CN112516294B - Application of cerium oxide nano stabilizer in preparation of medicine for preventing allergic diseases and medicine composition for preventing allergic diseases - Google Patents
Application of cerium oxide nano stabilizer in preparation of medicine for preventing allergic diseases and medicine composition for preventing allergic diseases Download PDFInfo
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Abstract
The invention discloses an application of a cerium oxide nano stabilizer in preparation of a medicament for preventing allergic diseases. The cerium oxide nano stabilizer provided by the invention can imitate the activity of phosphatase, can continuously and efficiently catalyze the hydrolysis dephosphorylation of a phosphorylated substrate in a mast cell, namely can catalyze the dephosphorylation of phosphorylated protein kinase to inactivate the phosphorylated protein kinase, regulate phosphorylation cascade reaction related to degranulation, and inhibit degranulation from releasing inflammatory mediators, so that the occurrence of allergic diseases can be prevented for a long time. In addition, the cerium oxide nano stabilizer provided by the invention can also simulate the activity of antioxidant enzyme, eliminate overhigh ROS in cells in mast cells and relieve oxidative stress pressure. The cerium oxide nano stabilizer provides a strategy for the development of preparing a long-acting medicament for preventing allergic diseases.
Description
Technical Field
The invention relates to application of a nano biological material, in particular to application of a cerium oxide nano stabilizer with phosphatase activity in preventing allergic diseases.
Background
The global incidence of allergic diseases is increasing and has become an epidemic disease. More than 25% of people worldwide suffer from various allergic diseases including asthma, rhinitis, atopic dermatitis, food allergies. Unfortunately, more than 90% of patients with allergic diseases are not treated well, so that the allergic diseases cause great pain and socio-economic burden to the patients.
Allergic diseases are pathological immune reactions, a process involving a series of immune cells. Mast cells, which are the major effector cells of this pathological immune response, activate intracellular phosphorylation-related signaling cascades to cause a series of "domino effects" upon stimulation by antigens, including ROS production, degranulation to release allergic mediators, pathological reactions (e.g., bronchodilation, increased vascular permeability, vasodilation, inflammation, etc.), and ultimately allergic disease.
To date, antihistamines, steroids, and non-steroidal anti-inflammatory drugs are the main drugs against allergic diseases, however, these drugs can only relieve symptoms and cannot prevent allergic diseases. For example, chinese patent publication No. CN1711092 discloses compositions containing certain antiallergic agents in combination with steroids for the treatment of rhinitis and methods of treating rhinitis therewith. For example, chinese patent publication No. CN109806271A discloses a pharmaceutical composition for treating allergic dermatitis and a preparation thereof, wherein the pharmaceutical composition for treating allergic dermatitis comprises an iridoid compound and optionally an antihistamine compound as active ingredients, and can effectively relieve various symptoms such as pruritus of allergic dermatitis, and the iridoid is used in combination with the antihistamine, so that the pharmaceutical composition has an excellent effect of treating allergic dermatitis, is helpful for reducing the administration dosage of the antihistamine and reducing side effects, can further reduce the administration frequency of the medicament after being prepared into a sustained-release preparation, maintains a stable blood concentration, and can further reduce the side effects of the medicament, thereby providing an effective and safe pharmaceutical composition for treating allergic dermatitis.
In addition to these drugs, mast cell stabilizers inhibit mast cell degranulation, a potential drug for preventing the development of allergic diseases. However, most mast cell stabilizers, including gold standard cromolyn sodium (DSCG), have a short treatment window and are effective in stabilizing mast cells only when administered immediately after the mast cells are stimulated by an antigen. These mast cell stabilizers are not clinically effective, subject to a short therapeutic window. Although many nanoenzymes with enzymatic activity (nanomaterials with enzymatic activity) are proved to relieve the inflammatory mediators released by mast cell degranulation by scavenging ROS, no direct prevention of allergic diseases by directly regulating phosphorylation related signallings has been reported so far. Because the development of allergic disease is unpredictable, allergic patients often miss the optimal time for administration of mast cell stabilizers. Therefore, it is of great importance to develop a mast cell stabilizer with a long prevention time window for the prevention of allergic diseases.
