Use of hydroxybenzoic acid compounds in the preparation of drugs for treating jellyfish stings

Abstract

The present invention discloses the application of hydroxybenzoic acid compounds in the preparation of drugs for treating jellyfish stings, and relates to the field of biotechnology. The present invention has verified through experiments that: a variety of hydroxybenzoic acid compounds can inhibit the activity of jellyfish toxin phospholipase A2 in vitro, and can also inhibit the cytotoxicity of jellyfish toxin. Some hydroxybenzoic acid compounds can regulate the cellular inflammatory response through the NF-κB and MAPK pathways, and inhibit the release of inflammatory factors interleukin 6 and tumor necrosis factor α by cells. Therefore, the hydroxybenzoic acid compounds provided by the present invention can be used as jellyfish toxin toxicity inhibitors to relieve the local skin reactions caused by jellyfish stings, and thus can be applied to the treatment of jellyfish stings.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the application of hydroxybenzoic acid compounds in the preparation of drugs for treating jellyfish stings. Background Art

[0002] Jellyfish are widely distributed in the seas around the world. They have cnidocytes unique to animals of the phylum Cnidaria, and the nematocysts contained therein can quickly and effectively inject the stored biological toxins into the organisms in contact with them. In recent years, due to reasons such as climate change, the growth and reproduction of jellyfish have shown an explosive trend worldwide, leading to a frequent occurrence of jellyfish sting incidents. It is estimated that there are 150 million jellyfish sting incidents globally every year, and the coastal areas of our country have also become a hard-hit area for jellyfish stings. Only in Qinhuangdao City, thousands of stung tourists are treated every year. After being stung by jellyfish, people will have local reaction symptoms such as skin swelling and pain, as well as systemic symptoms such as difficulty breathing and arrhythmia. In severe cases, it may even lead to death.

[0003] Nemopilema nomurai and Cyanea nozakii are common large jellyfish that can cause harm to humans. The main symptom after being stung by them is the local skin inflammatory reaction, specifically manifested as red stripe, blisters, etc. at the stung area, accompanied by burning pain and itching. The analysis and research on the components and activities of jellyfish toxins show that the main components in the toxins, phospholipase and metalloproteinase, will cause bleeding and tissue necrosis at the stung area, and play an important role in the local reaction caused by stings. As secondary metabolites of plants, polyphenolic compounds play an important role in the growth, reproduction, ultraviolet resistance and pathogen resistance of plants. As an important class of polyphenols, hydroxybenzoic acid compounds have various biological activities such as antioxidant, anti-inflammatory and anti-cancer, and are widely used in the fields of food, medicine and cosmetics, but their application in inhibiting the toxicity of jellyfish toxins and alleviating the local reaction after stings has not been reported. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of hydroxybenzoic acid compounds in the preparation of drugs for treating jellyfish stings, so as to solve the problems existing in the above-mentioned prior art and provide a new therapeutic drug for alleviating the local skin reaction caused by jellyfish stings.

[0005] To achieve the above purpose, the present invention provides the following solution:

[0006] The present invention provides the application of hydroxybenzoic acid compounds in the preparation of drugs for treating jellyfish stings, and the structural formula of the hydroxybenzoic acid compounds is shown as the following formula (I):

[0007]

[0008] In formula (I), R 1 to R 5 are each independently selected from the following groups: hydrogen, hydroxy, and methoxy, and at least one of R 1 to R 5 is hydroxy.

[0009] Preferably, the hydroxybenzoic acid compound is used as a jellyfish toxin toxicity inhibitor to treat jellyfish stings.

[0010] The present invention also provides the use of a hydroxybenzoic acid compound in the preparation of a jellyfish toxin toxicity inhibitor. The structural formula of the hydroxybenzoic acid compound is shown as formula (I) below:

[0011]

[0012] In formula (I), R 1 to R 5 are each independently selected from the following groups: hydrogen, hydroxy, and methoxy, and at least one of R 1 to R 5 is hydroxy.

[0013] Preferably, the hydroxybenzoic acid compound is an effective component of the jellyfish toxin toxicity inhibitor and inhibits the activity of jellyfish toxin phospholipase A 2 activity.

