Application of arachidonic acid in the preparation of medicine for treating burns

Abstract

The present invention discloses the application of arachidonic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating burns. The arachidonic acid proposed in the present invention is applied to the preparation of a medicine for treating burns, which expands the application of arachidonic acid in aspects other than the prevention and treatment of cardiovascular diseases, diabetes and tumors. The present invention experiments show that arachidonic acid can promote the migration and proliferation of fibroblasts and promote the formation of collagen fibers in burn wounds, thereby facilitating the healing of burn wounds and effectively treating deep second-degree burns. Therefore, developing arachidonic acid into a medicine for treating burns has great application potential. In addition, arachidonic acid is a pure natural plant chemical component with relatively small toxic and side effects, and has a large number of extraction sources, which can better reduce the cost of raw materials. Developing this component into a medicine for treating burns has better safety and market benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmacology and medicinal chemistry, and in particular relates to the application of arachidonic acid in the preparation of medicines for treating burns. Background Art

[0002] Burns are common and dangerous sudden diseases in daily life. Traditional burns can be divided into first degree, second degree, third degree or fourth degree, and can also be divided into superficial burns, superficial partial-thickness burns, deep partial-thickness burns and full-thickness burns according to the depth of the burn. For the drug treatment of deep second-degree burns, current research focuses on the development of cell therapy, new dressings, burn ointments, etc. The above drugs have significant therapeutic effects, but they also face problems such as complex materials, time-consuming production, high genetic risks, and immunogenicity.

[0003] Aloe vera contains a variety of active ingredients, mainly phenolic compounds, flavonoid compounds, terpenoids and steroid compounds, sugar compounds, organic acid chemicals, alkaloid compounds, etc. Arachidonic acid (AA) is the active ingredient in aloe vera, and its pharmacological effects reported today include: regulating cardiac excitability, participating in neuroendocrine, promoting cell division, and inhibiting platelet aggregation. The various active ingredients in aloe vera produce a series of special functions, among which aloe vera has certain antibacterial, anti-inflammatory, analgesic and healing effects, and can assist in the treatment of mild burns. However, there are currently no reports on the use of arachidonic acid in the treatment of burns. Summary of the invention

[0004] In order to overcome the problems existing in the prior art, one of the purposes of the present invention is to provide the use of arachidonic acid or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating burns. Another purpose of the present invention is to provide a drug for treating burns.

[0005] The invention prepares arachidonic acid into a spray for treating burn wounds, and experiments have found that arachidonic acid has a good effect in treating burns, thus expanding the new function of arachidonic acid. The invention uses simple materials, is economical and practical, and has a simple and convenient manufacturing process.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the use of arachidonic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating burns.

[0008] The molecular formula of arachidonic acid is C 20 H 32 O2, molecular weight 304.47, CAS registration number: 506-32-1, its structural formula is shown in formula (I):

[0009]

[0010] Preferably, the arachidonic acid is extracted from aloe vera.

[0011] Preferably, the drug for treating burns is a drug that promotes the repair and / or healing of skin wounds caused by burns.

[0012] Preferably, the drug for treating burns is a drug for treating the epidermis and / or dermis in burned skin.

[0013] Preferably, the drug for treating burns is a drug that promotes the formation of collagen fibers in burned skin.

[0014] Preferably, the drug for treating burns is a drug that promotes fibroblast migration;

[0015] Preferably, the drug for treating burns is a drug that promotes fibroblast proliferation.

