CN114425056A - A stem cell exosome composition for improving asthma - Google Patents

Abstract

The present invention relates to a stem cell exosome composition for improving asthma, which is characterized by comprising stem cell exosome as an active ingredient and 1% to 3% of ectoine. Compared with the prior art, the present invention has the advantages of The following advantages: the stem cell exosome and ectoine composition of the present application can reduce or block the release of TNF-α and IL-1β, and also can improve the synthesis of IL-10, and is expected to become an effective method for asthma treatment in the future .

Description

A stem cell exosome composition for improving asthma

technical field

The present invention relates to the technical field of pharmaceutical compositions for improving asthma, in particular to a specific stem cell exosome composition for improving asthma.

Background technique

Asthma is a chronic respiratory disease that affects approximately 300 million people worldwide and causes 250,000 deaths each year. Current asthma therapy, including corticosteroids and beta-agonists, provides relief of asthma symptoms in subjects treated with beta-agonist-based therapy, but underlying airway inflammation persists. Thus, subjects requiring long-term beta-agonist use are at greater risk for severe asthma exacerbations, resulting in hospitalization and death. It has been reported that patients with severe asthma often require high-dose corticosteroid therapy, which may be associated with systemic side effects and does not necessarily improve lung function or quality of life. In addition, patients with asthma continue to experience episodic asthma attacks, which are often related to triggering factors, including viral or bacterial infections, and ambient air pollution, and do not prevent asthma attacks in most patients. Long-term use can lead to an increase in blood sugar and blood pressure in the body, resulting in a decline in the body's resistance, which can stimulate the gastric mucosa and induce gastrointestinal bleeding. The elderly are also prone to osteoporosis. Therefore, this highlights the urgent need for effective treatment of asthma.

For example, a Chinese invention patent application with application number CN201880059290.3 (application publication number CN111093680 A), "Method for Treating Allergic Airway Disease (AAD)/Asthma", discloses the use of mesenchymal stem cells to treat allergic gas A method for airway disease/asthma, which has been demonstrated in a mouse model of allergic airway disease that mimics several features of human asthma, that mesenchymal stem cells exhibit immunomodulatory and paracrine factor secretion through direct cell contact and paracrine factor secretion. Anti-inflammatory properties. Administration of exogenous mesenchymal stem cells has been shown to reduce Th2 proliferation and reduce Th2 preference, which contributes to AAD, and inhibition of dendritic cell activation, migration and antigen presentation has been observed. Reductions in eosinophil-associated pro-inflammatory cytokines were observed in bronchoalveolar lavage fluid, and mesenchymal stem cells have been demonstrated in these models to actively reduce the presence and presence of inflammation-causing cells compared to corticosteroids that inhibit AI. active. However, since the exact cause of asthma is unknown, the therapeutic effect of a single component (eg, mesenchymal stem cells) may be temporary and may only occur in some patients, therefore, ongoing research and clinical trials are required. To overcome these limitations, there is a need to develop a new therapeutic agent for the treatment of asthma.

SUMMARY OF THE INVENTION

The first technical problem to be solved by the present invention is to provide a new pharmaceutical composition for improving asthma in view of the current state of the art.

The second technical problem to be solved by the present invention is to provide the use of stem cell exosomes isolated from umbilical cord-derived mesenchymal stem cells in the preparation of a drug for preventing or treating asthma in view of the current state of the art.

The technical solution adopted by the present invention to solve the above-mentioned first technical problem is: the pharmaceutical composition for improving asthma is characterized by comprising stem cell exosomes as an active ingredient.

Further, the stem cell exosomes are isolated from umbilical cord stem cells cultured under hypoxic conditions.

Further, the hypoxic conditions are induced by 2% to 9% oxygen or by a hypoxic cell sensitizer.

Further, the stem cell exosomes contain biologically active factors indoleamine 2,3 dioxygenase (IDO) and interleukin-10.

Further, the pharmaceutical composition comprises stem cell exosomes and ectoine.

Further, the concentration of the ectoine is 1% to 3%.

Further, the concentration of stem cell exosomes contained in the pharmaceutical composition is 150-200ug/ml.

Further, the pharmaceutical composition is for intratracheal administration or inhalation.

Further, the pharmaceutical composition is an aerosolized drug.

To solve the second technical problem, the present invention also provides the use of stem cell exosomes isolated from umbilical cord-derived mesenchymal stem cells in the preparation of a medicament for preventing or treating asthma.

