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

Abstract

The present invention belongs to the field of medical technology and discloses the use of a protein nano-selenium hydrogel in the preparation of a drug for the prevention and treatment of psoriasis. The protein nano-selenium hydrogel prepared by the present invention has better stability, stronger antioxidant and anti-inflammatory properties, and a better therapeutic effect on psoriatic dermatitis than nano-selenium gels prepared by traditional methods.

Description

Technical Field

[0001] The present 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 keratinocyte proliferation and inflammatory cell infiltration, affecting 2-3% of the global population. Commonly used psoriasis treatments include retinoids, methotrexate, calcipotriol, tacrolimus, and monoclonal antibodies. However, these drugs still suffer from issues such as first-pass effects, low bioavailability, insufficient drug accumulation in psoriatic skin, toxic side effects (such as nausea, vomiting, and liver damage), and high costs. Therefore, the development of highly effective psoriasis treatments with minimal side effects remains crucial.

[0003] Selenium is an essential trace element for the human body, possessing excellent antioxidant, antitumor, and immunomodulatory properties. Literature reports suggest that selenium levels are decreased in psoriasis patients, suggesting that selenium supplementation can help improve redox imbalance and, consequently, alleviate disease progression. However, the effective safe dose range for selenium is narrow, and excessive selenium intake can lead to serious adverse reactions. Studies have demonstrated that preparing selenium into inorganic elemental nanoparticles (SePs) can enhance their bioactivity and safety. Furthermore, studies have shown that SePs induce cell apoptosis, eliminate acanthosis and splenomegaly, and mitigate imiquimod (IMQ)-induced psoriasis-like phenotypes by generating excessive reactive oxygen species (ROS). However, SePs still have limitations, such as susceptibility to aggregation and oxidation. Summary of the Invention

[0004] In order to overcome the shortcomings and deficiencies of the prior art, the object 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:

[0006] 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).

[0007] The protein reducing agent is tris(2-carboxyethyl)phosphine (TCEP), and dithiothreitol, β-mercaptoethanol, cysteine ​​or glutathione can also be used.

[0008] The raw materials of the hydrogel matrix are carbomer gel, carboxyethyl cellulose, hyaluronic acid powder, chitosan or polyvinyl alcohol.

[0009] The albumin can 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.

[0010] Preferably, the protein nano-selenium hydrogel is prepared according to the following steps:

[0011] (1) Human serum albumin is dissolved in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, with a final concentration of human serum albumin of 0.01 to 200 mg / mL and a final concentration of the protein reducing agent of 0.1 to 60 mM; the mixture is reacted at 37°C for 90 to 110 min with stirring to obtain a homogeneous protein solution with a spatially unfolded structure; then, a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan is added to the homogeneous protein solution with a spatially unfolded structure, 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; the mixture is stirred at room temperature or 4°C for 3 to 12 h to obtain a crude protein nanoselenium solution; the crude protein nanoselenium solution is placed in a dialysis bag and dialyzed against a low-temperature PBS solution at 0 to 20°C overnight, or the crude protein nanoselenium solution is centrifuged 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);

[0012] (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;

[0013] 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.

[0014] 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.

[0015] 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;

[0016] 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 vibrated 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 mixture is stirred evenly to obtain a protein nano-selenium hydrogel.

[0017] The dialysis molecular retention of the dialysis bag in step (1) is not less than 1000.

[0018] The present invention has the following advantages and beneficial effects compared to the prior art:

[0019] (1) The present invention prepares a protein nanoselenium (HSPs) hydrogel that has better stability and biological activity than single-substance nanoselenium (SePs) gel.

[0020] (2) The protein nanoselenium particles (HSPs) prepared by the present invention have a more excellent anti-inflammatory effect than the elemental nanoselenium (SePs) gel and can be used as a drug treatment for inflammatory diseases such as psoriasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 5 is the preparation and characterization diagram 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 is the UV-visible spectrum and Fourier transform infrared spectrum.

[0022] 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-element 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.

[0023] Figure 3 Figure 3 is a graphic representation of the safety evaluation of HSPs Gel (protein nano-selenium hydrogel) and Se Ps Gel (elemental nano-selenium hydrogel) on healthy mice, where A and C are images of the dorsal skin and H&E-stained sections of mice after 6 days of treatment with different drugs; B is the epidermal thickness of H&E sections measured using Image J; D is the daily weight changes 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 the H&E staining of tissues of major organs (heart, liver, spleen, lung, kidney).

