CN112891539A - Application of intervention BOK in preparation of medicine for treating new coronary pneumonia - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and discloses an application of intervention BOK in preparation of a medicine for treating neocoronary pneumonia. The invention provides a new immunotherapy scheme for comprehensive treatment of new coronary pneumonia, and researches on lung tissues of patients with new coronary pneumonia show that the expression amount of BOK in the lung tissue cells of the patients with new coronary pneumonia is increased, the effect of inhibiting new coronary virus membrane protein from inducing apoptosis of human-derived lung cells and vascular endothelial cells can be realized by removing the BOK gene, the shRNA aiming at the BOK can be expressed by injecting lentivirus into a mouse gas-moving tube, and the effect of inhibiting the new coronary virus membrane protein from inducing apoptosis of the lung tissues can be realized by removing the BOK gene, so that the BOK is expected to be applied to comprehensive treatment of the new coronary pneumonia and prepared into a form of a medicament for treating the new coronary pneumonia to reduce lung injury and alveolar fluid infiltration of the patients with the new coronary pneumonia, and the invention is suitable for the protective effect of the lung tissue organs in the process of the new coronary pneumonia.

Description

Application of intervention BOK in preparation of medicine for treating new coronary pneumonia

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to application of intervention BOK in preparation of a medicine for treating new coronary pneumonia.

Background

Coronavirus disease 2019(COVID-19) is a respiratory disease caused by acute respiratory syndrome coronavirus 2(SARS-CoV-2), and most COVID-19 patients exhibit mild to moderate symptoms, but progress to severe pneumonia in about 15%, and eventually Acute Respiratory Distress Syndrome (ARDS), septic shock and/or multiple organ failure in about 5%. The clinical treatments mainly include symptomatic management and oxygen therapy, and provide mechanical ventilation for patients with respiratory failure. Although several antiviral drugs, including the nucleotide analog remdesivir, are being actively tested, no specific drug for treating COVID-19 exists. In addition to vaccine development and methods directed against viruses or blocking viral entry, treatments that address the pathological problem of infection have also become a major concern.

Infection of host cells by SARS-CoV-2 can lead to apoptosis, which further leads to organ tissue damage and lesions, especially alveolar epidermises and epithelial cells and immediately adjacent vascular endothelial cells. The death of these cells, which constitute the "line/barrier" of the host immunity, leads on the one hand to the spread and infection of the virus in the body, aggravates the patient's condition and even to secondary infections; on the other hand, apoptotic lung epithelial cells and vascular endothelial cells cause leakage of interstitial fluid, causing physiological dysfunction. The lung section of a patient with the new coronary pneumonia and the lung death is immunohistochemically, and lung epidermal cells are invaded by SARS-CoV-2 to cause respiratory tract pathological changes, including the exudation of bronchial and alveolar tissues, the destruction of alveolar walls, and mucus containing various desquamated cells in alveolar cavities, so that oxygen cannot reach the alveolar surfaces for oxygen exchange. The pathological report suggests that the medicament can relieve or even inhibit fragment shedding and alveolar effusion caused by apoptosis in bronchus and alveolus, improve oxygen supply of lung tissues and save the life of a patient.

Disclosure of Invention

The invention aims to overcome the defect that the prior art lacks a method or medicament capable of inhibiting apoptosis in alveoli, and firstly provides a medicament for treating new coronary pneumonia, wherein the medicament can relieve apoptosis and fragment shedding in alveoli caused by new coronary virus infection.

The purpose of the invention is realized by the following technical scheme:

a medicament for the treatment of neo-coronary pneumonia, said medicament being capable of inhibiting or antagonizing BOK protein expression.

B-lymphocytoma-2 gene ovarian cancer killer protein (BCL-2 ovarial killer, BOK) is a participating molecule of an important pathway in mediating cell endogenous apoptosis. The invention discovers that SARS-CoV-2 infected lung tissue cells present strong positive expression BOK cells and apoptosis activating enzyme co-infection through pathological section research of a new coronary pneumonia patient, and the molecular level research shows that SARS-CoV-2 membrane protein can mediate apoptosis through BOK and knock out the ability of the membrane protein to induce apoptosis through BOK, and animal in vivo experiments show that chronic virus infection expressing SARS-CoV-2 membrane protein can cause lung cell apoptosis and shedding, and the lung tissue apoptosis caused by the membrane protein can be reduced through knocking down the BOK.

