Use of securinine in preparation of drugs for preventing and treating aortic dissection

Abstract

The application discloses application of securinine in preparation of a drug for preventing and / or treating aortic dissection, and securinine can significantly reduce the incidence of aortic dissection caused by BAPN, and securinine can significantly reduce the expression levels of XAF1, Cleved-caspase3 and Cleved-caspase7 proteins, and can effectively inhibit the apoptosis of mouse aortic endothelial cells.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of eurythrin in the preparation of drugs for the prevention and treatment of aortic dissection. Background Technology

[0002] Aortic dissection is a catastrophic acute large vessel disease characterized by tearing of the aortic intima and the formation of a false lumen by blood flowing into the tunica media. It is often referred to as a "time bomb" in the cardiovascular field. Its characteristics include rapid onset, rapid disease progression, and extremely high rates of misdiagnosis and mortality. The mortality rate of aortic dissection is extremely high: in patients with untreated aortic dissection, the mortality rate rises sharply by approximately 1% per hour within the first 48 hours of onset. Type A aortic dissection (involving the ascending aorta) is particularly dangerous, with approximately 50% of patients potentially dying within minutes to hours of onset.

[0003] In aortic dissection, blood forcefully surges through the torn media, causing not only continuous expansion of the false lumen and compression of blood flow in the true lumen, but also potentially leading to rupture of the adventitia of the aortic wall, resulting in massive hemorrhage, or causing ischemia and obstruction of important branch vessels. Continuous pathological processes such as apoptosis, inflammatory response, oxidative stress, and phenotypic transformation of vascular smooth muscle cells collectively drive the progression and deterioration of the dissection. The ancient Chinese medical text *Xue Zheng Lun* also records aortic dissection: "Blood that has left its channels becomes stasis… When stagnant blood is present in the meridians and internal organs, it causes pain throughout the body." This demonstrates that people have deeply considered the pathogenesis and treatment of aortic dissection since ancient times.

[0004] *Hymenochloa crus-galli* is one of the oldest plants in my country used for both food and medicine. Its tender stems and leaves are edible and rich in nutrients, and it plays an irreplaceable role in traditional Chinese medicine for treating various diseases. In TCM theory, *Hymenochloa crus-galli* is sweet, bitter, neutral, and toxic; its branches, leaves, and flowers are used medicinally to invigorate blood circulation, strengthen the spleen and resolve stagnation, and tonify the kidneys and strengthen muscles, often used to treat cardiovascular diseases. In modern medicine, securinine is considered the main active ingredient of *Hymenochloa crus-galli*, commonly used to treat facial nerve paralysis, sequelae of poliomyelitis, neurasthenia, and narcolepsy; it is a mature and safe drug.

[0005] Endothelial cells are the core regulators of the structural integrity and functional homeostasis of the aortic wall. Endothelial dysfunction is the initiating factor and core pathological basis of aortic dissection. Endothelial cell apoptosis leads to the loss of their barrier function, allowing harmful substances to invade and ultimately forming a vicious cycle. XAF1 is a key pro-apoptotic factor that mainly promotes the execution of the apoptosis program by antagonizing the apoptosis-inhibiting protein XIAP to relieve the inhibition of caspase. It is induced to express under stress. In endothelial cells, it also responds to injury stimuli and has been shown to promote apoptosis, making it a core factor in endothelial dysfunction. Therefore, maintaining the integrity of the endothelial cell barrier by antagonizing the pro-apoptotic effect of XAF1 is one of the important means of preventing aortic dissection.

[0006] This invention is of great significance in exploring whether cyclophosphamide can prevent and treat aortic dissection by antagonizing XAF1 and reducing endothelial cell apoptosis. Summary of the Invention

[0007] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing the application of eurythrin in the preparation of drugs for the prevention and treatment of aortic dissection.

[0008] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0009] In a first aspect, the present invention discloses the use of cyclophosphamide in the preparation of drugs for the prevention and / or treatment of aortic dissection.

