Application of sulforaphane

Abstract

The invention discloses application of sulforaphane, and belongs to the field of medicine. Experiments with mouse AML-12 cells find that when the mouse AML-12 cells are jointly treated with sulforaphane and alpha-amatoxin, the inhibition effect of alpha-amatoxin on the activity of the AML-12 cells can be effectively relieved, the ROS level in the cells can be reduced, cell oxidative stress injury can be relieved, mitochondrial membrane potential reduction can be inhibited, and the mouse AML-12 cells can be treated with raphane and alpha-amatoxin. The sulforaphane can be used for relieving alpha-amatoxin induced mouse AML-12 cell mitochondrial injury, so that the sulforaphane can become a novel medicine for relieving alpha-amatoxin poisoning. The new application of the sulforaphane is found for the first time, and a new substance is provided for preparing the medicine for relieving alpha-amatoxin poisoning.

Description

TECHNICAL FIELD

[0001] The application relates to an application of sulforaphane and belongs to the medical field. BACKGROUND

[0002] Alpha-amanitin is the most toxic and abundant toxin in amanita phalloides and is the main component of fatal amanita phalloides poisoning. The toxic dose is only 0.1 mg / kg, and 1 g of amanita phalloides generally contains about 5 mg of alpha-amanitin. A single amanita phalloides mushroom can be fatal. The gastrointestinal tract of rats and mice has a relatively low absorption rate of alpha-amanitin, while cats, dogs and humans have a very high absorption rate of alpha-amanitin and show high sensitivity. Generally, there are no obvious symptoms within 12 hours after the human body ingests food containing alpha-amanitin. However, more than 12 hours after ingestion, gastrointestinal reactions may occur, mainly manifested as nausea, vomiting, diarrhea and abdominal pain. After ingesting food containing alpha-amanitin for 72 hours, the concentration of sodium ions in the body decreases, and the urea level rises. This stage is the liver toxicity stage, mainly causing damage to the liver. When the ingestion time exceeds 96 hours, patients may experience severe consequences such as liver and kidney failure and liver necrosis. Alpha-amanitin is resistant to heat, cold and acid, and can be stably stored in water and methanol regardless of temperature.

[0003] The retention time of alpha-amanitin in the human body is relatively short. According to research, it is still difficult to detect alpha-amanitin in the blood 24 hours after poisoning. The course of alpha-amanitin poisoning can generally be divided into the following stages: (1) incubation period: this stage usually lasts for 6-40 hours, the incubation period is long, and the patient almost shows no symptoms of poisoning; (2) gastrointestinal reaction period: immediately after the incubation period, this stage generally lasts for 8-48 hours, the patient will have symptoms such as vomiting, abdominal pain, diarrhea, dehydration and electrolyte imbalance, and liver and kidney function tests are normal; (3) apparent recovery period (false recovery period): 36-48 hours after ingesting the poison, the gastrointestinal symptoms disappear, and the patient's clinical symptoms improve, but the enzyme indicators of the liver show an upward trend; (4) liver toxicity period: about 72-96 hours after ingesting the poison, alpha-amanitin enters the liver, leading to acute liver and kidney failure; (5) liver and kidney damage period: in this stage, the liver and kidney are severely damaged, and progressive and irreversible dysfunction occurs, with a mortality rate of up to 90%. It is worth noting that different clinical manifestations of alpha-amanitin poisoning are closely related to the dose and time of ingesting the toxin. Alpha-amanitin poisoning needs to be detected and treated early, otherwise the patient often has poor prognosis. Unfortunately, there is no specific antidote for alpha-amanitin poisoning, and there is no satisfactory detoxification method. SUMMARY

[0004] In order to solve the problems in the prior art, the application provides an application of sulforaphane in preparation of a medicine for relieving α-amatoxin poisoning.

[0005] By performing cell experiments, it is confirmed that when cells are treated with α-amatoxin, the α-amatoxin can inhibit the activity of mouse AML-12 cells, increase the ROS level in the cells, induce oxidative stress damage of the cells, reduce the mitochondrial membrane potential, cause mitochondrial damage of the mouse AML-12 cells, and cause cell poisoning; when the mouse AML-12 cells are treated with sulforaphane and α-amatoxin, the inhibition of the activity of the AML-12 cells by the α-amatoxin can be effectively reduced, the ROS level in the cells can be reduced, the oxidative stress damage of the cells can be relieved, the decline of the mitochondrial membrane potential can be inhibited, and the mitochondrial damage of the mouse AML-12 cells induced by the α-amatoxin can be reduced, so it can be seen that the sulforaphane can become a new medicine for relieving α-amatoxin poisoning.

