A short peptide and its use in the preparation of a therapeutic product for radiotherapy-induced anemia

Abstract

The present application relates to a kind of short peptide and its application in the preparation of radiotherapy-induced anemia treatment product, belong to biological medicine technical field.The short peptide Ac-LSPD-CHO is prepared in the present application, in the short peptide of the present application, the aldehyde group modified on aspartic acid can be covalently combined with the thiol of cysteine residue in the active site of caspase, form thiohemiacetal structure, to irreversibly inhibit the activity of caspase 2 and 3.When caspase 2 and 3 are inhibited, its enzyme cleavage myo-inositol polyphosphate-5-phosphatase D is blocked, and the differentiation of red blood cells involved by myo-inositol polyphosphate-5-phosphatase D can be normally carried out.Therefore, Ac-LSPD-CHO can not only improve anemia after radiotherapy, but also it will not stimulate the production of EDMC cell like erythropoietin, even will inhibit the expansion of immunosuppressive cells, can be used as radiotherapy adjuvant to strengthen its anti-tumor effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a short peptide and its application in the preparation of therapeutic products for radiotherapy-induced anemia. Background Technology

[0002] Cancer patients often experience anemia to varying degrees. Studies have shown that tumors can induce a portion of CD45... + Erythroid precursor cells (EPCs) lose their ability to differentiate into erythroid cells and differentiate into a population of "erythroid-myeloid hybrids" called erythroid differentiated myeloid cells (EDMCs). In cancer patients and tumor-bearing mice, EDMCs develop into a myeloid-derived suppressor cell-like subset with increased immunosuppressive potential. They impair T cell function and are associated with negative responses to immune checkpoint inhibitors (ICIs) and tumor-associated anemia.

[0003] Radiation therapy (RT) and chemotherapy are the two pillars of modern cancer treatment. The incidence of cancer treatment-associated anemia (CTRA) is quite high. Studies show that the incidence of anemia in patients receiving both RT and chemotherapy, especially those receiving both simultaneously, can range from 40% to 90%, depending on the type of tumor and the treatment regimen. Anemia is particularly common in pelvic malignancies due to extensive bone marrow irradiation.

[0004] Erythropoietin (EPO), also known as red blood cell stimulating factor, is a glycoprotein that stimulates bone marrow progenitor cells to form red blood cells. Recombinant EPO has significantly improved the treatment of anemia by increasing serum hemoglobin levels and reducing the need for blood transfusions. However, preclinical background and some clinical data indicate that EPO can exert a detrimental effect in cancer by stimulating tumor growth, and therefore, EPO is not suitable for the treatment of anemia in cancer patients. Therefore, there is an urgent need to find a drug that can treat anemia caused by radiotherapy and chemotherapy without stimulating tumor growth. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem that drugs that relieve anemia caused by radiotherapy in the prior art can stimulate tumor growth and thus aggravate cancer.

[0006] To address the aforementioned technical problems, this invention provides a short peptide and its application in the preparation of therapeutic products for radiotherapy-induced anemia. This invention prepares a short peptide, Ac-LSPD-CHO. In this short peptide, the aldehyde group modified on the aspartic acid residue can covalently bind to the thiol group (-SH) of the cysteine ​​residue at the caspase active site, forming a thiohemiacetal structure, thereby irreversibly inhibiting the activity of caspases 2 and 3. When caspases 2 and 3 are inhibited, their cleavage of inositol polyphosphate-5-phosphatase D is blocked, allowing the normal erythrocyte differentiation mediated by inositol polyphosphate-5-phosphatase D to proceed. Therefore, Ac-LSPD-CHO can not only promote the differentiation of bone marrow stem cells into MEP (megakaryocyte-erythroid progenitor cell), EPC (erythroid progenitor cell), and erythrocytes, thus improving anemia after radiotherapy, but it also does not stimulate the production of EDMC cells like erythropoietin. In fact, it can even inhibit the proliferation of immunosuppressive cells, thus enhancing the anti-tumor effect of radiotherapy without aggravating the cancer condition.

[0007] The first object of the present invention is to provide a short peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Furthermore, SEQ ID NO.1: LSPD.

[0009] Furthermore, in the short peptide, leucine is modified by acetylation (Ac), and aspartic acid is modified by aldehyde group (CHO). Ac is an acetyl group (N-Acetyl), located at the N-terminus of the peptide, which blocks the amino terminus, enhances the cell membrane permeability and in vivo stability of the molecule, prevents the peptide chain from being rapidly degraded by aminopeptidase, and prolongs the reaction time of the reagent in the experimental system. CHO is an aldehyde group (aldehyde functional group), located at the C-terminus of the peptide, which can form a reversible covalent bond with the cysteine ​​residue at the active site of caspase, precisely binding to the catalytic center of the enzyme to achieve a strong and specific inhibitory effect. This is the core structural basis for the reagent's ability to efficiently block caspase cleavage.

[0010] Furthermore, the structural formula of the short peptide is shown in Formula I:

[0011] .

[0012] A second objective of this invention is to provide an application of the above-mentioned short peptide in the preparation of anemia treatment drugs.

[0013] Furthermore, the anemia includes anemia caused by irradiation (X-rays, gamma rays, protons and / or heavy ions).

