CN112494472B

CN112494472B - Use of 3-hydroxybutyric acid and derivatives thereof for the treatment or prevention of immune system mediated diseases

Info

Publication number: CN112494472B
Application number: CN202110150307.XA
Authority: CN
Inventors: 陈国强; 张书杰
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2021-07-20
Anticipated expiration: 2041-02-04
Also published as: CN112494472A

Abstract

The invention discloses application of 3-hydroxybutyric acid and derivatives thereof in treating or preventing immune system mediated diseases. The immune system mediated disease may be Th1 and/or Th17, or a cytokine mediated disease related thereto, for example, an autoimmune disease, an allergic disease, a transplant rejection, particularly an allergic disease such as allergic asthma. The invention opens up new application of the 3-hydroxybutyric acid and the derivatives thereof, provides a safer and more effective drug selection for preventing and treating immune system mediated diseases, and has high application value.

Description

Use of 3-hydroxybutyric acid and derivatives thereof for the treatment or prevention of immune system mediated diseases

Technical Field

The invention relates to the technical field of medicines, in particular to application of 3-hydroxybutyric acid and derivatives thereof in treating or preventing immune system mediated diseases (such as autoimmune diseases, allergic diseases and transplant rejection, particularly allergic diseases such as allergic asthma).

Background

The human immune system, including innate immunity and adaptive immunity, plays an important role in defending against body damage caused by various endogenous and exogenous factors. However, over-activation of immune function can also lead to severe tissue damage. Diseases associated with excessive activation of immune function are mainly inflammatory diseases, autoimmune diseases, allergic diseases (e.g. allergic asthma), transplant rejection (e.g. graft versus host disease). Innate immune overactivation mainly leads to inflammatory diseases. Adaptive immune overactivation leads to autoimmune diseases, allergic diseases, transplant rejection (e.g. graft versus host disease).

With the development of industry, in recent years, various allergic diseases have been increasing due to pollution of living environment and change of dietary life, and the incidence of asthma among these allergic diseases is increasing greatly. Allergic asthma is a chronic inflammatory disease that is involved by a variety of cells and cellular components. The immune function imbalance of (helper T cell 1) Th 1/(helper T cell 2) Th2 cells has been dominant in the pathogenesis of allergic asthma, namely, allergyAsthma sexually transmitted disease is an allergic disease caused by over-differentiation of Th2 cells. Th1 and Th2 cells are differentiated from primitive T cells, Th1 cell can secrete IFN-gamma, and Th2 cell can secrete IL-4 and IL-13. The imbalance of the ratio Th1/Th2 does not completely explain the pathogenesis of allergic asthma [ Wang Yanni, common small red Th1/Th2 function imbalance and bronchial asthma.Medical information ten days 024.006(2011):390-390.]. Regulatory T cells also play a key role in the development and progression of allergic asthma. Treg cells can reduce the secretion of IL-17 so as to play a role in regulating the immune negative, and the number and the function of the Treg cells are obviously reduced in the body of an asthma patient [ Pezongqiang.Zhengzhou university newspaper, 2012.12, 25-30]。

At present, the drugs for preventing or treating immune system diseases mainly comprise non-steroidal anti-inflammatory drugs represented by glucocorticoids and cyclooxygenase inhibitors. Although glucocorticoids and nonsteroidal anti-inflammatory drugs have good anti-inflammatory effects, adverse drug reactions severely limit their application. Long-term administration of glucocorticoids can lead to metabolic dysfunction, and long-term administration of nonsteroidal anti-inflammatory drugs can lead to gastric ulcers. For example, in allergic asthma, hormone drugs (such as dexamethasone, budesonide and the like) are mostly used clinically at present to control and relieve asthma symptoms, but the treatment effect is general, the side effect is strong, long-term administration is required, and the compliance of patients is poor.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention provides an application of a compound shown as the specification and pharmaceutically acceptable salts, esters and stereoisomers thereof in preparing a medicament for treating and/or preventing immune system mediated diseases,

（Ⅰ）

wherein R is₁Selected from: H. OH, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, halogen, alkoxy, alkoxyalkyl, -COR₃、-C(O)OR₃、-NR₃R₄、-C(O)NR₃R₄；

R₂Selected from: H. alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl;

n is an integer from 1 to 20 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20);

R₃and R₄Independently selected from: H. alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl.

In one embodiment of the invention, R₁Are alkyl radicals, for example C1-C6 alkyl radicals, in particular C1-C4 alkyl radicals, for example methyl, ethyl, n-propyl, isopropyl, n-butyl.

