Application of vitamin E compound in preparation of MACIR protein targeting agent

Abstract

The invention relates to application of a vitamin E compound in preparation of a MACIR protein targeting agent. The vitamin E compound comprises any one or a combination of at least two of tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives. The invention creatively discovers the interaction between tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives and MACIR protein, and proves the strong binding capacity of tocopherol or derivatives thereof and MACIR protein through experiments. The tocopherol, the tocopherol derivative, the tocotrienol or the tocotrienol derivative can be used for regulating the function of MACIR protein associated immune cells so as to influence the expression of inflammatory factors, play an important role in inflammation and anaphylaxis, and also can be used for improving skin injury repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and particularly relates to the application of a vitamin E compound in the preparation of a MACIR protein targeting agent. Background Art

[0002] Tocopherol, a type of vitamin E compound, is a type of fat-soluble vitamin that plays an important role in human growth and development and is an essential nutrient for the human body. Research shows that tocopherol has a wide range of functions such as antioxidant, anti-inflammatory, and delaying skin aging, so it is widely used in health foods and cosmetics.

[0003] The chemical structure of tocopherol contains a chromanol mother ring and a phytyl derivative side chain. According to the different substitutions of the chromanol mother ring, it can be divided into α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol. The side chains of tocopherol are the same, all being saturated phytyl.

[0004] In addition, in natural vitamin E, the four chromanol mother rings can also be connected to an unsaturated phytyl side chain, called tocotrienol. These natural tocotrienols are considered to be intermediates in the synthesis process of tocopherol derivatives and have physiological functions similar to those of tocopherol.

[0005] Macrophage Immunometabolism Regulator (MACIR) gene is derived from C5orf30, that is, open reading frame 30 on chromosome 5. It is a locus discovered during large-scale arthritis-related gene screening in the European and American populations; a single nucleotide polymorphism of this gene has a significant association with the occurrence and prognosis of arthritis in the population. Research shows that C5orf30 regulates the immunity and metabolism of related cells by interacting with various inflammation-related factors in cells at the transcriptional and expression levels, affects the expression of multiple cytokines, and plays an important role in inflammation and allergic reactions.

[0006] However, so far, no one has reported the correlation between tocopherol and its derivatives and MACIR protein, and it is not clear whether tocopherol can affect the physiological functions of MACIR protein-related cells by targeting MACIR protein. Summary of the Invention

[0007] In view of the deficiencies of the prior art, the purpose of the present invention is to provide an application of vitamin E compounds in the preparation of MACIR protein targeting agents. The present invention has experimentally demonstrated the strong binding ability of tocopherol or its derivatives to MACIR protein. Vitamin E compounds (tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives) can also be used to regulate the function of MACIR protein-related immune cells, thereby affecting the expression of inflammatory factors, playing an important role in inflammation and allergic reactions, and can also be used to improve skin injury repair.

[0008] To achieve the purpose of this invention, the following technical solutions are adopted:

[0009] In the first aspect, the present invention provides an application of vitamin E compounds in the preparation of MACIR protein targeting agents.

[0010] The vitamin E compounds include any one or a combination of at least two of tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives.

[0011] The present invention creatively discovers the interaction between tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives and MACIR protein. The schematic diagram of the binding of tocopherol or its derivatives to MACIR protein is as Figure 1 shown (wherein blue is tocopherol derivative and green is MACIR protein). It can be seen that the tocopherol derivative can dock well into the core domain of MACIR protein, contact both the α-helix and β-sheet regions therein, and experimentally demonstrates the strong binding ability of tocopherol or its derivatives to MACIR protein.

[0012] Preferably, the tocopherol includes any one or a combination of at least two of α-tocopherol, β-tocopherol, γ-tocopherol or δ-tocopherol.

[0013] Preferably, the tocotrienol includes any one or a combination of at least two of α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

[0014] In the present invention, tocopherol or its derivatives have the structure shown in Formula I, and tocotrienol or its derivatives have the structure shown in Formula II.

[0015]

[0016] In Formula I, for α-tocopherol, R 1 is CH 3 、R 2 is CH 3 、R 3 is H, and for β-tocopherol, R 1 is CH3 , R 2 is H, R 3 is H, and the R of γ-tocopherol 1 is H, R 2 is CH 3 , R 3 is H, and the R of δ-tocopherol 1 is H, R 2 is H, R 3 is H.

[0017]

[0018] In formula II, the R of α-tocotrienol 1 is CH 3 , R 2 is CH 3 , R 3 is H, and the R of β-tocotrienol 1 is CH 3 , R 2 is H, R 3 is H, and the R of γ-tocotrienol 1 is H, R 2 is CH 3 , R 3 is H, and the R of δ-tocotrienol 1 is H, R 2 is H, R 3 is H.

[0019] Preferably, the tocopherol derivative is obtained by binding the hydroxyl group of tocopherol with a glycosyl group (that is, in the structure shown in formula I, R 3 is a glycosyl group).

[0020] Preferably, the tocotrienol derivative is obtained by binding the hydroxyl group of tocotrienol with a glycosyl group (that is, in the structure shown in formula II, R 3 is a glycosyl group).

[0021] Each of the glycosyl groups independently includes any one or a combination of at least two of glucosyl group, galactosyl group or mannosyl group, preferably glucosyl group.

[0022] Preferably, the tocopherol derivative is δ-tocopherol glucoside, that is, in the structure shown in formula I, R 1 is H, R 2 is H, R 3 is glucosyl group.

[0023] In a second aspect, the present invention provides an application of a vitamin E compound in a MACIR protein receptor.

[0024] The vitamin E compound includes any one or a combination of at least two of tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives.