Preferably, the medicament is capable of inhibiting BOK protein-mediated apoptosis in lung tissue.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary agents. Through the cooperation of the composition and pharmaceutically acceptable auxiliary agents, the composition can ensure the treatment effect and simultaneously improve the safety, effectiveness and stability of the medicament.

More preferably, the medicament is in a form of injection or gastrointestinal administration, so that the application range of the medicament is increased; specifically, the administration form by gastrointestinal tract includes common powders, tablets, granules, capsules, solutions, emulsions, suspensions, and the like; the injection administration forms include commonly used injections (e.g., intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection). More preferably, when the preparation of the medicament is an injection preparation, the medicament is mixed with normal saline to prepare a solution when in use.

The invention discovers that: after SARS-CoV-2 infects host cells, membrane protein synthesis relies on BOK molecule to mediate apoptosis, which leads to alveolar cell shedding, and apoptosis inhibitor and BOK knock-down can inhibit membrane protein mediated lung tissue apoptosis.

Therefore, the invention also provides the application of the substance for specifically inhibiting the expression of the BOK protein in the preparation of the medicine for treating the new coronary pneumonia.

The invention treats the new coronary pneumonia by inhibiting the excessive apoptosis of cells after the body is infected with SARS-CoV-2 and inhibiting the permeability of alveolus through BOK related medicaments. Therefore, the invention also provides the application of the substance for specifically inhibiting the expression of the BOK protein in the preparation of the medicine for treating the lung cell apoptosis caused by the new coronavirus; preferably, the lung cells are alveolar cells.

Preferably, the substance specifically inhibiting the expression of the BOK protein comprises any one of the following:

(i) including but not limited to small interfering RNA, dsRNA, shRNA, microRNA and antisense nucleic acid which take DDX19A/NOX1 transcripts as target sequences and can inhibit expression of DDX19A/NOX1 gene expression products or gene transcription;

(ii) including but not limited to, those capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid constructs of (i);

(iii) including but not limited to constructs containing the complementary sequence DDX19A/NOX1 and capable of forming interfering molecules that inhibit the expression or gene transcription of the DDX19A/NOX1 gene expression product after transfer into the body;

(iv) an immune-related cell, differentiated cell or construct thereof after inhibiting or knocking out DDX19A/NOX1 gene sequence.

Compared with the prior art, the invention has the following beneficial effects:

according to the invention, the research on the lung tissue of a new coronary pneumonia patient discovers that the expression amount of BOK in the lung tissue cell of the new coronary pneumonia patient is increased, the removal of the BOK gene can inhibit the apoptosis of the human lung cell and the vascular endothelial cell induced by the new coronary virus membrane protein, the reduction of the BOK can inhibit the apoptosis of the lung tissue induced by the new coronary virus membrane protein by injecting the shRNA aiming at the BOK into the gas moving tube of a mouse, and the BOK is expected to be applied to the comprehensive treatment of the new coronary pneumonia and prepared into a form of a medicament for treating the new coronary pneumonia so as to reduce the lung injury and the alveolar fluid infiltration of the new coronary pneumonia patient, and the BOK is suitable for the protection effect of the lung tissue organ in the new coronary pneumonia process.

