Application of 6RK73 in preparation of medicine for preventing or treating atrial fibrillation

Abstract

The invention discloses application of 6RK73 in preparation of a medicine for preventing or treating atrial fibrillation and a medicine composition for preventing or treating atrial fibrillation. According to the application disclosed by the invention, by injecting the 6RK73 into the intraperitoneal cavity of an atrial fibrillation (especially Ang II induced) mouse for pretreatment, the 6RK73 is found to be capable of obviously reducing the atrial fibrillation area, improving inflammatory response and oxidative stress in atrial tissues and relieving atrial structure remodeling so as to prevent and treat atrial fibrillation, and a new medicine and a new treatment scheme are provided for clinical treatment of atrial fibrillation patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to use of 6RK73 in preparing a drug for preventing or treating atrial fibrillation. Background Art

[0002] Atrial fibrillation (also referred to as AF) is the most common persistent arrhythmia in clinical practice and an important cause of cardiovascular and cerebrovascular diseases including stroke and heart failure, as well as increased mortality. According to the "China Cardiovascular Health and Disease Report 2019", there are more than 300 million cardiovascular disease patients in my country, and the average prevalence of AF is about 0.77%. With the increase of age, its incidence rate gradually increases. The prevalence of AF in people over 65 years old can reach 6%. At present, the total number of people with AF in my country is about 10 million, which is equivalent to the incidence of coronary heart disease. It has become an important disease that seriously endangers the health of Chinese residents. Although surgical treatment and drug treatment of AF have developed rapidly in recent years, there are still problems such as relatively high drug side effects, recurrence rate and potential complications, and relatively limited beneficiary population. The main reason is that the molecular mechanism of AF is still not very clear and there is a lack of effective treatment methods. Therefore, it is urgent to study the pathological mechanism of AF, find effective new intervention targets, reduce the incidence and mortality of AF, and improve the health level of Chinese residents. It has important practical significance.

[0003] Atrial electrical remodeling and structural remodeling are the main pathological basis for the occurrence and maintenance of atrial fibrillation. It is currently believed that the etiology and pathophysiological mechanism of atrial fibrillation are complex. Various risk factors such as hypertension, elevated angiotensin II (Ang II), high salt and ischemia all lead to neuroendocrine and angiotensin-aldosterone systems, oxidative stress, systemic inflammatory response and metabolic abnormalities, which in turn cause atrial structural and electrical remodeling. Atrial structural remodeling is mainly manifested as atrial dilatation, increased extracellular matrix deposition and interstitial fibrosis, which is the main reason for the maintenance of atrial fibrillation. Among them, the size of the atrium is the determining factor for the maintenance of atrial fibrillation; while fibrosis causes the continuity of atrial muscle fiber bundles to be interrupted, interferes with local signal conduction, and then promotes the occurrence of atrial fibrillation, playing a key role in atrial electrical remodeling and atrial fibrillation. Atrial fibrosis is a common feature of patients with atrial fibrillation, and the degree of fibrosis is a predictive factor for the recurrence of atrial fibrillation after catheter ablation. The occurrence of atrial fibrillation at the molecular level is related to the abnormal expression or degradation of multiple proteins. Studies have found that abnormal protein degradation can cause atrial structural remodeling and various pathological changes, such as atrial fibrosis, oxidative stress and increased inflammatory response.

