Application of brown algae polysaccharide sulfate in the preparation of drugs for treating jellyfish stings

Abstract

The invention discloses the application of fucoidan sulfate in the preparation of a drug for treating jellyfish stings, and belongs to the technical field of biopharmaceuticals. The invention uses fucoidan sulfate to effectively inhibit the death of skin cells caused by jellyfish toxins and the expression of intracellular inflammatory factor mRNA, effectively inhibit the stimulation of jellyfish toxins on inflammatory cells and inflammatory factors in the body, and can effectively treat skin damage caused by jellyfish toxins, and can further be used as a drug for preparing jellyfish dermatitis, thereby broadening the application scope of fucoidan sulfate.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to the application of brown algae polysaccharide sulfate in the preparation of medicines for treating jellyfish stings. Background Art

[0002] In recent years, the global outbreak of jellyfish has not only caused disorder in the marine ecosystem, but also brought serious impacts on human production and life. Jellyfish belong to the phylum Cnidaria, which are marine aquatic animals with cnidocytes. Once humans come into contact with jellyfish, the toxins in the cnidocytes will quickly penetrate the skin and cause stings, causing local skin symptoms such as pain, burning, swelling and blisters. These skin symptoms are called jellyfish dermatitis, which is the most direct harm caused by jellyfish stings. Jellyfish stings affect daily life and work, and have become the most common marine biological injury.

[0003] Basically, jellyfish dermatitis is the result of the combined effects of various components in jellyfish toxins, accompanied by the production of inflammation in the body. Therefore, inhibiting the inflammatory stimulation of jellyfish toxins is the key to treating jellyfish dermatitis. At present, there is no drug specifically for jellyfish dermatitis at home and abroad. Most of them use folk remedies, proven prescriptions, or refer to other types of dermatitis for treatment, which has poor effects and even aggravates the condition. Therefore, there is an urgent need for a drug to treat jellyfish dermatitis. Summary of the invention

[0004] The purpose of the present invention is to provide the use of brown algae polysaccharide in the preparation of drugs for treating jellyfish stings, so as to solve the problems existing in the above-mentioned prior art. Through experimental verification, it is found that brown algae polysaccharide can reduce the number of inflammatory cells, reduce the mRNA expression level and protein expression amount of inflammatory factors, and reduce the skin damage caused by jellyfish stings, and can be used in the preparation of drugs for treating jellyfish dermatitis.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides application of brown algae polysaccharide sulfate in preparing a medicine for treating jellyfish dermatitis caused by jellyfish stings.

[0007] The present invention provides application of brown algae polysaccharide sulfate in preparing a medicine for treating skin damage caused by jellyfish stings.

[0008] Preferably, the above-mentioned brown algae polysaccharide can treat dermatitis or skin damage caused by jellyfish stings by reducing the number of inflammatory cells and reducing the mRNA expression level and protein expression amount of inflammatory factors.

[0009] The above-mentioned brown algae polysaccharide sulfate can inhibit the death of human embryonic skin fibroblasts (CCC-ESF-1) caused by jellyfish toxins, reduce the mRNA expression of inflammatory factors IL-6, TNF-α and MCP-1 in human embryonic skin fibroblasts (CCC-ESF-1) after the action of jellyfish toxins, reduce the number of inflammatory cell leukocytes and lymphocytes in the body after the action of jellyfish toxins, and reduce the protein expression of inflammatory factors IL-6, TNF-α and MCP-1 in the body after the action of jellyfish toxins, thereby reducing the skin irritation of jellyfish toxins to treat dermatitis or skin damage caused by jellyfish stings.

[0010] Preferably, the fucoidan sulfate is dissolved in sterile water and then diluted with 10 mM phosphate buffer.

[0011] Preferably, the concentration of the fucoidan sulfate is in the range of 1-5000 μg / mL.

[0012] The present invention also provides the use of fucoidan sulfate in preparing medicine for inhibiting jellyfish toxins.

