Application of antler protein targeting NF [kappa] B-p65 molecule in resisting jellyfish toxin death

Abstract

The invention provides application of an NF [kappa] B-p65 molecule-targeted antler protein in resisting jellyfish toxin death, and belongs to the technical field of biological medicines. Experiments prove that the cornu cervi pantotrichum protein has remarkable anti-hemolytic activity, the survival rate of mice can be remarkably increased, damage of jellyfish toxin to important viscera is effectively inhibited, meanwhile, the level of inflammation-related factors in serum of the mice is reduced, and the total antioxidant capacity and antioxidant enzyme activity in the serum are improved. Cell experiments show that after cells are treated by the jellyfish toxin, the antler protein can improve the cell viability, improve the oxidation resistance of the cells, reduce the phosphorylation level of NF kappa B-p65 in total protein, cytoplasm and cell nucleus protein in the cells, reduce the expression of inflammatory factors such as IL-15, Cxcl2, CCL4, TNF-alpha and the like, and relieve inflammatory response caused by the jellyfish toxin. The invention provides a new idea for treating jellyfish sting.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of velvet antler protein targeting NFκB-p65 molecules in resisting death caused by jellyfish toxins. Background Art

[0002] Sand jellyfish belongs to the phylum Cnidaria, class Scyphozoa, order Rhizostomata. It is a poisonous jellyfish widely distributed near the coastlines and estuaries of the western and central Pacific and Atlantic oceans. The stinging cells of sand jellyfish are highly toxic. Mild stings will cause local symptoms such as redness, swelling, heat, pain, epidermal necrosis, and unbearable pain. Severe injuries or those with allergic constitutions will immediately develop erythema, urticaria, blisters, ecchymosis, and even epidermal necrosis. Moderate or severe stings are rare, but can cause systemic poisoning symptoms such as difficulty breathing and shock, which are life-threatening. Therefore, it is particularly necessary to provide a sting treatment drug.

[0003] Deer antler (Cervus nippon Temminck) refers to the young antlers of male sika deer that are not ossified but with velvet. It is a precious Chinese medicinal material. Deer antler contains phospholipids, glycolipids, gelatin, hormones, fatty acids, amino acids, proteins, calcium, phosphorus, magnesium, sodium and other ingredients, of which amino acids account for more than half of the total ingredients. Ancient doctors believed that the essence of deer lies in the horns, and antlers are the tender buds of the horns. The qi is full but not released, so it has the strongest effect of replenishing yang and blood. Li Shizhen of the Ming Dynasty said in the "Compendium of Materia Medica" that deer antler "is good at replenishing the kidney and strengthening yang, producing essence and blood, replenishing marrow and strengthening bones." However, there are no reports on the application of deer antler in jellyfish stings and its mechanism of action. Summary of the invention

[0004] The purpose of the present invention is to provide application of velvet antler protein targeting NFκB-p65 molecule in resisting death caused by jellyfish toxin.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides application of velvet antler protein in preparing medicine for reducing organ damage after jellyfish stings, wherein the organ includes heart, liver or kidney.

[0007] The invention also provides the use of velvet antler protein in preparing a medicine for improving the total antioxidant capacity and antioxidant enzyme activity of the body after being stung by jellyfish.

[0008] Preferably, the antioxidant enzymes include superoxide dismutase and catalase.

[0009] The present invention also provides the use of velvet antler protein in preparing a medicine for reducing the phosphorylation level of NFκB-p65 in body organs after jellyfish stings.

[0010] The present invention also provides the use of velvet antler protein in the preparation of a drug for reducing inflammatory factors in cells, wherein the cells are cells treated with jellyfish toxins.

[0011] Preferably, the inflammatory factors include IL-6, IL-1β and TNF-α.

[0012] The present invention also provides the use of velvet antler protein in preparing a drug for reducing the phosphorylation level of NFκB-p65 in cells, wherein the cells are cells treated with jellyfish toxin.