In one embodiment of the invention, R₂Is H.

In another embodiment of the invention, R₂Are alkyl radicals, for example C1-C6 alkyl radicals, in particular C1-C4 alkyl radicals, for example methyl, ethyl, n-propyl, isopropyl, n-butyl.

Specifically, n is an integer of 1 to 10, particularly 1 to 6.

In one embodiment of the present invention, n = 1.

In another embodiment of the present invention, n = 2-6.

Specifically, the above compounds may be selected from the following structures:

。

specifically, the above compound may also be selected from the following structures:

。

specifically, the above compounds may be selected from a combination of one or more of the following structures:

。

in one embodiment of the present invention, the above-mentioned application is 3-hydroxybutyric acid (C

) And pharmaceutically acceptable salts, esters, and stereoisomers thereof, for use in the manufacture of a medicament for the treatment or prevention of immune system mediated diseases, e.g. ethyl 3-hydroxybutyrate

) The use thereof for the preparation of a medicament for the treatment or prevention of an immune system mediated disease.

Specifically, the compound may be in the D form or the L form, or may be a mixture of the D form and the L form.

Specifically, the compound can be obtained by various methods such as hydrolysis and alcoholysis of various polyhydroxyalkanoates PHA, and is purified by distillation, and is confirmed to have extremely high purity by GC analysis, and no by-products such as double bonds which harm cell growth (Chen GQ, Wu Q. Microbial Production and Applications of Chiral Hydroxyalkanoates. application Microbiol Biotechno l67(2005) 592-599).

Specifically, the immune system-mediated disease may be a disease mediated by T helper 1 (Th 1) or a disease mediated by a Th 1-related cytokine (e.g., γ -IFN).

Specifically, the immune system-mediated disease may be a disease mediated by T helper 17 (Th 17) or a disease mediated by a Th 17-related cytokine (e.g., IL-17).

Specifically, the immune system mediated disease may be an immunoglobulin e (ige) -mediated allergic disease.

Specifically, the immune system mediated disease may be a disease caused by adaptive immune overactivation, such as, but not limited to, autoimmune diseases, allergic diseases, transplant rejection.

Specifically, the autoimmune disease may be, for example, but not limited to, ankylosing spondylitis, autoimmune hepatitis, autoimmune mumps, crohn's disease, diabetes (type 1), dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, graves ' disease, guillain-barre syndrome, hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, spondyloarthropathies, thyroiditis, vasculitis, vitiligo, myxoedema, pernicious anemia, ulcerative colitis, psoriasis, uveitis, sjogren's syndrome, and the like.

Specifically, the allergic disease may be, for example, but not limited to, allergic asthma, anaphylactic shock, allergic purpura, allergic rhinitis, allergic dermatitis, food allergy.

Specifically, the transplantation may be, for example, but not limited to, kidney transplantation, liver transplantation, heart transplantation, small intestine transplantation, hematopoietic stem cell transplantation, cornea transplantation, skin transplantation, or the like.

Specifically, the above graft rejection reactions include host-versus-graft reaction (HVGR) and graft-versus-host reaction (GVHR).

In one embodiment of the invention, the immune system mediated disease in the above-mentioned use of the invention is an allergic disease, in particular allergic asthma.

In particular, the above-mentioned medicaments may also comprise other active ingredients for the prophylaxis and/or treatment of the same or different diseases.

Specifically, the medicine can also comprise one or more pharmaceutically acceptable auxiliary materials.

Specifically, the above-mentioned drugs may be in any suitable dosage form or administration form, and those skilled in the art may select them as appropriate, including, but not limited to, tablets (e.g., sugar-coated tablets, film-coated tablets, sublingual tablets, orally disintegrating tablets, buccal tablets, etc.), pills, powders, granules, capsules (including soft capsules, microcapsules), troches, syrups, liquids, emulsions, suspensions, controlled release preparations (e.g., immediate release preparations, sustained release microcapsules), aerosols, films (e.g., orally disintegrating films, oral mucosa-adhering films), injections (e.g., subcutaneous injection, intravenous injection, intramuscular injection, intraperitoneal injection), intravenous drip injections, transdermal absorption preparations, ointments, lotions, adhering preparations, suppositories (e.g., rectal suppositories, vaginal suppositories), pellets, pills, oral administration forms, etc.), and the like, Nasal preparations, pulmonary preparations (inhalants), eye drops, etc., especially in the form of preparations for administration via the gastrointestinal tract.

