Application of HCK agonist in resisting mycobacterium tuberculosis infection

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of a non-receptor protein tyrosine kinase agonist in resisting mycobacterium tuberculosis infection. Tyrosine phosphorylated protein is screened out through a bioinformatics technology to serve as a research target, cell and animal experiments are carried out, and it is found that the target agonist improves host immunity and inhibits growth and propagation of mycobacterium tuberculosis by activating immune cells and enhancing the antibacterial function of macrophages. Compared with the traditional antibiotic treatment depending on multi-drug combination, the agonist provided by the invention reduces the dependence on antibiotics through precise targeting immunoregulation, thereby reducing the generation risk of drug-resistant strains.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of a non-receptor protein tyrosine kinase agonist in resisting Mycobacterium tuberculosis infection. Background Art

[0002] Tuberculosis is a common infectious disease in clinical practice, characterized by a long course of illness and long course of medication. It is often treated with a combination of antimicrobial drugs. If the patient has poor immune function and multiple underlying diseases, repeated medication may cause dysbiosis and lead to multidrug-resistant bacteria (MDRO) infection (Qi Liying, Li Xuezheng, Guo Xue, et al. Application of bundled management strategy in the prevention and control of tuberculosis infection in tuberculosis specialty hospitals. Qilu Nursing Journal, 2021, 27(1): 165-167.). After infection with Mycobacterium tuberculosis, there are no typical clinical symptoms in the early stage. As the bacteria become clearer, the body's resistance gradually decreases. If the bacterial-mediated allergic reaction increases abnormally, it will be accompanied by obvious clinical symptoms, including fever, fatigue, poor appetite, weight loss, respiratory changes, etc., which seriously affect the patient's physical health and life safety (Zhou Jiaxian, Zhang Weifen, Zhou Guijiao. Application of T cell enzyme-linked immunosorbent assay in the investigation of latent tuberculosis infection (LTBI) status of nursing staff in general hospitals. Nursing Practice and Research, 2020, 17(4): 145-147; Zhang Lijun, Liu Yan, Song Yan. Effect of hospital-family joint supervision and continuity nursing model on family burden and social support of patients with drug-resistant tuberculosis. Nursing Practice and Research, 2022, 19(5): 631-634.). In 2020, approximately 9.9 million people worldwide will suffer from tuberculosis and approximately 1.3 million patients will die from tuberculosis. With the continuous emergence of anti-tuberculosis drugs, the prevalence of tuberculosis has been limited to a certain extent. However, there are still many shortcomings in anti-tuberculosis treatment, and the standard chemotherapy regimen requires at least 4 to 6 months. The economic pressure of long-term treatment of tuberculosis patients and the adverse reactions of anti-tuberculosis drugs have caused them to interrupt anti-tuberculosis treatment, thereby accelerating the development of drug-resistant tuberculosis. Drug-resistant tuberculosis is resistant to first-line anti-tuberculosis drugs such as isoniazid (INH) and rifampicin (RMP), and a few are resistant to second-line anti-tuberculosis drugs and fluoroquinolones, which has led to a significant decrease in the cure rate of tuberculosis. At the same time, the toxic side effects of anti-tuberculosis drugs and the increasing multidrug resistance have affected the treatment of tuberculosis patients. Therefore, it is urgent to develop new drugs and intervention programs to control infection. Host-directed therapy targets multiple molecules or pathways in the host immune system, which may help fight bacterial infections and improve the cure rate of tuberculosis.

[0003] Tyrosine kinase and tyrosine phosphorylation are closely related to a variety of biological processes and have become one of the most important drug targets. Tyrosine kinase is an important class of molecules widely present in the human body. The tyrosine kinase family is divided into two major categories: receptor tyrosine kinase family and non-receptor tyrosine kinase family. The receptor tyrosine kinase family is a class of cell membrane-bound proteins that receive signals transmitted by ligands. They include epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and mesenchymal epithelial transition factor (c-MET) and other molecular targets. Non-receptor tyrosine kinases include Ab1, Src family kinases, focal adhesion kinases, etc. The activation of these kinases then activates their downstream signaling molecules to regulate the growth and differentiation of tumor cells. Under normal circumstances, the activation of these molecules is strictly controlled. However, in tumor tissues, the activation of receptor and non-receptor tyrosine kinases is dysregulated, causing changes in cell proliferation and cell function. Protein phosphorylation is coordinated by protein kinases and protein phosphatases. It is a key step in cell-to-cell communication, cell growth and proliferation, cell cycle regulation, differentiation, malignant tumor changes, occurrence and metastasis, nerve conduction, angiogenesis, embryogenesis, and many genetic diseases and acquired human diseases. Therefore, the study of protein phosphorylation has become a hot topic in life sciences.