[0025] Preferably, the tocopherol includes any one or a combination of at least two of α-tocopherol, β-tocopherol, γ-tocopherol or δ-tocopherol.

[0026] Preferably, the tocotrienol includes any one or a combination of at least two of α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

[0027] In the present invention, the tocopherol or its derivative has the structure shown in Formula I, and the tocotrienol or its derivative has the structure shown in Formula II.

[0028]

[0029] In Formula I, for α-tocopherol, R 1 is CH 3 , R 2 is CH 3 , R 3 is H; for β-tocopherol, R 1 is CH 3 , R 2 is H, R 3 is H; for γ-tocopherol, R 1 is H, R 2 is CH 3 , R 3 is H; for δ-tocopherol, R 1 is H, R 2 is H, R 3 is H.

[0030]

[0031] In Formula II, for α-tocotrienol, R 1 is CH 3 , R 2 is CH 3 , R 3 is H; for β-tocotrienol, R 1 is CH 3 , R 2 is H, R 3 is H; for γ-tocotrienol, R 1 is H, R 2 is CH 3 , R 3 is H; for δ-tocotrienol, R 1 is H, R 2 is H, R 3 is H.

[0032] Preferably, the tocopherol derivative is obtained by binding the hydroxyl group of tocopherol with a glycosyl group (i.e., in the structure shown in Formula I, R 3 is a glycosyl group).

[0033] Preferably, the tocotrienol derivative is obtained by binding the hydroxyl group of tocotrienol with a glycosyl group (i.e., in the structure shown in Formula II, R 3 is a glycosyl group).

[0034] Each of the glycosyl groups independently includes any one or a combination of at least two of glucosyl group, galactosyl group or mannosyl group, preferably a glucosyl group.

[0035] Preferably, the tocopherol derivative is δ-tocopherol glucoside, that is, in the structure shown in Formula I, R 1 is H, R 2 is H, R 3 is a glucosyl group.

[0036] In a third aspect, the present invention provides an application of a vitamin E compound in the preparation of a MACIR protein-related immunocyte function regulator.

[0037] The vitamin E compound includes any one or a combination of at least two of tocopherol, tocopherol derivative, tocotrienol or tocotrienol derivative.

[0038] The immunocytes include macrophages and / or mast cells.

[0039] Previous studies have shown that the MACIR protein can regulate the immune functions of macrophages and / or mast cells, thereby affecting the expression of inflammatory factors and playing an important role in inflammation and allergic reactions. Therefore, any one or a combination of at least two of tocopherol, tocopherol derivative, tocotrienol or tocotrienol derivative involved in the present invention can be used to prepare a MACIR protein-related immunocyte function regulator.

[0040] Preferably, the tocopherol includes any one or a combination of at least two of α-tocopherol, β-tocopherol, γ-tocopherol or δ-tocopherol.

[0041] Preferably, the tocotrienol includes any one or a combination of at least two of α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

[0042] In the present invention, tocopherol or its derivative has the structure shown in Formula I, and tocotrienol or its derivative has the structure shown in Formula II.

[0043]

[0044] In formula I, for α-tocopherol, R 1 is CH 3 , R 2 is CH 3 , R 3 is H; for β-tocopherol, R 1 is CH 3 , R 2 is H, R 3 is H; for γ-tocopherol, R 1 is H, R 2 is CH 3 , R 3 is H; for δ-tocopherol, R 1 is H, R 2 is H, R 3 is H.

[0045]

[0046] In formula II, for α-tocotrienol, R 1 is CH 3 , R 2 is CH 3 , R 3 is H; for β-tocotrienol, R 1 is CH 3 , R 2 is H, R 3 is H; for γ-tocotrienol, R 1 is H, R 2 is CH 3 , R 3 is H; for δ-tocotrienol, R 1 is H, R 2 is H, R 3 is H.

[0047] Preferably, the tocopherol derivative is obtained by binding the hydroxyl group of tocopherol with a glycosyl group (that is, in the structure shown in formula I, R 3 is a glycosyl group).

[0048] Preferably, the tocotrienol derivative is obtained by binding the hydroxyl group of tocotrienol with a glycosyl group (that is, in the structure shown in formula II, R 3 is a glycosyl group).

[0049] Each of the glycosyl groups independently includes any one or a combination of at least two of glucosyl group, galactosyl group or mannosyl group, and is preferably a glucosyl group.

[0050] Preferably, the tocopherol derivative is δ-tocopherol glucoside, that is, in the structure shown in formula I, R 1are H, R 2 are H, R 3 is a glucosyl group.

[0051] Fourthly, the present invention provides an application of a vitamin E compound in the preparation of a MACIR protein-related anti-inflammatory drug and / or a MACIR protein-related anti-allergic drug.

[0052] The vitamin E compound includes any one or a combination of at least two of tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives.

[0053] Previous studies have shown that the MACIR protein can affect the secretion of macrophage inflammatory factors and mast cell inflammatory factors caused by stimulating factors. Therefore, any one or a combination of at least two of tocopherol, tocopherol derivatives, tocotrienol or tocotrienol derivatives involved in the present invention can bind to the MACIR protein to inhibit or relieve the body's inflammatory or allergic reactions, and thus be used as an anti-inflammatory or anti-allergic drug.

[0054] Preferably, the tocopherol includes any one or a combination of at least two of α-tocopherol, β-tocopherol, γ-tocopherol or δ-tocopherol.

[0055] Preferably, the tocotrienol includes any one or a combination of at least two of α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol.

[0056] In the present invention, tocopherol or its derivative has the structure shown in Formula I, and tocotrienol or its derivative has the structure shown in Formula II.

