Application of water microspheres of modified polysaccharide in promoting wound skin regeneration and inhibiting wound skin fibrosis

Abstract

The invention discloses application of water microspheres of modified polysaccharide in promoting wound skin regeneration and inhibiting wound skin fibrosis. In the invention, compared with a Control group (silicone oil) as a control and an unmodified polysaccharide water microsphere group (AVBEC), when the modified polysaccharide water microsphere group (AVBER) is externally applied to the skin of a bama miniature pig in a deep II-level scald model, the wound healing can be effectively promoted, the inflammation degree and duration can be controlled, and the extracellular matrix composition and plasticity can be regulated; expression of fibrosis marker protein is reduced, and the dual effects of promoting skin regeneration and inhibiting fibrosis are achieved. The problems that natural extracts and common polysaccharide microsphere products are low in biological activity and poor in stability are solved, and new functions of modified polysaccharide are found.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of application of modified polysaccharides, and particularly relates to the application of water microspheres of modified polysaccharides in promoting wound skin regeneration and inhibiting wound skin fibrosis. BACKGROUND

[0002] Polysaccharides are a class of biological macromolecules with structural diversity, which are widely sourced, rich in content, important in function, and good in biocompatibility. Natural polysaccharides have many excellent biological activities, such as immunomodulatory, antitumor, antiviral, antioxidant, and hypoglycemic effects, but often have limitations such as poor solubility, insufficient stability, and single function. The biological activity and physicochemical property of natural polysaccharides are closely related to their structure. In order to better exert the efficacy of polysaccharides and make them have more extensive applications in food, medicine, materials and other fields, researchers often use scientific means to modify or modify polysaccharide molecules.

[0003] Common modification methods include physical modification, chemical modification, biological modification, and composite modification. Physical modification does not require the addition of chemical reagents, has no toxic by-products, is simple to operate, and can preserve the natural structure and biological activity of polysaccharides while modifying the functional groups of polysaccharides by optimizing conditions. In addition, it can utilize the natural active enzymes in raw materials and synergize with enzyme modification. Pervaporation membrane technology is a new type of membrane separation technology. This membrane technology has special properties for screening substances, which is different from other membranes. Unlike membranes based on petroleum chemical polymers, we use a device for promoting aloe polysaccharide to form a pervaporation membrane (ZL202410397753.4) to stably and continuously obtain aloe polysaccharide pervaporation membrane with new functions and biological activity, which is used to prepare polysaccharide-water microspheres and study their functions.

[0004] Currently marketed and research stage drugs for promoting wound repair include the following categories: (1) growth factors; (2) antibacterial / anti-infective drugs; (3) anti-inflammatory / immune modulating drugs; (4) moisturizing / barrier repair; (5) traditional Chinese medicine / natural extracts; (6) new materials and cell therapy, etc. The means of inhibiting fibrosis include: (1) inflammation modulators, such as inflammatory factors, receptor activators / antagonists, etc.; (2) proliferation phase modulators, such as collagen post-translational modification inhibitors, growth factors, etc.; in addition, there are methods of surgical removal of scars, such as transdermal injection, laser, etc.

[0005] The disadvantages of the prior art are: (1) there is no double-effect drug for promoting skin regeneration and inhibiting fibrosis at present, and even some drugs for promoting regeneration (such as fibroblast growth factor) can aggravate the degree of fibrosis, or the use of drugs for inhibiting fibrosis at an early stage can cause poor skin regeneration; (2) it is currently a trend to add natural anti-inflammatory or metabolic regulation extracts to antibacterial and moisturizing materials, but it is difficult to maintain the natural structure and biological activity of the extracts; (3) the growth factor and antibiotic drugs have problems of poor stability, unstable efficacy, drug resistance and toxicity, allergy and carcinogenic risk; (4) the efficacy of the above drugs varies greatly for different individuals, and the efficacy on chronic wounds is limited. SUMMARY

[0006] Therefore, the technical problem to be solved by the present application is to provide an application of modified polysaccharide water microspheres in promoting skin regeneration and inhibiting skin fibrosis.