[0004] The SRC family of non-receptor protein tyrosine kinases (SFKs) consists of 9 members and can be divided into two groups according to the expression pattern of individual kinases in adults. Thus, SRC, YES and FYN are ubiquitously expressed, while the expression of LCK, FGR, BLK, LYN, YRK and HCK is limited to certain types of cells and tissues (Poh AR, O'Donoghue RJ, Ernst M. Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cells [J]. Oncotarget. 2015, 6 (18): 15752-15771.). Hematopoietic cell kinase (HCK) expression is limited to cells of the bone marrow and B lymphocyte lineages, and the protein exists in two isoforms. HCK can participate in macrophage activation, secretion of myeloid cell growth factor and proinflammatory cytokines, mediate cell development and intracellular signal transduction and other physiological and pathological processes (Kim J, Sinn DH, Choi MS, et al. Hepatocellular carcinoma with extrahepatic metastasis: Are there still candidates for transarterial chemoembolization as an initial treatment? [J]. PkS One, 2019, 14(3): e0213547.). HCK can participate in the course of acute myeloid leukemia by activating signaling pathways such as phosphatidylinositol 3-kinase / protein kinase B, mitogen-activated protein kinase / extracellular signal-regulated kinase, and the SH2 and SH3 domains of HCK affect the development of chronic myeloid leukemia by interacting with Bcr.Abl (Roseweir AK, Powell AGMT, Horstman SL, et al. Srefamily kinases. HCK and FGR, associate with local inflammation and turnout progression in colorectal cancer [J] Cell Sisnal, 2019 (56): 15-22.).HCK can promote the occurrence of colorectal cancer through the Star6-dependent Th2 cytokine signaling pathway, and the survival rate of patients with high expression is low (antermans HC, Minderman M, Kuil A, et al. Identification of the SRC-family tyrosine kinase HCK as a therapeutic target in mantle cell lymphoma[J]. Leukemia, 2021, 35(3): 881-886.). Moreover, HCK was also found to have sequence changes, copy number distortion and high expression in gastric cancer, and is a potential oncogene (Mei J, Zhou WJ, Zhu XY, et al. Suppression of autophagy and HCK signaling promotes PTGS2 high FCGR3-NK cell differentiation triggered by ectopic endometrial stromal cells[J]. Autophagy, 2018. 14(8): 1376-1397.). The occurrence of other solid malignancies such as breast cancer and pancreatic cancer is also closely related to the overexpression of HCK (Ochieng P, Nath S, Macarulay R, et al. Phospholipid transfer protein and alpha-1 antitrypsin regulate Hck kinase activity during neutrophil degranulation[J]. Sci Rep, 2018, 8(1): 15394; Poh AR,. Donoghue RJ, Ernst M. Hematopoietic cell kinase (HCK) as a therapeutic target in immune and cancer cells [J]. Oncotarget, 2015, 6(18): 15752-15771.). Summary of the invention

[0005] The present invention found that the HCK agonist EPQpYEEIPIYL can improve the clearance efficiency of macrophages against MTB infection and enhance the immune response. Based on this, the present invention was completed.

[0006] In a first aspect, the present invention provides a use of an HCK agonist in the preparation of a drug for resisting Mycobacterium tuberculosis infection, wherein the agonist can inhibit Mycobacterium tuberculosis infection, and the agonist is EPQpYEEIPIYL.

[0007] Furthermore, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

[0008] Furthermore, the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0009] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0010] Furthermore, the drug can be introduced into the body by injection, penetration, absorption, physical or chemical mediation.

[0011] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0012] In a second aspect, the present invention provides a pharmaceutical composition for use against Mycobacterium tuberculosis, wherein the pharmaceutical composition contains the HCK agonist EPQpYEEIPIYL.