[0057]

[0058] In Formula I, for α-tocopherol, R 1 is CH 3 , R 2 is CH 3 , R 3 is H, for β-tocopherol, R 1 is CH 3 , R 2 is H, R 3 is H, for γ-tocopherol, R 1 is H, R 2 is CH 3 , R 3 is H, for δ-tocopherol, R 1 is H, R 2 is H, R 3 is H.

[0059]

[0060] In formula II, R of α-tocotrienol 1 is CH 3 , R 2 is CH 3 , R 3 is H, R of β-tocotrienol 1 is CH 3 , R 2 is H, R 3 is H, R of γ-tocotrienol 1 is H, R 2 is CH 3 , R 3 is H, R of δ-tocotrienol 1 is H, R 2 is H, R 3 is H.

[0061] Preferably, the tocopherol derivative is obtained by binding the hydroxyl group of tocopherol with a glycosyl group (i.e., in the structure shown in formula I, R 3 is a glycosyl group).

[0062] Preferably, the tocotrienol derivative is obtained by binding the hydroxyl group of tocotrienol with a glycosyl group (i.e., in the structure shown in formula II, R 3 is a glycosyl group).

[0063] Each of the glycosyl groups independently includes any one or a combination of at least two of glucosyl group, galactosyl group or mannosyl group, preferably glucosyl group.

[0064] Preferably, the tocopherol derivative is δ-tocopherol glucoside, i.e., in the structure shown in formula I, R 1 is H, R 2 is H, R 3 is glucosyl group.

[0065] Fifthly, the present invention provides an application of a vitamin E compound in preparing a MACIR protein-related skin cell damage repair agent.

[0066] The vitamin E compound includes any one or a combination of at least two of tocopherol, tocopherol derivative, tocotrienol or tocotrienol derivative.

[0067] The cells include epidermal cells and / or fibroblasts.

[0068] Long-term inflammation and allergic reactions can cause damage to the skin. Previous studies have shown that the MACIR protein plays an important role in inflammation and allergic reactions, thus regulating skin injury repair. Therefore, any one or a combination of at least two of the tocopherols, tocotrienols, tocopherol derivatives, or tocotrienol derivatives involved in the present invention can promote the repair of damaged epidermal cells and / or fibroblasts by binding to the MACIR protein.

[0069] Preferably, the tocopherol includes any one or a combination of at least two of α-tocopherol, β-tocopherol, γ-tocopherol, or δ-tocopherol.

[0070] Preferably, the tocotrienol includes any one or a combination of at least two of α-tocotrienol, β-tocotrienol, γ-tocotrienol, or δ-tocotrienol.

[0071] In the present invention, the tocopherol or its derivative has the structure shown in Formula I, and the tocotrienol or its derivative has the structure shown in Formula II.

[0072]

[0073] In Formula I, for α-tocopherol, R 1 is CH 3 , R 2 is CH 3 , R 3 is H; for β-tocopherol, R 1 is CH 3 , R 2 is H, R 3 is H; for γ-tocopherol, R 1 is H, R 2 is CH 3 , R 3 is H; for δ-tocopherol, R 1 is H, R 2 is H, R 3 is H.

[0074]

[0075] In Formula II, for α-tocotrienol, R 1 is CH 3 , R 2 is CH 3 , R 3 is H; for β-tocotrienol, R 1 is CH 3 , R 2 is H, R 3 is H; for γ-tocotrienol, R 1 is H, R 2 is CH3 , R 3 is H, and R of δ-tocotrienol 1 is H, R 2 is H, R 3 is H.

[0076] Preferably, the tocopherol derivative is obtained by binding the hydroxyl group of tocopherol with a glycosyl group (i.e., in the structure shown in Formula I, R 3 is a glycosyl group).

[0077] Preferably, the tocotrienol derivative is obtained by binding the hydroxyl group of tocotrienol with a glycosyl group (i.e., in the structure shown in Formula II, R 3 is a glycosyl group).

[0078] Each of the glycosyl groups independently includes any one or a combination of at least two of glucosyl group, galactosyl group or mannosyl group, preferably glucosyl group.

[0079] Preferably, the tocopherol derivative is δ-tocopherol glucoside, that is, in the structure shown in Formula I, R 1 is H, R 2 is H, R 3 is glucosyl group.

[0080] Compared with the prior art, the present invention has the following beneficial effects:

[0081] The present invention creatively discovers the interaction between tocopherol, tocopherol derivative, tocotrienol or tocotrienol derivative and MACIR protein, and proves the strong binding ability of tocopherol or its derivative to MACIR protein.

[0082] Furthermore, in the binding experiment with MACIR protein, the tocopherol derivative, especially δ-tocopherol glucoside, exhibits a more excellent binding effect and can be used as a more effective MACIR protein targeting agent or MACIR protein receptor.

[0083] Furthermore, based on the regulatory effect of MACIR protein on the immune functions of macrophages and / or mast cells, the tocopherol, tocopherol derivative, tocotrienol or tocotrienol derivative involved in the present invention can be used to regulate the functions of MACIR protein-related immune cells, thereby affecting the expression of inflammatory factors, playing an important role in inflammation and allergic reactions, and can also be used to regulate skin injury repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 is a schematic diagram of the binding of a tocopherol derivative to MACIR protein.

[0085] Figure 2It is the mass spectrometry diagram of the polypeptide pep1 for research in Example 1.

[0086] Figure 3 It is the mass spectrometry diagram of the polypeptide pep2 for research in Example 1.

[0087] Figure 4 It is the circular dichroism spectrum diagram of the polypeptide for research in Example 1.