[0007] The aloe is made into modified polysaccharide water microspheres with natural biological activity, which has good biocompatibility and high safety, and does not need to add exogenous chemical reagents such as metal ions, photoinitiators, porogens and crosslinking agents; Compared with the Control group (silicone oil) and the unmodified polysaccharide water microsphere group (AVBEC), the modified polysaccharide water microsphere group (AVBER) can effectively promote wound healing, control the degree and duration of inflammation, regulate the composition and plasticity of extracellular matrix, and reduce the expression of fibrosis marker proteins when used on the skin of deep Ⅱ degree burn model Bama miniature pigs, achieving the dual effects of promoting skin regeneration and inhibiting fibrosis. The problems of low biological activity and poor stability of natural extracts and ordinary polysaccharide microsphere products are solved, and new functions of modified polysaccharides are discovered.

[0008] Therefore, compared with the prior art, the present application has the beneficial effects of: (1) The raw materials are simple and easy to obtain, and the natural biological activity of aloe polysaccharide is retained by using modification and emulsification technology to improve the properties of the microspheres; (2) The modified polysaccharide water microspheres have a special oil-modified polysaccharide-water multilayer structure, have good cell permeability, and are easy to be absorbed by the skin and play a role; (3) The modified polysaccharide water microsphere preparation has a dual effect of promoting skin regeneration and inhibiting fibrosis, and achieves better wound repair effect. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1Figure for the effect of Control group, AVBEC group and AVBER group on scalded wound; Wherein: (A) is the design flow chart of each group in the Bama miniature pig scalding experiment; (B) is the appearance photo of the left and right wound areas of Bama miniature pigs in Control group, AVBEC group and AVBER group at 14 days and 21 days; (C) is the statistical chart of the body weight change of Bama miniature pigs in Control group, AVBEC group and AVBER group at 2, 7, 14, 21 days; (D) is the statistical chart of the wound healing rate of Bama miniature pigs in Control group, AVBEC group and AVBER group at 3, 6, 9, 14, 21 days; (** indicates p<0.01 between groups).

[0010] Figure 2 Figure for AVBER inhibiting inflammation and dermal thickening after pig scalding; Wherein: (A) is the H&E staining chart of the left and right wound skin of Bama miniature pigs in Control group, AVBEC group and AVBER group at 14 days and 21 days; (B) is the statistical chart of the dermal thickness of the wound skin of Bama miniature pigs in Control group, AVBEC group and AVBER group at 14 days and 21 days, (* indicates p<0.05 between groups, ** indicates p<0.01 between groups).

[0011] Figure 3 Figure for AVBER inhibiting collagen deposition and adjusting collagen proportion; The left and right sides are the Masson staining chart and Sirius Red staining chart of the wound skin of Bama miniature pigs in Control group, AVBEC group and AVBER group at 14 days and 21 days.

[0012] Figure 4 Figure for the Western Blot experiment result showing the TGF-β, α-SMA protein expression difference of the wound of Bama miniature pigs in Control group, AVBEC group and AVBER group at 14 days and 21 days. DETAILED DESCRIPTION

[0013] In order to enable those skilled in the art to have a clearer and more intuitive understanding of the present application, the present application will be further described below with reference to the drawings.

[0014] Example 1 The preparation method of aloe polysaccharide comprises the following steps: (1) The fresh Aloe vera leaves are washed and peeled, and the long strip aloe gel is reserved; in order to ensure sufficient functional substances, the growth period of the aloe leaves is not less than 6 months; (2) Aloe vera gel is beaten to be free of obvious granularities to obtain an aloe vera gel turbid liquid; the rotation speed of the beater is preferably 1000 r / min, and the beating time is preferably 20-30 min, and then an ultrasonic probe is used to break the aloe vera gel turbid liquid at a power of 120-150 W for 15 min, during which the temperature is controlled to be no higher than 50℃; (3) The aloe vera gel turbid liquid broken by ultrasonic is added with hot purified water at 80-95℃ at a volume ratio of 1:5, and the temperature is controlled at 70-80℃, and then the aloe vera gel turbid liquid is concentrated to 1 / 3 of the original volume, filtered twice to remove impurities, and an aloe vera polysaccharide concentrated liquid is obtained.