[0006] The application has the following beneficial effects: The application first finds that the sulforaphane can relieve the mitochondrial damage of the AML-12 cells caused by the α-amatoxin, specifically, the inhibition of the activity of the AML-12 cells by the α-amatoxin is reduced, the ROS level is reduced, the decline of the mitochondrial membrane potential is relieved, and the change of the mitochondrial morphology and structure is weakened, which indicates that the sulforaphane can relieve the mitochondrial damage of the AML-12 cells induced by the α-amatoxin, inhibit the hepatotoxicity of the α-amatoxin, and the sulforaphane can be used for preparing a medicine for relieving AML-12 poisoning, and the application first finds a new use of the sulforaphane. BRIEF DESCRIPTION OF DRAWINGS

[0007] Figure 1 The influence of the α-amatoxin on the activity of the AML-12 cells in Example 1.

[0008] Figure 2 The influence of the sulforaphane on the activity of the AML-12 cells in Example 2.

[0009] Figure 3 The influence of the sulforaphane on the activity of the AML-12 cells induced by the α-amatoxin in Example 3.

[0010] Figure 4 The influence of the α-amatoxin on the ROS in the AML-12 cells after the intervention of the sulforaphane in Example 4, wherein Figure 4 A is a ROS fluorescence map of the cell intervened by the sulforaphane; Figure 4 B is the average fluorescence intensity of the cell intervened by the sulforaphane.

[0011] Figure 5 The influence of the α-amatoxin on the mitochondrial membrane potential of the AML-12 cells after the intervention of the sulforaphane in Example 5, whereinFigure 5 A is the fluorescence diagram of the mitochondrial membrane potential of the cell intervened by sulforaphane; Figure 5 B is the average fluorescence intensity of the mitochondrial membrane potential of the cell intervened by sulforaphane.

[0012] Figure 6 Figure 6 is the effect of α-amanitin on the mitochondrial morphology of AML-12 cells after intervention by sulforaphane in Example 6 (20000x). DETAILED DESCRIPTION

[0013] The technical solutions provided by the present application are described in detail below in conjunction with examples, but they should not be understood as limiting the scope of protection of the present application.

[0014] Example 1 The specific steps are as follows: (1) Take well-conditioned AML-12 cells to prepare a cell suspension, count them with a hemocytometer, adjust the concentration of the cell suspension, inoculate them in a 96-well plate, and culture them in a 37℃, 5% CO2 incubator. When the confluence of the cells is 80%-90%, perform cell challenge; (2) 2% fetal bovine serum protein (FBS), 1% double antibody, and DMEM / F-12 (1:1) are supplemented and mixed to prepare 2% cell maintenance solution, which is stored at 4℃. 1 mg of α-amanitin is dissolved in 1.0882 mL of DMSO to prepare 1 mM of α-amanitin stock solution, which is stored in a -80℃ refrigerator; (3) Set up a blank group, a control group, and α-amanitin groups (1 μM, 5 μM, 10 μM, 15 μM, and 20 μM), with 5 replicate wells in each group. Take 1 mM of the α-amanitin stock solution, add it to the corresponding 2% cell maintenance solution, and dilute it to the corresponding concentration of the α-amanitin working solution for drug treatment for 12 h and 24 h; (4) After drug treatment, remove the old solution, wash it with PBS buffer solution for 2 times, add 90 μL of fresh serum-free cell culture solution and 10 μL of CCK-8 reagent to each well, and incubate it in a 37℃, 5% CO2 incubator for 1.5 h in the dark; (5) Measure the absorbance value at 450 nm with a multifunctional enzyme label meter, and calculate the cell survival rate according to Formula 1 Formula 1: Cell survival rate (%) = (OD value of each test group - OD value of blank group) / (OD value of control group - OD value of blank group) x 100%.

[0015] The final cell survival rate is shown in Table 1. Figure 1 As can be seen from the figure, the activity of AML-12 cells gradually decreased with the increase of the concentration. After challenge for 12 h, the activity of AML-12 cells significantly decreased from 10 µM (** P<0.01), while the cell viability of AML-12 cells decreased significantly from 5 µM after 24 h of treatment P <0.05), indicating that α-amanitin had toxic effects on AML-12 cells, and the effects were enhanced with the increase of treatment time and drug concentration. Compared with 12 h of treatment, the toxic effects of α-amanitin on AML-12 cells were more obvious after 24 h of treatment, which was conducive to observing the damage of cells.

