutations, specifically manifested as promoting the differentiation of hematopoietic stem cells with erythroid differentiation disorder into multipotent progenitor cells, promoting the differentiation of common myeloid progenitor cells with erythroid differentiation disorder into megakaryocyte-erythroid progenitor cells, alleviating problems such as the reduction of the proliferation ability of erythroid phenotype cells and the increase of apoptosis in erythroid differentiation disorder caused by ribosomal protein gene mutations, so that the erythroid phenotype cells are overall alleviated or restored. Therefore, the present invention can provide ideas for drugs for treating erythroid differentiation disorder diseases such as DBA. Description of the Drawings

[0033] Figure 1 Analysis results of HSPC cells of DBA patients; among them, A is the population analysis result of CD34+ HSPC cells; B is the marker analysis result of CD34+ HSPC cells; C is the result of GSVA verification of the population analysis result against the known population gene set; D is the evaluation result of the proportion of CD34+ HSPC cells in normal healthy people (Ctrl) and DBA patients (Patient); E is the statistical result of the significant difference in the number of cells in the CD34+ HSPC cell population between normal healthy people (Ctrl) and DBA patients (Patient); F is the result of the pseudo-time differentiation trajectory analysis of four cell subsets (HSC / MPP, CMP, MEP, CLP) in normal healthy people (Ctrl) and DBA patients (Patient);

[0034] Figure 2 Analysis results of the relative expression levels of the RPS19 gene in the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random);

[0035] Figure 3 Expression of cell surface CD235a and CD71 antigens after 7 days of directed erythroid differentiation culture of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random); A shows the results of flow cytometry detection, and B shows the statistical results of the proportion of CD235a and CD71 double-positive cells.

[0036] Figure 4 Analysis results of the relative expression level of the TP53 gene after 7 days of directed erythroid differentiation culture of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random).

[0037] Figure 5 Giemsa staining of cells after 10 days of directed erythroid differentiation culture of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random).

[0038] Figure 6 Expression of cell surface CD11b antigen after 5 days of monocyte differentiation of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random); A shows the results of flow cytometry detection, and B shows the statistical results of the proportion of CD11b-positive cells.

[0039] Figure 7 Expression of cell surface CD11b antigen after 36 days of monocyte differentiation of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random); A shows the results of flow cytometry detection, and B shows the statistical results of the proportion of CD11b-positive cells.

[0040] Figure 8 Expression of cell surface CD15 and CD68 antigens after 7 days of granulocyte differentiation culture of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random); A shows the results of flow cytometry detection, and B shows the statistical results of the proportion of CD15 and CD68 double-positive cells.

[0041] Figure 9 Expression of cell surface CD15 after 22 days of granulocyte differentiation culture of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random); A shows the results of flow cytometry detection, and B shows the statistical results of the proportion of CD15-positive cells.

[0042] Figure 10Flow cytometry detection results of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random);

[0043] Figure 11 Population statistics results of the constructed DBA HSPC cell model (sh-RPS19) and control cells (sh-Random);

[0044] Figure 12 Statistical results of the proportion of hematopoietic stem and progenitor cell subsets after adding different concentrations of helichrysin during the proliferation of the DBA HSPC cell model (sh-RPS19);

[0045] Figure 13 Erythrocyte differentiation after adding different concentrations of helichrysin during the differentiation of the DBA HSPC cell model (sh-RPS19); among them, A is the flow cytometry detection result; B is the statistical result of the erythrocyte differentiation ratio.

[0046] In the above figure, * indicates p -value ≤ 0.05, with statistical significance; ** indicates p -value ≤ 0.01, with significant statistical significance; *** indicates p -value ≤ 0.001, with extremely significant statistical significance; ns indicates no statistical significance. Detailed implementation manners

[0047] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention, and they should not be construed as limiting the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. In the description of the present invention, it should be understood that the terms used are only for the purpose of description and cannot be construed as indicating or implying relative importance.

[0048] In the following embodiments, unless otherwise defined, the terms should be understood according to the conventional usage of those of ordinary skill in the relevant art. The experimental methods involved in the following embodiments are all conventional methods, unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0049] The StemSpan SFEM II culture medium used in the following examples was purchased from StemCell Technologies, USA; the cytokines SCF, TPO, Flt-3 Ligand, and IL-3 were purchased from PeproTech, USA; streptomycin and penicillin were purchased from Yantai Sails Biotechnology Co., Ltd.; Helichrysetin with the chemical structure shown in Formula 1 was purchased from MedChemExpress, USA, with the catalog number HY-N4058 and the CAS number 62014-87-3; the flow antibodies PE conjugated anti-CD34, APC conjugated anti-CD38, BV421 conjugated anti-CD90, AF700 conjugated anti-CD45RA, BV510 conjugated anti-CD10, PE-Cy7 conjugated anti-CD135, APC conjugated anti-CD235a, and PE conjugated anti-CD71 were purchased from BioLegend, USA; the flow antibodies APC-CD11b, APC-CD68, and PE-CD15 were purchased from Elabscience / EliteBio, China.