[0015] Example 2 The preparation method of the modified aloe vera polysaccharide comprises the following steps: (1) The aloe vera polysaccharide concentrated liquid prepared in Example 1 is placed in a light reaction tank for light treatment, the light reaction tank is made of stainless steel and has a circulating water jacket, and is connected with a cold water circulating unit, the circulating water temperature is 9-12℃, an adjustable color LED lamp is arranged above the light reaction tank, the distance between the light source and the liquid surface is 60-70 cm, and the light source is treated in the order of blue light, red light and no light, the alternating time is 40-80 min, the total treatment time is 24-48 h, the wavelength of the blue light LED is 450-470 nm, and the wavelength of the red light LED is 620-650 nm; (2) The aloe vera polysaccharide liquid treated in the light reaction tank is placed in a high-temperature film forming tank, and maintained at a high temperature of 80-90℃, and the aloe vera polysaccharide pervaporation membrane continuously generated on the surface of the liquid is separated and collected from the liquid layer.

[0016] In this embodiment, the device for promoting the formation of the aloe vera polysaccharide pervaporation membrane includes a light reaction tank and a high-temperature film forming tank, the aloe vera polysaccharide liquid is placed in the light reaction tank, the aloe vera polysaccharide liquid is stirred for a certain time while being alternately irradiated by the light reaction lamp at a specific illumination, the treated liquid is placed in the high-temperature film forming tank, and the aloe vera polysaccharide pervaporation membrane generated on the surface of the liquid is obtained under high temperature. The specific device and process flow of the modification are referred to the patent ZL202410397753.4, and the raw material circulation enrichment and reaction time are slightly different.

[0017] Example 3 The preparation method of the water microspheres of the modified aloe vera polysaccharide comprises the following steps: (1) The aloe vera polysaccharide pervaporation membrane prepared in Example 2 is sheared and homogenized with wheat germ oil at a mass ratio of 1:(1-1.5), and the treatment is performed for 20-30 h at a temperature of 40-50℃, and then the oil is removed and vacuum dried to obtain a first mixture; (2) The first mixture and pure water are sheared and homogenized at a mass ratio of 1:(1.5~2) for 20h~30h at a temperature of 40℃~50℃. After the process is completed, the oil is removed and the mixture is vacuum dried to obtain the second mixture. (3) The second mixture and silicone oil are sheared and homogenized at a mass ratio of 1:(0.1~0.5) for 20h~30h at a temperature of 40℃~50℃. After the process is completed, the oil is removed to obtain the third mixture. (4) The third mixture was subjected to high temperature and high pressure treatment to obtain stable modified aloe polysaccharide water microspheres. The specific parameters were 121℃, 0.1MPa, and 20min.

[0018] In homogenization, the rotation speed of the homogenizer is above 4000 r / min, preferably 4000 r / min to 10000 r / min.

[0019] In this embodiment, the modified aloe polysaccharide was sequentially homogenized with wheat germ oil, water, and silicone oil under high shear to obtain stable water microspheres of the modified polysaccharide for external use. Zeta potential analysis was then used to confirm the charge distribution and particle size of the microsphere system. High-temperature and high-pressure treatment confirmed the good stability of the microspheres and their ability to accelerate transdermal absorption and penetration.

[0020] To help those skilled in the art better understand the technical solution of this invention, the process of emulsifying water microspheres into polysaccharides can be referred to patents ZL202410395301.2, ZL202410021189.6, and ZL202510025281.4, but there are slight differences in the ratio of raw materials: oil: water and process parameters or steps.

[0021] Performance verification of modified polysaccharide water microspheres First, unmodified polysaccharide aqueous microspheres are prepared. The preparation method of unmodified polysaccharide aqueous microspheres includes the following steps: (1) The aloe polysaccharide concentrate prepared in Example 1 was not subjected to light modification treatment. Instead, it was directly placed in a high-temperature film-forming tank and maintained at a high temperature of 80°C to 90°C. The aloe polysaccharide pervaporation membrane was continuously generated on the liquid surface. The semi-solid aloe polysaccharide pervaporation membrane was separated from the liquid layer and collected. (2) Prepare unmodified aloe polysaccharide water microspheres from the aloe polysaccharide pervaporation membrane collected in step (1) according to the method in Example 3.