[0013] The preparation method of the velvet antler protein comprises the following steps:

[0014] (1) degreasing the velvet antlers using pure ether and ethanol solutions in sequence;

[0015] (2) subjecting the defatted velvet antler to reflux extraction with 30-40% by volume ethanol for 2-3 times, each time for 1.5-2.5 hours, and collecting the extract;

[0016] (3) mixing the extract with ethanol until the volume fraction of ethanol in the extract is 75 to 85%, and then standing for 8 to 12 hours to obtain a mixed solution;

[0017] (4) The mixed solution is centrifuged at 4000-5000 rpm for 8-12 min, and the precipitate is obtained, which is the velvet antler protein.

[0018] Preferably, the volume fraction of the ethanol solution is 90-98%, the mass volume ratio of the velvet antler and pure ether is 1 g: 8-12 mL, and the mass volume ratio of the velvet antler and the ethanol solution is 1 g: 8-12 mL.

[0019] By adopting the above technical scheme, the present invention has the following beneficial effects: the present invention has experimentally confirmed that velvet antler protein has significant anti-hemolytic activity, can significantly improve the survival rate of mice, and effectively inhibit the damage of sand jellyfish to important organs, while reducing the level of inflammatory-related factors in the blood of mice, and improving the total antioxidant capacity and antioxidant enzyme activity in serum. Cell experiments show that after the Raw 264.7 cells are treated with jellyfish toxin, velvet antler protein can improve cell viability, improve the antioxidant capacity of cells, and reduce the expression of inflammatory factors such as NFκB-p65, IL-15, Cxcl2, CCL4, TNF-α, etc. The present invention provides a new idea for the treatment of jellyfish stings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a survival analysis diagram of ICR mice;

[0021] Figure 2 This is the evaluation chart of creatine kinase (CK) index in ICR mice;

[0022] Figure 3 This is the evaluation chart of total bilirubin (TBIL) index in ICR mice;

[0023] Figure 4 This is the evaluation chart of direct bilirubin (DBIL) index in ICR mice;

[0024] Figure 5 This is the evaluation chart of the creatinine (CREA) index of ICR mice;

[0025] Figure 6 This is the evaluation chart of alanine aminotransferase (ALT) index in ICR mice;

[0026] Figure 7 This is the analysis diagram of MDA activity in ICR mouse serum;

[0027] Figure 8 This is a graph analyzing TSOD activity in serum of ICR mice;

[0028] Fig. 9 This is a graph analyzing CAT activity in ICR mouse serum;

[0029] Fig.10 This is an analysis of IL-6 content in serum of ICR mice;

[0030] Fig.11 This is an analysis of IL-1β content in serum of ICR mice;

[0031] Fig.12 This is an analysis of TNF-α content in serum of ICR mice;

[0032] Fig.13 HE staining of the heart, liver, and kidney of ICR mice;

[0033] Fig.14 This is the immunohistochemical staining of NFκB-p65 phosphorylation;

[0034] Fig.15 This is the result diagram of hemolysis analysis;

[0035] Fig.16 This is a statistical chart of RAW264.7 cell viability;

[0036] Fig.17 For the determination of MDA levels in RAW264.7 cells;

[0037] Fig.18 For the determination of TSOD levels in RAW264.7 cells;

[0038] Fig.19 For the determination of CAT levels in RAW264.7 cells;

[0039] Fig. 20 Images showing the detection of ROS levels in RAW264.7 cells using DCFH-DA staining;

[0040] Fig.21 Fluorescence quantitative statistics of ROS levels in RAW264.7 cells;

[0041] Fig. 22 Flow cytometry determination of ROS levels in RAW264.7 cells;

[0042] Fig.23 Flow cytometry statistics of ROS levels in RAW264.7 cells;

[0043] Fig.24 Statistics of differentially expressed genes in RAW264.7 cells affected by DAP;

[0044] Fig.25 Heat map analysis of inflammation-related differentially expressed genes in RAW264.7 cells suppressed by DAP;