[0016] Preferably, the pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

[0017] More preferably, the metal salt includes an alkali metal salt and an alkaline earth metal salt. More preferably, the alkali metal salt includes at least one of a sodium salt and a potassium salt. More preferably, the alkaline earth metal salt includes at least one of a calcium salt, a magnesium salt, a barium salt and an aluminum salt. More preferably, the salt formed with an organic base includes a salt formed with the following organic bases: at least one of trimethylamine, triethylamine, pyridine, picoline, 2,6-lutidine, ethanolamine, diethanolamine, triethanolamine, cyclohexylamine, dicyclohexylamine and N,N'-dibenzylethylenediamine. More preferably, the salt formed with an inorganic acid includes a salt formed with the following inorganic acids: at least one of hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid and phosphoric acid. More preferably, the salt formed with an organic acid includes a salt formed with the following organic acids: at least one of formic acid, acetic acid, trifluoroacetic acid, phthalic acid, fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid, succinic acid, malic acid, methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid. More preferably, the salt formed with the basic amino acid includes a salt formed with at least one of the following basic amino acids: arginine, lysine, ornithine. More preferably, the salt formed with the acidic amino acid includes a salt formed with at least one of the following acidic amino acids: aspartic acid, glutamic acid.

[0018] The second aspect of the present invention provides a medicine for treating burns, comprising arachidonic acid or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.

[0019] Preferably, the dosage form of the drug for treating burns is selected from one of solutions, lotions, spirits, tinctures, suspensions, creams, ointments, creams, gels, and medicated excipients.

[0020] Preferably, the auxiliary material includes at least one of a desiccant, a stabilizer, an antioxidant, a disintegrant, a lubricant, a colorant, and a dispersant.

[0021] Substances that can be used as pharmaceutically acceptable excipients include, but are not limited to, ion exchangers; aluminum; aluminum stearate; lecithin; serum proteins, such as human serum albumin; buffer substances such as phosphates; glycine; sorbic acid; potassium sorbate; partial glyceride mixtures of saturated vegetable fatty acids; water; salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts; colloidal silicon; magnesium trisilicate; polyvinyl pyrrolidone; polyacrylates; waxes; polyethylene-polyoxypropylene-blocking polymers; lanolin; sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives Biological materials such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetate; gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycol compounds such as propylene glycol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; and other non-toxic suitable lubricants such as sodium lauryl sulfate and magnesium stearate; coloring agents; release agents; preservatives and antioxidants.

[0022] Preferably, arachidonic acid is used as an active ingredient.

[0023] More preferably, the arachidonic acid is the sole active ingredient.

[0024] The beneficial effects of the present invention are:

[0025] 1. The arachidonic acid proposed in the present invention is used to prepare a drug for treating burns, which expands the application of arachidonic acid in areas other than the prevention and treatment of cardiovascular diseases, participation in neuroendocrine, diabetes and tumors. The experiments of the present invention show that arachidonic acid can promote the migration and proliferation of fibroblasts and promote the formation of collagen fibers in mouse burn wounds, thereby facilitating the healing of mouse burn wounds and effectively treating mice with deep second-degree burns. Therefore, developing arachidonic acid as a drug for treating burns has great application potential.

[0026] 2. Arachidonic acid is extracted from aloe vera and is a pure natural phytochemical component with relatively small toxic side effects. It can also be extracted from different animals and plants, and has many sources, which can better reduce the cost of raw materials. Therefore, developing arachidonic acid into a drug for the treatment of burns has better safety and market benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The target network of aloe vera for treating burns; Figure a shows the common target genes obtained by the intersection of aloe vera target genes and burns target genes; Figure b shows the visualization result of the interaction between aloe vera and burns common target genes; Figure c shows the network diagram of aloe vera active ingredients - aloe vera and burns common target genes;

[0028] Figure 2 The heat map of the binding energy matrix of arachidonic acid docking with the 11 most potential target molecules;

[0029] Figure 3 The molecular docking diagrams of arachidonic acid and gene targets; among them, Figure a is the gene target ESR1; Figure b is the gene target AKT1; Figure c is the gene target SRC; Figure d is the gene target MDM2;

[0030] Figure 4 Figure 1 is a HE staining picture of tissues; Figure a is a HE staining control picture of normal skin tissue on the back of mice; Figure b is a HE staining model picture of burned skin tissue on the back of mice;