The invention also provides application of the compound and pharmaceutically acceptable salts, esters and stereoisomers thereof in preparing functional food.

Specifically, the functional food is a food having a specific nutritional health function, which can regulate body functions (e.g., building up body, regulating body rhythm, delaying aging, etc.), but is not intended for the treatment of diseases, and is sometimes referred to as a health product. In the present invention, the functional food may take any suitable form, such as tablets, pills, capsules (e.g., soft capsules, microcapsules), candies (e.g., tabletted candies, gel candies, gum candies, etc.), solid beverages (e.g., powders, granules, etc.), liquid beverages, and the like.

The present invention also provides a method for preventing and/or treating a disease, which comprises the step of administering a prophylactically effective amount or a therapeutically effective amount of the above-described compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, or the above-described medicament of the present invention to a subject in need thereof.

In particular, the diseases, compounds and products in the above methods have the corresponding definitions of the invention as described above.

In particular, the subject is an animal, in particular a mammal, e.g. a mouse, rabbit, cat, dog, monkey, cow, horse, sheep, pig, human, in particular a human.

In particular, the mode of administration in the above methods may be gastrointestinal or parenteral, in particular gastrointestinal.

In one embodiment of the present invention, the above method is a method for treating allergic asthma, which comprises the step of administering to a subject in need thereof a therapeutically effective amount of the above compound of the present invention or a pharmaceutically acceptable salt, ester, stereoisomer, or above medicament of the present invention.

In particular, the above therapeutic effect includes an improvement in one or more of the following pathological conditions: increased IgE levels, airway thickening, irregular alveolar structure, increased airway mucus, increased eosinophil count, increased levels of inflammatory factors (e.g., gamma-IFN, IL-17), increased levels of immune cells with pro-inflammatory effects (e.g., Th1, Th 17) caused by the disease.

In particular, the above-mentioned therapeutic effect includes an improvement in one or more of the following symptoms: sneezing, lacrimation, nasal itching, itchy eyes, cough, chest distress, airflow obstruction, and repeated wheezing.

The invention opens up new application of the 3-hydroxybutyric acid and the derivatives thereof, provides a safer and more effective drug selection for preventing and treating immune system mediated diseases (such as Th1 and/or Th17, or related cytokine mediated diseases thereof, such as autoimmune diseases, allergic diseases, transplant rejection reactions, particularly allergic diseases, such as allergic asthma) which is suitable for long-term administration, and has high application value.

Drawings

FIG. 1 shows the results of experiments on the effect of 3-hydroxybutyric acid (3-HB) and its derivative ethyl 3-hydroxybutyrate (3-HBE) on body weight in mice with allergic asthma.

FIG. 2 shows the experimental results of the effect of ethyl 3-hydroxybutyrate on the level of immunoglobulin E (IgE) in serum of mice with allergic asthma of 3-hydroxybutyrate and its derivative.

FIG. 3 shows the experimental results of the effect of 3-hydroxybutyrate and its derivative, ethyl-3-hydroxybutyrate, on airway thickness in allergic asthmatic mice: representative results of mouse airway H & E staining.

FIG. 4 shows the experimental results of the effect of 3-hydroxybutyrate and its derivative, ethyl-3-hydroxybutyrate, on airway thickness in allergic asthmatic mice: mouse airway H & E staining pathology score results.

FIG. 5 shows the experimental results of the effect of 3-hydroxybutyric acid and its derivative ethyl 3-hydroxybutyrate on airway remodeling in allergic asthma mice: representative results for mouse airway PAS staining.

FIG. 6 shows the experimental results of the effect of 3-hydroxybutyric acid and its derivative ethyl 3-hydroxybutyrate on airway remodeling in allergic asthma mice: mouse airway PAS staining pathology staining results.

FIG. 7 shows the experimental results of the effect of 3-hydroxybutyrate and the derivative ethyl 3-hydroxybutyrate on the proportion of eosinophils in alveolar lavage fluid of allergic mice.

FIG. 8 shows experimental results of the effect of ethyl 3-hydroxybutyrate and its derivative on the levels of inflammatory factors in serum of allergic asthma mice.

FIG. 9 shows the experimental results of the effect of ethyl 3-hydroxybutyrate on the ratio of (helper T cell 1) Th1 cells and (helper T cell 2) Th2 cells in spleen cells of mice with allergic asthma.

