Application of licorice protein drug-loaded nanoparticles in the preparation of drugs for the treatment of psoriasis

Abstract

The present invention relates to the application of glycyrrhizin drug-loaded nanoparticles for preparing drugs for treating psoriasis, and belongs to the technical field of nano traditional Chinese medicine. The multi-component drug includes baicalin, paeoniflorin, and liquiritin. Baicalin can regulate NF-κB and JAK / STAT signaling pathways, inhibit skin inflammatory reactions, and improve atopic dermatitis lesions; it can also relieve skin inflammation in psoriasis mice by inhibiting the IL-17 / IL-23 axis. Peony glycosides can regulate a variety of signaling pathways, including the GPCR pathway and the JAK2 / STAT3 pathway, etc., can significantly reduce the transcription level of STAT3, inhibit the secretion of cytokines by Th17 cells, and thus alleviate the symptoms of psoriasis. As one of the main active ingredients of licorice, liquiritin has a good therapeutic effect on acute lung injury, cancer, and cell damage caused by inflammation. The present invention utilizes glycyrrhizin nanoparticles to load the above-mentioned drugs, and the corresponding drug-loaded nanoparticles can be better absorbed by intestinal cells and inflammatory cells, and exert a good therapeutic effect on psoriasis.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-traditional Chinese medicine, and more specifically, relates to the application of licorice protein-loaded nanoparticles for preparing drugs for treating psoriasis, and in particular to a traditional Chinese medicine protein nanoformulation loaded with multi-component drugs. The multi-component traditional Chinese medicine protein nanoparticles can be particularly used in the treatment of psoriasis. Background Art

[0002] Although medicine has achieved rapid development, psoriasis still cannot be completely cured. Psoriasis, commonly known as psoriasis, is an autoimmune disease that affects the health of about 125 million people in the world. The main characteristics of psoriasis are thickening of the skin, erythema, and white scales. Psoriasis is generally considered to be caused by a cascade reaction of immune cells (such as dendritic cells, T cells and macrophages), which are abnormally activated and release inflammatory factors, and then attack skin cells including keratinocytes. Psoriasis patients are at a higher risk of heart disease, stroke, hypertension and diabetes. Psoriasis can significantly reduce the quality of life of patients and is even more serious than cancer. Currently, effective therapeutic drugs mainly include biologics, transdermal hormone administration and traditional oral drugs (such as methotrexate). These drugs are difficult to meet the requirements of economy, safety and good medication compliance at the same time, so it is of practical significance to develop new therapeutic drug strategies to overcome the above shortcomings.

[0003] As one of the main active ingredients of licorice, liquiritin has a good therapeutic effect on acute lung injury, cancer and cell damage caused by inflammation. The detoxification, anti-inflammatory, anti-ulcer, antiviral and anti-cancer effects of licorice have been confirmed. For glycyrrhizin (GP), studies have shown that it can inhibit the proliferation of colon cancer cells and induce apoptosis, but the role of GP in inflammation has not been reported. In glycyrrhizin-loaded nanoparticles, glycyrrhizin can be used as a safe drug carrier. Baicalin can regulate NF-κB and JAK / STAT signaling pathways, inhibit skin inflammation, and improve atopic dermatitis lesions; in addition, baicalin can alleviate skin inflammation in psoriasis mice by inhibiting the IL-17 / IL-23 axis, among which the activation of JAK / STAT is an important inflammatory mechanism leading to autoimmune diseases, and inflammation is related to the IL-6 / STAT3 pathway in DC cells and Th17 cells. Paeoniflorin can regulate multiple signaling pathways, including the GPCR pathway and the JAK2 / STAT3 pathway. Studies have shown that paeoniflorin can significantly reduce the transcription and protein levels of STAT3, inhibit the secretion of cytokines by Th17 cells, and thus relieve the symptoms of psoriasis. All of the above substances have the potential to relieve psoriasis and are economical and applicable, but their poor water solubility, difficulty in drug formulation, low bioavailability, and large drug usage limit their clinical application.

