Application of oxymatrine in preparation of medicine for inhibiting jellyfish toxin

Abstract

The invention provides application of oxymatrine in preparation of a medicine for inhibiting jellyfish toxin, and belongs to the technical field of biological medicine. Screening tests are carried out on multiple drugs, and it is found that the oxymatrine OMT has a better antagonistic effect on TE toxin than other drugs, and cytotoxicity is not exposed. Further research finds that oxymatrine can reduce H9C2 cell apoptosis, antagonize cell apoptosis and oxidative damage caused by the ormopilema esculentum toxin, increase the survival rate of mice injected with the ormopilema esculentum toxin and improve mouse heart tissue damage caused by the ormopilema esculentum toxin, has huge potential in preparation of TE antagonistic drugs, and can be used for preparing TE antagonistic drugs. The medicine can be used as a candidate medicine for treating the stings of the sandfish.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of oxymatrine in the preparation of drugs for inhibiting jellyfish toxins. Background Art

[0002] Jellyfish stings are one of the most common types of marine life injuries, which have a great impact on tourists or marine operations and may cause serious consequences. In recent years, many cases of jellyfish stings have been reported in coastal areas. Cardiotoxicity caused by jellyfish stings is considered to be the main cause of death, but the mechanism of cardiac toxicity is still unclear, which also brings difficulties to the development of drugs for the treatment of jellyfish stings.

[0003] Nemopilema nomurai is a poisonous jellyfish widely distributed in my country's coastal waters. In recent years, this jellyfish has caused some serious fatal and injury incidents. Studies have found that stings from Nemopilema nomurai can cause acute heart failure in a short period of time. The toxins induce inflammatory responses, oxidative damage, and hemolysis. In vivo models show that the toxins seriously affect the cardiovascular system, causing dysfunction of cardiac tissue and exposing selective toxicity to the heart. Although the cardiotoxicity of Nemopilema nomurai toxins has been confirmed by in vivo and in vitro models, related research is still limited. Similar to most jellyfish-related studies, there is currently a lack of further elaboration on the cardiotoxic effects and mechanisms of Nemopilema nomurai, which to a certain extent increases the difficulty of diagnosing jellyfish stings and creates a dilemma of a lack of emergency treatment options.

[0004] Oxymatrine (OMT, CAS NO: 16837-52-8) is a natural alkaloid with multiple pharmacological properties, including antiviral, antibacterial and antitumor activities. It has good antioxidant and anti-inflammatory abilities and has a good protective effect on the heart. However, there is no relevant research on the use of alkaloids to antagonize jellyfish toxins. Summary of the invention

[0005] The purpose of the present invention is to provide a new medicine for inhibiting jellyfish toxin.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The invention provides application of oxymatrine in preparing medicine for inhibiting jellyfish toxin.

[0008] Preferably, the jellyfish toxin is produced by sand jellyfish.

[0009] The present invention also provides the use of oxymatrine in preparing medicine for treating or preventing the functional disorder of cardiac tissue caused by jellyfish toxin poisoning.

[0010] Preferably, the dysfunction of the cardiac tissue is cardiac tissue damage.

[0011] Preferably, the cardiac tissue damage includes cardiac tissue edema, myocardial cell apoptosis, myofiber lysis, rupture or inflammatory cell infiltration.

[0012] Beneficial Effects

[0013] The present invention studies the inhibitory effects of different compounds on TE cytotoxicity and confirms that oxymatrine (OMT) is an effective TE antagonist, which provides a good idea for developing therapeutic drugs for inhibiting jellyfish stings from natural compounds, and is of great significance for developing innovative drugs for jellyfish stings with high efficiency and low toxicity.

[0014] The present invention finds that oxymatrine can reduce H9C2 cell apoptosis, antagonize cell apoptosis and oxidative damage caused by sand jellyfish toxin, improve the survival rate of mice injected with sand jellyfish toxin, and improve the heart tissue damage of mice caused by sand jellyfish toxin. It has great potential in preparing TE antagonist drugs and can be used as a candidate drug for treating sand jellyfish stings. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The changes in cell morphology of H9C2 cells after TE treatment for 1 h in Example 1 (a) and the changes in cell viability of H9C2 cells after TE treatment of different concentrations for 1 h (n=3) (b).

