Application of compounds in the preparation of drugs for proliferation and damage repair of hematopoietic stem / progenitor cells

Abstract

The present invention relates to the field of biomedicine, and in particular to the use of compounds in the preparation of hematopoietic stem / progenitor cell proliferation and damage repair drugs. The baicalin compounds provided by the present invention have the effect of inhibiting the activity of STS1 and STS2 phosphatases, thereby increasing the phosphorylation levels of FLT3 and cKIT in hematopoietic stem / progenitor cells, and can promote the proliferation and differentiation of mouse bone marrow and human umbilical cord blood hematopoietic stem / progenitor cells in vitro. In vivo application can also enhance the proportion of hematopoietic stem / progenitor cells in normal mouse bone marrow and their colony-forming ability, and at the same time have a significant improvement effect on bone marrow suppression / damage caused by chemotherapy, radiotherapy, etc. Therefore, baicalin compounds can be used for the expansion of hematopoietic stem / progenitor cells in vitro, and can also be used for the prevention and treatment of pathological diseases such as bone marrow suppression / damage caused by chemotherapy, radiotherapy, etc. in vivo that need to enhance or improve the physiological conditions of bone marrow hematopoiesis and chemotherapy, radiotherapy, etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of compounds in the preparation of drugs for the proliferation and damage repair of hematopoietic stem / progenitor cells. Background Art

[0002] Hematopoietic stem cells (HSCs) are multipotent stem cells that have the ability to self-renew and differentiate into mature blood cells of various lineages. They play an important role in maintaining the homeostasis of the blood system under physiological conditions and in rebuilding the blood system under pathological conditions. Hematopoietic stem cell transplantation is one of the most important clinical applications of HSCs and is the only effective treatment for patients with a variety of malignant blood tumors. The HSCs required for transplantation generally come from bone marrow or mobilized peripheral blood. In recent years, human umbilical cord blood HSCs have gradually become the source of HSCs due to their advantages such as convenient collection, weak immunogenicity, and low expression of human leukocyte antigens. However, transplantation is often limited by the small number of HSCs. On the other hand, HSCs in the body are extremely sensitive to chemotherapy drugs and ionizing radiation. Patients will experience symptoms such as bone marrow suppression after receiving radiotherapy and chemotherapy, which seriously affects the patient's quality of life and treatment effect, and becomes the main obstacle to effective radiotherapy and chemotherapy for patients with malignant tumors. Therefore, how to improve the proliferation ability of HSCs in vivo and in vitro, effectively promote the activity of hematopoietic function, and enhance its ability to resist external stress is a research hotspot in the field of hematopoietic stem cells. Since small molecule compounds have the advantages of low cost, stable properties, and precise regulation, the development of small molecule compounds that can promote the function of hematopoietic stem cells has more application prospects in clinical practice.

[0003] Suppressor of T-cell receptor signaling 1 (STS1) is widely present in various tissues, while STS2 is specifically present in hematopoietic tissues. Both have phosphatase activity and can negatively regulate the signal transduction of some intracellular protein phosphorylation levels. Previous studies have shown that the MPPs and LMPPs cell populations in the bone marrow of STS1 and STS2 knockout mice significantly expanded and their colony-forming ability was enhanced, indicating that the loss of STS1 and STS2 promoted the expansion of mouse hematopoietic progenitor cells. Although the number of HSCs did not change, the long-term hematopoietic reconstruction ability of mouse bone marrow HSPC was significantly improved; in vitro studies have shown that the characteristically expressed markers FMS-like tyrosine kinase 3 (FLT3) and stem cell factor receptor cKIT in hematopoietic stem / progenitor cells (HSPC) are substrates of STS1 and STS2 phosphatases. The loss of STS1 and STS2 can lead to the hyperphosphorylation of FLT3 and cKIT in cells, thereby enhancing downstream signal transduction. Combined with the above results, it shows that knocking out STS1 / STS2 in mice may improve the proliferation and differentiation ability of mouse HSPCs and the hematopoietic recovery ability after transplantation by promoting the phosphorylation and downstream signal transduction of FLT3 and cKIT in HSPCs. This shows that STS1 and STS2 may be potential drug targets for enhancing the functional activity of hematopoietic stem cells.

[0004] Baicalein is mainly found in Scutellaria baicalensis and is a flavonoid compound. Its structural characteristics refer to a class of compounds containing a flavonoid nucleus. Studies have shown that baicalein has pharmacological effects such as anti-inflammatory, antibacterial, and antiplatelet aggregation. So far, no research has been reported on baicalein's ability to inhibit the activity of STS1 and STS2 phosphatases, promote HSPC proliferation and differentiation, and especially improve bone marrow suppression. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a class of small molecule compounds, namely baicalin compounds, as phosphatase inhibitors of STS1 and STS2 in the preparation of drugs for promoting the proliferation and damage repair of hematopoietic stem / progenitor cells, which can be used for the expansion of hematopoietic stem / progenitor cells in vivo and in vitro and for the treatment of diseases related to bone marrow suppression / damage caused by chemotherapy or radiotherapy. In view of this, the present invention provides the use of compounds in the preparation of drugs for the proliferation and damage repair of hematopoietic stem / progenitor cells.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of flavonoid compounds in the preparation of STS1 inhibitors and / or STS2 inhibitors.

[0008] In some specific embodiments of the present invention, the flavonoids include one or more of baicalin compounds, derivatives, analogs, pharmaceutically acceptable salts, prodrugs or metabolites thereof. The baicalin compounds include but are not limited to one or more of baicalin, wogonin, scutellarin, melaleuca, chrysin or scutellarin.

[0009] In some specific embodiments of the present invention, the flavonoid compound

[0010] (I), inhibiting STS1 and / or STS2 phosphatase activity; and / or

[0011] (II) increasing the phosphorylation level of the substrate, wherein the substrate includes but is not limited to FLT3 and / or cKIT.

[0012] The present invention provides the use of flavonoid compounds in preparing any of the following medicines:

[0013] (I) promoting the proliferation and / or differentiation of hematopoietic stem / progenitor cells in vivo; and / or

[0014] (II), promoting the proliferation and / or differentiation of hematopoietic stem / progenitor cells in vitro; and / or

[0015] (III) enhancing the proportion of hematopoietic stem / progenitor cells and / or colony-forming ability in the bone marrow; and / or

[0016] (IV) Prevent, improve and / or treat bone marrow suppression and / or damage caused by radiotherapy; and / or

[0017] (V) Prevent, improve and / or treat bone marrow suppression and / or damage caused by radiotherapy; and / or

[0018] (VI) reduce radiation mortality; and / or

[0019] (VII) Preventing and / or treating hematopoietic damage.

[0020] In some specific embodiments of the present invention, the hematopoietic stem / progenitor cells are derived from bone marrow or cord blood.

[0021] In some specific embodiments of the present invention, the inhibition and / or damage is caused by 5-FU and / or radiation.

[0022] In some specific embodiments of the present invention, the flavonoid compound includes one or more of baicalein compounds, their derivatives, their analogs, their pharmaceutically acceptable salts, their prodrugs or their metabolites.

[0023] In some specific embodiments of the present invention, the baicalein compounds include but are not limited to one or more of baicalein, wogonin, baicalein, melaleuca, chrysin or scutellarin.

[0024] The present invention provides an application of a baicalein compound in preparing a drug capable of promoting the proliferation and differentiation of hematopoietic stem / progenitor cells in vivo and in vitro and improving bone marrow suppression. The invention first clones and expresses recombinant proteins of STS1 and STS2 phosphatase domains, and establishes an in vitro phosphatase activity detection method thereof. The detection method is used to screen out baicalein (BC) which can significantly inhibit the phosphatase activities of STS1 and STS2. It is then proved that BC can bind to STS1 and STS2 and inhibit the phosphatase activities of STS1 and STS2 in cells, thereby increasing the phosphorylation levels of FLT3 and cKIT. BC can significantly promote the expansion and function of mouse and human hematopoietic stem / progenitor cells by treating isolated mouse bone marrow nucleated cells and human umbilical cord blood hematopoietic stem / progenitor cells (HSPC) in vitro. In vivo injection of BC can also significantly increase the proportion of hematopoietic stem / progenitor cells in the bone marrow of normal mice and their differentiation ability, and can also improve the bone marrow suppression caused by 5-fluorouracil (5-Fluorouracil, 5-FU) and radiation, increase the proportion of hematopoietic stem / progenitor cells in the bone marrow of 5-FU-injured mice and enhance their differentiation function, thereby reducing the death of mice caused by 5-FU and radiation. The above evidences all suggest that baicalin, as an inhibitor of STS1 and STS2 phosphatases, has the pharmacological effect of improving the proliferation and differentiation ability of hematopoietic stem / progenitor cells and preventing and treating bone marrow damage. Therefore, baicalin compounds have the potential to be developed as drugs to promote the proliferation, differentiation and treatment of hematopoietic stem cells and prevent hematopoietic damage in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0026] Figure 1 Figures showing the results of SDS-PAGE analysis of the expression and purification of the recombinant proteins of the STS1 and STS2 phosphatase domains, wherein A: the analysis result of the STS1 recombinant protein, B: the analysis result of the STS2 recombinant protein;

[0027] Figure 2 The results of in vitro phosphatase activity detection of STS1 and STS2 are shown, wherein A: in vitro phosphatase reaction curve of STS1 recombinant protein, B: in vitro phosphatase reaction curve of STS2 recombinant protein, and the data are expressed as mean ± standard deviation (n = 3);

[0028] Figure 3 The results of screening of STS1 and STS2 phosphatase inhibitors are shown, wherein A: STS1 phosphatase inhibitor screening results, the red circles in the figure represent the screened target small molecules, B: STS2 phosphatase inhibitor screening results, data are expressed as mean ± standard deviation (n = 6);

[0029] Figure 4 The figure shows the results of baicalein inhibiting the phosphatase activities of STS1 and STS2 in vitro, and the data are expressed as mean ± standard deviation (n = 6);

[0030] Figure 5 The diagrams show the molecular docking results of baicalein with the phosphatase domains of STS1 and STS2, wherein A: the molecular docking results of baicalein with the phosphatase domain of STS1 (left: 3D display of the binding model; right: 2D display of the hydrophobic interaction), B: the molecular docking results of baicalein with the phosphatase domain of STS2 (left: 3D display of the binding model; right: 2D display of the hydrophobic interaction);

[0031] Figure 6 The capillary electrophoresis analysis results of baicalein (BC) binding to STS1 and STS2 recombinant proteins are shown, wherein A: capillary electrophoresis analysis results of baicalein binding to STS1 recombinant protein, the red arrow represents the STS1 protein peak, B: capillary electrophoresis analysis results of baicalein binding to STS2 recombinant protein, the blue arrow represents the STS2 protein peak;

[0032] Figure 7 The Western blot results show that baicalein (BC) inhibits the intracellular STS1 and STS2 phosphatase activities and increases the FLT3 phosphorylation level, wherein A: BC increases the phosphorylation level of exogenously expressed FLT3 in cells, B: BC relieves the inhibitory effect of overexpressed STS1 on the FLT3 phosphorylation level, and C: BC relieves the inhibitory effect of overexpressed STS2 on the FLT3 phosphorylation level;