[0004] In recent years, nanoformulations have been used in clinical practice. For example, the use of liposomes to encapsulate doxorubicin hydrochloride can increase the accumulation of drugs in lesions and prolong the half-life of drugs in the blood circulation. In addition, nanotechnology can be used to improve some drugs with poor drugability, such as enhancing drug solubility and improving drug stability. The use of nanotechnology to transform the active ingredients of traditional Chinese medicine into drugs can achieve the purpose of improving the treatment effect of diseases and reducing toxic side effects. Therefore, the use of nanopharmaceutical technology to treat the above drugs is expected to improve their bioavailability and obtain simple and easy-to-obtain nanoformulations. Licorice protein can promote the assembly of nanostructures and encapsulate active ingredients at the same time. The denatured protein has a certain inflammatory targeting effect. Therefore, the use of licorice protein denaturation to form nanoparticles is expected to better encapsulate the above drugs, achieve the co-delivery of the three, and play a good role in alleviating psoriasis. Summary of the invention

[0005] In view of the above defects or improvement needs of the prior art, the purpose of the present invention is to provide a kind of Chinese medicine drug assembly nanoparticles for the treatment of psoriasis, using glycyrrhizin to load baicalin, paeoniflorin and liquiritin with anti-inflammatory effects, and the corresponding glycyrrhizin nanoparticles can efficiently encapsulate the above hydrophobic Chinese medicine drugs, promote cell uptake, and exert good anti-inflammatory effects, especially for the treatment of psoriasis. The glycyrrhizin drug-loaded nanoparticles in the present invention have good biocompatibility, and solve the problems of poor solubility, poor drugability, and poor stability of some Chinese medicine drugs, realize the co-delivery of three Chinese medicine components, and show significant therapeutic effects on psoriasis. The drug combination of the present invention is novel and effective, the preparation method is simple and convenient, and has good clinical transformation and application prospects.

[0006] According to a first aspect of the present invention, there is provided a use of glycyrrhizin-loaded nanoparticles for preparing a drug for treating psoriasis, wherein the glycyrrhizin-loaded nanoparticles use glycyrrhizin as a carrier, and the carrier at least encapsulates baicalin, paeoniflorin and glycyrrhizin.

[0007] Preferably, the licorice protein drug-loaded nanoparticles are used to downregulate the expression level of inflammatory factors, thereby inhibiting skin inflammatory response.

[0008] Preferably, the inflammatory factors are TNF-α, IL-17 and IL-6.

[0009] According to another aspect of the present invention, a method for preparing glycyrrhizin drug-loaded nanoparticles is provided, comprising the following steps:

[0010] (1) dissolving glycyrrhizin, then adding a mixed solution of baicalin, paeoniflorin and liquiritin, mixing thoroughly, and then removing the solvent by rotary evaporation;

[0011] (2) adjusting the pH of the mixed solution obtained in step (1) to acidic to denature the glycyrrhizin, and encapsulate baicalin, paeoniflorin and liquiritin, and discarding the supernatant after centrifugation to obtain glycyrrhizin drug-loaded nanoparticles.

[0012] Preferably, in the mixed solution of baicalin, paeoniflorin and liquiritin, the mass concentration of baicalin is 1-6 mg / mL, the mass concentration of paeoniflorin is 1-4 mg / mL, and the mass concentration of liquiritin is 1-4 mg / mL.

[0013] Preferably, in step (2), the pH of the mixed solution obtained in step (1) is adjusted to 3-5.

[0014] Preferably, in step (2), the centrifugal speed is 10000-14000 rpm, and the time is 30-60 min.

[0015] According to another aspect of the present invention, a glycyrrhizin drug-loaded nanoparticle is provided. The glycyrrhizin drug-loaded nanoparticle uses glycyrrhizin as a carrier, and the carrier at least encapsulates baicalin, paeoniflorin and glycyrrhizin.

[0016] Preferably, the particle size of the nanoparticles is 250-420 nm.

[0017] Preferably, the mass ratio of baicalin, paeoniflorin and liquiritin in the nanoparticles is (1-6):(1-4):(1-4).