[0031] Figure 5 Figure 1 is a schematic diagram of the mouse experimental process and the burn experiment results; Figure a is a schematic diagram of the mouse experimental process; Figure b is photos of burn wounds of mice in different experimental groups at different times; Figure c is a line graph of the diameter of burn wounds of mice; Figure d is a line graph of the healing rate of burn wounds of mice;

[0032] Figure 6 HE staining and local magnification of the tissue sections of the burn wound of mice after 25 days of drug treatment;

[0033] Figure 7 The collagen fiber formation in the mouse burn experiment; Figure a is the Masson staining and local magnification of the mouse burn wound tissue section after 25 days of drug treatment; Figure b is the collagen volume fraction bar graph;

[0034] Figure 8 The results of cell experiments; Figure a is a cell viability bar graph; Figure b is a cell migration bar graph; Figure c is a representative picture of cell scratch experiment;

[0035] In the figures, *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns means not significant. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below through specific examples. The raw materials used in the following examples, unless otherwise specified, can be obtained from conventional commercial sources or prepared and separated by simple synthesis; the processes used, unless otherwise specified, are conventional processes in the art.

[0037] Experimental analysis-pharmacological analysis

[0038] 1. Screening of active ingredients of aloe vera

[0039] The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to analyze the chemical components of aloe vera. The screening condition DL (drug-like property) ≥ 0.18 was set to screen the chemical components of aloe vera. Considering that aloe vera is an external medicine and has extremely low toxicity, WM (molecular size) and OB (oral bioavailability) were not used as screening conditions.

[0040] After screening and prediction, 30 active ingredients of aloe vera were found. After TCMSP screening, 30 active ingredients of aloe vera were found, including arachidonic acid, beta-carotene, lupeol, sitosterol, rutin, aloe-emodin, aloesin, tocopherol, cholesterol (CLR), aloin, quercetin, etc., as shown in Table 1:

[0041] Table 1 Active ingredients of aloe vera

[0042]

[0043]

[0044] Mol ID indicates the number corresponding to the ingredient in the TCMSP database; Molecule Name indicates the full name of the ingredient; DL value, drug-like property, indicates drug-like properties, which refers to the similarity of the compound to known drugs.

[0045] Among them, MOL000471, MOL005053, and MOL005058 were discarded because no target was predicted, and MOL005037, MOL005042, MOL005048, MOL005041, and MOL002328 were discarded because they did not meet the target screening conditions (parameterProbability>0.1). Considering the different algorithms of multiple databases and the standardization and scientificity of the data, other databases were not used for supplementation, and a total of 22 aloe active ingredients were obtained after screening.

[0046] 2. Construction of target network for aloe vera in treating burns

[0047] Aloe target prediction: TCMSP database and Swiss Target Prediction database were used to predict the targets of aloe, and then parameter probability>0.1 was set to screen out targets that met the conditions.

[0048] Collection of burn targets: The human genome database GeneCards, Therapeutic Target Database (TTD) and Online Mendelian Inheritance in Man (OMIM) were used to analyze target information related to Burn.

[0049] The common targets of aloe vera and burns were screened out using Venny2.1.0 website mapping and imported into the STRING database. The protein target type was set to "Homo sapiens" to construct a PPI network. The PPI network was visualized with the help of Cytoscape software, where the "CytoNCA" plug-in was selected and the key targets of aloe vera for treating burns were screened out according to the degree value "Degree" (Degree can reflect the number of links between a node and other nodes in the network, and the betweenness reflects the ratio of the number of paths passing through the node in all the shortest paths in the network to the total number of shortest paths). Similarly, aloe vera, burns, aloe vera active ingredients, and aloe vera burns common intersection targets were imported into Cytoscape software to construct a drug-disease-ingredient-target network diagram, and the effective active ingredients of aloe vera were screened out according to the degree size.