[0088] Figure 5 It is the test diagram of the α-helix content of the binding of the pep1 polypeptide with tocopherol and its derivatives in Example 1.

[0089] Figure 6 It is the test diagram of the α-helix content of the binding of the pep2 polypeptide with tocopherol and its derivatives in Example 1.

[0090] Figure 7 It is the test diagram of the expression level of the inflammatory factor IL-6 secreted by macrophages in different experimental groups in Example 2.

[0091] Figure 8 It is the test diagram of the expression level of the inflammatory factor TNF-α secreted by mast cells in different experimental groups in Example 3.

[0092] Figure 9 It is the test diagram of the healing ability of different tocopherols and their derivatives on the scratch injury of HaCat cells in Example 4.

[0093] Figure 10 It is the test diagram of the healing ability of different tocopherols and their derivatives on the scratch injury of HSF cells in Example 5. Detailed implementation manners

[0094] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention.

[0095] Example 1

[0096] In this example, the interaction between tocopherol and its derivatives and the polypeptide was determined through circular dichroism experiments to evaluate the interaction between tocopherol and the MACIR protein.

[0097] (1) Synthesize the polypeptide for research of the MACIR protein:

[0098] (1.1) Design the polypeptide for research of the MACIR protein:

[0099] The MACIR protein is too long to be directly synthesized. To facilitate experimental studies on the interaction between tocopherols and their derivatives and the MACIR protein, polypeptides for research were designed based on the structure of the MACIR protein. First, the structure of the MACIR protein was retrieved from the RCSB PDB database. Next, two polypeptides for research (named pep1 and pep2, respectively) were designed according to the core domain, subdomain, secondary subdomain, and loop region of the MACIR protein.

[0100] Among them, pep1 contains the core domain, subdomain, and the middle loop region of the MACIR protein. The amino acid sequence of pep1 is shown in SEQ ID NO:1. The specific sequence of SEQ ID NO:1 is: TVSGYQILHMDSNYLVGFTTGEELLKLAQKCTGGEESKAEAMPSLRSKQLDAGLARSSRLYKTRSRYYQ P; pep2 contains the main domain, secondary subdomain, and a partial loop region at the front of the secondary subdomain of the MACIR protein. The amino acid sequence of pep2 is shown in SEQ ID NO:2. The specific sequence of SEQ ID NO:2 is: GYQILHMDSNYLVGFTTGEELLKLAQKCTGGKRAHSKSLDLDKMIKEPADTEVLQYQLQHL.

[0101] (1.2) Verify the structure of the polypeptides for research on the MACIR protein:

[0102] The above-mentioned pep1 and pep2 polypeptides were synthesized by solid-phase peptide synthesis, and the polypeptide structures were verified by mass spectrometry. The mass spectrometry results of pep1 and pep2 polypeptides are shown in Figure 2 and Figure 3 respectively, showing that the molecular weights are consistent with the expected molecular weights of the designed polypeptides, proving that the above-mentioned pep1 and pep2 polypeptides were accurately synthesized.

[0103] The above-mentioned pep1 and pep2 polypeptides were diluted with water to a final concentration of 0.2 mg / mL, and the configurations of the above-mentioned polypeptides were verified by circular dichroism spectroscopy.

[0104] By scanning the circular dichroism spectrum from 190 to 270 nm, the secondary structure of the polypeptide can be determined. The negative peaks near 208 and 220 nm correspond to the typical α-helix structure, and the proportion of α-helix is estimated according to the ratio of the peak values at 208 and 220 nm. The results are shown in Figure 4 As shown, it can be seen that pep1 and pep2 show a partial α-helix structure, and the α-helix content of the two is estimated to be about 30% according to the ratio of the circular dichroism signals near 220 nm and 208 nm.

[0105] (2) Evaluate the interaction between tocopherols and their derivatives and polypeptides through circular dichroism experiments:

[0106] Polypeptides exhibit certain secondary structures, including α-helix structure, β-sheet structure, and random coil. When polypeptides specifically bind to drug molecules, changes in secondary structure often occur, and through these changes, the binding between polypeptides and drug molecules can be determined.

[0107] (2.1) Verify the interaction between tocopherols and their derivatives and Pep1 polypeptide:

[0108] (2.1.1) Experimental grouping and treatment methods (dissolve the polypeptide in water and dissolve tocopherol in absolute ethanol):

[0109] ① pep1 group: Dilute the pep1 polypeptide with water to a final concentration of 0.2 mg / mL;

[0110] ② pep1 + α-tocopherol group (abbreviated as pep1 + α-VE group): Mix the α-tocopherol ethanol solution (the concentration of tocopherol is 0.25 mg / mL) and the pep1 polypeptide aqueous solution (the concentration of polypeptide is 0.25 mg / mL) according to a volume ratio of 1:3 to prepare a complex;

[0111] ③ pep1 + α-tocopherol glucoside group (abbreviated as pep1 + α-VE-G group): Mix the α-tocopherol glucoside ethanol solution (the concentration of tocopherol glucoside is 0.25 mg / mL) and the pep1 polypeptide aqueous solution (the concentration of polypeptide is 0.25 mg / mL) according to a volume ratio of 1:3 to prepare a complex;

[0112] ④ pep1 + δ-tocopherol group (abbreviated as pep1 + δ-VE group): Mix the δ-tocopherol ethanol solution (the concentration of tocopherol is 0.25 mg / mL) and the pep1 polypeptide aqueous solution (the concentration of polypeptide is 0.25 mg / mL) according to a volume ratio of 1:3 to prepare a complex;

[0113] ⑤ pep1 + δ-tocopherol glucoside group (abbreviated as pep1 + δ-VE-G group): Mix the δ-tocopherol glucoside ethanol solution (the concentration of tocopherol glucoside is 0.25 mg / mL) and the pep1 polypeptide aqueous solution (the concentration of polypeptide is 0.25 mg / mL) according to a volume ratio of 1:3 to prepare a complex.