[0022] Unmodified aloe vera polysaccharide water microspheres (AVBEC) and modified aloe vera polysaccharide water microspheres (AVBER) were used in a deep grade II burn model skin experiment in Bama miniature pigs. The experiment was approved by the institution's animal ethics committee; anesthesia and euthanasia complied with the "Guidelines for Laboratory Animal Welfare". Figure 1 As shown in Figure A, the experimental design is as follows: ①Select 24 Bama mini-pigs with similar body weight as experimental objects, half of them are male and the other half are female; Bama mini-pigs are adapted for 7 days in SPF level environment before the experiment, free water and standard feed; ②Randomly divide the 24 Bama mini-pigs into AVBEC group, AVBER group and Control group, n=8 for each group, half of them are male and the other half are female; ③Deep Ⅱ degree burn model construction: after anesthesia, fix the Bama mini-pigs on the constant temperature operating table, disinfect the skin on both sides of the spine with conventional sterile method, use gauze or thermal insulation film to block the parts that do not need to be burned, select two 40-layer gauzes soaked in hot water, when the water temperature is constant at 95℃ or above, take out and lay flat on the area to be burned, burn for 28s to construct the model, cover the wound with sterile Vaseline gauze after modeling, observe the infection / healing situation every day; ④Apply the unmodified aloe polysaccharide water microspheres to the burn site of the Bama mini-pigs in the AVBEC group, apply the modified aloe polysaccharide water microspheres to the burn site of the Bama mini-pigs in the AVBER group, and apply silicon oil to the burn site of the Bama mini-pigs in the Control group, the amount of each group is equal; record and count the body weight, wound healing rate and other indicators during the period, and at the midpoint of treatment (14 days) and the endpoint (21 days), respectively, anesthetize and sacrifice the animals and take samples. The number of Bama mini-pigs sacrificed in each group at the midpoint of treatment (14 days) is 4, half of them are male and the other half are female.

[0023] According to the experimental results, it is found that the modified aloe polysaccharide water microspheres (AVBER group) have the dual functions of promoting skin regeneration and inhibiting fibrosis, while silicon oil (Control group) and unmodified aloe polysaccharide water microspheres (AVBEC group) do not have the function of inhibiting fibrosis, the specific results are as follows: (1) From the appearance, compared with the Control group and the AVBEC group, the AVBER group can promote wound healing and skin regeneration after 14 days and 21 days of treatment, and the healing site is smoother (A) Figure 1 B), significantly improves the wound healing rate (C) Figure 1 C) without affecting the body weight of the animals (D) Figure 1 D); (2) The H&E staining results of skin sections show that, compared with the Control group and the AVBEC group, the AVBER group can reduce the infiltration of inflammatory cells in the skin damage area, reduce the degree of inflammation, and accelerate the recovery of skin structure (A) Figure 2 A); (3) One of the signs of fibrosis is the increase in dermal thickness, which may be related to inflammation. The statistical results show that, compared with the Control group, the AVBER group can significantly reduce the dermal thickness at 14 days and 21 days of treatment, and the dermal thickness of the AVBER group at 21 days is also significantly lower than that of the AVBEC group, and the dermal thickness of the AVBEC treatment group is similar to that of the Control group Figure 2 B); (4) The second sign of fibrosis is collagen fiber deposition. Masson staining results show that the Control group and the AVBEC group have more collagen fiber (green) deposition in the skin after 14 days and 21 days of treatment, compared with these two groups, the AVBER group has less collagen fiber deposition and more muscle fibers (red) (left) Figure 3 ), indicating good skin function recovery; Sirius Red staining results show that the Control group and the AVBEC group have more type I collagen (yellow and red) and less type III collagen (green) related to wound repair under polarized light after 14 days and 21 days of treatment, compared with these two groups, the AVBER group has more type III collagen at 14 days and gradually changes to type I collagen at 21 days (right) Figure 3 ), consistent with the wound repair model, indicating that AVBER can regulate the proportion of wound collagen fibers.

[0024] (5) The third sign of fibrosis is the increase in the expression of characteristic proteins TGF-β and a-SMA. Western blot results show that, compared with the Control group and the AVBEC group, the AVBER group can reduce the protein levels of TGF-β and a-SMA after 14 days and 21 days of treatment Figure 4 ), indicating that AVBER can inhibit the fibrosis-related signaling pathway and reduce the protein expression of TGF-β and a-SMA in skin wounds.