[0016] Example 2 Effect of sulforaphane on the viability of AML-12 cells AML-12 cells were inoculated in 96-well plates according to the procedure in Example 1. 5 mg of sulforaphane was dissolved in 5.6405 mL of DMSO to prepare a 1 mM sulforaphane stock solution, which was stored at -20 °C. The 1 mM sulforaphane stock solution was diluted with 2% cell maintenance solution to prepare sulforaphane working solutions with corresponding concentrations, which were stored in a 4 °C refrigerator. Blank, control, and sulforaphane groups (1 µM, 5 µM, 10 µM, 15 µM, 20 µM, 30 µM, and 40 µM) were set up, each with 5 replicates. The cells were treated with drugs for 3 h, and then the old medium was removed and the cells were washed twice with PBS buffer. Then, 100 µL of 2% cell maintenance solution was added to each well, and the cells were cultured in a 37 °C, 5% CO2 incubator for 24 h. The subsequent procedures were the same as those in Example 1.

[0017] The final cell survival rate is shown in Table 2. Figure 2 As shown in the figure, compared with the control group, the cell viability of the sulforaphane treatment group decreased slightly. The maximum non-influence concentration of sulforaphane on AML-12 cells was 30 µM. Therefore, in the subsequent experiments, the cells were pretreated with 30 µM sulforaphane for 3 h to observe the effect of sulforaphane on the AML-12 cell damage induced by α-amanitin.

[0018] Example 3 Effect of sulforaphane on the viability of AML-12 cells induced by α-amanitin AML-12 cells were inoculated in 96-well plates according to the procedure in Example 1. Blank, control, sulforaphane (30 µM), α-amanitin (10 µM), and sulforaphane + α-amanitin (30 µM sulforaphane + 10 µM α-amanitin) groups were set up, each with 5 replicates. The sulforaphane and sulforaphane + α-amanitin groups were pretreated with 30 µM sulforaphane for 3 h, and then the old medium was removed and the cells were washed once with PBS buffer. Then, 100 µL of 2% cell maintenance solution was added to the control and sulforaphane groups, and the same amount of 10 µM α-amanitin working solution was added to the α-amanitin and sulforaphane + α-amanitin groups. The subsequent procedures were the same as those in Example 1.

[0019] Cell survival rate, such as Figure 3 As shown in the figure, compared with the control group, the cell viability of the α-amanita peptide group was significantly decreased (****). P <0.0001), compared with the α-amatoxin group, the cell viability of the sulforaphane + α-amatoxin group was significantly increased (^ P <0.05). The results indicate that sulforaphane can alleviate the decrease in AML-12 cell viability induced by α-amanita peptide.

[0020] Example 4 Effects of sulforaphane on intracellular ROS levels in α-amanita peptide-induced AML-12 cells (1) Cell seeding: Place cell spreaders into 6-well plates, seeding 1.2 × 10⁶ cells per well. 6 Use a figure-eight shaking motion to distribute the cells evenly in the cell culture plate. Incubate at 37°C in a 5% CO2 incubator until the cells reach 80%–90% confluence before proceeding to the next step.

[0021] (2) Cell challenge: A control group, sulforaphane group (30 μM), α-amatoxin group (10 μM), sulforaphane + α-amatoxin group (30 μM sulforaphane + 10 μM α-amatoxin), and a positive control group were set up. 30 minutes before the end of the 24-hour challenge, the positive control group was washed twice with serum-free fresh culture medium and 1 mL of serum-free culture medium diluted with Rosup reagent was added. The dilution ratio was 1:500.

[0022] (3) In-situ probe loading: Dilute 10 mM of the probe with PBS buffer at a dilution ratio of 1:500. The DCFH-DA stock solution is the DCFH-DA loading working solution. Remove the old solution, wash twice with PBS buffer, add 1 mL of DCFH-DA loading working solution to each well, incubate at 37°C in a 5% CO2 incubator in the dark for 25 min, remove the old solution, wash three times with serum-free medium to thoroughly remove any unloaded DCFH-DA.

[0023] (4) Mounting: Add 1 drop of anti-fluorescence attenuation mounting medium to the glass slide, remove the cell smear and dry the liquid, then invert the smear onto the glass slide, avoiding the formation of air bubbles during the process, and then observe under a fluorescence microscope.

[0024] The results are as follows Figure 4 As shown in the figure, compared with the control group, the α-amanita peptide group exhibited bright green fluorescence, and the average fluorescence intensity of ROS in AML-12 cells was significantly increased (***). P<0.001), and the mean fluorescence intensity of ROS in AML-12 cells in the sulforaphane + alpha-amanitin group was significantly lower than that in the alpha-amanitin group (^^ P <0.01), indicating that sulforaphane can inhibit the generation of ROS in AML-12 cells induced by alpha-amanitin and reduce the oxidative stress damage of AML-12 cells caused by alpha-amanitin.

[0025] Example 5 Effect of sulforaphane on mitochondrial membrane potential of AML-12 cells induced by alpha-amanitin (1) Cell plating: as in step (1) of Example 4.