[0014] Preferably, the hydroxybenzoic acid compound includes at least one compound selected from o-hydroxybenzoic acid (A), 3,4-dihydroxybenzoic acid (B), 3,4,5-trihydroxybenzoic acid (C), 2,5-dihydroxybenzoic acid (D), 4-hydroxy-3-methoxybenzoic acid (E), 3-hydroxy-4-methoxybenzoic acid (F), 2-hydroxy-4-methoxybenzoic acid (G), and 3,5-dimethoxy-4-hydroxybenzoic acid (H); the structural formulas of the above compounds are shown as follows:

[0015]

[0016] In the above structural formulas, R is a carboxyl group.

[0017] Preferably, the inhibitor includes a solution, solid, or semi-solid dosage form with the hydroxybenzoic acid compound as an effective component.

[0018] The present invention discloses the following technical effects:

[0019] (1) The present invention provides a new use of hydroxybenzoic acid compounds. Experiments have verified that hydroxybenzoic acid compounds can inhibit jellyfish toxin phospholipase A 2The activity can significantly inhibit the cytotoxicity of jellyfish toxin and can inhibit the inflammatory response induced by jellyfish toxin in cells by regulating related pathways. It can be used as a jellyfish toxin toxicity inhibitor and applied to the treatment of jellyfish stings.

[0020] (2) Various hydroxybenzoic acid compounds such as 3,4-dihydroxybenzoic acid (protocatechuic acid) and 3,4,5-trihydroxybenzoic acid (gallic acid) have pharmacological activities and applications in aspects such as anti-inflammation and antibacterial, and are easy to achieve clinical application transformation, providing new therapeutic drugs for alleviating local skin reactions caused by jellyfish stings. Brief Description of the Drawings

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 For the inhibitory rates of different hydroxybenzoic acid compounds and jellyfish toxin after co-incubation in Examples 1-8 on the PLA 2 activity of jellyfish toxin, A to H respectively represent the experimental groups after co-incubation of o-hydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 4-hydroxy-3-methoxybenzoic acid, 3-hydroxy-4-methoxybenzoic acid, 2-hydroxy-4-methoxybenzoic acid, 3,5-dimethoxy-4-hydroxybenzoic acid with the toxin;

[0023] Figure 2 For the changes in the survival rates of HaCaT cells after pre-incubation of 3,4-dihydroxybenzoic acid (A) and 2,5-dihydroxybenzoic acid (B) at different concentrations with jellyfish toxin in Examples 9-12;

[0024] Figure 3 For the release amounts of IL-6 and TNF-α in HaCaT cells after treatment with 3,4-dihydroxybenzoic acid (A, C) and 2,5-dihydroxybenzoic acid (B, D) at different concentrations after pre-incubation with jellyfish toxin in Examples 13-17. The Control group is the blank group, and the NnV group is the jellyfish toxin control group;

[0025] Figure 4 For the blotting diagrams of the effects of 3,4-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid on the protein expressions in the NF-κB and MAPK pathways in Examples 18-19. The MEM group is the blank group cultured with pure MEM medium, the NnV group is the jellyfish toxin control group, and β-actin is the internal reference protein. Detailed Embodiments

[0026] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as limiting the present invention, but rather as a more detailed description of certain aspects, features, and implementation manners of the present invention.

[0027] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0028] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0029] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of this application are merely exemplary.

[0030] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0031] Example 1 Inhibition of the Activity of Phospholipase A of Jellyfish Toxin by Hydroxybenzoic Acid Compounds 2 Activity

[0032] The cryopreserved jellyfish toxin was thawed at 4°C. 50 μL of a 2 mg / mL jellyfish toxin sample was mixed with 50 μL of a 10 mg / mL o-hydroxybenzoic acid solution and incubated at 25°C for 30 min. Meanwhile, 50 μL of the jellyfish toxin sample was mixed with 50 μL of PBS buffer (10 mM, pH 7.3) and incubated as a positive control; 50 μL of PBS buffer (10 mM, pH 7.3) was mixed with 50 μL of a 10 mg / mL o-hydroxybenzoic acid solution and incubated as a blank control.