[0010] In a second aspect, the present invention discloses a drug for the prevention and / or treatment of aortic dissection.

[0011] The drug mentioned includes eugenol.

[0012] Thirdly, the present invention discloses a pharmaceutical preparation for the prevention and / or treatment of aortic dissection.

[0013] The pharmaceutical preparation includes i) cyclophosphamide; ii) pharmaceutically acceptable excipients.

[0014] The content of component i) in the pharmaceutical preparation is 1-95%, such as 2-20%, 25-45%, 50-70%, or 75-95%.

[0015] The dosage form of the drug preparation is an oral dosage form, such as tablets or capsules.

[0016] Fourthly, this invention discloses the use of eugenol in the preparation of XAF1 inhibitors.

[0017] Fifthly, this invention discloses the use of eugenol in the preparation of drugs that inhibit endothelial cell apoptosis.

[0018] In this invention, the therapeutically effective dose of the drug or drug preparation is 8-16 mg per dose for adults.

[0019] In this invention, the drug or drug preparation has any one or more of the following effects:

[0020] (1) Reduce the mortality rate caused by aortic dissection;

[0021] (2) Inhibit aortic dilation and / or the occurrence of aortic dissection;

[0022] (3) Inhibit intramural hematoma and false lumen of the aorta;

[0023] (4) Reduce liver and kidney damage caused by aortic dissection;

[0024] (5) Reduce the mRNA expression level of XAF1 in aortic tissue;

[0025] (6) Inhibit the XAF1-Caspase pathway;

[0026] (7) Inhibits endothelial cell apoptosis.

[0027] The inhibition of the XAF1-Caspase pathway includes reducing the expression of any one or more proteins among XAF1, Cleved-caspase3, and Cleved-caspase7.

[0028] In the technical solution provided by this invention, eugenol exerts an anti-endothelial cell apoptosis effect by regulating the XAF1-CASPASE pathway, thereby alleviating the pathogenesis of reduced apoptosis of normal endothelial cells in the aorta.

[0029] Beneficial effects:

[0030] This invention significantly reduced the incidence of aortic dissection induced by BAPN through gavage treatment with eurythromycin, and eurythromycin can significantly reduce the expression levels of XAF1, Cleved-caspase3, and Cleved-caspase7 proteins, effectively inhibiting apoptosis of mouse aortic endothelial cells. Attached Figure Description

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.

[0032] Figure 1 Effects of gavage administration of basiline on a mouse aortic dissection model.

[0033] Figure 2 This is a representative gross image of the mouse aorta.

[0034] Figure 3 Aortic ultrasound images and aortic diameter statistics were obtained for representative mice from each group at two weeks.

[0035] Figure 4 These are representative images of mouse aorta stained with HE and EVG.

[0036] Figure 5 These are representative HE-stained images of mouse liver and kidney sections.

[0037] Figure 6 The effect of cypermethrin on XAF1 mRNA. *P<0.05, **P<0.01, ***P<0.001.

[0038] Figure 7 This study examines the effects of eugenol on apoptosis pathway proteins.

[0039] Figure 8 The effects of cypermethrin on apoptosis pathway proteins: XAF1, Cleved-caspase 3, and Cleved-caspase 7. *P<0.05, **P<0.01, ***P<0.001. Detailed Implementation

[0040] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0042] Example 1

[0043] By administering basiline via gavage to mice, aortic dissection induced by BAPN was alleviated at the tissue level. Furthermore, by extracting mRNA and protein from mouse aortic tissue, the molecular mechanism by which basiline prevents and treats aortic dissection through anti-apoptotic effects was verified. Details are as follows:

[0044] 1. Modeling and grouping

[0045] To clarify the role of securinine in aortic dissection, 60 mice were randomly divided into four groups: a control group, a high-dose securinine group (SCR) with normal drinking water, a drinking water group (BAPN) containing 0.5% BAPN (3-aminopropionitrile fumarate), a low-dose securinine group (BAPN+SCR-L, 2 mg / kg / day) with 0.5% BAPN, a medium-dose securinine group (BAPN+SCR-M, 5 mg / kg / day) with 0.5% BAPN, and a high-dose securinine group (BAPN+SCR-H, 10 mg / kg / day) with 0.5% BAPN (SCR-H, 10 mg / kg / day) (n = 10 / group). Mice were given 0.5% BAPN in water and securinine at different concentrations daily for 4 weeks.