[0018] In general, the above technical solution conceived by the present invention has the following technical advantages compared with the prior art:

[0019] (1) The multi-component drug in the present invention includes baicalin, paeoniflorin, and liquiritin. Baicalin can regulate NF-κB and JAK / STAT signaling pathways, inhibit skin inflammatory reactions, and improve atopic dermatitis lesions; it can also relieve skin inflammation in psoriasis mice by inhibiting the IL-17 / IL-23 axis. Paeoniflorin can regulate multiple signaling pathways, including the GPCR pathway and the JAK2 / STAT3 pathway, etc., can significantly reduce the transcription level of STAT3, inhibit the secretion of cytokines by Th17 cells, and thus relieve the symptoms of psoriasis. Liquiritin, as one of the main active ingredients of licorice, has a good therapeutic effect on acute lung injury, cancer, and cell damage caused by inflammation. The present invention uses licorice protein nanoparticles to load the above-mentioned drugs, and the corresponding drug-loaded nanoparticles can be better absorbed by intestinal cells and inflammatory cells, and exert a good therapeutic effect on psoriasis.

[0020] (2) The drug combination strategy provided by the present invention is a novel and effective form of psoriasis treatment. Co-encapsulation of drugs through nanotechnology can achieve synergistic and effective relief of psoriasis.

[0021] (3) The glycyrrhizin drug-loaded nanoparticles of the present invention have good stability; solve the problem of poor drug solubility and difficulty in preparing preparations; have efficient drug loading capacity for drugs of different physical and chemical properties; and have a simple preparation process, which is easy for clinical transformation and expanded production. The nanoparticle preparation method of the present invention is not limited to the combination of glycyrrhizin and the above-mentioned drugs, and other hydrophobic and hydrophilic drugs are also suitable for this method, but the drugs described in this invention are preferred.

[0022] (4) The glycyrrhizin drug-loaded nanoparticles of the present invention have good cellular uptake ability and can also reduce the dosage of administration, thereby avoiding systemic toxic and side effects caused by high concentrations of free drugs.

[0023] (5) After oral administration of the glycyrrhizin-loaded nanoparticles of the present invention, the psoriasis mouse status, pathological changes and inflammatory factor levels were significantly improved, indicating that the glycyrrhizin-loaded nanoparticles have good biological activity, close to or even better than the positive control drug methotrexate, and have a good therapeutic effect on psoriasis.

[0024] (6) The licorice protein drug-loaded nanoparticles of the present invention can effectively encapsulate three hydrophobic substances, namely baicalin, paeoniflorin and liquiritin, improve drug solubility, and promote cellular uptake, thereby exerting a good alleviating effect on psoriasis and reducing the levels of inflammatory factors in the skin and blood of psoriasis mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the polyacrylamide gel electrophoresis characterization of extracted licorice protein.

[0026] Figure 2 The particle size, electron microscopy and stability of the licorice protein drug-loaded nanoparticles of the present invention are characterized; wherein, Figure 2 (a) is the result. Figure 2 (b) is the electron microscope morphology characterization result. Figure 2 (c) in the figure is the stability monitoring result.

[0027] Figure 3 The drug loading characterization of the glycyrrhizin protein nanoparticles of the present invention is shown in FIG. Figure 3 (a) is the HPLC chromatogram of licorice protein drug-loaded nanoparticles and standard substances. Figure 3 (b) in the figure is the characterization of drug loading of different substances.

[0028] Figure 4 The results of in vitro cellular uptake of the licorice protein drug-loaded nanoparticles of the present invention include in vitro uptake models of mouse colon cancer cells CT26 and mouse mononuclear macrophages Raw264.7.

[0029] Figure 5This is a study on the efficacy of the licorice protein drug-loaded nanoparticles of the present invention at the level of psoriasis animals; it includes a drug administration timeline and a diagram of changes in the back skin of psoriasis mice.

[0030] Figure 6 The results of the Psoriasis Area and Severity Index (PASI) of mice after treatment with the licorice protein drug-loaded nanoparticles of the present invention include erythema score, scaling score, skin thickness score and total score.

[0031] Figure 7 The invention discloses the effects of the licorice protein drug-loaded nanoparticles on various physiological indices of mice, including changes in the thickness of the back skin, the thickness of the ears, the weight and the spleen weight index of the mice.

[0032] Figure 8 The histological changes of mouse skin after treatment with the licorice protein drug-loaded nanoparticles of the present invention.

[0033] Fig. 9 This is an immunofluorescence section analysis of mouse skin after treatment with the licorice protein drug-loaded nanoparticles of the present invention, including two indicators: IL-17 and Ki-67.