FIG. 10 shows the experimental results of the effect of ethyl 3-hydroxybutyrate on the ratio of (regulatory T cells) Treg cells, (helper T cells 17) Th17 in spleen cells of mice with allergic asthma.

FIG. 11 shows the results of statistical analysis of the ratios of Th1 cells, Th2 cells and Treg cells in spleen cells of mice with allergic asthma of ethyl 3-hydroxybutyrate and its derivative 3-hydroxybutyrate.

Detailed Description

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

"immune system mediated disease" refers to a disease caused by the body's immune response to self-antigens resulting in damage to its tissues.

The term "alkyl" refers to a hydrocarbon chain radical that is straight or branched and free of unsaturation, and that is attached by a single bond to the rest of the molecule. Typical alkyl groups contain 1 to 12 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, t-pentyl, n-hexyl, isohexyl, and the like. If an alkyl group is substituted with a cycloalkyl group, it corresponds to a "cycloalkylalkyl" radical, such as cyclopropylmethyl, cyclopropylethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl, and the like. If an alkyl group is substituted with an aryl group, it is correspondingly an "aralkyl" radical, such as benzyl, benzhydryl or phenethyl.

The term "alkoxy" refers to a substituent formed by substituting a hydrogen of a hydroxyl group with an alkyl group, and the alkoxy group having C1-C6 refers to an alkoxy group having 1-6 carbon atoms, such as methoxy, ethoxy, propoxy, butoxy, etc.

The term "cycloalkyl" refers to alicyclic hydrocarbons, such as those containing 1 to 4 single and/or fused rings, having 3 to 18 carbon atoms, preferably 3 to 10 (e.g., 3, 4, 5, 6, 7, 8, 9, 10) carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, and the like.

The term "aryl" refers to a monocyclic or polycyclic radical, including polycyclic radicals containing monoaryl groups and/or fused aryl groups, e.g., containing 1-3 monocyclic or fused rings and 6-18 (e.g., 6, 8, 10, 12, 14, 16, 18) carbon ring atoms, e.g., phenyl, naphthyl, biphenyl, indenyl, and the like.

The term "halogen" refers to bromine, chlorine, iodine, fluorine.

The term "salt" is to be understood as any form of the corresponding compound of the invention, wherein the compound is in ionic form or is charged and coupled to an oppositely charged ion (cation or anion) or is in solution. This definition specifically includes pharmaceutically acceptable salts. Salts include acid addition salts and base addition salts. Acid addition salts include, but are not limited to, salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, and phosphonic acids, and salts derived from organic acids such as aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Thus, these salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, iodate, acetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, tosylate, phenylacetate, citrate, lactate, maleate, tartrate, and mesylate, and salts of amino acids such as arginate, gluconate, galacturonate, and the like. Acid addition salts may be prepared by contacting the free base form with a sufficient amount of the desired acid to form the salt in a conventional manner. The free base form can be regenerated by contacting the salt form with a base and isolating the free base in a conventional manner. Base addition salts refer to salts formed with metals or amines, such as hydroxides of alkali and alkaline earth metals, or with organic amines. Examples of metals used as cations include, but are not limited to, sodium, potassium, magnesium, calcium. Examples of suitable amines include, but are not limited to, N' -dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine (ethane-1, 2-diamine), N-methylglucamine, and procaine. Base addition salts can be prepared by contacting the free acid form with a sufficient amount of the desired base to form the salt in a conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner.

The term "ester" is understood to mean a compound formed by the reaction of an acid with the hydroxy group (if present) of the corresponding compound of the invention. This definition specifically includes pharmaceutically acceptable esters. This definition specifically includes hydrolyzable esters.

Furthermore, any of the compounds referred to in the present invention may exist in tautomeric forms. In particular, the term tautomer refers to one of two or more structural isomers of a compound, which isomers are in equilibrium and may be interconverted. Common tautomeric pairs are enamine-imine, amide-imidic acid, keto-enol, lactam-lactam imide, and the like.

The term "pharmaceutically acceptable" means that the indicated material is not of a nature that would cause reasonable caution to the practitioner in avoiding administration of the material to the patient, is theoretically non-toxic, irritating and allergic to the patient, and is capable of achieving or providing clinically acceptable pharmacokinetic, absorption, distribution and metabolic properties of the drug molecule to achieve the intended purpose, given the disease or condition to be treated and the corresponding route of administration.

The term "treating" includes eradicating, removing, reversing, alleviating, altering or controlling the disease after its onset.