[0025] Masson staining introduction: Connective tissue staining is narrowly defined as the differentiation staining between the three fibers contained in it, i.e. the differentiation staining between collagen fibers, reticular fibers and elastic fibers or between other tissues. Masson's trichrome staining, also known as Masson staining, is the most classic method of connective tissue staining, and is the authoritative and classic technical method for collagen fiber staining. The so-called trichrome staining usually refers to staining of cell nuclei and selectively showing collagen fibers and muscle fibers. The staining principle of this method is related to the size of anionic dye molecules and the permeability of tissues: the size of the molecule is reflected by the molecular weight, and small molecular weight can penetrate the structure of dense and low permeability tissues, while large molecular weight can only enter the structure of loose and high permeability tissues.

[0026] Specific steps: 1. Paraffin section dewaxing to water: Put the required section into TO biological sectioning transparent agent (ACME C, item number: AYA0150, referred to as TO) for the first time (10 min) - TO for the second time (10 min) - TO for the third time (10 min) - anhydrous ethanol for the first time (5 min) - anhydrous ethanol for the second time (5 min) - 95% ethanol (prepared with ultrapure water, the same below) (5 min) - 85% ethanol (5 min) - 75% ethanol (5 min) dewaxing treatment, and ultrapure water washing for 2 min; 2. Weigert iron hematoxylin (Solebo, item number: G1142) staining: mix Weigert dye A1 and A2 in equal volume to obtain Weigert iron hematoxylin staining solution, which is prepared and used immediately, and staining for 5-10 min; 3. Acidic ethanol differentiation: differentiate for 3 s with acidic ethanol differentiation solution, and ultrapure water washing for 3-5 s. Hydrochloric acid: 70% ethanol = 1:99; 4. Masson blue solution blueing: blueing for 2 min with 1% lithium carbonate aqueous solution blueing solution, and ultrapure water washing for 1 min; 5. Light green (Solebo, item number G1347) staining: light green staining solution staining for 5-10 min, and weak acid working solution washing for 1 min. Weak acid solution: prepare 0.2% acetic acid solution, and then prepare weak acid working solution with ultrapure water according to the ratio of distilled water: weak acid solution = 2:1; 6. Phosphomolybdic acid solution (Solebo, item number G1348) cleaning: phosphomolybdic acid solution washing for 1-2 min, and weak acid working solution washing for 1 min; 7. Fast green staining: directly into fast green staining solution (0.5%) for 1-2 min. Weak acid working solution washing for 1 min; 8. Dehydration: 95% ethanol rapid dehydration for 2-3 s, and anhydrous ethanol dehydration for 3 times, each time for 5-10 s; 9. Transparency: TO transparency for 3 times, each time for 2 min; 10. Mounting: neutral gum (Biyun Tian, C0173) mounting; 11. Staining result: collagen fibers / protein are blue, cytoplasm, muscle, red blood cells are red, and cell nucleus is blue-brown. Collagen fibers, mucus, and cartilage are blue (such as bright green liquid staining is green), cytoplasm, muscle, cellulose, and nerve gelatin are red, and cell nucleus is black blue.

[0027] Introduction of Sirius red staining: Collagen fiber is the most widely distributed and most abundant fiber in connective tissue, widely distributed in various organs, among which the skin, sclera and tendon are the most abundant. Type I collagen fiber is mainly in bone, skin and tendon fiber; type II collagen fiber is mainly in cartilage collagen; type III collagen fiber is mainly in embryonic tissue, adult blood vessels and gastrointestinal tract; type IV collagen fiber is mainly in basement membrane.

[0028] Sirius red staining solution is mainly used for the study of collagen fiber abnormalities or fibrosis in various tissue lesions, and the study of typing and grading of various fibrosis under polarizing microscope. Under ordinary optical microscope, collagen fibers in heart blood vessels and other tissues are stained red, muscle fibers and blood cells are stained yellow. Under polarizing optical microscope, type I collagen fiber is stained strong orange yellow or bright red, and type III collagen fiber is stained green.