[0034] Fig.10 The figure shows the changes of inflammatory factors IL-6 and TNF-α in the blood of mice after treatment with the licorice protein drug-loaded nanoparticles of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0036] The present invention discloses a plant protein nanoparticle for encapsulating a multi-component hydrophobic drug, comprising a drug combination and plant protein nanoparticles loaded with the drug combination, wherein the drug combination comprises three hydrophobic traditional Chinese medicine active ingredients, and the drug combination nanoparticles can relieve psoriasis symptoms and lower the level of inflammatory factors.

[0037] The nanoparticles of the Chinese medicine composition of the present invention have three hydrophobic Chinese medicine active ingredients of baicalin, paeoniflorin and liquiritin; and the plant protein is licorice protein (molecular weight of 28-31 kDa).

[0038] The method for preparing glycyrrhizin in the present invention comprises the following steps:

[0039] (1) Mix the licorice slices with ultrapure water at a ratio of 1:5 (w / v), sonicate in a water bath for 30 min, and then stir in a water bath at 60°C for 30 min;

[0040] (2) After the sample obtained in step (1) is cooled to room temperature, the residue is filtered out using a double-layer filter cloth; the filtrate is centrifuged at 4000 rpm for 15 min, 1.5 times the volume of acetone is added to the supernatant, the mixture is shaken well, and the mixture is allowed to stand at -20°C for 30 min;

[0041] (3) The sample in step 2 was centrifuged at 4000 rpm for 30 min at 4°C, the supernatant was discarded, the acetone was evaporated, and the sample was freeze-dried to obtain licorice crude protein (GP) for subsequent experiments.

[0042] The method for preparing the pharmaceutical composition nanoparticles for treating psoriasis of the present invention comprises the following steps:

[0043] (1) Dissolve licorice protein, take 2 mL of the protein solution and add 350 μL of a mixed methanol solution of baicalin, paeoniflorin and liquiritin;

[0044] (2) Ultrasonication was performed 10 times at 60% power using an ultrasonic crusher, and methanol was removed by rotary evaporation;

[0045] (3) After rotary evaporation, the sample was adjusted to pH 3-5 while being ultrasonicated in a water bath, and then centrifuged at 13000 rpm for 30 min to discard the supernatant and collect the nanoparticles to obtain licorice protein drug-loaded nanoparticles.

[0046] Preferably, the mass concentration of baicalin is 1-6 mg / mL, preferably 4 mg / mL, the mass concentration of paeoniflorin is 1-4 mg / mL, preferably 2 mg / mL, and the mass concentration of liquiritin is 1-4 mg / mL, preferably 2 mg / mL.

[0047] Example 1

[0048] Extraction of licorice protein: Licorice protein was extracted from licorice pieces by acetone precipitation. In short, licorice pieces were mixed with ultrapure water at a ratio of 1:5 (w / v), ultrasonicated in a water bath for 30 minutes, and then stirred in a water bath at 60°C for 30 minutes. After the sample was dried to room temperature, the residue was filtered out with a double-layer filter cloth; the filtrate was centrifuged at 4000rpm for 15min, 1.5 times the volume of acetone was added to the supernatant and shaken, and then allowed to stand at -20°C for 30min. Finally, the sample was centrifuged at 4000rpm for 30min at 4°C, the supernatant was discarded, the acetone was evaporated, and the licorice crude protein (GP) was obtained by freeze-drying.

[0049] SDS-PAGE characterization of licorice protein: After obtaining the above licorice protein, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to characterize it. The specific operation steps are as follows:

[0050] ① Weigh an appropriate amount of lyophilized licorice protein powder, dissolve it in ultrapure water, mix it evenly with protein loading buffer (Loading buffer, 5×) at a volume ratio of 1:4, heat it at 95℃ for 10min to denature the protein, and store it at -20℃;

[0051] ② Preparation of 10% separation gel: ultrapure water, 30% acrylamide, 1.5M Tris-HCl (pH8.8), 10% SDS, and 10% ammonium persulfate (AP) are prepared in a ratio of 40:33:25:1:1 to form a solution with a total volume of 10 mL. After adding 5 μL of TEMED, quickly pour it into the assembled glass plate, smooth the gel surface with isopropanol, and let it stand to solidify to obtain the separation gel;