[0114] (2.1.2) Verification results:

[0115] Verify the α-helix content of each of the above experimental groups through circular dichroism (the test method refers to step (1.2)), and the test results of each experimental group are as Figure 5As shown, it can be seen that after tocopherol and its derivatives act on pep1, obvious changes occur in the secondary structure. The α-helix content of the polypeptide in the pep1 + α-tocopherol group increases from 30% to 50%, the α-helix content of the polypeptide in the pep1 + α-tocopherol glucoside group increases from 30% to 60%, the α-helix content of the polypeptide in the pep1 + δ-tocopherol group increases from 30% to 60%, and the α-helix content of the polypeptide in the pep1 + δ-tocopherol glucoside group increases from 30% to 65%.

[0116] The above results indicate that tocopherol and its derivatives can all bind to the MACIR protein, causing obvious changes in the secondary structure; further, compared with tocopherol, tocopherol glycoside and the MACIR protein have a more excellent binding effect.

[0117] (2.2) Verify the interaction between tocopherol and its derivatives and the pep2 polypeptide:

[0118] (2.2.1) Experimental grouping and treatment methods (dissolve the polypeptide in water and dissolve tocopherol in ethanol):

[0119] ① pep2 group: Dilute the pep2 polypeptide with water to a final concentration of 0.2 mg / mL;

[0120] ② pep2 + α-tocopherol glucoside group (abbreviated as pep2 + α-VE-G group): Mix the α-tocopherol glucoside ethanol solution (the concentration of tocopherol glucoside is 0.25 mg / mL) and the pep2 polypeptide aqueous solution (the concentration of the polypeptide is 0.25 mg / mL) in a volume ratio of 1:3 to prepare a complex;

[0121] ③ pep2 + δ-tocopherol group (abbreviated as pep2 + δ-VE group): Mix the δ-tocopherol ethanol solution (the concentration of tocopherol is 0.25 mg / mL) and the pep2 polypeptide aqueous solution (the concentration of the polypeptide is 0.25 mg / mL) in a volume ratio of 1:3 to prepare a complex;

[0122] ④ pep2 + δ-tocopherol glucoside group (abbreviated as pep2 + δ-VE-G group): Mix the δ-tocopherol glucoside ethanol solution (the concentration of tocopherol glucoside is 0.25 mg / mL) and the pep2 polypeptide aqueous solution (the concentration of the polypeptide is 0.25 mg / mL) in a volume ratio of 1:3 to prepare a complex.

[0123] (2.2.2) Verification results:

[0124] Referring to the method in step (2.1.2), verify the α-helix content of each of the above experimental groups by circular dichroism spectroscopy. The test results of each experimental group are as Figure 6As shown, it can be seen that after tocopherol and its derivatives act on pep2, obvious changes in the secondary structure occur. The α-helix content of the polypeptide in the pep2 + α-tocopherol group increases from 30% to 60%, the α-helix content of the polypeptide in the pep2 + δ-tocopherol group increases from 30% to 50%, and the α-helix content of the polypeptide in the pep2 + δ-tocopherol glucoside group increases from 30% to over 80%.

[0125] The above results indicate that tocopherol and its derivatives can all bind to the MACIR protein, causing obvious changes in the secondary structure; further, compared with tocopherol, tocopherol glycosides and the MACIR protein have a more excellent binding effect.

[0126] Example 2

[0127] This example verifies the inhibitory effect of tocopherol and its derivatives on the macrophage inflammatory response associated with the MACIR protein.

[0128] Under resting state, macrophages have low expression levels of cytokines such as TNF and IL-6. Stimulated by inflammatory molecules such as LPS, macrophages can cause a large amount of expression of cytokines related to inflammation, leading to the activation of macrophages and thus causing an inflammatory response. Anti-inflammatory drugs can effectively inhibit the overexpression of macrophage cytokines caused by inflammatory molecules such as LPS, thereby playing an anti-inflammatory role. We used mouse macrophage RAW264.7 cells as model cells, induced macrophage activation through LPS, and determined macrophage activation and inhibition by measuring the IL-6 expression level.

[0129] (1) Experimental grouping and intervention methods:

[0130] ① Medium control group (abbreviated as M group): Replace the drugs added subsequently with an equal volume of DMEM medium (1% of the medium volume), and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0131] ② PBS control group (abbreviated as blank group): Replace the drugs added subsequently with an equal volume of PBS buffer (1% of the medium volume), and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0132] ③ Unstimulated macrophage group (abbreviated as N group): Mouse macrophages RAW264.7 without LPS stimulation, and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0133] ④ Macrophage group after LPS stimulation (abbreviated as C group): Stimulate the cell group with LPS at a final concentration of 0.001 μg / μL, and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0134] ⑤ α-tocopherol group (abbreviated as α-VE group): Cells were treated with α-tocopherol at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), and incubated at 37 °C in a 5% CO 2 incubator for 1 h. Then, the cell group was stimulated with LPS at a final concentration of 0.001 μg / μL and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0135] ⑥ α-tocopherol glucoside group (abbreviated as α-VE-G group): Cells were treated with α-tocopherol glucoside at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), and incubated at 37 °C in a 5% CO 2 incubator for 1 h. Then, the cell group was stimulated with LPS at a final concentration of 0.001 μg / μL and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0136] ⑦ δ-tocopherol group (abbreviated as δ-VE group): Cells were treated with δ-tocopherol at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), and incubated at 37 °C in a 5% CO 2 incubator for 1 h. Then, the cell group was stimulated with LPS at a final concentration of 0.001 μg / μL and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0137] ⑧ δ-tocopherol glucoside group (abbreviated as δ-VE-G group): Cells were treated with δ-tocopherol glucoside at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), and incubated at 37 °C in a 5% CO 2 incubator for 1 h. Then, the cell group was stimulated with LPS at a final concentration of 0.001 μg / μL and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0138] ⑨ Spermidine group: Cells were treated with spermidine at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), and incubated at 37 °C in a 5% CO 2 incubator for 1 h. Then, the cell group was stimulated with LPS at a final concentration of 0.001 μg / μL and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0139] ⑩ Centella asiatica group: Cells were treated with Centella asiatica tissue culture extract at a final concentration of 30 μg / mL (dissolved in DMSO, with a volume of 1% of the culture medium volume, purchased from Canon Biotechnology Co., Ltd.), and incubated at 37 °C in a 5% CO 2Incubate in an incubator for 1 h, add LPS with a final concentration of 0.001 μg / μL to stimulate the cell group, at 37 °C, 5% CO 2 Incubate overnight in an incubator;