[0016] Figure 2 This is the cell apoptosis condition of H9C2 cells after TE treatment for 1 hour in Example 1.

[0017] Figure 3 The data are for the statistics of apoptosis of H9C2 cells after TE treatment for 1 h in Example 1 (n=3).

[0018] Figure 4 The changes in cellular ROS levels in H9C2 cells after TE treatment for 1 hour in Example 1.

[0019] Figure 5 The statistics of cellular ROS levels in H9C2 cells after TE treatment for 1 h in Example 1 (n=3).

[0020] Figure 6 The changes of MDA and T-SOD levels in H9C2 cells after TE treatment for 1 hour in Example 1 (n=3)

[0021] Figure 7 The changes in cell viability of H9C2 cells after being treated with different concentrations of OMT for 1 h in Example 2.

[0022] Figure 8The changes in cell viability after H9C2 cells were simultaneously treated with different concentrations of OMT and TE for 1 hour in Example 2.

[0023] Fig. 9 The changes in cell morphology of H9C2 cells after being treated with 5 μg / mL TE and 1 mM OMT for 1 h in Example 2 (n=3).

[0024] Fig.10 This is the cell apoptosis status and statistics of H9C2 cells treated with 5 μg / mLTE and 1 mM OMT group for 1 hour in Example 2 (n=3).

[0025] Fig.11 The changes and statistics of cellular ROS levels in H9C2 cells treated with 5 μg / mL TE and 1 mM OMT groups for 1 hour in Example 2 (n=3).

[0026] Fig.12 The changes in cellular MDA and T-SOD levels in H9C2 cells after being treated with 5 μg / mLTE and 1 mM OMT groups for 1 h in Example 2 (n=3).

[0027] Fig.13 The figure shows the decrease in cell viability caused by different drugs antagonizing TE in Example 3.

[0028] Fig.14 The changes in the translation levels of ERK, JNK and p38 proteins and phosphorylated proteins after treatment in each group in Example 4 (n=3).

[0029] Fig.15 The changes of TNFα, IL1β and IL6 after treatment in each group in Example 4 (n=4).

[0030] Fig.16 The figure shows the survival of mice within 24 hours after tail vein injection of different doses of TE in Example 5 (left figure) and the survival of mice within 24 hours after tail vein injection of 15 mg / kg TE and different doses of OMT (right figure) (n=12).

[0031] Fig.17 The figure shows the survival of mice within 24 hours after tail vein injection of 15 mg / kg TE and 100 mg / kg OMT under different administration methods in Example 5 (left figure) and the survival of mice within 24 hours after treatment with OMT at doses of 100 mg / kg and below (right figure) (n=12).

[0032] Fig.18 This is the HE staining image of the mouse heart 1 hour after the tail vein injection of 14 mg / kg TE and 100 mg / kg OMT in Example 5 (n=3).

[0033] Fig.19 The changes in the levels of CK, CK-MB and LDH in the mouse serum 1 hour after the tail vein injection of 14 mg / kg TE and 100 mg / kg OMT in Example 5 (n=4). DETAILED DESCRIPTION

[0034] Experimental materials used in the embodiments of the present invention:

[0035] 1. Preparation of Sand Jellyfish Toxin: After catching Sand Jellyfish from Bohai Sea, Dalian City, Liaoning Province, its fresh tentacles were manually removed while alive, and the tentacles were placed in a 1,000mL plastic box and quickly frozen with liquid nitrogen. After being transported to Shanghai with dry ice, they were stored in a -80℃ refrigerator for a long time. The tentacle part was placed in a 4℃ refrigerator and stirred continuously for 72h until completely dissolved, and then filtered twice with a 200-mesh sieve to remove the undissolved tissue. The filtrate was collected and centrifuged at 4℃, 1,000g for 15min, and the supernatant was collected as the Sand Jellyfish Toxin Extract (TE). The TE concentration was determined to be 2.0mg / mL using the BCA protein concentration assay kit (Yeasen, China). It was stored in liquid nitrogen.

[0036] 2. Oxymatrine: purchased from Shanghai Taoshu Biotechnology Co., Ltd. (TargetMol, China), product number T2754.