Disclosure of Invention
The invention aims to provide an application of a cerium oxide nano stabilizer in preparation of a medicine for preventing allergic diseases, which can effectively regulate a signal cascade related to phosphorylation in mast cells by utilizing the sustainable phosphatase activity of the cerium oxide nano stabilizer, inhibit the degranulation of the mast cells caused by antigen stimulation to release inflammatory mediators, and thus play a role in preventing the allergic diseases for a long time.
The invention provides the following technical scheme:
an application of a cerium oxide nano stabilizer in preparing a medicament for preventing allergic diseases is disclosed, wherein the cerium oxide nano stabilizer is composed of cerium oxide nano enzyme with a phospholipid polyethylene glycol modified on the surface.
Further, the size of the cerium oxide nanoenzyme is 1-8nm, and the size of the cerium oxide nanoenzyme of which the surface is modified with phospholipid polyethylene glycol is 9-30nm.
Further, the allergic disease is a mast cell mediated allergic disease. The mast cell mediated allergic disease is atopic dermatitis, rhinitis, asthma, food allergy, allergic purpura, anaphylactic shock, urticaria, eczema or drug allergy.
Furthermore, the allergic disease is induced when the organism contacts with the allergen. The allergic diseases induced when the organism contacts with the allergen are atopic dermatitis, rhinitis, asthma, food allergy, allergic purpura, anaphylactic shock, urticaria, eczema or drug allergy.
The cerium oxide nano stabilizer provided by the invention has phosphatase activity, can dephosphorize a phosphorylated substrate, namely can hydrolyze and break phosphorus-oxygen bonds of the phosphorylated substrate. The cerium oxide nano stabilizer provided by the invention can inhibit the phosphorylation cascade reaction in mast cells stimulated by allergen, stabilize the mast cells and prevent degranulation from releasing allergic mediators.
The cerium oxide nano stabilizer is nano enzyme which has phosphatase activity and can hydrolyze phosphorylation substrate for dephosphorylation, namely, the phosphorus-oxygen bond of the phosphorylation substrate can be hydrolyzed and broken. After the cerium oxide nano stabilizer is taken up by mast cells into cytoplasm, the phosphatase activity can be continuously and stably exerted. When the mast cells are stimulated by the allergen, the cerium oxide nano stabilizer can regulate and control signal cascades related to phosphorylation, inhibit degranulation to release allergic mediums, and achieve the effect of preventing allergic diseases. Secondly, the cerium oxide nano stabilizer also has antioxidant activity, and can remove excessive ROS (Reactive oxygen species) generated in the signal cascade process, thereby relieving oxidative stress. In addition, the cerium oxide nano stabilizer is non-toxic and non-irritant, and has high biological safety.
The size of the cerium oxide nano enzyme is 1-8nm, and the size of the modified phospholipid polyethylene glycol is 9-30nm. The surface of the cerium oxide nanoenzyme has a large number of oxygen holes, and the surface part Ce is used for maintaining the surface charge balance 4+ Is reduced to Ce 3+ So that the surface of the prepared cerium oxide nano enzyme simultaneously contains Ce 3+ And Ce 4+ . In the catalytic process, ce 3+ Can be combined with water molecules, ce 4+ Can be combined with P = O bond, namely with phosphorylation substrate, and further SN is generated on the surface 2 Hydrolysis reaction, high efficiency of the phosphatase activity. In addition, due to Ce 3+ And Ce 4+ Can be mutually and rapidly converted, and the cerium oxide nano enzyme also shows antioxidant activity.
The cerium oxide nano stabilizer can be easily taken up by mast cells, so that phosphatase activity can be efficiently and continuously exerted in the mast cells, phosphorylation-related cascade reactions generated in the mast cells during antigen stimulation can be effectively regulated, and the degranulation of the mast cells can be effectively inhibited to release inflammatory mediators.
Specifically, the cerium oxide nano stabilizer can act on phosphorylation cascade reaction which causes degranulation to release inflammatory mediators, and hydrolysis dephosphorylation of phosphorylated protein kinase is catalyzed, so that the protein kinase loses activity, and phosphorylation-related cascade reaction is inhibited, so that degranulation of mast cells is inhibited from the source.
Meanwhile, the cerium oxide nano stabilizer can effectively eliminate ROS generated in phosphorylation cascade reaction and relieve oxidative stress in mast cells, thereby helping to inhibit degranulation of the mast cells.