[0045] Fig.26 This is a WB analysis of DAP inhibiting p-IKK / IKK and p-p65 / p65 in RAW264.7 cells;

[0046] Fig. 27 The quantitative graph is for DAP inhibition of p-IKK / IKK in RAW264.7 cells;

[0047] Fig.28 The quantitative graphs are for DAP inhibition of p-p65 / p65 in RAW264.7 cells;

[0048] Fig.29 This is a statistical diagram of the relative expression of IL-6 in RAW264.7 cells inhibited by DAP;

[0049] Fig.30 This is a statistical diagram of the relative expression of IL-1β in RAW264.7 cells inhibited by DAP;

[0050] Fig.31 This is a statistical graph showing the relative expression of TNF-α in RAW264.7 cells inhibited by DAP. DETAILED DESCRIPTION

[0051] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0052] The instruments used in the embodiments of the present invention are as follows:

[0053] High-speed centrifuge, pipette, and Vortex-5 vortex vortexer were purchased from Eppendorf AG, Germany;

[0054] HH-501(A) super constant temperature water bath was purchased from Baita Xinbao Instrument Factory, Jintan District;

[0055] The carbon dioxide incubator CLM-170B-8-CN was purchased from Jiapo Yisi High Technology Co., Ltd.;

[0056] Clean bench W-CJ-D was purchased from Zhengzhou North-South Instrument Equipment Co., Ltd.

[0057] Constant temperature water bath DK-8B was purchased from Shanghai Jinghong Experimental Equipment Co., Ltd.;

[0058] Analytical electronic balance FA1004 was purchased from Shanghai Hengping Scientific Instrument Co., Ltd.;

[0059] Fluorescence microscope Axio Vert.A1 was purchased from Zeiss Technology Co., Ltd.;

[0060] Snow ice machine SZB-20 was purchased from Shanghai Kuncheng Scientific Instrument Co., Ltd.;

[0061] Low speed centrifuge: Xiangyi L500;

[0062] Handheld centrifuge: SCILOGEX S1010E;

[0063] Ultra-low temperature refrigerator: SANYO MDF-U32V(N);

[0064] Inverted biological microscope: BM-37XC;

[0065] Ultrapure water machine: Think-Lab Labonova UltraPro;

[0066] Vortex mixer: SCILOGEX SCI-VS;

[0067] Horizontal shaker: Biyuntian TS-200;

[0068] Low temperature refrigerator: Haier BCD-532WGHSS8EL9U1;

[0069] ICR mice were purchased from the Animal Center of Shanghai Naval Medical University;

[0070] RAW264.7 cells were purchased from Shanghai Kuisai Biotechnology Co., Ltd.

[0071] Example 1

[0072] Preparation of sand jellyfish toxin: Jellyfish samples were mainly collected from sand jellyfish (Nemopilema nomurai) in Wafangdian City, Dalian City, Liaoning Province. The tentacles were cut off and placed in dry ice and quickly transported back to the laboratory. Thawed at 4°C, during which time the jellyfish samples were placed on a magnetic stirrer and stirred continuously for 72 hours to autolyze. After the jellyfish samples were autolyzed, they were filtered with a 200-mesh sieve, and the filtrate was centrifuged at 4°C and 1,000g for 15 minutes. The supernatant was dialyzed in 1×PBS for 8 hours and the protein concentration was determined. Finally, it was divided into 15mL centrifuge tubes and stored at -80°C for use, which is the prepared jellyfish toxin (TE).

[0073] Preparation of velvet antler protein: The samples used in the present invention are all purchased from the two-bar velvet antlers of Shuangyang Deer Farm in Jilin Province. The ratio of velvet antler raw material to extraction reagent is 1:10 during protein extraction. The samples are defatted for 3 hours using ether and 95% ethanol in turn, and the ethanol is evaporated from the residue. Then, 35% ethanol is used for 3 times of reflux extraction, 2 hours each time, and the filtrates are combined. The alcohol concentration is adjusted to 80% (v / v), and the solution is kept at 4°C overnight. The precipitate is centrifuged at 4500rpm and 4°C for 10 minutes. The alcohol on the surface of the precipitate is evaporated until no alcohol remains. The total protein of sika deer, referred to as DAP, is obtained by freeze-drying.