[0037] 3. Cardiomyocytes: Rat H9C2 cardiomyocytes were purchased from Shanghai QuiCell Biotechnology Co., Ltd. (QuiCell, China). The cells were cultured in DMEM low sodium bicarbonate medium (QuiCell, China) containing 10% FBS (Qida, China) at 37°C in a humidified 5% CO2 incubator.

[0038] 4. ICR mice: ICR mice (Institute of Cancer Research Mice) were purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd. The mice were housed in ventilated cages at a temperature of 22-23°C, a relative humidity of 55-60%, and a 12-hour light / 12-hour dark cycle. Water and standard food pellets were available ad libitum.

[0039] 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.

[0040] Example 1 TE induces apoptosis and oxidative damage in H9C2 cells

[0041] 1. Experimental groups:

[0042] Control group: no treatment;

[0043] Toxin group (TE group): 5 μg / m LTE was selected and incubated with H9C2 cells for 1 h;

[0044] 2. Experimental Methods

[0045] (1) Cell morphology observation

[0046] After culturing H9C2 cells in a 6-well plate for 24 h, they were treated according to the above grouping. After treatment, the culture medium was discarded, and the cells were washed twice with PBS without calcium or magnesium. 500 μL of PBS without calcium or magnesium was added to each well, and the cell morphology was observed under a microscope and images were recorded.

[0047] (2) Cell viability assay

[0048] Take out the 10 cm cell culture dish with H9C2 cell density of 80-90% from the incubator and discard the culture medium. Wash the cells once with complete culture medium. Digest with trypsin (Gibco, USA) until the cells become round and can be detached by gently tapping (1 min), and add complete culture medium to terminate digestion. Transfer the cell suspension into a 15 mL enzyme-free centrifuge tube, centrifuge at 1,000 rpm for 3 min, discard the supernatant, dilute the cells with complete culture medium, and dilute with 100 μL cell suspension and 8×10 3 96-well plates were plated with 100 μL of cells (no cells were plated in the edge wells, and 100 μL of PBS was added), and the plates were placed in an incubator for 24 h. The 96-well plates were tilted and the culture medium in the wells was carefully aspirated with a pistol. 100 μL of TE solution, drug solution, or a mixed solution of TE and drug diluted with DMEM medium was added to each well and cultured for 1 h. DMEM medium and cck8 solution (TargetMol, China) were mixed in a pipette tank at a ratio of 9:1. The 96-well plates were tilted and the liquid in the wells was carefully aspirated with a pistol. The diluted cck8 solution was added to the wells with a pistol and placed in an incubator. The absorbance was detected at 450 nm at 1 h, 2 h, 3 h, and 4 h, respectively. The maximum value was between 0.8 and 1.2, and the observation was stopped and the cell viability was calculated using the formula [(OD experimental group-OD blank group) / (OD control group-OD blank group)].

[0049] (3) Flow cytometry detection of cell apoptosis

[0050] H9C2 cells were cultured in 6-well plates for 24 h and then grouped and treated. The treated cells were digested with EDTA-free trypsin and collected by centrifugation at 4°C, 300 g for 5 min. The cells were washed twice with pre-cooled PBS without calcium or magnesium, each time by centrifugation at 4°C, 300 g for 5 min. 5×105 Cells. Aspirate PBS and add 100μL 1×Binding Buffer to resuspend the cells. Add 5μL Annexin V-FITC and 10μL PI Staining Solution (Yeasen, China), mix gently, and react at room temperature for 15 minutes in the dark. Add 400μL 1×Binding Buffer, mix well and place on ice. The samples were detected using a flow cytometer (Beckman Coulter, USA) within 1 hour.

[0051] (4) Flow cytometry detection of cellular reactive oxygen species

[0052] H9C2 cells were cultured in 6-well plates for 24 hours and then grouped. Serum-free DMEM cell culture medium containing drugs and DCFH-DA (Beyotime, China) (final concentration of 10 μmol / L) was added to each well of the six-well plate and incubated in a cell culture incubator for 1 hour. For reactive oxygen fluorescence, the cells were washed three times with serum-free cell culture medium after the incubation to fully remove the DCFH-DA that did not enter the cells. After the incubation, the cells were washed three times with serum-free cell culture medium to fully remove the DCFH-DA that entered the cells. The cells were digested and collected in a 1.5 mL centrifuge tube, centrifuged at 1,000 rpm for 3 minutes, the supernatant was discarded, 1 mL of PBS without calcium and magnesium was added to wash the cells, centrifuged at 1,000 rpm for 3 minutes, PBS was discarded, and 500 μL of PBS without calcium and magnesium was added to resuspend. The samples were detected by flow cytometry within 1 hour. The fluorescence spectrum of DCF is very similar to that of FITC, and DCF can be detected using the parameter settings of FITC.

