Application of atorvastatin in the preparation of drugs for the prevention or treatment of heatstroke

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of atorvastatin in the preparation of drugs for the prevention or treatment of heatstroke. This invention establishes an in vivo animal model of heatstroke and its induced myocardial injury, and observes related indicators such as rectal temperature, blood biochemistry and routine blood tests, and cardiac function in mice. The findings reveal that atorvastatin can reduce the levels of rectal temperature and creatine kinase isoenzyme in mice after heatstroke injury, and increase the levels of white blood cells, lymphocytes, and intermediate cells. Therefore, the invention proposes the application of atorvastatin in the preparation of drugs for the prevention or treatment of heatstroke.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine, and particularly relates to application of atorvastatin in preparation of a medicine for preventing or treating heat stroke. BACKGROUND

[0002] Heat stroke (HS), also known as severe heatstroke, is a fatal acute disease caused by long-term exposure of the body to a high-temperature and high-humidity environment. Its main pathological features are dysfunction of the body temperature regulation center, failure of sweat glands, and severe loss of water and electrolytes, which lead to rapid rise of core temperature to above 40℃. The clinical manifestations are centered on abnormality of the central nervous system, often accompanied by multiple organ dysfunction, such as acute liver and kidney failure, arrhythmia, brain edema, and shock, and severe cases can develop into multiple organ failure syndrome, and the prognosis of the disease is extremely poor.

[0003] The cardiovascular system plays a key role in the process of body temperature regulation and heat adaptation, and the heart is one of the most vulnerable target organs in heat stroke. Studies have shown that the proportion of heat stroke-induced cardiac dysfunction is 43.4%-65.2%. With the increase of high-temperature exposure time and intensity, cardiac function decreases progressively, and cardiac output decreases significantly, which further reduces blood perfusion of the skin and other tissues and organs, and further weakens the heat dissipation capacity, forming a vicious cycle of "high temperature-cardiac dysfunction-heat dissipation disorder", which eventually leads to continuous rise of core body temperature, multiple organ hypoperfusion, and even shock or sudden death. In the prior art, physical cooling, fluid resuscitation, replacement therapy, and some anti-arrhythmic drugs or vasoactive drugs are used for the comprehensive treatment of heat stroke, and the effect of atorvastatin on heat stroke is unknown. SUMMARY

[0004] To solve the above technical problems, the application provides application of atorvastatin in preparation of a medicine for preventing or treating heat stroke.

[0005] Application of atorvastatin in preparation of a medicine for preventing or treating heat stroke.

[0006] Preferably, the medicine is used for reducing anal temperature rise caused by heat stroke.

[0007] Preferably, the medicine is used for improving cardiac dysfunction caused by heat stroke.

[0008] Preferably, the medicine is an oral preparation or an injection preparation.

[0009] Preferably, the medicine is prepared from the atorvastatin and a pharmaceutically acceptable excipient.

[0010] Preferably, the pharmaceutically acceptable excipient comprises a solubilizing agent or a diluent.

[0011] Preferably, the diluent comprises dimethyl sulfoxide.

[0012] Preferably, when the drug is an injection preparation, the concentration of atorvastatin is 1 µM~10 µM.

[0013] Compared with the prior art, the present application has the beneficial effects that:

[0014] The present application found that ATOR can improve the cell survival rate of HL-1 myocardial cells after HS injury, reduce the generation of intracellular reactive oxygen species, and reduce the release of LDH, by constructing a cell HS (42.5℃, 9h) injury model and detecting cell survival rate, DCFH-DA staining, LDH level, and JC-1 staining. Meanwhile, ATOR can improve the mitochondrial membrane potential. The maximum safe concentration of ATOR in HL-1 myocardial cells is 10 µM.

[0015] The present application found that ATOR can reduce the anal temperature and creatine kinase isoenzyme (CK-MB) levels of mice after HS injury, and increase the levels of white blood cells (WBC), lymphocytes (LYM), and middle cells (MID), by constructing an in vivo HS (39.5℃, 1.5h) and its induced myocardial injury animal model, and observing the anal temperature, blood biochemistry, blood routine related indicators, and heart function related indicators of mice.

[0016] ATOR can improve cardiac dysfunction caused by HS injury, as evidenced by the increase in ejection fraction (EF), fractional shortening (FS), and stroke volume (SV).