[0014] Furthermore, the anemia includes aplastic anemia caused by irradiation (X-rays, gamma rays, protons and / or heavy ions).

[0015] Furthermore, the anemia includes anemia caused during cancer radiotherapy.

[0016] Furthermore, the cancers mentioned include colorectal cancer.

[0017] A third object of the present invention is to provide a pharmaceutical composition for treating anemia, the pharmaceutical composition comprising the aforementioned short peptides.

[0018] A fourth objective of this invention is to provide an application of the above-mentioned short peptide in the preparation of antitumor drugs.

[0019] A fifth object of the present invention is to provide a product for preventing the enzymatic cleavage of inositol polyphosphate-5-phosphatase D, the product comprising the aforementioned short peptide, the product being intended for non-disease treatment and diagnostic purposes.

[0020] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0021] This invention presents for the first time a short peptide, Ac-LSPD-CHO, which irreversibly inhibits the activity of caspases 2 and 3. When caspases 2 and 3 are inhibited, their cleavage of inositol polyphosphate-5-phosphatase D is blocked, allowing erythrocyte differentiation mediated by inositol polyphosphate-5-phosphatase D to proceed normally. Therefore, Ac-LSPD-CHO can not only promote the differentiation of bone marrow stem cells into MEPs (megakaryocyte-erythroid progenitor cells), EPCs (erythroid precursor cells), and erythrocytes, thus improving anemia after radiotherapy, but also avoids stimulating the production of EDMC cells like erythropoietin, thereby preventing the aggravation of cancer symptoms by anemia medications. Attached Figure Description

[0022] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0023] Figure 1 This is the mass spectrum of Ac-LSPD-CHO of the present invention;

[0024] Figure 2 This is the high-performance liquid chromatogram of Ac-LSPD-CHO of the present invention;

[0025] Figure 3These are electrophoresis images obtained from Western blot analysis. Image A shows the inhibition results of Ac-LSPD-CHO on Inpp5d protein cleavage by caspase 3; Image B shows the inhibition results of Ac-LSPD-CHO on Inpp5d protein cleavage by caspase 2.

[0026] Figure 4 This is a graph showing the results of Ac-LSPD-CHO improving irradiated anemia and bone marrow MEP cells in normal mice. In the graph, ns indicates no significant difference, and * indicates... P <0.05, ** indicates P <0.01, *** indicates P <0.001;

[0027] Figure 5 This is an experimental flowchart and results diagram of the AOM / DSS-induced orthotopic colorectal cancer mouse model. A is the experimental flowchart, and B is an image of the colorectal tumor in the orthotopic colorectal cancer mouse model and an HE staining image of the colorectal tissue. The red arrow points to the colorectal tumor.

[0028] Figure 6 The results of Ac-LSPD-CHO improving anemia and bone marrow MEP cells after radiotherapy in mice with orthotopic colorectal cancer are shown in Figure A, which shows the results of blood routine index detection in each group, and Figure B shows the proportion of MEP cells in the bone marrow of mice in each group.

[0029] Figure 7 This study shows the effect of Ac-LSPD-CHO on the tumor proliferation activity (Ki67) of mice with orthotopic colorectal cancer after radiotherapy. A is a representative image of Ki67 immunohistochemical staining in tumor tissues of each group. Ki67 positive signals are brownish-yellow and located in the cell nucleus. Scale bar = 100 μm. B is a statistical graph of Ki67 proliferation index.

[0030] Figure 8 These are images of the spleen and spleen area of ​​mice after radiotherapy with Ac-LSPD-CHO in situ for colorectal cancer. A is an external image of the spleen, and B is a statistical image of the spleen area.

[0031] Figure 9 This is a flow cytometry diagram of EPC cells (A), TER cells (B), and EDMC cells (C), where Q1-Q4 represent the four quadrants, FSC-A (forward scattered light, Area): represents the relative size of the cell, SSC-A (side scattered light, Area): represents the internal complexity and granularity of the cell, FSC-H represents the height of the forward scattered light pulse signal, and Comp represents fluorescence compensation;

[0032] Figure 10This is a statistical chart showing the proportions of TER cells and EPC cells in the bone marrow, and TER cells and EDMCs in the spleen. **** indicates... P <0.0001. Detailed Implementation

[0033] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0034] The cells and their characteristics involved in the following examples are shown in Table 1.

[0035] Table 1

[0036]

[0037] Example 1: Synthesis and Characterization of Ac-LSPD-CHO

[0038] 1. Synthesis of Ac-LSPD-CHO

[0039] Ac-LSPD-CHO was synthesized by Hangzhou Gutuo Biotechnology Co., Ltd. The specific synthesis steps are shown below:

[0040] (1) Materials and Instruments:

[0041] Resin: A solid-phase support with special connecting arms (such as skeletal amide connecting arms (BAL connecting arms) or N-methoxy-N-methylamide connecting arms (Weinreb amide connecting arms)) is required to directly generate C-terminal aldehyde groups after pyrolysis. If standard p-alkoxybenzyl alcohol resin (Wang resin) is used, an additional liquid-phase reduction reaction is required after pyrolysis.