[0053] 2. Experimental results

[0054] After TE treatment for 1 h, the cell density decreased and severe shrinkage occurred. A large number of cells became round, the contact between each other decreased, the cytoplasm condensed, and some cells swelled and ruptured ( Figure 1 a). TE showed concentration-dependent toxicity to H9C2 cells. Cell viability was detected by CellCounting Kit-8 (CCK-8) assay. As the concentration of TE increased, the cell viability decreased (IC 50 =5.339 μg / mL)( Figure 1 b). After the cells were treated with 5μg / mL TE for 1 hour, a large number of cells underwent apoptosis, and the apoptosis rate increased from 6.06±1.30% in the control group to 37.94±3.58% ( Figure 2 and Figure 3); The intracellular ROS level in the treated group was significantly higher than that in the control group, and the geometric mean of fluorescence intensity increased from 43818.94±2405.36 in the control group to 57648.76±3924.90 ( Figure 4 and Figure 5 ); the intracellular MDA level in the treated group was significantly higher than that in the control group, from 2.30±0.07nmol / mg protein in the control group to 4.40±0.06nmol / mg protein ( Figure 6 Left); the intracellular T-SOD level in the treated group was significantly lower than that in the control group, from 39.79±1.98U / mg protein in the control group to 26.88±0.24U / mg protein ( Figure 6 Right) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001, ns means no significance).

[0055] Example 2 OMT antagonizes TE-induced cytotoxicity and oxidative damage in sand jellyfish

[0056] 1. Experimental Grouping

[0057] Control group: no treatment;

[0058] Drug group (OMT group): H9C2 cells were treated with 0, 0.01, 0.03, 0.06, 0.1, 0.3, 0.6, and 1 mM OMT for 1 h;

[0059] Toxin group (TE group): 5 μg / m LTE was selected and incubated with H9C2 cells for 1 h;

[0060] Drug intervention group (TE and OMT co-treatment group): 5 μg / mLTE was selected and co-incubated with 0.3, 0.6, and 1 mM OMT for 1 hour with H9C2 cells.

[0061] 2. Experimental method: Same as Example 1

[0062] 3. Experimental results:

[0063] After the cells were treated with different concentrations of OMT for 1 h, there was no significant change in cell viability, and 0.01-1 mM OMT had no significant cytotoxicity to H9C2 cells ( Figure 7 ). The cells were treated with 5μg / mL TE and different concentrations of OMT for 1h. The results showed that compared with the cells treated with TE, the cell viability of the cells treated with 0.3, 0.6, and 1mM OMT and TE was significantly improved (EC 50 =0.2824mM), among which 1mM OMT has the strongest effect ( Figure 8 ).

[0064] The drug intervention group selected 1mM OMT for subsequent experiments. The cells were treated with 5μg / mL TE and 1mM OMT for 1h. The cell morphology was significantly restored compared with the TE group. The cell shrinkage and rounding phenomenon were significantly reduced, and the cell swelling and rupture phenomenon disappeared, indicating that OMT can significantly antagonize the cell morphological changes caused by TE ( Fig. 9 ). When the cells were treated with 1mM OMT for 1h, cell apoptosis was significantly reduced compared with the control group, and the apoptosis rate decreased from 6.06±1.30% in the control group to 4.90±0.33%, indicating that OMT has the function of inhibiting cell apoptosis; when H9C2 cells were treated with 5μg / mL TE and 1mM OMT for 1h, cell apoptosis was significantly reduced compared with the TE group, and the apoptosis rate decreased from 37.94±3.58% in the TE group to 10.60±3.29%, indicating that 1mM OMT can significantly inhibit cell apoptosis caused by TE ( Fig.10 ). The cells were treated with 5μg / mL TE and 1mM OMT for 1h. The results showed that OMT could significantly antagonize the increase in cellular ROS levels caused by TE. Compared with the TE group, the geometric mean of fluorescence intensity in the OMT and TE co-treatment group decreased from 57648.76±3924.90 to 41541.89±4530.99 ( Fig.11 ); the intracellular MDA level was significantly lower than that in the TE group, from 4.40±0.06nmol / mg protein in the TE group to 4.03±0.14nmol / mg protein ( Fig.12 Left); the intracellular T-SOD level was significantly higher than that of the TE group, from 26.88±0.24U / mg protein in the TE group to 31.91±1.14U / mg protein ( Fig.12 Right) (*p<0.05, **p<0.01, ***p<0.001, ****p<0.001, ns means no significance).