[0052] ③ Preparation of 5% concentrated gel: ultrapure water, 30% acrylamide, 1.0M Tris-HCl (pH6.8), 10% SDS, and 10% ammonium persulfate (AP) were mixed in a ratio of 40:5:5:4:3, 8μL TEMED was added and then quickly added to the upper layer of the solidified separation gel, and a 1.5mm sample comb was inserted. After solidification, it was the concentrated gel;

[0053] ④ Preparation of electrophoresis solution: Dissolve 18.8g of glycine, 3.02g of Tris-Base, and 1g of SDS in 1L of UP water to obtain electrophoresis solution;

[0054] ⑤ Preparation of staining solution: Take 50mL, 10mL, and 40mL of methanol, acetic acid (glacial acetic acid), and ultrapure water respectively, mix well, and then add 0.05g of Coomassie Brilliant Blue R-250;

[0055] ⑥ Preparation of decolorizing solution: Mix methanol, acetic acid (glacial acetic acid) and ultrapure water in a volume ratio of 2:1:7 to obtain the decolorizing solution;

[0056] ⑦ Electrophoresis: Place the prepared gel plate into the electrophoresis tank and add an appropriate amount of electrophoresis solution; remove the comb and add about 30 μg of protein sample, connect the electrodes in a standardized manner, and separate at 80V for about 30 minutes. After the sample enters the separation gel, maintain a voltage of 120V until the Maker protein bands are completely separated;

[0057] ⑧ Staining: After protein separation, remove the gel from the glass plate, discard the concentrated gel part, soak it in staining solution, place it on a shaker for staining at room temperature for 4 hours, and recover the staining solution after staining;

[0058] ⑨ Destaining: Elute the stained gel with destaining solution until the protein bands are clearly visible.

[0059] The results are as follows Figure 1 , the molecular weight of licorice protein is 28~31kDa,

[0060] Example 2

[0061] The present invention further assembles and loads the glycyrrhizin in Example 1. The glycyrrhizin was dissolved in ultrapure water (2 mg / mL), and 2 mL of the protein solution was added to a mixed solution of about 350 μL of baicalin (4 mg / mL), paeoniflorin (2 mg / mL) and liquiritin (2 mg / mL) dissolved in methanol. After ultrasonication with an ultrasonic crusher at 60% power for 10 times, the methanol was removed by rotary evaporation; the remaining sample was adjusted to pH = 3 to 5 while ultrasonicating in a water bath, and then centrifuged at 13000 rpm for 30 min to discard the supernatant, and the nanoparticles were collected and named glycyrrhizin drug-loaded nanoparticles (GP-NPs).

[0062] The GP-NPs sample obtained by the above assembly was stored in a 4°C refrigerator after the particle size was measured, and its particle size was measured for seven consecutive days; the newly prepared GP-NPs were diluted with ultrapure water to 100-200 kcps, and the dispersion (10 μL) was slowly dripped onto the electron microscope copper grid. After standing for 30 minutes, the residual liquid was sucked off, and the copper grid was evaporated to dry. The morphological characteristics of GP-NPs were observed under a transmission electron microscope (TEM, JEM-1230) at 100 kV. The results are as follows Figure 2 As shown. Figure 2 As can be seen from (a), the particle size is concentrated in the range of 300-400 nm. Figure 2 The electron microscope image in (b) shows that the GP-NPs are relatively regular and spherical and relatively uniform. The particle size was measured for 7 consecutive days. Figure 2 In (c), the particle sizes fluctuate between 400-500 nm with a small variation range, indicating that the assembled GP-NPs have good physical stability.

[0063] Example 3

[0064] Preparation of reference standard curve: Weigh appropriate amount of baicalin, paeoniflorin and liquiritin standards, dissolve in chromatographic grade methanol, prepare 0.1, 0.5, 1, 2.5, 5, 10, 20, 50, 100, 200, 500, 1000 μg / mL standard solutions, perform HPLC analysis under the following liquid phase conditions, and draw a concentration-peak area standard curve. The mobile phase is acetonitrile (A) and 0.05% phosphoric acid (B) solution, and the HPLC analysis is performed by gradient washing. The mobile phase examples are shown in Table 1-1:

[0065] Table 1-1 Liquid chromatography conditions (time-mobile phase ratio correspondence table)

[0066]

[0067] The detection wavelength was 230 nm, the column temperature was 30 °C, the flow rate was 1 mL / min, and the injection volume was 10 μL.