[0042] Protected amino acids: Fmoc-Leu(Ac)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Asp(OtBu)-OH. Where Fmoc represents fluorene methoxycarbonyl, tBu represents tert-butyl, OtBu represents tert-butyl ester, Leu represents leucine (L), Ser represents serine (S), Pro represents proline (P), Aspartic acid (D), and Ac represents acetylation.

[0043] Condensation system: HBTU / HOBt / DIEA (molar ratio 1:1:2) or PyBOP. HBTU represents O-(benzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate; HOBt represents 1-hydroxybenzotriazole; DIEA represents N,N-diisopropylethylamine; PyBOP represents (benzotriazol-1-yloxy)tripyrrolidinylphosphine hexafluorophosphate.

[0044] The pyrolysis buffer is TFA / TIS / H2O (95:2.5:2.5, v / v / v). TFA stands for trifluoroacetic acid, and TIS represents triisopropylsilane.

[0045] Purification: Preparative high performance liquid chromatography.

[0046] 2. Synthesis Steps

[0047] Phase 1: Peptide chain assembly

[0048] Weigh out Fmoc-Weinreb linker resin with a substitution degree of 0.3-0.5 mmol / g and swell it in N,N-dimethylformamide (DMF). Remove Fmoc using 20% ​​piperidine / DMF. Couple Fmoc-Asp(OtBu)-OH, Fmoc-Pro-OH, Fmoc-Ser(tBu)-OH, and Fmoc-Leu(Ac)-OH sequentially. Each coupling uses a 4-molar excess of amino acid with HBTU / HOBt / DIEA, reacting at room temperature for 40-60 minutes. After coupling, N-terminal acetylation capping is performed using Ac2O / Pyridine.

[0049] Phase Two: Cleavage and Aldehyde Release

[0050] The resin was washed with DCM and methanol (MeOH) and dried. Pre-cooled lysis buffer was added, and the mixture was stirred at room temperature for 2-3 hours. The lysis buffer was collected by filtration, and the resin was washed with TFA. The filtrates were combined, precipitated with diethyl ether, and the crude peptide was collected by centrifugation. During this process, the linker arm structure on the resin broke, directly converting to a C-terminal aldehyde group (-CHO).

[0051] Phase 3: Purification and Lyophilization

[0052] The crude peptide was dissolved in 0.1% TFA aqueous solution / acetonitrile. Gradient elution was performed using a C18 reversed-phase column (typically 5%-40% acetonitrile / 0.1% TFA, 30 min). The target peaks were collected (identified by electrospray ionization mass spectrometry (ESI-MS) or high-performance liquid chromatography (HPLC), combined, and lyophilized.

[0053] 2. Characterization of Ac-LSPD-CHO

[0054] The electrospray mass spectrometry conditions for Ac-LSPD-CHO are shown in Table 2. The results are as follows: Figure 1 As shown, the observed molecular weight is close to the theoretical molecular weight, indicating that Ac-LSPD-CHO was successfully synthesized.

[0055] Table 2

[0056]

[0057] The HPLC detection conditions for Ac-LSPD-CHO are shown in Table 3. The purity was determined to be 97.24% by HPLC (Table 3 and...). Figure 2 ).

[0058] Table 3 HPLC detection conditions

[0059]

[0060] Table 4 Peak Results

[0061]

[0062] Example 2: Enzymatic digestion experiment of Ac-LSPD-CHO on inositol polyphosphate-5-phosphatase D (Inpp5d) protein

[0063] Caspase 3 protein was purchased from MedChemexpress Biotechnology, Inc., USA; Caspase 2 protein was purchased from Wuhan Yunkelong Technology Co., Ltd.; and Inpp5d protein (gene ID: 3635) was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0064] Inositol polyphosphate-5-phosphatase D (Inpp5d) protein is a member of the inositol polyphosphate-5-phosphatase (INPP5) family, encoding a protein with an N-terminal SH2 domain, an inositol phosphatase domain, and two C-terminal protein-protein interaction domains.

[0065] Caspases are a class of cysteine ​​proteases whose active sites contain cysteine ​​residues, enabling them to specifically cleave peptide bonds following aspartic residues in target proteins. In this embodiment, the aldehyde group of Ac-LSPD-CHO can covalently bind to the sulfhydryl (-SH) group of the cysteine ​​residue at the caspase active site, forming a thiohemiacetal structure, thereby irreversibly inhibiting the activities of caspase 2 and caspase 3. When caspases 2 and 3 are inhibited, their cleavage of Inpp5d molecules is blocked, allowing Inpp5d-mediated erythrocyte differentiation to proceed normally.

[0066] In vitro enzymatic digestion of Inpp5d protein was performed. Proteins were added according to the following groups, and each group of proteins was reacted in PBS buffer at 37°C for 1 hour.

[0067] (1) Inpp5d+Caspase 3 group: The reaction system contains 500 ng of Inpp5d protein and 500 ng of caspase 3 protein;

[0068] (2) Inpp5d+Caspase3+Ac-LSPD-CHO group: The reaction system contained 500 ng of Inpp5d protein, 500 ng of caspase3 protein and 1 μg of Ac-LSPD-CHO.