Drawings

FIG. 1 is a graph of flow analysis of FIG. 1A and a histogram of statistics of the effect of transient expression of SARS-CoV-2 membrane protein on apoptosis of human lung cells and vascular endothelial cells, FIG. 1B;

FIG. 2 is a graph showing the detection of the effect of transient expression of SARS-CoV-2 membrane protein on the expression of apoptosis-related factors in cells by WB;

FIG. 3A is a graph showing the effect of SARS-CoV-2 membrane protein and BOK knockdown on apoptosis and apoptosis-related factors in mouse lung cells by slice observation and fluorescent staining; FIGS. 3B and 3C are graphs showing the effect of WB detection of SARS-CoV-2 membrane protein and BOK knockdown on apoptosis-related factors in mouse lung cells;

FIG. 4A is immunohistochemical staining (HE) of pathological tissue sections of the lungs of patients with new coronary pneumonia, FIG. 4B, FIG. 4C and FIG. 4D show intracytoplasmic viral inclusions, alveolar epidermal desquamation, bronchially blocked exfoliated cells and alveolar structures filled with eosinophilic mucus;

FIG. 5 shows the detection of strong positive BOK cells and activated caspase-3 in lung tissues of healthy and patients with new coronary pneumonia by fluorescent staining.

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The test methods used in the following experimental examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1 detection of apoptosis after NCl-H292 and Ea.hy926 transient plasmid expression of the Membrane protein of SARS-CoV-2

1. Detecting an object:

NCl-H292 and Ea.hy926: transient transformation of the unloaded plasmid and the plasmid expressing the membrane protein, respectively.

2. The detection method specifically comprises the following steps:

(1) 1X 10^5 cells were plated per well overnight, every other day 1.5. mu.g of plasmid (Vector for no-load expression plasmid, M-Flag for expression of Flag-tagged SARS-CoV-2 membrane protein) was transferred into cells using transfection reagent lipo2000, and the cells were changed after 6 hours.

(2) The cells were trypsinized for 48 hours, centrifuged at 1000rpm for 5 minutes and washed 1X PBS 1 time.

(3) Resuspending cells with apoptosis staining buffer, staining with Annexin-V/PI (Annexin-V-FITC, PI-PE, staining at room temperature for 25min, detecting on computer, and detecting apoptosis with Annexin-V⁺PI^-Cells and Annexin-V⁺PI⁺Percentage statistics for Cells).

3. The experimental results are as follows:

as shown in the results of FIG. 1, the increased cellular ratios of Annexin-V and PI of H292 and Ea.hy926 transiently expressing membrane proteins compared to the Vector group indicate that expression of membrane proteins in host cells leads to apoptosis. Thus, transient expression of the membrane protein of SARS-CoV-2 resulted in a significant increase in the number of H292 and Ea.hy926 cells that were apoptotic.

Example 2 knock-out and recovery of BOK affects the ability of SARS-CoV-2 membrane protein to induce apoptosis

1. Detecting a cellular subject:

BOK-knocked-out H292 cell lines (KO1 and KO2), and cell lines (KO1+ BOK + GFP and KO2+ BOK + GFP) expressing BOK are recovered by adopting a lentivirus stable transfer mode on the basis of the BOK-knocked-out cell line H292, and the KO + Vector + GFP cell line represents a lentivirus no-load stable transfer cell line.

2. The detection method specifically comprises the following steps:

(1) 1X 10^5 cells were plated per well overnight, every other day 1.5. mu.g of the corresponding plasmid (Vector for no-load expression plasmid, M-Flag for expression of Flag-tagged SARS-CoV-2 membrane protein) was transferred into the cells using transfection reagent lipo2000, and the cells were changed after 6 hours.

(2) And (3) carrying out trypsinization for 48 hours, centrifuging at 1000rpm for 5 minutes, cracking cells by adopting protein lysate, detecting the expression condition of apoptosis-related protein by using Western blot, and increasing the contents of sheared caspase-9, caspase-3 and PARP during apoptosis.

3. The experimental results are as follows:

as shown in the results of FIG. 2, compared with the Vector treated group, the apoptosis-related enzyme of the WT cell line with transient membrane protein conversion is activated, after BOK knockout, the membrane protein cannot activate apoptosis-related enzymes Caspase-3 and Caspase-9 and cause the cleavage of downstream PARP, and the H292 knockout cell line recovering BOK has the expression of the apoptosis-related enzyme again after transient membrane protein conversion, which indicates that the expression of BOK is recovered on the basis of the knockout cell line to recover the activation of the apoptosis-related enzyme induced by the membrane protein, and indicates that the activation of the apoptosis enzyme induced by the SARS-CoV-2 membrane protein needs the participation of the BOK.