[0004] The ubiquitin-proteasome system (UPS) is an important and highly selective protein degradation pathway in eukaryotic cells. It participates in a variety of biological processes such as cell cycle, differentiation, apoptosis, oxidative stress and inflammatory response, and plays an important role in maintaining protein homeostasis and function. UPS mainly includes ubiquitin activating enzyme (E1), ubiquitin transporter (E2), ubiquitin ligase (E3), 26S proteasome and deubiquitinating enzyme (DUBs). UPS abnormalities are associated with a variety of diseases. As the reverse regulatory enzyme of ubiquitination, DUBs mainly hydrolyze ubiquitin molecules from substrate proteins or precursor proteins by hydrolyzing the ester bond, peptide bond or isopeptide bond at the carboxyl terminal of ubiquitin. DUBs are mainly divided into five families, namely the ubiquitin carboxyl terminal hydrolase (UCH) family, the ubiquitin-specific protease (USP / UBP) family, the Otubaim (OTU) family, the Josephin domain protein family and the JAMM family. As an important member of the DUBs family, UCHL1 not only has the function of deubiquitination, but also has the ATPase-independent ligase activity and the function of stabilizing intracellular ubiquitin monomers. In addition, UCHL1 has been found to be associated with a variety of diseases, including type 1 diabetes, type 2 diabetes, Huntington's disease, metabolic diseases (such as 3-hydroxyacyl-CoA dehydrogenase deficiency), Parkinson's disease, acute myeloid leukemia, chronic pancreatitis, breast cancer, prostate cancer, cataracts, leukemia, fibrosis (such as congenital extraocular muscle fibrosis), and acute pancreatitis. Although 6RK73 is a covalently irreversible specific inhibitor of UCHL1, 6RK73 has only been used in cells and zebrafish to inhibit breast cancer. There is no research showing whether 6RK73 is effective in other diseases, and there is no research showing whether 6RK73 is effective in mammals (such as mice). Although previous studies by our research group have shown that the expression of UCHL1 in atrial tissue is significantly increased, it is still unclear whether 6RK73 can improve atrial fibrillation by inhibiting UCHL1, and it is even less clear which signaling pathways 6RK73 can affect the occurrence of atrial fibrillation. Summary of the invention

[0005] Based on the problems existing in the above-mentioned prior art, after research, the present invention proposes the following technical solutions:

[0006] The first aspect of the present invention provides use of 6RK73 in preparing a medicament for preventing or treating atrial fibrillation.

[0007]

[0008] In the present invention, the molecular formula of 6RK73 is C 13 H 17 N5O2S has the structure shown in the above formula.

[0009] Furthermore, the atrial fibrillation is angiotensin Ang II-induced atrial fibrillation.

[0010] Furthermore, the drug comprises an effective amount of 6RK73. Furthermore, the effective amount is 0.1-1 mg / kg, and is administered at least twice a week.

[0011] Furthermore, the drug is prepared from 6RK73 and at least one pharmaceutical carrier and / or at least one pharmaceutical excipient via a general pharmaceutical method.

[0012] Furthermore, the pharmaceutical excipients include at least one of a diluent, a binder, a disintegrant, a lubricant and a flavoring agent.

[0013] Furthermore, the dosage form of the drug includes tablets, capsules, granules, injections or sprays.

[0014] Furthermore, the method of taking the drug includes oral administration or parenteral administration, wherein the parenteral administration method includes but is not limited to oral administration or one or more administration routes of intravenous injection, intravenous drip, intramuscular injection, subcutaneous injection, and intraventricular direct administration injection.

[0015] The second aspect of the present invention provides a pharmaceutical composition for preventing or treating atrial fibrillation, comprising an effective amount of 6RK73, at least one pharmaceutically acceptable carrier and / or at least one pharmaceutically acceptable excipient.

[0016] Furthermore, the effective amount of 6RK73 is 0.1-1 mg / kg, administered at least twice a week.

[0017] Furthermore, the atrial fibrillation is angiotensin Ang II-induced atrial fibrillation.

[0018] The endpoints and any values ​​of the ranges disclosed herein are not limited to the exact ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be regarded as specifically disclosed in this article. It should be noted that in this article, specific numerical values ​​can vary within ±2% as the numerical protection range of this application.

[0019] In the description of this application, unless otherwise specified, "multiple / multiple" and other similar words mean two / kinds or more than two / kinds. In addition, "include", "including", "have", "contain" and any variations thereof used in the present invention are open terms, i.e., including but not limited to.

