Application of protein nano-selenium hydrogel in preparation of medicine for preventing and treating psoriasis

Abstract

The invention belongs to the technical field of medicines, and discloses application of protein nano-selenium hydrogel in preparation of a medicine for preventing and treating psoriasis. Compared with nano-selenium gel prepared by a traditional method, the protein nano-selenium hydrogel prepared by the preparation method disclosed by the invention is better in stability, higher in oxidation resistance and anti-inflammatory capacity and better in treatment effect on psoriasis dermatitis.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and particularly relates to use of a protein nano-selenium hydrogel in preparing a drug for preventing and treating psoriasis. Background Art

[0002] Psoriasis is a common chronic skin disease characterized by excessive proliferation of keratinocytes and infiltration of inflammatory cells, affecting 2-3% of the world's population. Commonly used drugs for the treatment of psoriasis in clinical practice include retinoids, methotrexate, calcipotriol, tacrolimus and monoclonal antibodies, but there are still problems such as first-pass effect, low bioavailability, insufficient drug accumulation in psoriatic skin, toxic side effects (such as nausea, vomiting and liver damage) and high prices. Therefore, it is still very necessary to develop highly effective drugs for the treatment of psoriasis with fewer side effects.

[0003] Selenium is one of the important trace elements necessary for the human body, and has good antioxidant, anti-tumor and immunomodulatory effects. Literature reports that the selenium content in patients with psoriasis decreases, and selenium supplementation helps to improve the redox imbalance in patients, thereby alleviating the progression of the disease to a certain extent; however, the effective safe dose range of selenium is narrow, and excessive intake of selenium by the human body will produce serious adverse reactions. Studies have shown that the preparation of selenium into inorganic nano-elemental selenium (also known as nano-elemental selenium, inorganic nano-selenium or nano-selenium, Selenium nanoparticles, Se Ps) can improve its biological activity and safety. In addition, studies have shown that Se Ps induces cell apoptosis by producing excessive reactive oxygen species (ROS), eliminates acanthosis and splenomegaly, and alleviates the psoriasis-like phenotype induced by imiquimod (IMQ); however, Se Ps still have shortcomings such as easy aggregation and easy oxidation. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of the present invention is to provide a use of a protein nano-selenium hydrogel in the preparation of a drug for preventing and treating psoriasis; based on the fact that albumin can maintain good stability at a certain temperature and pH, and has the advantages of being non-toxic, low immunogenic and biodegradable, the present invention constructs protein nano-selenium (also known as protein-bound selenium nano, protein-bound nano-elemental selenium, protein-bound nano-elemental selenium, HSPs) through protein self-assembly technology, which has better stability and biological activity than Se Ps, and its efficacy in improving imiquimod-induced psoriasis by scavenging ROS and having anti-inflammatory effects is better than that of Se Ps.

[0005] The purpose of the present invention is achieved through the following technical solutions: A use of a protein nano-selenium hydrogel in preparing a drug for preventing and treating psoriasis. The preparation steps of the protein nano-selenium hydrogel include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan, stirring and reacting at room temperature or 4°C for 3-12 hours, dialyzing or centrifuging to remove unreacted substances to obtain protein nano-selenium particles; then, adding the protein nano-selenium particles to a hydrogel matrix raw material, and preparing the protein nano-selenium hydrogel under sufficient stirring and the action of a cross-linking inducer (such as triethanolamine).

[0006] The protein reducing agent is tris(2-carboxyethyl)phosphine (TCEP), and dithiothreitol, β-mercaptoethanol, cysteine ​​or glutathione can also be used.

[0007] The raw material of the hydrogel matrix is ​​carbomer gel, carboxyethyl cellulose, hyaluronic acid powder, chitosan or polyvinyl alcohol.

[0008] The albumin may be one or more of human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin, donkey serum albumin, transferrin or similar disulfide bond-rich proteins.