[0017] Based on the above findings, the present application provides the use of ATOR for preparing a drug for treating and / or preventing HS injury and its induced myocardial injury.

[0018] Meanwhile, a drug for treating and / or preventing HS injury is provided, which is prepared from ATOR and pharmaceutical excipients, and is a preparation for intravenous injection and / or oral administration. The administration dose of the drug is 5mg, 10mg, or 20mg of ATOR per kilogram of body weight. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figures 1-3 Figure 1 is a graph showing the effect of different concentrations of ATOR on the survival rate of HL-1 myocardial cells. HL-1 myocardial cells were cultured in a basic medium containing different concentrations of ATOR for 24h, and the cell survival rate was detected. Figure 1 Figure 2 is a cell morphology chart.Figure 2 Typical flow cytometry plot of cell viability, Figure 3 Statistical plot of cell viability, results expressed as "mean ± standard deviation", **** P< 0.0001, ns is no statistical difference.

[0020] Figures 4-6 Cell viability after HL-1 cardiomyocytes were treated with different concentrations of ATOR for 3 h and then subjected to HS injury, wherein, Figure 4 Cell morphology under microscope, Figure 5 Typical flow cytometry plot of cell viability, Figure 6 Statistical plot of cell viability, results expressed as "mean ± standard deviation", **** P< 0.0001.

[0021] Figures 7-8 Cell DCFH-DA staining results, wherein, Figure 7 Green fluorescence represents ROS level, Figure 8 Statistical plot of ROS level, results expressed as "mean ± standard deviation", **** P< 0.0001.

[0022] Figure 9 Statistical plot of cell LDH release level results, results expressed as "mean ± standard deviation", **** P< 0.0001.

[0023] Figures 10-11 Cell JC-1 staining results, wherein, Figure 10 Staining plot, Figure 11 Statistical plot of red / green fluorescence ratio, results expressed as "mean ± standard deviation", **** P< 0.0001.

[0024] Figure 12 Effect of ATOR on anal temperature of mice after HS injury, results expressed as "mean ± standard deviation", n = 10; * P< 0.05, **** P< 0.0001, ns is no statistical difference.

[0025] Figure 13 Effect of ATOR on each index of blood biochemistry and blood routine of mice after HS injury for 1.5 h, results expressed as "mean ± standard deviation", n = 10; * P< 0.05, ** P<0.01, *** P <0.001, **** P< 0.0001, ns is no statistical difference.

[0026] Figure 14 Figure is the typical picture of heart long axis M mode and the statistical analysis graph of each index of heart function, the results are expressed as "mean ± standard deviation", n = 10, * P< 0.05, **** P< 0.0001, ns is no statistical difference. DETAILED DESCRIPTION

[0027] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of protection of the present application is not limited by the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application. The experimental methods described in each embodiment of the present application are conventional methods unless otherwise specified.

[0028] The structural formula of atorvastatin in the present application is: , abbreviated as ATOR.

[0029] The purity of atorvastatin used in the present application is not less than 98% by high performance liquid chromatography analysis; the cell line is derived from the ATCC cell library, and the experimental animals are purchased from the Experimental Animal Center of Air Force Military Medical University. Unless otherwise specified, the experimental methods and detection means are conventional methods in the art.

[0030] Example 1: ATOR can reduce the anal temperature of mice after HS injury.

[0031] HS injury is used to construct a HS injury model in mice at the in vivo level, and ATOR is given for pre-protection treatment.

[0032] (1) Grouping: BALB / c mice were used as research objects and divided into Control group, ATOR+HS group (5, 10, 20 mg / kg dose), HS group, a total of 5 groups, 10 in each group;

[0033] (2) Medicine: DMSO (dimethyl sulfoxide) was used to dissolve ATOR to prepare ATOR solutions with concentrations of 5 mg / mL, 10 mg / mL and 20 mg / mL;

[0034] (3) Administration: Six days before HS modeling, mice in each group were given pre-protective treatment (intraperitoneal injection). The Control group was given DMSO (intraperitoneal injection) at a concentration of 5 mg / kg (body weight). The ATOR+HS group was given ATOR at doses of 5 mg / kg, 10 mg / kg, and 20 mg / kg, by intraperitoneal injection, once every 2 days, once 0.5 h before heat injury, for a total of 4 times, ensuring that the time of administration for each administration was the same as the time of heat injury for each group.