[0140] Dexamethasone group: Treat the cells with dexamethasone at a final concentration of 50 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), at 37 °C, 5% CO 2 Incubate in an incubator for 1 h, add LPS with a final concentration of 0.001 μg / μL to stimulate the cell group, at 37 °C, 5% CO 2 Incubate overnight in an incubator;

[0141] IL-6 standard group: Treat the cells with the standard of IL-6 protein diluted with DMSO (the standard of IL-6 protein provided in the ELISA kit is diluted 16 times, with a final concentration of 31.25 pg / mL), at 37 °C, 5% CO 2 Incubate in an incubator for 1 h, add LPS with a final concentration of 0.001 μg / μL to stimulate the cell group, at 37 °C, 5% CO 2 Incubate overnight in an incubator.

[0142] (3) Detection of the inflammatory factor IL-6 secreted by macrophages:

[0143] Detect the inflammatory factor IL-6 secreted by macrophages in each of the above experimental groups by ELISA respectively. Taking the 1 / 16 IL-6 standard group as a reference, evaluate the inhibitory effect of each experimental group on the macrophage inflammatory response associated with the MACIR protein through the relative expression level of IL-6 in each experimental group. The results are as Figure 7 shown.

[0144] Among them, the IL-6 expression levels in the culture medium control group (M group) and the PBS control group (blank group) are both close to 0, indicating that the ELISA detection has high specificity under this experimental condition; the IL-6 expression level in the unstimulated macrophage group (N group) is very low, indicating that the inflammatory factor IL-6 is at a very low expression level in resting macrophages; while under the stimulation of LPS, the inflammatory factor is highly expressed in macrophages (C group), and the expression level is increased by more than 20 times compared with the resting state.

[0145] Meanwhile, α-tocopherol, α-tocopheryl glucoside, δ-tocopherol, and δ-tocopheryl glucoside involved in this application all have significant inhibitory effects on the secretion of inflammatory factors in macrophages stimulated by LPS. Among them, tocopherol glycoside has a stronger inhibitory effect on inflammatory factors, which is comparable to that of dexamethasone, a glucocorticoid drug widely used as an anti-inflammatory drug, and spermidine and asiaticoside tissue culture extract, commonly used drugs with anti-inflammatory effects. It shows that tocopherol and its derivatives targeting the MACIR molecule can effectively inhibit the inflammatory response of MACIR-related cells, thereby exerting an anti-inflammatory effect in the body.

[0146] Example 3

[0147] This example verifies the inhibitory effect of tocopherol and its derivatives on the inflammatory response of MACIR protein-related mast cells.

[0148] Mast cells are cells closely related to allergic reactions, and their activation mechanism is similar to that of macrophages. Under resting conditions, mast cells have low expression levels of cytokines such as TNF-α and IL-6. Stimulation by inflammatory molecules such as LPS can lead to a large amount of expression of cytokines related to inflammation in mast cells, causing the activation of mast cells, thereby triggering allergies and resulting in a systemic inflammatory response. Anti-inflammatory drugs can effectively inhibit the overexpression of mast cell cytokines caused by inflammatory molecules such as LPS, thereby playing a role in inhibiting allergies. We used P815 mouse mast cells as model cells, induced mast cell activation by LPS, and determined the activation and inhibition of mast cells by measuring the expression level of TNF-α.

[0149] (1) Experimental grouping and intervention methods:

[0150] ① Medium control group (abbreviated as M group): Replace the drugs added later with an equal volume of DMEM medium (1% of the medium volume), and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0151] ② PBS control group (abbreviated as blank group): Replace the drugs added later with an equal volume of PBS (1% of the medium volume), and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0152] ③ Unstimulated mast cell group (abbreviated as N group): Mouse mast cells P815 without LPS stimulation, and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0153] ④ Mast cell group after LPS stimulation (abbreviated as C group): Stimulate the cell group with LPS at a final concentration of 0.001 μg / μL, and incubate overnight in an incubator at 37°C and 5% CO 2 2.