[0074] Example 2 Evaluation of the protection of velvet antler protein against jellyfish toxin-induced death in mice

[0075] ICR mice were randomly divided into 4 groups, namely PBS group, DAP high-dose group (50 mg / kg), DAP medium-dose group (30 mg / kg) and DAP low-dose group (15 mg / kg), with 5 mice in each group. The body weight of each ICR mouse was weighed and recorded every day, and then 0.2 mL of DAP solution of different concentrations was gavaged, while the control group was gavaged with the same amount of PBS. The body weight and gavage were continuously weighed and gavaged for one week. The ICR mice that were gavaged with DAP for 7 consecutive days were injected with TE through the tail vein, and the survival rate of the mice was counted.

[0076] The results are as follows Figure 1 As shown. Figure 1 It can be seen that the survival rate of mice in the different doses of DAP gavage groups was significantly higher than that of the single TE injection group. The most significant doses were 50 mg / kg>30 mg / kg>15 mg / kg, and 50 mg / kg of DAP could even increase the survival rate by more than 50%. Therefore, 50 mg / kg of DAP was used in the subsequent TE antagonism experiment.

[0077] Example 3

[0078] The experiment was divided into Con group, DAP group, TE group and DAP+TE group, with 6 mice in each group. The ICR mice in the DAP group and DAP+TE group were gavaged with 50 mg / kg DAP solution for one week in advance, and the mice in the TE group and DAP+TE group were injected with 1.52 mg / kg TE by tail vein on the 8th day, and the Con group was injected with an equal amount of 1×PBS solution. Fresh blood was collected 6 hours later for blood biochemical index detection.

[0079] 1. Determination of blood biochemical indicators in mice

[0080] The heart indexes: CK, LDH; the liver indexes: AST, ALT; the kidney index: Crea were measured.

[0081] The results are as follows Figures 2 to 6 The results showed that after DAP intervention, the cardiac function indexes CK, LDH, liver function indexes AST, ALT and renal function Crea showed a downward trend compared with the TE group, and it was statistically significant.

[0082] 2. Determination of Antioxidant Capacity in Mice

[0083] Centrifuge part of the mouse serum at 3,000 rpm for 15 min to obtain the supernatant, which is the serum, and place it on ice for testing.

[0084] Catalase (CAT) activity assay: Catalase assay kit (ELISA) was used. 10 μL of sample and 190 μL of working solution were added to a 96-well UV plate, mixed, and the initial absorbance value A1 at 240 nm and the absorbance value A2 after 1 min were recorded. Calculate ΔA=A1-A2, the blank control group was defined as 100%, and the ratio of each group was calculated.

[0085] Lipid oxidation (MDA) activity assay:

[0086] The lipid oxidation detection kit (Shanghai Biotech Co., Ltd.) was used for determination. 0.1 mL of 1×PBS was added to the centrifuge tube as a blank control, and 0.1 mL of TBA standard of different concentrations was added to prepare the standard curve. In the experimental group, 0.1 mL of each sample lysate was added for determination. Then 0.2 mL of MDA detection working solution was added and mixed, and heated at 100°C for 15 min. Cooled to room temperature in a water bath, centrifuged at 1,000 g for 10 min at room temperature. 200 μL of supernatant was added to a 96-well plate, and then the absorbance was measured at A532 nm using an enzyme reader. After calculating the molar concentration of MDA according to the standard curve, it was divided by the tissue weight to represent the MDA content in the sample.