[0013] The present invention discloses the following technical effects:

[0014] The present invention uses fucoidan sulfate to effectively inhibit the death of skin cells (human embryonic skin fibroblasts CCC-ESF-1) caused by jellyfish toxins and the expression of inflammatory factor mRNA, and effectively inhibits the stimulation of jellyfish toxins on the body's inflammatory cells and inflammatory factors. In addition, fucoidan sulfate may promote the proliferation of human embryonic skin fibroblasts and has a promoting effect on the treatment of mouse skin damage caused by jellyfish stings. It can then be used as a drug for preparing jellyfish dermatitis, broadening the application range of fucoidan sulfate, and also laying a foundation for the development of drugs for treating jellyfish stings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0016] Figure 1is the effect of fucoidan sulfate on the survival rate of human embryonic skin fibroblasts (CCC-ESF-1) under different mass ratios of fucoidan sulfate and jellyfish toxin (calculated as protein), wherein Control represents the blank group, NnNV represents the jellyfish toxin control group, 1 represents the 1 μg / mL fucoidan sulfate treatment group, 5 represents the 5 μg / mL fucoidan sulfate treatment group, and 10 represents the 10 μg / mL fucoidan sulfate treatment group;

[0017] Figure 2 The inhibition of jellyfish toxin on the expression of inflammatory factor mRNA in human embryonic skin fibroblasts (CCC-ESF-1) by fucoidan sulfate, wherein Control represents the blank group, NnNV represents the jellyfish toxin control group, and NnNV+FPS represents the jellyfish toxin and fucoidan sulfate treatment group;

[0018] Figure 3 The effect of fucoidan sulfate on the inhibition of jellyfish toxin on the number of inflammatory cells in the body, where Control represents the blank group, NnNV represents the jellyfish toxin control group, and NnNV+FPS represents the jellyfish toxin and fucoidan sulfate treatment group;

[0019] Figure 4 The fucoidan sulfate inhibits the expression of inflammatory factor proteins in the body induced by jellyfish toxins, wherein Control represents the blank group, NnNV represents the jellyfish toxin control group, and NnNV+FPS represents the jellyfish toxin and fucoidan sulfate treatment group;

[0020] Figure 5 The brown algae polysaccharide sulfate is effective in treating skin damage caused by jellyfish toxins, wherein a is the control group, b is the sting group, and c is the treatment group. DETAILED DESCRIPTION

[0021] Now, various exemplary embodiments of the present invention are described in detail. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared by conventional methods unless otherwise specified. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, features and embodiments of the present invention.

[0022] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] In the following description, the methods involved are conventional methods in the art unless otherwise specified, and the raw materials involved are raw materials that can be obtained from public commercial channels unless otherwise specified.

[0027] Example 1 Experimental Materials

[0028] 1.1 Preparation of Fucoidan Sulfate Solution

[0029] Take 6 mg of fucoidan sulfate and dissolve it with 1 mL of sterile water to prepare a 6 mg / mL fucoidan sulfate solution; then dilute the 6 mg / mL fucoidan sulfate solution with 10 mM phosphate buffer (NaH2PO4-Na2HPO4, pH 7.4) to prepare fucoidan sulfate solutions with concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL and 3 mg / mL, respectively.

[0030] 1.2 Preparation of jellyfish toxin solution

[0031] After the frozen jellyfish tentacle tissue (sampled from Laoshan District, Qingdao City) was taken out of the -80℃ refrigerator, it was placed in a 4℃ refrigerator to thaw and autolyze for 2-4 days. During this period, in order to accelerate the dissolution of the jellyfish tentacles, the upper layer of seawater was carefully poured out every other day and fresh seawater was added to accelerate the shedding of the nematocyst cells from the tentacles. After the jellyfish tissue was completely autolyzed, the upper self-solution was slowly poured out, and the lower self-solution with precipitate was collected, and then filtered with 60 mesh and 100 mesh sieves respectively. The filtrate was collected, centrifuged at 3000g for 15 minutes, the lower precipitate was collected, and then washed with clean seawater 2-3 times and centrifuged to obtain a relatively pure nematocyst. The nematocyst cells were placed in a -80℃ refrigerator for later use.