[0154] ⑤ α - tocopherol group (abbreviated as α - VE group): Cells were treated with α - tocopherol at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), incubated at 37 °C in a 5% CO 2 incubator for 1 h, then stimulated with LPS at a final concentration of 0.001 μg / μL, and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0155] ⑥ δ - tocopherol group (abbreviated as δ - VE group): Cells were treated with δ - tocopherol at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), incubated at 37 °C in a 5% CO 2 incubator for 1 h, then stimulated with LPS at a final concentration of 0.001 μg / μL, and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0156] ⑦ δ - tocopherol glucoside group (abbreviated as δ - VE - G group): Cells were treated with δ - tocopherol glucoside at a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the culture medium volume), incubated at 37 °C in a 5% CO 2 incubator for 1 h, then stimulated with LPS at a final concentration of 0.001 μg / μL, and incubated at 37 °C in a 5% CO 2 incubator overnight;

[0157] ⑧ TNF - α standard group: Cells were treated with the diluted TNF - α protein standard (the TNF - α protein standard provided in the ELISA kit was diluted 16 - fold, with a final concentration of 32.15 pg / mL), incubated at 37 °C in a 5% CO 2 incubator for 1 h, then stimulated with LPS at a final concentration of 0.001 μg / μL, and incubated at 37 °C in a 5% CO 2 incubator overnight.

[0158] (2) Detection of inflammatory factor TNF - α secreted by mast cells:

[0159] The inflammatory factor TNF - α secreted by mast cells in each of the above experimental groups was detected by ELISA. Taking the 1 / 16 standard group as a reference, the inhibitory effects of each experimental group on the macrophage inflammatory response associated with the MACIR protein were evaluated through the relative expression levels of TNF - α in each experimental group. The results are as Figure 8 shown.

[0160] Among them, the TNF-α expression levels in both the culture medium control group (Group M) and the PBS control group (blank group) were close to 0, indicating that ELISA detection had high specificity under the experimental conditions; the TNF-α expression level in the unstimulated mast cell group (Group N) was very low, indicating that the inflammatory factor TNF-α was at a very low expression level in mast cells in the resting state; while under the stimulation of LPS, the inflammatory factors were highly expressed in mast cells (Group C), and the expression level increased by more than 20 times compared with the resting state.

[0161] Meanwhile, α-tocopherol, δ-tocopherol, and δ-tocopherol glucoside involved in this application all had obvious inhibitory effects on the secretion of inflammatory factors in macrophages stimulated by LPS. Among them, α-tocopherol could inhibit about 50% of TNF-α expression, while δ-tocopherol and its glucoside showed stronger inhibitory effects, and the inhibitory effect of δ-tocopherol was greater than 80%. It was shown that tocopherol and its derivatives targeting the MACIR molecule could effectively inhibit the allergic reactions of MACIR-related cells, thereby playing an anti-allergic effect in the body.

[0162] Example 4

[0163] This example verified the effect of tocopherol and its derivatives on the repair of MACIR protein-related epidermal cell damage.

[0164] HaCat is a human epidermal cell line with immortalization characteristics. We detected the effects of tocopherol and its derivatives on the scratch injury repair ability of human skin HaCat cells.

[0165] (1) Test method:

[0166] By scratching in a culture plate filled with HaCat cells, the wound repair function of the compound was determined by observing the closing time of the scratch. This method is a widely accepted in vitro evaluation model for evaluating the wound healing ability of compounds.

[0167] ① Activate, count, and dilute Hacat cells to 4×10 4 cells / well. Add 2 mL of cell suspension to each well in a 6×3 well plate, and culture overnight at 37°C in a 5% CO 2 incubator until the cells cover the entire bottom of the culture dish;

[0168] ② Gently discard the upper layer of the culture medium, and add 2 mL of culture medium containing 2% serum for starvation treatment for 24 h;

[0169] ③ Use a 1 mL pipette tip to make a scratch. Do not pause or repeat the scribing during the process to make the scratch as flat as possible;

[0170] ④ Rinse the loose cells with 2 mL of PBS, repeat 3 times until the excess suspended cells are washed away, add 2 mL of medium containing 2% serum with the drugs of each experimental group in step (2), and incubate in an incubator at 37°C and 5% CO 2 Incubate in an incubator, take pictures at 0 h and 24 h after scratching, and calculate the healing rate.

[0171] (2) Experimental grouping and intervention methods:

[0172] ① Blank group (abbreviated as C+DMSO group): Add 1% DMSO of the medium volume in step (1)-④;

[0173] ② α-Tocopherol group (abbreviated as α-VE group): Add α-tocopherol with a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the medium volume) in step (1)-④;

[0174] ③ δ-Tocopherol group (abbreviated as δ-VE group): Add δ-tocopherol with a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the medium volume) in step (1)-④;

[0175] ④ δ-Tocopherol glucoside group (abbreviated as δ-VE-G group): Add δ-tocopherol glucoside with a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the medium volume) in step (1)-④.

[0176] (3) Test results:

[0177] After scratching for 24 hours, the healing rate results of each experimental group for the healing ability of tocopherol and its derivatives on the scratch injury of HaCat cells are as Figure 9 shown. The results show that δ-tocopherol and its glucoside have a significant effect on promoting cell injury healing, indicating that tocopherol and its derivatives with anti-inflammatory and anti-allergic effects can effectively promote the repair of MACIR protein-related skin cell injuries, thus playing a role in protecting skin injuries and promoting wound healing.

[0178] Example 5

[0179] This example verifies the effect of tocopherol and its derivatives on the repair of MACIR protein-related fibroblast injuries.

[0180] HSF is a kind of human skin fibroblast, which can synthesize and secrete a large amount of matrix components such as elastin, collagen, glycosaminoglycans and glycoproteins, and then generate elastic fibers, collagen fibers and reticular fibers, and secrete a variety of cell repair factors, making the skin have a strong renewal and self-repair ability. We detected the effect of tocopherol and its derivatives on the scratch injury repair ability of human skin HSF cells.