[0033] After the incubation, take 25 μL of the incubation mixture and add it to 200 μL of a solution containing 10 mM Tris, 10 mM CaCl 2 , 100 mM NaCl, pH 8.0, and set up three parallel wells. After mixing, add 25 μL of NOBA (1 mg / mL, dissolved in acetonitrile) to make the final concentration of NOBA 0.32 mM. After reacting at 37 °C for 60 min, measure the absorbance of the reaction system at 405 nm. Calculate according to formula (1):

[0034]

[0035] In the formula, A 0 is the absorbance value of the blank group after mixing PBS buffer and the solution of hydroxybenzoic acid compounds;

[0036] A 1 is the absorbance value of the positive control group after mixing the sample of jellyfish toxin and PBS buffer;

[0037] A 2 is the absorbance value of each experimental group after incubating the sample of jellyfish toxin with the solution of hydroxybenzoic acid compounds.

[0038] The results are as Figure 1 shown. The inhibition rate of o-hydroxybenzoic acid on phospholipase A of jellyfish toxin 2 is 83.19 ± 0.56%, indicating that o-hydroxybenzoic acid can significantly inhibit the specific activity of phospholipase A of jellyfish nematocyst toxin 2 .

[0039] Example 2 Inhibition of the activity of phospholipase A of jellyfish toxin by hydroxybenzoic acid compounds 2 The difference from Example 1 is that: replace the o-hydroxybenzoic acid solution with 3,4-dihydroxybenzoic acid solution, and the other steps are the same.

[0040] The results are as

[0041] shown. The inhibition rate of 3,4-dihydroxybenzoic acid on phospholipase A of jellyfish toxin Figure 1 is 61.01 ± 1.31%, indicating that 3,4-dihydroxybenzoic acid can significantly inhibit the specific activity of phospholipase A of jellyfish nematocyst toxin 2 2 .

[0042] Example 3 Inhibition of the activity of phospholipase A of jellyfish toxin by hydroxybenzoic acid compounds 2 The difference from Example 1 is that: replace the o-hydroxybenzoic acid solution with 3,4,5-trihydroxybenzoic acid solution, and the other steps are the same.

[0043] The results are as

[0044] shown. Figure 1 ​As shown, the inhibition rate of 3,4,5-trihydroxybenzoic acid on jellyfish toxin phospholipase A 2 was 64.49 ± 6.34%, indicating that 3,4,5-trihydroxybenzoic acid could significantly inhibit the specific activity of jellyfish nematocyst phospholipase A 2 .

[0045] Example 4 Inhibition of jellyfish toxin phospholipase A by hydroxybenzoic acid compounds 2 activity

[0046] The difference from Example 1 was that the o-hydroxybenzoic acid solution was replaced with a 2,5-dihydroxybenzoic acid solution, and the other steps were the same.

[0047] The results were as Figure 1 shown, and the inhibition rate of 2,5-dihydroxybenzoic acid on jellyfish toxin phospholipase A 2 was 84.05 ± 0.47%, indicating that 2,5-dihydroxybenzoic acid could significantly inhibit the specific activity of jellyfish nematocyst phospholipase A 2 .

[0048] Example 5 Inhibition of jellyfish toxin phospholipase A by hydroxybenzoic acid compounds 2 activity

[0049] The difference from Example 1 was that the o-hydroxybenzoic acid solution was replaced with a 4-hydroxy-3-methoxybenzoic acid solution, and the other steps were the same.

[0050] The results were as Figure 1 shown, and the inhibition rate of 4-hydroxy-3-methoxybenzoic acid on jellyfish toxin phospholipase A 2 was 44.34 ± 3.13%, indicating that 4-hydroxy-3-methoxybenzoic acid could significantly inhibit the specific activity of jellyfish nematocyst phospholipase A 2 .

[0051] Example 6 Inhibition of jellyfish toxin phospholipase A by hydroxybenzoic acid compounds 2 activity

[0052] The difference from Example 1 was that the o-hydroxybenzoic acid solution was replaced with a 3-hydroxy-4-methoxybenzoic acid solution, and the other steps were the same.

[0053] The results were as Figure 1 shown, and the inhibition rate of 3-hydroxy-4-methoxybenzoic acid on jellyfish toxin phospholipase A 2 was 35.86 ± 2.95%, indicating that 3-hydroxy-4-methoxybenzoic acid could significantly inhibit the specific activity of jellyfish nematocyst phospholipase A 2 .

[0054] Example 7 Inhibition of jellyfish toxin phospholipase A by hydroxybenzoic acid compounds 2Activity

[0055] The difference from Example 1 is that the o-hydroxybenzoic acid solution is replaced with a 2-hydroxy-4-methoxybenzoic acid solution, and the other steps are the same.