[0024] Figure 4 Figure 5 shows the signs of psoriasis in IMQ-induced mice after various treatments. Figures A and B show images of the dorsal skin, H&E-stained sections, and spleen of mice after 6 days of treatment with different drugs. Figure 5 shows the daily weight changes of mice. Figure 5 shows the daily PASI scores of psoriasis mice, reflecting the severity of erythema, scaling, and skin thickening on the back of mice after treatment. Figure 5 shows the epidermal thickness of H&E sections measured using Image J. Figure 5 shows the spleen index recorded and calculated after 6 days of treatment with different drugs. Figure 5 shows the changes in STAT3, p-STAT3, and PCNA protein levels in psoriasis mouse models after treatment with different drug gels.

[0025] Figure 5HSPs Gel (protein nanoselenium hydrogel) improves IMQ-induced inflammation in psoriasis mice. 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 routine blood 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.

[0026] Figure 6 HSPs improve LPS-induced HaCaT inflammation and inhibit HaCaT cell hyperproliferation, where A is the cytotoxicity of HSPs and SePs 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 the inhibition of LPS-induced HaCaT hyperproliferation by HSPs; D is the inhibition of LPS-induced excessive production of reactive oxygen species in HaCaT cells by HSPs; E is the inhibition of LPS-induced production of inflammatory factors such as IL-6, IL-1β and TNF-α; F and G are the inhibition of STAT3, p-STAT3 and PCNA protein levels in HaCaT cells by HSPs, 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

[0027] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0028] Example 1: Preparation of protein nanoselenium (HSPs) and its hydrogel, elemental nanoselenium (SePs) and its hydrogel

[0029] (1) Preparation of protein nanoselenium (HSPs):

[0030] Human serum albumin was dissolved in a phosphate buffered saline (PBS) solution containing tris(2-carboxyethyl)phosphine (TCEP) at a pH of 5.0, with a final albumin concentration of 10 mg / mL and a final protein reducing agent concentration of 20 mM. The solution was reacted at 37°C for 100 min with stirring to obtain a homogeneous protein solution with a spatially unfolded structure. Subsequently, a sodium selenite solution containing chitosan was added to the homogeneous protein solution with a final chitosan concentration of 0.05 mg / mL and a final sodium selenite concentration of 5 mM. The solution was stirred at 4°C for 12 h to obtain a crude protein nanoselenium solution. The crude protein nanoselenium 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 nanoselenium particles (HSPs).

[0031] (2) Preparation of elemental nanoselenium (SePs):

[0032] Weigh 736.8 mg of glutathione (GSH) and 480 mg of human serum albumin (BSA) into 22.92 mL of water, stir evenly, then add 103.2 mg of Na2SeO3 solution, and finally add an appropriate amount of 1 mol / L NaOH solution and stir evenly to prepare a red elemental nanoselenium (SePs) solution with a concentration of 1520 μg / mL.

[0033] (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 it, after stirring it 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 (elemental nano-selenium carbomer gel).

[0034] (4) Preparation of HSPs / SePs 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 it swells and dissolves, add 20 mg of HSPs obtained in step (1) or SePs 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 nanoselenium carboxymethyl cellulose gel) or SePs Gel (elemental nanoselenium carboxymethyl cellulose gel).

[0035] (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).

[0036] (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 HSPs obtained in step (1) or 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 (elemental nano-selenium sodium alginate gel).

[0037] (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 mixed evenly; finally, according to the mass ratio of sodium hyaluronate / β-tricalcium phosphate of 7:3, the 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).

[0038] Example 2: Characterization of protein nanoselenium (HSPs) and elemental nanoselenium (Se Ps)

[0039] In order to determine the morphology and size, a transmission electron microscope (FEI Tecnai G212, USA) was used to examine the appearance and size of the HSPs and Se Ps prepared in Example 1. A UV spectrophotometer was used to examine the ultraviolet absorption of the HSPs and Se Ps in the range of 600 nm to 200 nm. A Fourier transform infrared spectrometer (Nicolet iS50, Thermo Fisher Scientific) was used to examine the infrared absorption of the HSPs and Se Ps.

[0040] 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).

[0041] Example 3: Characterization of protein nanoselenium (HSPs) hydrogel and elemental nanoselenium (Se Ps) hydrogel

[0042] 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.

[0043] 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 B); Amplitude sweep, frequency sweep and linear viscoelastic region measurements showed that the elasticity of each gel was greater than its viscosity, making it easy to spread and retain on the skin, and having 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.

[0044] Example 4: Safety evaluation of HSPs Gel and SePs Gel prepared using carbomer gel as the hydrogel matrix raw material in Example 1 after application to healthy mouse skin

[0045] Male C57BL / 6 mice, weighing 18-20 g, were acclimated for one week. Before treatment, the back of each mouse was shaved with a depilatory cream, covering an area of ​​approximately 2 × 2 cm. The mice were randomly divided into three groups (n = 5). Each group was treated as follows:

[0046] Control group: treated with blank hydrogel.