The term "prevention" refers to the ability to prevent, minimize or make difficult the onset or progression of a disease or condition by treatment prior to the onset of the disease.

Thus, "treating" and/or "preventing" as a whole means at least achieving an inhibition or improvement of the symptoms associated with the subject's painful condition, wherein inhibition or improvement is meant in a broad sense to include at least a decrease in the size of a parameter, such as symptoms associated with the condition being treated, such that the methods of the invention also include situations where the condition is completely inhibited, such as prevention or cessation of occurrence, such that the subject no longer experiences the condition.

The disclosures of the various publications, patents, and published patent specifications cited herein are hereby incorporated by reference in their entirety.

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available.

Experimental Material

Mice: the C57BL/6J mice were purchased from the center of laboratory animals at Qinghua university and were normally bred for a total of 30 mice at 8 weeks of age, and were grown in the same environment and fed the same diet.

Establishing a model: the asthma model is divided into two phases, the first phase being the sensitization phase: the model group and the administration group (2% 3-HB or 2% 3-HBE dissolved in the mouse daily drinking water) were subcutaneously injected on days 0 and 14, and were sensitized with 10. mu.g of OVA and 10mg of aluminum hydroxide adjuvant as a mixed emulsion (0.2 ml/mouse), while the normal control group was injected with the same dose of physiological saline as a control. The second stage is an excitation stage: two groups of mice, model and administration groups, were placed in an atomization chamber on days 21 to 28, and 2% OVA solution was continuously atomized for 7 days with the aid of an atomizer, while normal groups were atomized for 30min per day with physiological saline as a blank control.

Collecting samples:

blood collection: after the last excitation, after fasting for 12-16h, taking eyeball and blood, placing into a blood collection tube, centrifuging at 3000rpm for 30min, taking supernatant blood, and storing in a refrigerator at-80 deg.C.

Alveolar Lavage (BALF): after the mice are bled and euthanized, the mice are fixed on a foam board, the skin and hairs of the mice are cut off by surgical scissors, tissues are separated to expose an air outlet pipe, an air pipe is inserted into a syringe connected with an air pipe cannula, the air pipe cannula is fixed by a surgical line, 1ml of precooled PBS is sucked into the syringe, the PBS in the syringe is pushed into the lungs, lavage is carried out repeatedly, and lavage fluid is sucked out to obtain alveolar lavage fluid. Alveolar lavage fluid was taken, giemsa staining was performed, and cells were counted by classification. Taking down the lung tissue, and putting the lung tissue into 4% paraformaldehyde for fixed preservation. Carrying out H

E dyeing and PAS dyeing.

Example 1: 3-hydroxybutyric acid and its derivative ethyl 3-hydroxybutyrate can inhibit OVA-induced weight loss in mice

Experimental methods

Mice were molded as described above and the mice were weighed on day 3 after the last nebulization with 2% OVA solution.

Results of the experiment

The results of the body weight changes of asthmatic mice are shown in fig. 1, wherein the model groups: OVA group, administration group OVA +3-HB and OVA +3-HBE, positive reference group OVA + Dex (dexamethasone).

Statistics and data analysis: the numerical value adopts the average value +/-SD; statistical comparisons using one way ANOVA, p < 0.001, showed significant statistical differences.

The results in FIG. 1 show that: compared with a control group, the weight of the mice in the group of chicken Ovalbumin (OVA) is obviously reduced, and the weight of the mice in the group of 3-hydroxybutyric acid (3-HB) and the derivative thereof, namely ethyl 3-hydroxybutyrate (3-HBE), is obviously increased compared with the mice in the group of OVA treatment, which shows that the weight of the mice caused by OVA can be inhibited by the 3-hydroxybutyric acid (3-HB) and the derivative thereof, namely ethyl 3-hydroxybutyrate (3-HBE).

Example 2: 3-hydroxybutyric acid and its derivative ethyl 3-hydroxybutyrate can reduce IgE level in serum

Experimental methods

Serum was collected according to the above sample collection method and the level of IgE in serum was detected using an ELISA kit (BioLegend) according to the manufacturer's instructions.

Results of the experiment

The results of the IgE content in the serum of asthmatic mice are shown in fig. 2, wherein the model groups: OVA group, administration group OVA +3-HB and OVA +3-HBE, positive reference group OVA + Dex (dexamethasone).