[0069] (3) Inpp5d+Caspase 2 group: The reaction system contains 500 ng of Inpp5d protein and 500 ng of caspase 2 protein;

[0070] (4) Inpp5d+Caspase2+Ac-LSPD-CHO group: The reaction system contains 500 ng of Inpp5d protein, 500 ng of caspase2 protein and 1 μg of Ac-LSPD-CHO.

[0071] The results are as follows Figure 3 As shown in Figures A and B, the band of Inpp5d digested by caspase 3 is 13 kDa, while the band of Inpp5d digested by caspase 2 is 28 kDa in addition to 13 kDa. Ac-LSPD-CHO can completely inhibit the production of the 28 kDa band and partially inhibit the production of the 13 kDa band.

[0072] Example 3: Ac-LSPD-CHO improves X-ray-induced anemia in normal mice

[0073] I. Establishment and grouping of a mouse local irradiation model

[0074] The animals used in this embodiment were 6-8 week old male C57BL / 6J mice. Normal C57BL / 6J mice were randomly divided into the following 4 groups:

[0075] NC group: normal control group, normal C57BL / 6J mice did not receive any treatment.

[0076] IR (Ionizing Radiation) group: Irradiated using an X-ray biological irradiator. Mice were anesthetized before irradiation and fixed on an irradiation plate, with only the lower abdomen (colorectal and anal regions) exposed, while the rest of the body was shielded with lead plates. Only the colorectal and anal regions were irradiated locally (2 Gy × 3 times).

[0077] IR + LSPD group: Irradiated using an X-ray biological irradiator, receiving only local irradiation of the colorectal and anal areas. One hour before each irradiation, an Ac-LSPD-CHO inhibitor (40 μg / Kg) was injected intraperitoneally.

[0078] IR + EPO group: Irradiated using an X-ray biological irradiator, receiving only local irradiation of the colorectal and anal areas. One hour before each irradiation, erythropoietin (EPO) (40 μg / Kg) was injected intraperitoneally.

[0079] In the above treatment, the irradiation parameters were adjusted to a voltage of 160 kV and a current of 25 mA. The irradiation protocol was to administer local irradiation of 2 Gy × 3 times, once a day for 3 consecutive days. Samples were taken on the 4th day, 24 hours after the last irradiation. 24 hours after the last irradiation, blood was collected from the orbital cavity in EDTA anticoagulant tubes for routine blood tests.

[0080] Mice were euthanized, and the femurs and tibias of both sides were rapidly separated for bone marrow cell analysis. The femurs and tibias were quickly dissected, removing as much attached muscle tissue as possible. The bone tissue at both ends was removed, and 1 mL of FACS solution (phosphate-buffered saline (PBS) containing 2% fetal bovine serum) was drawn into a syringe and inserted into the bone marrow cavity. The bone was repeatedly rinsed until it turned white. The cell suspension was collected, filtered through a 200-mesh filter to remove residue, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were washed once with FACS solution.

[0081] Resuspend the cells in 1 mL of erythrocyte lysis buffer and incubate on ice in the dark for 3 minutes to lyse the erythrocytes. Centrifuge again at 1000 rpm for 5 minutes and discard the supernatant. Resuspend the cells in 1 mL of FACS solution and count the viable cells using a counting chamber.

[0082] Take 5×10 6 Bone marrow cells were resuspended in flow cytometry tubes and blocked with an anti-mouse anti-Fc receptor monoclonal antibody (FcRmAb, diluted 1:200). The mixture was thoroughly incubated at 4°C in the dark for 15 minutes. After washing twice with 1 ml of FACS solution, viable cells were labeled with 7-aminoactinomycin D staining solution (7-AAD staining solution, diluted 1:200), incubated for 20 minutes, and washed twice with FACS solution. Flow cytometry antibody staining solution was then prepared using FACS solution, incubated at 4°C in the dark for 30 minutes, and washed twice with FACS solution. The flow cytometry antibody staining solution included antibodies 7-AAD, Lin, Sca-1, c-Kit, CD150, CD127, CD48, CD34, CD16 / 32, and FIK2 (Chinese explanations are shown in Table 5), all prepared with FACS solution at a ratio of 1:200.

[0083] Table 5

[0084]

[0085] Data acquisition was performed using flow cytometry, and data analysis was performed using FlowJo software.

[0086] II. Experimental Results

[0087] Red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) are important indicators for assessing anemia. MEP (megakaryocyte-erythrocyte progenitor cells) is defined as Lin... - c-Kit + Sca-1 - CD34 - and CD16 / 32 - The result is as follows Figure 4 As shown, compared with the NC group, the IR group mice showed significantly decreased red blood cell count (RBC), hemoglobin concentration (HGB), and hematocrit (HCT). The above indicators in the IR+LSPD and IR+EPO groups were significantly higher than those in the IR group, indicating that Ac-LSPD-CHO can effectively alleviate irradiation-induced anemia in normal mice. Flow cytometry analysis showed that the proportion of megakaryocyte-erythroid progenitor cells (MEP) in the bone marrow of the IR group mice was significantly lower than that in the NC group. The MEP in the IR+LSPD and IR+EPO groups was significantly higher than that in the IR group, indicating that Ac-LSPD-CHO can effectively protect against irradiated MEP.