[0020] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0021] The pharmaceutical composition of the present invention is used to prevent and / or treat atrial fibrillation. In the present application, the terms "drug" and "pharmaceutical composition" can be used interchangeably, and "drug / pharmaceutical composition" refers to a preparation of a compound of the present invention and a medium generally accepted in the art for delivering biologically active compounds to mammals (e.g., humans). The purpose of the pharmaceutical composition is to promote administration to an organism, facilitate the absorption of the active ingredient, and then exert biological activity. The term "pharmaceutical" as used herein refers to a substance (such as a pharmaceutical carrier or a pharmaceutical excipient) that does not affect the biological activity or properties of the compound of the present invention, and is relatively non-toxic, that is, the substance can be administered to an individual without causing adverse biological reactions or interacting with any component contained in the composition in an adverse manner.

[0022] In the present invention, the pharmaceutical excipients include at least one of a diluent, a binder, a disintegrant, a lubricant and a flavoring agent.

[0023] In the present invention, the pharmaceutical carrier includes at least one of 1,3-butanediol, castor oil, corn oil, cottonseed oil, glucose, germ oil, peanut oil, liposomes, oleic acid, olive oil, peanut oil, Ringer's solution, safflower oil, sesame oil, soybean oil or sodium chloride solution, water or a mixture thereof.

[0024] In the present invention, "treatment" should be understood in the broadest sense and may include the meaning of "prevention".

[0025] In the present invention, the term "prevent" includes reducing the likelihood of a disease or condition occurring or becoming worse in a patient.

[0026] In the present invention, the term "treatment" and other similar synonyms include the following meanings:

[0027] (i) preventing a disease or condition from occurring in a mammal, particularly where such mammal is susceptible to the disease or condition but has not yet been diagnosed as having the disease or condition;

[0028] (ii) inhibiting a disease or condition, i.e. arresting its development;

[0029] (iii) alleviate the disease or condition, that is, cause regression of the disease or condition; or

[0030] (iv) alleviating the symptoms caused by the disease or condition.

[0031] In the present invention, the term "effective amount" or "pharmaceutically effective amount" refers to an amount that can produce a preventive or therapeutic effect on humans and / or other animals and can be accepted by humans and / or other animals. The result can be the reduction and / or alleviation of signs, symptoms or causes of disease, or any other desired changes in biological systems. For example, an "effective amount" for treatment is the amount of a composition comprising a compound disclosed herein that is required to provide a significant symptom alleviation effect clinically. Techniques such as dose escalation tests can be used to determine the effective amount suitable for any individual case.

[0032] In the present invention, the terms "administering", "dosing", "medication" and the like refer to methods capable of delivering a compound or composition to a desired site for biological action.

[0033] Administration of a compound of the present invention comprising an effective amount of 6RK73 is used for mammals (including humans and other animals) in need of preventing or treating atrial fibrillation, wherein the dosage at the time of administration is an effective dosage for pharmaceutical prevention or treatment. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians. In some embodiments, if it is treatment, three times a week, each dosage is usually 0.1-1 mg / kg. In some preferred embodiments, if it is treatment, three times a week, each dosage is usually 0.5 mg / kg.

[0034] The beneficial effects of the present invention include at least:

[0035] The present invention pre-treats mice with atrial fibrillation (especially Ang II-induced) by intraperitoneal injection of 6RK73, and finds that 6RK73 can significantly reduce the area of ​​atrial fibrosis, improve inflammatory response and oxidative stress in atrial tissue, and alleviate atrial structural remodeling, so as to prevent and treat the occurrence of atrial fibrillation, and provide new drugs and treatment plans for the clinical treatment of patients with atrial fibrillation.

[0036] The present invention verifies for the first time the role of 6RK73 in atrial fibrillation, and verifies for the first time in mice that 6RK73 affects the occurrence of atrial fibrillation by improving the activation of Ang II-induced atrial ion channels, inflammatory responses, oxidative stress and fibrosis signaling pathways. At the same time, 6RK73 has high potency, small dosage, and significantly fewer adverse reactions (especially effects on the gastrointestinal tract), providing a basis for the preparation of therapeutic drugs suitable for popularization and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1The effect of 6RK73 pretreatment on atrial fibrillation induced by hypertensive factors (Ang II) in mice: the left figure shows the electrocardiogram image of atrial fibrillation or sinus rhythm in mice recorded by an electrophysiological recorder; the right figure shows a comparison of the incidence of atrial fibrillation in each group of mice and the duration of atrial fibrillation in mice.