[0033] Figure 8 The Western blot results show that baicalein (BC) inhibits the phosphatase activities of STS1 and STS2 in cells and increases the phosphorylation level of cKIT, wherein A: BC increases the phosphorylation level of exogenously expressed cKIT in cells, B: BC relieves the inhibitory effect of overexpressed STS1 on the phosphorylation level of cKIT, and C: BC relieves the inhibitory effect of overexpressed STS2 on the phosphorylation level of cKIT;

[0034] Fig. 9 The figure shows the result of baicalein (BC) promoting the proliferation of mouse primary bone marrow mononuclear cells in vitro, the data are expressed as mean ± standard deviation (n = 3), ** indicates P < 0.01 compared with the DMSO group;

[0035] Fig.10 The results of baicalein (BC) increasing the proportion of LSK cells in primary mouse bone marrow mononuclear cells in vitro, A: representative figure of LSK cell flow cytometry analysis, B: statistical figure of LSK cell proportion data, data are expressed as mean ± standard deviation (n = 3), **P < 0.01;

[0036] Fig.11 The figure shows the result that baicalein (BC) increases the number of colony-forming units of primary bone marrow hematopoietic stem / progenitor cells of mice in vitro, CFU-GM: granulocyte-macrophage colony-forming unit, BFU-E: blast-forming unit, CFU-GEMM: granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit, data are expressed as mean ± standard deviation (n = 3), * indicates P < 0.05 compared with the Control group, ** indicates P < 0.01 compared with the Control group;

[0037] Fig.12 Figure 2 shows the results of baicalin (BC) increasing the proportion of hematopoietic stem / progenitor cells in human umbilical cord blood in vitro, where A: CD34 + Hematopoietic stem / progenitor cell population (CD34 + CD38 - ), hematopoietic stem cell population (CD34 + CD38 - CD45RA - CD90 + ) Representative flow cytometry analysis, B: Statistical analysis of hematopoietic stem / progenitor cell populations

[0038] (CD34 + CD38 - ) accounted for the CD34 + C: Statistical analysis of hematopoietic stem cell population (CD34 + CD38 - CD45RA - CD90 + ) accounted for the CD34 + The proportion of cells, data are expressed as mean ± SD (n = 3), *P < 0.05, **P < 0.01;

[0039] Fig.13The figure shows the result that baicalein (BC) increases the number of colony-forming cells of human umbilical cord blood hematopoietic stem / progenitor cells in vitro, CFU-GM: granulocyte-macrophage colony-forming unit, BFU-E: blast-forming unit, CFU-GEMM: granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit, data are expressed as mean ± standard deviation (n = 3), * indicates P < 0.05 compared with the Control group, ** indicates P < 0.01 compared with the Control group;

[0040] Fig.14 Figure 2 shows the results of increasing the proportion of hematopoietic stem / progenitor cells in the bone marrow of normal mice after intraperitoneal injection of baicalein (BC) for 7 consecutive days, where A: representative figure of flow cytometry analysis of hematopoietic stem / progenitor cells in mouse bone marrow, B: statistical figure of the proportion of hematopoietic stem / progenitor cells, hematopoietic stem / progenitor cells (LSK, Lin-Sca1 + cKit + ), long-term hematopoietic stem cells (LT-HSCs, Lin-Sca1 + cKit + CD34 - FLT3 - ), short-term hematopoietic stem cells (ST-HSCs, Lin - Sca1 + cKit + CD34 + FLT3 - ), multipotent progenitor cells (MPPs, Lin - Sca1 + cKit + CD34 + FLT3 + ), data are expressed as mean ± standard deviation (n = 3), * indicates P < 0.05 compared with the Control group, ** indicates P < 0.01 compared with the Control group;

[0041] Fig.15 The figure shows the result of 7-day continuous intraperitoneal injection of baicalein (BC) to enhance the colony-forming ability of normal mouse bone marrow hematopoietic stem / progenitor cells, CFU-GM: granulocyte-macrophage colony-forming unit, BFU-E: blast-forming unit, CFU-GEMM: granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit, the data of each colony number are expressed as mean ± standard deviation (n = 3), * indicates P < 0.05 compared with the control group, ** indicates P < 0.01 compared with the control group;

[0042] Fig.16The figure shows the results of in vivo injection of baicalein to protect mice injured by 5-FU, where A: daily weight monitoring of mice, B: survival rate curve of mice, data are expressed as mean ± standard deviation (n = 10), * indicates P < 0.05 compared with control;

[0043] Fig.17 The results of in vivo injection of baicalein (BC) to alleviate the bone marrow damage of mice induced by 5-FU are shown in Figure 1, where A: unilateral femoral bone marrow nucleated cell counts, data are expressed as mean ± standard deviation (n = 5), ## indicates P < 0.01 compared with the control group, ** indicates P < 0.01 compared with the 5-FU group, B: HE staining of femoral bone marrow tissue, scale bar = 100 μm;

[0044] Fig.18 Figures showing the results of in vivo injection of baicalein (BC) to increase the proportion of hematopoietic stem / progenitor cells in the bone marrow of mice injured by 5-FU, where A: representative figure of flow cytometry analysis of hematopoietic stem / progenitor cells in the bone marrow of mice injured by 5-FU, B: statistical figure of the proportion of hematopoietic stem / progenitor cells, C: statistical figure of the number of hematopoietic stem / progenitor cells, hematopoietic stem / progenitor cells (LSK, Lin-Sca1 + cKit + ), long-term hematopoietic stem cells (LT-HSCs, Lin - Sca1 + cKit + CD34 - FLT3 - ), short-term hematopoietic stem cells (ST-HSCs, Lin - Sca1 + cKit + CD34 + FLT3 - ), multipotent progenitor cells (MPPs, Lin - Sca1 + cKit + CD34 + FLT3 + ), data are expressed as mean ± SD (n = 5), ## indicates P < 0.01 compared with the control group, ** indicates P < 0.01 compared with the 5-FU group;

[0045] Fig.19The figure shows the result of in vivo injection of baicalein (BC) to enhance the colony-forming ability of bone marrow hematopoietic stem / progenitor cells in 5-FU-injured mice, CFU-GM: granulocyte-macrophage colony-forming unit, BFU-E: blast-forming unit, CFU-GEMM: granulocyte-erythrocyte-macrophage-megakaryocyte colony-forming unit, the data of each colony number are expressed as mean ± standard deviation (n = 3), ## indicates P < 0.01 compared with the control group, ** indicates P < 0.01 compared with the 5-FU group;

[0046] Fig. 20 The graphs show the results of in vivo injection of baicalein (BC) protecting mice from lethal radiation doses, wherein A: daily weight monitoring of mice, data are expressed as mean ± standard deviation (n = 8); B: mouse survival rate curve (n = 8). DETAILED DESCRIPTION

[0047] The present invention discloses the use of compounds in the preparation of hematopoietic stem / progenitor cell proliferation and damage repair drugs. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the purpose. It should be particularly noted that all similar substitutions and

[0048] The modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant persons can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0049] The present invention provides an application of a baicalein compound in preparing a drug capable of promoting the proliferation and differentiation of hematopoietic stem / progenitor cells in vivo and in vitro and improving bone marrow suppression.

[0050] The present invention first clones and expresses recombinant proteins of STS1 and STS2 phosphatase domains, and establishes an in vitro phosphatase activity detection method thereof. The detection method is used to screen out baicalein (BC) which can significantly inhibit the phosphatase activities of STS1 and STS2. It is then proved that BC can bind to STS1 and STS2 and inhibit the phosphatase activities of STS1 and STS2 in cells, thereby increasing the phosphorylation levels of FLT3 and cKIT. BC can be used to treat isolated mouse bone marrow mononuclear cells and human umbilical cord blood hematopoietic stem / progenitor cells (HSPC) in vitro, and can significantly promote the expansion and function of HSPC in mouse bone marrow and human umbilical cord blood. In vivo injection of BC can also significantly increase the proportion of HSPC in normal mouse bone marrow and enhance its differentiation ability, and can also improve the bone marrow suppression caused by 5-fluorouracil (5-Fluorouracil, 5-FU) and radiation, increase the proportion of HSPC in the bone marrow of 5-FU-damaged mice and enhance its differentiation function, thereby reducing the death of mice caused by 5-FU and radiation. The above evidences suggest that baicalein, as an inhibitor of STS1 and STS2 phosphatases, has the pharmacological effect of improving HSPC proliferation and differentiation ability and preventing and treating bone marrow damage. Therefore, baicalein has the potential to be developed as a drug for promoting hematopoietic stem cell proliferation, differentiation and treatment, and preventing hematopoietic damage in the future.

[0051]

[0052] In the present invention, the structural analogues of baicalein refer to a class of compounds containing a flavonoid core.

[0053] In the present invention, the class of compounds containing flavonoid nuclei refers to one or more of wogonin, baicalein, melaleuca, chrysin and scutellaria baicalensis.

[0054] In the present invention, the baicalein compounds serve as STS1 and STS2 inhibitors, which means that they inhibit the phosphatase activity of STS1 and STS2, thereby increasing the phosphorylation levels of their substrates including FLT3 and cKIT.

[0055] In the present invention, the baicalein compounds can increase the proportion of hematopoietic stem / progenitor cells in in vitro cultured mouse bone marrow and human umbilical cord blood and enhance their colony-forming ability in vitro.

[0056] In the present invention, the baicalin compounds increase the proportion of hematopoietic stem / progenitor cells in the bone marrow of normal mice and enhance their colony-forming ability when used in vivo; at the same time, their in vivo application can alleviate bone marrow suppression caused by chemotherapy or radiation, including increasing the proportion of hematopoietic stem / progenitor cells and colony-forming ability in damaged bone marrow, and reducing the mortality rate of mice.

[0057] The present invention found that the baicalein compound represented by formula (I) has the effect of promoting the proliferation and differentiation of hematopoietic stem / progenitor cells and improving bone marrow suppression, mainly based on the following research results:

[0058] (1) Expression and purification of recombinant proteins of STS1 and STS2 phosphatase domains Figure 1 ): Using prokaryotic expression plasmid to express STS1 in prokaryotic cells by IPTG induction ( Figure 1 A) and STS2( Figure 1 B) Phosphatase domain recombinant protein,

[0059] Then, the recombinant protein with high purity was purified by Ni-NTA column. SDS-PAGE combined with Coomassie Brilliant Blue staining proved that we obtained STS1 with high protein concentration and purity ( Figure 1 A) and STS2( Figure 1 B) Recombinant protein, which can be used for subsequent research.