[0013] Furthermore, the Mycobacterium tuberculosis includes a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

[0014] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0015] Furthermore, the carrier material is selected from one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0016] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0017] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0018] Furthermore, the pharmaceutical composition can be introduced into the body by injection, penetration, absorption, physical or chemical mediation.

[0019] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0020] Beneficial Effects

[0021] 1. The present invention screened out the tyrosine phosphorylated protein HCK through bioinformatics technology, and carried out cell and animal experiments using siRNA, HCK inhibitor and HCK agonist EPQpYEEIPIYL. It was found that the agonist can significantly improve the clearance efficiency of macrophages against Mycobacterium tuberculosis (MTB) and enhance the immune response.

[0022] 2. Compared with existing anti-tuberculosis treatments, HCK agonists activate immune cells and enhance the antibacterial function of macrophages, thereby improving host immunity and inhibiting the growth and spread of Mycobacterium tuberculosis.

[0023] 3. The present invention also has the advantage of reducing the risk of drug resistance. Compared with traditional antibiotic treatment that relies on multi-drug combination, HCK agonists reduce dependence on antibiotics through precise targeted immune regulation, thereby reducing the risk of drug-resistant strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 : Temporal clustering (CA) analysis of 128 differentially phosphorylated proteins.

[0025] Figure 2 :Protein-protein interaction network (PPI) of HCK in MTB infection.

[0026] Figure 3 :Effect of MTB infection on HCK tyrosine phosphorylation level in macrophages.

[0027] Figure 4 :Analysis of the total number of colonies in MTB-infected macrophages at 0h, 4h, 8h, and 24h after iHCK treatment. Note: ****P<0.0001.

[0028] Figure 5 :Effect of siHCK on the regulation of autophagy in macrophages infected with MTB.

[0029] Figure 6 : RT-qPCR detection of TNF-α transcription (A) and ELISA detection of TNF-α secretion (B). Note: **P<0.01, ***P<0.001, ****P<0.0001.

[0030] Figure 7 : RT-qPCR detection of IL-1β transcription (A) and ELISA detection of IL-1β secretion (B). Note: *P<0.05, **P<0.01, ****P<0.0001.

[0031] Figure 8 : RT-qPCR detection of IL-6 transcription (A) and ELISA detection of IL-6 secretion (B). Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0032] Fig. 9 :CFU identification of the antibacterial effect of iHCK-37. Note: **P<0.01.

[0033] Fig.10 : Evaluation of the inhibitory effect of iHCK-37 on HCK tyrosine phosphorylation and the impact on the autophagy pathway.

[0034] Fig.11 : Evaluation of the inhibitory effect of iHCK-37 on HCK tyrosine phosphorylation, autophagy pathway and ERK activation.

[0035] Fig.12 : RT-qPCR detection of TNF-α transcription (A) and ELISA detection of TNF-α secretion (B). Note: *P<0.05, ***P<0.001, ****P<0.0001.

[0036] Fig.13 : RT-qPCR detection of IL-1β transcription (A) and ELISA detection of IL-1β secretion (B). Note: *P<0.05, ****P<0.0001.

[0037] Fig.14 : RT-qPCR detection of IL-6 transcription (A) and ELISA detection of IL-6 secretion (B). Note: **P<0.01, ****P<0.0001.

[0038] Fig.15 :The antibacterial effect of EPQpYEEIPIYL identified by CFU. Note: *P<0.05, ****P<0.0001.

[0039] Fig.16 :Evaluation of the inhibitory effect of EPQpYEEIPIYL on HCK tyrosine phosphorylation and the impact on the autophagy pathway.

[0040] Fig.17:Evaluation of the effects of EPQpYEEIPIYL on HCK tyrosine phosphorylation agonism, autophagy and ERK activation.

[0041] Fig.18 : RT-qPCR detection of TNF-α transcription (A) and ELISA detection of TNF-α secretion (B). Note: *P<0.05, ***P<0.001, ****P<0.0001.

[0042] Fig.19 : RT-qPCR detection of IL-1β transcription (A) and ELISA detection of IL-1β secretion (B). Note: *P<0.05, ***P<0.001, ****P<0.0001.