[0038] Figure 2 The figure below shows the effect of 6RK73 pretreatment on the enlargement of the atrial diameter in mice with atrial fibrillation. The figure on the left shows the diameter of the left atrium of mice measured by echocardiography. The figure on the right shows the comparison of the diameter of the left atrium of four groups of mice.

[0039] Figure 3 The figure below shows the effect of 6RK73 pretreatment on atrial fibrosis in mice with atrial fibrillation. The figure on the left shows Masson's staining micrographs of the atria of the four groups of mice. The figure on the right shows a comparison of the degree of atrial fibrosis in the four groups of mice.

[0040] Figure 4 This is the effect of 6RK73 pretreatment on the atrial tissue fibrosis markers collagen I (COL1A1) and collagen III (COL3A1) in mice with atrial fibrillation.

[0041] Figure 5 The figure below shows the effect of 6RK73 pretreatment on atrial inflammatory response in mice with atrial fibrillation. The figure on the left shows the microscopic image of immunofluorescence staining (CD68) of the atrial tissues of the four groups of mice. The figure on the right shows the comparison of the degree of inflammatory response in the atrial tissues of the four groups of mice.

[0042] Figure 6 Effects of 6RK73 pretreatment on inflammatory markers IL-1β (IL1β) and IL-6 (IL6) in atrial tissue of mice with atrial fibrillation.

[0043] Figure 7 The figure below shows the effect of 6RK73 pretreatment on oxidative stress in the atria of mice with atrial fibrillation. The figure on the left shows the microscopic image of superoxide anion fluorescent probe (dihydroethidium, DHE) staining in the atrial tissue of four groups of mice. The figure on the right shows the comparison of relative fluorescence intensity of DHE in the atria of four groups of mice.

[0044] Figure 8 Effects of 6RK73 pretreatment on oxidative stress markers NOX2 (CYBB) and NOX4 (NOX4) in atrial tissue of mice with atrial fibrillation.

[0045] Fig. 9 This is the effect of 6RK73 pretreatment on atrial tissue ion channels, oxidative stress, inflammatory response and fibrosis signaling pathways in mice with atrial fibrillation. DETAILED DESCRIPTION

[0046] The present invention is further described below in conjunction with specific embodiments. It should be understood that the following contents are only used to explain the present invention and cannot be understood as limiting the present invention.

[0047] Without departing from the scope or spirit of the present invention, various modifications and variations may be made to the specific embodiments of the present invention description, which will be apparent to those skilled in the art. Other embodiments obtained from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only. The experimental methods in the following examples that do not specify specific conditions are generally tested under conventional conditions or under conditions recommended by the manufacturer.

[0048] Example 1

[0049] 1 Experimental design and methods:

[0050] 1.1 Animal husbandry and pretreatment:

[0051] Experimental animals: 8-10 week old, male mice weighing 22-25 g with a C57BL / 6 background were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. They were housed in separate cages at a constant temperature (23-25° C.) and humidity (55-70%).

[0052] The animal experiments were divided into 4 groups: healthy control group (WT group), normal drug group (6RK73 group), model group (Ang II infusion group) and treatment group (6RK73+Ang II infusion group), with at least 6 animals in each group.

[0053] Preparation of 6RK73 working solution: For each mouse (23 g), 0.0115 mg of 6RK73 powder was dissolved in 5 μl of DMSO, and 95 μl of 0.9% sodium chloride solution was added and mixed until clear.

[0054] The mice in the WT group and the Ang II infusion group were injected intraperitoneally with 0.9% sodium chloride solution (i.e., normal saline) at 0.1 mL / mouse. The mice in the 6RK73 group and the 6RK73+Ang II infusion group were pretreated with the 6RK73 inhibitor working solution (0.5 mg / kg, 3 times a week, intraperitoneal injection) for the experimental mice.

[0055] The four groups of mice were treated with drugs one week before Ang II infusion until the end of the experiment or the mice were collected. One week after 6RK73 treatment, atrial fibrillation models were established in the mice in the Ang II infusion group and the 6RK73+Ang II infusion group.