[0050] Example 1. Single-cell transcriptome analysis of HSPC (hematopoietic stem and progenitor cells) in DBA patients

[0051] As Figure 1 shown in A below, by using single-cell transcriptome sequencing data, a cell atlas of CD34+ HSPC cells was constructed, and the CD34+ HSPC cells were divided into 16 populations: HSC / MPP (hematopoietic stem cell / multipotent progenitor cell), CMP (common myeloid progenitor cell), CLP (common lymphoid progenitor cell), GMP (granulocyte-monocyte progenitor cell), MEP (megakaryocyte-erythroid progenitor cell), ERR-Early (early erythroid progenitor cell), ERP (erythroid progenitor cell), Erythroblast (erythroblast), MKP (megakaryocyte progenitor cell), MDP-1 (monocyte-dendritic progenitor cell type 1), MDP-2 (monocyte-dendritic progenitor cell type 2), Eo / B / Mast (eosinophil / basophil / mast cell progenitor cell), Lymphoid-UNK (NK cell progenitor cell), pre_PC (pre-plasma cell), pre_B (B cell progenitor cell), and pre_T (T cell progenitor cell). For Figure 1 the biomarkers of the 16 cell subtypes shown in A below were analyzed, and the analysis results are as Figure 1As shown in B, it can be seen that the specific biomarkers of the 16 types of cells are significant, and the cells are effectively distinguished. The clustering results are verified by GSVA against the known clustering gene set, as Figure 1 shown in C of this example. The clustering results provided in this example can be well matched with the cell populations known in the public data, reflecting the reliability of the clustering results in this example.

[0052] According to the above clustering results, the clustering ratios of CD34+ HSPC cell subtypes in normal healthy people (Ctrl) and DBA patients (Patient) are evaluated, and the evaluation results are as Figure 1 shown in D. It can be seen that the ratios of progenitor cell stages (MEP, ERP-early, EPR) related to erythroid differentiation in DBA patients are down-regulated, while the ratios of lymphoid progenitor cells (CLP, Lymphoid-UNK, pre_T) related to lymphoid differentiation are up-regulated. Then, a significant difference statistic is performed on the cell numbers of CD34+ HSPC cell populations in normal healthy people (Ctrl) and DBA patients (Patient), and the statistical results are as Figure 1 shown in E. It can be seen that the erythroid lineages MEP and ERP-Early in the HSPC cell population are significantly reduced, other lineages such as the lymphoid lineage CLP are significantly increased, the early HSC / MPP is reduced to a certain extent, and the GMP early myeloid progenitor cells related to myeloid differentiation are increased to a certain extent, indicating that the cell ratios of each progenitor cell subset of hematopoietic stem / progenitor cells have changed. A pseudo-time differentiation trajectory analysis is performed on four cell subsets (HSC / MPP, CMP, MEP, CLP) of normal healthy people (Ctrl) and DBA patients (Patient), and the analysis results are as Figure 1 shown in F. It can be seen that the number of seed cells in DBA patients (Patient) decreases towards the cells at the MEP stage of early erythroid-megakaryocyte progenitors.

[0053] The above results show that the HSPC cell ratio in DBA patients is abnormal, suggesting that there is an abnormality in hematopoietic stem progenitors, and it is specifically the down-regulation of the ratio of early erythroid-related cells.