[0087] Superoxide dismutase (SOD) activity assay:

[0088] The superoxide dismutase detection kit (Yisheng Biotechnology (Shanghai) Co., Ltd.) was used for determination. Evenly mix 151μL SOD detection buffer, 8μL WST-8 and 1μL enzyme solution to obtain the WST-8 / enzyme working solution. Dilute by adding 39μL SOD detection buffer to every 1μL reaction start solution (40×), and mix to obtain the reaction start working solution. Refer to Table 1 to set up sample wells and various blank control wells using a 96-well plate. Add the sample to be tested and other various solutions in sequence according to the table below. After adding the reaction start working solution, mix thoroughly. After incubation at 37°C for 30 minutes, measure the absorbance at A450 nm. Inhibition percentage = (A blank control 1-A sample) / (A blank control 1-A blank control 2)×100%.

[0089] Table 1 Settings of different treatment groups

[0090]

[0091] The results are as follows Figures 7 to 9 As shown in the figure, TE can reduce the activity of antioxidants CAT and SOD in mouse serum, and induce the release of lipid peroxide MDA. In the DAP gavage group, the activity of antioxidants CAT and SOD increased, while the content of lipid peroxide MDA decreased, showing good antioxidant activity.

[0092] 3. Mouse serum ELISA analysis

[0093] Set up standard wells and sample wells, add 50μL of detection standards of different concentrations to each standard well, add 10μL of the sample to be tested to the sample well first, then add 40μL of sample diluent, and do not add to the blank well. Except for the blank wells, add 100μL of horseradish peroxidase (HRP)-labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate in a 37°C water bath for 60min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution and let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat 5 times. Add 50μL of substrates A and B to each well and incubate at 37°C in the dark for 15min. Add 50μL of stop solution to each well, and within 15min, add A 450nm The OD value of each well was measured at the wavelength, and the content of IL-6, IL-1β and TNF-α was determined.

[0094] The results are as follows Figures 10-12 The results showed that the representative inflammatory factors IL-6, IL-1β and TNF-α in the blood of mice in the TE group were significantly upregulated, while those in the DAP group were weakened, indicating that DAP can reduce the content of inflammatory factors IL-6, IL-1β and TNF-α in mice.

[0095] 4. Histopathological examination of mouse heart, liver and kidney

[0096] The hearts, livers, and kidneys of mice in the Con, TE, DAP, and DAP+TE groups were collected and fixed in paraformaldehyde fixative, and the sections were routinely dewaxed to water. Hematoxylin stain and stain were used in sequence for 5 to 8 minutes, and then rinsed with water. Then, eosin stain was used for 1 to 5 minutes and rinsed with water again. After differentiation and anti-blue treatment, the sections were dehydrated by gradient ethanol (70%, 80%, 90%, 100%) and anhydrous ethanol, with each step taking about 10 to 20 seconds and 1 to 2 minutes. The sections were dehydrated by gradient ethanol (70%, 80%, 90%, 100%) and anhydrous ethanol, with each step taking about 10 to 20 seconds and 1 to 2 minutes, and finally sealed with neutral gum sealant.

[0097] Results from Fig.13 It can be seen that the heart tissue of mice in the TE group had obvious pathological damage compared with the Con group, with myocardial fiber degeneration and bleeding; the pathological changes in liver tissue were the most obvious, with broken cord-like structures, vacuolar deformation, inflammatory cell infiltration, and diffuse bleeding; the kidney tissue was least affected by pathological changes, with bleeding in the glomeruli. However, after DAP administration, the heart, liver, and kidneys all showed significant relief and recovery.