[0043] Fig. 20 : RT-qPCR detection of IL-6 transcription (A) and ELISA detection of IL-6 secretion (B). Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0044] Fig.21 : CFU counts in lung tissues of four groups of mice on day 3 after infection with BCG via tail vein injection.

[0045] Fig. 22 : CFU counts in the right lung and spleen tissues of the four groups of mice four weeks after infection with BCG via tail vein injection. Note: **P<0.01, ****P<0.0001.

[0046] Fig.23 :ELISA identified the secretion of cytokines in the four groups of mice after 4 weeks of BCG infection. Note: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0047] Fig.24 :The effect of tyrosine kinase HCK identified by acid-fast staining on the clearance of BCG infection in mice. Note: EPQ: EPQpYEEIPIYL.

[0048] Fig.25 :HE staining to identify the effect of tyrosine kinase HCK on the clearance of BCG infection in mice. Note: EPQ: EPQpYEEIPIYL. DETAILED DESCRIPTION

[0049] The specific embodiments of the present invention are further described below. It should be noted that the description of these embodiments is used to help understand the present invention, but does not constitute a limitation of the present invention. In addition, the technical features involved in the embodiments described below can be combined with each other as long as they do not conflict with each other.

[0050] The experimental methods in the following examples are conventional methods unless otherwise specified, and the experimental materials used in the following examples are commercially available unless otherwise specified.

[0051] Example 1 Mass spectrometry analysis to screen HCK as a research target

[0052] 1.1 Macrophages

[0053] The U937 cell line (human histiocytic lymphoma cell line) was obtained from the Bacterial Immunology Laboratory of Beijing Chest Hospital Affiliated to Capital Medical University (hereinafter referred to as Beijing Chest Hospital).

[0054] 1.2 Mycobacterium tuberculosis strains

[0055] The standard strain of Mycobacterium tuberculosis H37Rv (ATCC 27298) was preserved by the Bacterial Immunology Laboratory of Beijing Chest Hospital.

[0056] 1.3 Preparation of Mycobacterium tuberculosis suspension

[0057] (1) Add Tween-80 (20%) to the grinding bottle, scrape the colonies from the solid culture medium, transfer them to the grinding bottle, shake them, and let them stand.

[0058] (2) Dilute the bacterial solution with RPMI 1640 medium and adjust the OD600 to 1 (1×10 8 The concentration of the bacterial solution was then adjusted according to the ratio of multiple of infection (MOI) of 1:5. 1.4 Mycobacterium tuberculosis infection macrophage model

[0059] (1) Preparation of sodium pervanadate activation solution

[0060] Sodium metavanadate (500 mM) was melted, activated by heating, and then cooled. Activated sodium vanadate (500 mM) and an equal amount of H2O2 (500 mM) were mixed in a 1:1 ratio to prepare a sodium pervanadate activation solution with a final concentration of 250 mM.

[0061] (2) H37Rv infection macrophage model

[0062] Prepare OD 600 =1(1×10 8 / mL) bacterial solution, and the OD 600= 1 was diluted 10 times with RPMI 1640 containing 10% FBS, and the bacterial solution was added to the culture medium, mixed, divided, and marked 4-12. At 0h, cells 10, 11, and 12 were infected; the old culture medium of 10-12 was discarded, washed, and RPMI 1640 culture medium containing bacteria and 10% FBS was added to culture dishes 10, 11, and 12 respectively; culture dishes 1-12 were placed in a cell culture incubator. The same method was used to infect No. 7-9 at 0.5h; No. 4-6 was infected at 45min, and at 1h, cells 1-12 were collected to obtain macrophage models infected at 0h, 15min, 45min, and 1h.

[0063] 1.5 Macrophage collection

[0064] Prepare 12 centrifuge tubes, take out No. 1-12, first suck out the bacterial solution, then wash; then add RPMI 1640 (1; 100) containing sodium pervanadate activation solution to 1-12 culture dishes and incubate. After the incubation, remove the liquid in the culture dish, add trypsin containing OTDA to each dish, shake it well, let it stand, wait for the specified time, flick the bottom, and then add complete culture medium RPMI 1640 containing 10% FBS to stop the digestion, blow the cells away with a dropper, and absorb the mixed cell suspension at the same time; transfer the cell suspension of No. 1-12 into 12 pre-labeled new centrifuge tubes, centrifuge, and discard the supernatant. Wash, resuspend, centrifuge again, and then remove the supernatant. After completing the packaging of samples No. 1-12, carry out the cell lysis and protein extraction operation, and save the extracted protein.