[0009] Preferably, the protein nano-selenium hydrogel is prepared according to the following steps: (1) Dissolve human serum albumin in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, wherein the final concentration of human serum albumin is 0.01 to 200 mg / mL and the final concentration of the protein reducing agent is 0.1 to 60 mM; react at 37°C for 90 to 110 min, and stir to obtain a homogeneous protein solution with a spatially unfolded structure; then, add a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan to the homogeneous protein solution with a spatially unfolded structure, wherein the final concentration of chitosan is 0.05 to 5 mg / mL and the final concentration of sodium selenite or sodium selenate is 0.1 to 30 mM; stir and react at room temperature or 4°C for 3 to 12 h to obtain a crude protein nano-selenium solution; place the crude protein nano-selenium solution in a dialysis bag and dialyze it overnight in a low-temperature PBS solution at 0 to 20°C, or centrifuge the crude protein nano-selenium solution at 8,000 to 30,000 rpm for 10 to 60 min. min and repeatedly resuspended with deionized water and centrifuged several times to finally obtain protein nanoselenium particles (HSPs); (2) adding the carbomer gel into water and stirring to fully swell it, and then adding the protein nano-selenium particles obtained in step (1) and deionized water; finally, adding triethanolamine to induce carbomer cross-linking, and stirring evenly to obtain the protein nano-selenium hydrogel; Alternatively, hydroxyethyl cellulose is added to water and stirred evenly, followed by the addition of sodium carboxymethyl cellulose, and after it swells and dissolves, the protein nano-selenium particles obtained in step (1) are added; finally, aluminum sulfate is added to induce cellulose cross-linking, and the mixture is stirred evenly to obtain the protein nano-selenium hydrogel.

[0010] Alternatively, chitosan is dissolved in acetic acid, and then the protein nano-selenium particles obtained in step (1) are added and stirred evenly. Finally, glutaraldehyde is added to induce chitosan cross-linking, and the protein nano-selenium hydrogel is obtained after stirring evenly. Alternatively, sodium alginate is added to water and stirred evenly, and then the protein nano-selenium particles obtained in step (1) are added; finally, calcium chloride is added to induce cross-linking of the sodium alginate, and the protein nano-selenium hydrogel is obtained after stirring evenly; Alternatively, adipic acid dihydrazide is dissolved in ultrapure water, and sodium hyaluronate powder is added and stirred to dissolve; the protein nano-selenium particles obtained in step (1) are added thereto, and stirred evenly to obtain a mixed solution; then, a mixed solution of N-succinimide and carbodiimide is ultrasonically shaken to remove bubbles and then added to the above mixed solution; finally, β-tricalcium phosphate powder is added to induce cross-linking of sodium hyaluronate, and the protein nano-selenium hydrogel is obtained after stirring evenly.

[0011] The dialysis molecular retention of the dialysis bag in step (1) is not less than 1000.

[0012] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0013] (1) The present invention prepares a protein nanoselenium (HSPs) hydrogel that has better stability and biological activity than single-substance nanoselenium (SePs) gel.

[0014] (2) The protein nano-selenium particles (HSPs) prepared by the present invention have a more excellent anti-inflammatory effect than single-substance nano-selenium (SePs) gel, and can be used as a drug for the treatment of inflammatory diseases such as psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 are the preparation and characterization diagrams of HSPs and Se Ps, where A is the preparation process diagram of HSPs; BC are TEM images of HSPs and Se Ps (scale, 100 nm); D is the dispersibility of HSPs and SePs at 0, 14 and 28 days; EF are UV-visible spectra and Fourier transform infrared spectra.

[0016] Figure 2are characterization diagrams of the morphology and colloidal properties of different gels, where A is the optical photograph of Blank Gel (blank hydrogel), Vehicle Gel (pure matrix hydrogel containing protein), 500 μg / g Se Ps Gel (single nano-selenium hydrogel) and 500 μg / g HSPs Gel (protein nano-selenium hydrogel); B is the SEM image of Blank Gel, Vehicle Gel, 500 μg / g Se Ps Gel and 500 μg / g HSPs Gel; CJ are the rheological analyses of Blank Gel, Vehicle Gel, 500 μg / g SePs Gel and 500 μg / g HSPs Gel, including amplitude sweep test, frequency sweep test and linear viscoelastic region; K is the water absorption and swelling rate of Blank Gel, Vehicle Gel, 500 μg / g Se Ps Gel and 500 μg / g HSPs Gel.

