Use of oxymatrine in preparation of medicine for inhibiting jellyfish toxin

Abstract

This invention provides the application of oxymatrine in the preparation of drugs that inhibit jellyfish toxins, belonging to the field of biomedical technology. Screening experiments were conducted on various drugs, revealing that oxymatrine (OMT) was more effective than other drugs in antagonizing jellyfish toxins without exhibiting cytotoxicity. Further research showed that oxymatrine can reduce H9C2 cell apoptosis, antagonize apoptosis and oxidative damage induced by jellyfish toxins, improve the survival rate of mice after injection of jellyfish toxins, and improve cardiac tissue damage caused by jellyfish toxins in mice. It shows great potential in the preparation of jellyfish toxin antagonistic drugs and can serve as a candidate drug for treating jellyfish stings.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of oxymatrine in the preparation of drugs that inhibit jellyfish toxins. Background Technology

[0002] Jellyfish stings are one of the most common types of marine organism injuries, posing a significant threat to tourists and those working at sea, and can have serious consequences. In recent years, numerous cases of jellyfish stings have been reported in coastal areas. Cardiotoxicity caused by jellyfish stings is considered a major cause of death, but the mechanism of this toxicity remains unclear, hindering the development of treatments for jellyfish stings.

[0003] The sand jellyfish (Nemopilema nomurai) is a venomous jellyfish widely distributed in the coastal waters of my country. In recent years, this jellyfish has caused several serious fatal and injury incidents. Studies have found that sand jellyfish stings can cause acute heart failure within a short period of time. The toxin induces inflammatory responses, oxidative damage, and hemolysis. In vivo models show that the toxin severely affects the cardiovascular system, causing cardiac tissue dysfunction and revealing selective cardiotoxicity. Although the cardiotoxicity of sand jellyfish toxin has been confirmed through in vitro and in vivo models, related research remains limited. Similar to most jellyfish-related studies, there is currently a lack of further elucidation of the cardiotoxic effects and mechanisms of sand jellyfish stings. This, to some extent, increases the difficulty of diagnosing jellyfish stings and contributes to the lack of emergency treatment options.

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

[0005] The purpose of this invention is to provide a new drug for inhibiting jellyfish toxins.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides the application of oxymatrine in the preparation of drugs that inhibit jellyfish toxins.

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

[0009] This invention also provides the use of oxymatrine in the preparation of medicaments for treating or preventing cardiac tissue dysfunction caused by jellyfish toxin poisoning.

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

[0011] Preferably, the cardiac tissue injury includes cardiac tissue edema, cardiomyocyte apoptosis, myofibril dissolution, rupture, or inflammatory cell infiltration.

[0012] Beneficial effects

[0013] This invention, through the study of the inhibitory effects of different compounds on TE cytotoxicity, confirms that oxymatrine (OMT) is an effective TE antagonist, providing a good approach for developing anti-jellyfish sting drugs from natural compounds, and is of great significance for developing innovative drugs for jellyfish stings that are highly effective and low in toxicity.

[0014] This invention discovers that oxymatrine can reduce H9C2 cell apoptosis, antagonize cell apoptosis and oxidative damage caused by jellyfish toxin, improve the survival rate of mice after injection of jellyfish toxin, and improve cardiac tissue damage in mice caused by 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 jellyfish stings. Attached Figure Description

[0015] Figure 1 The changes in cell morphology of H9C2 cells after 1 hour of TE treatment in Example 1 are shown in (a) and the changes in cell viability of H9C2 cells after 1 hour of treatment with different concentrations of TE are shown in (b).

[0016] Figure 2 This shows the apoptosis of H9C2 cells after 1 hour of TE treatment in Example 1.

[0017] Figure 3 The apoptosis of H9C2 cells treated with TE for 1 hour in Example 1 was statistically analyzed (n=3).

[0018] Figure 4 The changes in ROS levels in H9C2 cells after 1 hour of TE treatment in Example 1 are shown.

[0019] Figure 5 The statistical analysis of ROS levels in H9C2 cells after 1 hour of TE treatment in Example 1 (n=3).

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

[0021] Figure 7 The changes in cell viability of H9C2 cells after 1 hour of treatment with different concentrations of OMT in Example 2 are shown.