[0065] 1.6 Macrophage lysis

[0066] (1) Sample lysis

[0067] The frozen samples were taken out, and RIPA tissue cell lysis buffer was added to each sample, and then mixed to obtain a viscous liquid.

[0068] (2) Tissue fragmentation

[0069] Put the supernatant into a tissue crushing tube, crush it, and let it stand.

[0070] (3) Sample centrifugation

[0071] After centrifugation, the supernatant was transferred to an EP tube.

[0072] 1.7 Macrophage protein precipitation

[0073] (1) Add precipitation reagent, mix well, and let stand.

[0074] (2) Centrifuge and discard the supernatant.

[0075] (3) Add pre-cooled ET (75% ethanol), let stand, centrifuge again, and discard the supernatant.

[0076] (4) Let it stand to allow ET to evaporate.

[0077] 1.8 Tyrosine phosphorylation detection

[0078] (1) Use lysis buffer to lyse 12 cell samples and extract whole protein.

[0079] (2) The protein is alkylated and acetylated, and then digested with Trypsin to recover the peptide fragments.

[0080] (3) SH2 superphile enriches and recovers tyrosine phosphopeptides.

[0081] (4) Tyrosine phosphopeptide LC-MS / MS mass spectrometry detection.

[0082] (5) The mass spectrometry data were used to search the protein database and perform peptide and protein quantification using the software Maxquant_1.6.7.0.

[0083] (6) Bioinformatics analysis of tyrosine (Tyr) phosphopeptide data.

[0084] 1.9 Results Analysis

[0085] Figure 1 Temporal expression clustering (CA) showed that the proteins in each cluster had consistent fluctuation trends in phosphorylation abundance at different times. After macrophages were infected with MTB, protein Tyr phosphorylation and dephosphorylation occurred rapidly in the cells, suggesting that protein Tyr phosphorylation plays a key role in host cell antagonism to MTB infection. The protein interaction network revealed that ( Figure 2 ), HCK is located in the central position, which shows that HCK may play a core role in macrophages' resistance to MTB infection.

[0086] WB verification found that MTB invasion of macrophages did not cause significant changes in HCK protein expression, while the phosphorylation level of HCKTyr410 site increased first and then decreased over time ( Figure 3 ).

[0087] Example 2 Experiments with HCK inhibitors and agonists

[0088] 2.1 Macrophages

[0089] The source of macrophages is the same as in 1.1.

[0090] 2.2 Mycobacterium tuberculosis strains

[0091] The source of Mycobacterium tuberculosis standard strain H37Rv is the same as 1.2.

[0092] 2.3 Knockdown of HCK gene in induced differentiated macrophages using siRNA

[0093] (1) Before infection, the HCK gene was knocked down using siRNA and transfected.

[0094] (2) In each well of the culture plate, siRNA + Opti-MEMTM medium was prepared at a ratio of 1:25, and Lipofectamine RNAiMAX transfection reagent + Opti-MEMTM medium was prepared at a ratio of 3:25.

[0095] (3) Prepare a mixture of Opti-MEM™ medium containing siRNA and Opti-MEM™ medium containing Lipofectamine RNAiMAX transfection reagent, mix thoroughly, and add the mixture to the culture plate.

[0096] (4) After 48 h of transfection, the cell proteins were collected and the gene knockdown effect was evaluated by Western Blot assay.

[0097] (5) Evaluate the experimental data and establish an MTB infection macrophage model. The specific operation method is the same as 1.4.

[0098] (6) CFU sample collection: After the culture medium containing bacteria is aspirated and discarded, the culture plate is washed with PBS, then lysed with 0.05% SDS and pipetted into an EP tube for subsequent CFU experiments.

[0099] (7) Protein sample collection: add 1:100 phosphatase inhibitor to RIPA and mix well. Then take the culture plate and add RIPA solution to each well. After the reaction, blow the culture plate and collect the protein sample. Then take Loading Buffer and add it to the plate. Mix well, boil the sample, centrifuge and store.