[0060] (2) In vitro phosphatase activity assay of recombinant proteins of STS1 and STS2 phosphatase domains Figure 2 ): Different concentrations of PNPP were selected as substrates and enzymatically reacted with 25 nmol / L STS1 and 2.5 μmol / L STS2, respectively. The reactions were carried out at room temperature. The reaction time between STS1 and PNPP was 10 min, and the reaction time between STS2 and PNPP was 20 min. After the reaction, the absorbance of the product at a wavelength of 405 nm was measured, and the absorbance was calculated according to the formula Velocity (mmol / L / min)

[0061] =OD405 / time / lightpath(cm) / molar extinction coefficient(Molarextinction coefficient for pNP is 18mmol / L -1 cm -1 ) The reaction rate V under different substrate concentrations was calculated, and then the substrate concentration and rate V were nonlinearly fitted using Graphpad Prism 8.0 to obtain the Michaelis-Menten equation for the enzymatic reaction of STS1 and STS2. The results showed that the maximum reaction rate Vmax of the STS1 enzymatic reaction was 0.1015mmol / L / min, and Km was 3.512mmol / L( Figure 2 A), the Vmax of STS2 phosphatase enzymatic reaction is 0.04053mmol / L / min, and the Km is 5.364mmol / L ( Figure 2 B). This indicates that the recombinant STS1 and STS2 phosphatases obtained by prokaryotic expression and purification have good enzymatic activity and can be used for the subsequent screening of phosphatase inhibitors.

[0062] (3) Screening of STS1 and STS2 phosphatase inhibitors Figure 3 ): Using the above-established STS1 and STS2 in vitro phosphatase reaction system, each Chinese medicine monomer compound (final concentration of 10 μg / mL) was added to screen for candidate compounds that inhibit STS1 or STS2 phosphatase. The screening results showed that a total of 2 compounds (circled) had an inhibition rate of greater than 20% on STS1 phosphatase activity and reached statistical significance ( Figure 3 A), one of which was baicalein (BC), with an inhibition rate of about 25%. However, due to the relatively low activity of STS2 phosphatase, no compound with an inhibitory effect that reached the judgment standard was obtained ( Figure 3 B).

[0063] (4) Baicalein inhibits STS1 and STS2 phosphatase activities in vitro ( Figure 4 ): The above-established STS1 and STS2 in vitro phosphatase reaction systems were used again, and baicalin (final concentration of 10 μg / mL) was added to confirm the effect of baicalin on the in vitro phosphatase activity of STS1 or STS2. The results showed that baicalin had an inhibition rate of 25% on the STS1 phosphatase activity, which was similar to the results of the screening. It also had a certain inhibitory effect on the STS2 phosphatase activity, with an inhibition rate of 13.77%. This result shows that baicalin has a certain inhibitory effect on the phosphatase activity of both STS1 and STS2.

[0064] (5) Baicalein binds to the STS1 and STS2 phosphatase domains ( Figures 5-6 ): Molecular docking was performed using GOLD5.2 software, with the three-dimensional conformation of baicalin in the PubChem database as the ligand and the crystal structure of the STS1 or STS2 histidine phosphatase domain in the RCSB PDB database as the receptor. The direct binding ability of baicalin with STS1 or STS2 protein was evaluated according to the GOLDscore main scoring function and the ASPscore repeated scoring function. Table 7 shows that the Goldscore of the molecular docking of baicalin with STS1 and STS2 is 51.64 and 54.33, respectively, and the scoring functions are all greater than 50, suggesting that there may be interactions between baicalin and the STS1 and STS2 phosphatase domains. Figure 5 Shown is the expression of baicalin and STS1 ( Figure 5 A) and STS2( Figure 5 B) Simulation diagram of protein molecule docking. As can be seen from the figure, baicalin can be docked with baicalin through hydrogen bonds ( Figure 5 A left) and hydrophobic force ( Figure 5 A right) interacts with STS1 protein; at the same time, it also interacts with STS1 protein through hydrogen bonds ( Figure 5 B left) and hydrophobic force ( Figure 5B (right) Interaction with STS2 protein. The above computer simulation results suggest that baicalein has good binding with both STS1 and STS2 phosphatase domains.

[0065] Based on the above computer simulation, we used capillary electrophoresis to directly detect the in vitro binding of baicalein (BC) with STS1 or STS2 recombinant proteins ( Figure 6 ),from Figure 6 A shows that after adding baicalein, the peak of STS1 protein migrated later than that without adding the compound; similarly, Figure 6 B shows that after adding baicalein, STS2 protein

[0066] The peak of white also obviously shifted later. Therefore, this result further proves that baicalein can bind to STS1 and STS2 proteins.

[0067] (6) Baicalein inhibits intracellular STS1 and STS2 phosphatase activities ( Figures 7-8 ): To further clarify whether baicalein inhibits the phosphatase activity of STS1 and STS2 in cells, we transfected 293T cells with plasmids expressing STS1 and STS2 substrate proteins FLT3 and cKIT, and then used western blot to detect the phosphorylation levels of FLT3 and cKIT. First, 293T cells that only exogenously expressed FLT3 or cKIT were treated with baicalein (BC). The results showed that after 2 to 10 minutes of baicalein treatment, FLT3 ( Figure 7 A) and cKIT( Figure 8 A) The phosphorylation level increased significantly; and after 15 minutes, the phosphorylation level of cKIT can still maintain a high phosphorylation level ( Figure 8 A). After that, the FLT3 or c-KIT expression plasmid was co-transfected with the STS1 or STS2 expression plasmid into the cells and then treated with baicalin. The results showed that when STS1 or STS2 was overexpressed, FLT3 ( Figure 7 B, C) and cKIT ( Figure 8 B, C) phosphorylation level was significantly decreased, while after baicalein treatment, FLT3 ( Figure 7 B, C) and cKIT ( Figure 8 The above results indicate that baicalein can also inhibit the phosphatase activity of STS1 and STS2 in cells and thus promote the phosphorylation level of FLT3 and cKIT.

[0068] (7) Baicalein promotes proliferation and differentiation of mouse bone marrow hematopoietic stem / progenitor cells in vitro ( Figures 9-11): Mouse bone marrow mononuclear cells (BMMNCs) were isolated and cultured in vitro, and baicalin was treated to observe the effect of baicalin on the proliferation and differentiation of hematopoietic stem / progenitor cells (HSPC) in mouse bone marrow in vitro. First, the cell counting results were as follows Fig. 9 The results showed that at 14 days of culture, the number of BMMNCs in the baicalein-treated group was significantly higher than that in the untreated group, and at 17 days of culture, the number of cells in the baicalein-treated group was still higher than that in the untreated group. The above results indicate that baicalein promotes the proliferation of HSPCs in BMMNCs and keeps them in a stable growth state for a long time. + ckit + ) The proportion of hematopoietic stem / progenitor cells, the results are as follows Fig.10 As shown in the figure, on the 4th day of culture, the proportion of LSK cells in the BMMNCs treated with baicalin was significantly higher than that in the control group; on the 7th day of culture, the proportion of LSK cells in the baicalin-treated group was also significantly higher than that in the control group. The above results indicate that baicalin can promote the proliferation of HSPCs in mouse BMMNCs under in vitro culture conditions. Finally, the differentiation ability of mouse HSPCs cultured in vitro was analyzed by colony formation experiment. The results are shown in the figure. Fig.11 As shown in the figure, compared with the control group cells, the total number of mouse BMMNCs colonies and the number of colonies of each lineage in baicalin-treated mice increased significantly, and there was no obvious lineage-biased differentiation, indicating that baicalin can improve the differentiation ability of mouse HSPCs. The above results show that baicalin can improve the proliferation and differentiation ability of mouse hematopoietic stem / progenitor cells in vitro.

[0069] (8) Baicalein promotes the expression of human umbilical cord blood CD34 in vitro + Cell proliferation and differentiation ( Figures 12-13 ):Purification of CD34 from human umbilical cord blood by magnetic bead separation + The cells were cultured in vitro and treated with baicalin. + The effect of baicalin on the proliferation and differentiation of human umbilical cord blood HSPCs in vitro was observed. Fig.12 The results showed that after 4 days of in vitro culture, compared with the control group, the CD34 + CD34 + CD38 - Cell population ( Fig.12 B) and CD34 + CD38 - CD45RA - CD90 + Cell population ( Fig.12 C) were significantly increased; on the 7th day of culture, although the CD34 +CD38 - The proportion of hematopoietic stem / progenitor cells was lower than that of the control group ( Fig.12 B), but a more primitive hematopoietic stem cell population (CD34 + CD38 - CD45RA - CD90 + ) was significantly higher than that of the control group ( Fig.12 C), representative flow cytometry analysis Fig.12 As shown in A. This shows that baicalein can promote the expansion of human cord blood HSPC under in vitro culture conditions, and the effect is most obvious at 4 days of culture. Fig.13 The results showed that human cord blood CD34 treated with baicalein in vitro + The total number of cells, colonies and colonies of each lineage increased significantly, and were statistically significant, indicating that baicalin can improve the differentiation ability of human hematopoietic stem / progenitor cells. The above results prove that baicalin can promote the proliferation and differentiation of human umbilical cord blood hematopoietic stem / progenitor cells in vitro.

[0070] (9) Baicalein promotes the proliferation and differentiation of normal mouse bone marrow hematopoietic stem / progenitor cells in vivo Figures 14-15 ): Normal mice were intraperitoneally injected with baicalin (BC) for 7 consecutive days, and then the proliferation and differentiation ability of hematopoietic stem / progenitor cells (HSPC) in the mouse bone marrow was analyzed. First, Fig.14 It showed that after 7 consecutive days of intraperitoneal injection of baicalin, the proportion of hematopoietic stem / progenitor cells LSK in mouse BMMNCs was significantly increased compared with the control group; a deeper analysis showed that compared with the control group mice, the proportions of long-term HSCs (LT-HSCs), short-term HSCs (ST-HSCs) and multipotent progenitor cells (MPPs) were significantly increased after baicalin treatment. This result suggests that baicalin may increase the proportion of more primitive cells in mouse bone marrow hematopoietic stem cells to a greater extent, and may promote their ability to self-renew. At the same time, Fig.15 The results showed that 7 days after the injection of baicalein, the total number of colonies and the number of colonies of each lineage formed by mouse bone marrow HSPC were significantly higher than those of the control group, and no lineage differentiation occurred. The number of more primitive progenitor cell colonies CFU-GEMM was also higher than that of the control group. The above results show that in vivo injection of baicalein can significantly improve the proliferation and differentiation ability of normal mouse bone marrow hematopoietic stem / progenitor cells.