Compared with the prior art, the present invention has the following advantages: the stem cell exosome and ectoine composition of the present application can reduce or block the release of TNF-α and IL-1β, and can also increase the level of IL-10. Synthetic, is expected to be an effective method for asthma treatment in the future.

Description of drawings

Fig. 1 is the preparation flow chart of the pharmaceutical composition for improving asthma in Example 1 of the present invention;

Fig. 2 is the step reference diagram of utilizing the pharmaceutical composition to carry out atomization treatment to different groups of asthmatic rats in the embodiment of the present invention 3;

3 is a bar graph of the relative expression levels of TNF-α in lung tissue in normal group rats and different groups of asthmatic rats in Example 3 of the present invention;

4 is a bar graph of the relative expression levels of serum TNF-α in normal group rats and different groups of asthmatic rats in Example 3 of the present invention;

5 is a bar graph of the relative expression levels of IL-1β in lung tissue in normal group rats and different groups of asthmatic rats in Example 3 of the present invention;

6 is a bar graph of the relative expression levels of serum IL-1β in normal group rats and different groups of asthmatic rats in Example 3 of the present invention;

7 is a bar graph of the relative expression levels of IL-10 in lung tissue in normal group rats and different groups of asthmatic rats in Example 3 of the present invention;

Figure 8 is a bar graph of the relative expression levels of serum IL-10 in normal group rats and asthmatic rats in different groups in Example 3 of the present invention.

Detailed ways

The present invention will be further described below with reference to the accompanying drawings and embodiments.

Example 1 Preparation of stem cell exosome composition for improving asthma

Stem cell exosome (Exosome) is a lipid inclusion structure, which is encapsulated with cytokines, mRNA and other substances. It can regulate the body's immunity through immune cells, and can inhibit inflammation through cytokines to improve and protect lung function. . Ectoin (Ectoin) is an amino acid derivative belonging to the extreme enzyme component. Extreme electrolytes are protective molecules with little stress that protect extremophiles and plants from living in salt lakes, hot springs, ice blocks, deep seas or deserts Ectoin protects these organisms from harmful habitats environmental impact. The anti-pollution efficacy of Ectoin has been confirmed by numerous studies (in vitro and in vivo clinical). To this day, it is also the only anti-pollution active ingredient that is also approved for use in medical products and medical applications, including the prevention of pollution-induced lung diseases such as asthma. Exosomes are tiny vesicles secreted by umbilical cord stem cells, with a diameter of about 30-200nm, and 5um-sized particles can reach bronchioles and alveoli. Therefore, the volume of exosomes is very suitable for atomization.

details as follows:

①The primary bronchial epithelial cells/tracheal epithelial cells derived from asthma patients were purchased from LONZA, the product number (LONZAcc2932s), with LONZA's medium CC-3171 (BEBM bronchial epithelial cell growth basal medium) 500mL, CC-4175 (BEGM SingleQuots additive) and growth factors) were cultured until day 22 and differentiated into transepithelial resistance, mucin production, and cilia production. When the cell confluence reaches 70%-80%, wash the cells twice with normal saline, and perform starvation culture with compound electrolyte injection; The cell supernatant was collected, centrifuged at 3000-4500g for 5-8min to remove the precipitate, and the supernatant was centrifuged for use (A);

②Umbilical cord mesenchymal stem cell culture: select 3 tubes of P3-generation mesenchymal stem cells from the stem cell bank for recovery, a total of 1 × ^10e7 cells, and the seeding density is 30%-60% of the maximum confluence; the cells used for culturing cells are fully cultured with T4 Base plus 10% human blood platelet lysate; after cell inoculation is completed, place it in an incubator with a temperature of 37°C, a hypoxia concentration of 2%-9%, and a concentration of 5% carbon dioxide volume ratio for hypoxia culture, simulating the lack of oxygen in human asthma. In this case, stem cells are activated to release hypoxia-inducible factor HIF and related factors; when the cell confluency reaches 70%-80%, the cells are washed twice with normal saline, and compound electrolyte injection is added to the supernatant obtained in the first step (A) , perform starvation culture; after 12-24 hours of starvation culture, the cell confluency reaches 90%-95%, collect 2-3×10e7 stem cell culture supernatant, centrifuge at 3000-4500g for 5-8min to remove the precipitate, take the centrifugation The supernatant was filtered through ultracel-10 ultrafiltration membrane, and finally a 200ul volume of concentrated supernatant containing various cytokines and exosomes was obtained (B);

③ The composition of specific stem cell exosomes and ectoine: Concentrated supernatant exosomes (B) were added to 10ml of normal saline containing 1%-3% ectoine and resuspended to prepare an aerosol. The combination of specifically stimulating umbilical cord stem cell exosomes and 1-3% edocine is used to improve asthma by atomization. The specific preparation process is shown in Figure 1.