[0029] Specific steps: 1. Paraffin section deparaffinization to water: place the required sections in TO first treatment (10 min) - TO second treatment (10 min) - TO third treatment (10 min) - anhydrous ethanol first treatment (5 min) - anhydrous ethanol second treatment (5 min) - 95% ethanol (5 min) - 85% ethanol (5 min) - 75% ethanol (5 min) deparaffinization treatment, and ultrapure water washing for 2 min; 2. Drop stain 3-10 min with hematoxylin staining solution (Biyun, item number: C0107), and wash with water for 30-120 s; 3. Drop stain 10-60 min with Sirius red staining solution (Yingsheng Biological, item number: 60415ES50), and stain 10-20 min for tissues that are easy to color; 4. Wash with running water to remove the staining solution on the surface of the sections; 5. Dehydrate with 95% ethanol for 1 min, and dehydrate with anhydrous ethanol twice, each for 1 min; 6. TO transparency twice, each for 2 min; 7. Neutral gum sealing.

[0030] Hematoxylin-Eosin staining Introduction: H&E staining is the most commonly used staining technique for pathological tissue sections. The originally transparent tissue sections can clearly reflect the tissue structure, tissue type, cell level, etc. after HE staining, and the general morphological structure characteristics of various tissue or cell components and lesions can be clearly observed under a microscope. Hematoxylin and eosin are mainly used as two staining solutions. Hematoxylin is an alkaline staining solution that is blue, which can stain the acidic structure of the tissue into blue-purple (nuclei appear blue; cartilage matrix, calcium salt particles appear dark blue; mucus appears gray-blue). Eosin is a chemically synthesized acid dye that dissociates into negatively charged anions in water, which easily combines with the positive charges in the amino groups of proteins to stain the cytoplasm. Cytoplasm, muscle, connective tissue, eosinophilic granules, etc. are stained into different degrees of red or pink.

[0031] Specific steps: 1. Paraffin dissolution: Place the section in a 70°C in situ hybridization instrument and bake the section for 30 min. 2. Dewaxing: Place the baked section into TO quickly, 2 times, 5 min each time. 3. Gradient hydration: Place in 100%, 90%, 80%, and 70% ethanol for 5 min each time, and place in distilled water for 5 min. 4. Nucleus staining: Dry the water, add hematoxylin staining solution, and stain for 5 min. Rinse off the hematoxylin staining solution with running water, and observe the staining degree under a microscope. 5. Differentiation: Differentiate with 1% hydrochloric acid alcohol for 1-3 s, wash with water, and immerse in tap water for 5-10 min for blue return. Observe the color under a microscope. If the color is not deep enough, directly add hematoxylin staining solution for 1 min, then differentiate and wash with water immediately. If it is too deep, increase the differentiation time appropriately. 6. Counterstaining: Add 0.5% eosin staining solution drop by drop, and stain for 10-15 s. After washing with distilled water, observe under a microscope. 7. Gradient dehydration: Dehydrate with 80%, 90%, 95%, and anhydrous ethanol. 8. TO transparency, 2 times, 5 min each time. 9. Mounting: Add one drop of neutral gum to the tissue, cover with a glass slide, and observe under a microscope for photography. 10. Results: Blue: normal cell nuclei appear bright blue, apoptotic cell nuclei appear blue-black, necrotic cells appear light blue or blue disappears, cartilage matrix, calcium salt particles appear dark blue, and mucus appears gray-blue; red: cytoplasm is different shades of pink to peach, acidophilic granules in cytoplasm appear bright red, collagen fibers appear light pink, elastic fibers appear bright pink, red blood cells appear orange-red, and proteinaceous fluid appears pink.

[0032] Western blot procedure: Western blot is a technique that separates proteins according to their molecular weight using gel electrophoresis, and then identifies these proteins using specific antibodies. Immunoassay typically uses membranes made of nitrocellulose or PVDF (polyvinylidene fluoride). The gel is placed tightly against the membrane, and proteins migrate from the gel to the membrane under the influence of an electric current. The membrane is then further treated with target-specific antibodies, and then colored with secondary antibodies and detection reagents. Analysis of the colored bands allows for quantitative comparison of the concentration of specific proteins. Western blot is one of the most commonly used biochemical techniques for the separation and identification of proteins.