[0056] The results are as Figure 1 shown. The inhibition rate of 2-hydroxy-4-methoxybenzoic acid on jellyfish toxin phospholipase A 2 is 34.25 ± 1.58%, indicating that 2-hydroxy-4-methoxybenzoic acid can significantly inhibit the specific activity of jellyfish nematocyst phospholipase A 2 .

[0057] Example 8 Inhibition of the activity of hydroxybenzoic acid compounds on jellyfish toxin phospholipase A 2 Activity

[0058] The difference from Example 1 is that the o-hydroxybenzoic acid solution is replaced with a 3,5-dimethoxy-4-hydroxybenzoic acid solution, and the other steps are the same.

[0059] The results are as Figure 1 shown. The inhibition rate of 3,5-dimethoxy-4-hydroxybenzoic acid on jellyfish toxin phospholipase A 2 is 40.70 ± 4.72%, indicating that 3,5-dimethoxy-4-hydroxybenzoic acid can significantly inhibit the specific activity of jellyfish nematocyst phospholipase A 2 .

[0060] Example 9 Effect of hydroxybenzoic acid compounds on the cytotoxicity of jellyfish toxin

[0061] The MTT method was used to test the effect of hydroxybenzoic acid compounds on the cytotoxicity of the toxin. The cells used were human immortalized keratinocyte HaCaT cells.

[0062] After filtering the jellyfish toxin sample and the 3,4-dihydroxybenzoic acid solution with a 0.22 μm filter membrane respectively, they were mixed at a volume ratio of 1:1 (the final concentration of 3,4-dihydroxybenzoic acid was 200 μg / mL, and the final concentration of jellyfish toxin was 12 μg / mL), and pre-incubated in an ice box for 30 minutes. At the same time, the jellyfish toxin sample was mixed and incubated with PBS buffer (10 mM, pH 7.3) as a positive control; only the MEM complete medium was used for cell culture in the well plate as a blank group.

[0063] After the cells in the cell culture flask grew to the logarithmic phase, they were digested with trypsin or scraped off with a cell scraper, and after being evenly pipetted, the cells were seeded into a 96-well plate to make the final concentration 1×10 6cells / mL, and three parallels were set for each group of samples. After culturing the cells in a cell incubator at a constant temperature for 24 h, 100 μL of medium containing incubation solution with different volumes was added to each well. After acting for 24 h, the supernatant was aspirated and discarded with a pipette. 0.5 mg / mL of MTT solution was added to each well, and after continuing to culture in the cell incubator for 4 h, 100 μL of dimethyl sulfoxide (DMSO) was added with a pipette, and the well plate was shaken to continue the reaction until the crystals in the well dissolved. Finally, the absorbance of the sample at 490 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The cell survival rate was calculated according to formula (2):

[0064]

[0065] In the formula, A 1 is the absorbance value of the blank group in which only complete medium was used for culturing cells in the well plate;

[0066] A 2 is the absorbance value of the positive control group incubated with PBS buffer and each experimental group incubated with hydroxybenzoic acid compounds.

[0067] The results are as Figure 2 shown. When only jellyfish toxin acted, the cell survival rate was 40.05 ± 4.37%; after co-incubation of jellyfish toxin with 200 μg / mL of 3,4-dihydroxybenzoic acid, the cell survival rate was 92.03 ± 9.88%, indicating that 3,4-dihydroxybenzoic acid could inhibit the cytotoxicity of jellyfish toxin to HaCaT cells.

[0068] Example 10 Effect of Hydroxybenzoic Acid Compounds on the Cytotoxicity of Jellyfish Toxin

[0069] The difference from Example 9 was that 3,4-dihydroxybenzoic acid with a final concentration of 100 μg / mL was replaced by 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL; other steps were the same.

[0070] The results are as Figure 2 shown. When only jellyfish toxin acted, the cell survival rate was 40.05 ± 4.37%; after co-incubation of jellyfish toxin with 100 μg / mL of 3,4-dihydroxybenzoic acid, the cell survival rate was 85.27 ± 7.71%, indicating that 3,4-dihydroxybenzoic acid could inhibit the cytotoxicity of jellyfish toxin to HaCaT cells.

[0071] Example 11 Effect of Hydroxybenzoic Acid Compounds on the Cytotoxicity of Jellyfish Toxin

[0072] The difference from Example 9 was that 2,5-dihydroxybenzoic acid with a final concentration of 200 μg / mL was replaced by 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL; other steps were the same.