The cerium oxide nano enzyme stabilizer is cerium oxide nano enzyme with phosphatase activity, and can stably and efficiently play the phosphatase activity, so that the cerium oxide nano enzyme stabilizer can play a stabilizing role in cells for a long time once being taken by mast cells, thereby having a longer treatment window and being capable of preventing allergic diseases for a long time.
The invention proves the application of the cerium oxide nano stabilizer in preventing allergic diseases through two aspects of animal experiments and in-vitro cell experiments, and provides a potential nano material for clinically preventing allergic diseases for a long term.
Compared with the prior art, the invention has the beneficial effects that:
(1) The phospholipid polyethylene glycol modified cerium oxide nanoenzyme has extremely small size, high specific surface area and rich oxygen cavities on the external surface (Ce exists around the phospholipid polyethylene glycol modified cerium oxide nanoenzyme simultaneously) 3+ And Ce 4+ ) A large number of reaction sites are provided to exert phosphatase activity.
(2) The cerium oxide nano stabilizer can remarkably inhibit mast cells from degranulating and releasing inflammatory mediators through regulating phosphorylation signal cascade reaction related to degranulation and releasing inflammatory mediators through phosphatase activity, thereby preventing allergic diseases.
(3) The cerium oxide nano stabilizer is essentially a cerium oxide nano stabilizer with phosphatase activity, has high efficiency and stability in the phosphatase activity and the like, and can continuously exert the phosphatase activity in mast cells, thereby realizing the prevention of allergic diseases with a long treatment time window.
(4) The cerium oxide nano stabilizer disclosed by the invention can be used for relieving oxidative stress pressure by eliminating ROS generated in a phosphorylation signal cascade process.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) image of cerium oxide nanoenzyme prepared in example 1;
FIG. 2 is a graph showing a dynamic light scattering particle size distribution of the phospholipid polyethylene glycol-modified cerium oxide nano-stabilizer prepared in example 1;
FIG. 3 is a characterization of phosphatase activity of phospholipid polyethylene glycol-modified cerium oxide nanostabilizer prepared in example 1;
FIG. 4 is a representation of the ROS scavenging performance of phospholipid polyethylene glycol modified cerium oxide nanostabilizers prepared in example 1;
FIG. 5 is a diagram of the results of quantitative analysis of phospholipid polyethylene glycol-modified cerium oxide nano-stabilizers inhibiting mast cell degranulation using beta-hexosidase as an indicator;
FIG. 6 shows the Western blot to detect the inhibition of phosphorylation signal cascade of inflammatory mediators released by degranulation of mast cells by cerium oxide nano-stabilizer;
fig. 7 is a graph showing the effect of cerium oxide nano-stabilizer on the prevention of allergic diseases, using a passive skin allergy test.
Detailed Description
The invention is further described below with reference to specific embodiments and the attached drawings.
Example 1: synthesis of cerium oxide nano-stabilizer with phosphatase activity
(1) Synthesis of cerium oxide nanoenzyme:
0.4g of cerium acetate hydrate and 3.2g of oleylamine were added to 15ml of xylene, and stirred at room temperature for 12 hours to form a complex. Then vacuumizing, introducing argon to make the reaction system be in an inert atmosphere, heating the reactant to 90 ℃ at the heating rate of 2 ℃ per minute, injecting 1ml of deionized water into the reaction system, keeping the temperature at 90 ℃ and aging for 3 hours, precipitating with acetone, and centrifuging to obtain the cerium oxide nanoenzyme.
The result is shown in figure 1, the morphology of the cerium oxide nanoenzyme is characterized by a transmission electron microscope, and the particle size is about 1-8 nm.
(2) Synthesis of phospholipid polyethylene glycol modified cerium oxide nanoenzyme:
1ml of cerium oxide nanoenzyme and 15mg of phospholipid polyethylene glycol were added to 5ml of chloroform and sonicated in a water bath sonication pan for 10min. And (3) carrying out rotary evaporation on the mixed solution at the temperature of 60 ℃ for 1 hour, and then adding 1ml of deionized water for hydration to obtain phospholipid polyethylene glycol modified cerium oxide nano enzyme, thereby obtaining the cerium oxide nano stabilizer for a biological system.