Example 2 Establishment of Asthma Rat Animal Model

(1) Methods and materials

Female SD rats, weighing 120-180 g, were selected and housed in a controlled environment on a 12-hour light/12-hour dark light cycle with free access to water and laboratory food; all mice were provided with During the 4-5 day adaptation period, the rats were divided into normal group (group A), asthmatic rats (group B), asthmatic rats (group C), and asthmatic rats (group D), with 13 rats in each group.

(2) Experimental reagents and instruments

Normal saline; ovalbumin 25g/bottle; inactivated pertussis bacillus vaccine 2ml/bottle; aluminum hydroxide 500g/bottle; ultrasonic nebulizer.

(3) Modeling method

①Before the modeling of the rats, they were adaptively reared for a week.

②The rats in groups B to D were sensitized by intraperitoneal injection of 1ml of antigen solution (100mg of ovalbumin per ml of antigen solution, 100mg of aluminum hydroxide dry powder and 5×10e9 inactivated pertussis vaccine). Two weeks later, when asthma was provoked, the rats in each group were placed in semi-closed rat cages, and nebulized inhaled 5% ovalbumin normal saline for 40 min, provoked once a day for 8 consecutive days. The mice in group A were injected with normal saline instead of antigen solution. The injection site and dose were the same as those in groups B, C, and D. The ovalbumin was inhaled into normal saline by atomization, and the concentration of ovalbumin was 0.05 mg/mL.

(4) Observation of asthma rat model

The asthmatic rats showed the following main characteristics: asthmatic rats (group B), asthmatic rats (group), and asthmatic rats (group D) had shortness of breath, slow breathing or irregular rhythm in severe cases, and mild cyanosis , quadriplegia, delayed action, unresponsiveness, and is accepted in the art as a clinical model of asthma.

Embodiment 3 utilizes pharmaceutical composition to carry out atomization treatment to asthmatic rat animal

After establishing an asthmatic rat animal model, the drug composition is used to perform aerosol treatment on the asthmatic rat animal, and the specific steps are as follows:

①Rats in the normal group (group A) were replaced with normal saline instead of the antigen solution;

②Asthmatic rats (group B) received the ultrasonic nebulizer in the box for 7 days, and sprayed the specific exosome (containing stem cell factor) and ectoine composition for 30 minutes every day;

③Asthmatic rats (group C) received the ultrasonic nebulizer in the box for 7 days, and sprayed normal saline for 30 minutes every day;

④Asthmatic rats (group D) received an ultrasonic nebulizer in the box for 7 days, and sprayed normal saline + 1-3% ectoine for 30 minutes every day. The specific nebulization treatment step diagram can refer to Figure 2;

⑤ On the 8th day, the normal group (group A) and asthmatic rats (B), asthmatic rats (C), and asthmatic rats (D) were anesthetized and collected 20 ml of blood to obtain serum by centrifugation, and dissected to obtain 50 mg of lung tissue in Homogenize tissue in frozen lysis buffer, centrifuge at 12,000 rpm for 5 minutes at 4°C to obtain 50 ul of supernatant;

⑥ Serum and supernatant were subjected to enzyme-linked immunosorbent assay (ELISA) to analyze the levels of IL-1β, TNF-α, and IL-10, and the results can be found in Figures 3 to 8;

Among them, from Figure 3 to Figure 6, it can be seen that the effect of the pharmaceutical composition on TNF-α and IL-1β in lung tissue, TNF-α and IL-1β were semi-quantified by HE-stained lung sections, both of which play a role in asthma. With a key role, it is the initiator of the inflammatory cytokine network, the acceleration and strengthening factor and the tissue damage factor, the asthmatic rats (group B) received nebulization of specific stem cell exosomes and ectoine composition for 7 consecutive days After treatment, TNF-α and IL-1β in lung tissue and serum were the same as those in normal group (group A) rats, but asthmatic rats (group C) received normal saline for 7 consecutive days after TNF-α and IL-1β were increased. The levels in lung tissue and serum were significantly higher than those in control SD rats (group A) and asthmatic rats (group B). - Alpha and IL-1beta levels were slightly elevated in lung tissue and serum.