[0050] The predicted aloe targets and BURN targets were intersected using Venny2.1.0 to obtain 85 common target genes, which were used to construct a Venn diagram (see Figure 1a). The obtained common target genes were imported into the STRING database to obtain the PPI network. Data analysis showed that the PPI network had 85 nodes, 180 edges, and an average node degree of 4.45. The Cytoscape software was then used for visualization analysis (see Figure 1 b) According to the degree value, the top 11 targets were selected, namely HSP90AA1, SRC, TP53, HRAS, PIK3CA, EGFR, AKT1, ESR1, MDM2, PTK2, and AR. Aloe vera, burns, aloe vera active ingredients, and common target genes of aloe vera and burns were imported into Cytoscape software to construct a drug-disease-ingredient-target network diagram (see Figure 1 c) Using the CytoNCA plug-in, the aloe vera active ingredient MOL001439arachidonic acid with a high degree value was screened according to the degree value.

[0051] 3. Molecular docking

[0052] Molecular docking of arachidonic acid with HSP90AA1, SRC, TP53, HRAS, PIK3CA, EGFR, AKT1, ESR1, PTK2, AR, and MDM2 targets was performed, and the results of the binding energy matrix thermal analysis were as follows: Figure 2 . The binding energy reflects the possibility of binding between the receptor and the ligand. The lower the binding energy, the higher the affinity between the receptor and the ligand, and the more stable the conformation. The docking binding energies of SRC, ESR1, AKT1, MDM2 and arachidonic acid are -6.8kcal / mol, -7.7kcal / mol, -6.9kcal / mol, and -6.5kcal / mol, which are all lower than the average binding energy of -6.2kcal / mol. Molecular docking analysis showed that arachidonic acid treatment of burns may be related to the gene targets ESR1, AKT1, SRC, and MDM2. The molecular docking diagram of arachidonic acid and gene targets ESR1, AKT1, SRC, and MDM2 is shown in the figure. Figure 3 shown.

[0053] Experimental analysis II Animal experiment and cell experiment analysis

[0054] 1. Experimental supplies

[0055] (1) Animal experiment supplies

[0056] Experimental animals: C57BL / 6J male mice, aged 3-8 weeks, SPF level, purchased from Zhuhai BesTest Biotechnology Co., Ltd. (BesTest), catalog number R-2021-07-C5702-2.

[0057] Arachidonic acid: purity ≥99% (GC), purchased from Guangzhou Shuopu Biotechnology Co., Ltd., brand Aladdin, item number A131025-100mg, CAS number CAS number: 506-32-1. 100mg arachidonic acid was dissolved in 2mL DMSO solution to prepare a mother solution, and then 244μL of the mother solution was added to 100mL 0.9% saline to prepare a 400μmol / L arachidonic acid working solution.

[0058] Burn spray: purchased on JD.com, the approval number is National Medicine Standard Z42021897, and the manufacturer is Jingzhou Jinfeng Pharmaceutical Development Co., Ltd.

[0059] Aloe vera: purchased on Taobao platform, the variety is Aloe vera.

[0060] Jinfeng burn spray: purchased from JD.com, manufactured by Jingzhou Jinfeng Pharmaceutical Development Co., Ltd., product specification 40mL / bottle, product number 10085140262596, approval number: National Medicine Standard Z420211897.

[0061] (2) Cell experiment supplies

[0062] Arachidonic acid: purity ≥99% (GC), purchased from Guangzhou Shuopu Biotechnology Co., Ltd., brand Aladdin, item number A131025-100mg, CAS number CAS number: 506-32-1. 100mg arachidonic acid was dissolved in 2mL DMSO solution to prepare a mother solution, and the mother solution was added to DMEM medium containing 10% FBS to prepare 5μmol / L, 10μmol / L, 20μmol / L, 40μmol / L, 60μmol / L, 80μmol / L, 100μmol / L arachidonic acid working solutions for CCK8 experiments, and the mother solution was added to DMEM containing 2% FBS to prepare 60μmol / L arachidonic acid working solution for cell scratch experiments.

[0063] Premium fetal bovine serum (South America): purchased from Guangzhou Gude Biotechnology Co., Ltd., the brand is Aofan serum, item number FBSKM0503.