The results are shown in FIG. 2, which shows the morphology of the cerium oxide nano stabilizer, and the particle size is about 9-30nm.
The results are shown in fig. 3, which characterizes the phosphatase activity of the prepared cerium oxide nano stabilizer, and the cerium oxide nano stabilizer can efficiently hydrolyze phosphorylated substrates.
The results are shown in fig. 4, and the ability of the prepared cerium oxide nano stabilizer to scavenge ROS is characterized, and the cerium oxide nano stabilizer can scavenge hydrogen peroxide, superoxide and hydroxyl free radicals.
Example 2: stabilizing effect of cerium oxide nano stabilizer on allergen-stimulated mast cells
Preparation of the medicament: the phospholipid polyethylene glycol-modified cerium oxide nano stabilizer prepared in example 1 was dispersed in an aqueous solution to prepare an aqueous solution of a cerium oxide nano stabilizer.
Establishing a cell model: mast cells were incubated with anti-DNP antibody (500 ng/mL) for 12h to give primed mast cells.
Group setting:
a. control group: normal cells were given fresh medium.
b. Model group: DNP-HAS is added into sensitized mast cells to make the concentration of the DNP-HAS be 100ng/mL, and degranulation of the mast cells is stimulated to release inflammatory mediators.
c. Treatment groups: adding cerium oxide nano stabilizer into sensitized mast cells to enable the final concentration to be 20 mu g/mL, respectively, pre-incubating for 0.5,1,2,4,6 hours, and then adding DNP-HAS to enable the concentration to be 100ng/mL to stimulate degranulation of the mast cells to release inflammatory mediators.
After 1 hour of DNP-HAS stimulation, the supernatant was collected, added to an equal volume of substrate solution (prepared by dissolving 1.29mM p-nitrophenyl-N-acetyl- β -D-glucosaminide in 0.1M citric acid solution) and reacted at 37 ℃ for 1.5 hours, followed by addition of 75. Mu.L glycine buffer (0.2M, pH 10.7) to interrupt the reaction and detection of absorbance using a microplate reader at 405nm wavelength.
The result is shown in fig. 5, the cerium oxide nano stabilizer can stabilize the allergen-stimulated mast cells for a long time and inhibit the degranulation of the mast cells to release inflammatory mediators.
Example 3: the cerium oxide nano stabilizer has cascade regulation effect on phosphorylation signals in mast cells.
Preparation of the medicament: dispersing the phospholipid polyethylene glycol modified cerium oxide nano stabilizer prepared in the example 1 into an aqueous solution to prepare a cerium oxide nano stabilizer aqueous solution; and dispersing the DSCG in the aqueous solution to obtain the DSCG aqueous solution.
Establishing a cell model: mast cells were incubated with anti-DNP antibody (500 ng/mL) for 12h to give primed mast cells.
Group setting:
a. control group: normal cells were given fresh medium.
b. Model group: DNP-HAS was added to the sensitized mast cells to a concentration of 100ng/mL, and the mast cells were stimulated.
Dscg group: the mast cells were stimulated by preincubation with DSCG in water to a final concentration of 400 μ g/mL followed by DNP-HAS to a concentration of 100 ng/mL.
d. Treatment groups: the sensitized mast cells were incubated in advance with cerium oxide nanostabilizer to final concentrations of 5, 10, 20. Mu.g/mL, followed by DNP-HAS to 100ng/mL to stimulate the mast cells.
After 1 hour of DNP-HAS stimulation, the cells were lysed and cell lysates were collected for electrophoretic analysis. The protein was quantified using a BCA protein assay kit and separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The membrane was transferred to a polyvinylidene fluoride membrane, blocked with 5% skim milk, incubated overnight at 4 ℃ while adding antibodies against p-Syk, p-PLC γ, p-LAT, gab 2. The membrane was then washed 3 times and incubated with secondary antibody for 1h imaging at room temperature. The intensity of the bands was determined by density analysis.
The results are shown in fig. 6, the cerium oxide nano-stabilizer can effectively regulate and control phosphorylation cascade reaction related to the release of inflammation mediators by degranulation.