[0022] Figure 8The changes in cell viability of H9C2 cells after 1 hour of simultaneous treatment with different concentrations of OMT and TE in Example 2 are shown.

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

[0024] Figure 10 The apoptosis status and statistics of H9C2 cells treated with 5 μg / mLTE and 1 mM OMT for 1 h in Example 2 are presented (n=3).

[0025] Figure 11 The changes and statistics of ROS levels in H9C2 cells after 1 hour of treatment with 5 μg / mL TE and 1 mM OMT in Example 2 are presented (n=3).

[0026] Figure 12 The changes in MDA and T-SOD levels in H9C2 cells after 1 hour of treatment with 5 μg / mLTE and 1 mM OMT groups in Example 2 (n=3).

[0027] Figure 13 This shows the cell viability decline caused by different drugs antagonizing TE in Example 3.

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

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

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

[0031] Figure 17 The left 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, and the right figure shows the survival of mice within 24 hours after treatment with OMT at doses of 100 mg / kg and below (n=12).

[0032] Figure 18 HE staining images of mouse hearts 1 hour after tail vein injection of 14 mg / kg TE and 100 mg / kg OMT in Example 5 (n=3).

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

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

[0035] 1. Preparation of Tentacle Toxin from Sand Jellyfish: Sand jellyfish were captured from the Bohai Sea in Dalian, Liaoning Province. Fresh tentacles were manually removed while the jellyfish were still alive. The tentacles were placed in a 1000 mL plastic container and flash-frozen in liquid nitrogen. After being transported to Shanghai on dry ice, they were stored at -80°C for long-term preservation. The tentacles were then placed in a 4°C freezer and stirred continuously for 72 hours until completely dissolved. The solution was then filtered twice through a 200-mesh sieve to remove any non-autolyzed tissue. The filtrate was collected and centrifuged at 1000g for 15 min at 4°C. The supernatant was collected as the sand jellyfish toxin extract (TE). The TE concentration was determined to be 2.0 mg / mL using a BCA protein assay kit (Yeasen, China). The extract was then 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). Cells were cultured in DMEM low-carbonate medium (QuiCell, China) containing 10% FBS (Qida, China) and incubated 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 with 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 readily available.

[0039] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

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

[0041] 1. Experimental Groups:

[0042] Control group: No treatment received;

[0043] Toxin group (TE group): H9C2 cells were co-incubated with 5 μg / mLTE for 1 h;

[0044] 2. Experimental Methods

[0045] (1) Cell morphology observation

[0046] After H9C2 cells were cultured in 6-well plates for 24 hours, they were treated according to the above grouping. After treatment, the culture medium was discarded, and the cells were washed twice with calcium- and magnesium-free PBS. 500 μL of calcium- and magnesium-free PBS was added to each well, and the cell morphology was observed under a microscope and the images were recorded.

[0047] (2) Cell viability detection

[0048] Remove 10cm cell culture dishes containing 80-90% H9C2 cells 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 rounded and easily detached by gentle tapping (1 min), then add complete culture medium to stop digestion. Transfer the cell suspension to 15mL enzyme-free centrifuge tubes, centrifuge at 1000rpm for 3 min, discard the supernatant, and dilute the cells with complete culture medium, using 100μL of cell suspension per well at 8×10⁻⁶ cells / well. 3 Seed cells into 96-well plates (excluding the edge wells, which contain 100 μL PBS) and incubate for 24 h. Tilt the 96-well plate and carefully aspirate the culture medium from the wells using a pipette. Add 100 μL of TE solution, drug solution, or a mixture of TE and drug diluted in DMEM medium to each well and incubate for 1 h. Mix DMEM medium with cck8 solution (TargetMol, China) 9:1 in a pipette. Tilt the 96-well plate and carefully aspirate the liquid from the wells using a pipette. Add the diluted cck8 solution to the wells using a pipette and incubate. Measure the absorbance at 450 nm at 1 h, 2 h, 3 h, and 4 h. Stop observation when the maximum value is between 0.8 and 1.2 and calculate cell viability using the formula [(OD experimental group - OD blank group) / (OD control group - OD blank group)].