[0073] The results are as follows Figure 2 As shown, when only jellyfish toxin acts, the cell survival rate is 40.05±4.37%; after jellyfish toxin is co-incubated with 200 μg / mL of 2,5-dihydroxybenzoic acid, the cell survival rate is 113.00±12.07%, indicating that 2,5-dihydroxybenzoic acid can inhibit the cytotoxicity of jellyfish toxin to HaCaT cells and has a weak stimulating effect on the proliferation of HaCaT cells.

[0074] Example 12 Effect of hydroxybenzoic acid compounds on the cytotoxicity of jellyfish toxin

[0075] The difference from Example 9 is that 2,5-dihydroxybenzoic acid with a final concentration of 100 μg / mL is replaced by 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL; other steps are the same.

[0076] The results are as follows Figure 2 As shown, when only jellyfish toxin acts, the cell survival rate is 40.05±4.37%; after jellyfish toxin is co-incubated with 100 μg / mL of 2,5-dihydroxybenzoic acid, the cell survival rate is 90.97±14.87%, indicating that 2,5-dihydroxybenzoic acid can inhibit the cytotoxicity of jellyfish toxin to HaCaT cells.

[0077] Example 13 Hydroxybenzoic acid compounds inhibit the secretion of inflammatory factors IL-6 and TNF-α by jellyfish toxin-stimulated cells. The regulation of the release of inflammatory factors by hydroxybenzoic acid compounds in jellyfish toxin-stimulated cells was tested by ELISA method. The cells used were human immortalized keratinocyte HaCaT cells. The specific method is as follows:

[0078] First, standard products of IL-6 and TNF-α at 1000 pg / mL were respectively diluted to 500, 250, 125, 62.5, 31.25, and 15.625 pg / mL according to concentration gradients to measure the standard curves of the two inflammatory factors. Then, HaCaT cells were cultured with the jellyfish toxin sample and the sample pretreated with the mixture of jellyfish toxin and the compound. The above compound was 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL. The cells were plated in a 6-well plate, and the cell dilution factor was the same as that in the 96-well plate (see Example 9), and cultured in an incubator at 37 °C for 24 h; before adding the drugs, the jellyfish toxin sample and the drug sample were thawed at 4 °C and diluted to the target concentration with PBS buffer (10 mM, pH 7.3); 300 μL of jellyfish toxin and 300 μL of the drug were mixed at a volume ratio of 1:1 and incubated at 4 °C for 30 min (for the jellyfish toxin negative control group, 300 μL of toxin was mixed with 300 μL of PBS buffer and incubated); after incubation, the samples of each group were mixed and diluted with MEM medium in proportion, added to the well plate, and cultured in an incubator at 37 °C for 24 h; after 24 h, the cell culture medium was transferred to a sterile centrifuge tube, centrifuged at 1000×g for 10 min at 4 °C, and then the supernatant was equally divided and aliquoted into small EP tubes and stored at -20 °C; the required number of well plate strips were taken out of the kit, washed 3 times and dried by shaking, then 100 μL of the standard product and the test samples were added to the reaction wells, after sealing the plate, incubated at 37 °C for 90 min, then washed the plate successively, added the specific biotinylated antibody for IL-6 or TNF-α, washed the plate, added 100 μL of the specific enzyme conjugate for IL-6 or TNF-α, washed the plate, and finally added 100 μL of the specific chromogenic substrate for IL-6 or TNF-α to the reaction wells, after sealing the plate, developed color at 37 °C in the dark for 15 min; 50 μL of the termination solution was added, and the OD value was measured at a wavelength of 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader within 5 min, and the corresponding concentration levels were calculated from the standard curve, and then the concentration values of each group were compared and analyzed.

[0079] The results are as Figure 3 shown. When only the jellyfish toxin acted, the concentration level of IL-6 was 1271 ± 113.10 pg / mL, and the concentration level of TNF-α was 23.61 ± 3.03 pg / mL; after co-incubation of 200 μg / mL of 3,4-dihydroxybenzoic acid with the jellyfish toxin, the concentration level of IL-6 was 401.60 ± 12.61 pg / mL, and the concentration level of TNF-α was 9.818 ± 0.59 pg / mL; it was indicated that 3,4-dihydroxybenzoic acid could significantly inhibit the secretion of IL-6 and TNF-α by the cells stimulated by the jellyfish toxin.