[0054] Example 2. Construction of an HSPC cell differentiation model with RPS19 deficiency

[0055] 2.1. Extraction of modeling cells:

[0056] CD34+ HSPC cells were isolated from human umbilical cord blood obtained from Beijing Obstetrics and Gynecology Hospital. The specific steps are as follows: 1.1. Use a sterilized scissors to cut the catheter of the blood bag containing human umbilical cord blood and disinfect the cut end again with the outer flame of an alcohol lamp. 1.2. Transfer the umbilical cord blood to a 50 mL centrifuge tube, with each tube containing approximately 15 mL of umbilical cord blood. 1.3. Add RosetteSep TM Cocktail at a rate of 5 μL / mL, that is, approximately 80 μL per tube, and let it stand at room temperature for 20 min. 1.4. Take 50 mL of SepMate TM density gradient centrifuge tubes (the number is the same as the 50 mL centrifuge tubes containing umbilical cord blood), and add 15 mL of lymphocyte separation solution to each tube, paying attention to avoiding light. 1.5. Add 1×PBS solution containing 2% serum and 1 mM EDTA with the same volume as the umbilical cord blood (about 15 mL) to the 50 mL centrifuge tube containing umbilical cord blood, invert it up and down to mix evenly, and then gently add it to the density gradient centrifuge tube containing lymphocyte analysis solution. 1.6. Set the break of the centrifuge to the lowest, and centrifuge the mixture in step 1.5 at 1200 g for 20 min. 1.7. Discard the supernatant 1 cm outside the cloudy cell layer with a 10 mL pipette, quickly pour the remaining liquid containing the cloudy cell layer into a new 50 mL centrifuge tube, and finally add PBS to make up to 50 mL. 1.8. Set the break to 3300 g and centrifuge for 10 min. 1.9. Remove all the supernatant, resuspend the precipitate with 750 μL of 1×PBS, and transfer it to a 14 mL round-bottom tube with a polypropylene snap cap. Add 75 μL of EasySep TM Positive Selection Cocktail, and let it stand at room temperature for 10 min. 1.10. Add 50 μL of EasySep TM Dextran Rapid Spheres, let it stand at room temperature for 3 min, then make up to 3 mL with 1×PBS and mix well by pipetting with a dropper. 1.11. Insert the test tube into the EasySep™ Magnet. After 3 min, pour out the liquid in the tube, add another 3 mL of 1×PBS and mix well. After 3 min, discard the liquid. Repeat this washing process 4 times, and the round-bottom tube cannot be taken out during the whole process. 1.12. After the washing is completed, take out the test tube, add a certain amount of proliferation medium, resuspend the cells adsorbed on the tube wall, count the cells, and culture the cells in a 6-well plate at a certain density to obtain HSPC (CD34+) cells.

[0057] 2.2. Culture of modeling cells:

[0058] The extracted HSPC (CD34+) cells were cultured in a proliferation medium for 7 days of proliferation culture to expand a certain number of cells to meet the subsequent experiments. The proliferation medium was SFEM II medium containing 50 ng / mL of SCF, 50 ng / mL of Flt3 Ligand, 50 ng / mL of TPO, and 1% double antibody, and the cell density was 5×10 4 cells / mL, and placed in a cell incubator (37 °C, 5% (V / V) CO 2 2) to prepare for the subsequent construction of the cell model.

[0059] 2.3. Construction of lentivirus (shRPS19)-infected HSPC cells:

[0060] 2.3.1. Design shRNA for knocking down the RPS19 gene online through the website (the website address is http: / / rnaidesigner.thermofisher.com / rnaiexpress / sort.do), and synthesize the oligonucleotide chains required for expressing sh-RPS19. The nucleotide sequences of the oligonucleotide chains are shown in SEQ ID NO:1-2 respectively.

[0061] SEQ ID NO:1:

[0062] 5’-GATCC gagatctggacagaatcgc cttcctgtcaga gcgattctgtccagatctc tttttg-3’;

[0063] SEQ ID NO:2:

[0064] 5’-AATTCAAAAA gagatctggacagaatcgc TCTGACAGGAAG gcgattctgtccagatctc G-3’.

[0065] 2.3.2. Mix 1 μl of single-stranded DNA molecule with the nucleotide sequence of SEQ ID NO:1 at 20 μM, 1 μl of single-stranded DNA molecule with the nucleotide sequence of SEQ ID NO:2 at 20 μM, 2 μl of 10×T4 Polynucleotide KinaseBuffer, 2 μl of 10 mM ATP, and 1 μl of T4 Polynucleotide Kinase (10 U / μl), and use ddH2O to make up the total volume to 20 μl. Heat the mixed system at 95 °C for 2 min, and then reduce the temperature at a rate of 1 °C every 30 s until the temperature drops to 25 °C to obtain double-stranded DNA.

[0066] 2.3.3. Use BamH1 and EcoR1The plasmid pSHI-H1-copGFP-T2A-Puro (#SI501A-1, System Biosciences) was digested with a restriction endonuclease to obtain a digested plasmid; 1 μl of 10× DNA Ligase buffer, 1 μl of the digested plasmid, 7 μl of double-stranded DNA, and 3 μl of T4 DNA Ligase were mixed to ligate the digested plasmid and the double-stranded DNA, resulting in a ligation product.