[0098] Example 4 Effect of velvet antler protein on the phosphorylation level of NFκB-p65 in multiple organs induced by jellyfish toxin

[0099] The hearts, livers and kidneys of mice in the Con group, TE group, DAP group and DAP+TE group were collected and fixed in paraformaldehyde fixative, and the sections were routinely dewaxed to water. The sections were incubated in Hydrogen Peroxide Block for 10-15 minutes and washed with buffer for 5 minutes / 2 times. After adding Ultra V Block, incubate at room temperature for 5 minutes to block nonspecific background staining. After blocking, add an appropriate amount of p-NFκB-p65 primary antibody working solution and incubate at 37℃ for 1-2 hours. After washing twice, add Primary Antibody Enhancer and incubate at room temperature for 20 minutes. Add enzyme-labeled secondary antibody and incubate at room temperature for 30 minutes, wash with buffer for 5 minutes / 2 times, add 1 drop of DAB Plus Chromogen to 1mL DAB Plus Substrate, mix well and add to the sections, and incubate for 3-15 minutes. Rinse thoroughly with tap water, counterstain for 1 minute, dehydrate until transparent after color development, seal with neutral gum, and finally observe and photograph under a fluorescence microscope.

[0100] The results showed that compared with the control group, the p65 phosphorylation levels in the heart, liver, and kidney of the TE group were greatly aggravated, while the mice in the DAP group showed significant improvement or even recovery ( Fig.14 ).

[0101] Example 5 Evaluation of the effect of velvet antler protein on antagonizing the hemolytic activity of jellyfish TE

[0102] 1×PBS was used to prepare DAP solutions of different concentration gradients (5mg / mL, 3mg / mL, 1.5mg / mL, 0.5mg / mL, 0.15mg / mL, 0.05mg / mL and 0mg / mL). 100μL of red blood cell suspension and 100μL of DAP solutions of different concentrations were added to each tube and gently blown evenly, and then placed in a 37℃ incubator for 1h. The blank group was an equal amount of 1×PBS group. Then, an equal amount of 5mg / mL TE solution was added and continued to be incubated in a 37℃ incubator for 1h, and then centrifuged at 2,000g for 5min, and 100μL of supernatant was taken to a 96-well plate, and its optical absorption value (OD) at 415nm was measured. 415nm The calculation formula of hemolytic activity is: hemolytic activity (%) = (A experimental group - A blank group) / (A control group - A blank group) × 100%.

[0103] The results are as follows Fig.15 As shown by Fig.15 It can be seen that DAP can significantly alleviate TE-induced erythrocyte hemolysis, showing an obvious dose-effect relationship.

[0104] Example 6 Evaluation of the Antler Protein in Antagonizing the Toxicity and Oxidative Damage of Jellyfish Toxins on RAW264.7 Cells

[0105] Select Raw 264.7 cells with good growth for cell experiments, and dilute DAP to 5mg / mL, 3mg / mL, 1.5mg / mL, 0.5mg / mL, 0.15mg / mL, 0.05mg / mL and 0mg / mL using DMEM gradient. The culture medium in the 96-well plate was aspirated and replaced with 100μL DAP solution of different concentration gradients, where the blank control group was 100μL DMEM solution without cells, and the negative control group was 100μL DMEM group containing cells. After incubation for 2h, it was discarded, and the experimental group was replaced with TE solution, and the control group was an equal amount of DMEM solution. After incubation for another 2h, it was discarded and replaced with 100μL 10% cck8 solution. After 1h, the light absorption value at 450nm was detected by an ELISA instrument, where the blank group was DMEM culture medium without cells, the control group was DMEM culture medium containing cells, and the 0mg / mL group was the TE group.

[0106] The formula for calculating cell viability is: cell viability (%) = (A experimental group - A blank group) / (A control group - A blank group) × 100%. At the same time, the TSOD, CAT and MDA contents of each group of cells were detected.

[0107] The results are as follows Figures 16 to 19 The results showed that the viability of RAW264.7 cells increased with the increase of DAP concentration, TSOD and CAT activities decreased after TE stimulation and then increased in the DAP group, while lipid peroxide MDA showed the opposite trend.