[0026] (2) Cell challenge: set up control group, sulforaphane group (30 μM), alpha-amanitin group (10 μM), sulforaphane + alpha-amanitin group (30 μM sulforaphane + 10 μM alpha-amanitin), and positive control group. The challenge was performed 30 min before the end of the 24 h challenge. The positive control group was washed twice with fresh serum-free medium, and 1 mL of CCCP reagent diluted with serum-free medium at a dilution ratio of 1:500 was added.

[0027] (3) TMRE staining working solution preparation: TMRE (1000x) was diluted with the detection buffer provided in the kit to 2x, which was the TMRE staining working solution.

[0028] (4) TMRE staining: after the challenge, the old liquid in the 6-well plate was removed, and PBS was washed once. 1 mL of TMRE staining working solution was added to each well, and incubated at 37°C in a 5% CO2 incubator for 35 min in the dark. The old liquid was removed, and washed with serum-free medium for 3 times.

[0029] (5) Mounting: as in step (4) of Example 4.

[0030] The results are shown in Figure 5 Compared with the control group, the mean fluorescence intensity of mitochondrial membrane potential in AML-12 cells in the alpha-amanitin group was significantly decreased (**** P <0.0001), and the mean fluorescence intensity of mitochondrial membrane potential in AML-12 cells in the sulforaphane + alpha-amanitin group was significantly higher than that in the alpha-amanitin group (^^^ P <0.001), indicating that sulforaphane can reduce the decrease in mitochondrial membrane potential of AML-12 cells induced by alpha-amanitin.

[0031] Example 6 Effect of sulforaphane on mitochondrial morphology of AML-12 cells induced by alpha-amanitin (1) Cell plating: select AML-12 cells in good condition to prepare a cell suspension, count with a hemocytometer, adjust the cell suspension to the appropriate concentration, transfer the cell suspension to a T75 cell culture flask containing fresh complete medium, supplement the medium to 14 mL, shake the cell culture flask in the 8 shape to evenly distribute the cells, and place it in a 37°C, 5% CO2 incubator. When the cell confluence is 80%-90%, the cells are infected.

[0032] (2) Cell infection: set up a control group, a sulforaphane group (30 μM), an α-amanitin group (10 μM), and a sulforaphane + α-amanitin group (30 μM sulforaphane + 10 μM α-amanitin), and incubate them in a 37°C, 5% CO2 incubator for 24 hours.

[0033] (3) Electron microscope sample preparation: after the infection is completed, wash the cells once with completely warmed 2% maintenance solution, gently scrape off the cells with a cell scraper, collect and centrifuge the cells at low speed to enrich them, resuspend the cells in room temperature electron microscope fixing solution, transfer the cell suspension to a 1.5 mL sharp-bottom centrifuge tube, and centrifuge it at 10,000 rpm for 12 minutes. After centrifugation, the cell mass is about the size of a half green bean, carefully discard the supernatant, carefully add new electron microscope fixing solution along the wall of the tube to avoid impacting the cell mass, and let it stand at room temperature for 30 minutes, then fix it at 4°C for 1 hour, and send the sample at low temperature.

[0034] (4) The subsequent operations are all completed by Sichuan Sayinste Company.

[0035] The results are shown in Table 1. Figure 6 Most of the mitochondria in the control group and the sulforaphane group were normal in shape, the mitochondrial membrane was complete, clear mitochondrial cristae were visible, the matrix density was uniform, and the nuclear membrane was clear and complete. The mitochondria in the α-amanitin group were swollen, some mitochondrial membranes were damaged, the mitochondrial matrix was thin, and a large number of autophagosomes were visible. The nuclear membrane was blurred. Compared with the sulforaphane group, the mitochondria in the sulforaphane + α-amanitin group did not change significantly in shape, the matrix density was uniform, and the nuclear membrane was complete. This indicates that α-amanitin can cause mitochondrial damage in AML-12 cells, and sulforaphane can alleviate the mitochondrial damage caused by α-amanitin.

[0036] The experimental results of Examples 1-6 show that α-amanitin can cause a decrease in AML-12 cell viability, an increase in ROS levels, a decrease in mitochondrial membrane potential, mitochondrial swelling and rupture, and mitochondrial damage in AML-12 cells. After pretreatment with sulforaphane, the mitochondrial damage caused by α-amanitin in AML-12 cells was alleviated, which was manifested by a decrease in the inhibitory effect of α-amanitin on AML-12 cell viability, a decrease in ROS levels, an alleviation of mitochondrial membrane potential, and a decrease in mitochondrial morphological changes. This indicates that sulforaphane can alleviate the mitochondrial damage induced by α-amanitin in AML-12 cells and inhibit the hepatotoxicity of α-amanitin.

Claims

1. A sulforaphane, characterized in that: The application of sulforaphane in the preparation of drugs to alleviate α-amanita peptide poisoning.

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