[0035] (4) HS modeling: thermal injury was performed 0.5 h after the last intraperitoneal administration.

[0036] Mice were placed in an artificial climate incubator for heat injury. The incubator was set to a constant temperature of 39.5℃ (±1℃) and a humidity of 60±5%. Once all parameters stabilized, the mice were immediately placed back in the incubator for heat injury for 1.5 hours. During the experiment, the mice were not given food or water. After 1.5 hours, the mice were removed, and their rectal temperature was measured using a digital thermometer.

[0037] result:

[0038] Rectal temperature was measured in mice 1.5 h after HS treatment, and the results were as follows: Figure 12 As shown, compared with the Control group, the rectal temperature of mice in the HS group was significantly increased ( P< 0.0001), after administration of ATOR at various concentrations, the rectal temperature of mice in the 20 mg / kg administration group was significantly reduced ( P< 0.05), while there were no significant differences among the other concentration groups ( P＞ (0.05), but there is a downward trend.

[0039] Example 2: ATOR can improve the decreased survival rate of HL-1 cardiomyocytes caused by HS injury.

[0040] (1) Basic cell culture conditions: HL-1 cardiomyocytes were used as the research object. The cells were cultured in a 37°C, 5% CO2 incubator with complete medium containing 10% FBS. The cells adhered to the wall and grew. The medium was changed every other day. When the cells adhered to the wall for 70%-85%, they were digested and passaged with 0.25% trypsin. HL-1 cardiomyocytes in good growth status during the logarithmic growth phase were used for experiments.

[0041] (2) Grouping: HL-1 cardiomyocytes were divided into Control group and ATOR group (1 µM, 2.5 µM, 5 µM, 10 µM, 20 µM, 40 µM) to explore the safe drug dosage:

[0042] Control group: HL-1 cardiomyocytes were cultured in a basal culture (cells were cultured in a 37°C, 5% CO2 cell culture incubator with complete medium containing 10% FBS, adherent growth, and medium was changed every other day) for 24 hours.

[0043] ATOR-treated group: In addition to the basic culture conditions, cells were cultured for 24 hours in complete medium containing 10% FBS with ATOR concentrations of 1 µM, 2.5 µM, 5 µM, 10 µM, 20 µM, and 40 µM, and the cell viability was then detected.

[0044] HS group: Under the basal culture conditions, the HL-1 cardiomyocyte culture medium was replaced with serum-free basal culture medium, and the cells were placed in a cell culture incubator at 42.5℃ for heat injury for 9 hours. After the end of the heat injury, the cell viability was measured.

[0045] ATOR+HS group: HL-1 cardiomyocytes were cultured in complete medium containing 10% FBS at ATOR concentrations of 1 µM, 2.5 µM, 5 µM, and 10 µM for 3 h before being subjected to heat injury, and cell viability was detected.

[0046] Further analysis was performed on the cells in each group as follows:

[0047] Cell viability assay: The Muse Count & Viability Kit contains nuclear dyes and cell viability dyes. Nuclear dyes, being membrane permeable, can distinguish between cells and cell debris. Cell viability dyes are non-permeable, allowing them to enter damaged, dead, or apoptotic cells and bind to nuclear DNA for color development. The combination of these two dyes allows for the differentiation of live cells, apoptotic cells, dead cells, and cell debris.

[0048] The specific method is as follows: After digesting the cells with trypsin without EDTA, centrifuge and prepare a single-cell suspension (1 mL PBS); bring the Muse Count & Viability reagent to room temperature, aspirate 450 µL of the reagent into a 1.5 mL EP tube, add 50 µL of the cell suspension, and mix well; incubate at room temperature in the dark for 5 min, gently shake, and then perform the detection as soon as possible.

[0049] result:

[0050] HL-1 cardiomyocyte survival rate, such as Figures 1-3 As shown, compared with the control group, there was no significant change in cell viability in the 1 µM, 2.5 µM, 5 µM, and 10 µM ATOR drug administration groups ( P >0.05), cell viability decreased in the 20 µM and 40 µM ATOR groups ( P<0.0001). Therefore, the maximum safe concentration of ATOR in HL-1 cardiomyocytes is 10 µM.