[0033] Specific steps: 1. Total protein extraction from skin tissue: Cut a small amount of tissue into small pieces and place them in a 2ml homogenizing EP tube. Add 1-2mL of RIPA buffer (150mM sodium chloride, 1% IGEPA CA-630, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl sulfate), 50mM Tris-HCl, pH 8.0, protease inhibitor). Homogenize at low temperature using a homogenizer. After lysis for 30 minutes, transfer the lysate to a 1.5ml centrifuge tube using a pipette. Centrifuge at 12000rpm for 5 minutes at 4℃. Aliquot the supernatant into 0.5ml centrifuge tubes and store at -20℃. 2. BCA Protein Concentration Assay (Beyotime, Catalog No.: P0009): Prepare BSA standards and prepare an appropriate amount of BC working solution by mixing BCA reagent A solution and B solution at a ratio of 50:1. Dilute the sample with pre-cooled PBS. Add the standards and samples to 96-well plates respectively. Add BCA working solution to each well and incubate at 37°C for 30 min. Read the OD value of each well at 562 nm using a microplate reader. Calculate the protein concentration of the sample based on the standard curve and the sample volume used. Adjust all protein samples to the same concentration using RIPA buffer, mix thoroughly, add loading buffer, and then load the samples directly. 3. Electrophoresis: Prepare a 10%–15% SDS-PAGE gel for the lower separating layer. Add the gel to the wells according to the protein concentration of the sample, generally 20–40 μg. Load the sample into the lanes on both sides with an equal volume of 1× loading buffer. Adjust the marker volume to be equal to the sample volume using 1× loading buffer. Perform steady-current electrophoresis at an initial voltage of 45V. When the voltage reaches 65V, switch to steady-voltage electrophoresis. Stop electrophoresis when the target protein has migrated to a distance of more than 1 cm from the bottom edge of the gel. Electrophoresis buffer formulation: Tris-base 3g, glycine 14.4g, 0.1% SDS (10ml 10% SDS) / L; 4. Membrane transfer: Prepare pre-cooled electrotransfer liquid and methanol-activated PVDF membrane in advance. Remove the completed gel block, and place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in the transfer clamp in the order from bottom to top. After fixing, insert the clamp into the transfer electrophoresis core, and ensure that the black plate faces the black plate. Then, place the transfer electrophoresis core into the membrane transfer tank, and add enough transfer buffer to ensure that the clamp is completely immersed in the buffer. Set the transfer current to constant current, 220 mA, and set the transfer time according to the size of the protein. Wet transfer uses a conventional electrotransfer liquid: Tris 3.0 g, Gly 14.4 g, M-OH 200 ml, and deionized water to 1,000 ml; 5. Blocking: At room temperature, block the transferred membrane on a shaker with 5% BSA (TBST prepared) buffer for 1-2 h. TBST: 10x TBS buffer (Savillex, item number: G0015) is diluted with deionized water to 1x, and 1 ml of Tween-20 is added to each liter of 1x TBS buffer to prepare the TBST buffer; 6. Incubation of primary antibody: Dilute the protein antibody (1:1000~1:3000) with the primary antibody diluent (Biyun Tian, item number: P0023A), and incubate overnight at 4 DEG C. Membrane washing: Wash the membrane three times with TBST, each time for 5 min; 7. Incubation of secondary antibody: Prepare the secondary antibody of the corresponding species of the primary antibody (1:10000) using 5% BSA solution dissolved in TBST, and incubate at room temperature for 1-2 h. Membrane washing: Wash the membrane three times with TBST and once with TBS, each time for 10 min; 8. ECL development (Savillex, item number: G2161): In a light-proof environment, prepare the ECL chemiluminescence reagent A liquid and B liquid at a ratio of 1:1, and mix thoroughly. Place the PVDF membrane on the chemical luminescence imager loading platform. Use a pipette to drop an appropriate amount of ECL mixed liquid on the PVDF membrane, and ensure that the working solution uniformly covers the entire blotting membrane. Push the loading platform, set the exposure time, and collect the picture.

[0034] From the above results, it can be seen that the modified polysaccharide water microspheres of the application not only have the function of promoting skin regeneration when applied to a wound, but also have the function of inhibiting wound fibrosis. Therefore, the modified polysaccharide water microspheres simultaneously stimulate epidermis / dermis regeneration + inhibit myofibroblast overactivation, which can accelerate wound healing and reduce scars for burns, significantly improve the appearance and functional recovery of burn or postoperative wounds; block the TGF-beta / Smad pathway, reduce abnormal collagen deposition, reduce the risk of chronic wounds, avoid repeated infections and prolonged wounds; have the dual functions of promoting regeneration + anti-fibrosis, and can overcome the limitations of single mechanism drugs.