[0065] Example 3 Screening of Sand Jellyfish TE Antagonists

[0066] The screened drugs shown in Table 1 were co-treated with 5 μg / mL TE at different concentrations (30, 60, 100 μM) for 1 h in H9C2 cells, and then the cell viability was detected by CCK-8 assay to compare the antagonistic ability of the drugs on TE cytotoxicity.

[0067] Table 1 Antagonistic ability of different drugs on TE cytotoxicity

[0068]

[0069]

[0070] like Fig.13 As shown: the ability of 0.03mM drugs to inhibit TE and cause a decrease in cell viability is ranked (the colored bubbles in the figure are all p<0.0001, and the colors are distinguished based on the p value as the degree of credibility), showing that drugs No. 139, 172, and 356 cannot improve cell viability; drug No. 380 cannot improve cell viability as the drug concentration increases, and causes cell viability to decrease; other drugs can significantly improve cell viability, among which No. 251 has the strongest ability to improve cell viability, and the viability increases from 36.14±4.00% in the TE group to 106.71±3.67%, and can increase to 110.40±2.70% at high concentrations.

[0071] In summary, OMT(251) has a better antagonistic effect than other drugs and is not cytotoxic, so it can be further studied as a potential TE antagonist.

[0072] Example 4 OMT inhibits TE-induced upregulation of inflammatory factors and MAPK activation

[0073] 1. Experimental Grouping

[0074] Control group: no treatment;

[0075] Drug group (OMT group): 1 mM OMT was selected and incubated with H9C2 cells for 1 h;

[0076] Toxin group (TE group): 5 μg / m LTE was selected and incubated with H9C2 cells for 1 h;

[0077] Drug intervention group (TE and OMT co-treatment group): 5 μg / mLTE and 1 mM OMT were co-incubated with H9C2 cells for 1 hour.

[0078] 2. Experimental Methods

[0079] (1) Western Blot

[0080] MAPK nuclear transcription factors ERK, JNK and p38 were analyzed by immunoblotting: Protein samples of H9C2 cells after OMT incubation and TE treatment were extracted with a mixture of RIPA (strong) lysis buffer (Epizyme, China), 50× protease inhibitor (Epizyme, China), and 50× phosphatase inhibitor (Epizyme, China). Protein concentration was determined using a BCA protein assay kit. 15 μg of protein was loaded onto a 10% SDS-PAGE gel and then transferred to a 0.45 μm NC membrane (Cytiva, USA). The NC membrane was blocked with a protein-free fast blocking buffer (Epizyme, China) for 2 h and then washed three times with TBST (Epizyme, China) for 10 min each time. Then, the cells were incubated with antibodies against pERK (Cell Signaling Technology, USA) (1:2000), ERK (Proteintech, USA) (1:5000), pJNK (Huabio, China) (1:1000), JNK (Proteintech, USA) (1:5000), p38 (Nature Biosciences, China) (1:1000), pp38 (Nature Biosciences, China) (1:1000) and β-Tubulin (Nature Biosciences, China) (1:1000) overnight, and then washed three times with TBST for 10 min each time. Secondary antibodies (Epizyme, China) (1:5000) were used for incubation at room temperature for 2 h, and then washed three times with TBST for 10 min each time. Protein expression levels were determined using enhanced chemiluminescence (ECL) reagent (Beyotime, China), and protein expression intensity was analyzed using Image J software.