[0051] Following HS injury, the HL-1 cardiomyocyte survival rate was as follows: Figures 4-6 As shown: Compared with the Control group, the cell viability in the HS group was significantly decreased ( P <0.0001); compared with the HS group, the cell viability of the ATOR-treated groups was significantly increased ( P <0.0001).

[0052] Example 3: ATOR can reduce the generation of reactive oxygen species in HL-1 cardiomyocytes caused by HS injury.

[0053] The Reactive Oxygen Species Assay Kit (ROS Assay Kit) is a kit for detecting reactive oxygen species using the cell-permeable fluorescent probe DCFH-DA. DCFH-DA itself is non-fluorescent and can freely cross the cell membrane. Once inside the cell, it is hydrolyzed by intracellular esterases to generate DCFH. DCFH is impermeable to the cell membrane, making it easy for the probe to be loaded into the cell. Intracellular reactive oxygen species oxidize the non-fluorescent DCFH to generate fluorescent DCF. Detecting the fluorescence of DCF indicates the level of intracellular reactive oxygen species. Follow the kit instructions: Prepare the working solution for the fluorescent detection probe by dissolving 6 µL of the stock solution in 12 mL of DMEM; discard the old cell culture medium, add the working solution to a 60 mm cell culture dish, and incubate at 37°C for approximately 30 minutes to load the fluorescent probe; after loading, wash three times with DMEM for 5 minutes each time; after washing, acquire images under a fluorescence microscope.

[0054] result:

[0055] DCFH-DA staining results are as follows Figures 7-8 The results showed that compared with the Control group, the HS group had increased ROS generation, which was reflected in the increased green fluorescence intensity. P <0.0001); Compared with the HS group, the ROS generation in each ATOR+HS treatment group was reduced, which was reflected in the decrease in green fluorescence intensity (P<0.0001).

[0056] Example 4: ATOR can reduce LDH release from HL-1 cardiomyocytes caused by HS injury.

[0057] Prepare the necessary reagents (coenzyme I, 0.4 mol / L NaOH solution, and 0.2 μmol / mL pyruvate standard working solution) according to the lactate dehydrogenase (LDH) assay kit, and then perform sample addition and analysis according to the following steps:

[0058] Table 1. Procedures for measuring LDH levels

[0059]

[0060] result:

[0061] LDH detection results of cell supernatant are as follows Figure 9 The results show that LDH release is increased in the HS group compared to the Control group. P <0.0001); compared with the HS group, LDH release was reduced in each ATOR+HS treatment group ( P <0.0001).

[0062] Example 5: ATOR can improve mitochondrial membrane potential imbalance in HL-1 cardiomyocytes caused by heatstroke.

[0063] Prepare the necessary reagents according to the JC-1 assay kit. Resuspend cells from each group in 0.5 ml of cell culture medium containing serum and phenol red, add 0.5 ml of JC-1 staining working solution, invert several times to mix, and incubate at 37°C for 20 min in a cell culture incubator. Then centrifuge at 600 g for 3-4 min to pellet the cells and discard the supernatant. Wash twice with JC-1 staining buffer: resuspend cells in 1 ml of JC-1 staining buffer, centrifuge at 600 g for 3-4 min to pellet the cells and discard the supernatant; resuspend cells in 1 ml of JC-1 staining buffer again, centrifuge at 600 g for 3-4 min to pellet the cells and discard the supernatant. After resuspending with JC-1 staining buffer again, observe under a fluorescence microscope or laser confocal microscope.

[0064] result:

[0065] JC-1 staining results are as follows Figures 10-11 As shown: Compared with the Control group, the HS group showed significantly weakened red fluorescence, significantly enhanced green fluorescence, and a significantly reduced red fluorescence / green fluorescence ratio. P <0.0001); After treatment with each dose of ATOR, compared with the HS group, the red fluorescence of each dose treatment group was significantly enhanced, the green fluorescence was significantly weakened, and the red fluorescence / green fluorescence ratio was significantly increased ( P <0.0001).

[0066] Example 6: ATOR can improve changes in blood biochemistry and routine blood indicators caused by HS injury.