[0046] Four-week-old wild-type male C57BL / 6J mice were fed via gavage to each group. Blood pressure and cardiac ultrasound were measured in the mice before, two weeks after, and four weeks after modeling. Modeling was completed at four weeks, and the mice were euthanized and their aortic vascular tissue was harvested.

[0047] 2. Survival curves of each group of mice, and statistical analysis of the incidence of aortic dissection.

[0048] After 4 weeks of modeling, no mice in the normal drinking water group and the normal drinking water + high-dose basiline group showed mortality or aortic dilation after gavage. The BAPN group showed a 60% mortality rate due to aortic rupture, and surviving mice generally developed aortic dissection / aneurysm (AAD). Mice fed BAPN-containing water and simultaneously administered basiline via gavage showed fewer cases of AAD. Figure 1 )

[0049] 3. The occurrence of aortic dilation and dissection in mice of each group.

[0050] After the model was established, the mice in each group were euthanized and the aortic tissue was dissected, observed and photographed under a microscope. Figure 2 The formation of aortic aneurysm / dissection was observed in mice of each group at week four using echocardiography. The maximum internal diameter of the aorta was measured. Results ( Figure 3 We found that no mice in the Control and SCR groups developed aortic dissection or significant dilation. BAPN administration via drinking water significantly induced aortic dilation and dissection in mice, while basiline significantly reduced the incidence of these events. Among them, medium and high doses of basiline were more effective.

[0051] 4. Mouse aortic blood vessels, liver and kidney HE and EVG staining

[0052] (1) After obtaining mouse aortic tissue through dissection, it was immediately immersed in 4% PFA and fixed for 4-24 hours. After fixation, the tissue was dehydrated so that it could be embedded in water-insoluble paraffin. The tissue was gradually dehydrated by immersing it in a dehydrating agent of increasing concentration, such as ethanol. Then, it was incubated in xylene to replace the dehydrating agent and to make the tissue clear. The paraffin was heated to 60°C to embed the tissue, and then allowed to harden overnight. The tissue was then sectioned using a microtome.

[0053] (2) HE staining: Paraffin sections were dewaxed to water, rinsed with ddH2O, and then immersed in hematoxylin staining solution for 3 minutes to stain the nuclei. Afterward, they were rinsed in running water for 1 minute. Subsequently, they were immersed in 1% hydrochloric acid-ethanol solution for 8 seconds to induce differentiation, then immersed in ammonia water for 8 seconds for inversion blue staining, and finally rinsed in running water for 1 minute. The cytoplasm was then stained with 1% eosin solution for 1 minute, followed by rinsing in running water for 1 minute. The sections were dehydrated with anhydrous ethanol, cleared with xylene, and mounted with neutral resin.

[0054] (3) EVG staining: Dewax sections to water, stain with prepared Verhoeff's stain at room temperature for 1-3 minutes until the color is deep black. Rinse with ddH2O, differentiate with 2% ferric chloride solution for 10-20 seconds. Under a microscope, the elastic fibers appear black against a gray background. After a brief rinse with ddH2O, counterstain with Van Gieson's solution for 10-15 seconds, and wash with anhydrous ethanol. Dehydrate with anhydrous ethanol, clear with xylene, and mount with neutral resin.

