Use of exogenous mitochondrial inhalation in treatment of novel coronavirus infections

Abstract

The invention relates to application of exogenous mitochondria in a medicine for treating novel coronavirus infection. The exogenous mitochondria is applied to preparation of the medicine for treating novel coronavirus infection, after the medicine enters the human respiratory system, the exogenous mitochondria can be endocytosed by olfactory epithelial cells, alveolar epithelial cells and alveolar macrophages, and the endocytosed mitochondria not only provides more ATP for the cells, but also can excite the internal antiviral mechanism of the cells, so that the novel coronavirus infection can be treated. The traditional Chinese medicine composition can be used for regulating the metabolic state and phagocytic ability of macrophages, promoting the macrophages to be polarized towards the M2 direction, reducing the release of proinflammatory factors and increasing the release of repairing factors, so that the remarkable treatment effect on olfactory loss, pulmonary edema, lung function decline and lung inflammation caused by novel coronavirus infection is achieved; the application disclosed by the invention not only can be used for preparing a medicine for treating new coronavirus infection, but also can be used for preparing a medicine for treating respiratory diseases caused by other virus infection.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to the use of exogenous mitochondria in drugs for treating novel coronavirus infection. Background Art

[0002] Novel coronavirus infection (COVID-19) is an acute severe respiratory syndrome caused by a novel coronavirus strain that has never appeared in humans. The pathogen causing the disease was named "SARS-CoV-2" by the Coronavirus Research Group of the International Committee on Taxonomy of Viruses. Elderly people and those with underlying cardiopulmonary diseases are more likely to develop critical illness and have a higher mortality rate when infected with the novel coronavirus. Moreover, many people are experiencing repeated infection with the novel coronavirus (secondary or multiple infections). Studies have shown that repeated infection with the novel coronavirus increases the risk of mortality and sequelae during and after the acute phase of infection. With the continuous emergence of variants, new variants have led to stronger immune escape responses, so new antiviral drugs are urgently needed. To date, there is still a lack of efficient and safe treatment measures for the proliferation of the novel coronavirus and the inflammation it causes.

[0003] After infection with the new coronavirus, it can rapidly proliferate in the olfactory mucosal epithelial cells and alveolar epithelial cells, causing necrosis and shedding of the olfactory mucosal epithelial cells and alveolar epithelial cells, causing olfactory dysfunction and respiratory failure. Neutrophil and macrophage infiltration releases a variety of inflammatory factors, causing an inflammatory storm. In the process of severe disease, the occurrence of an inflammatory storm plays an important role and is also an important cause of acute respiratory distress syndrome and multiple organ failure.

[0004] The inflammatory response characterized by the inflammatory storm is an important pathological feature of pneumonia caused by the new coronavirus infection. The polarization of macrophages plays an important role in this: M1 macrophages secrete a large number of proinflammatory factors and chemokines, aggravating lung inflammation, and M2 macrophages play an anti-inflammatory role. In the development of pneumonia caused by the new coronavirus infection, the phagocytic ability of macrophages decreases, and the M1 / M2 ratio continues to increase, which is one of the important reasons for the aggravation of pneumonia and the formation of the inflammatory storm.

[0005] There is a lack of effective treatment for novel coronavirus infection, and current treatments are mostly symptomatic. The mechanisms of commonly used antiviral drugs in clinical practice are: ritonavir inhibits HIV-1 protease; azithromycin inhibits viral reverse transcriptase; monolavir inhibits RNA polymerase. These mechanisms also have a certain inhibitory effect on the corresponding enzymes in human cells, and therefore have certain toxic side effects on normal cells. Anti-inflammatory drugs mainly use glucocorticoids and IL-6 inhibitors. It is widely known in clinical practice that glucocorticoids have great side effects. IL-6 inhibitors only target inflammatory responses caused by IL-6 and are ineffective against inflammatory responses caused by other cytokines. Therefore, there is an urgent need for a safer treatment plan with a broader spectrum of antiviral and anti-inflammatory effects.