Example 3 Effect of SARS-CoV-2 Membrane protein expression on mouse Lung tissue

1. Detecting an object:

mouse lung tissue expressing the SARS-CoV-2 membrane protein lentivirus was transduced.

2. The detection method specifically comprises the following steps:

(1) preparation of lentivirus expressing SARS-CoV-2 Membrane protein: the membrane protein sequence of SARS-CoV-2 is connected to pLVX target vector, the connection is verified by sequencing, and target plasmid and auxiliary plasmid (pMD2G and psPAX2) are amplified in trans-competence. According to the objective plasmid: pMD 2G: transfecting HEK293T cell packaging virus with a PEI transfection reagent (PsPAX 2) at a ratio of 10:7:5, changing the liquid for 8h, collecting the virus particle supernatant for 48h, collecting the virus particles by ultra-high speed centrifugation, and re-suspending PBS to prepare the content of the tracheal injection;

(2) mice were randomized into eight groups, and tracheal injection of the corresponding contents: PBS group (50. mu.l PBS), Lenti-Mock (no-load lentivirus), Lenti-M (lentivirus expressing membrane protein), Lenti-M + Q-VD-OPh (lentivirus expressing membrane protein, post-operative tail vein injection Q-VD-OPh), PBS + Lenti-M (PBS injection, lentivirus expressing membrane protein injected after 3 days), ShCtrl. + Lenti-M (lentivirus expressing no-load interfering shRNA injected, lentivirus expressing membrane protein injected after 3 days), ShBOK1+ Lenti-M (lentivirus expressing BOK interfering shRNA knocked down, lentivirus expressing membrane protein injected after 3 days), ShBOK2+ Lenti-M (lentivirus expressing BOK interfering shRNA knocked down, lentivirus expressing membrane protein injected after 3 days).

(3) Mice were euthanized 3 days after surgery, lungs were fixed with 4% paraformaldehyde, paraffin sectioned, or ground for protein extraction.

(4) Immunohistochemical staining: paraffin section tissues are subjected to dewaxing, hydration and antigen retrieval, HE staining is carried out, immunofluorescence staining is carried out after decoloration, and pictures are observed and taken under an immunofluorescence microscope after mounting.

(5) Western blot protein detection: and carrying out protein electrophoresis on each group of animal lung tissue proteins, transferring to a PVDF membrane, and then adopting corresponding antibody incubation to detect the expression change condition of corresponding proteins.

3. The experimental results are as follows:

as shown in the results of FIG. 3A and FIG. 3B, the cell expressing SARS-CoV-2 membrane protein exhibited apoptosis execution enzyme (C-C3) and shedding, and the expression of membrane protein lentivirus resulted in increased levels of mouse lung tissue BOK protein, and apoptosis shedding, and the apoptosis-related enzyme inhibitor Q-VD-OPh inhibited activation of SARS-CoV-2 membrane protein-induced lung tissue apoptosis enzyme, compared to PBS or unloaded lentivirus treated group, and the results of FIG. 3C showed that reduction of lung tissue expression level by knocking down mouse lung tissue reduced activation of SARS-CoV-2 membrane protein-induced lung tissue apoptosis enzyme, indicating that inhibition of BOK inhibited induction of SARS-CoV-2 membrane protein.

Example 4 pathological injury of the Lung of New coronary pneumonia patients by SARS-CoV-2 infection

1. Detecting an object:

lung tissue of patient with new coronary pneumonia

2. The detection method specifically comprises the following steps:

(1) fixing the lung of a patient who dies from the new coronary pneumonia by 10% paraformaldehyde, and slicing the patient after paraffin embedding

(2) Immunohistochemical staining: dewaxing and hydrating paraffin section tissues, carrying out HE staining, dehydrating and transparentizing, and carrying out observation and photographing under a common optical microscope after sealing.

3. The experimental results are as follows:

as shown in the results of fig. 4A, the presence of viral inclusion bodies in bronchial epidermal cells of patients with new coronary pneumonia indicates that viral activity still exists in the lungs when the lungs of the patients die.