[0100] (8) RNA sample collection: Add Trizol reagent to the culture plate. After the reaction, collect the RNA sample in an EP tube and store it for future use.

[0101] (9) Collecting cell supernatant samples: Aseptically filter the supernatant and collect the cell supernatant without tuberculosis bacteria into an EP tube for future use.

[0102] 2.4 Inhibition or stimulation of macrophage HCK gene using iHCK-37 or EPQpYEEIPIYL

[0103] (1) 2 h before infection, add 0.1 μM concentration of iHCK-37 (C 30 H 32N4O2S2) or EPQpYEEIPIYL in RPMI1640 medium (containing 10% FBS);

[0104] (2) Prepare bacterial suspension, MOI = 5, and construct macrophage infection model, same as 1.3-1.4;

[0105] (3) After 24 hours, the samples were collected and processed in the same way as in 2.3. The samples were used for subsequent CFU, WB, PCR and ELISA experiments.

[0106] 2.5MTB colony forming unit (CFU) count

[0107] (1) Add sterile PBS to each tube and pipette the cell lysate to the mark 10. -1 Mix well and pipette the tube labeled 10 -1 In the tube to mark 10 -2 pipette to mix, then pipette the -2 In the tube to mark 10 -3 tubes, mix well, and dilute 10 times in sequence to make dilutions of different concentrations.

[0108] (2) Mark on 7H10 solid medium for 10 -1 , 10 -2 , 10 -3 , take different concentrations of dilutions and add them to the culture dishes, and then apply them on the culture medium.

[0109] (3) Count the cells using a CFU counter after approximately 2-3 weeks.

[0110] 2.6 Western Blot

[0111] After sample extraction and protein quantification, the samples were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes, followed by blocking, antibody incubation, and chemiluminescence detection to analyze protein expression.

[0112] 2.7RT-qPCR Real-time fluorescence quantitative PCR technology

[0113] (1) RNA extraction and reverse transcription cDNA synthesis

[0114] Use RNA extraction kit to extract RNA, measure RNA sample concentration with Nanodrop, and store in -80 refrigerator for subsequent experiments.

[0115] The reverse transcription was performed using the full-form gold kit. The PCR instrument was programmed to reverse transcribe all RNA into cDNA.

[0116] (2) Primer design

[0117] The primer sequences of human HCK, ACTB, TNF-α, IL-1β, and IL-6 were searched on the Primer Bank website and verified by Primer-Blast on the NCBI website. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The primer sequences are shown in Table 1.

[0118] Table 1 Primer sequences

[0119]

[0120] (3) Establish the following RT-qPCR reaction system (20 μL), see Table 2.

[0121] Table 2 RT-qPCR reaction system

[0122]

[0123] (4) After preparing the RT-qPCR reaction system, perform the reaction according to the conditions in Table 3.

[0124] Table 3 RT-qPCR reaction conditions

[0125]

[0126] (5) Results analysis

[0127] Adoption 2 -△△Ct The formula processes the data and calculates the relative expression level of the target gene.

[0128] 2.8 ELISA enzyme-linked immunosorbent assay to detect the expression levels of TNF-α, IL-1β and IL-6

[0129] The cell culture supernatants obtained in steps 2.3 and 2.4 were thawed, and the expression levels of TNF-α, IL-1β, and IL-6 in the cell culture supernatants of the two groups at each time point in the above steps were detected using the enzyme-linked immunosorbent assay kit of Xinbosheng Biotechnology Co., Ltd.

[0130] 2.9 Statistical analysis and graphics

[0131] All statistical analyses were performed using GraphPad Prism Version 9.0 software. The measured data that conformed to the normal distribution were expressed as mean ± standard deviation (Mean ± SD). The t-test (Student t-test) was used for statistical analysis when comparing the data between two groups. One-way analysis of variance (Analysis of Variance, ANOVA) was used to evaluate the statistical differences when comparing multiple groups of data. The measured data that did not conform to the normal distribution were expressed as M (1 / 4, 3 / 4). The Wilcoxon test was used for statistical analysis when comparing the data between two groups. The Kruskal-Wallis H test was used to evaluate the statistical differences when comparing multiple groups of data. P < 0.05 was defined as statistically significant.