[0006] Mitochondria are semi-autonomous intracellular organelles formed by the endocytosis of archaea and their symbiosis with cells during evolution. Mitochondria are generally considered to be mainly the "energy factory" of cells. Recent studies have found that mitochondria play an important role in triggering antiviral responses in cells and also play an important regulatory role in the polarization of macrophages. An important effect of the new coronavirus in cells is to damage mitochondrial function, resulting in insufficient ATP production, affecting cell activity, and causing continuous expression of pro-inflammatory factors in macrophages. Recent studies have shown that mitochondria can pass through the cell membrane and can be transferred between cells. Therefore, the use of intracellular antiviral effects and inflammatory regulation induced by exogenous mitochondria may play an important role in the treatment of pneumonia caused by new coronavirus infection. Summary of the invention

[0007] The purpose of the present invention is to provide the use of exogenous mitochondria in drugs for treating novel coronavirus infection. After entering the human body, the drug can inhibit the intracellular replication of the novel coronavirus and the M1 polarization of macrophages, promote the polarization of macrophages to M2, reduce the inflammatory storm, and have a significant therapeutic effect on loss of smell, pulmonary edema, decreased lung function, and lung inflammation caused by novel coronavirus infection.

[0008] To achieve the above-mentioned purpose, the present invention provides the following technical solution: the use of exogenous mitochondria in the preparation of drugs for treating novel coronavirus infection.

[0009] Furthermore, the exogenous mitochondria include mitochondria from the infected person's own blood cells, platelets, mitochondria from other tissues, and human mitochondria from non-infected persons, and the other tissues include liver, fat and mesenchymal stem cells.

[0010] Preferably, the exogenous mitochondria are mitochondria derived from the infected person's own blood cells or platelets, and the specimens are easy to obtain, facilitating future promotion to clinical practice.

[0011] The present invention also provides a drug for treating novel coronavirus infection, wherein the drug comprises exogenous mitochondria.

[0012] Furthermore, the drug inhibits the intracellular replication of the new coronavirus and the M1 polarization of macrophages, promotes the polarization of macrophages to M2, and alleviates the inflammatory storm to treat the new coronavirus infection.

[0013] Furthermore, the drug also includes a pharmaceutical excipient, which is a pharmaceutically acceptable salt, excipient or carrier. By using the drug in combination with the pharmaceutically acceptable excipient, the safety, effectiveness and stability of the drug can be improved while ensuring the therapeutic effect.

[0014] Furthermore, the drug administration route is selected from any one or more of intravenous injection, local injection, and respiratory inhalation administration. Preferably, the drug administration route is atomization inhalation administration or nasal inhalation administration, which has high absorption efficiency and high safety.

[0015] The beneficial effects of the present invention are: The present invention applies exogenous mitochondria to the preparation of drugs for treating novel coronavirus infection. After the drug enters the human respiratory system, the exogenous mitochondria can be internalized by olfactory epithelial cells, alveolar epithelial cells, and alveolar macrophages. The internalized mitochondria not only provide more ATP to the cells, but also stimulate the cells' intrinsic antiviral mechanism, regulate the metabolic state and phagocytic ability of macrophages, promote the polarization of macrophages in the M2 direction, reduce the release of proinflammatory factors, and increase the release of reparative factors, thereby playing a significant therapeutic role in loss of smell, pulmonary edema, decreased lung function, and lung inflammation caused by novel coronavirus infection; the application described in the present invention can not only be used for the preparation of drugs for treating novel coronavirus infection, but also can be used for the preparation of drugs for treating respiratory diseases caused by other viral infections.

[0016] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, some of the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 A technical roadmap used for embodiments of the present invention; Figure 2 is a mouse model infected with the new coronavirus: A: lung tissue, B: olfactory epithelial tissue, green is rVSV-SARS-CoV-2, C: CT imaging of mouse lung tissue, D: HE staining of lung tissue; Figure 3 This is a microscopic image of exogenous mitochondria entering mouse lung tissue; Figure 4 Comparison of lung tissues of mice in the exogenous mitochondria inhalation group and the control group after infection with the new coronavirus: Compared with the control group, the lung inflammation of the exogenous mitochondria inhalation group was reduced; Figure 5 is a comparison of cell changes after infection with the new coronavirus in mice in the exogenous mitochondria group and the control group: A: Compared with the control group, the exogenous mitochondria group had reduced COVID-19 (GFP) replication and no change in total macrophages (Iba-1). B: Compared with the control group, the exogenous mitochondria group had reduced COVID-19 (GFP) replication and increased M2 macrophages (CD206). C: Statistical results of A and B, ****p<0.0001; Figure 6 Comparison of the number of viruses in alveolar cells of mice infected with the new coronavirus after inhalation of exogenous mitochondria and mice in the control group; Figure 7 Comparison of the changes in pro-inflammatory factors in mice after inhalation of exogenous mitochondria and mice in the control group were infected with the new coronavirus: Compared with the control group, the IL-6 level was reduced in the exogenous mitochondria group, while IL-1A and IL-1B remained unchanged, *p<0.05. DETAILED DESCRIPTION

[0019] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0020] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art; the reagents or materials described, unless otherwise specified, are all from commercial channels.