[0032] The nematocysts were extracted with 20 mM phosphate buffer (NaH2PO4-Na2HPO4, pH 7.4) pre-cooled at 4°C at a ratio of 1:3 (w / v). After the nematocysts were broken, they were centrifuged at 4°C and 15,000 g for 30 min, and the supernatant was the jellyfish toxin solution.

[0033] The determination of jellyfish toxins was carried out by the Folin-phenol method (Zhang Lei, Liu Yu, Jiang Dahe, Yang Mingyuan, Cao Zhijian. Biochemical Experiment Guide [M]. Wuhan: Wuhan University Press, 2011.95-98.), with bovine serum albumin (BSA) as the standard. The toxins were then diluted to 50 μg / mL and 1 mg / mL with 20 mM phosphate buffer (NaH2PO4-Na2HPO4, pH 7.4) precooled at 4°C.

[0034] 1.3 Preparation of human embryonic skin fibroblast (CCC-ESF-1) culture medium

[0035] Add 125 mL of fetal bovine serum and 6.3 mL of cell culture double antibody (penicillin-streptomycin mixture) to 500 mL of DMEM high-glucose medium.

[0036] 1.4 Preparation of cell reaction termination solution

[0037] Weigh 20 g of sodium dodecyl sulfate (SDS) and dissolve it in 200 mL of distilled water, then add 0.176 mL of hydrochloric acid with a mass concentration of 37%.

[0038] Example 2: Sulfated Fucoidan Inhibits the Mortality of Human Embryonic Skin Fibroblasts (CCC-ESF-1) Induced by Jellyfish Toxins

[0039] Take 1 μg / mL of the fucoidan sulfate solution in Example 1, mix them evenly with 50 μg / mL of jellyfish toxin in a volume ratio of 1:1, react at 37° C. for 30 minutes, and use them as mixed solution A for later use;

[0040] 100 μL of 1×10 5100 μL of mixed solution A, 100 μL of a 1:1 mixture of jellyfish toxin and human embryonic skin fibroblast culture medium (control), and 100 μL of human embryonic skin fibroblast culture medium (blank) were added to a 96-well plate containing 100 μL / mL human embryonic skin fibroblasts. After reacting for 24 h, the supernatant was removed, MTT was added, and after reacting for 4 h, the stop solution was added. A was measured with an ELISA reader within 24 h. 490nm The reaction was set up in triplicate and the cell viability was calculated according to the following formula:

[0041] Cell survival rate (%) = A 490nm，样品 / A 490nm，空白 ×100

[0042] The results showed (see Figure 1 ), when the mass ratio of brown algae polysaccharide sulfate to jellyfish toxin (calculated as protein) was 1:50, the cell survival rates were 92.28%, 87.97%, and 87.26%, respectively, with an average value of 89.17%±1.81%, which was higher than the cell survival rate of 66.17% in the control group, indicating that brown algae polysaccharide sulfate can inhibit the death of human embryonic skin fibroblasts (CCC-ESF-1) caused by jellyfish toxin.