[0064] High glucose culture medium DMEM: purchased from Thermo Fisher Life Sciences Products and Services flagship store, brand is GIBCO, product number C11995500BT.

[0065] Trypsin TRYPSIN 0.25% EDTA: purchased from Thermo Fisher Life Science Products and Services Flagship Store, brand GIBCO, product number 25200072.

[0066] 2. Animal Experimental Methods

[0067] (1) Establishment of deep second-degree burn model

[0068] C57BL / 6J male mice were fed in an SPF environment and kept normally for one week to adapt to the environment. The mice were continuously inhaled with isoflurane by means of a small animal anesthesia machine. After the mice were sedated, the hair on the back of the mice was removed with a razor and then the remaining hair was removed with a depilatory cream. The skin was wiped clean with saline and fully exposed. A brass rod heated to 120°C in a constant temperature metal bath was quickly placed on the back skin area for 12 seconds to establish a deep second-degree burn model.

[0069] (2) Experimental groups

[0070] 40 mice with deep second-degree burns were randomly divided into Ctrl, AA, Av, and SSPWJ groups, as shown in Table 2. Ten mice in each group (n=10) were treated with medication. The wounds were washed with saline every day, the weight of the mice was weighed and recorded, the wounds of the mice were photographed and recorded, the diameter of the wounds of the mice was measured, and the medication was given once a day until the wounds of the mice healed. In the Av group, aloe vera was applied to the wounds of the mice using a disposable sterilized cotton swab, and the medication in the other groups was administered in the form of spray. The samples were collected on the 1st, 5th, 10th, 15th, 20th, and 25th days, and the specimens were fixed and stained with HE and Masson.

[0071] Table 2 Experimental groups

[0072] Group Quantity / pcs Dosing times / day Drug forms Ctrl Group 10 3 0.9% saline AA group 10 3 Arachidonic acid solution Av Group 10 3 Aloe Vera Solution SSPWJ Group 10 3 Jinfeng burn spray

[0073] (3) Drug preparation

[0074] The drug concentration was optimized according to the drug effect of the pre-experiment and prepared into a solution, which was made into a spray for administration. The Ctrl group was administered with 0.9% saline; the AA group dissolved 100 mg of arachidonic acid in 2 mL of DMSO solution to prepare a mother solution, and then took 244 μL of the mother solution and added it to 100 mL of 0.9% saline to prepare a 7.4 μmol / L arachidonic acid working solution; the Av group placed fresh Aloe vera leaves in a blender and stirred them into a clean culture dish; the SSPWJ group was administered with a burn spray drug on the market (Jinfeng Burn Spray, National Medicine Standard Z420211897) as a positive control to compare the therapeutic effects.

[0075] (4) Histomorphological experiments

[0076] Tissue paraffin sections: ① Tissue fixation: Obtain the full-thickness skin tissue of surgically removed mice, add 4% paraformaldehyde, and fix at room temperature for 24 hours; ② Discard the 4% paraformaldehyde, rinse repeatedly with running water, and dehydrate step by step with gradient alcohol (50% ethanol (1 hour) - 75% ethanol (1 hour) - 85% ethanol (1 hour) - 95% ethanol (1 hour) - anhydrous ethanol (1 hour)); ③ Anhydrous ethanol: xylene (1:1) (1 hour) - xylene I (30 minutes) - xylene II (30 minutes) n), make the tissue transparent at room temperature; ④ embed the tissue in paraffin with paraffin: xylene (1:1) (2h)-paraffin I (2h)-paraffin II (2h); ⑤ After the embedded tissue blocks are trimmed and numbered, use a microtome to cut wax strips with a thickness of about 4μm; use a dry brush and elbow tweezers to gently transfer the slices to a slide spreader filled with warm water to fully unfold the slices, and then use an anti-shedding slide to pick up the slices; ⑥ Place the paraffin slices prepared above in a 65℃ oven for 3h.