[0017] Figure 3 Figure 3 is a graphic representation of the safety evaluation of HSPs Gel (protein nano-selenium hydrogel) and Se Ps Gel (single-element nano-selenium hydrogel) on healthy mice, where A and C are images of the back skin and H&E-stained sections of mice after 6 days of treatment with different drugs; B is the measurement of epidermal thickness of H&E sections using Image J; D is the daily weight change of mice; E is the blood routine indicators WBC, RBC, HGB and PLT, liver function indicators (AST and ALT) and kidney function indicators (SCr and UREA); F is H&E staining of tissues of major organs (heart, liver, spleen, lung, kidney).

[0018] Figure 4 Figure 5 is the psoriasis sign of IMQ-induced mice after various treatments. A-C is the images of the back skin, H&E-stained sections and spleen of mice after 6 days of treatment with different drugs. D is the daily weight change of mice. E is the daily PASI score of psoriasis mice, reflecting the severity of erythema, scaling and skin thickening on the back of mice after drug treatment. F is the measurement of epidermal thickness of H&E sections using Image J. G is the recording and calculation of spleen index after 6 days of different drug treatments. H is the changes in STAT3, p-STAT3 and PCNA protein levels after different drug gels were treated in psoriasis mouse models.

[0019] Figure 5HSPs Gel (protein nano-selenium hydrogel) improves IMQ-induced inflammation in psoriasis mice, where A is the PCR method used to detect the expression of inflammatory cytokines IL-6, IL-23, IL-1β, TNF-α and IL-17A in the skin after drug treatment; B is the blood routine indicators WBC, RBC, HGB and PLT; C is liver function (AST and ALT), renal function (SCr and UREA); D is H&E staining of major organ (heart, liver, spleen, lung, kidney) tissues.

[0020] Figure 6 HSPs improve LPS-induced HaCaT inflammation and inhibit the excessive proliferation of HaCaT cells, where A is the cytotoxicity of HSPs and Se Ps on HaCaT cells at 24 h and 48 h; B is the cell viability of HaCaT induced by LPS at 24 h and 48 h; C is HSPs inhibiting LPS-induced excessive proliferation of HaCaT; D is HSPs inhibiting LPS-induced excessive production of reactive oxygen species in HaCaT cells; E is HSPs inhibiting LPS-induced production of inflammatory factors such as IL-6, IL-1β and TNF-α; F and G are that HSPs inhibit the protein levels of STAT3, p-STAT3 and PCNA in HaCaT cells, and statistical analysis was performed using Image J software; H is the observation of the live and dead status of HaCaT cells after HSPs treatment by AM / PI staining. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0022] Example 1: Preparation of protein nanoselenium (HSPs) and its hydrogel, elemental nanoselenium (Se Ps) and its hydrogel

[0023] (1) Preparation of protein nanoselenium (HSPs):

[0024] Human serum albumin was dissolved in a phosphate buffered saline (PBS) solution containing tri(2-carboxyethyl)phosphine (TCEP), a protein reducing agent, at a pH of 5.0, to a final concentration of 10 mg / mL albumin and 20 mM protein reducing agent; the mixture was reacted at 37°C for 100 min with stirring to obtain a homogeneous protein solution with a spatially unfolded structure; then, a sodium selenite solution containing chitosan was added to the homogeneous protein solution with a spatially unfolded structure, to obtain a final concentration of 0.05 mg / mL chitosan and 5 mM sodium selenite, and the mixture was stirred at 4°C for 12 h to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution was placed in a dialysis bag and dialyzed against a low-temperature PBS solution at 0-20°C overnight to obtain human serum albumin nano-selenium particles (HSPs).

[0025] (2) Preparation of single-element nano-selenium (Se Ps):

[0026] Weigh 736.8 mg of glutathione (GSH) and 480 mg of human serum albumin (BSA) into 22.92 mL of water, stir evenly, add 103.2 mg of Na2SeO3 solution, and finally add an appropriate amount of 1 mol / L NaOH solution, stir evenly, and prepare a red elemental nanoselenium (SePs) solution with a concentration of 1520 μg / mL.

[0027] (3) Preparation of HSPs / Se Ps carbomer gel: First, add 3 g of carbomer into water and stir to fully swell it to obtain a 3% carbomer solution; take an appropriate amount of 3% carbomer solution and add it into a beaker, then add deionized water and stir, after stirring evenly, add 20 mg of HSPs obtained in step (1) or Se Ps obtained in step (2), continue stirring, and finally add an appropriate amount of triethanolamine to prepare 500 μg / g HSPs Gel (protein nano-selenium carbomer gel) or Se Ps Gel (single-element nano-selenium carbomer gel).