[0043] Example 3: Sulfated Fucoidan Inhibits the Mortality of Human Embryonic Skin Fibroblasts (CCC-ESF-1) Induced by Jellyfish Toxins

[0044] Take 5 μg / mL of the fucoidan sulfate solution in Example 1, mix them evenly with 50 μg / mL of jellyfish toxin in a volume ratio of 1:1, react at 37°C for 30 minutes, and use them as mixed solution B for later use;

[0045] 100 μL of 1×10 5 100 μL of mixed solution B, 100 μL of a 1:1 mixture of jellyfish toxin and human embryonic skin fibroblast culture medium (control), and 100 μL of human embryonic skin fibroblast culture medium (blank) were added to a 96-well plate containing 100 μL / mL human embryonic skin fibroblasts. After reacting for 24 h, the supernatant was removed, MTT was added, and after reacting for 4 h, the stop solution was added. A was measured with an ELISA reader within 24 h. 490nm The reaction was set up in triplicate and the cell viability was calculated according to the following formula:

[0046] Cell survival rate (%) = A 490nm，样品 / A 490nm，空白 ×100

[0047] The results showed (see Figure 1), when the mass ratio of fucoidan sulfate to jellyfish toxin (calculated as protein) was 1:10, the cell survival rates were 106.21%, 89.73%, and 97.97%, respectively, with an average value of 97.97%±5.49%, which was higher than the cell survival rate of 66.17% in the control group, indicating that fucoidan sulfate can inhibit the death of human embryonic skin fibroblasts (CCC-ESF-1) caused by jellyfish toxins. At the same time, the cell survival rate of 106.21% suggests that fucoidan sulfate may promote the proliferation of human embryonic skin fibroblasts and has a promoting effect on the treatment of mouse skin damage caused by jellyfish stings.

[0048] Example 4: Sulfated Fucoidan Inhibits the Mortality of Human Embryonic Skin Fibroblasts (CCC-ESF-1) Induced by Jellyfish Toxins

[0049] Take 10 μg / mL of the fucoidan sulfate solution in Example 1, mix them evenly with 50 μg / mL of jellyfish toxin in a volume ratio of 1:1, react at 37° C. for 30 minutes, and prepare the mixture as mixed solution C for standby use;

[0050] 100 μL of 1×10 5 100 μL of mixed solution C, 100 μL of a 1:1 mixture of jellyfish toxin and human embryonic skin fibroblast culture medium (control), and 100 μL of human embryonic skin fibroblast culture medium (blank) were added to a 96-well plate containing 100 μL / mL human embryonic skin fibroblasts. After reacting for 24 h, the supernatant was removed, MTT was added, and after reacting for 4 h, the stop solution was added. A was measured with an ELISA reader within 24 h. 490nm The reaction was set up in triplicate and the cell viability was calculated according to the following formula:

[0051] Cell survival rate (%) = A 490nm，样品 / A 490nm，空白 ×100

[0052] The results showed (see Figure 1 ), when the mass ratio of brown algae polysaccharide sulfate to jellyfish toxin (calculated as protein) was 1:5, the cell viability were 85.46%, 82.86%, and 80.23%, respectively, with an average value of 82.85%±1.75%, which was higher than the cell viability of 66.17% in the control group, indicating that brown algae polysaccharide sulfate can inhibit the death of human embryonic skin fibroblasts (CCC-ESF-1) caused by jellyfish toxin.

[0053] Example 5: Fucoidan sulfate inhibits the expression of inflammatory factors mRNA in human embryonic skin fibroblasts (CCC-ESF-1) induced by jellyfish toxins

[0054] 5 μg / mL of fucoidan sulfate solution and 50 μg / mL of jellyfish toxin were mixed evenly in a volume ratio of 1:1, and reacted at 37°C for 30 minutes to prepare the mixed solution D for later use;

[0055] 1 mL of 1×10 5 1 mL of mixed solution E, 1 mL of a 1:1 volume ratio of jellyfish toxin and human embryonic skin fibroblast culture medium (control), and 1 mL of human embryonic skin fibroblast culture medium (blank) were added to a culture dish containing 100 cells / mL human embryonic skin fibroblasts. After reacting at 37°C for 24 h, the supernatant was removed, the cell RNA was extracted and reverse transcribed into cDNA, and the mRNA expression levels of inflammatory factors IL-6, TNF-α, and MCP-1 in human embryonic skin fibroblasts were determined according to the qRT-PCR method (Pfaffl, MW (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 29(9), 45e–45. https: / / doi.org / 10.1093 / nar / 29.9.e45). The reaction was set up in triplicate.