As shown by the results in FIG. 4B, there was a significant amount of cell death and desquamation of alveolar epithelial cells in patients with new coronary pneumonia, indicating that SARS-CoV-2 infection leads to lung tissue destruction.

As shown in the results of FIGS. 4C and 4D, the lung bronchi of the patients with new coronary pneumonia were blocked with exfoliated cells and the alveoli were blocked with eosinophilic mucus.

Example 5 relationship between the expression of BOK and the apoptosis-activating enzyme Caspase-3 in Lung tissue of SARS-CoV-2 infected patients with New coronary pneumonia

1. Detecting an object:

lung tissue of patient with new coronary pneumonia

2. The detection method specifically comprises the following steps:

(1) the lung of a patient who dies from the new coronary pneumonia is fixed by 10% paraformaldehyde, and is sliced after being embedded by paraffin.

(2) Paraffin section tissues are subjected to dewaxing, hydration and antigen retrieval, HE staining is carried out, immunofluorescence staining is carried out after decoloration, and pictures are observed and taken under an immunofluorescence microscope after mounting.

3. The experimental results are as follows:

as shown in the results of FIG. 5, compared with the normal lung tissue of SARS-CoV-2 human (health control), the lung tissue of the patient with new coronary pneumonia expresses more BOK strong positive cells and activated caspase-3, and BOK and activated caspase-3 are co-expressed, indicating that the activation of the pulmonary cell death enzyme caspase-3 of the patient is related to the high expression of BOK.

The results show that the BOK protein is related to the enhancement of the apoptosis of the lung cells of patients with COVID-19, and the knock-out and recovery experiments and animal level experiments of the cells prove that the inhibition of the BOK can abolish the capability of SARS-CoV-2 membrane protein to induce the apoptosis.

The invention detects the high expression of BOK by immunohistochemical staining in pathological sections of new coronary pneumonia patients, and is related to the activation of apoptosis enzyme, and secondly, the invention observes that the membrane protein of SARS-CoV-2 depends on host cell BOK mediated apoptosis to cause the shedding of mouse alveolar cells and other reasons to cause lung injury, thereby indicating that the intervention BOK is expected to be applied to the comprehensive treatment of the new coronary pneumonia to prepare a medicament for treating the new coronary pneumonia so as to relieve the physiological function damage caused by the pathological change of the new coronary pneumonia in the lung.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Claims (7)

1. A medicament for the treatment of neo-coronary pneumonia, wherein said medicament is capable of inhibiting or antagonizing BOK protein expression.

2. The medicament for treating neocoronary pneumonia according to claim 1, wherein the medicament is capable of inhibiting BOK protein mediated apoptosis in lung tissue.

3. The medicament for treating neocoronary pneumonia according to claim 2, further comprising pharmaceutically acceptable adjuvants.

4. The application of a substance for specifically inhibiting the expression of BOK protein in the preparation of a medicament for treating neocoronary pneumonia.

5. The application of the substance for specifically inhibiting the expression of the BOK protein in the preparation of the medicine for treating the lung cell apoptosis caused by the new coronavirus.

6. The use of claim 5, wherein the pulmonary cells are alveolar cells.

7. The use according to claim 6, wherein the substance that specifically inhibits the expression of the BOK protein comprises any one of:

(i) including but not limited to small interfering RNA, dsRNA, shRNA, microRNA and antisense nucleic acid which take DDX19A/NOX1 transcripts as target sequences and can inhibit expression of DDX19A/NOX1 gene expression products or gene transcription;

(ii) including but not limited to, those capable of expressing or forming the small interfering RNA, dsRNA, shRNA, microRNA, antisense nucleic acid constructs of (i);

(iii) including but not limited to constructs containing the complementary sequence DDX19A/NOX1 and capable of forming interfering molecules that inhibit the expression or gene transcription of the DDX19A/NOX1 gene expression product after transfer into the body;

(iv) an immune-related cell, differentiated cell or construct thereof after inhibiting or knocking out DDX19A/NOX1 gene sequence.

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