[0071] (10) Baicalein has a protective effect on 5-FU bone marrow injury in mice ( Figures 16 to 19): A mouse model of chemotherapy-induced bone marrow injury was established by injecting a lethal dose of 5-FU once a week. Baicalein was injected intraperitoneally for three consecutive days before modeling to observe its protective effect on mice with 5-FU bone marrow injury. First, Fig.16 It showed that after the third round of 5-FU injection, the weight of mice in the baicalein group was significantly higher than that in the control group ( Fig.16 A); By the end of the experiment, all mice in the control group had died from 5-FU injury, while the survival rate of mice in the baicalin dose group was 40-60% ( Fig.16 B) This experiment proves that the preventive use of baicalin can significantly improve the health status and survival rate of 5-FU-injured mice. The results of further analysis of the number of cells in the bone marrow of each group of mice are as follows Fig.17 As shown in the figure, after 5-FU injection, the number of bone marrow cells in the unilateral femur of mice decreased sharply, while the number of bone marrow cells in the baicalin-treated group was significantly higher than that in the model control group, indicating that baicalin treatment partially restored the number of nucleated cells in the bone marrow of 5-FU-injured mice ( Fig.17 A); HE staining results of femoral bone marrow of mice also showed that compared with the model group, the bone marrow structure of mice in the baicalin treatment group was significantly improved, and the number of bone marrow cells and microstructural integrity were also significantly improved ( Fig.17 B) The results of subsequent bone marrow cell flow cytometry analysis are as follows Fig.18 As shown in the figure, compared with the normal control group mice, after two lethal doses of 5-FU injections, the proportion of LSK in bone marrow cells decreased significantly, while the proportion of LSK in the baicalein-treated group mice was significantly higher than that in the model group ( Fig.18 B); Further analysis of the proportions of different cell populations in LSK cells showed that the proportions of LT-HSCs, ST-HSCs and MPPs in the baicalein-treated group were significantly higher than those in the untreated model group ( Fig.18 C) Finally, Fig.19 The results showed that after the injection of a lethal dose of 5-FU, the number of colonies formed by mouse bone marrow hematopoietic stem / progenitor cells was significantly lower than that of the normal control group, while the total number of colonies in the baicalin-treated group, as well as the number of BFU-E, CFU-GM and CFU-GEMM colonies were significantly higher than those in the model group. The above results indicate that the preventive use of baicalin can improve the proliferation and differentiation ability of mouse hematopoietic stem / progenitor cells to resist the bone marrow damage caused by 5-FU, thereby improving the survival rate of mice.

[0072] (11) Baicalin reduces the mortality of mice irradiated with a lethal dose ( Fig. 20 ): The mice were irradiated with a lethal dose of X-rays to establish a radiation-induced bone marrow suppression mouse model, and baicalin was injected intraperitoneally before irradiation. Fig. 20 As shown in Figure 2, compared with the control group, the body weight of mice in the baicalein group increased ( Fig. 20A). By the end of the experiment, the survival rate of mice in the radiation control group was 25%, while that of the baicalin group was 37.5-50%, indicating that the preventive use of baicalin can improve the survival rate of radiation-damaged mice to a certain extent ( Fig. 20 B). In summary, baicalein has a preventive effect on bone marrow damage caused by radiation.

[0073] (12) Baicalein analogs inhibit the phosphatase activity of STS1 and STS2: The previously confirmed STS1 and STS2 in vitro phosphatase activity detection method was used to detect the effects of baicalein analogs on the phosphatase activity of STS1 and STS2. The results are shown in Table 20. Baicalein analogs, including wogonin, baicalein, melaleuca, chrysin, and scutellariae, all showed different degrees of inhibition on the phosphatase activity of STS1 and STS2. Therefore, it is believed that

[0074] It is the common part in their structure that plays this role. It is inferred that other baicalein structural analogs with this common structure should also have the same effect of inhibiting the activity of STS1 or STS2 phosphatase, and can be used as drugs to promote the proliferation and differentiation of hematopoietic stem / progenitor cells and improve bone marrow suppression.

[0075] This patent cloned and expressed recombinant proteins of the phosphatase domains of STS1 and STS2, and established an in vitro phosphatase activity detection method. Using this detection method, baicalein (BC) was screened out to significantly inhibit the phosphatase activity of STS1 and STS2. It was later proved that BC could bind to STS1 and STS2 and inhibit the phosphatase activity of STS1 and STS2 in cells, thereby increasing the phosphorylation levels of FLT3 and cKIT; BC in vitro treatment of isolated mouse bone marrow nucleated cells and human umbilical cord blood HSPCs could significantly promote the expansion and function of HSPCs in mice and humans; and in vivo injection of BC could also significantly promote the proliferation and differentiation ability of HSPCs in the bone marrow of normal mice, and at the same time improve the bone marrow suppression caused by 5-fluorouracil (5-Fluorouracil, 5-FU) and radiation, increase the proportion of HSPCs in the bone marrow of 5-FU-damaged mice and enhance their differentiation function, thereby reducing the death of mice caused by 5-FU and radiation.

[0076] The baicalein compounds mentioned in the present invention, alone or in combination, as inhibitors of STS1 and STS2 phosphatase activity, are also used in other physiological or pathological conditions requiring strengthening or improving bone marrow hematopoiesis, which are also within the scope of the present invention.

[0077] The baicalein used in the present invention can be purchased from a commercial product (Source Leaf Biotechnology, product number: B20571) or prepared by the following method: Fan Jinghui, Li Haiyan, Zhou Yang. Research progress on the preparation and anti-tumor pharmacological effects of baicalein [J]. Heilongjiang Medicine. 2015, 28(04): 783-384;

[0078] Zhou Jian, Du Yongfeng, Zhang Wenguang. A new method for preparing baicalin. Journal of the Fourth Military Medical University[J]. Journal of the Fourth Military Medical University. 2009, 30(18): 1825-1827.

[0079] The structural analogs of baicalein used in the present invention, wogonin (source leaf biological, item number: B20489), baicalein (source leaf biological, item number: B21479), melaleuca (source leaf biological, item number: B20958), chrysin (source leaf biological, item number: B20063), and scutellaria baicalensis (source leaf biological, item number: B20761) can all be purchased.

[0080] In the application of the compound provided by the present invention in the preparation of hematopoietic stem / progenitor cell proliferation and damage repair drugs, the raw materials and reagents used can be purchased from the market.

[0081] The present invention will be further described below in conjunction with embodiments:

[0082] Example 1 Expression and purification of recombinant proteins of STS1 and STS2 phosphatase domains

[0083] Main materials: pNIC28-Bsa4-STS1 and pNIC28-Bsa4-STS2 recombinant protein prokaryotic expression plasmids were kindly donated by Professor Zhang Jing from the School of Hematology / Oncology, Goethe University, Germany (Zhang J, et al. Stem Cell Reports, 2015, 5(4): 633-646.); Coomassie Brilliant Blue was purchased from Sigma-Aldrich; IPTG was purchased from Beijing Solebao Technology Co., Ltd.; Ni Sepharose TM high performance was purchased from GE Healthcare, imidazole was purchased from Acros Organics; Bradford protein concentration determination kit was purchased from Beyotime Biotechnology Co., Ltd., and protein ultrafiltration tube was purchased from Millipore, USA.

[0084] Methods: 50 μL E.coli BL21 (DE3) competent cells were added to a sterile 1.5 mL centrifuge tube. After melting on ice, pNIC28-Bsa4-STS1 or pNIC28-Bsa4-STS2 recombinant plasmids were transferred according to conventional transformation methods. The obtained clones were cultured overnight by single cloning inoculation and expanded again to the logarithmic growth phase, and then induced by IPTG. The conditions for inducing expression of STS1 were IPTG 0.8 mM, 37 ° C for 12 h; and the conditions for inducing expression of STS2 were IPTG 0.1 mmol / L, 37 ° C for 4 h. After the induced recombinant bacteria were lysed by conventional methods, the recombinant protein was purified by nickel column affinity chromatography, and the eluted recombinant protein was desalted and concentrated using a protein ultrafiltration tube. After the protein concentration was determined by Bradford method, SDS-PAGE and Coomassie Brilliant Blue staining were performed.

[0085] Results: The results are as follows Figure 1 As shown, compared with the uninduced group, the induced group showed the target molecular weight of STS1 ( Figure 1 A) or STS2( Figure 1 B) Positive recombinant protein bands, molecular weight is about 30kDa, and protein concentration and purity are high. Based on this, we obtained the ideal STS1 and STS2 recombinant proteins.

[0086] Example 2 In vitro phosphatase activity detection of STS1 and STS2 phosphatase domain recombinant proteins

[0087] Main materials: p-Nitrophenyl phosphate (PNPP) was purchased from Sigma-Aldrich.

[0088] Methods: Different concentrations of PNPP were selected as substrates and enzymatically reacted with 25 nmol / L STS1 and 2.5 μmol / L STS2, respectively. The reactions were carried out at room temperature. The reaction time of STS1 and PNPP was 10 min, and the reaction time of STS2 and PNPP was 20 min. After the reaction, the absorbance of the product at a wavelength of 405 nm was measured, and the absorbance was calculated according to the formula Velocity (mmol / L / min) = OD405 / time / light path (cm) / molar extinction coefficient (Molar extinction coefficient for pNP is 18mmol / L -1 cm -1 ) was used to calculate the reaction velocity V at different substrate concentrations, and then Graphpad Prism 8.0 was used to perform a nonlinear fit between the substrate concentration and the velocity V to obtain the Michaelis-Menten equation for the enzymatic reaction of STS1 and STS2.

[0089] Results: The absorbance values ​​of the STS1 and STS2 phosphatase reaction products at different PNPP substrate concentrations are shown in Table 1 (STS1) and Table 3 (STS2), and the reaction rates are shown in Table 2 (STS1) and Table 4 (STS2). Based on this, the Michaelis-Menten equation curve of the enzymatic reaction is obtained as follows: Figure 2 As shown, Figure 2 A is the Michaelis-Menten curve of the STS1 enzymatic reaction. From the figure, we can know that the maximum reaction rate Vmax of the STS1 enzymatic reaction is 0.1015mmol / L / min, and Km is 3.512mmol / L. Figure 2 B is the Michaelis-Menten curve of the STS2 enzymatic reaction. From the figure, we can know that the Vmax of the STS2 phosphatase enzymatic reaction is 0.04053mmol / L / min and the Km is 5.364mmol / L. This shows that the recombinant STS1 and STS2 phosphatases obtained by prokaryotic expression and purification have good enzymatic activity and can be used for the subsequent screening of phosphatase inhibitors.

[0090] Table 1: Absorbance values ​​of STS1 phosphatase reaction products at different PNPP substrate concentrations

[0091]

[0092] Table 2: STS1 phosphatase reaction rate at different PNPP substrate concentrations

[0093]

[0094]

[0095] Table 3: Absorbance values ​​of STS2 phosphatase reaction products at different PNPP substrate concentrations

[0096]

[0097] Table 4: STS2 phosphatase reaction rate at different PNPP substrate concentrations

[0098]

[0099] Example 3 Screening of inhibitors of STS1 and STS2 phosphatase activity

[0100] Main materials: The 292 Chinese medicine monomer compounds used for screening were extracted from different Chinese herbal medicines by the National Engineering Laboratory for Drug Gene and Protein Screening of Northeast Normal University, with a purity greater than 98%; the positive control inhibitor Na3VO4 and the substrate p-nitrophenyl phosphate (PNPP) were purchased from Sigma-Aldrich.