[0077] (5) HE staining

[0078] ① Take the paraffin sections out of the oven, dewax them regularly in water, and soak them in xylene I (8min) - xylene II (8min) - anhydrous ethanol (3min); ② Put the sections in a wet box and soak them in PBS for 5min. ③ Add hematoxylin to the specimen, and rinse with running water until the water is clear after staining for 6min; ④ Differentiate with hydrochloric acid alcohol for 3s and then rinse with running water; ⑤ Stain with eosin for 2min and rinse with running water until the water is clear; ⑥ Rapidly dehydrate with gradient alcohol (75% ethanol (3s) - 95% ethanol (3s) - anhydrous ethanol (3s) - anhydrous ethanol (3s)); ⑦ Transparent with xylene three times, 1-2min each time; ⑧ Put the sections in a fume hood to dry and seal with neutral gum.

[0079] (6) Masson staining

[0080] Masson staining is mainly used to distinguish collagen fibers from muscle fibers, making collagen fibers appear blue (stained with aniline blue) and muscle fibers appear red (stained with Ponceau red and acid fuchsin). It is the most classic connective tissue staining method. The main steps are as follows: ① Dewaxing and hydration: soak in xylene I, II, and III for 10 minutes respectively, for a total of 30 minutes; soak in gradient alcohol (concentration from high to low 100% ~ 100% ~ 95% ~ 75%) for 2 minutes each, for a total of 8 minutes, rinse with distilled water for 30 seconds × 2 times; ② If necessary, use Bouin solution at room temperature for one night or place in a 37℃ incubator for 2 hours for mordant dyeing, and then rinse with running water until the yellow color on the slice disappears; ③ Dye with lapis lazuli blue solution for 3 minutes, rinse with water slightly; ④ Dye with Mayer's hematoxylin solution for 3 minutes, rinse with water slightly; ⑤ Hydrochloric acid and ethanol differentiation for 5 seconds, rinse with running water for 10 minutes; ⑥ Dye with ponceau acid fuchsin solution for 15 minutes, rinse with distilled water slightly; ⑦ Treat with phosphomolybdic acid for about 10 minutes, and pour off the upper liquid; ⑧ Do not wash the slice with water, and directly drop it into aniline blue solution for 4 minutes; ⑨ Treat with glacial acetic acid for 1-2 minutes, dehydrate quickly with 95% ethanol, dehydrate with anhydrous ethanol, make xylene transparent, and seal with neutral gum. Image J was used to calculate the blue collagen fiber area and the total area of ​​the entire tissue. The percentage of the blue collagen fiber area to the total tissue area was used to obtain the collagen volume fraction (CVF), that is, CVF = S 胶原纤维 / S 组织面积 ×100%.

[0081] 3. Cell Experiment Methods

[0082] (1) Cell culture

[0083] Take out the cryopreserved tube containing NIH 3T3 cells from the liquid nitrogen tank, and immediately place it in a 37℃ water bath and shake it slightly to accelerate thawing. After thawing, aspirate the cell suspension, transfer it to a 2mL centrifuge tube, and centrifuge it at 1000rpm for 5min. Discard the supernatant, add 1ml of complete culture medium to the cell pellet after centrifugation, mix it by pipetting, and then transfer it to a T25 culture flask, shake it in an "8" shape until the cell suspension can evenly cover the bottom of the culture flask, and place it in a cell culture incubator (37℃, 5% CO2) for culture.

[0084] (2) Cell passaging and cryopreservation

[0085] Observe the cell growth under an inverted microscope. When the cells grow to 80%, they are subcultured. Discard the old culture medium, add 2ml PBS to wash away the residual culture medium and detached cells, wash twice, add 1ml 0.25% trypsin, digest in the cell culture incubator for 1min, wait until most of the cells fall off, quickly add 1ml complete culture medium to terminate the digestion, and gently blow repeatedly with a pipette, then transfer the cell suspension to a 2ml centrifuge tube and centrifuge at 1000rpm for 5min. Discard the supernatant, add 3ml complete culture medium, inoculate the cell suspension in the culture bottle at a ratio of 1:2, add 1ml cell suspension to each bottle, and place it in the incubator for culture. Repeat the digestion and centrifugation operations during the subculture process, and add 1ml serum-free freezing solution to the centrifuged cell pellet to resuspend it. Pipette the cell suspension into a cryopreservation tube, seal it with a sealing film and mark it, place it in a -80℃ refrigerator overnight, and then transfer it to liquid nitrogen for long-term storage.