[0021] 1.1 Design strategy of novel coronavirus based on VSV vector, replace the G protein of VSV with the S protein of novel coronavirus, and obtain recombinant novel coronavirus (rVSV-SARS-CoV-2). The surface of recombinant novel coronavirus (rVSV-SARS-CoV-2) is covered with the S protein of novel coronavirus and has the ability to replicate. The S protein mediates the infection of recombinant virus and is also an immunogen, so the infection route of rVSV-SARS-CoV-2 is similar to that of novel coronavirus.

[0022] 1.2 Adult C57 mice were selected to construct the model. The mice were anesthetized by inhalation of 2% isoflurane gas, and 15ul (15,000 pfu) of recombinant new coronavirus (rVSV-SARS-CoV-2) was inhaled through the nose. After 24 hours, the samples were collected by perfusion. It can be seen that the new coronavirus stably replicates in the lung tissue and olfactory epithelium of C57 mice. The results are as follows Figure 2A , Figure 2B .

[0023] 1.3 In vivo CT imaging of small animals was performed 5 days after infection with the recombinant novel coronavirus (rVSV-SARS-CoV-2), and the imaging manifestations of pneumonia were visible. The results are as follows Figure 2C .

[0024] 1.4 Five days after infection with the recombinant novel coronavirus (rVSV-SARS-CoV-2), perfusion sampling and HE staining of lung tissue revealed the pathological manifestations of pneumonia caused by novel coronavirus infection. The results are as follows: Figure 2D .

[0025] 2.1 Blood mitochondria purification method: Blood was collected from the heart of C57 mice, added with mitochondria / cytoplasm separation solution, and homogenized to obtain a homogenate.

[0026] 2.2 Transfer the homogenate to a 2 ml Eppenduf tube, centrifuge at 1000 g for 5 min at 4°C, discard the precipitate and retain the supernatant; transfer the supernatant to another new 2 ml Eppenduf tube, centrifuge at 10000 g for 10 min at 4°C, retain the precipitate, which is the crude mitochondrial extract, resuspend the precipitate in 500 uL IB solution, record it as solution A, and set aside.

[0027] 2.3 Take 500 uL of ice-bathed 15% Percoll solution in a 1.5 mL EP tube, slowly add the A solution onto the 15% Percoll solution, centrifuge at 21000g at 4℃ for 8 minutes, discard the supernatant, and retain the precipitate.

[0028] 15% Percoll solution: 10% 0.25M L-sucrose, 75% IB solution, 15% Percoll (GE Healthcare, 17-00891-02), prepare before use, filter, retain the filtrate, and store at 4°C for later use.

[0029] 0.25M L-sucrose: Dissolve 0.855 g L-sucrose in 10 ml distilled water, filter, retain the filtrate, and store at 4°C.

[0030] 2.4 Add 800ul IB solution to the precipitate, centrifuge at 15000g at 4℃ for 5min, discard the supernatant and retain the precipitate to obtain purified mitochondria.

[0031] 2.5 Adult C57 mice were transplanted with Mito-tracker by inhalation. 24 hours later, the mice were perfused and samples were collected. Frozen sections of lung tissue were taken. Mitochondria were observed to enter the lung tissue under an immunofluorescence microscope. The results are as follows Figure 3 .

[0032] 3.1 Eight adult C57 mice were randomly numbered 1-8. On the first day, all mice were nasally inhaled with COVID-19 (i.e., recombinant virus rVSV-SARS-CoV-2) (15ul / mouse).

[0033] 3.2 On the next day, mitochondria were prepared by extraction and purification. After preparation, the mitochondria were dissolved in PBS and stored on ice. After preparation, the mitochondria were used immediately and transplanted into mice infected with the new coronavirus within 1 hour (the longer the mitochondria remain in vitro, the worse their activity).