[0055] After paraffin embedding, sectioning, and HE / EVG staining of aortic tissue from different groups of mice, we found that the number of elastic fiber ruptures in the aortic media of mice that drank water containing BAPN was significantly higher than that in the group that drank ordinary drinking water. Many of these mice also showed obvious intramural hematoma and false lumen in the aorta. Gavage with basiline significantly improved this phenomenon. Figure 4 ).

[0056] To determine whether basiline damages liver and kidney function in mice, we dissected liver and kidney tissues from each group of mice, embedded them in paraffin, sectioned them, and performed HE staining and scanning to observe whether basiline could prevent aortic dissection without causing liver and kidney damage. The results showed no significant difference in HE staining morphology of the liver and kidneys between the normal drinking water group and the normal drinking water + high-dose basiline treatment group. However, the 0.5% BAPN group showed varying degrees of liver and kidney damage, and the basiline treatment groups all reversed the liver and kidney damage caused by 0.5% BAPN to varying degrees. Figure 5 ).

[0057] 5. RNA extraction and RT-qPCR experiment from mouse aortic tissue

[0058] To clarify whether cypermethrin exerts its anti-apoptotic effect and thus prevents aortic dissection by inhibiting XAF1, we conducted validation at both the mRNA and protein levels. After the aorta was dissected following animal modeling, we extracted RNA from the aortic tissue and performed RT-qPCR experiments.

[0059] 5.1. RNA extraction from mouse aortic tissue

[0060] (1) Fresh mouse aortic tissue was quickly frozen with liquid nitrogen and then rapidly transferred to a mortar pre-cooled with liquid nitrogen. It was then ground with a pestle while continuously adding liquid nitrogen until it was ground into powder.

[0061] (2) Transfer the aortic tissue, which has been ground into powder, to a centrifuge tube. Add 500 μl of FreeZolReagent (Vazyme) to every 50 mg of tissue. Vortex the tissue to fully lyse it and then let it stand at room temperature for 5 min.

[0062] (3) Add Dilution Buffer (Vazyme) to the above lysis buffer. For every 500 μl of FreeZol Reagent, add 100 μl of Dilution Buffer. Tighten the centrifuge tube cap, vortex until the solution is fully mixed, and then let it stand at room temperature for 5 min.

[0063] (4) After centrifuging at 11,200 rpm (12,000 × g) at room temperature for 15 min, carefully aspirate the supernatant into a new centrifuge tube, add an equal volume of isopropanol, mix thoroughly by inverting the tube, and let stand at room temperature for 10 min.

[0064] (5) Centrifuge at 11,200 rpm (12,000 × g) at room temperature for 10 min and carefully discard the supernatant.

[0065] (6) Add 1 ml of 75% ethanol (prepared with RNase-free ddH2O). Gently tap the bottom of the tube to suspend the precipitate, and invert it several times. Centrifuge at 9,100 rpm (8,000 × g) at room temperature for 3 min, and discard the supernatant.

[0066] (7) Repeat step (6), discard the supernatant and let it air dry at room temperature. Add 100 μl RNase-free ddH2O to dissolve the precipitate and vortex at room temperature to fully dissolve the RNA precipitate.

[0067] 5.2. RT-qPCR

[0068] (1) Genomic DNA removal

[0069]

[0070] (2) Reverse transcription reaction

[0071]

[0072] (3) SYBR Green I chimeric fluorescence qPCR reaction

[0073]

[0074]

[0075] Note: Primers were designed using Primer Premier 5 software, and all primers were synthesized by Qingke Biotechnology Co., Ltd.

[0076] We found that compared to the control group, the mRNA expression level of XAF1 in the aortic tissue of mice in the BAPN group was significantly increased, while this trend was significantly inhibited in the basiline gavage group. Figure 6 ).

[0077] 6. Tissue protein homogenate lysis and Western blotting

[0078] To further verify that eugenol can inhibit the XAF1-Caspas pathway, we conducted experiments at the protein level.