Statistics and data analysis: the numerical value adopts the average value +/-SD; statistical comparison adopts one way ANOVA; p < 0.01, p < 0.001; p < 0.0001, indicating that the results were statistically significantly different.

Increased IgE is a major immunological feature of allergic asthma. Figure 2 results show that: compared with a blank control group, the serum IgE level of mice in the OVA group is obviously improved, and the serum IgE level of 3-hydroxybutyric acid (3-HB) and the derivative ethyl-3-hydroxybutyrate (3-HBE) of the administration group is obviously reduced compared with the serum IgE level of the mice in the OVA treatment group, which shows that the serum IgE level of the asthma mice can be reduced by the 3-hydroxybutyric acid (3-HB) and the derivative ethyl-3-hydroxybutyrate (3-HBE).

Example 3: 3-hydroxybutyric acid and its derivative ethyl 3-hydroxybutyrate can improve pathological characteristics of asthmatic mice

Experimental methods

After the mice are successfully modeled, lung tissues are taken and fixed in 4% paraformaldehyde to prepare paraffin sections, and H is adopted&E staining is used for observing the thickness of the airway after 3-HB and 3-HBE treatment, so that the influence of 3-HB and 3-HBE on airway thickness in the remodeling of the mouse asthma model is judged. H

E, dyeing steps are as follows:

1) paraffin section dewaxing to water: sequentially placing the slices in xylene I10 min-xylene II 10 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5min-95% ethyl alcohol 5min-90% ethyl alcohol 5min-80% ethyl alcohol 5min-70% ethyl alcohol 5 min-distilled water washing;

2) hematoxylin staining of cell nucleus: slicing into Harris hematoxylin, dyeing for 3-8min, washing with tap water, differentiating with 1% hydrochloric acid alcohol for several seconds, washing with tap water, returning blue with 0.6% ammonia water, and washing with running water;

3) eosin staining of cytoplasm: placing the slices in eosin dye solution for dyeing for 1-3 min;

4) dewatering and sealing: placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent, taking out the slices from xylene, air drying, and sealing with neutral gum;

5) microscopic examination and image acquisition and analysis.

Results of the experiment

Mouse airway H&Representative results of E staining are shown in figure 3; mouse airway H

The results of the E-staining pathology scores are shown in fig. 4, where model groups: OVA group, administration group OVA +3-HB and OVA +3-HBE, positive reference group OVA + Dex (dexamethasone).

Statistics and data analysis: the numerical value adopts the average value +/-SD; statistical comparison adopts one way ANOVA; p < 0.01, with statistical significance.

The results in FIG. 3 show that, compared with the control group, the mice in the OVA group have obviously thickened airways and irregular alveolar structures, and the mice in the OVA treatment group have reduced airway thickness, complete alveolar structures and lower pathological scores compared with the mice in the OVA treatment group to which 3-hydroxybutyrate (3-HB) and the derivative thereof, namely ethyl-3-hydroxybutyrate (3-HBE) is administered. This suggests that 3-hydroxybutyric acid (3-HB) and its derivative ethyl 3-hydroxybutyrate (3-HBE) can improve asthma.

Example 4: 3-hydroxybutyric acid and its derivative ethyl 3-hydroxybutyrate can improve pathological characteristics of asthmatic mice

Experimental methods

After the model building of the mouse is successful, lung tissues are taken and fixed in 4% paraformaldehyde, paraffin sections are prepared, PAS dyeing is adopted to observe sugar substances which are mauve near the air passages of the 3-HB and 3-HBE treatment group and the control group, and then the influence of the 3-HB and 3-HBE on the mucus secretion of the air passages in the air passage remodeling of the mouse asthma model is judged. PAS staining procedure was as follows:

1) paraffin section dewaxing to water: sequentially placing paraffin section into xylene I20 min-xylene II 20 min-absolute ethyl alcohol I5 min-absolute ethyl alcohol II 5min-75% alcohol 5min, rinsing with tap water;

2) dyeing: the slices are dyed in periodic acid dye liquor for 15min, washed by tap water and then rinsed twice by distilled water;

3) and (3) performing snow dyeing: the section is placed into a Xuefer dye solution to be dyed for 30min in a dark place, washed for five minutes by running water and examined by a microscope;

4) hematoxylin staining of cell nucleus: staining the slices with hematoxylin for 1-3min, rinsing with tap water, differentiating with hydrochloric acid water solution for several seconds, rinsing with tap water, turning the ammonia water solution to blue, and rinsing with running water;

5) dewatering and sealing: placing the slices in anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, anhydrous ethanol III for 5min, n-butanol for 5min, xylene I for 5min, and xylene II for 5min, air drying, and sealing with neutral gum;

6) microscopic examination and image acquisition and analysis.