[0067] 2.3.4. Take 5 μl of the ligation product and add it to 50 μl of Escherichia coli stbl3 competent cells (#CD521-01, TransGen Biotech). Incubate on ice for 30 min, heat shock at 42°C for 1 min, and then incubate on ice again for 3 min. The transformed competent cells were inoculated and spread on a solid LB medium containing ampicillin (AMP) (#CA0006-10ml, LEAGENE), and cultured overnight at 37°C.

[0068] 2.3.5. Select colonies with good growth status, put them into 5 mL of LB medium containing Amp, and culture them in a shaker at 37°C for 16 h. Extract the plasmid and sequence it. After identification, the fragment was successfully inserted into the vector, and the vector expressing sh-RPS19 was obtained and named PSIHI-H1-shRNA.

[0069] 2.3.6. Lentivirus packaging

[0070] PSIHI-H1-shRNA and its auxiliary packaging plasmids pMDLg (#12251, Addgene), pRSV-Rev (#12253, Addgene), and pVSV-G (#138479, Addgene) were co-transfected into 293T cells. The culture medium was replaced with a complete culture medium 6 - 8 h after transfection. After culturing for 48 and 72 h, the supernatant culture media rich in lentivirus particles were collected respectively, mixed and concentrated to obtain lentivirus.

[0071] 2.3.7. Infect HSPC cells

[0072] The concentrated lentivirus was used to infect HSPC cells for 24 - 48 h, and then a new proliferation medium was added. The cells were continuously amplified until the 7th day. The obtained cells were the constructed HSPC cell model with RPS19 deficiency, named sh-RPS19, to simulate the HSPC cells of DBA patients.

[0073] Meanwhile, a random sequence was used to construct lentivirus by the same method as above and infect HSPC cells, named sh-Random, as a control cell for sh-RPS19.

[0074] Among them, the nucleotide sequences of the oligonucleotide chains corresponding to sh-Random are shown in SEQ ID NO: 3-4 respectively.

[0075] SEQ ID NO:3:

[0076] 5’-GATCC GACTCCAGTGGTAATCTAC cttcctgtcaga GTAGATTACCACTGGAGTC tttttg-3’;

[0077] SEQ ID NO:4:

[0078] 5’-AATTCAAAAA CTGAGGTCACCATTAGATG TCTGACAGGAAG CATCTAATGGTGACCTCAG G-3’.

[0079] The sh-RPS19 cells and sh-Random cells were respectively detected by qPCR, and the detection results are as Figure 2 shown. The relative expression level of RPS19 in sh-RPS19 cells was significantly decreased, indicating that RPS19 was successfully knocked down.

[0080] 2.4. Verification that the HSPC cell model with RPS19 deficiency shows erythroid lineage differentiation disorder, the method is as follows:

[0081] a. The constructed HSPC cell model with RPS19 deficiency was subjected to directed erythroid induction differentiation culture in a differentiation medium. The differentiation medium was SFEM II medium containing 10 ng / ml of SCF, 10 ng / ml of IL3, 3 U / ml of EPO and 1% double antibody. The cell density was 5×10 4 cells / mL, and it was placed in a cell culture incubator (37 °C, 5% (V / V) CO 2 2). After differentiating for 7 days, it was incubated with APC conjugated anti-CD235a and PE conjugated anti-CD71 antibodies, and then the fluorescence was detected by flow cytometry. By detecting the expression levels of cell surface CD235a and CD71 antigens, the erythroid differentiation degree of the cells was judged.

[0082] The results are as Figure 3 shown. The CD71 cell surface antigen begins to be expressed at the early stage of erythroid lineage differentiation, while CD235a begins to be expressed at the late stage of erythroid lineage differentiation. Generally, the double positive expression of the two antigens represents the maturity degree of erythroid differentiation. Compared with the control group (sh-Random), after knocking down RPS19, the erythroid phenotype (CD71+CD235a+) was significantly inhibited, indicating that knocking down RPS19 caused erythroid differentiation disorder.

[0083] b. TP53 is a molecule that is commonly abnormally activated in DBA. The expression of TP53 in the RPS19-deficient HSPC cell model was further detected by qPCR. The results are shown in Figure 4 As shown, after RPS19 knockdown, TP53 was abnormally activated, which was consistent with the DBA phenotype.