[0088] Example 4: Ac-LSPD-CHO improves radiotherapy-induced anemia in tumor-bearing mice

[0089] I. Establishment of AOM / DSS-induced colorectal cancer mouse model

[0090] Azomethane (AOM) was dissolved in sterile physiological saline to prepare a 1 mg / mL stock solution. The dosage was calculated based on the mouse's body weight before use. On day 1 of the experiment, mice were weighed and then injected intraperitoneally with AOM (10 mg / kg). The mice were observed for 3 consecutive days after injection.

[0091] Following the AOM injection, normal drinking water was provided for 7 days. On the eighth day, the drinking water was replaced with sterilized water containing 2.5% (w / v) sodium dextran sulfate (DSS). This sterilized water was consumed for one week, then replaced with normal water on the eighth day for two weeks, constituting one cycle. This process was repeated four times, for a total of 13 weeks. Figure 5 A).

[0092] Throughout the induction period, the mice's weight should be measured weekly, and the characteristics of their feces and any blood in their stool should be observed. After the fourth DSS cycle, 2-3 mice should be randomly selected and sacrificed to observe the formation of intestinal tumors. Subsequent experiments can only proceed after the model has been successfully established.

[0093] II. Experimental Grouping and Processing

[0094] Mice with colorectal cancer were randomly divided into the following 5 groups:

[0095] Tumor group: Colorectal cancer mice that did not receive irradiation or drug treatment.

[0096] Tumor + IR group: Colorectal cancer mice that received local irradiation of the colorectal and anal areas (2 Gy × 3 times).

[0097] Tumor + IR + LSPD group: Colorectal cancer mice were injected intraperitoneally with Ac-LSPD-CHO inhibitor (dose as above) 1 hour before each irradiation.

[0098] Tumor + IR + EPO group: Colorectal cancer mice were injected intraperitoneally with EPO (dose as above) 1 hour before each irradiation.

[0099] In the above grouping, irradiation refers to irradiation using an X-ray biological irradiator, with irradiation parameters adjusted to a voltage of 160 kV and a current of 25 mA. Mice were anesthetized before irradiation and fixed to an irradiation plate, exposing only the lower abdomen (colorectal and anal regions), while the remaining areas were shielded with lead plates. The irradiation protocol consisted of local irradiation of 2 Gy × 3 times, once daily for 3 consecutive days. Samples were collected on the 4th day, 24 hours after the last irradiation.

[0100] Twenty-four hours after the last irradiation, blood was collected from the orbital cavity in EDTA anticoagulant tubes for routine blood tests. Mice were euthanized, and the bilateral femurs and tibias were rapidly separated for bone marrow cell analysis. Part of the spleen was used for flow cytometry to determine cell type and quantity. Colorectal and anal tissues from mice were collected, Swiss rolls were prepared, fixed in 4% paraformaldehyde for pathological analysis (hematoxylin-eosin staining (HE staining)), and paraffin sections were prepared, some of which were frozen at -80°C for later use.

[0101] III. Experimental Results

[0102] 1. Establishment of an AOM / DSS-induced mouse model of colorectal cancer (CRC)

[0103] like Figure 5 As shown in Figure B, on day 70 after AOM / DSS induction, typical multiple tumors appeared in the colorectal tissue of mice. Macroscopic observation revealed multiple raised nodules or polypoid masses scattered across the colorectal mucosa, with some tumors exhibiting cauliflower-like growth. HE staining results showed typical adenocarcinoma structures in the colorectal mucosa and submucosa of the colorectal cancer model mice, with tumor cells exhibiting atypical proliferation, disordered glandular structure, large and deeply stained nuclei, and pathological mitotic figures. These results indicate that the AOM / DSS induction protocol successfully established an orthotopic colorectal cancer mouse model, which can be used for subsequent radiotherapy and drug intervention experiments.

[0104] 2. Ac-LSPD-CHO increases bone marrow MEP and improves anemia in CRC mice.

[0105] (1) Peripheral blood routine and bone marrow flow cytometry analysis of CRC mice

[0106] Male C57BL / 6J mice with successfully induced colorectal cancer were selected. Blood was collected from the orbital cavity in EDTA anticoagulant tubes 24 hours after the last irradiation for routine blood tests. Mice were sacrificed at various time points (24 hours after irradiation / drug administration), and the bilateral femurs and tibias were quickly removed, removing as much attached muscle tissue as possible. The bone tissue at both ends was removed, and 1 mL of FACS solution (phosphate-buffered saline (PBS) containing 2% fetal bovine serum) was drawn into the medullary cavity using a syringe. The cavity was repeatedly flushed until the bone turned white.

[0107] Collect the cell suspension, filter through a 200-mesh filter to remove residue, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Wash once with FACS solution. Resuspend the cells in 1 mL of erythrocyte lysis buffer and incubate on ice in the dark for 3 minutes to lyse the erythrocytes. Centrifuge again at 1000 rpm for 5 minutes and discard the supernatant. Resuspend the cells in 1 mL of FACS solution and count the viable cells using a counting chamber.