[0028] (4) Preparation of HSPs / Se Ps carboxymethyl cellulose gel: First, add 0.32 g of carboxyethyl cellulose into water and stir. Then, weigh 1.6 g of sodium carboxymethyl cellulose and add it. After swelling and dissolution, add 20 mg of HSPs obtained in step (1) or Se Ps obtained in step (2). Finally, add 0.32 g of aluminum sulfate into the above solution and stir evenly to obtain 500 μg / g HSPs Gel (protein nano-selenium carboxymethyl cellulose gel) or Se Ps Gel (elemental nano-selenium carboxymethyl cellulose gel).

[0029] (5) Preparation of HSPs / Se Ps chitosan hydrogel: First, weigh 0.71 g of chitosan into a clean beaker, add 2% acetic acid solution and stir to dissolve; then add 20 mg of HSPs obtained in step (1) or Se Ps obtained in step (2) and stir evenly; finally, add 2% glutaraldehyde and continue stirring to obtain 500 μg / g HSPs Gel (protein nano-selenium chitosan gel) or Se Ps Gel (single nano-selenium chitosan gel).

[0030] (6) Preparation of HSPs / Se Ps sodium alginate hydrogel: First, prepare a sodium alginate solution with a mass fraction of 4.0%; then, add 20 mg of the HSPs obtained in step (1) or the Se Ps obtained in step (2) and stir evenly; finally, add a calcium chloride solution with a mass fraction of 4.0% and stir evenly to obtain 500 μg / g HSPs Gel (protein nano-selenium sodium alginate gel) or Se Ps Gel (single nano-selenium sodium alginate gel).

[0031] (7) Preparation of HSPs / Se Ps sodium hyaluronate / β-tricalcium phosphate composite hydrogel: First, 0.115 g of adipic acid dihydrazide was dissolved in 10 mL of ultrapure water, and then 0.5 g of sodium hyaluronate powder was added thereto and fully dissolved; then, 20 mg of HSPs obtained in step (1) or Se Ps obtained in step (2) was added and stirred evenly; then, 1 mL of a solution containing 0.765 g of N-succinimide (NHS) and 0.32 g of carbodiimide (EDC) was added to the above mixed solution after ultrasonic vibration to remove bubbles, and slowly stirred until the mixture was uniformly mixed; finally, according to the mass ratio of sodium hyaluronate / β-tricalcium phosphate of 7:3, a corresponding amount of β-tricalcium phosphate powder was added to the above sodium hyaluronate solution; the two were fully stirred to mix to obtain 500 μg / g HSPsGel (protein nanoselenium sodium hyaluronate / β-tricalcium phosphate composite gel) or Se Ps Gel (elemental nano-selenium sodium hyaluronate / β-tricalcium phosphate composite gel).

[0032] Example 2: Characterization of protein nanoselenium (HSPs) and elemental nanoselenium (Se Ps)

[0033] In order to determine the morphology and size, a transmission electron microscope (FEI Tecnai G212, USA) was used to detect the appearance and size of the HSPs and Se Ps prepared in Example 1; a UV spectrophotometer was used to detect the ultraviolet absorption of HSPs and Se Ps at 600 nm-200 nm; and a Fourier transform infrared spectrometer (Nicolet iS50, Thermo Fisher Scientific) was used to detect the infrared absorption of HSPs and Se Ps.

[0034] We constructed HSPs by protein self-assembly technology ( Figure 1 A), according to transmission electron microscopy analysis, the particle size of Se Ps is about 60 nanometers, and the particle size of HSPs is about 25 nanometers ( Figure 1 BC). It can be seen that HSPs are less likely to aggregate than Se Ps ( Figure 1D), which may be due to the smaller particle size and better dispersion of albumin-modified HSPs. UV-visible spectroscopy and Fourier transform infrared spectroscopy were used to analyze HSPs and Se Ps. UV-visible spectroscopy and Fourier transform infrared spectroscopy showed that HSPs showed a peak shift relative to Se Ps ( Figure 1 EF).