[0081] (2) Detection of oxidative stress indicators

[0082] Sample preparation: After treatment, the culture medium was collected in a 15 mL centrifuge tube and centrifuged at 4°C and 1,000 rpm for 3 min to collect non-adherent cells. The adherent cells were washed three times with PBS without calcium and magnesium, and the cells were collected using a cell scraper. The non-adherent cells and adherent cells were resuspended and mixed using the extract in the kit (D799762 malondialdehyde content detection kit, D799594 superoxide dismutase activity detection kit, Sangon Biotech, China), and the cells were ultrasonically disrupted. The supernatant was taken and placed on ice for testing. The sample concentration was determined using the BCA protein concentration determination kit. Sample detection: The above samples were tested according to the instructions of the kit.

[0083] (3) RNA extraction and RT-qPCR

[0084] RNA was extracted using a total RNA rapid extraction kit (Fastagen, China), the RNA concentration was determined and a reverse transcription system (20 μL) was prepared. The mRNAs were reverse transcribed using a PCR instrument to obtain cDNA. The obtained 20 μL cDNA was diluted with 180 μL of DEPC water, and the accuracy of the differential expression of the transcriptome mRNA was verified by real-time quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR heating scheme was as follows: initial denaturation at 95°C for 5 min, denaturation at 94°C for 10 s, annealing at 60°C for 20 s, and extension at 72°C for 30 s for 40 cycles. All samples in the reaction were diluted with DEPC water. Detection was performed on an Eppendorf Master Cycler realplex 2 (Eppendorf). The termination value of the cycle number threshold (Ct) value was analyzed, and the comparative Ct value (2 –ΔΔCt ) method to estimate gene expression levels.

[0085] 3. Experimental results

[0086] The results showed that TE could induce a significant increase in the phosphorylation levels of ERK, JNK and p38 proteins in H9C2 cells and decreased under the intervention of OMT ( Fig.14 ). To further verify the damage mechanism caused by TE and the antagonistic mechanism of OMT, the cytokines TNFα, IL1β, and IL6 were detected at the transcriptional level. The results of RT-qPCR showed that TE induced an increase in the transcriptional levels of proinflammatory factors TNFα, IL1β, and IL6 in H9C2 cells, and all of them could be inhibited by OMT. At the same time, TNFα upstream of JNK and IL1β upstream of p38 were induced to be upregulated by TE, but were inhibited by OMT ( Fig.15 )(*p<0.05, **p<0.01, ***p<0.001, ****p<0.001, ns means nosignificance).

[0087] Example 5 OMT antagonizes TE-induced mouse death and cardiac injury

[0088] 1. Experimental groups:

[0089] The ICR mice used in the experiment were all male, weighing 20-22 g.

[0090] (1) Survival of mice

[0091] The mice were divided into the following groups (12 in each group): tail vein injection of normal saline group (Control group, 0.1 mL normal saline was injected for every 10 g body weight of mice), tail vein injection of TE group (TE group), tail vein injection of OMT followed by tail vein injection of TE group, tail vein injection of OMT and TE mixture group, tail vein injection of TE followed by OMT group. The injection volume of each drug solution group was: 0.1 mL of drug solution was injected for every 10 g body weight of mice. The drug solutions of each group were diluted with normal saline as diluent to the corresponding dose for injection;

[0092] Among them, when studying the effects of different doses of TE on the survival of mice, the TE doses in the TE group were 1, 3, 6, 10, 12, 14, 14.5, 15, and 20 mg / kg;

[0093] When studying the effects of different doses of OMT and TE co-treatment on mice, the TE dose was 15 mg / kg, and the OMT doses were 10, 30, 60, and 100 mg / kg;

[0094] When studying different administration methods, the OMT dose was 100 mg / kg and the TE dose was 15 mg / kg.

[0095] (2) Heart damage in mice

[0096] Control group: injection of normal saline via tail vein, injection volume was 0.1mL / 10g (body weight);

[0097] Drug group (OMT group): OMT was injected into the tail vein at a dose of 100 mg / kg;

[0098] Toxin group (TE group): TE was injected into the tail vein at a dose of 14 mg / kg;

[0099] Drug intervention group (TE+OMT co-treatment group): 14 mg / kg TE and 100 mg / kg OMT were injected into the tail vein, and the injection volume was the same as above.

[0100] 2. Experimental Methods

[0101] (1) Survival analysis of experimental mice

[0102] The injections were performed according to the above groupings. The mice were observed for 24 hours after the injections, the time of death of the mice was recorded, and the survival curves were drawn and survival analysis was performed.