[0084] c. The cell morphology of erythroid differentiation was examined by Giemsa staining. As Figure 5 shown, it could be seen that after RPS19 knockdown, most cells lost their differentiation ability and remained in the HPCs stage (large cell nuclei).

[0085] 2.5 Verification of the ability of the RPS19-deficient HSPC cell model to differentiate into the myeloid lineage - monocytes. The method is as follows:

[0086] a. The constructed RPS19-deficient HSPC cell model was inoculated into the monocyte differentiation medium. The differentiation medium was SFEM II medium containing 50 ng / ml of M-CSF and 1% double antibody, and the cell density was 5×10 4 cells / mL. It was placed in a cell culture incubator (37 °C, 5% (V / V) CO2), and the medium was changed every 4 days. The cells differentiated for 5 days (early stage) and 36 days (late stage) were collected, incubated with APC-CD11b antibody, and then the fluorescence was detected by flow cytometry. Among them, SSC-A: A is area, and SSC-A represents the area of the pulse signal waveform curve detected. Its signal intensity is proportional to the cell complexity. By measuring the expression level of the CD11b antigen on the cell surface, the differentiation degree of monocytes in the RPS19-deficient HSPC cell model was judged.

[0087] The results are shown in Figures 6 - 7 As shown, compared with the control group, after RPS19 knockdown, the early myeloid (CD11b+) differentiation ability was significantly enhanced. However, as the culture time increased, the control group and the knockdown group tended to be the same, indicating that RPS19 knockdown would stimulate myeloid differentiation in the short term but had no significant effect on myeloid in the long term.

[0088] 2.6 Verification of the ability of the RPS19-deficient HSPC cell model to differentiate into the myeloid lineage - granulocytes. The method is as follows:

[0089] a. The constructed RPS19-deficient HSPC cell model was inoculated into the granulocyte differentiation medium. The granulocyte differentiation medium was SFEM II medium containing 50 ng / ml of FLT3, 100 ng / ml of G-CSF and 1% double antibody, and the cell density was 5×10 4Cells were placed in a cell incubator (37°C, 5% (V / V) CO2) at a density of cells / mL, and the culture medium was changed every 4 days. Cells collected on the 7th day (early stage) and the 22nd day (late stage) of differentiation culture were incubated with APC-CD68 and PE-CD15 antibodies, and then fluorescence was detected using a flow cytometer. By detecting the expression levels of CD68 and CD15 antigens on the cell surface, the degree of granulocyte differentiation of the cells was determined.

[0090] The results are as Figures 8 - 9 shown. Compared with the control group, after RPS19 knockdown, the ability of early myeloid (CD68+CD15+) differentiation was significantly enhanced. However, as the culture time increased (CD15+), the control group and the knockdown group became consistent, indicating that RPS19 knockdown would stimulate myeloid differentiation in the short term but had no significant effect on myeloid in the long term.

[0091] In summary, through further induced differentiation of the constructed RPS19-deficient HSPC cell model, it was found that this cell model specifically inhibited the ability of erythroid differentiation, resulting in abnormal activation of TP53 and having no effect on myeloid differentiation ability in the long term, which was consistent with the DBA phenotype.

[0092] Example 3 Lineage differentiation bias in the hematopoietic stem / progenitor cell component of the RPS19-deficient HSPC cell model

[0093] a. Control (sh-Random) and RPS19-knockdown HSPC cell samples (sh-RPS19) were separately collected in 1.5 mL centrifuge tubes, centrifuged at 300 g for 5 min. After completely discarding the supernatant, the precipitate was resuspended with 1 ml of DPBS buffer containing FBS and centrifuged at 300 g for 5 min.

[0094] b. After completely discarding the supernatant, 60 μL of DPBS buffer was added to each tube to resuspend the precipitate.

[0095] c. 10 μL of the cell suspension was left unstained as a blank control. For the remaining 50 μL of the cell suspension, when using multi-color flow dyes to detect HSPC cell components, 2.5 μL of PE conjugated anti-CD34, APC conjugated anti-CD38, BV421 conjugated anti-CD90, BV510 conjugated anti-CD10, PE-Cy7 conjugated anti-CD135, and 1.5 μl of AF700 conjugated anti-CD45RA fluorescent dyes were added to the same 1.5 mL EP tube and incubated at 4°C in the dark for 15 min.

[0096] d. Add 1 ml of DPBS buffer containing FBS to the cell suspension after staining and incubation, pipette and mix well, centrifuge at 300 g for 5 min, and repeat the washing 2 times.