[0108] At the same time, according to 6×10 5 Each well was evenly spread on a 12-well plate and cultured overnight at 37°C and 5% CO2. The culture medium in the 12-well plate was replaced with 5 mg / mL DAP and incubated for 2 hours, then discarded and replaced with 0.04 mg / mL TE solution and continued to be cultured for 2 hours. The culture medium was discarded and DCFH-DA was diluted with serum-free culture medium at 1:1000 to a final concentration of 10 μM. The cell culture medium was removed and the cells were gently washed twice with a pre-cooled 1× PBS solution, each time for 5 minutes. After adding 1 mL of diluted DCFH-DA to evenly cover the cells, they were incubated in a 37°C cell culture incubator for 20 minutes. The cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that did not enter the cells. The cells in each group were collected and ROS were detected by flow cytometry, while another cell well plate was observed and photographed under a fluorescence microscope.

[0109] The results are as follows Figures 20-23 The results showed that after TE stimulated Raw264.7 cells, Raw264.7 cells produced a large amount of reactive oxygen species, the intensity of which was even close to that of the positive control Rosup. After DAP intervention, the fluorescence value of ROS was reduced to varying degrees, and it had a certain statistical significance.

[0110] Example 7 Transcriptomic analysis of antler protein against jellyfish toxin-induced cytotoxicity in Raw264.7 cells

[0111] Preparation of 5 × 10 5 / 2mL RAW264.7 cell suspension was evenly spread in a 6-well plate. After culturing for 24 hours, the culture medium was replaced with 2mL 5mg / mL DAP and incubated for 2 hours, and then replaced with 2mL 0.04mg / mL TE and incubated for 2 hours. The control group was replaced with DMEM solution without fetal bovine serum and cultured at 37°C for 2 hours. Then, total RNA was extracted, sequenced and analyzed for cell samples in each group.

[0112] The results are as follows Figures 24-25Compared with the TE group, DAP intervention led to the upregulation of 689 genes and the downregulation of 495 genes in RAW264.7 cells, and these differentially expressed genes included inflammatory factors such as NFκB-p65, IL-15, Cxcl2, CCL4, and TNF-α.

[0113] Example 8 Western blot analysis of the effect of velvet antler protein on the apoptosis of Raw264.7 cells induced by jellyfish toxin

[0114] In order to further prove the antagonistic effect of DAP on TE-induced apoptosis of Raw264.7 cells, this example uses protein immunoblot analysis to explore the classic apoptosis indicators. The cells in the 6-well plate were successively incubated with DAP and TE solutions for total protein extraction. First, the culture medium in the culture plate was discarded, and after washing 3 times with pre-cooled 1×PBS, 100 μL of SDS lysis mixture containing protease inhibitors, phosphatase inhibitors and PMSF was added to each well, and the mixture was blown several times with a gun. After uniform stirring, the lysate was aspirated into a 1.5mL centrifuge tube and immediately inserted into an ice box. The cell lysate was broken for 3s under a 65W cell ultrasonic disruptor. Continue to stand on ice for 5 minutes, then centrifuge at 4°C 14,000g for 5 minutes, and take the supernatant, which is the total cell protein. After BCA quantitative analysis of the protein, 10% SDS polyacrylamide gel electrophoresis was used, and the constant voltage was 80V first, and 120V after 30 minutes until bromophenol blue ran to the bottom of the gel 1cm for equal separation. Then transfer the gel to the NC membrane in the order of positive electrode-sponge-NC membrane-gel-sponge-negative electrode and transfer the membrane at 100V for 90 minutes. Keep it in an ice bath during the transfer process. After the transfer, incubate with protein-free fast blocking solution at room temperature for 1 hour. Add the primary antibody of the target protein including GAPDH, Bax, and Bcl-2 and incubate overnight at 4°C. Then recover the primary antibody, wash it 3 times with 1×TBST, add the secondary antibody and incubate it at room temperature for 2 hours. Finally, use the ECL luminescence kit to detect the protein expression level, and use imag J software for band analysis.

[0115] The results are as follows Figures 26-28 The results showed that TE led to increased phosphorylation of NFκB-p65 and upstream IKK protein, while the total protein levels of NFκB-p65 and IKK did not change significantly. However, after DAP intervention, the phosphorylation of NFκB-p65 and upstream IKK protein was alleviated.