Example 4: effect of cerium oxide nano-stabilizer on prevention of skin passive allergy
Preparation of the medicament: the phospholipid polyethylene glycol-modified cerium oxide nano stabilizer prepared in example 1 was dispersed in an aqueous solution to prepare an aqueous solution of a cerium oxide nano stabilizer.
Establishing an animal model: a7-8 week female white mouse was taken, dorsal hair was removed, and the mouse was sensitized on the back by intradermal injection of 20. Mu.L of an anti-DNP antibody (6. Mu.g/mL).
Group setting:
a. control group: physiological saline.
b. Model group: the tail vein was injected with 0.2mL of DNP-HAS solution (1 mg/mL) containing 0.5% Evans blue dye.
Dscg group: mice were injected intradermally with 20. Mu.L of DSCG in water (100. Mu.g/mL) at the dorsal sensitization site, and 6 hours later, followed by tail vein injection of 0.2mL of DNP-HAS solution (1 mg/mL) containing 0.5% Evans blue dye.
d. Treatment groups: the mice were injected intradermally with 20. Mu.L of an aqueous solution of cerium oxide nano-stabilizer (25, 50, 75. Mu.g/mL) at the dorsal sensitization site, and after 6 hours, were subsequently injected in the tail vein with 0.2mL of a DNP-HAS solution (1 mg/mL) containing 0.5% Evans blue dye.
Half an hour later, the mice were sacrificed, the back skin was collected, evans blue dye was extracted with formamide at 65 ℃ for 12 hours, and quantitative analysis was performed using an enzyme-labeling instrument.
The results are shown in fig. 7, and the cerium oxide nano stabilizer can effectively prevent the occurrence of allergic diseases.
Claims (5)
1. An application of a cerium oxide nano stabilizer in preparing a medicament for preventing allergic diseases, wherein the cerium oxide nano stabilizer is composed of cerium oxide nano enzyme with phospholipid polyethylene glycol modified on the surface;
the size of the cerium oxide nanoenzyme is 1-8nm, and the size of the cerium oxide nanoenzyme with phospholipid polyethylene glycol modified on the surface is 9-30nm;
the allergic disease is a mast cell mediated allergic disease;
the preparation method of the cerium oxide nano stabilizer comprises the following steps:
adding 0.4g of cerium acetate hydrate and 3.2g of oleylamine into 15ml of dimethylbenzene, stirring at room temperature for 12 hours to form a complex, vacuumizing, introducing argon to enable a reaction system to be in an inert atmosphere, heating the reactant to 90 ℃ at a heating rate of 2 ℃ per minute, injecting 1ml of deionized water into the reaction system, keeping the temperature of 90 ℃ for aging for 3 hours, precipitating with acetone, and centrifuging to obtain cerium oxide nanoenzyme;
adding 1ml of cerium oxide nanoenzyme and 15mg of phospholipid polyethylene glycol into 5ml of chloroform, performing ultrasound treatment in a water bath ultrasonic pot for 10min, performing rotary evaporation on the mixed solution at 60 ℃ for 1 hour, and adding 1ml of deionized water for hydration to obtain the phospholipid polyethylene glycol modified cerium oxide nanoenzyme serving as a cerium oxide nano stabilizer.
2. The use of the cerium oxide nanostabilizer of claim 1 for the preparation of a medicament for preventing allergic diseases, wherein the mast cell mediated allergic diseases are atopic dermatitis, rhinitis, asthma, food allergy, allergic purpura, anaphylactic shock, urticaria, eczema or drug allergy.
3. The use of the cerium oxide nano-stabilizer according to claim 1 in the preparation of a medicament for preventing allergic diseases, wherein the allergic diseases are induced when an organism contacts an allergen.
4. The use of the cerium oxide nano-stabilizer according to claim 3, in the preparation of a medicament for preventing allergic diseases, wherein the allergic diseases induced when the body contacts an allergen are atopic dermatitis, rhinitis, asthma, food allergy, allergic purpura, anaphylactic shock, urticaria, eczema or drug allergy.
5. The pharmaceutical composition for preventing allergic diseases is characterized by comprising a cerium oxide nano stabilizer, wherein the cerium oxide nano stabilizer is composed of cerium oxide nano enzyme with phospholipid polyethylene glycol modified on the surface; the size of the cerium oxide nano enzyme with the surface modified phospholipid polyethylene glycol is 9-30nm.
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