[0056] like Figure 2 As shown, when 5 μg / mL of fucoidan sulfate solution was mixed with 50 μg / mL of jellyfish toxin, the expression level of inflammatory factor mRNA in human embryonic skin fibroblasts (CCC-ESF-1) after the action of jellyfish toxin was reduced.

[0057] Example 6: Fucoidan sulfate inhibits jellyfish toxins from stimulating inflammatory cells in the body

[0058] 100 μg / mL of fucoidan sulfate solution and 50 μg / mL of jellyfish toxin were mixed evenly in a volume ratio of 1:1, and reacted at 37°C for 30 minutes to prepare the mixed solution E for later use;

[0059] One day before the experiment, the back skin of Kunming mice was depilated with 8% sodium sulfide. The mice were subcutaneously injected with 40 μL of mixed solution F, 40 μL of a mixture of jellyfish toxin and PBS mixed in a volume ratio of 1:1 (control), and 40 μL of PBS (blank). Blood was taken from the mice 6 hours later to determine the number of inflammatory cell leukocytes and lymphocytes in the blood. The reaction was set up in 5 parallels.

[0060] like Figure 3 As shown, when 100 μg / mL of fucoidan sulfate solution is mixed with 50 μg / mL of jellyfish toxin, the number of inflammatory cells in the body after the action of jellyfish toxin can be reduced.

[0061] Example 7: Sulfated Fucoidan Inhibits the Expression of Inflammatory Factor Proteins Induced by Jellyfish Toxins

[0062] 100 μg / mL of fucoidan sulfate solution and 50 μg / mL of jellyfish toxin were mixed evenly in a volume ratio of 1:1, and reacted at 37°C for 30 minutes to prepare a mixed solution F for later use;

[0063] One day before the experiment, the back skin of Kunming mice was depilated with 8% sodium sulfide. Mice were subcutaneously injected with 40 μL of mixed solution G, 40 μL of a mixture of jellyfish toxin and PBS mixed in a volume ratio of 1:1 (control), and 40 μL of PBS (blank). After 6 hours, the blood of the mice was taken and centrifuged (4000×g, 5min) to obtain the supernatant, and the protein expression of inflammatory factors IL-6, TNF-α and MCP-1 in the supernatant was determined. The reaction was set up in 5 parallels.

[0064] like Figure 4 As shown, when 100 μg / mL of fucoidan sulfate solution is mixed with 50 μg / mL of jellyfish toxin, the protein expression of inflammatory factors in the body after the action of jellyfish toxin can be reduced.

[0065] Example 8 Fucoidan sulfate effectively treats skin damage caused by jellyfish stings

[0066] One day before the experiment, the back skin of Kunming mice was shaved with a shaver, and care was taken not to damage the skin. The mice were subcutaneously injected with 100 μL of 1 mg / mL jellyfish toxin. After the stinging symptoms appeared, 500 μL of 3 mg / mL fucoidan sulfate was applied to the sting site of the mice for a total of 5 days, twice a day, 30 minutes each time, with an interval of 6 hours. After the treatment, photos were taken and recorded, and the skin tissue on the back of the mice was sliced ​​to observe the microscopic manifestations of the skin.

[0067] like Figure 5 As shown, after 5 days of treatment with fucoidan sulfate, the symptoms of jellyfish stings were significantly alleviated, indicating that fucoidan sulfate can effectively reduce the skin irritation of jellyfish toxins, and the results of tissue sections also showed that fucoidan sulfate can significantly reduce the stimulating effect of jellyfish toxins on inflammatory cells in mouse skin tissue.

[0068] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of brown algae polysaccharide sulfate in the preparation of drugs for inhibiting jellyfish toxins.

Images