[0116] Example 9 RT-qPCR Verification

[0117] Total RNA of Raw264.7 cells after DAP incubation and TE treatment was extracted for nucleic acid quantification. Reverse transcription system was configured: the RNA volume required for 1 μg was calculated based on the unified RNA concentration (1 / sample concentration × 1000). 4 μL 5×RT mix and 1 μg RNA were added to each tube, and 20 μL of the total system was made up with DEPC water for reverse transcription. The reaction parameters were set as: 37°C for 15 min, 98°C for 5 min, and 4°C for 11 min. Subsequently, cDNA was diluted with 480 μL DEPC water for qPCR determination. 8 μL cDNA, 1 μL 10 μM F (Forward) primer, 1 μL 10 μM R (Reverse) primer, and 10 μL SYBR were added to eight rows and mixed evenly. The primer sequences are shown in Table 2. GAPDH was used as an internal reference, and n = 8. The qPCR reaction system is: pre-denaturation at 95°C for 5 min, amplification 40 times: 94°C for 20 s, 60°C for 20 s, 72°C for 30 s, and finally click Start run.

[0118] The calculation formula of RT-qPCR is 2^-△△C t , where △C t =C t目的基因 -C t内参基因 , △△C t =△C t实验组 -△C t对照组 .

[0119] Table 2 Primer sequences

[0120]

[0121] The results are as follows Figures 29 to 31 As shown in the results, the mRNA expression levels of IL-6, IL-1β and TNFα in the TE group were significantly upregulated compared with the control group, and the mRNA expression levels of IL-6, IL-1β and TNFα were decreased after DAP intervention, which were all statistically significant.

[0122] It can be seen from the above examples that in the present invention, the velvet antler protein has significant anti-hemolytic activity, can significantly improve the survival rate of the body, and effectively inhibit the damage of jellyfish toxins to important organs, while reducing the level of inflammatory-related factors in serum and improving the total antioxidant capacity and antioxidant enzyme activity in serum.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of velvet antler protein in the preparation of a drug for reducing organ damage after jellyfish stings, wherein the organ includes the heart, liver or kidney.

2. Application of velvet antler protein in the preparation of drugs for improving the body's total antioxidant capacity and antioxidant enzyme activity after jellyfish stings.

3. The use according to claim 2, characterized in that: The antioxidant enzymes include superoxide dismutase and catalase.

4. Application of velvet antler protein in the preparation of drugs for reducing the phosphorylation level of NFκB-p65 in body organs after jellyfish stings.

5. Application of velvet antler protein in the preparation of drugs for reducing inflammatory factors in cells, wherein the cells are cells treated with jellyfish toxins.

6. The use according to claim 5, characterized in that The inflammatory factors include IL-6, IL-1β and TNF-α.

7. Use of velvet antler protein in the preparation of a drug for reducing the phosphorylation level of NFκB-p65 in cells, wherein the cells are cells treated with jellyfish toxin.

8. The method for preparing velvet antler protein according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) degreasing the velvet antlers using pure ether and ethanol solutions in sequence; (2) subjecting the defatted velvet antler to reflux extraction with 30-40% by volume ethanol for 2-3 times, each time for 1.5-2.5 hours, and collecting the extract; (3) mixing the extract with ethanol until the volume fraction of ethanol in the extract is 75 to 85%, and then standing for 8 to 12 hours to obtain a mixed solution; (4) The mixed solution is centrifuged at 4000-5000 rpm for 8-12 min, and the precipitate is obtained, which is the velvet antler protein.

9. The preparation method according to claim 8, characterized in that: The volume fraction of the ethanol solution is 90-98%, the mass volume ratio of the velvet antler and pure ether is 1g:8-12mL, and the mass volume ratio of the velvet antler and the ethanol solution is 1g:8-12mL.

Images