[0108] Take 5×10 6 Bone marrow cells were resuspended in flow cytometry tubes, blocked with anti-mouse FcR mAb (1:200), thoroughly mixed, and incubated at 4°C in the dark for 15 minutes. After washing twice with 1 mL of FACS solution, live cells were labeled with 7-AAD (1:200), incubated for 20 minutes, and washed twice with FACS solution. Flow cytometry antibody staining solution was then prepared using FACS solution, incubated at 4°C in the dark for 30 minutes, and washed twice with FACS solution. The flow cytometry antibody staining solution included antibodies 7-AAD, Lin, Sca-1, c-Kit, CD150, CD127, CD48, CD34, CD16 / 32, and FIK2, all prepared with FACS solution at a ratio of 1:200.

[0109] Data acquisition was performed using flow cytometry, and data analysis was performed using FlowJo software.

[0110] like Figure 6As shown, the RBC, HGB, and HCT levels in the tumor group mice were significantly lower than those in normal mice, suggesting that the tumor itself can cause anemia. These indicators further decreased in the tumor + IR group. Erythrocyte parameters in both the tumor + IR + LSPD group and the tumor + IR + EPO group were significantly higher than those in the tumor + IR group, indicating that Ac-LSPD-CHO can effectively improve irradiation-induced anemia even in tumor-bearing environments. Bone marrow flow cytometry results showed that the MEP ratio in the tumor + IR group was significantly lower than that in the tumor group, while the erythroid progenitor cells in both the tumor + IR + LSPD group and the tumor + IR + EPO group significantly increased. These results confirm that Ac-LSPD-CHO, an inhibitor targeting the caspase 2 / 3-Inpp5d cleavage axis, has a clear therapeutic effect on anemia after tumor radiotherapy, laying the foundation for further evaluation of its safety and impact on the tumor microenvironment.

[0111] 3. Effects of Ac-LSPD-CHO on tumor proliferation in CRC mice

[0112] Immunohistochemical staining of tumor tissue with nuclear proliferation antigen Ki-67 (Ki67): Paraffin sections were placed in the following order: xylene I: 5 minutes, xylene II: 5 minutes, 100% ethanol: 5 minutes, 95% ethanol: 5 minutes, 85% ethanol: 5 minutes, 75% ethanol: 5 minutes, deionized water: 5 minutes, and then dewaxed.

[0113] Thermal retrieval was performed using an alkaline retrieval solution. The antigen retrieval solution was prepared as follows: Tris(hydroxymethyl)aminomethane (Tris) 2.42 g, EDTA 0.74 g, Tween-20 1 mL, distilled water 2 L, pH 9.0. The alkaline retrieval solution was placed in an autoclave and heated to boiling. The slides were then placed in the autoclave, and after boiling and steam was released, autoclaving was performed for 90 seconds. The slides were then cooled to room temperature on ice for 30 minutes. The slides were washed three times with phosphate-buffered saline (PBST) solution for 5 minutes each time.

[0114] Draw water-blocking rings on the slices, add 3% H2O2, and incubate at room temperature in the dark for 10 minutes to block endogenous peroxidase activity. Wash three times with PBST for 5 minutes each time.

[0115] Add 5% bovine serum albumin (BSA) dropwise and incubate at room temperature in the dark for 30 minutes.

[0116] Add rabbit anti-Ki67 monoclonal antibody and incubate overnight in a humidified chamber at 4°C. Wash three times with PBST the next day, 5 minutes each time.

[0117] Add HRP-labeled goat anti-rabbit immunoglobulin G (IgG) polymer and incubate at room temperature in the dark for 60 minutes. Wash three times with phosphate-buffered saline (PBS) for 5 minutes each time.

[0118] Add the diaminobenzidine (DAB) chromogenic solution prepared according to the kit instructions. Under a microscope, control the chromogenic time (usually 1-5 minutes). A positive signal will appear brownish-yellow. Rinse with tap water to stop the chromogenic process.

[0119] The slices were placed in hematoxylin staining solution for 8 seconds for counterstaining, rinsed with tap water, and then bathed in a 55-degree water bath for 1 minute to return to blue.

[0120] Dehydration, clearing, and mounting: 75% ethanol, 85% ethanol, 95% ethanol, 100% ethanol, xylene I, and xylene II for 5 minutes each, then mount with neutral resin and observe under a microscope.

[0121] Experimental Results: To evaluate the effect of Ac-LSPD-CHO on tumor growth, this study investigated the expression of Ki67 in colorectal tumor tissues from mice with orthotopic carcinoma using immunohistochemical staining to reflect the proliferative activity of tumor cells. After fixation and embedding, tissues were subjected to Ki67 immunohistochemical staining, and the Ki67 proliferation index (number of Ki67-positive cells / total cell number × 100%) was calculated. Results are as follows: Figure 7 As shown, in the tumor group, the Ki67 proliferation index was 60.1 ± 3.7%, indicating active tumor cell proliferation. The Ki67 index in the tumor + IR group significantly decreased to 45.0 ± 2.1%, suggesting that local irradiation effectively inhibited tumor cell proliferation. Notably, the Ki67 index in the tumor + IR + LSPD group further decreased to 35.7 ± 2.8%, significantly lower than the tumor + IR group, indicating that the combined Ac-LSPD-CHO radiotherapy had a better inhibitory effect on tumor proliferation than radiotherapy alone. Conversely, the Ki67 index in the tumor + IR + EPO group was 53.5 ± 1.0%, still lower than the tumor group, but significantly higher than the tumor + IR group, suggesting that EPO may have partially weakened the inhibitory effect of radiotherapy on tumor proliferation.