[0101] Methods: Each monomer compound was dissolved in dimethyl sulfoxide (DMSO) to prepare a 1 mg / mL stock solution and stored at -20°C. A 96-well plate was used for screening. Each screening plate was set up with 6 replicate wells per group, and there must be a negative control group (DMSO group) and a positive control group (Na3VO4 group, 100 mmol / L, pH: 10.0). Each screening group was added with each Chinese medicine monomer compound to be screened. First, 3 μL of Chinese medicine monomer compound (final concentration of 10 μg / mL) or control solution was added to the 96-well plate, and then 50 μL of PNPP was added to it and mixed evenly. Then 10 μL of STS1 (150 nmol / L) or STS2 protein (15 μmol / L) working solution was added, and incubated at room temperature for 10 min (STS1) and 20 min (STS2). After the reaction, 40 μL of stop buffer was added to terminate the reaction, and the absorbance at 405 nm was detected by an ELISA instrument. The inhibition rate was calculated according to the following formula: inhibition rate (100%) = (1-OD405nm of the drug-treated group / OD405nm of the negative control group) × 100%). Finally, SPSS and Graphpad Prism 8.0 were used to perform statistics and analysis on the inhibition rate and p-value, and the Chinese medicine monomer compounds with an inhibition rate of more than 20% and a significant difference from the negative control group were screened as target compounds.

[0102] Results: The results of STS1 phosphatase inhibitor screening (a total of 292 natural small molecule compounds were screened) are shown in Table 5, and the results of STS2 phosphatase inhibitor screening (a total of 263 natural small molecule compounds were screened) are shown in Table 6. The screening summary results are as follows Figure 3 show, Figure 3 A is the screening result of STS1 phosphatase activity inhibitors. The one in bold black is baicalein (BC), and the inhibition rate is 25%, which is statistically significant. Figure 3 B is the screening result of STS2 phosphatase activity inhibitors. Due to the relatively low activity of STS2 phosphatase, no compounds that met the inhibitory effect criteria were obtained.

[0103] Table 5: Screening results of STS1 phosphatase activity inhibitors

[0104]

[0105]

[0106]

[0107] Note: Compound No. 162 in the table is BC, showing its inhibition rate and P value on STS1.

[0108] Table 6: Screening results of STS2 phosphatase activity inhibitors

[0109]

[0110]

[0111]

[0112]

[0113] Example 4 Baicalein inhibits STS1 and STS2 phosphatase activities in vitro

[0114] Main materials: p-Nitrophenyl phosphate (PNPP) was purchased from Sigma-Aldrich.

[0115] Methods: The inhibitory effect of baicalin on the phosphatase activity of STS1 and STS2 was confirmed in 96-well plates using the previously confirmed in vitro phosphatase activity detection method of STS1 and STS2. Six replicate wells were set up in each group. First, 3 μL of baicalin (final concentration of 10 μg / mL), negative control solution DMSO or positive inhibitor Na3VO4 (100 mmol / L, pH: 10.0) were added to the 96-well plate, and then 50 μL of PNPP was added to it and mixed evenly. Then 10 μL of STS1 (150 nmol / L) or STS2 (15 μmol / L) protein working solution was added, and incubated at room temperature for 10 min (STS1) and 20 min (STS2). After the reaction, 40 μL of stop buffer was added to terminate the reaction, and the absorbance at 405 nm was detected by microplate reader. The inhibition rate was calculated according to the following formula: inhibition rate (100%) = (1-OD405nm of drug-added group / OD405nm of negative control group) × 100%.

[0116] Results: Figure 4 As shown, the inhibition rate of baicalein on STS1 phosphatase activity was 25%, which was similar to the screening result. It also had a certain inhibitory effect on STS2 phosphatase activity, with an inhibition rate of 13.77%, indicating that baicalein had a certain inhibitory effect on the phosphatase activities of STS1 and STS2.

[0117] Example 5 Baicalein binds to the phosphatase domains of STS1 and STS2

[0118] (1) Molecular docking verification of the interaction between baicalein and STS1 or STS2 protein

[0119] Main materials: Use SYBYL-X and GOLD5.2 software for molecular docking, PyMol software for docking mapping, LigPlot to analyze the interacting amino acid residues, and perform 2D display.

[0120] Methods: The ligands were processed by SYBYL-X to generate the potential three-dimensional conformation of baicalein as a ligand set, and the molecular docking study was performed using GOLD5.2 software. The crystal structure of the STS1 histidine phosphatase domain was derived from the RCSB PDB database (ID: 5W5G), and the crystal structure of the STS2 histidine phosphatase domain was derived from the RCSB PDB (ID: 5WDI). The protein crystal structure was used as the receptor, and the structure of baicalein was derived from the PubChem database (CID: 5281605) as the ligand. First, the crystal structure of the STS1 / STS2 histidine phosphatase domain was opened in the GOLD5.2 software, the ligand in the crystal structure was extracted, hydrogen atoms were added, and water molecules were deleted. Centered on the original ligand, Define active sites within the range: Set 30 as the number of genetic algorithm runs for the ligand, turn off the early termination command, and re-dock the compound with the protein. Use the scoring function as the evaluation standard, GOLDscore as the main scoring function, ASPscore as the repeated scoring function, retain the top 15 docking conformations, and keep the default settings for the rest. Based on the above parameters, baicalein was docked with STS1 and STS2, and the strength of the binding ability between the small molecule compound and the protein was evaluated according to the scoring function. The higher the scoring function, the stronger the binding ability, and vice versa.

[0121] Results: Table 7 shows the scoring function of the docking of baicalein with STS1 and STS2 protein molecules. It can be seen from the table that the Goldscore of the docking of baicalein with STS1 and STS2 molecules are 51.64 and 54.33, respectively, and the scoring functions are all greater than 50, suggesting that there may be interaction between baicalein and the phosphatase domains of STS1 and STS2. Figure 5 A on the left and 5B on the left show the 3D images of baicalin binding to STS1 and STS2 proteins. The images show that baicalin forms hydrogen bond interactions with the amino acid residues Ala194, Arg7, Ser195, and Arg11 of the STS1 protein ( Figure 5 A left), forming hydrogen bond interactions with amino acid residues Arg12, Glu124, Arg91, His194, and Arg8 of the STS2 protein ( Figure 5 B left); Figure 5 A (right) and 5B (right) show the 2D images of baicalein binding to STS1 and STS2 proteins. It can be seen from the figure that baicalein generates hydrophobic interactions with the amino acid residues Trp122, Glu118, So4538, and His193 of the STS1 protein ( Figure 5 A right), forming hydrophobic interactions with Lys284, Trp123, Gly283, Val1220, Arg8, and Glu119 amino acid residues of the STS2 protein ( Figure 5B right). The above results indicate that baicalein has good binding to both STS1 and STS2 phosphatase domains.

[0122] Table 7: Scoring function values ​​for docking of baicalein with STS1 and STS2 protein molecules

[0123]

[0124] (2) Capillary electrophoresis verification of baicalein binding to STS1 and STS2 proteins

[0125] Main materials: Capillary electrophoresis device was purchased from Beijing Cailu Scientific Instrument Co., Ltd.

[0126] Methods: The voltage was adjusted to 20 kV, and the injection method was 10 cm height difference injection for 5 s; 0.1 mol / L NaOH (5 min), ddH2O (2 min) and PBS (5 min) were injected in sequence to pretreat the capillary; the concentration of baicalein was 1 mg / mL, and the injection conditions were 10 cm height difference, 5 s, and after the STS1 or STS2 histidine phosphatase domain recombinant protein sample was injected into the capillary, a separation potential of 20 kV was applied for separation. In the interval between each electrophoresis, the capillary was rinsed with 0.1 mol / L NaOH (5 min), distilled water for 2 min, and PBS for 5 min, and the change of migration time was determined by the appearance of protein peak.

[0127] Results: Figure 6 As shown, from Figure 6 A shows that after adding baicalein, the peak of STS1 protein has a delayed migration compared with that without adding the compound. According to the principle of capillary electrophoresis, due to the binding of baicalein to STS1 protein, the charge-to-mass ratio of STS1 protein is changed, resulting in the delayed migration of the protein peak, which indicates that baicalein can bind to STS1 protein; Figure 6 B shows that after the addition of baicalein, the peak of STS2 protein also obviously lagged behind. This result indicates that baicalein can bind to the phosphatase domains of STS1 and STS2.

[0128] Example 6 Baicalein inhibits intracellular STS1 and STS2 phosphatase activities

[0129] Main materials: Human embryonic kidney cells 239T were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences; DMEM medium and fetal bovine serum (FBS) were purchased from Gibco; 2000 transfection kit was purchased from Invitrogen; pcDNA-FLT3, pcDNA-STS1 and pcDNA-STS2 expression plasmids were synthesized by Nanjing GenScript Biotechnology Co., Ltd.; pcDNA-cKIT plasmid was synthesized by Wuhan Miaoling Biotechnology Co., Ltd.; anti-GAPDH (CAT#10494-1-AP), anti-STS1 (CAT#19563-1-AP), anti-STS2 (CAT#15823-1-AP) antibodies were purchased from Proteintech Group (CAT#: KC-5G4), anti-FLT3 (8F2) (CAT#3462S), anti-phospho (p) FLT3 Tyr591 (54H1) (CAT#3466S), anti-p-FLT3 Tyr842 (10A8) (CAT#4577S) and anti-p-cKIT Tyr719 (CAT#3391S) antibodies were purchased from Cell Signaling Technology Company; anti-Flag (CAT#F1804) antibody was purchased from Sigma; PVDF membrane was purchased from Roche; protein standard molecular weight was purchased from Thermo Company; ultra-sensitive ECL luminescence kit was purchased from Beyotime Biotechnology Co., Ltd.

[0130] Methods: 293T cells with good growth status cultured in 10% FBS high-glucose DMEM medium were inoculated into 6-well culture plates, and the cell density reached 80-90% on the second day. After 24 hours, Lipofectamine 2000 liposome transfection reagent was used to co-transfect the cells with the target plasmids according to the different combinations marked in the figure according to the instructions. The total amount of plasmids used for transfection was 4μg / well. After 48 hours of transfection, baicalin (final concentration 10μg / mL) was added for different treatment times, and DMSO was used as a control. After the treatment, wash the cells three times with pre-cooled PBS, then gently scrape the cells in the plate with a cell scraper, centrifuge at 5000rpm for 5 minutes, discard the supernatant, add whole-cell lysis buffer (50mmol / L Tris-HCl pH7.5, 150mmol / LNaCl, 1mmol / LNaF, 0.5% NP-40, 2μg / mL Aprotinin, 1mmol / L PMSF), let it stand on ice for 10 minutes, centrifuge at 12000rpm at 4℃ for 5 minutes, take the supernatant, i.e. the whole-cell protein extract, to another centrifuge tube, add one-third of the supernatant volume of 4× protein loading buffer, boil at 100℃ for 10 minutes, and perform electrophoresis directly after cooling.

[0131] The whole-cell lysates treated as above were separated by 12% SDS-PAGE electrophoresis and transferred to a membrane according to the conventional method, and then subjected to Western blot analysis. The PVDF membrane was washed twice with TBST (0.2% Tween20), 5 minutes each time, and then blocked in 5% skim milk powder blocking solution at room temperature for 2 hours; washed twice in TBST buffer, 5 minutes each time; the PVDF membrane was placed in a hybridization box containing a primary antibody and labeled at 4°C overnight; the PVDF membrane was washed 3 times with TBST, 15 minutes each time; the secondary antibody was labeled at room temperature for 50 minutes; the membrane was washed 3 times with TBST, 10 minutes each time; and ultra-sensitive ECL was added to develop the PVDF membrane.