[0080] Example 14 Inhibition of the Secretion of Inflammatory Factors IL-6 and TNF-α by Hydroxybenzoic Acid Compounds in Cells Stimulated by Jellyfish Toxin

[0081] It is different from Example 13 in that 3,4-dihydroxybenzoic acid with a final concentration of 100 μg / mL is used to replace 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL; other steps are the same.

[0082] The results are as Figure 3 shown. When only the jellyfish toxin acts, the concentration level of IL-6 is 1271 ± 113.10 pg / mL, and the concentration level of TNF-α is 23.61 ± 3.03 pg / mL; after co-incubation with 100 μg / mL of 3,4-dihydroxybenzoic acid and the toxin, the concentration level of IL-6 is 458.50 ± 9.15 pg / mL, and the concentration level of TNF-α is 12.67 ± 1.91 pg / mL; it shows that 3,4-dihydroxybenzoic acid can significantly inhibit the secretion of IL-6 and TNF-α by the cells stimulated by the jellyfish toxin.

[0083] Example 15 Inhibitory effect of hydroxybenzoic acid compounds on the secretion of inflammatory factors IL-6 and TNF-α by cells stimulated by jellyfish toxin

[0084] It is different from Example 13 in that 2,5-dihydroxybenzoic acid with a final concentration of 400 μg / mL is used to replace 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL; other steps are the same.

[0085] The results are as Figure 3 shown. When only the jellyfish toxin acts, the concentration level of IL-6 is 1271 ± 113.10 pg / mL, and the concentration level of TNF-α is 23.61 ± 3.03 pg / mL; after co-incubation with 400 μg / mL of 2,5-dihydroxybenzoic acid and the toxin, the concentration level of IL-6 is 235.90 ± 12.59 pg / mL, and the concentration level of TNF-α is 10.21 ± 2.38 pg / mL; it shows that 2,5-dihydroxybenzoic acid can significantly inhibit the secretion of IL-6 and TNF-α by the cells stimulated by the jellyfish toxin.

[0086] Example 16 Inhibitory effect of hydroxybenzoic acid compounds on the secretion of inflammatory factors IL-6 and TNF-α by cells stimulated by jellyfish toxin

[0087] It is different from Example 13 in that 2,5-dihydroxybenzoic acid with a final concentration of 200 μg / mL is used to replace 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL; other steps are the same.

[0088] The results are as Figure 3As shown, when only jellyfish toxin acts, the concentration level of IL-6 is 1271±113.10 pg / mL, and the concentration level of TNF-α is 23.61±3.03 pg / mL; after co-incubation of 2,5-dihydroxybenzoic acid at 200 μg / mL with the toxin, the concentration level of IL-6 is 265.50±2.81 pg / mL, and the concentration level of TNF-α is 8.61±1.02 pg / mL; indicating that 2,5-dihydroxybenzoic acid can significantly inhibit the secretion of IL-6 and TNF-α by jellyfish toxin-stimulated cells.

[0089] Example 17 Inhibition of the secretion of inflammatory factors IL-6 and TNF-α by jellyfish toxin-stimulated cells by hydroxybenzoic acid compounds

[0090] The difference from Example 13 is that 2,5-dihydroxybenzoic acid with a final concentration of 100 μg / mL is used to replace 3,4-dihydroxybenzoic acid with a final concentration of 200 μg / mL; other steps are the same.

[0091] The results are as Figure 3 As shown, when only jellyfish toxin acts, the concentration level of IL-6 is 1271±113.10 pg / mL, and the concentration level of TNF-α is 23.61±3.03 pg / mL; after co-incubation of 2,5-dihydroxybenzoic acid at 100 μg / mL with the toxin, the concentration level of IL-6 is 298.70±2.16 pg / mL, and the concentration level of TNF-α is 9.79±1.35 pg / mL; indicating that 2,5-dihydroxybenzoic acid can significantly inhibit the secretion of IL-6 and TNF-α by jellyfish toxin-stimulated cells.