[0079] (1) The aortic tissue of mice was dissected using clean instruments. The tissue was placed in centrifuge tubes and flash-frozen in liquid nitrogen, and then homogenized immediately on ice. 300 μL of lysis buffer was added to every 5 mg of tissue, and the tissue was homogenized with an electric homogenizer and shaken continuously at 4°C for 2 hours.

[0080] (2) Centrifuge at 11,200 rpm (12,000 × g) at room temperature for 15 min. Gently remove the centrifuge tube from the centrifuge and place it on ice. Aspirate the supernatant and transfer it to a new tube that has been pre-cooled on ice. Discard the precipitate.

[0081] (3) Take a small amount of lysis buffer for protein quantitative analysis, and add 1 / 4 volume of 5× loading buffer to the remaining lysis buffer and boil at 100℃ for 5 minutes for reduction and denaturation.

[0082] (4) Gel running: Load equal amounts of protein and molecular weight markers into the wells of an SDS-PAGE gel. The total protein loading amount of aortic tissue homogenate is 20-30 μg, and the gel is run at 100V for 1-2 hours.

[0083] (5) Transfer: PVDF was activated with methanol for 1 minute and rinsed with transfer buffer before preparing the transfer sandwich structure. Transfer time and voltage were optimized according to the molecular weight of the target protein.

[0084] (6) Sealing: After the transfer is completed, remove the PVDF membrane and immerse it in TBST (TBS with Tween-20 added, concentration of 0.05%) containing 5% skim milk powder for 1 hour.

[0085] (7) Primary antibody incubation: After blocking, incubate the PVDF membrane in blocking buffer with an appropriate concentration of primary antibody and shake overnight at 4°C.

[0086] (8) Secondary antibody binding: Wash the membrane with TBST for 10 min × 3 times. Incubate the membrane with the conjugated secondary antibody at the recommended dilution in blocking buffer at room temperature for 1 hour. Wash the membrane again with TBST for 10 min × 3 times.

[0087] (9) ECL color development: Mix ECL color development solution A and B and add them evenly to the membrane surface. Expose the membrane to light and observe the results.

[0088] We found that the levels of XAF1, Cleved-caspase 3, and Cleved-caspase 7 in the BAPN group were significantly higher than those in the normal drinking water group, and this upward trend could be significantly inhibited by basiline. Figure 7 , 8 There were no significant differences in any protein levels between the normal drinking water group and the normal drinking water + cypermethrin group. These experimental results demonstrate that cypermethrin can effectively inhibit the XAF1-Caspase pathway, thereby preventing endothelial cell apoptosis in aortic dissection.

[0089] The above experimental results sufficiently demonstrate that activation of the XAF1-Caspase pathway promotes aortic endothelial cell apoptosis and participates in the development of aortic dissection, while eurythrin can effectively slow down and inhibit this process. Increasing eurythrin intake can effectively prevent endothelial cell apoptosis and inhibit the occurrence of aortic dissection. Therefore, we believe that eurythrin can serve as a new and important drug for the clinical prevention of aortic dissection, and has potential clinical application value in the prevention and treatment of aortic dissection.

[0090] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. Application of cyclophosphamide in the preparation of drugs for the prevention and / or treatment of aortic dissection.

2. The application according to claim 1, characterized in that, The therapeutically effective dose of the drug is 8-16 mg per dose for adults.

3. The application according to claim 1, characterized in that, The drug described herein has one or more of the following effects: (1) Reduce the mortality rate caused by aortic dissection; (2) Inhibit aortic dilation and / or the occurrence of aortic dissection; (3) Inhibit intramural hematoma and false lumen of the aorta; (4) Reduce liver and kidney damage caused by aortic dissection; (5) Reduce the mRNA expression level of XAF1 in aortic tissue; (6) Inhibit the XAF1-Caspase pathway; (7) Inhibits endothelial cell apoptosis.

4. The application according to claim 3, characterized in that, The inhibition of the XAF1-Caspase pathway includes reducing the expression of any one or more of the proteins XAF1, Cleved-caspase3, and Cleved-caspase7.

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