Results of the experiment

Representative results of PAS staining in mouse airways are shown in fig. 5; the pathological staining results of PAS staining of mouse airways are shown in fig. 6, wherein the model groups: OVA group, administration group OVA +3-HB and OVA +3-HBE, positive reference group OVA + Dex (dexamethasone).

Statistics and data analysis: the numerical value adopts the average value +/-SD; statistical comparison adopts one way ANOVA; p < 0.01.

FIGS. 5-6 show that the airway mucus of mice in the OVA group is significantly increased, and the airway mucus of mice in the OVA-treated group is significantly decreased compared to the control group, and the 3-hydroxybutyrate (3-HB) and its derivative ethyl-3-hydroxybutyrate (3-HBE) administered to the mice in the OVA-treated group. This suggests that 3-hydroxybutyric acid (3-HB) and its derivative ethyl 3-hydroxybutyrate (3-HBE) can improve asthma.

Example 5: 3-hydroxybutyrate and its derivative ethyl 3-hydroxybutyrate can reduce the proportion of eosinophilic granulocyte in alveolar lavage fluid

Experimental methods

Alveolar lavage fluid was collected according to the above sample collection method, centrifuged, cell pellets were collected, resuspended in 100. mu.l PBS, and the cell suspension was uniformly spread on a slide glass, stained with Giemsa, and the number of pink (eosinophils), violet (lymphocytes), and light purple (neutrophils) cells on the slide glass was recorded. The giemsa staining experiment procedure was as follows:

1) smearing and fixing: uniformly coating the cells on a clean glass slide, and fixing for 10min by using 4% paraformaldehyde after the cells are dried;

2) dyeing: fixing, airing gout at room temperature, covering Giemsa with the glass slide, dyeing for about 3min, washing off excessive dye liquor with running water, punching, and airing at room temperature;

3) sealing: mounting with neutral resin, and observing the dried glass slide under a microscope.

Results of the experiment

The statistical analysis of inflammatory cells in mouse alveolar lavage fluid is shown in fig. 7, where the statistical and data analysis: the numerical value adopts the average value +/-SD; statistical comparison was performed using one way ANOVA. Eosinophils: administration group (OVA +3-HB and 3-HBE) vs model group (OVA), p < 0.0001; macrophages, neutrophils, lymphocytes: the administration groups (OVA +3-HB and 3-HBE) vs model group (OVA) had no statistical significance.

The results in FIG. 7 show that the number of eosinophils in alveolar lavage fluid of mice in the OVA group was significantly increased, and that the number of eosinophils in alveolar lavage fluid was significantly decreased in the group to which ethyl 3-hydroxybutyrate (3-HBE) and its derivative was administered, compared to the OVA-treated mice, as compared to the control group, which indicates that 3-hydroxybutyrate (3-HB) and its derivative ethyl 3-hydroxybutyrate (3-HBE) are the main effector cells in allergic asthma, indicating that 3-hydroxybutyrate (3-HB) and its derivative ethyl 3-hydroxybutyrate (3-HBE) can improve asthma.

Example 6: 3-hydroxybutyric acid and its derivative ethyl 3-hydroxybutyrate can reduce the level of inflammatory factor in the serum of asthmatic mice

Experimental methods

Mice were bled from the orbit before sacrifice, serum was centrifuged and assayed for the inflammatory factors γ -IFN, IL-5, and IL-17A using an ELISA kit (BioLegend) according to the manufacturer's instructions.

Results of the experiment

The results of the levels of inflammatory factors in mouse serum are shown in fig. 8, where statistics and data analysis: the numerical value adopts the average value +/-SD; statistical comparison was performed using one way ANOVA. Statistical analysis of inflammatory factors in mouse serum: the gamma-IFN/IL-17A administration group (OVA +3-HB and 3-HBE) vs model group (OVA), p is less than 0.05, and the statistical significance is achieved; IL-5 administration groups (OVA +3-HB and 3-HBE) vs model group (OVA) had no statistical significance.