[0092] Example 18 Western blot analysis of the effects of hydroxybenzoic acid compounds on the activation of NF-κB and MAPK pathways in HaCaT cells by jellyfish toxin

[0093] Using the HaCaT cell lysate as a sample, the p65 (RelA) protein in the NF-κB pathway and the p38 and ERK1 / 2 proteins in the MAPK pathway were selected for analysis. The cell culture method is the same as that of "Inhibition of phospholipase A by hydroxybenzoic acid compounds in jellyfish toxin" 2For the "activity" part, the hydroxybenzoic acid compound was 3,4-dihydroxybenzoic acid at 200 μg / mL. After the culture ended, the cell culture medium was removed, and the cells in the 6-well plate were washed twice with PBS buffer (10 mM, pH 7.3). 200 μL of cell lysate (mixed proportionally according to the instructions using Ripa lysate, PMSF protease inhibitor, and phosphatase inhibitor) was added to each well, and the adherent cells were shaken evenly, then placed flat in an ice box for 30 min. After that, the bottom of the well was repeatedly pipetted, and the mixture was transferred to a 1 mL EP tube. The centrifuge was pre-cooled to 4 °C and centrifuged at 12,000 rpm for 10 min. The supernatants of parallel samples in the same group were mixed and then sub-packed, with 30 μL sub-packed in each tube. The protein content of each group of samples was measured and stored at -80 °C; the sample volume was calculated according to the loading amount and sample concentration, and the corresponding volume of loading buffer (5×) was added, and it was boiled for 15 minutes. After loading the precast gel of 12% separating gel, electrophoresis was carried out; then transfer, blocking, and washing were performed. The membrane was placed in a rabbit-derived primary antibody dilution solution diluted 1:1000 and incubated overnight at 4 °C. After washing again, a rabbit anti-secondary antibody dilution solution diluted 1:800 was added and incubated on a shaker at room temperature for 60 minutes. After washing again, the washing solution was discarded, and 0.05 mL of ECL working solution was added to each 1 cm 2 membrane, and it was kept in the dark for 2 minutes, then placed in an imaging system for observation and photography. The corresponding bands of each protein were intercepted to compare the color depth and area size. A darker color and a larger area represent a larger amount of protein.

[0094] The results are as Figure 4 shown. In the 3,4-dihydroxybenzoic acid experimental group, the bands corresponding to the proteins NF-κB p65, p38 MAPK, phosphorylated p38 MAPK, ERK1 / 2, and phosphorylated ERK1 / 2 were lighter in color and smaller in area than the corresponding protein bands in the toxin control group, indicating that the expression levels of the above proteins were lower. This shows that 3,4-dihydroxybenzoic acid can inhibit the expression of NF-κB p65 and the p38 and ERK1 / 2 proteins in the MAPK pathway.

[0095] Example 19 Western blot analysis of the effect of hydroxybenzoic acid compounds on the activation of NF-κB and MAPK pathways in jellyfish toxin-activated HaCaT cells

[0096] The difference from Example 18 was that 2,5-dihydroxybenzoic acid at a final concentration of 200 μg / mL was used to replace 3,4-dihydroxybenzoic acid at a final concentration of 200 μg / mL; other steps were the same.

[0097] The results are as Figure 4As shown, the band intensity and area of the β-actin internal reference protein are basically the same. In the 2,5-dihydroxybenzoic acid experimental group, the bands corresponding to NF-κB p65, phosphorylated p38 MAPK, ERK1 / 2, and phosphorylated ERK1 / 2 proteins are lighter in color and smaller in area than the corresponding protein bands in the toxin control group, that is, the expression levels of the above proteins are lower, indicating that 2,5-dihydroxybenzoic acid can inhibit the expression of NF-κB p65 and the p38 and ERK1 / 2 proteins in the MAPK pathway.

[0098] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of hydroxybenzoic acid compounds in the preparation of drugs for treating jellyfish stings, characterized in that, the hydroxybenzoic acid compounds are 3,4-dihydroxybenzoic acid or 2,5-dihydroxybenzoic acid; the hydroxybenzoic acid compounds are used as active components in the drugs.

2. Use of hydroxybenzoic acid compounds in the preparation of jellyfish toxin toxicity inhibitors, characterized in that, the hydroxybenzoic acid compounds are 3,4-dihydroxybenzoic acid or 2,5-dihydroxybenzoic acid; The hydroxybenzoic acid compound is an active component of the jellyfish toxin toxicity inhibitor, inhibiting the phospholipase A of jellyfish toxin 2 activity.

3. The use according to claim 2, characterized in that, the inhibitor includes a solution, solid or semi-solid dosage form with the hydroxybenzoic acid compounds as active components.

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