[0086] (3) CCK-8 cell proliferation assay

[0087] NIH 3T3 cells in logarithmic growth phase were cultured at 5 × 10 3 The cells were added to a 96-well plate at a density of 100 μl of cell suspension per well, and then placed in an incubator for 24 hours until the cells adhered to the wall. The culture medium was discarded, and 100 μl of basal culture medium was added to each well for starvation treatment for 12 hours. The culture medium was discarded, washed with PBS, and the liquid was discarded. 100 μL of culture medium containing different concentrations of AA solution (5 μmol / L, 10 μmol / L, 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L, 100 μmol / L) was added to each well for continued culture, and the drug was administered and cultured for 24 hours. The drug was discarded, 90 μL of basal culture medium was added to each well, and then 10 μL of CCK-8 was added to each well. After incubation for 1 hour, the absorbance of each well at 450 nm was tested with an enzyme marker. The drug-containing medium without cells was used as the blank well, and the drug-free medium with cells was used as the control well. Cell viability (%) = (OD of experimental group - OD of blank well) / (OD of control group - OD of blank well) × 100%.

[0088] (4) Cell scratch test

[0089] NIH 3T3 cells were cultured at 1 × 10 5The cells were inoculated at a density of 100 cells per well in a 6-well plate, grown to 80% confluence, and starved overnight. Then a 200μL sterile pipette tip was used to scratch the well plate perpendicularly, with uniform force, trying to keep the width of the scratch in each well consistent. The old culture medium was then aspirated, 2mL PBS was slowly added to the wall, and the cells were washed twice to remove floating cell debris. Subsequently, the cells were cultured in a low serum (2% FBS) culture medium containing an AA concentration of 60μmol / L, and then cultured in a 37°C incubator and photographed once at 0, 6, 12, and 24h using a microscope. Six fields of view were selected for each group to take pictures, and the scratch area was measured using Image J. The cell migration rate was calculated according to the following formula: Migration rate (%) = (A0-A t ) / A0×100%, where A0 represents the area of ​​the initial scratch, A t The remaining scratch area at each time point is indicated.

[0090] 4. Experimental results

[0091] (1) The mouse model of deep second-degree burns was successfully established. HE staining results showed that the Ctrl group (see Figure 4 a) The epidermis is clearly structured, with a large number of basal cells and normal morphology, normal morphology of dermal collagen fibers and neat arrangement, and normal morphology and number of skin appendages. Figure 4 b) The epidermis was locally thinned, the structural stratification was not obvious, the cells were degenerated and atrophied, the collagen fibers in the dermis were degenerated and arranged in disorder, the skin accessory glands were degenerated and atrophied, and the number was reduced, indicating that the mouse deep second-degree burn model was successfully established.

[0092] (2) AA group has the effect of treating deep second-degree burns in mice. Figure 5Figure 1 shows the experimental process and burn experimental results of mice; Figure a is a schematic diagram of the experimental process of mice; Figure b is a photo of burn wounds of mice in different experimental groups at different times; Figure c is a line graph of the diameter of burn wounds of mice; Figure d is a line graph of the healing rate of burn wounds of mice. After the model was established, the wound skin of the mice was white and pink, and the periphery of the wound was congested and swollen. On the 5th day, the wound of the mouse was congested, the color became darker, and some wound edges began to scab. On the 10th day, the wound of the mouse began to shrink, and some wound edges began to scab. From the changes in wound diameter and wound healing rate, it can be seen that the AA group, Av group and SSPWJ group were statistically significant compared with the Ctrl group. On the 15th day, the wound of the mouse shrank significantly, and the AA group and Av group had completely scabbed. Compared with the Ctrl group, it can be seen that the wound healing rate of the AA group and Av group was significantly increased. After that, the wound healing of the mouse tended to be flat, but it was better than Ctrl. On the 20th day, the Ctrl group had completed scabbing, while the wounds of the other groups had basically completed epithelialization. On the 25th day, the wounds in the AA, Av and SSPWJ groups had completely healed, while the wounds in the Ctrl group had not completely healed, indicating that the AA group had the effect of treating deep second-degree burns in mice.