[0056] 1.2 Methods of Ang II infusion to induce hypertension and atrial fibrillation in mice

[0057] 1.2.1 Filling scheme of micro sustained-release pump and preparation method of Ang II working solution for mouse hypertension and atrial fibrillation model:

[0058] Take an Alzet micro-sustained release pump (1004, Alzet Osmotic Pumps) and immerse it in physiological saline solution and place it in a cell culture incubator (37°C) for 48 hours to activate it. Calculate the dosage of Ang II (A107852, Aladdin) (the volume of the Alzet micro-sustained release pump is 100 μL, and the pumping speed is 0.11 μL / h): measure the body weight (g) of each mouse, and the amount of Ang II perfused per mouse per hour = 2000 ng / kg / min × mouse body weight (kg) × 10 -3 ×60min; Ang II working solution concentration = the amount of Ang II infused per hour / 0.11 μL / h. In this example, the mouse weighed about 25 g. According to the above method, 2.73 mg of Ang II powder was weighed for each mouse and dissolved in 100 μl of saline solution, which was then injected into the micro pump. Similarly, the saline solution was injected into the Alzet micro sustained-release pump as a control group.

[0059] 1.2.2 Implantation of micro sustained-release pump

[0060] The four groups of experimental mice mentioned above were taken and anesthetized by intraperitoneal injection of saturated tribromoethanol (T48402, Sigma) solution. The mice were placed in a prone position with their limbs fixed. The surgical area was disinfected with 75% alcohol. The skin of the back of the neck of the mice was cut about 4 to 5 mm. The back skin layer and muscle layer were bluntly separated using scissors or a needle holder. A micro sustained-release pump filled with agonist Ang II or saline solution was placed subcutaneously in the back, and the incision was sutured.

[0061] 1.3 Imaging evaluation of atrial function in mice

[0062] Mice were anesthetized with isopentane to maintain spontaneous breathing and heart rate at about 600 bpm. Vevo 1100 ultrasound instrument and high-frequency probe were used to collect two-dimensional images of the left atrium, and the left atrial end-diastolic diameter (LAD) was recorded using M-mode ultrasound mode.

[0063] 1.4 High-frequency programmed electrical stimulation to induce atrial fibrillation

[0064] After 3 weeks of Ang II perfusion, the experimental mice were anesthetized by intraperitoneal injection of saturated tribromoethanol, fixed on the operating table in a supine position, and placed under a stereomicroscope. The skin on the right side of the neck was cut longitudinally for about 2 cm, and the submandibular gland, thyroid gland, neck muscles, etc. were bluntly separated to fully expose the right jugular vein to the surgical field. The distal end of the right jugular vein was ligated, and the vein was cut obliquely at the proximal end of the ligation point with microscissors. A Millar 1.1F EP catheter (Scisense) was inserted into the opening of the right jugular vein and entered the right heart system along the direction of the blood vessel. The position and angle of the guide wire were adjusted according to the real-time electrocardiogram displayed by the electrophysiological recorder. Continuous pacing was performed from 40ms to 20ms, with a pacing cycle decreasing by 2ms each time. Each mouse received a round of pacing stimulation at 3.5V, 5V and 8V respectively. Atrial fibrillation in mice was defined as the occurrence of rapid and irregular atrial fibrillation waves lasting more than 1000ms. Atrial fibrillation was induced at least twice, which was defined as positive. The incidence of induced atrial fibrillation and the duration of atrial fibrillation in each group of mice were statistically analyzed.

[0065] 1.5 Myocardial pathological examination

[0066] After fully anesthetizing the experimental mice with tribromoethanol, the mice were fixed and the hearts were exposed. A small incision was made in the inferior vena cava near the right atrium. A 20mL syringe was used to draw heparinized saline solution and injected into the left ventricle from the apex of the heart. After the heart was rinsed with blood, the heart was removed, the atrial tissue was separated, placed in a labeled cryotube, quickly placed in a liquid nitrogen tank, and then stored in a -80℃ refrigerator for molecular biology detection.