[0035] Example 3: Characterization of protein nanoselenium (HSPs) hydrogel and elemental nanoselenium (Se Ps) hydrogel

[0036] The morphology of the gel was observed using a scanning electron microscope, and the elasticity and viscosity of the gel were tested using a rheometer.

[0037] The morphologies of HSPs Gel and Se Ps Gel, pure matrix hydrogel (containing only protein) and blank hydrogel (containing only carbomer) prepared in Example 1 using carbomer gel as the hydrogel matrix raw material were observed. It was found that the blank hydrogel was clear and transparent, the pure matrix hydrogel (containing only protein) was light yellow, the HSPs Gel and Se Ps Gel were orange-red, and the color of Se PsGel was darker ( Figure 2 A). The SEM image of the gel shows that the gel has a network structure ( Figure 2 Amplitude scanning, frequency scanning and linear viscoelastic region measurements showed that the elasticity of each gel was greater than its viscosity, and that it was easy to spread and retain on the skin, and had the water absorption properties of a gel ( Figure 2 In summary, the present invention successfully prepared a hydrogel containing Se nanoparticles suitable for external use on the skin.

[0038] Example 4: Safety evaluation of HSPs Gel and SePs Gel prepared by using carbomer gel as the hydrogel matrix raw material in Example 1 after application on healthy mouse skin

[0039] Male C57BL / 6 mice weighing 18-20 g were fed adaptively for one week. Before treatment, the back of each mouse was depilated with a depilatory cream, and the depilated area on the back was approximately 2×2 cm. The mice were randomly divided into 3 groups (n=5). The treatment of mice in each group was as follows:

[0040] Control group: treated with blank hydrogel.

[0041] Drug-treated groups: HSPs Gel (500 μg / g), Se Ps Gel (500 μg / g), and matrix group (gel containing albumin only) were all smeared with 80 mg / mouse of drug-containing gel on the back of mice (4 cm 2), once a day for 6 consecutive days. The body weight of mice was recorded every day before drug treatment, and skin images were taken on days 2, 4, and 6. On day 7, blood was collected from mice for blood routine, liver function, and kidney function tests; the back skin and major organs of mice were collected for H&E staining.

[0042] in conclusion:

[0043] The results showed that neither HSPs Gel nor Se Ps Gel caused redness or swelling of the mouse skin, nor did they affect the thickness of the skin or body weight ( Figure 3 There were no significant differences in liver and kidney function indicators (alanine transaminase ALT, aspartate transaminase AST, creatinine Scr, urea nitrogen UREA), platelets (PLT), red blood cells (RBC), white blood cells (WBC) and hemoglobin levels (HGB) in the blood compared with the blank gel group ( Figure 3 E). H&E staining showed that HSPs Gel and Se Ps Gel had no effect on the tissue structure, cell morphology and immune cell infiltration of healthy mice ( Figure 3 F).

[0044] Example 5: Establishment of an IMQ-induced psoriasis-like mouse model and verification of the use of HSPs Gel (prepared using carbomer gel as a hydrogel matrix raw material) to alleviate the IMQ-induced psoriasis-like phenotype in mice

[0045] Male C57BL / 6 mice weighing 18-20 g were fed adaptively for one week. Before treatment, the back of each mouse was depilated with a depilatory cream, and the depilated area on the back was about 2×2 cm. The mice were randomly divided into 10 groups (5 mice in each group). The treatment of each group of mice was as follows:

[0046] Control group: no treatment.

[0047] Model group: 62.5 mg imiquimod cream (IMQ) was applied on the back of mice once a day for 6 consecutive days.

[0048] Treatment group: After 6 hours of application of IMQ, the high, medium and low HSPs Gels (containing Se concentrations of 500, 250 and 125 μg / g, respectively) obtained in Example 1, the high, medium and low Se Ps Gels (containing Se concentrations of 500, 250 and 125 μg / g, respectively) obtained in Example 1, the matrix gel (gel containing only albumin), the blank gel (containing only carbomer) and the positive control drug (0.02% clobetasol propionate cream) were applied to the back of mice (80 mg / mouse, about 4 cm 2 ), once a day for 6 consecutive days.