[0093] (3) AA has the effect of promoting wound healing in mice with burns. Figure 6 HE staining results of paraffin sections treated with different experimental groups. The results showed that after 25 days of drug treatment, the epidermis of the Ctrl group recovered well with clear layers. However, the wound recovery was not complete, the epidermis was thicker, the keratinized layer was relatively thicker, and the number of blood vessels in the dermis was relatively small. Compared with the Ctrl group, the wounds of the AA group, Av group and SSPWJ group recovered completely, the epidermis recovered well with clear layers, granulation tissue formed in the dermis, the number of blood vessels increased, the collagen fibers were neatly arranged, and the skin accessory glands and nerves recovered well.

[0094] (4) AA promotes the formation of collagen fibers in burn wounds of mice. Masson staining results showed that the collagen fiber content in the Ctrl group was less than that in the AA group, Av group, and SSPWJ group (see Figure 7 a), while the collagen volume fraction (CVF) results showed that the AA group, Av group and SSPWJ group were significantly higher than those of the Ctrl group (P values ​​were 0.0014, 0.0007 and 0.0011, respectively), among which the CVF of the AA group was not significantly different from that of the Av group and the SSPWJ group (see Figure 7 b). This indicates that AA can promote the formation of collagen fibers in burn wounds of mice, that is, AA has the effect of promoting the healing of burn wounds of mice.

[0095] (5) CCK8 results showed that cell viability was best when the AA concentration reached 60 μM. The gradient range of AA was set between 0 μmol / L and 100 μmol / L. The CCK-8 experimental results (see Figure 8 a) It can be found that with the increase of AA concentration, cell viability also gradually increased. When AA reached 60μM, cell viability was the best, and then cell viability gradually decreased. Compared with the Ctrl group, cell viability increased after adding AA (except AA = 100μmol / L), indicating that AA may have the function of promoting fibroblast proliferation.

[0096] (6) The results of the cell migration experiment showed that the AA (60 μmol / L) group promoted the migration of fibroblasts compared with the Ctrl group. The cell scratch experiment was used to observe the migration effect of AA on fibroblasts (NIH 3T3). At the 6th hour, the cell migration rate of the AA group was higher than that of the Ctrl group, but there was no statistical difference (P=0.1756). At the 12th and 24th hours, it can be seen that the cell migration rate of the AA group was significantly higher than that of the Ctrl group and there was a statistical difference (P=0.0005, P<0.0001). This indicates that after the addition of AA, the migration ability of fibroblasts is enhanced (see Figure 8 b and c).

[0097] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. Use of arachidonic acid or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating burns, characterized in that: The medicine for treating burns is a medicine for promoting the repair and / or healing of burned skin wounds; the medicine for treating burns is a medicine for promoting the proliferation and migration of fibroblasts.

2. The use according to claim 1, characterized in that: The arachidonic acid is extracted from aloe vera.

3. The use according to claim 1, characterized in that: The medicine for treating burns is a medicine for treating the epidermis and / or dermis in burned skin.

4. The use according to claim 1, characterized in that: The medicine for treating burns is a medicine that promotes the formation of collagen fibers in burned skin.

5. The use according to claim 1, characterized in that: The pharmaceutically acceptable salt includes at least one of a metal salt, an ammonium salt, a salt formed with an inorganic acid, a salt formed with an organic base, a salt formed with an organic acid, a salt formed with a basic amino acid, and a salt formed with an acidic amino acid.

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