[0132] Results: Figure 7 and 8 As shown. FLT3 and c-KIT are substrates of STS1 and STS2 phosphatases. In order to detect the inhibitory effect of baicalein on STS1 and STS2 phosphatases in blood cells, the phosphorylation levels of FLT3 and c-KIT in cells were detected as evaluation indicators. First, pcDNA-FLT3( Figure 7 A) or pcDNA-cKIT ( Figure 8 A) The expression plasmid was transfected into 293T cells alone and treated with baicalin for different time periods. Western Blot analysis of the phosphorylation levels of FLT3 or c-KIT was performed. The results showed that after 2 minutes of baicalin treatment, the phosphorylation level of FLT3 increased significantly and reached a peak. After 10 minutes, FLT3 still maintained a high phosphorylation level, and then gradually weakened ( Figure 7 A). After 2 minutes of baicalein treatment, the phosphorylation level of cKIT increased significantly. After 10 minutes of treatment, the phosphorylation level of cKIT reached a peak and remained at a high level after 15 minutes of treatment ( Figure 8 A). Afterwards, the FLT3 or c-KIT expression plasmid was co-transfected with the STS1 or STS2 expression plasmid into the cells and then treated with baicalin. The results showed that when STS1 or STS2 was overexpressed, the phosphorylation level of FLT3 was significantly reduced ( Figure 7 B, C), while after 2 min of baicalein treatment, the phosphorylation level of FLT3 was restored ( Figure 7 B, C). Similarly, when STS1 or STS2 was overexpressed, the phosphorylation level of cKIT was also significantly reduced ( Figure 8 B, C), while after 10 min of baicalein treatment, the phosphorylation level of cKIT was restored ( Figure 8 B, C). The above results indicate that baicalein can also inhibit the phosphatase activity of STS1 and STS2 in cells and thus promote the phosphorylation levels of FLT3 and cKIT.

[0133] Example 7 Baicalein promotes proliferation and differentiation of mouse bone marrow hematopoietic stem / progenitor cells in vitro

[0134] Main materials: 6-8 week old male C57BL / 6J mice (SPF grade) were purchased from Liaoning Changsheng Biotechnology Co., Ltd.; Ammonium Chloride Solustion (CAT#07800), MethoCult TM GF M3434 culture medium (CAT#03434) was purchased from Stemcell; mouse SCF (CAT#250-03), TPO (CAT#AF-315-14) and IL-3 (CAT#213-13) were purchased from PeproTech; BD Cytofix / Cytoperm TM (CAT#554714) was purchased from BD Bioscience, 200-mesh copper mesh was purchased from Hebei Huanyu Metal Mesh Products Co., Ltd.; anti-mouse Ly-6A / E (Sca-1) PE (D7) (CAT#12-5981-83), anti-mouse CD34 Alexa Fluor 700 (RAM34) (CAT#56-0341-82), anti-mouse CD117 (c-Kit) PE-Cyanine7 (2B8) (CAT#25-1178-42), anti-mouse CD135 (FLT3) APC (BV 10A4H2) (CAT#17-1351-82), anti-mouse CD150 APC (mShad 150) (CAT#17-1502-80) and other antibodies were purchased from eBioscience.

[0135] Methods: After mice were killed by cervical dislocation, they were soaked in 75% ethanol for 15 minutes, and then the femurs and tibias on both sides were taken and placed in pre-cooled PBS. The two ends of the femurs and tibias were cut with sterile scissors to expose the bone marrow cavity. The bone marrow cavity was flushed with PBS several times until the bone was flushed white. The flushed bone marrow cells were blown away with a pipette, and then the bone marrow cell suspension was filtered with a 200-mesh copper mesh. After obtaining the bone marrow single cell suspension, it was centrifuged at 1200rpm for 10min; the supernatant was discarded, and the cells were resuspended with 1mL of pre-cooled PBS, and 1× red blood cell lysis buffer was added at a ratio of 1:4, and the cells were allowed to stand at 4℃ for 20min and centrifuged at 1200rpm for 10min; the cell pellet was washed with 5mL of PBS and the cells were resuspended with 1mL of culture medium (IMDM+10% FBS+100ng / mL SCF+25ng / mL TPO+25ng / mL IL-3), which are mouse primary bone marrow mononuclear cells (BMMNCs);

[0136] Cell count: 1×10 per well7 Primary mouse BMMNCs were inoculated into 6-well plates, and two groups of baicalin concentrations were set, 10 μg / mL and 20 μg / mL, respectively. A control group (i.e., DMSO group) was set as a control. After drug addition, the cells were cultured in a 37°C, 5% CO2 incubator according to the normal BMMNCs culture method. The cells in each group were stained with trypan blue and counted on the 4th, 7th, 11th, and 14th days of culture, and the cell proliferation rate was calculated.

[0137] Flow cytometry: BMMNCs cultured and treated as described above were collected on the 4th and 7th days of culture. 5×10 primary mouse bone marrow cells were collected from each group. 6 cells, centrifuged at 1500 rpm for 5 min, discarded the supernatant, washed with 500 μL PBS, added with 100 μL 2% FBS staining solution containing different antibody combinations (LSK: 5 μL Lineage-FITC, 1 μL Sca-1-PE, 1 μL cKit-PE-Cy7; LT-HSCs, ST-HSCs and MPPs: 5 μL Lineage-FITC, 1 μL Sca-1-PE, 1 μL cKit-PE-Cy7, 1 μL FLT3-APC, 1 μL CD34), incubated at 4°C in the dark for 30 min, washed with PBS, resuspended in 200 μL PBS, and detected by flow cytometry;

[0138] Colony formation experiment: BMMNCs from mice treated with baicalin were collected on the 7th day of in vitro culture. 3 cells, add the cells to MethoCult TM After GF M3434 culture medium, vortex to mix the cells and culture medium, then use a spiral syringe and a blunt needle to absorb 1.2 mL of culture medium, and inject it into a 6-well plate at a uniform speed to make the culture medium evenly and stably spread in the 6-well plate; culture in a 37°C, 5% CO2 incubator, and mouse hematopoietic stem / progenitor cell colonies can be observed on the 10th to 12th day of culture; observe according to the atlas of mouse hematopoietic stem / progenitor cell colonies: granulocyte-macrophage colony forming units (colony forming units for granulocytes and macrophages, CFU-GM), burst-forming unit-erythroid (burst-forming unit-erythroid, BFU-E); granulocyte-erythrocyte-macrophage-megakaryocyte colony formation (colony forming unit-granulocyte, erythrocyte, monocyte and megakaryocyte, CFU-GEMM) and count.

[0139] Experimental results:

[0140] (1) Baicalein promotes the proliferation of mouse BMMNCs in vitro

[0141] Table 8 shows the ratio of the number of cells in the BMMNCs isolated by baicalein treatment for different time periods to that of the untreated group. Fig. 9 As shown in the figure, the proliferation of BMMNCs cells in the baicalin treatment group was not obvious in the first 4 days of culture. As the culture time prolonged, the cell proliferation rate gradually accelerated and reached a peak on the 14th day of culture. The cell numbers of the 10μg / mL and 20μg / mL baicalin treatment groups were 5.35 times and 6.11 times of the initial cell number, respectively. At this time, the cell number of the untreated group was 4.18 times of the initial cell number, which was significantly lower than that of the baicalin treatment group. After continued culture, the cell number of the baicalin treatment group did not decrease significantly. At the 17th day of culture, the cell number was still higher than that of the untreated group. The cell numbers of the 10μg / mL and 20μg / mL baicalin treatment groups were 4.73 times and 5.61 times of the original cell number, respectively, while the cell number of the untreated group was 3.32 times of the original cell number. The above results show that baicalin promotes cell proliferation at a slow rate in the early stage of culture, but can keep cells in a stable growth state for a long time, and can still keep the cell number at a high level in the later stage of culture.

[0142] Table 8: The number of BMMNCs cells treated with baicalin for different time periods relative to the untreated group

[0143]

[0144] (2) Baicalein increases the proportion of hematopoietic stem / progenitor cells in mouse BMMNCs in vitro

[0145] Lin - For immature cells, LSK (Lin - sca1 + ckit + ) are hematopoietic stem / progenitor cells. Fig.10As shown, Figure A is a representative figure of LSK cell flow cytometry analysis, and Figure B is a statistical diagram of the proportion of LSK cells. It can be seen from the figure that, overall, with the extension of in vitro culture time, the proportion of LSK cells in the cells gradually increased. When cultured to the 4th day, the proportion of LSK cells in the control group accounted for 0.49% of BMMNCs, while the proportion of LSK cells in BMMNCs treated with baicalein on the 4th day of culture was significantly higher than that in the control group. The proportions of LSK after treatment with 10μg / mL and 20μg / mL baicalein were 0.69% and 0.65%, respectively; when cultured to the 7th day, the proportion of LKS in the control group increased to 0.94%, and the proportions of LSK cells in the 10μg / mL and 20μg / mL baicalein treatment groups were 1.46% and 2.71%, respectively, which were significantly higher than those in the control group. The above results indicate that baicalein can increase the proportion of hematopoietic stem / progenitor cells in mouse BMMNCs under in vitro culture conditions, and when cultured to the 7th day, the improvement effect of 20μg / mL baicalein is better.

[0146] (3) Baicalein improves the colony-forming ability of mouse hematopoietic stem / progenitor cells in vitro

[0147] The number of colonies formed by each lineage in three repeated experiments is shown in Table 9, and the results are analyzed as follows Fig.11 As shown in the figure, after treatment with 20μg / mL baicalin, the total number of colonies increased significantly, 1.44 times and 1.36 times that of the control group, respectively. At the same time, the number of colonies of each lineage also increased to varying degrees, and there was no obvious lineage-biased differentiation, indicating that baicalin can improve the differentiation ability of mouse HSPC. The above results are basically consistent with the results of the phenotypic analysis of mouse BMMNCs cells after in vitro culture, indicating that baicalin can improve the proliferation and differentiation ability of mouse hematopoietic stem / progenitor cells.