[0132] 2.10 Results Analysis

[0133] After siRNA transfection, HCK expression and phosphorylation levels were significantly decreased, including the reduction of autophagy-related protein LC3 I / II expression ( Figure 5 ) and the ability of macrophages to clear MTB was significantly reduced ( Figure 4 ), while inhibiting the cytokine TNF-α ( Figure 6 ), IL-1β( Figure 7 ) and IL-6 ( Figure 8 ) secretion. The effect of 0.1μM concentration of iHCK-37 on inhibiting HCK phosphorylation ( Fig.10 ), and by inhibiting HCK phosphorylation, reducing the expression of autophagy-related protein LC3 I / II and the activation of the ERK signaling pathway ( Fig.11 ), inhibiting the immune function of macrophages and increasing the colony forming unit (CFU) count after MTB infection ( Fig. 9 ), while inhibiting the cytokine TNF-α ( Fig.12 ), IL-1β( Fig.13 ) and IL-6 ( Fig.14 ) secretion. In contrast, the HCK agonist EPQpYEEIPIYL at a concentration of 0.1 μM promoted the phosphorylation of HCK ( Fig.16 ), and by promoting HCK phosphorylation, enhancing the expression of autophagy-related proteins LC3 I / II and the activation of the ERK signaling pathway ( Fig.17 ), enhance macrophage immune function, and reduce colony forming unit (CFU) counts after MTB infection ( Fig.15 ) and significantly promoted TNF-α( Fig.18 ), IL-1β( Fig.19 ) and IL-6 ( Fig. 20 ) secretion.

[0134] Example 3 Mouse model verification

[0135] 3.1 BCG strains

[0136] The BCG strain (strain number: ATCC35737) was preserved by the Bacterial Immunology Laboratory of Beijing Chest Hospital.

[0137] 3.2 Preparation of BCG strain lysate

[0138] The frozen BCG was taken out for resuscitation, and an appropriate amount of bacterial liquid was aspirated from it, and then smeared on LowensteinJensen (LJ) medium for culture. BCG cells in the logarithmic growth phase were collected and cultured using Fast -245G lyses the collected bacteria, and the supernatant after centrifugation is the BCG whole cell lysate, which is stored after measuring its concentration.

[0139] 3.3 Infection of mice with BCG by tail vein injection

[0140] (1) Experimental groups: ① blank control group, ② rifampicin treatment group (RIF), ③ iHCK-37 treatment group, ④ EPQpYEEIPIYL treatment group, 9 mice in each group.

[0141] (2) Infectious strain: BCG.

[0142] (3) Infection method: In a negative pressure animal infection laboratory, mice were infected by tail vein injection of BCG using a mouse tail vein injection imaging device. The specific steps were to collect BCG cells in the logarithmic growth phase and adjust the BCG bacterial solution to OD 600 =0.5, about 5×10 7 Each mouse was injected with 200 μL of bacterial solution through the tail vein. After the tail vein injection, the mice were housed in separate cages for subsequent experiments.

[0143] 3.4 Processing of spleen and lung tissues after BCG infection in mice

[0144] (1) Validation of mouse infection model

[0145] On the third day after the infection by tail vein injection, three mice in each of the four groups were killed, the lung tissues of the mice were separated, the lung tissues were ground, and the stock solution was diluted to 10 -3 , take 10 -2 and 10 -3 The dilution is plated and then cultured for 3-4 weeks before the CFU is counted using a counter to determine whether the infection is successful.

[0146] (2) Counting of CFU in mouse lungs

[0147] Four weeks after the mouse tail vein injection, six mice in each group were killed, and the mouse lung tissue was separated. The lung tissue was placed in a cell filter, and the tissue was ground and broken. The stock solution was serially diluted to 10 -3 , then take 10 -1 , 10 -2 , 10 -3 The dilutions were plated, cultured, and CFU counted after 4 weeks.

[0148] (3) Counting of CFU in mouse spleen

[0149] Four weeks after the mouse tail vein injection, six mice in each of the four groups were killed, and the spleens were placed in a cell filter. Sterile PBS was added and fully ground. Then the stock solution was taken and serially diluted to 10 -3 , take 10 -1 , 10 -2 , 10 -3 The diluted solution was plated, cultured at 37°C, and CFU counted after 4 weeks.