[0122] The above results indicate that Ac-LSPD-CHO, while improving anemia, not only does not promote tumor growth, but may also enhance the anti-tumor effect of radiotherapy, exhibiting superior anti-tumor characteristics compared to EPO.

[0123] Example 5: Unlike EPO, Ac-LSPD-CHO does not increase splenic immunosuppressive TER cells and EDMC cells.

[0124] I. Flow Cytometry Analysis of Bone Marrow and Spleen

[0125] 1. Bone marrow flow cytometry protocol

[0126] Male C57BL / 6J mice aged 6-8 weeks were selected and sacrificed at various time points (24 hours after irradiation / drug administration). The femurs and tibias of both sides were quickly removed, and as much attached muscle tissue as possible was removed. The bone tissue at both ends was removed, and 1 mL of FACS solution (phosphate-buffered saline (PBS) containing 2% fetal bovine serum) was drawn into a syringe and inserted into the medullary cavity. The bone was repeatedly rinsed until it turned white.

[0127] Collect the cell suspension, filter it through a 200-mesh filter to remove residue, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Wash once with FACS solution.

[0128] Resuspend the cells in 1 mL of erythrocyte lysis buffer and incubate on ice in the dark for 3 minutes to lyse the erythrocytes. Centrifuge again at 1000 rpm for 5 minutes and discard the supernatant. Resuspend the cells in 1 mL of FACS solution and count the viable cells using a counting chamber.

[0129] Take 5×10 6 Bone marrow cells were resuspended in flow cytometry tubes, blocked with anti-mouse FcR mAb (1:200), thoroughly mixed, and incubated at 4°C in the dark for 15 minutes. After washing twice with 1 mL of FACS solution, live cells were labeled with 7-AAD (1:200), incubated for 20 minutes, and washed twice with FACS solution. Flow cytometry antibody staining solution was then prepared using FACS solution, incubated at 4°C in the dark for 30 minutes, and washed twice with FACS solution. The flow cytometry antibody staining solution included antibodies 7-AAD, Lin, Sca-1, c-Kit, CD150, CD127, CD48, CD34, CD16 / 32, and FIK2, all prepared with FACS solution at a ratio of 1:200.

[0130] Data acquisition was performed using flow cytometry, and data analysis was performed using FlowJo software.

[0131] 2. Spleen collection and single-cell suspension preparation

[0132] Twenty-four hours after the last irradiation, the mice were euthanized, fixed, and the abdominal cavity was opened. The spleen was separated, and the surrounding connective tissue and fat were removed. The spleen was rinsed in pre-cooled PBS to remove blood, placed on graph paper, photographed, measured, and recorded.

[0133] Transfer the spleen to a culture dish, soak it in 2 mL of PBS, and gently grind the spleen with the rubber end of a 1 mL syringe plunger until no obvious tissue fragments remain. Filter the cell suspension through a 70 μm cell filter into a 5 mL microcentrifuge tube, rinse the culture dish and filter with PBS, and centrifuge at 4°C and 1000 rpm for 5 minutes to collect all cells. Discard the supernatant.

[0134] Add 2 mL of erythrocyte lysis buffer to the cell pellet, gently pipette to resuspend, incubate at room temperature for 3 minutes, centrifuge at 4°C and 1000 rpm for 5 minutes, and discard the supernatant. Repeat the lysis once.

[0135] Resuspend cells in FACS solution, take 10 μL and count using a cell counting chamber, then adjust the cell density to 1 × 10⁶ cells / mL. 7 cells / mL available for later use.

[0136] Add 100 μL of cell suspension to each flow cytometry tube, and add 1 μL of Fc receptor blocker (CD16 / 32 antibody) to block the cell suspension. Incubate at 4°C for 10 minutes to block non-specific binding. After washing once, prepare flow cytometry antibody staining solution with FACS solution, incubate at 4°C in the dark for 30 minutes, and wash twice with FACS solution. The flow cytometry antibody staining solution includes antibodies CD45, TER119, CD71, CD11b (integrin αM chain), Gr-1 (granulocyte marker-1), and Fvd506 (Fvd506 is an active dye that irreversibly labels dead cells before cryopreservation, fixation, and / or permeabilization procedures). All of these are prepared with FACS solution, with the ratio of Fvd506 being 1:1000 and the others being 1:200. Add 2 mL of staining buffer to wash the cells, centrifuge at 1000 rpm for 5 minutes at 4°C, and discard the supernatant. Repeat the washing once.

[0137] Cells were resuspended in 200 μL of FACS solution, fixed with an equal volume of 4% paraformaldehyde for 15 minutes, and stored at 4°C in the dark. Detection was performed within 24 hours. Data were acquired using flow cytometry.