[0148] Table 9: Number of colony formation of each lineage of BMMNCs treated with baicalein in vitro

[0149]

[0150] Example 8 Baicalein promotes human umbilical cord blood CD34 + Cell proliferation and differentiation

[0151] Main materials: Human umbilical cord blood samples were collected from healthy normal delivery women in the Department of Obstetrics and Gynecology of the Second Clinical Hospital of Jilin University. The donations were voluntary and in compliance with ethical regulations. The umbilical cord blood was collected into a sodium heparin anticoagulation blood collection bag under sterile conditions, stored at 4°C, and sorted within 24 hours; Ammonium Chloride Solution (CAT#07800), MethoCult TM H4034optimum medium (CAT#04034), StemSpan TMSFEM II serum-free hematopoietic stem cell medium (CAT#09605), EasySep Human CD34 Positive selection Kit II (CAT#17856) and Lymphoprep (CAT#07801) were purchased from Stemcell; human SCF (CAT#30007), TPO (CAT#300-18) and IL-6 (CAT#20006) were purchased from PeproTech; BD Cytofix / Cytoperm TM purchased from BD Bioscience; anti-human CD45 APC (HI30) (CAT#17-0459-42) antibody was purchased from eBioscience, anti-human CD34 PE (581) (CAT#343506), anti-human CD90 (Thy 1) (CAT#328108), anti-human CD38 (HIT2) FITC (CAT#980304) and anti-human CD45RA-PE / Cy7 (HI100) (CAT#983006) antibodies were purchased from Biolegend, and 200-mesh copper mesh was purchased from Hebei Huanyu Metal Mesh Products Co., Ltd.

[0152] Methods: Umbilical cord blood samples were collected in a 50 mL centrifuge, with 15 mL of sample in each tube; 75 μL RosetteSep was added to the blood sample, mixed and incubated at room temperature for 20 min; an equal volume of EasySep Buffer was added and density gradient centrifugation was performed; the cells after centrifugation were washed with EasySep Buffer and resuspended with 2 mL EasySep Buffer, and then red blood cell lysis buffer was added at a ratio of 1:4 to lyse the red blood cells; the remaining nucleated cells were added with 0.75 mL EasySep Buffer and transferred to a 5 mL flow tube, 75 μL Selection Cocktail was added, mixed and incubated at room temperature for 10 min, and then 50 μL magnetic beads were added for CD34 + Cell magnetic bead sorting;

[0153] Human umbilical cord blood CD34 + Cell culture: sorted human cord blood CD34 + The cells were cultured at 1×10 5100 cells / mL were inoculated in a 12-well plate. The culture conditions were StemSpan SFEMⅡ hematopoietic stem cell serum-free medium, supplemented with human SCF (100 ng / mL), human TPO (100 ng / mL) and human IL-6 (100 ng / mL). No treatment was performed in the first 3 days of culture. The color of the culture medium was observed on the 4th day of culture, and it changed from pink to slightly yellowish. At this time, half-volume medium replacement was adopted, and an equal volume of fresh culture medium containing cytokines was added. After that, half-volume medium replacement was performed every 2 days, and culture was carried out in a 37°C, 5% CO2 incubator. When baicalein was treated, a control group and baicalein 10μg / mL and 20μg / mL drug-added groups were set up. On the 0th day of culture, the cells were cultured with culture medium containing the corresponding concentration of compounds. After that, fresh culture medium containing baicalein was added every half-volume medium replacement.

[0154] Flow cytometry: Human cord blood CD34 cultured and processed as described above + Cells, 1×10 per group 5 Cells were collected on the 4th and 7th day of culture and stained with 100 μL antibody staining solution (i.e., 1 μL CD34-PE, 1 μL CD38-FITC, 1 μL CD45RA-Cy7, and 1 μL CD90-APC were added to 100 μL 2% FBS PBS) to mark the hematopoietic stem / progenitor cell population with CD34 and CD38 as markers, and CD45RA and CD90 were used to further distinguish the more primitive hematopoietic stem cell population. After washing, 200 μL PBS was added to resuspend the cells and detected by flow cytometry;

[0155] Colony formation experiment: CD34 cells of umbilical cord blood treated with baicalein were collected on the 7th day of in vitro culture. + Cells were collected at 2 × 10 3 cells, add the cells to MethoCult TM After H4034 culture medium, vortex and then use a spiral syringe and a blunt needle to draw 1.2 mL of culture medium and inject it into a 6-well plate at a uniform speed so that the culture medium is evenly and stably spread in the 6-well plate; culture in a 37°C, 5% CO2 incubator, and human hematopoietic stem / progenitor cell colonies can be observed on the 10th to 12th day of culture; observe and count the colonies according to the atlas of human hematopoietic stem / progenitor cell colonies.

[0156] Experimental results:

[0157] (1) Baicalein increases the proportion of hematopoietic stem / progenitor cells in human umbilical cord blood in vitro

[0158] like Fig.12 As shown in the figure, on the 4th day of culture, both 10 μg / mL and 20 μg / mL baicalein significantly increased the proportion of hematopoietic stem / progenitor cells, CD34 + CD38- The cell population accounted for CD34 + The proportion of cells reached 14.429% and 13.291%, respectively, which was 1.4 times and 1.29 times that of the control group ( Fig.12 B), CD34 + CD38 - CD45RA - CD90 + The proportions of the cell populations were 4.465% and 4.306%, respectively, which were 1.9 times and 1.83 times that of the control group ( Fig.12 C); on the 7th day of culture, although the CD34 + CD38 - The proportion of hematopoietic stem / progenitor cells was lower than that of the control group ( Fig.12 B), but a more primitive hematopoietic stem cell population (CD34 + CD38 - CD45RA - CD90 + ) was significantly higher than that of the control group. The proportions of the 10μg / mL and 20μg / mL baicalein treatment groups were 0.793% and 1.106%, respectively, which were 1.48 times and 2.08 times that of the control group ( Fig.12 C), representative flow cytometry analysis Fig.12 As shown in A. This shows that baicalein can increase the proportion of hematopoietic stem / progenitor cells in human umbilical cord blood under in vitro culture conditions, and the effect is most obvious at 4 days of culture.

[0159] (2) Baicalein improves the colony-forming ability of human umbilical cord blood hematopoietic stem / progenitor cells in vitro

[0160] The number of colonies formed by each lineage in three repeated experiments is shown in Table 10, and the results are analyzed as follows Fig.13 As shown in the figure, after treatment with 10μg / mL baicalein, the total number of colonies increased significantly, which was 1.72 times that of the control group. At the same time, the number of colonies of each lineage also increased to varying degrees, and all were statistically significant. This result shows that baicalein can improve the differentiation ability of human hematopoietic stem / progenitor cells, and the number of CFU-GEMM also increased significantly, indicating that baicalein can promote the proliferation and differentiation ability of human umbilical cord blood hematopoietic stem / progenitor cells.

[0161] Table 10: Number of colony formation of each lineage of human umbilical cord blood hematopoietic stem / progenitor cells treated with baicalein in vitro

[0162]

[0163] Example 9 Baicalein promotes proliferation and differentiation of normal mouse bone marrow hematopoietic stem / progenitor cells in vivo

[0164] Main materials: See the material introduction in Example 7.

[0165] Methods: Male 8-week-old C57BL / 6 mice were selected and baicalein was continuously administered for 7 days at concentrations of 10 mg / kg and 20 mg / kg. A control group (i.e., no-drug group) was set up at the same time, with 6 mice in each group. The mice were killed after 7 days, and then relevant tests were performed according to the methods described in the method section of Example 8 regarding mouse BMMNCs isolation, flow cytometry analysis, and clone formation ability detection.

[0166] Experimental results:

[0167] (1) Baicalein increases the proportion of hematopoietic stem / progenitor cells in the bone marrow of normal mice in vivo

[0168] Fig.14 A shows a representative diagram of the proportion of hematopoietic stem / progenitor cells analyzed by flow cytometry. Fig.14 B is a statistical chart of the proportion of hematopoietic stem / progenitor cells, and the specific data are shown in Table 11. The results showed that after 7 consecutive days of intraperitoneal injection of baicalin, the proportion of hematopoietic stem / progenitor cells LSK in mouse BMMNCs increased significantly, and the proportions of mice injected with 10mg / kg and 20mg / kg baicalin were 0.43% and 0.47%, respectively, which were 1.19 times and 1.3 times that of the control group; after a more in-depth analysis of the hematopoietic stem / progenitor cell population, it was found that the proportion of long-term HSCs (LT-HSCs) increased significantly after treatment with 10mg / kg and 20mg / kg baicalin, and the proportions were 0.049% and 0.047%, respectively, which were 1.63 times and 1.57 times that of the control group, and the proportions of short-term HSCs (ST-HSCs) and multi-pluoripoent progenitor cells (LT-HSCs) increased significantly. The proportion of ST-HSCs in the 10mg / kg baicalin treatment group was significantly higher than that in the control group, 1.3 times that of the control group, and the proportion of MPPs in the 20mg / kg baicalin treatment group was significantly higher than that in the control group, 1.39 times that of the control group. The above results show that after healthy mice were injected with baicalin, the proportion of LSK in the bone marrow increased significantly, and the proportions of the three cell subsets increased, but the increase in the proportion of LT-HSCs was the main factor. In short, the results show that baicalin may increase the proportion of more primitive cells in the mouse bone marrow hematopoietic stem cells to a greater extent, and may promote their ability to self-renew.

[0169] Table 11: Flow cytometry analysis of the proportion of hematopoietic stem / progenitor cells in the bone marrow of mice treated with baicalin

[0170]

[0171]

[0172] (2) Baicalein enhances the colony-forming ability of normal mouse bone marrow hematopoietic stem / progenitor cells in vivo

[0173] The number of colonies formed by each lineage in three repeated experiments is shown in Table 12, and the results are analyzed as follows Fig.15 As shown in the results, after continuous intraperitoneal injection of baicalein for 7 days, the total number of colonies formed by mouse bone marrow hematopoietic stem / progenitor cells was significantly higher than that of the control group, which was 1.17 times and 1.37 times of that of the control group, respectively. At the same time, there was no lineage differentiation, the number of colonies of each lineage increased, and the number of more primitive progenitor cell colonies CFU-GEMM was also higher than that of the control group. The above results show that after continuous intraperitoneal injection of baicalein for 7 days, the proliferation and differentiation ability of mouse bone marrow hematopoietic stem / progenitor cells was significantly improved, and the injection dose of 20 mg / kg had a better effect on improving the proliferation and differentiation ability of hematopoietic stem / progenitor cells than the injection dose of 10 mg / kg.

[0174] Table 12: Number of colonies formed by each lineage of hematopoietic stem / progenitor cells in the bone marrow of mice treated with baicalin

[0175]

[0176] Example 10 Baicalein has a protective effect on 5-FU bone marrow injury in mice

[0177] Materials: See the materials introduction in Example 7.

[0178] Methods: 8-week-old male C57BL6 / J mice were selected. The 5-FU model group was intraperitoneally injected with a lethal dose of 150 mg / kg 5-FU once a week. The survival period was observed for 3-4 weeks until all groups died. The bone marrow suppression was analyzed for two weeks. The baicalein treatment group was observed for survival with two doses of 20 mg / kg and 40 mg / kg. The bone marrow suppression was analyzed with a dose of 20 mg / kg. Another group was the normal control group. The baicalein group was intraperitoneally injected with baicalein (20 mg / kg) for 3 consecutive days, once a day. The first intraperitoneal injection of a lethal dose of 5-FU was started 24 hours after the last administration.