[0049] (3) Flow cytometry detection of apoptosis

[0050] H9C2 cells were cultured in 6-well plates for 24 h, then divided and treated. After treatment, the cells were digested with EDTA-free trypsin and collected by centrifugation at 300g for 5 min at 4°C. The cells were washed twice with pre-chilled, calcium- and magnesium-free PBS, each time by centrifugation at 300g for 5 min at 4°C. 5 × 10⁶ cells were collected.5 Cells. Discard PBS and resuspend cells in 100 μL of 1×Binding Buffer. Add 5 μL Annexin V-FITC and 10 μL PI Staining Solution (Yeasen, China), mix gently, and incubate in the dark at room temperature for 15 min. Add 400 μL of 1×Binding Buffer, mix well, and place on ice. Analyze samples using flow cytometry (Beckman Coulter, USA) within 1 h.

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

[0052] H9C2 cells were cultured in 6-well plates for 24 hours and then divided into groups. Serum-free DMEM cell culture medium containing the drug and DCFH-DA (Beyotime, China) (final concentration 10 μmol / L) was added to each well of the 6-well plate, and the plates were incubated for 1 hour. For reactive oxygen species fluorescence, the cells were washed three times with serum-free cell culture medium after incubation to thoroughly remove any DCFH-DA that had not entered the cells. Cells were digested and collected in 1.5 mL centrifuge tubes, centrifuged at 1,000 rpm for 3 min, the supernatant was discarded, and 1 mL of calcium- and magnesium-free PBS was added to wash the cells. The cells were then centrifuged again at 1,000 rpm for 3 min, the PBS was discarded, and the cells were resuspended in 500 μL of calcium- and magnesium-free PBS. Samples were analyzed 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 same parameters as FITC.

[0053] 2. Experimental Results

[0054] After TE treatment for 1 hour, cell density decreased and severe shrinkage occurred, with many cells becoming rounded, reduced contact between them, cytoplasmic shrinkage, and some cells swelling and rupturing. Figure 1 a) TE exhibits concentration-dependent toxicity to H9C2 cells. Cell viability was assessed using a Cell Counting Kit-8 (CCK-8) assay; cell viability decreased with increasing TE concentration (IC50). 50 =5.339μg / mL)( Figure 1 b). After treating cells with 5 μg / mL TE for 1 h, a large number of cells underwent apoptosis, with the apoptosis rate increasing from 6.06±1.30% in the control group to 37.94±3.58%. Figure 2 and Figure 3Intracellular ROS levels were significantly higher in the treatment group compared to the control group, with the geometric mean fluorescence intensity increasing from 43818.94±2405.36 in the control group to 57648.76±3924.90. Figure 4 and Figure 5 Intracellular MDA levels were significantly higher in the treatment group compared to the control group, increasing from 2.30 ± 0.07 nmol / mg protein in the control group to 4.40 ± 0.06 nmol / mg protein. Figure 6 (Left); Intracellular T-SOD levels in the treatment group were significantly lower than those in the control group, decreasing from 39.79±1.98 U / mg protein in the control group to 26.88±0.24 U / 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 effects of sand jellyfish.

[0056] 1. Experimental Grouping

[0057] Control group: No treatment received;

[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, respectively;

[0059] Toxin group (TE group): H9C2 cells were co-incubated with 5 μg / mLTE 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 with H9C2 cells for 1 h.

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

[0062] 3. Experimental Results:

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

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

[0065] Example 3: Screening of TE antagonists for jellyfish

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

[0067] Table 1. Antagonistic effect of different drugs on TE cell toxicity

[0068]

[0069]

[0070] like Figure 13 As shown: the ranking of the ability of 0.03 mM drugs to inhibit the decrease in cell viability caused by TE (colored bubbles in the figure represent p < 0.0001, and the color is used to distinguish the confidence level based on the p value). It shows that drugs 139, 172, and 356 cannot increase cell viability; drug 380 cannot increase cell viability with increasing drug concentration and even causes a decrease in cell viability; other drugs can significantly increase cell viability, among which drug 251 has the strongest ability to increase cell viability, increasing the viability from 36.14 ± 4.00% in the TE group to 106.71 ± 3.67%, and at high concentrations it can increase it to 110.40 ± 2.70%.

[0071] In summary, OMT(251) shows better antagonistic effects compared to other drugs and has not been exposed to cytotoxicity, making it a potential TE antagonist for further research.