[0138] II. Experimental Results and Analysis

[0139] Tumors can induce TER cells (CD45) in the spleen. - TER119 + CD71 + The expansion of erythroid progenitor cells (ERCs) and EDMCs (tumor-induced erythroid precursor-differentiated myeloid cells) mediates immunosuppression and can attenuate the efficacy of radiotherapy and immunotherapy. To assess the potential impact of Ac-LSPD-CHO on the tumor immune microenvironment, this study measured the size of mouse spleens and used flow cytometry to detect the proportions of TER cells and EPC cells in the bone marrow and TER cells and EDMCs in the spleen of tumor-bearing mice in each group. (See diagram below.) Figure 9 TER cells are defined as CD45 - TER119 + CD71 + EDMC cells are defined as CD45 + TER119 + CD71 + CD11b+ Gr1 + EPC cells are defined as TER119. + CD71 + . Figure 9 In A, Comp-VL2-A::L_DBV510-A represents the signal area value of Brilliant Violet 510 dye from channel 2 (525nm detection site) of the violet laser after fluorescence compensation correction; Comp-BL2-H::Ter119 PE-H represents the pulse signal height value of Ter119-PE antibody (detecting erythrocyte markers) from channel 2 (PE detection site) of the blue laser after fluorescence compensation correction; and Comp-VL1-A::CD71 BV421-A represents the pulse signal area value of CD71-BV421 antibody (detecting transferrin receptors) from channel 1 (BV421 detection site) of the violet laser after fluorescence compensation correction. Figure 9 In B, Comp-BL1-A::Ter119 FITC-A represents the pulse signal area value of the Ter119-FITC antibody (detecting erythrocyte markers) from channel 1 (FITC detection site) of the blue laser after fluorescence compensation correction, and Comp-PL3-A::Gr-1 APC-Cγ7-A represents the pulse signal area value of the Gr-1-APC-Cy7 antibody (detecting mouse myeloid cell markers) from channel 3 (APC-Cy7 detection site) of the red laser after fluorescence compensation correction. Figure 9 In C, Comp-RL2-A::CD45 AF700-A represents the pulse signal area value of the CD45-AF700 antibody (detecting all common leukocyte antigens) from the second channel (AF700 detection site) of the red laser after fluorescence compensation correction.

[0140] like Figure 8 As shown in Figures A and B, the spleen size in the tumor + IR group was not significantly different from that in the tumor group, but the spleen size in the tumor + IR + LSPD group tended to decrease compared to the tumor + IR group, and the spleen size in the tumor + IR + EPO group was significantly larger than that in the tumor + IR + LSPD group. Figure 10As shown, compared with normal mice, the proportions of TER cells and EDMC cells in the spleen of CRC tumor group (tumor group) mice were significantly increased, confirming that tumors can induce the expansion of these immunosuppressive cell populations, such as TER cells and EDMCs, in the spleen. The proportions of TER cells and EDMCs in the spleen of tumor + IR group mice were not significantly different from those in the tumor group, indicating that local irradiation alone (2 Gy × 3 times) cannot reverse the tumor-induced accumulation of these cells. Notably, the proportion of EDMCs in the spleen of tumor + IR + EPO group mice was significantly higher than that in tumor + IR group mice, while the proportion of TER cells also showed an increasing trend, but the difference was not statistically significant. This result is consistent with previous reports, suggesting that EPO may exacerbate the immunosuppressive microenvironment by promoting the differentiation of erythroid precursor cells into myeloid cells. Unlike EPO, the proportion of TER cells in the spleen of tumor + IR + LSPD group mice did not increase, but instead showed a decreasing trend. Although this did not reach statistical significance, it indicates that Ac-LSPD-CHO at least does not promote the expansion of these immunosuppressive cell populations, and the proportion of EDMC cells in the spleen of tumor + IR + LSPD group mice was significantly decreased. This embodiment also examined TER cells and EPC cells in the bone marrow. Erythroid progenitor cells (EPCs) differentiate from MEPs. Under physiological conditions, erythroid progenitor cells are primarily responsible for maintaining normal erythrocyte production to meet the body's oxygen transport needs. The trends of TER cells and EPC cells in the bone marrow are similar to... Figure 4 The trend of MEP in the study is consistent with this, further demonstrating that the inhibitor Ac-LSPD-CHO has a clear therapeutic effect in the treatment of post-radiotherapy anemia. Figure 10 These results reveal the potential advantages of Ac-LSPD-CHO compared to EPO, as it alleviates post-radiotherapy anemia without promoting or even inhibiting the proliferation of immunosuppressive cells, providing a theoretical basis for combined immunotherapy.

[0141] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A short peptide, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO.

1. In the short peptide, leucine is acetylated and aspartic acid is modified with an aldehyde group.

2. The use of the short peptide according to claim 1 in the preparation of anemia treatment drugs.

3. The application according to claim 2, characterized in that, The anemia includes anemia caused by exposure to X-rays, gamma rays, protons, and / or heavy ions.

4. The application according to claim 2, characterized in that, The anemia mentioned includes aplastic anemia caused by X-ray, gamma-ray, proton and / or heavy ion irradiation.

5. The application according to claim 2, characterized in that, The anemia mentioned includes anemia caused during cancer radiation therapy.

6. The application according to claim 5, characterized in that, The cancers mentioned include colorectal cancer.

7. A pharmaceutical composition for treating anemia, characterized in that, The pharmaceutical composition comprises the short peptide of claim 1.

8. The use of the short peptide according to claim 1 in the preparation of antitumor drugs.

9. A product for preventing the enzymatic cleavage of inositol polyphosphate-5-phosphatase D, characterized in that, The product includes the short peptide as described in claim 1.

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