[0047] The drug-containing gel was applied to the back of mice (4 cm) with 80 mg / mouse of HSPs Gel (500 μg / g), SePs Gel (500 μg / g), and matrix group (gel containing only albumin). 2), once daily for 6 consecutive days. Mice were weighed daily before drug treatment, and skin images were taken on days 2, 4, and 6. On day 7, blood was collected for routine blood tests, liver function tests, and kidney function tests. Dorsal skin and major organs were collected for H&E staining.

[0048] in conclusion:

[0049] 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), platelet (PLT), red blood cell (RBC), white blood cell (WBC) and hemoglobin (HGB) levels in the blood compared with the blank gel group ( Figure 3 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).

[0050] 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 material) to alleviate the IMQ-induced psoriasis-like phenotype in mice

[0051] Male C57BL / 6 mice, weighing 18-20 g, were acclimated for one week. Before treatment, the back of each mouse was shaved with a depilatory cream, covering an area of ​​approximately 2 × 2 cm. The mice were randomly divided into 10 groups (5 mice per group). Each group was treated as follows:

[0052] Control group: no treatment.

[0053] Model group: 62.5 mg of imiquimod cream (IMQ) was applied to the back of mice once a day for 6 consecutive days.

[0054] Treatment group: After 6 hours of IMQ application, 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, matrix gel (gel containing only albumin), blank gel (containing only carbomer), and positive control drug (0.02% clobetasol propionate cream) were applied to the back of mice (80 mg / mouse, approximately 4 cm 2 ), once a day for 6 consecutive days.

[0055] Erythema, thickness, and scaling were independently graded daily (0: none; 1: mild; 2: moderate; 3: severe; 4: very severe) to generate the Psoriasis Area Severity Index (PASI). On day 7, mice were weighed and photographed, and eye bleeding was performed for subsequent experiments. Skin tissue samples, as well as samples of the heart, liver, spleen, lung, and kidney, were collected and stored at -80°C. For histopathological analysis, samples were fixed in 10% formalin. The PASI score was used to assess the severity of the three indicators (erythema, thickness, and scaling) in the mouse skin lesions. The sum of the scores for these three indicators constituted the total score.

[0056] PASI scoring criteria:

[0057] None (0): no erythema on the surface;

[0058] Mild (1): Part of the lesion is covered with scales, mainly fine scales, slightly above the normal skin surface, and red;

[0059] Moderate (2): Most lesions are completely or incompletely covered with scales, which are flaky and moderately raised, with rounded or slanted edges and red color;

[0060] Severe (3): almost all lesions are covered with scales, with thick scales, thick lesions, prominent and raised, and dark red;

[0061] Extremely severe (4): All lesions. The total score is the sum of the three index scores (0 to 16).

[0062] Spleen / body mass index

[0063] Animal body weight was recorded daily 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 to body weight, and the results were expressed as mg / g body weight.

[0064] Hematoxylin and eosin (H&E) staining

[0065] Dorsal skin was removed from mice and fixed with 4% paraformaldehyde. The fixed skin was dehydrated and embedded in paraffin. Paraffin blocks were cut into 4 μm sections and stained with hematoxylin and eosin (H&E). Microscopic images of the skin sections were obtained, avoiding hair follicles. Five selected sites were selected, and epidermal thickness was measured in each skin section using Image J software.

[0066] Western blot experiments

[0067] Proteins were extracted from skin tissue, and total protein samples were electrophoresed on SDS-PAGE gels and then transferred to PVDF membranes. The membranes were blocked with 5% nonfat dry milk 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 for 1 hour at room temperature. Finally, the membranes were washed three times with TBST and photographed using a gel imaging system. Protein expression was analyzed using Image J software.

[0068] Real-time quantitative PCR

[0069] RNA was extracted from mouse skin using Beyozol reagent (Biyuntian). RNA was quantified using a nucleic acid analyzer, and cDNA was amplified using HiScrip QRT SuperMix reverse transcriptase. Real-time quantitative polymerase chain reaction was used to measure the expression of IL-6, IL-23, IL-1β, TNF-α, and IL-17A. GAPDH levels 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.

[0070] in conclusion:

[0071] (1) After mice were treated with imiquimod and drugs, dark red patches appeared on the back skin of mice in the imiquimod-treated group, covered with thick scales, and the skin was obviously infiltrated and thickened. Compared with the HSPs Gel-treated groups with different concentrations, 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 was also significantly reduced. Its effect was better than 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). Excessive HaCaT proliferation can promote the development and progression of psoriasis. Therefore, immunoblotting assays were performed to detect several proliferation-related proteins in the skin, revealing that HSPs Gel inhibited the IMQ-induced increase in STAT3, p-STAT3, and PCNA protein levels. In summary, HSPs Gel is more effective than Se Ps Gel in improving IMQ-induced psoriatic dermatitis, and its efficacy is comparable to that of the active agent clobetasol propionate cream. Furthermore, the psoriasis-improving effect of HSPs Gel is concentration-dependent.