[0034] 3.3 The experimental group (No. 1-4) inhaled mitochondria (50ul / mouse) through the nose, while the control group (No. 5-8) inhaled PBS (50ul / mouse) through the nose. On the 5th day, the lung tissue sections were perfused and HE stained, and CD206 and Iba-1 immunofluorescence stained (the results are shown in Figure 4 , 5).

[0035] Figure 6 The effect of inhaled exogenous mitochondria on the proliferation of the new coronavirus in alveolar epithelial cells. The new coronavirus used in this example is a green fluorescent protein-labeled new coronavirus. The left picture shows the green fluorescence of alveolar cells in mice in the control group infected with the new coronavirus, and the right picture shows the green fluorescence of mice in the exogenous mitochondria inhalation group. It can be seen that inhaled exogenous mitochondria have a significant inhibitory effect on the new coronavirus.

[0036] 4.1 Eight adult C57 mice were randomly numbered 1-8. On the first day, all mice inhaled COVID-19 (i.e., recombinant novel coronavirus rVSV-SARS-CoV-2) (15ul / mouse) through the nose.

[0037] 4.2 On the second day, mitochondria were prepared by mitochondrial extraction and purification. After preparation, the mitochondria were dissolved in PBS and stored on ice. After preparation, the mitochondria were used immediately and transplanted into mice infected with the new coronavirus within 1 hour (the longer the mitochondria remain in vitro, the worse their activity).

[0038] 4.3 The experimental group (No. 1-4) inhaled mitochondria (50ul / mouse) through the nose, while the control group (No. 5-8) inhaled PBS (50ul / mouse) through the nose. On the 5th day, lung tissue was directly collected, tissue RNA was extracted, and the levels of inflammatory factors (IL-1α, IL-1β and IL-6) were measured by qPCR method.

[0039] See also Figure 7 Compared with the control group, the replication of COVID-19 (GFP) in the exogenous mitochondria group was reduced, p<0.0001; the number of M2 macrophages (CD206) increased, p<0.0001; and the total macrophages (Iba-1) remained unchanged. The IL-6 level in the control group was reduced, p<0.05, while the levels of IL-1A and IL-1B did not change in the two groups. We found that inhalation of exogenous mitochondria can significantly inhibit the proliferation of the new coronavirus in lung epithelial cells, regulate the M1 / M2 polarization of macrophages, and have an inhibitory effect on the inflammatory storm.

[0040] In summary, the present invention applies exogenous mitochondria to the preparation of drugs for treating novel coronavirus infection. After the drug enters the human respiratory system, the exogenous mitochondria can be internalized by olfactory epithelial cells, alveolar epithelial cells, and alveolar macrophages. The internalized mitochondria not only provide more ATP to the cells, but also stimulate the cells' intrinsic antiviral mechanism, regulate the metabolic state and phagocytic ability of macrophages, promote the polarization of macrophages in the M2 direction, reduce the release of proinflammatory factors, and increase the release of reparative factors, thereby playing a significant therapeutic role in loss of smell, pulmonary edema, decreased lung function, and lung inflammation caused by novel coronavirus infection; the application described in the present invention can not only be used for the preparation of drugs for treating novel coronavirus infection, but also can be used for the preparation of drugs for treating respiratory diseases caused by other viral infections.

[0041] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0042] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims.

Claims

1. The use of exogenous mitochondria in the preparation of drugs for the treatment of novel coronavirus infection.

2. The use according to claim 1, characterized in that The exogenous mitochondria include mitochondria from the infected person's own blood cells, platelets, mitochondria from other tissues, and human mitochondria from non-infected persons. The other tissues include liver, fat and mesenchymal stem cells.

3. A drug for treating novel coronavirus infection, characterized in that: The drug comprises the exogenous mitochondria according to claim 2.

4. The drug for treating novel coronavirus infection according to claim 3, characterized in that: The drug treats novel coronavirus infection by inhibiting the intracellular replication of the new coronavirus and the M1 polarization of macrophages, promoting the polarization of macrophages to M2, and alleviating the inflammatory storm.

5. The drug for treating novel coronavirus infection according to claim 3, characterized in that: The medicine also includes pharmaceutical excipients, which are pharmaceutically acceptable salts, excipients or carriers.

6. The drug for treating novel coronavirus infection according to claim 5, characterized in that: The administration route of the drug is selected from any one or more of intravenous injection, local injection, and respiratory inhalation.

7. The drug for treating novel coronavirus infection according to claim 6, characterized in that: The administration route of the drug is atomization inhalation or nasal inhalation.

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