[0179] In the experiment of observing the survival period, the mortality rate in each time period was counted until all the mice in the model group died; when analyzing the bone marrow suppression, one week after the second 5-FU injection, the mice were anesthetized by intraperitoneal injection of an appropriate amount of 4% chloral hydrate and 5 mg / kg carprofen, and then the relevant tests were performed according to the methods described in the method section of Example 8, such as the separation of mouse BMMNCs, flow cytometry analysis and clone formation ability detection.

[0180] Experimental results:

[0181] (1) Baicalin reduces the mortality of mice with 5-FU bone marrow injury

[0182] The weight of each mouse in each group is recorded in Table 13, and the results are analyzed as follows: Fig.16 As shown in A, after the lethal dose of 5-FU was injected, the weight of the mice also showed a trend of first decreasing and then increasing. After the first two rounds of 5-FU injection, there was no significant difference in the weight of the mice in the baicalin group compared with the control group. By the third round of 5-FU injection, that is, 15 days after the first injection of 5-FU, the weight of the mice in the 20 mg / kg baicalin group was significantly higher than that of the control group, while there was no significant difference in the weight of the mice in the 40 mg / kg baicalin group compared with the control group. The weight results show that low-dose baicalin may be more helpful for 5-FU-injured mice to recover to a healthy state. The death records of mice in each group are shown in Table 14. At the end of the experiment, when all the mice in the control group had died from 5-FU injury, the survival rate of mice in the 20 mg / kg baicalin group was 60%, and the survival rate of mice in the 40 mg / kg group reached 40%, that is, the survival rate of the 20 mg / kg group was higher than the survival rate of ( Fig.16 B), which also shows that low-dose baicalin has a more significant protective effect on mice with bone marrow damage, which is consistent with the results of body weight detection. This experiment proves that preventive use of baicalin can significantly improve the health status and survival rate of mice with 5-FU damage.

[0183] Table 13: Body weight of 5-FU-injured mice treated with baicalin

[0184] Days 5-FUControl BC20mg / kg+5-FU BC40mg / kg+5-FU 1 22.885 22.452 21.775 2 22.701 22.576 22.405 3 22.317 21.948 21.655 4 23.4 23.067 23.315 5 21.351 20.913 21.094 6 20.588 20.339 20.344 7 20.363 20.571 20.399 8 20.239 21.136 20.563 9 20.508 21.683 21.143 10 21.527 22.062 21.601 11 22.799 22.886 22.631 12 20.297 21.059 20.573 13 19.598 20.181 20.486 14 20.45667 20.945 20.199 15 20.58556 21.111 20.291 16 19.81444 21.104 20.26333 17 19.13222 20.98444 19.27444 18 18.82857 20.895 18.4675 19 17.86286 19.84625 17.34143 20 17.74167 19.29625 16.405 21 18.1475 20.61167 18.57667 22 17.1 18.75222 17.53 23 16.43 17.95222 17.03 24 16.31 18.04 17.49

[0185] Table 14: Analysis of mortality in 5-FU-injured mice treated with baicalin

[0186]

[0187]

[0188] Note: 1 means death, 0 means survival

[0189] (2) Baicalein improves 5-FU-induced bone marrow damage and increases the number of bone marrow nucleated cells

[0190] The results are as follows Fig.17 After 5-FU injection, the number of bone marrow cells in the unilateral femur of mice was 0.79×10 7 The number of cells decreased sharply, only 56% of the number of nucleated cells in the bone marrow of the normal control group, while the number of cells in the unilateral bone marrow of the mice treated with baicalin was 1.2432×10 7 , which was significantly higher than that of the model control group, indicating that baicalin treatment partially restored the number of nucleated cells in the bone marrow of 5-FU-injured mice ( Fig.17A); HE staining results of femoral bone marrow of mice showed that the pathological phenomena such as the significant decrease in the number of cells in the bone marrow, irregular arrangement of trabeculae, disordered structure, incomplete microstructure and the presence of a large number of cavities caused by 5-FU were significantly reduced. The bone marrow structure of mice in the baicalin treatment group was significantly improved, and the number of bone marrow cells and microstructure integrity were also significantly improved ( Fig.17 B). The above results indicate that baicalein pretreatment has a certain protective effect on the destruction of bone marrow structure in the femur of mice injured by 5-FU.

[0191] (3) Effect of baicalin treatment on the proportion of hematopoietic stem / progenitor cells in the bone marrow of mice injured by 5-FU

[0192] The results are as follows Fig.18 As shown, Fig.18 A is a representative diagram of flow cytometry analysis of hematopoietic stem / progenitor cells. Fig.18 B and Fig.18 C is a statistical chart of the proportion and absolute number of hematopoietic stem cells and progenitor cells. The results show that LSK accounts for about 0.34% of the total bone marrow cells in normal mice, which is similar to the previous results. After two injections of lethal doses of 5-FU, the proportion of LSK decreased significantly to only 0.046%, while the proportion of LSK in the baicalein-treated group was 0.209%, significantly higher than that in the untreated group ( Fig.18 B); Further analysis of the proportions of different cell populations in LSK cells showed that the proportions of LT-HSCs, ST-HSCs and MPPs in the baicalein treatment group were 0.05%, 0.065% and 0.09%, respectively, while those in the untreated group were 0.009%, 0.013% and 0.023%, respectively. The proportions of the three cell populations were significantly higher than those in the untreated group ( Fig.18 B); the absolute number of each cell group in the bone marrow of mice treated with baicalin was also significantly higher than that of the untreated group ( Fig.18 C). Specific data are shown in Table 15 and Table 16. The above results indicate that baicalein pretreatment can partially alleviate bone marrow failure caused by a lethal dose of 5-FU.

[0193] Table 15: Flow cytometry analysis of the proportion of hematopoietic stem / progenitor cells in the bone marrow of 5-FU-injured mice treated with baicalin

[0194]

[0195] Table 16: Flow cytometry analysis of the total number of hematopoietic stem / progenitor cells in the bone marrow of 5-FU-injured mice treated with baicalin

[0196]

[0197] (4) Baicalin treatment improves the colony-forming ability of bone marrow hematopoietic stem / progenitor cells in 5-FU-injured mice

[0198] The number of colonies formed by each lineage in three repeated experiments is shown in Table 17, and the results are analyzed as follows: Fig.19 As shown in the results, after the injection of a lethal dose of 5-FU, the number of colonies formed by mouse bone marrow hematopoietic stem / progenitor cells was 21, which was significantly lower than that of the normal control group, while the total number of colonies in the baicalin treatment group was 44, which was 2.09 times the total number of the untreated group. At the same time, the number of BFU-E, CFU-GM and CFU-GEMM colonies also increased due to baicalin treatment, and the number of each colony was significantly higher than that of the untreated group. The above results show that the preventive use of baicalin can resist the bone marrow damage caused by 5-FU by improving the proliferation and differentiation ability of mouse hematopoietic stem / progenitor cells.

[0199] Table 17: Number of colony formation of each lineage of hematopoietic stem / progenitor cells in the bone marrow of 5-FU-injured mice treated with baicalin

[0200]

[0201] Example 11 Baicalin reduces the mortality rate of mice irradiated with a lethal dose

[0202] Materials: 8-week-old male C57BL / 6J mice (SPF grade) were purchased from Liaoning Changsheng Biological Company; X-ray radiotherapy machine was purchased from Dandong Kangjia Instrument Equipment Co., Ltd.

[0203] Methods: Mice were randomly divided into groups. Baicalein was administered at two concentrations, 20 mg / kg and 40 mg / kg, and the mice were irradiated with a dose of 6.5 Gy. A control group (irradiated and given solvent group) was set up, with 8 mice in each group. Baicalein was intraperitoneally injected for 3 consecutive days. 24 hours after the last administration, the mice were irradiated with 6.5 Gy. The body weight was recorded every day and the survival rate was observed.

[0204] Results: The body weight of each mouse in each group is shown in Table 18. Fig. 20 As shown in A, compared with the control group, the weight of mice in the baicalin group increased, but the overall weight still showed a downward trend and was always lower than before irradiation; while the weight change of mice in the 20 mg / kg baicalin group was no different from that of the control group ( Fig. 20 A). The conditions of each group of mice are recorded as shown in Table 19, and the results are analyzed as shown in Fig. 20 As shown in B, at the end of the experiment, the survival rate of the control group was 25%, and the survival rates of the 20mg / kg and 40mg / kg baicalein groups were 50% and 37.5%, respectively, indicating that the 20mg / kg and 40mg / kg baicalein groups can improve the survival rate of radiation-damaged mice to a certain extent ( Fig. 20 B). In summary, baicalein has a preventive effect on bone marrow damage caused by radiation.

[0205] Table 18: Body weight of mice treated with baicalin and subjected to lethal dose of radiation

[0206]

[0207]

[0208] Table 19: Analysis of mortality in mice treated with lethal dose of radiation by baicalein

[0209] Days Control BC20mg / kg BC40mg / kg 23 0 —— —— 23 0 —— —— 18 1 —— —— 15 1 —— —— 15 1 —— —— 14 1 —— —— 11 1 —— —— 11 1 —— —— 23 —— 0 —— 23 —— 0 —— 23 —— 0 —— 23 —— 0 —— 22 —— 1 —— 15 —— 1 —— 13 —— 1 —— 11 —— 1 —— 23 —— —— 0 23 —— —— 0 23 —— —— 0 22 —— —— 1 17 —— —— 1 14 —— —— 1 13 —— —— 1 10 —— —— 1

[0210] Note: 1 means death, 0 means survival

[0211] Example 12 Baicalein structural analogs inhibit STS1 and STS2 phosphatase activities

[0212] Main materials: p-Nitrophenyl phosphate (PNPP) was purchased from Sigma-Aldrich.

[0213] Methods: The inhibitory effect of baicalein analogs on STS1 and STS2 phosphatase activity was confirmed in 96-well plates using the previously confirmed in vitro phosphatase activity detection method of STS1 and STS2. Six replicate wells were set up in each group. First, 3 μL of each baicalein analog (final concentration of 10 μg / mL), negative control solution DMSO or positive inhibitor Na3VO4 (100 mmol / L, pH: 10.0) were added to the 96-well plate, and then 50 μL of PNPP was added and mixed evenly. Then 10 μL of STS1 (150 nmol / L) or STS2 (15 μmol / L) protein working solution was added, and incubated at room temperature for 10 min (STS1) and 20 min (STS2). After the reaction, 40 μL of stop buffer was added to terminate the reaction, and the absorbance at 405 nm was detected by microplate reader. The inhibition rate was calculated according to the following formula: inhibition rate (100%) = (1-OD405nm of drug-treated group / OD405nm of negative control group) × 100%).

[0214] Results: As shown in Table 20, each baicalein structural analogue showed varying degrees of inhibitory effect on the phosphatase activity of STS1 and STS2.

[0215] Table 20: Inhibitory effect of baicalein analogs on STS1 and STS2 phosphatase activities

[0216]

[0217] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of baicalin in the preparation of drugs for promoting proliferation and / or differentiation of hematopoietic stem / progenitor cells in vitro.

2. The use according to claim 1, characterized in that The hematopoietic stem / progenitor cells are derived from bone marrow or cord blood.

Images