[0049] Erythema, thickness, and scaling (0: none; 1: mild; 2: moderate; 3: severe; 4: very severe) were graded independently every day to obtain the Psoriasis Area Severity Index (PASI). On the seventh day, the mice were weighed and photographed, blood was collected from the eyeballs for subsequent experiments, and skin tissue samples and heart, liver, spleen, lung, and kidney were collected and stored at -80°C. For histopathological analysis, the samples were fixed in 10% formalin. The PASI scoring method was used to score the degree of the three indicators (erythema, thickness, and scaling) in the mouse skin lesions, and the sum of the scores of the three indicators was the total score.

[0050] PASI scoring criteria:

[0051] None (0): no erythema on the surface;

[0052] Mild (1): Part of the lesion is covered with scales, mainly fine scales, slightly higher than the normal skin surface, and red;

[0053] Moderate (2): Most lesions are completely or incompletely covered with scales, which are flaky and moderately raised, with rounded or slanted, red edges;

[0054] Severe (3): almost all lesions are covered with scales, with thick layers of scales, thick lesions, prominent and raised, and dark red;

[0055] Extremely severe (4): All lesions. The total score is the sum of the three index scores (0 to 16).

[0056] Spleen / BMI

[0057] Animal body weight was recorded every day until the end of the experiment, and spleen morphology was observed and collected after sacrifice. To obtain spleen weight index, spleen weight was measured and normalized with body weight, and the results were expressed as mg / g body weight.

[0058] Hematoxylin and eosin (H&E) staining

[0059] The skin from the back of the mouse was removed and fixed with 4% paraformaldehyde, and the fixed skin was dehydrated and embedded in paraffin. The paraffin blocks were cut into 4 μm sections and stained with hematoxylin and eosin (H&E). Images of the skin sections were obtained using a microscope, avoiding the location of the hair follicles, and 5 sites were selected to measure the epidermal thickness in each skin section using Image J software.

[0060] Western blot

[0061] Proteins were extracted from skin tissues, and total protein samples were electrophoresed using SDS-PAGE gels and then transferred to PVDF membranes. The membranes were blocked with 5% skim milk powder for 1 hour and incubated with primary antibodies overnight at 4°C. After washing three times with TBST, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Finally, the membranes were washed three times with TBST, and exposed and photographed using a gel imaging system, and the expression of each protein was analyzed using Image J software.

[0062] Real-time quantitative PCR

[0063] RNA was extracted from mouse skin using Beyozol reagent (Biyuntian). After RNA was quantified using a nucleic acid concentration analyzer, cDNA was amplified using HiScrip QRT SuperMix reverse transcriptase. The expression of IL-6, IL-23, IL-1β, TNF-α, and IL-17A was detected using real-time quantitative polymerase chain reaction. GAPDH values ​​were used as a control. Standard 2 -ΔΔCt Methods Gene expression relative to control was determined. Results were plotted using GraphPad Prism software and statistical analysis was performed using GraphPad Prism software.

[0064] in conclusion:

[0065] (1) After the mice were treated with imiquimod and drugs, dark red patches appeared on the back skin of the mice in the imiquimod group, covered with thick scales, and the skin infiltration and hypertrophy were obvious. Compared with the groups treated with different concentrations of HSPs Gel, the erythema in the high-dose group was significantly lighter, the scales were less, the skin infiltration was significantly alleviated, and the thickness of the epidermis of the skin was also significantly reduced. Its effect was better than that of SePs and comparable to that of the positive reference drug ( Figure 4 A, B, and F). IMQ-induced mice showed increasing PASI scores as their body weight decreased, and HSPs Gel administration significantly alleviated these symptoms ( Figure 4 D and E). It was also found that HSPs Gel treatment could reduce IMQ-induced splenomegaly ( Figure 4 C and G). HaCaT overproliferation can promote the occurrence and development of psoriasis. Therefore, Western blotting detected several proliferation-related proteins in the skin, and found that HSPs Gel could inhibit the increase of STAT3, p-STAT3, and PCNA protein levels induced by IMQ. In summary, HSPs Gel can improve IMQ-induced psoriatic dermatitis better than Se Ps Gel, and its efficacy is equivalent to that of the positive drug clobetasol propionate cream, and the effect of HSPs Gel in improving psoriasis is concentration-dependent.