The results in FIG. 8 show that the level of inflammatory factors in serum of mice in the OVA group was significantly increased, the level of inflammatory factors in serum was significantly decreased in the group to which 3-hydroxybutyrate (3-HB) and its derivative ethyl-3-hydroxybutyrate (3-HBE) were administered, gamma-IFN was mainly secreted from Th1 cells, IL-17 was mainly secreted from Th17 cells, and IL-5 was mainly secreted from mast cells, compared to the OVA-treated group, compared to the control group, suggesting that ethyl-3-hydroxybutyrate (3-HB) and its derivative ethyl-3-hydroxybutyrate (3-HBE) may affect T cells to alleviate asthma.

Example 7: 3-hydroxybutyrate and its derivative ethyl 3-hydroxybutyrate can reduce (helper T cell 1) Th1, increase (helper T cell 2) Th2 proportion and increase (regulatory T cell) Treg proportion

Experimental methods

The operation is carried out according to the following steps:

1) euthanizing the treated mice, taking out spleen tissues of the mice, shearing the spleens, grinding the spleens into single-cell suspensions, filtering the single-cell suspensions by adopting a 100-micron cell screen to remove redundant tissues, centrifuging the cell suspensions for 5min according to 500g, pouring cell supernatant, adding erythrocyte lysate into cell precipitates, cracking the cell precipitates at 4 ℃ for 3min, and adding cold PBS to stop cracking;

2) centrifuging to remove lysate, culturing the cell pellet in 200 μ l RPMI medium (containing 1 μ l ionomycin) at 37 deg.C for 3 hr, and shaking to mix the cells at intervals;

3) adding PBS to wash off ionomycin, then adding 200 mul of antibody blocking solution (1: 500), incubating for 10min at room temperature, respectively adding 1 mul of FITC-CD4+ (Th1, Th2) and APC-CD4+ (Th 17, Treg) antibody into the sample, incubating for 10min at room temperature, and washing off redundant antibody;

4) after a fixed membrane breaking agent is added into a sample to treat cells for 1h, the cells are washed for 2 times by PBS, 1 mul of PE-IFN-gamma +/APC-IL-4 + (Th1, Th2) or PE-IL-17+/FITC-Foxp3+ (Th 17, Treg) antibody is respectively added to incubate for 10min, after surplus antibody is washed away, 200 mul of PBS is added to resuspend the cells, and the proportion of the (Th1, Th2) or (Th 17, Treg) cells is respectively detected by a flow-type computer.

The experimental results are as follows:

the results of the variation of the ratios of Th1 and Th2 cells in the mouse spleen are shown in fig. 9, the results of the variation of the ratios of Th17 and Treg cells in the mouse spleen are shown in fig. 10, and the results of the statistical analysis of the ratios of Th1/Th2 and Th17/Treg cells in the mouse spleen are shown in fig. 11, wherein the statistical and data analysis: the numerical value adopts the average value +/-SD; statistical comparison was performed using one way ANOVA. P < 0.05 had significant differences.

PE-IFN-. gamma. +/FITC-CD4+ in the results of FIGS. 9-11 represent Th1 cells; APC-IL-4+/FITC-CD4+ represents Th2 cells; APC-CD4+/FITC-Foxp3+ represents Treg cells; APC-CD4+/PE-IL-17+ represents Th17 cells.

OVA can increase Th1 and Th17 cells with proinflammatory effect in mice, and from the results in FIGS. 9-11, it can be seen that 3-HB and 3-HBE can obviously reduce the proportion of Th1 and Th17 cells with proinflammatory effect in the 3-HB and 3-HBE administration group compared with the model control group.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

The foregoing embodiments and methods described in this disclosure may vary based on the abilities, experience, and preferences of those skilled in the art.

The mere order in which the steps of a method are listed in the present invention does not constitute any limitation on the order of the steps of the method.

Claims

1. An application of a compound shown as the following in preparing a medicament for treating and/or preventing allergic asthma,

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2. the use of claim 1, wherein the compound is in form D or L, or a mixture of form D and L.

3. The use of claim 1, wherein the medicament further comprises one or more pharmaceutically acceptable excipients.

4. The use of claim 1, wherein the medicament is in a dosage form selected from the group consisting of: tablet, pill, powder, granule, capsule, lozenge, syrup, emulsion, suspension, controlled release preparation, aerosol, pellicle, injection, intravenous drip, transdermal absorption preparation, ointment, lotion, adhesive preparation, nasal preparation, and pulmonary preparation.

5. The use of claim 1, wherein the treatment comprises an improvement in one or more of the following pathological conditions: the disease causes elevated IgE levels, airway thickening, irregular alveolar structure, increased airway mucus.