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

[0073] 1. Experimental Grouping

[0074] Control group: No treatment received;

[0075] Drug group (OMT group): H9C2 cells were co-incubated with 1mM OMT for 1h;

[0076] Toxin group (TE group): H9C2 cells were co-incubated with 5 μg / mLTE for 1 h;

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

[0078] 2. Experimental Methods

[0079] (1) Western Blot

[0080] Analyzing MAPK nuclear transcription factors ERK, JNK, and p38 by Western blotting: Protein samples from H9C2 cells after OMT incubation and TE treatment were extracted using 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 protein-free rapid blocking buffer (Epizyme, China) for 2 h, followed by washing three times with TBST (Epizyme, China) for 10 min each time. The protein was then incubated overnight 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), followed by three 10-minute washes with TBST. The protein was then incubated with secondary antibody (Epizyme, China) (1:5000) at room temperature for 2 hours, followed by three 10-minute washes with TBST. Protein expression levels were determined using enhanced chemiluminescence (ECL) reagent (Beyotime, China), and protein expression intensity was analyzed using ImageJ software.

[0081] (2) Detection of oxidative stress indicators

[0082] Sample Preparation: After processing, collect the culture medium in 15 mL centrifuge tubes and centrifuge at 4°C and 1,000 rpm for 3 min to collect non-adherent cells. Adherent cells are washed three times with calcium- and magnesium-free PBS, collected using a cell scraper, and resuspended in the extract of the kits (D799762 Malondialdehyde Content Assay Kit, D799594 Superoxide Dismutase Activity Assay Kit, Sangon Biotech, China) to disrupt the cell structure. Centrifuge at 4°C and 8000 g for 10 min, collect the supernatant, and place on ice for analysis. The sample concentration is determined using a BCA protein concentration assay kit. Sample Detection: The samples are analyzed according to the kit instructions.

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

[0084] RNA was extracted using a total RNA rapid extraction kit (Fastagen, China). RNA concentration was determined, and a reverse transcription system (20 μL) was prepared. mRNAs were reverse transcribed using a PCR instrument to obtain cDNA. The obtained 20 μL of cDNA was diluted with 180 μL of DEPC water. The accuracy of differential expression of transcriptome mRNA was verified using real-time quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR heating protocol was: 40 cycles of initial denaturation at 95℃ for 5 min, denaturation at 94℃ for 10 s, annealing at 60℃ for 20 s, and extension at 72℃ for 30 s. All samples were diluted with DEPC water. Detection was performed on an Eppendorf MasterCycler realplex 2 (Eppendorf). The termination value of the cycle number threshold (Ct) was analyzed, and the Ct value was compared with the 2-1 value. –ΔΔCt The method of estimating gene expression levels is used.

[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, which decreased under the intervention of OMT. Figure 14 To further verify the damage mechanism caused by TE and the antagonistic mechanism of OMT, the transcriptional levels of cytokines TNFα, IL1β, and IL6 were detected. RT-qPCR results showed that TE induced an increase in the transcriptional levels of pro-inflammatory factors TNFα, IL1β, and IL6 in H9C2 cells, all of which were inhibited by OMT. Simultaneously, TNFα upstream of JNK and IL1β upstream of p38 were upregulated by TE, but were inhibited by OMT. Figure 15 )(*p<0.05, **p<0.01, ***p<0.001, ****p<0.001, ns means nosignificance).

[0087] Example 5: OMT antagonism of TE-induced mouse death and cardiac injury

[0088] 1. Experimental Groups:

[0089] All ICR mice used in the experiment were male and weighed 20–22g.

[0090] (1) Survival status of mice

[0091] Mice were divided into the following groups (n=12 per group): tail vein injection of saline group (Control group, 0.1 mL of saline per 10 g body weight), 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, and tail vein injection of TE followed by OMT group. The injection volume for each drug group was 0.1 mL of drug per 10 g body weight. The drug in each group was diluted with saline to the corresponding dose before injection.

[0092] In the study of the effect 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, respectively.

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

[0094] The OMT dose was 100 mg / kg and the TE dose was 15 mg / kg when different administration methods were studied.

[0095] (2) Heart damage in mice

[0096] Control group: normal saline was injected via tail vein at a dose of 0.1 mL / 10 g (body weight);

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

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

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

[0100] 2. Experimental Methods

[0101] (1) Survival analysis of experimental mice

[0102] Mice were injected according to the above grouping. After injection, mice were observed for 24 hours, and the time of death was recorded. Survival curves were plotted and survival analysis was performed.