[0067] Using HS injury, an in vivo model of HS and its induced myocardial injury was constructed in mice. ATOR preprotective treatment was administered, and the specific steps were the same as in Example 1.

[0068] Mice were anesthetized with 2% isoflurane, and their eyeballs were removed with sterile surgical forceps. More than 0.5 mL of blood was collected in a centrifuge tube and centrifuged at 3000 rpm for 10 min. About 150 μL of the supernatant serum was collected and placed in a fully automated blood biochemistry analyzer for CK-MB index detection.

[0069] Mice were anesthetized with 2% isoflurane, their eyeballs were removed with sterile surgical forceps, and blood was collected into sterile heparin sodium tubes. After incubation on ice for 2 minutes, blood routine tests were performed, including WBC, LYM, and MID.

[0070] result:

[0071] Changes in blood biochemistry and routine blood count parameters were detected in mice 1.5 h after HS treatment. The results are as follows: Figure 13 As shown, compared with the control group, CK-MB significantly increased 1.5 h after HS injury ( P< 0.001), WBC, LYM and MID levels were significantly reduced ( P< 0.05); After treatment with various concentrations of ATOR, CK-MB decreased significantly ( P< 0.01), LYM levels increased significantly ( P< 0.05), WBC and MID levels increased significantly in the 5 mg / kg and 20 mg / kg administration groups ( P< Although there was no significant difference in the 0.05 mg / kg group, the 10 mg / kg group ( P＞ (0.05), but there is an upward trend.

[0072] Example 7: ATOR can improve cardiac dysfunction caused by HS.

[0073] Using HS injury, an in vivo model of HS and its induced myocardial injury was constructed in mice. ATOR preprotective treatment was administered, and the specific steps were the same as in Example 5.

[0074] Small animal ultrasound examination of HS injury 1.5 h later: The left chest area of ​​mice was shaved off the day before the ultrasound examination. The mice were anesthetized with 3% isoflurane at a flow rate of 1 L / min. After isoflurane inhalation anesthesia, the mice were fixed on a 37℃ constant temperature plate to fully expose the left thoracic cavity. M-mode cardiac ultrasound images were recorded using a 30MHz probe. The measured parameters included EF, FS and SV.

[0075] During the testing process, the following details should be noted as they may affect the test results: First, the anesthesia should not be too deep, otherwise it will affect the mouse's heart rate and contractile function; second, the mouse's body position should be correct, and its limbs should not be fixed too long or too tight, otherwise it will compress the mouse's heart and ultimately affect the accuracy of the cardiac function test; third, the area of ​​the mouse near the heart should be shaved at least one day in advance. Shaving the hair too early will cause new hair to grow during the test, generating artifacts during imaging and affecting the ultrasound results, while shaving the hair too late will put the mouse in a state of stress and interfere with the cardiac function results.

[0076] result:

[0077] Small animal ultrasound was used to assess cardiac function in mice 1.5 h after HS injury. The results are as follows: Figure 14 (As shown in the parasternal left ventricular long-axis ultrasound results), compared with the control group, HS injury significantly reduced the EF, FS, and SV of the mouse heart. P <0.05; After ATOR protection, the EF, FS, and SV of the mouse heart were significantly increased in the 5 mg / kg group ( P <0.05%, while there were no significant differences in other dosage groups ( P＞ The concentration of ATOR was 0.05, but there was an upward trend. These results suggest that cardiac function in mice was significantly improved after administration of different concentrations of ATOR.

[0078] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0079] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0080] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of atorvastatin for the preparation of a medicament for the prophylaxis or treatment of heat stroke, characterized in that, The medicine is used for reducing anal temperature rise caused by heat stroke, and the medicine is used for improving heart function disorder caused by heat stroke.

2. Use according to claim 1, characterized in that, The medicine is an oral preparation or an injection preparation.

3. Use according to claim 2, characterized in that, The medicine is prepared from the atorvastatin and a pharmaceutically acceptable auxiliary.

4. Use according to claim 3, characterized in that, The pharmaceutically acceptable auxiliary is a cosolvent or a diluent.

5. Use according to claim 4, characterized in that, The diluent is dimethyl sulfoxide.

6. Use according to claim 5, characterized in that, When the medicine is an injection preparation, the concentration of atorvastatin is 1 µM~10 µM.

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