[0181] (1) Test method:

[0182] Scratch the culture plate filled with HSF cells, and determine the wound repair function of the compound by observing the closing time of the scratch. This method is a widely accepted in vitro evaluation model for evaluating the wound healing ability of compounds.

[0183] ① Activate, count, and dilute HSF cells to 4×10 4 cells / well. Add 2 mL of cell suspension to each well of a 6-well plate, and culture overnight at 37 °C in a 5% CO2 incubator until the cells cover the bottom of the culture dish.

[0184] ② Gently discard the upper medium, and add 2 mL of serum-free medium for starvation treatment for 24 h.

[0185] ③ Use a 1 mL pipette tip to make a scratch. Do not pause or repeat the scribing during the process to make the scratch as flat as possible.

[0186] ④ Rinse the loose cells with 2 mL of PBS, repeat 3 times until the excess suspended cells are washed away, and add 2 mL of serum-free medium containing the drugs of each experimental group in step (2). Incubate in a 37 °C, 5% CO 2 incubator, and take pictures at 0 h after scratching and 24 h after scratching respectively.

[0187] (2) Experimental grouping and intervention methods:

[0188] ① Blank group (abbreviated as C+DMSO group): Add 1% DMSO of the medium volume in step (1)-④.

[0189] ② α-Tocopherol group (abbreviated as α-VE group): Add α-tocopherol with a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the medium volume) in step (1)-④.

[0190] ③ α-Tocopherol glucoside group (abbreviated as α-VE-G group): Add α-tocopherol glucoside with a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the medium volume) in step (1)-④.

[0191] ④ δ-Tocopherol group (abbreviated as δ-VE group): Add δ-tocopherol with a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the medium volume) in step (1)-④.

[0192] ⑤ δ-Tocopherol glucoside group (abbreviated as δ-VE-G group): Add δ-tocopherol glucoside with a final concentration of 30 μM (dissolved in DMSO, with a volume of 1% of the medium volume) in step (1)-④.

[0193] (3) Test results:

[0194] The healing ability of tocopherol and its derivatives on the scratch injury of HSF cells was tested 24 hours after scratching, and the results are as Figure 10 shown. The results showed that the scratch sizes of each experimental group were at the same level at 0 h, while after 24 h, the scratches in the α-tocopherol and α-tocopherol glucoside groups were alleviated to a certain extent, and the scratches in the δ-tocopherol and δ-tocopherol glucoside groups almost disappeared, indicating that δ-tocopherol and its glucoside have a very significant effect on promoting cell injury healing, and can effectively promote the repair of MACIR protein-related fibroblast injury, thus playing a role in protecting skin injury and promoting wound healing.

[0195] The applicant declares that the technical solution of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of the present invention.

[0196] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0197] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. In order to avoid unnecessary repetition, the present invention will not describe various possible combination methods separately.

Claims

1. Application of vitamin E compounds in the preparation of MACIR protein targeting agents; The vitamin E compound includes any one of tocopherol, tocopherol derivatives, tocotrienols or tocotrienol derivatives, or a combination of at least two thereof.

2. The use according to claim 1, characterized in that: The tocopherol includes any one of α-tocopherol, β-tocopherol, γ-tocopherol or δ-tocopherol or a combination of at least two thereof; Preferably, the tocotrienol includes any one of α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol, or a combination of at least two thereof.

3. The use according to claim 1 or 2, characterized in that: The tocopherol derivative is obtained by combining the hydroxyl group of tocopherol with the glycoside group; Preferably, the tocotrienol derivative is obtained by combining the hydroxyl group of tocotrienol with a glycoside group.

4. The use according to claim 3, characterized in that: The glycoside groups each independently include any one or a combination of at least two of glucoside, galactoside or mannoside, preferably glucoside; Preferably, the tocopherol derivative is delta-tocopheryl glucoside.

5. Application of vitamin E compounds as MACIR protein receptors; The vitamin E compound includes any one of tocopherol, tocopherol derivatives, tocotrienols or tocotrienol derivatives, or a combination of at least two thereof.

6. The use according to claim 5, characterized in that: The tocopherol includes any one of α-tocopherol, β-tocopherol, γ-tocopherol or δ-tocopherol or a combination of at least two thereof; Preferably, the tocotrienol includes any one of α-tocotrienol, β-tocotrienol, γ-tocotrienol or δ-tocotrienol, or a combination of at least two thereof.

7. The use according to claim 5 or 6, characterized in that: The tocopherol derivative is obtained by combining the hydroxyl group of tocopherol with the glycoside group; Preferably, the tocotrienol derivative is obtained by combining the hydroxyl group of tocotrienol with a glycoside group; Preferably, the glycoside groups each independently include any one or a combination of at least two of glucoside, galactoside or mannoside, preferably glucoside; Preferably, the tocopherol derivative is delta-tocopheryl glucoside.

8. Application of vitamin E compounds in the preparation of MACIR protein-associated immune cell function regulators; The vitamin E compound includes any one or a combination of at least two of tocopherol, tocopherol derivatives, tocotrienols or tocotrienol derivatives; The immune cells include macrophages and / or mast cells.

9. Use of vitamin E compounds in the preparation of MACIR protein-associated anti-inflammatory drugs and / or MACIR protein-associated anti-allergic drugs; The vitamin E compound includes any one of tocopherol, tocopherol derivatives, tocotrienols or tocotrienol derivatives, or a combination of at least two thereof.

10. Application of vitamin E compounds in the preparation of MACIR protein-associated skin cell damage repair agents; The vitamin E compound includes any one or a combination of at least two of tocopherol, tocopherol derivatives, tocotrienols or tocotrienol derivatives; The cells include epidermal cells and / or fibroblasts.

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