[0150] (4) Acid-fast bacilli staining of mouse lung tissue

[0151] Four weeks after the tail vein injection of mice, the four groups of mice were killed by cervical dislocation, and the lung tissues were taken for flushing and fixed overnight in 4% paraformaldehyde / universal tissue fixative to ensure the inactivation of bacteria. After that, the openings were sealed, externally disinfected, and sent to the company for acid-fast staining.

[0152] (5) HE staining of mouse lung tissue

[0153] Mouse lung tissue was fixed with 4% paraformaldehyde / universal tissue fixative, embedded in paraffin, and sliced. The tissue sections were then dewaxed and hydrated, and then stained with hematoxylin for nuclei and eosin for cytoplasm. The sections were then dehydrated and transparentized and sealed to observe the structure and morphology of tissue cells.

[0154] 3.5 Peripheral blood collection after BCG infection in mice

[0155] Four weeks after the tail vein injection of mice, peripheral blood was collected from the four groups of mice, kept static, and centrifuged. The peripheral serum of the mice after centrifugation was frozen and used for the detection of inflammatory cytokines in subsequent experiments.

[0156] 3.6 Detection of TNF-α, IL-1β and IL-6 expression levels by enzyme-linked immunosorbent assay

[0157] The mouse peripheral serum obtained in the above steps was thawed, and the expression levels of TNF-α, IL-1β and IL-6 in the peripheral serum of the four groups of mice in the above steps were detected using the ELISA kit of Xinbosheng Biotechnology Co., Ltd.

[0158] 3.7 Statistical analysis and graphics

[0159] Animal experiment data analysis and graph drawing are the same as 2.9.

[0160] 3.8 Results Analysis

[0161] In a mouse model of BCG infection, Figure 21-23 It was demonstrated that the HCK inhibitor iHCK-37 inhibited the clearance of MTB in the lungs and spleen, and reduced the secretion of inflammatory factors TNF-α, IL-1β, and IL-6; the HCK agonist EPQpYEEIPIYL enhanced the immune response in mice by activating HCK phosphorylation, promoted the clearance of BCG, and increased the levels of inflammatory factors TNF-α, IL-1β, and IL-6.

[0162] The results of acid-fast staining of mouse lung tissue showed that ( Fig.24 ), compared with the DMSO group, the bacterial load of acid-fast bacilli in the lung tissue of mice in the rifampicin group was the lowest, the bacterial load in the iHCK-37 group increased, and the bacterial load in the EPQpYEEIPIYL group decreased, which was consistent with the CFU count results of lung tissue. HE staining results ( Fig.25 ) showed that the infiltration of inflammatory cells decreased in the rifampicin group and iHCK-37 group, while the infiltration of inflammatory cells increased in the EPQpYEEIPIYL group. This indicates that the HCK inhibitor iHCK-37 inhibited the infiltration of inflammatory cells and hindered the clearance of BCG in mice, while the HCK agonist EPQpYEEIPIYL promoted the infiltration of inflammatory cells and facilitated the clearance of BCG in mice.

Claims

1. Use of an HCK agonist in the preparation of a drug for resisting Mycobacterium tuberculosis infection, wherein the agonist can inhibit Mycobacterium tuberculosis infection, and the agonist is EPQpYEEIPIYL.

2. The use according to claim 1, wherein the Mycobacterium tuberculosis comprises a standard strain of Mycobacterium tuberculosis, a clinical isolate of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

3. The use according to any one of claims 1 or 2, wherein the drug can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

4. The use according to any one of claims 1 to 3, wherein the drug can be introduced into the body by injection, penetration, absorption, physical or chemical mediation.

5. The use according to any one of claims 1 to 4, wherein the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

6. A pharmaceutical composition for use against Mycobacterium tuberculosis, comprising the HCK agonist EPQpYEEIPIYL.

7. The pharmaceutical composition according to claim 6, further comprising one or more pharmaceutically acceptable carriers.

8. The pharmaceutical composition according to any one of claims 6 or 7, which can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

9. The pharmaceutical composition according to any one of claims 6 to 8, wherein the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

10. The pharmaceutical composition according to any one of claims 6 to 9, which can be introduced into the body by injection, penetration, absorption, physical or chemical mediation.

Images