[0068] The GP-NPs sample obtained by the above assembly was dissolved in 500 μL of chromatographic grade methanol, and after ultrasonication for 30 min, centrifuged at 15000 g for 10 min. The supernatant was taken for fingerprint analysis and quantification of the main components in the NPs. The results are as follows: Figure 3 As shown in the figure, baicalin, paeoniflorin and liquiritin were all entrapped in the glycyrrhizin nanoparticles. The peak area normalization method was used to determine the contents of baicalin, paeoniflorin and liquiritin in glycyrrhizin nanoparticles, and the relative encapsulation rates of baicalin, paeoniflorin and liquiritin were calculated to be 31.70%, 22.56% and 23.80%, respectively.

[0069] Example 4

[0070] In order to investigate the cellular uptake of the licorice protein nanoparticles, nanoparticles (C6@GP-NPs) labeled with the fluorescent dye Coumarin 6 (C6) were first prepared. Then, a cell uptake model in vitro was established: the mouse mononuclear macrophage leukemia cell line Raw 264.7 and the mouse colon cancer cell line CT26 were both from the cell bank of the Chinese Academy of Sciences. Raw264.7 or CT26 was cultured at 1×10 5 Each dish of cells was inoculated in a confocal microscope culture dish overnight. Free coumarin-6 (C6) and licorice protein assembled nanoparticles (C6@GP-NPs) diluted in DMEM were added, and the coumarin concentration was 5μg / mL, and incubated with the cells for 4h. The cells were then fixed in 4% paraformaldehyde for 10min (300μL / dish) and rinsed twice with cold PBS. 300μL DAPI (10μg / mL) was added to each dish of cells for staining for 8 minutes, washed 3 times with PBS, and the cell uptake was observed under a confocal laser scanning microscope. The results are shown in Figure 4 As shown, the green fluorescence intensity of the glycyrrhizin protein nanoparticle group in Raw 264.7 and CT26 cells was significantly stronger than that of the free coumarin group, indicating that glycyrrhizin protein-loaded nanoparticles can be effectively absorbed by mouse intestinal epithelial-like cells and macrophages.

[0071] Example 5

[0072] The in vivo therapeutic effect of GP-NPs on psoriasis was observed in a psoriasis mouse model.

[0073] The psoriasis mouse modeling and drug administration methods are as follows: the mice are divided into a normal group, a model group, a methotrexate (MTX) group, and a licorice protein drug-loaded nanoparticle group. The BALB / c mice were shaved and depilated on the back (area of ​​approximately 3cm×4cm), and 5% imiquimod cream was applied topically daily starting from the second day after depilation. Except for the normal control group mice, which were given an equal amount of vaseline ointment, the mice in other groups were all given imiquimod cream (62.5 mg) on ​​the skin and ears after depilation, and the modeling was continued for 8 consecutive days. The mice were gavage-administered on the second day after the start of the modeling. The specific implementation plan is shown in Figure 5 The MTX group was given a dose of 0.62 mg / kg, the other treatment groups were given 18 mg of the corresponding drug, and the normal group and model group were given ultrapure water, with an intragastric volume of 200 μL. The back skin of the mice was photographed every day, the thickness of the skin and ears was measured, and the weight changes were recorded.

[0074] Skin changes such as Figure 5 As shown in the figure, the skin of mice in the model group gradually turned red and dark, accompanied by the production of scales. By the 7th day, the scales covered about 80% of the exposed skin. In the drug treatment group, only a small amount of white scales appeared, and the skin was red and slightly wrinkled. In general, the MTX and licorice protein drug-loaded nanoparticles group had a significant improvement in the psoriasis-like skin on the back of mice after 7 consecutive days of treatment.

[0075] The Psoriasis Area and Severity Index (PASI) score is a tool for assessing the severity of psoriasis. The scoring indicators include skin thickness, erythema and desquamation. The score ranges from 0 to 4 points. The subdivision criteria are as follows: 0 points, no symptoms; 1 point, mild symptoms; 2 points, moderate symptoms; 3 points, severe symptoms; 4 points, very severe. The total PASI score is the sum of the skin thickness, erythema and desquamation scores. The results are as follows: Figure 6 As shown in the four scoring graphs, the model group always showed a rapid growth trend. The trends shown by mice treated with MTX and licorice protein-loaded nanoparticles were consistent. From the third day, the treatment group and the model group gradually showed differences; by the seventh day, the erythema score of the GP-NPs group was close to that of the normal group, and its scaling score and PASI total score were lower than those of the MTX group.