[0035] The foregoing description of the embodiments has been presented for the purpose of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed. Modifications and variations are possible in light of the above teachings or can be acquired from practice of the application. As well, the description is presented in the context of the preferred embodiments as of their onset and might not represent the only or even the dominant form of the application. It is intended that the scope of the application embrace all techniques capable of attaining the same results caused by the embodiments described herein, including all equivalents thereof within the scope of the appended claims.

Claims

1. Application of modified aloe polysaccharide water microspheres in the preparation of drugs that inhibit wound skin fibrosis.

2. Application of modified aloe polysaccharide microspheres in the preparation of drugs that promote wound skin regeneration and inhibit wound skin fibrosis.

3. The application as described in claim 1 or 2, characterized in that, The wound is a thermal burn wound on the skin.

4. The application as described in claim 1 or 2, characterized in that, The modified aloe polysaccharide microspheres are obtained by peeling and pulping aloe vera, followed by ultrasonic crushing and concentration to obtain aloe polysaccharide concentrate, and then subjecting it to alternating blue light, red light, and no light light irradiation and emulsification treatment. The alternation time between blue light, red light, and no light is 40-80 minutes, and the total processing time is 24-48 hours.

5. The application as described in claim 4, characterized in that, The illumination process includes the following steps: The concentrated aloe polysaccharide solution obtained by pulping and ultrasonic crushing was placed in a photo-reaction tank for photo-treatment. The distance between the light source and the liquid surface was 60cm~70cm. The photo-treatment was carried out by alternating blue light, red light and no light. The photo-reaction tank was equipped with a circulating water jacket and connected to an external cold water circulation unit. The circulating water temperature was 9℃~12℃. The aloe polysaccharide liquid obtained after light treatment was placed in a high-temperature film-forming tank and the aloe polysaccharide pervaporation film on the surface of the liquid was collected under the high temperature conditions of 80℃~90℃.

6. The application as described in claim 4, characterized in that, Blue LEDs have a wavelength of 450-470nm, while red LEDs have a wavelength of 620-650nm.

7. The application as described in claim 4, characterized in that, The emulsification process includes the following steps: Aloe polysaccharide pervaporation membrane and wheat germ oil were sheared and homogenized at a mass ratio of 1:(1~1.5) for 20h~30h at a temperature of 40℃~50℃. After the process, the oil was removed and the mixture was vacuum dried to obtain the first mixture. The first mixture was sheared and homogenized with pure water at a mass ratio of 1:(1.5~2) for 20h~30h at a temperature of 40℃~50℃. After the process, the oil was removed and the mixture was vacuum dried to obtain the second mixture. The second mixture was sheared and homogenized with silicone oil at a mass ratio of 1:(0.1~0.5) for 20h~30h at a temperature of 40℃~50℃. After the process was completed, the oil was removed and the mixture was vacuum dried to obtain the third mixture. The third mixture is subjected to high temperature and high pressure treatment; In the shear homogenization process, the homogenizer speed is above 4000 r / min.

8. The application as described in claim 7, characterized in that, The rotation speed of the homogenizer in shear homogenization is 4000 r / min to 10000 r / min.

9. The application as described in claim 4, characterized in that, The pulping process includes the following steps: Wash and peel fresh aloe vera leaves, keeping the long strips of aloe vera gel. The aloe vera gel was beaten until there were no obvious particles, resulting in an aloe vera gel turbidity.

10. The application as described in claim 9, characterized in that, Ultrasonic fragmentation includes the following steps: Insert an ultrasonic probe under the surface of the aloe vera gel turbidity and break it up with a power of 120w~150w for 15 minutes, during which the temperature is controlled not to exceed 50℃. The ultrasonically broken aloe vera gel turbidity is added to hot purified water at a volume ratio of 1:5 (80℃~95℃), and the temperature is controlled at 70℃~80℃. The mixture is concentrated to 1 / 3 of its original volume, filtered to remove impurities, and aloe vera polysaccharide concentrate is obtained.

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