[0103] (2) Measurement of blood biochemical indicators

[0104] Each group of mice was deeply anesthetized with isoflurane and blood was collected. The blood volume was about 0.5 mL per mouse and then euthanized. The heart was quickly obtained, washed with physiological saline and placed in tissue fixative (Servicebio, China). After the blood was allowed to stand for 2 hours, it was centrifuged at 4°C, 3,000 rpm for 15 minutes, and the supernatant was collected and stored in a -80°C refrigerator. The levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) in mouse serum were measured at Shanghai Ru'an Biological Technology Co., Ltd.

[0105] 3. Experimental results

[0106] (1) Survival status

[0107] After mice were treated with different doses of TE, different numbers of mice died within 24 hours. Among them, no mice died after being treated with TE below 12 mg / kg; the mortality rate of mice was 8.33% after being treated with 14 mg / kg TE; the mortality rate of mice was 83.33% after being treated with 14.5 and 15 mg / kg TE; when the TE dose was increased to 20 mg / kg, the mortality rate of mice was 100% ( Fig.16 Left).

[0108] Compared with 15mg / kg TE treatment, OMT can improve the survival rate of mice with increasing doses. After 10mg / kg OMT and TE co-treated mice, the mortality rate of mice was still 83.33%; after 30mg / kg OMT and TE co-treated mice, the mortality rate of mice dropped to 75%; after 60mg / kg OMT and TE co-treated mice, the mortality rate of mice further dropped to 50%; when the OMT dose was increased to 100mg / kg, it could completely interfere with the lethal toxicity of TE, and the survival rate within 24 hours reached 100% ( Fig.16 right).

[0109] Next, we added a preventive or therapeutic administration form, giving 100 mg / kg OMT 15 minutes before and after TE injection. The results showed that administration 15 minutes in advance or 15 minutes after TE injection could improve the survival rate of mice, reducing the TE lethality from 83.3% to 33.3% ( Fig.17 Left). After mice were treated with OMT at a dose of 100 mg / kg or less, no mice died within 24 hours ( Fig.17 right).

[0110] (2) Heart damage in mice

[0111] After 14 mg / kg TE was treated for 1 hour, the mouse heart tissue had a certain degree of edema, myocardial cell apoptosis, myofiber dissolution and rupture, and inflammatory cell infiltration, indicating that TE caused certain damage to the heart tissue; OMT can effectively antagonize the heart tissue damage caused by TE ( Fig.18 ). After 14mg / kg TE was used to treat mice for 1 hour, the levels of CK, CK-MB, and LDH in the mouse serum increased significantly, among which CK increased from 3046.85±246.62U / L in the control group to 4302.82±614.82U / L; CK-MB increased from 1390.48±80.34U / L in the control group to 2304.93±355.41U / L; LDH increased from 2516.62±128.41 in the control group to 9211.23±1325.57U / L. The levels of CK-MB and LDH in the group treated with TE and OMT were significantly decreased compared with those in the TE group, among which CK-MB decreased to 1566.16±208.67U / L, and LDH decreased to 2371.56±259.16U / L, indicating that OMT can significantly antagonize the changes in CK-MB and LDH levels caused by TE ( Fig.19 )(*p<0.05, **p<0.01, ***p<0.001, ****p<0.001, ns means no significance).

[0112] In summary, oxymatrine can reduce H9C2 cell apoptosis, antagonize cell apoptosis and oxidative damage caused by sand jellyfish toxin, improve the survival rate of mice after injection of sand jellyfish toxin, and improve the heart tissue damage of mice caused by sand jellyfish toxin. It has great potential in the preparation of TE antagonist drugs and can be used as a candidate drug for the treatment of sand jellyfish stings.

[0113] 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. The application of oxymatrine in the preparation of drugs for inhibiting jellyfish toxins.

2. The use according to claim 1, characterized in that: The jellyfish toxin is produced by sand jellyfish.

3. Use of oxymatrine in the preparation of a drug for treating or preventing cardiac tissue dysfunction caused by jellyfish toxin poisoning.

4. The use according to claim 3, characterized in that: The dysfunction of the cardiac tissue is cardiac tissue damage.

5. The use according to claim 4, characterized in that: The cardiac tissue damage includes cardiac tissue edema, myocardial cell apoptosis, myofiber lysis, rupture or inflammatory cell infiltration.

Images