[0076] The changes of various indicators of mice are as follows Figure 7 As shown. The growth rate of the back skin thickness and ear thickness of the model group mice was significantly higher than that of the other groups, and the change value of the back skin reached 0.25mm on the 7th day. The weight loss of mice treated with licorice protein-loaded nanoparticles slowed down, indicating that it has a positive effect on maintaining the physiological function of mice, and the spleen weight index showed an overall downward trend.

[0077] Example 6

[0078] On the 8th day of the administration timeline, the skin was fixed with 4% paraformaldehyde and then stained with hematoxylin-eosin. H&E sectioning services were provided by Bolf Biotech Co., Ltd. The skin of mice in the normal group had intact hair and other skin appendages, while the skin of mice in the imiquimod-induced model group was significantly thickened, accompanied by microabscesses and elongated spinous processes. Figure 8 It can be seen that after treatment with different drugs, the above symptoms were significantly improved. The thickness of the epidermis in the two treatment groups was significantly thinner than that in the model group, no microabscesses appeared, and some hair follicles were retained in the licorice protein-loaded nanoparticle group. These data also show that licorice protein-loaded nanoparticles have a good therapeutic effect on psoriasis from the tissue level.

[0079] Example 7

[0080] Immunofluorescence staining analysis was performed on the back skin sections of mice to determine the level of inflammatory factor IL-17 in the skin lesions. Fig. 9 It can be seen that a large amount of inflammatory factor IL-17 infiltrated the lesions on the back of mice in the model group. The fluorescence brightness of IL-17 in the MTX group and GP-NPs group was significantly reduced, indicating that the secretion of IL-17 in these two groups of mice was less than that in the model group. Ki-67, as an antigen that marks cells in the proliferation cycle, was infiltrated in large quantities in the basal layer of the lesions on the back of mice in the model group, while the Ki-67 signal intensity in the epidermis of the drug treatment group was lower than that in the model group, indicating that the model group mice had epidermal hyperproliferation. In the normal group, MTX group and GP-NPs group, the hair follicles of the Ki-67 immunofluorescence sections of the mouse skin all had strong red fluorescence, indicating that the epithelial cells of the hair follicle tissue retained normal division and proliferation functions.

[0081] The ELISA method was used to determine the levels of TNF-α and IL-6 in mouse serum. After enucleation of each group of mice, an equal volume of blood was collected and centrifuged at 2000 rpm for 5 min. The upper layer of serum was taken as the test sample and stored in a -80°C refrigerator. The concentration of inflammatory factors in mouse serum was determined according to the kit instructions. Fig.10 Results shown. There was still a statistical difference in the TNF-α level in the blood of mice in the model group and the licorice protein drug-loaded nanoparticle group. The IL-6 and TNF-α levels in the licorice protein drug-loaded nanoparticle group were closest to those in the normal group, indicating that the efficacy of licorice protein drug-loaded nanoparticles was superior to that of other treatment groups.

[0082] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of licorice protein drug-loaded nanoparticles in the preparation of drugs for the treatment of psoriasis, It is characterized in that The glycyrrhizin drug-loaded nanoparticles use glycyrrhizin as a carrier, and the carrier at least contains baicalin, paeoniflorin and glycyrrhizin; The mass ratio of baicalin, paeoniflorin and liquiritin in the nanoparticles is 4:2:2; The relative encapsulation rates of baicalin, paeoniflorin and liquiritin were 31.7%, 22.56% and 23.8%, respectively; The licorice protein drug-loaded nanoparticles are prepared by adjusting the pH to 3-5; The licorice protein drug-loaded nanoparticles are used to improve drug solubility and promote cell uptake.

2. The use according to claim 1, It is characterized in that The licorice protein drug-loaded nanoparticles are used to downregulate the expression level of inflammatory factors, thereby inhibiting skin inflammatory reactions.

3. The use according to claim 2, It is characterized in that The inflammatory factors are TNF-α, IL-17 and IL-6.

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