Effects of specific pharmaceutical compositions on TNF-α and IL-1β in lung tissue:

Note: TNF-α in lung tissue in group C, compared with group A, **P<0.01; compared with group B, **P<0.01; compared with group D, *P<0.05;

IL-1β in lung tissue in group C, compared with group A, ***P<0.001; compared with group B, **P<0.01.

Effects of specific pharmaceutical compositions on serum TNF-α and IL-1β:

Note: In serum TNF-α, group C, compared with group A, **P<0.01; compared with group B, **P<0.01; compared with group D, *P<0.05;

In serum IL-1β, group C, compared with group A, ***P<0.001; compared with group B, ***P<0.001.

Effects of specific pharmaceutical compositions on pulmonary IL-10 and serum IL-10:

Note: IL-10 in the lungs of group C, compared with group A, **P<0.01; compared with group B, **P<0.01; compared with group D, *P<0.05;

In serum IL-1β, group C, compared with group A, **P<0.01; compared with group B, **P<0.01.

Serum interleukin IL-10 is also known as cytokine synthesis inhibitor. The decrease of IL-10 is an important factor in the pathogenesis of asthma. It can be seen from the results in Figure 7 and Figure 8 that IL-10 is a cytokine discovered in recent years. Mononuclear macrophages, T cells, B cells, etc., are powerful immunosuppressive factors with pleiotropic biological activities, and have anti-inflammatory effects. IL-10 has potential application prospects for the treatment of bronchial asthma. The levels of IL-10 in lung tissue and serum of asthmatic rats (group C) after receiving normal saline for 7 consecutive days were significantly lower than those of control SD rats (group A) and asthmatic rats (group B). After asthmatic rats (group B) received nebulization of a combination of specific stem cell exosomes and ectoine for 7 consecutive days, IL-10 levels in lung tissue and serum were the same as control SD rats (group A) , IL-10 levels in lung tissue and serum were slightly decreased in asthmatic rats (group D) after receiving 1-3% ectoine for 7 consecutive days. Therefore, the composition of stem cell exosomes and 1-3% ectoine of the present application can trigger the production of IL-10 and inhibit inflammation-promoting factors including TNF-a and IL-1bβ, which is expected to be an effective method for asthma treatment in the future. Treatment of asthma with stem cell exosomes in combination with ectoine, although one advantage of the present invention is that stem cell exosomes in combination with ectoine can be used alone to treat asthma, it should be understood that stem cell exosomes are combined with ectoine to treat asthma. The combination can be combined with another asthma therapy, e.g., when the asthmatic subject has an existing asthma treatment regimen that includes, for example, corticosteroid or beta-agonist therapy, and is subsequently treated with stem cell exosomes in combination with ectoine. Physicians can still treat asthmatic subjects with another asthma therapy.

Claims (10)

1. A stem cell exosome composition for ameliorating asthma, characterized by: comprises a stem cell exosome as an active ingredient.

2. The stem cell exosome composition according to claim 1, characterized in that: the stem cell exosomes are isolated from inter-umbilical stem cells cultured under hypoxic conditions.

3. The stem cell exosome composition according to claim 2, characterized in that: the hypoxic conditions are induced by 2% to 9% oxygen or by hypoxic cell sensitizers.

4. The stem cell exosome composition according to claim 1, characterized in that: the stem cell exosomes comprise the bioactive factors indoleamine 2,3 dioxygenase (IDO) and interleukin-10.

5. A stem cell exosome composition according to any one of claims 1 to 4, characterised in that: the stem cell exosome preparation composition comprises stem cell exosomes and ectoin.

6. The stem cell exosome composition according to claim 5, characterized in that: the concentration of the ectoin is 1% -3%.

7. The stem cell exosome composition according to claim 6, characterized in that: the pharmaceutical composition contains stem cell exosomes at a concentration of 150-200 ug/ml.

8. A stem cell exosome composition according to claim 5 or 6, characterised in that: the pharmaceutical composition is for intratracheal administration or inhalation.

9. The stem cell exosome composition according to claim 8, characterized in that: the pharmaceutical composition is an aerosolized medicament.

10. Use of a stem cell exosome isolated from umbilical cord-derived mesenchymal stem cells in the preparation of a medicament for the prevention or treatment of asthma.

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