[0103] (2) Blood biochemical index determination

[0104] Mice in each group were deeply anesthetized with isoflurane, and blood was collected from each mouse, approximately 0.5 mL. The mice were then euthanized. Hearts were rapidly obtained, washed with physiological saline, and placed in tissue fixative (Servicebio, China). After allowing the collected blood to stand for 2 hours, it was centrifuged at 3,000 rpm for 15 minutes at 4°C, and the supernatant was collected and stored at -80°C. The levels of creatine kinase (CK), creatine kinase-MB (CK-MB), and lactate dehydrogenase (LDH) in mouse serum were determined at Shanghai Ru'an Biological Technology Co., Ltd.

[0105] 3. Experimental Results

[0106] (1) Survival status

[0107] Different doses of TE resulted in varying numbers of mouse deaths within 24 hours. Specifically, no mice died after treatment with TE below 12 mg / kg; the mortality rate was 8.33% after treatment with TE at 14 mg / kg; the mortality rate was 83.33% after treatment with TE at 14.5 and 15 mg / kg; and the mortality rate was 100% when the TE dose was increased to 20 mg / kg. Figure 16 Left).

[0108] Compared with 15 mg / kg TE treatment, OMT improved mouse survival rate with increasing dose. After co-treatment of mice with 10 mg / kg OMT and TE, the mortality rate remained at 83.33%; after co-treatment with 30 mg / kg OMT and TE, the mortality rate decreased to 75%; after co-treatment with 60 mg / kg OMT and TE, the mortality rate further decreased to 50%; when the OMT dose was increased to 100 mg / kg, it completely neutralized the lethal toxicity of TE, achieving a 100% survival rate within 24 hours. Figure 16 right).

[0109] Next, we increased the dosage form for prophylaxis or treatment, administering 100 mg / kg OMT 15 minutes before and after TE injection, respectively. The results showed that administering the medication 15 minutes before or after TE injection improved mouse survival, reducing the TE lethality from 83.3% to 33.3%. Figure 17 Left). Mice treated with OMT at doses of 100 mg / kg or lower did not die within 24 hours. Figure 17 right).

[0110] (2) Heart damage in mice

[0111] After treating mice with 14 mg / kg TE for 1 hour, the mouse heart tissue showed a certain degree of edema, cardiomyocyte apoptosis, myofibrillation and breakage, and inflammatory cell infiltration, indicating that TE caused some damage to the heart tissue; OMT can effectively antagonize the heart tissue damage caused by TE. Figure 18 After treating mice with 14 mg / kg TE for 1 hour, the serum levels of CK, CK-MB, and LDH significantly increased. Specifically, CK increased from 3046.85±246.62 U / L in the control group to 4302.82±614.82 U / L; CK-MB increased from 1390.48±80.34 U / L in the control group to 2304.93±355.41 U / L; and LDH increased from 2516.62±128.41 U / L in the control group to 9211.23±1325.57 U / L. The CK-MB and LDH levels in the TE and OMT co-treatment group were significantly lower than those in the TE group, with CK-MB decreasing to 1566.16±208.67 U / L and LDH decreasing to 2371.56±259.16 U / L, indicating that OMT can significantly antagonize the changes in CK-MB and LDH levels induced by TE. Figure 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 apoptosis and oxidative damage caused by jellyfish toxin, improve the survival rate of mice after injection of jellyfish toxin, and improve cardiac tissue damage caused by jellyfish toxin in mice. It has great potential in the preparation of TE antagonist drugs and can be used as a candidate drug for the treatment of jellyfish stings.

[0113] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of oxymatrine in the preparation of drugs that inhibit jellyfish toxins, characterized in that, The jellyfish toxin is produced by the sand jellyfish.

2. The application of oxymatrine in the preparation of drugs for treating or preventing cardiac tissue dysfunction caused by jellyfish toxin poisoning, characterized in that, The jellyfish toxin is produced by the sand jellyfish; The dysfunction of the heart tissue is heart tissue damage.

3. The application as described in claim 2, characterized in that, The cardiac tissue damage includes cardiac tissue edema, cardiomyocyte apoptosis, myofibril dissolution, rupture, or inflammatory cell infiltration.

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