[0097] e. After completely discarding the supernatant in the staining group, resuspend the precipitate with 200 μL of DPBS buffer containing FBS. In the blank control group, add DPBS buffer containing FBS to make up to 200 μL. Detect the expression levels of different antigens on the cell surface by flow cytometry fluorescence detection method to determine each cell component in HSPC.

[0098] The results are as Figures 10 - 11 shown. It can be seen that compared with the control group, in the HSPC of the RPS19 knockdown group, the cell components changed, including a significant decrease in the proportions of MPP and MEP, and a significant increase in the proportion of GMP, indicating that the knockdown of RPS19 has an impact on HSPC.

[0099] Example 4 Helichrysin relieves the hematopoietic stem / progenitor cell differentiation deviation caused by RPS19 deficiency

[0100] Inoculate the constructed HSPC cell model with RPS19 deficiency into the proliferation medium. The proliferation medium is SFEM II medium containing 50 ng / ml of SCF, 50 ng / ml of Flt3 Ligand, 50 ng / ml of TPO and 1% double antibody, and the cell density is 5×10 4 cells / mL, and place it in a cell incubator (37 °C, 5% (V / V) CO 2 ₂) for culture. On day 0, add helichrysin solutions with final concentrations of 5 μM, 10 μM, 20 μM, and 40 μM (the solvent is DMSO) to the proliferation medium respectively, place it in a cell incubator (37 °C, 5% (V / V) CO 2 ₂), continue the proliferation culture for 72 h, and detect by flow cytometry. The results are as Figure 12 shown.

[0101] According to Figure 12 it can be known that compared with the control group (sh-Random), the generation of MPP and MEP in the cells of the sh-RPS19 group is significantly reduced, while the treatment with helichrysin can promote the generation of multipotent progenitor cells (MPP) and megakaryocyte-erythroid progenitor cells (MEP) and significantly inhibit the expansion of myeloid progenitor cells GMP, indicating that helichrysin can, to a certain extent, relieve the abnormal expansion of stem / progenitor cells caused by the decrease of RPS19 and relieve the lack of early erythroid-related cells.

[0102] Example 5 Helichrysin relieves the erythroid lineage differentiation arrest caused by RPS19 deficiency

[0103] The constructed HSPC cells with RPS19 deficiency were inoculated in a differentiation medium for directed erythroid induction and differentiation culture. The differentiation medium was SFEM II medium containing 10 ng / ml of SCF, 10 ng / ml of IL3, 3 U / ml of EPO and 1% double antibody, and the cell density was 5×10 4 cells / mL. Starting from day 0 of differentiation, helenalin solutions with final concentrations of 5 μM, 10 μM, 20 μM, and 40 μM (the solvent was DMSO) were added, and the cells were placed in a cell culture incubator (37 °C, 5% (V / V) CO2) and differentiated until day 7. The cells were collected and detected by flow cytometry.

[0104] The results are as Figure 13 shown. Compared with the control group (sh-Random), the generation of red blood cells (CD71+CD235a+) in the sh-PRS19 group of cells was significantly inhibited, while the addition of helenalin treatment restored the erythroid phenotype (CD71+CD235a+), indicating that helenalin can alleviate the erythroid defects caused by the decrease of RPS19 to a certain extent.

[0105] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of the compound represented by Formula 1 or a pharmaceutically acceptable salt thereof in the preparation of a drug for alleviating congenital pure red cell aplasia in a subject; ； The congenital pure red cell aplasia is caused by a mutation in a cellular ribosomal protein gene, and the mutation in the cellular ribosomal protein gene is an RPS19 mutation, and the RPS19 mutation is a mutation that causes a decrease in the expression level of the RPS19 gene.

2. The use according to claim 1, characterized in that: The alleviation of the subject's congenital pure red cell aplasia is manifested in increasing the number and / or proportion of the subject's erythroid cells.

3. The use according to claim 1, characterized in that: The congenital pure red cell aplasia at least includes erythroid differentiation disorder of hematopoietic stem and progenitor cells.

4. The use according to claim 1, characterized in that: The relief of the subject's congenital pure red cell aplasia is manifested by at least one of the following: 1) Promote the differentiation of hematopoietic stem cells with erythroid differentiation disorders into multipotent progenitor cells; 2) Promote the differentiation of common myeloid progenitor cells with erythroid differentiation disorder into megakaryotic-erythroid progenitor cells; 3) Increase the number and / or proportion of CD71-positive and CD235a-positive erythroid cells.

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