[0066] (2) HSPs Gel improves IMQ-induced inflammation in psoriasis mice

[0067] Four hours after topical application of 62.5 mg 5% imiquimod, the skin was treated with different concentrations (125, 250, 500 μg / g) of HSPs Gel and Se Ps Gel, and Dermovate (0.02%) for 6 days. Blood was collected from the eyeballs for blood routine and liver and kidney function tests. Pathological sections were made for the heart, liver, spleen, lungs, and kidneys of each group of mice. RNA was extracted from the mouse skin, and the RNA was quantified using a nucleic acid concentration analyzer, and cDNA was amplified using HiScrip QRT SuperMix reverse transcriptase. The expression of IL-6, IL-23, IL-1β, TNF-α, and IL-17A was detected by real-time quantitative polymerase chain reaction. The results showed that compared with Se Ps Gel, HSPs Gel could reduce the increase of inflammatory factors such as IL-6, IL-23, IL-1β, TNF-α, and IL-17A in the skin induced by IMQ ( Figure 5 A); it can also reduce the increase of WBC in serum caused by IMQ, and improve the disorder of spleen tissue cells induced by IMQ, and reduce splenomegaly ( Figure 5 of BD).

[0068] Example 6: Verification of HSPs improving LPS-induced HaCaT inflammation and inhibiting excessive proliferation of HaCaT cells

[0069] (1) Cytotoxicity

[0070] HaCaT cells in the logarithmic growth phase were obtained and trypsin digested and then plated at 3.0×10 3 Cells / well were inoculated in a 96-well cell culture plate, 100 μL of cell suspension was added to each well, and the plate was placed in a cell culture incubator at 37°C and 5% CO2 for overnight culture. Six replicate wells were set up for each group. After the cells were stably attached to the wall, the original culture medium was aspirated the next day, and DMEM culture medium containing HSPs and Se Ps obtained in Example 1 at drug concentrations of 2.5, 5, 10, 20, 40, and 80 μM was added to each well. After culturing in a cell culture incubator for 24 and 48 hours, 10 μL of CCK8 reagent was added to each well. After culturing in a cell culture incubator for 0.5-4 h, the absorbance was measured at 450 nm to indirectly reflect the number of living cells. The data were recorded, and the cells treated with the solvent control were used as the control group. The cell survival rate was calculated using the following formula:

[0071] Cell survival rate% = (OD value of drug group - OD value of blank group) / OD value of blank group × 100%

[0072] (2) Calcein-AM / PI staining

[0073] An appropriate number of HaCaT cells were seeded in a 12-well plate, and the next day, the cells were treated with LPS (20 mg / ml) and 5 μM and 10 μM HSPs and Se Ps obtained in Example 1 for 48 hours. After the treatment, the cells were stained with Calcein-AM and PI, and observed under a fluorescence microscope using green and red light.

[0074] (3) DCFDA staining

[0075] ROS formation in cells can be determined by DCFDA staining. An appropriate number of HaCaT cells were seeded in a 6-well plate, and the next day, the cells were treated with LPS (20 mg / ml) and HSPs and Se Ps obtained in Example 1 at a concentration of 5 μM for 48 hours. To detect ROS levels, 1 ml PBS (containing DCFDA 10 μM) was added to the cells and incubated at 37 °C in the dark for 30 minutes and observed under a fluorescence microscope with green light.

[0076] in conclusion:

[0077] (1) After treating cells with 2.5, 5, 10, 20, 40 and 80 μM HSPs and Se Ps for 24 h and 48 h, the safe dose range of HSPs and Se Ps on cells was evaluated. Figure 6 A). Then, different concentrations of LPS were used to stimulate the cells, indicating that 20 μg / ml could better stimulate the over-proliferation of HaCaT. Compared with Se Ps, 5 μM HSPs could better inhibit the over-proliferation ( Figure 6 B and C). Since excessive proliferation of HaCaT can promote the occurrence and development of psoriasis, 5 μM HSPs were selected for subsequent experiments. It was found that HSPs can remove the excessive production of ROS in cells and inhibit the production of inflammatory factors such as IL-6, IL-1β and TNF-α, and the effect of reducing inflammatory factors is better than Se Ps ( Figure 6 D and E).