[0072] (2) HSPs Gel improves IMQ-induced inflammation in psoriasis mice

[0073] Four hours after topical application of 62.5 mg of 5% imiquimod, the skin was treated with HSPs Gel and Se Ps Gel at different concentrations (125, 250, and 500 μg / g) and Dermovate (0.02%) for 6 days. Blood was collected from the eyeballs for routine blood tests and liver and kidney function tests. Pathological sections were made from the heart, liver, spleen, lung, and kidney of each group of mice. RNA was extracted from the mouse skin, quantified using a nucleic acid concentration analyzer, and cDNA was amplified using HiScrip QRT SuperMix reverse transcriptase. Real-time quantitative polymerase chain reaction was used to detect the expression of IL-6, IL-23, IL-1β, TNF-α, and IL-17A. The results showed that compared with Se Ps Gel, HSPs Gel was more effective in reducing 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 BD).

[0074] Example 6: Verification of HSPs ameliorating LPS-induced HaCaT inflammation and inhibiting HaCaT cell hyperproliferation

[0075] (1) Cytotoxicity

[0076] HaCaT cells in the logarithmic growth phase were obtained and trypsinized and then plated at 3.0×10 3 Cells / well were seeded in a 96-well cell culture plate, 100 μL of cell suspension was added to each well, and the plate was cultured overnight in a cell culture incubator at 37°C and 5% CO2. Six replicates 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 hours, the absorbance was measured at 450 nm to indirectly reflect the number of viable 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:

[0077] Cell survival rate % = (OD value of the drug group - OD value of the blank group) / OD value of the blank group × 100%

[0078] (2) Calcein-AM / PI staining

[0079] An appropriate number of HaCaT cells were seeded in a 12-well plate. The next day, the cells were treated with LPS (20 mg / ml) and 5 μM and 10 μM concentrations of HSPs and SePs obtained in Example 1 for 48 hours. After treatment, the cells were stained with Calcein-AM and PI and observed under a fluorescence microscope using green and red light.

[0080] (3) DCFDA staining

[0081] ROS formation in cells can be determined by DCFDA staining. An appropriate number of HaCaT cells were seeded in a 6-well plate. The next day, the cells were treated with LPS (20 mg / ml) and 5 μM HSPs and SePs obtained in Example 1 for 48 hours. To measure ROS levels, 1 ml of PBS (containing 10 μM DCFDA) was added to the cells and incubated in the dark at 37°C for 30 minutes. The cells were then observed under a fluorescence microscope using green light.

[0082] in conclusion:

[0083] (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 Then, different concentrations of LPS were used to stimulate the cells, indicating that 20 μg / ml could better stimulate the excessive proliferation of HaCaT. Compared with Se Ps, 5 μM HSPs could better inhibit this excessive proliferation ( Figure 6 B and C). Because 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 eliminate the excessive production of ROS in cells and inhibit the production of inflammatory factors such as IL-6, IL-1β and TNF-α. The effect of reducing inflammatory factors is better than that of Se Ps ( Figure 6 D and E).

[0084] (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 the cell death of 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.

[0085] 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 that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A use of a 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, stirring and reacting at room temperature or 4°C for 3 to 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; the protein reducing agent is tris(2-carboxyethyl)phosphine, and the hydrogel matrix raw material is carbomer gel.

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 nano-selenium hydrogel is prepared specifically according to the following steps: (1) Human serum albumin is dissolved in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, with a final concentration of human serum albumin of 0.01 to 200 mg / mL and a final concentration of the protein reducing agent of 0.1 to 60 mM; the mixture is reacted at 37°C for 90 to 110 min with stirring to obtain a homogeneous protein solution with a spatially unfolded structure; then, sodium selenite containing chitosan is added 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 of 0.1 to 30 mM, and the mixture is stirred at room temperature or 4°C for 3 to 12 h to obtain a crude protein nanoselenium solution; the crude protein nanoselenium solution is placed in a dialysis bag and dialyzed against a low-temperature PBS solution at 0 to 20°C overnight, or the crude protein nanoselenium solution is centrifuged at 8000 to 30000 rpm for 10 to 60 min and repeatedly resuspended with deionized water and centrifuged several times to obtain protein nanoselenium particles; (2) Add the carbomer gel into water and stir to fully swell it, then add the protein nano-selenium particles obtained in step (1); finally, add triethanolamine to induce carbomer cross-linking, and stir evenly to obtain the protein nano-selenium hydrogel.

3. The use according to claim 2, characterized in that: The dialysis molecular retention of the dialysis bag in step (1) is not less than 1000.

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