[0078] (2) We detected proliferation-related proteins at the cellular level and found that HSPs could inhibit the upregulation of STAT3, p-STAT3, and PCNA protein levels in HaCaT induced by LPS, thereby promoting cell death in some keratinized cells ( Figure 6 In summary, HSPs may inhibit the excessive proliferation of HaCaT by scavenging ROS and reducing the production of inflammatory factors, and its effect is better than that of Se Ps.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. A use of protein nano-selenium hydrogel in the preparation of a drug for preventing and treating psoriasis, characterized in that: The preparation steps of the protein nano-selenium hydrogel include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan, stirring and reacting at room temperature or 4°C for 3-12 hours, dialyzing or centrifuging to remove unreacted substances, and obtaining protein nano-selenium particles; then, adding the protein nano-selenium particles to a hydrogel matrix raw material, and preparing the protein nano-selenium hydrogel under sufficient stirring and the action of a cross-linking inducer.

2. Use of a protein nano-selenium hydrogel according to claim 1 in preparing a drug for preventing and treating psoriasis, characterized in that: The protein reducing agent is tris(2-carboxyethyl)phosphine.

3. Use of a protein nano-selenium hydrogel according to claim 1 in preparing a drug for preventing and treating psoriasis, characterized in that: The raw material of the hydrogel matrix is ​​carbomer gel, carboxyethyl cellulose, hyaluronic acid powder, chitosan or polyvinyl alcohol.

4. Use of a protein nano-selenium hydrogel according to claim 1 in preparing a drug for preventing and treating psoriasis, characterized in that: The protein nano-selenium hydrogel is prepared specifically according to the following steps: (1) Dissolve human serum albumin in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, wherein the final concentration of human serum albumin is 0.01 to 200 mg / mL and the final concentration of the protein reducing agent is 0.1 to 60 mM; react at 37°C for 90 to 110 min, stir, and obtain a homogeneous protein solution with a spatially unfolded structure; then, add a sodium selenite or sodium selenate solution containing chitosan to the homogeneous protein solution with a final concentration of chitosan of 0.05 to 5 mg / mL and a final concentration of sodium selenite or sodium selenate of 0.1 to 30 mM; stir and react at room temperature or 4°C for 3 to 12 h to obtain a crude protein nano-selenium solution; place the crude protein nano-selenium solution in a dialysis bag and dialyze it overnight in a low-temperature PBS solution at 0 to 20°C, or centrifuge the crude protein nano-selenium solution at 8,000 to 30,000 rpm for 10 to 60 min. min and repeatedly resuspended with deionized water and centrifuged several times, finally obtaining protein nano-selenium particles; (2) adding the carbomer gel into water and stirring to fully swell it, and then adding the protein nano-selenium particles obtained in step (1); finally, adding triethanolamine to induce carbomer cross-linking, and stirring evenly to obtain the protein nano-selenium hydrogel; Alternatively, hydroxyethyl cellulose is added to water and stirred evenly, followed by adding sodium carboxymethyl cellulose, and after sodium carboxymethyl cellulose swells and dissolves, the protein nano-selenium particles obtained in step (1) are added; finally, aluminum sulfate is added to induce cellulose cross-linking, and the mixture is stirred evenly to obtain the protein nano-selenium hydrogel; Alternatively, chitosan is dissolved in acetic acid, and then the protein nano-selenium particles obtained in step (1) are added and stirred evenly; finally, glutaraldehyde is added to induce chitosan cross-linking, and the protein nano-selenium hydrogel is obtained after stirring evenly; Alternatively, sodium alginate is added to water and stirred evenly, and then the protein nano-selenium particles obtained in step (1) are added; finally, calcium chloride is added to induce cross-linking of the sodium alginate, and the protein nano-selenium hydrogel is obtained after stirring evenly; Alternatively, adipic acid dihydrazide is dissolved in ultrapure water, and sodium hyaluronate powder is added and stirred to dissolve; the protein nano-selenium particles obtained in step (1) are added thereto, and stirred evenly to obtain a mixed solution; then, a mixed solution of N-succinimide and carbodiimide is ultrasonically shaken to remove bubbles and then added to the above mixed solution; finally, β-tricalcium phosphate powder is added to induce cross-linking of sodium hyaluronate, and the protein nano-selenium hydrogel is obtained after stirring evenly.

5. The use according to claim 4, characterized in that: The dialysis molecular retention of the dialysis bag in step (1) is not less than 1000.

Images