[0067] Mouse atria were fixed, dehydrated, and paraffin-embedded in sections for pathological staining, including Masson staining (D026, Nanjing Jiancheng), immunofluorescence, and DHE fluorescence staining (BMD00001, Abbkine). The specific steps are as follows.

[0068] 1.5.1 Masson staining

[0069] Place the tissue in a 70℃ oven for 20-30min, take it out and wait for it to cool naturally before dewaxing; dewaxing steps: immerse the tissue completely in xylene I (15min), xylene II (15min), anhydrous ethanol I (5min), anhydrous ethanol II (5min), 95% ethanol (3min), and 85% ethanol (3min) in sequence. Take out the tissue and soak it in double distilled water and change it every 5 minutes, for a total of three times; place it in Bouin's solution (LA0976, Labcoms) at 4℃ overnight. The next day, return Bouin's solution to room temperature and gently rinse the tissue sections with tap water until the yellow color fades. Stain with Ponceau red for 5-10min, rinse off the Ponceau red with phosphomolybdic acid differentiation solution, stain with aniline blue for 1-2min, and rinse with dd H2O. Dehydrate, permeabilize, seal with neutral gum, and take pictures in time.

[0070] 1.5.2 Immunofluorescence staining

[0071] Tissue sections were dewaxed to the aqueous phase, as in 1.5.1. Sodium citrate working solution was prepared using 50× sodium citrate antigen retrieval solution (SL1870, Coolaber) and double distilled water at a ratio of 1:49. The tissue sections were immersed in the sodium citrate working solution, heated at low heat in a microwave oven for 15 minutes, and cooled naturally to room temperature. A tissue pen was used to draw circles around the tissue, and the sections were washed three times with PBS buffer for 5 minutes each time. 0.5% Triton X-100 (Sigma-Aldrich) punching solution was prepared, permeabilized at room temperature for 15 minutes, and washed three times with PBS for 5 minutes each time. Serum blocking solution (ZLI-9056, Zhongshan Jinqiao) was used to block at room temperature for 30 minutes, and the primary antibody (rabbit polyclonal CD68 antibody, dilution ratio of 1:80, #97778, CST; mouse monoclonal cTNT antibody, dilution ratio of 1:150, 68300-1-Ig, Wuhan Tri-Tak) was incubated overnight at 4°C. The sections were taken out the next day and returned to room temperature, and washed three times with PBS for 5 minutes each time. Fluorescently labeled secondary antibodies (dilution ratio 1:100; A0453 / A0428, Bio-Tech) were incubated at room temperature for 2 h, and washed 3 times with PBS, 5 min each time. The slides were sealed with DAPI anti-fluorescence quenching sealing medium, and images were collected in time.

[0072] 1.5.3 Dihydroethidium (DHE) fluorescence staining

[0073] Take the frozen tissue of mouse heart and place it in a cryostat. Use a cryostat to cut the tissue into 10μm slices and adhere them to a cationic anti-shedding slide. Drop a 1μmol / L DHE fluorescent probe solution on the tissue slices and incubate at 37℃ in the dark for 30min. Wash the slices three times with PBS, 5min / time, and seal them with anti-fluorescence quenching sealing agent. Observe the red fluorescence intensity under a fluorescence microscope.

[0074] 1.6 Real-time quantitative PCR

[0075] Total mRNA was extracted and quantified by Trizol phenol-chloroform extraction method, and mRNA was reverse transcribed into single-stranded cDNA using a reverse transcription kit (11141ES60, Yeasen). Green fluorescence PCR kit (RR820A, Takara) and corresponding primers were used to amplify the target genes, and the expression levels of myocardial fibrosis markers Collagen I (COL1A1), CollagenIII (COL3A1), inflammatory response markers IL-1β (IL1β), IL-6 (IL6) and oxidative stress markers NOX2 (CYBB), NOX4 (NOX4) mRNA were detected.

[0076] The primer sequences are as follows:

[0077]

[0078] The reaction program of real-time quantitative PCR is as follows: Holding Stage: 95℃ (30s); Cycling Stage: 95℃ (5s)-60℃ (34s) for 40 cycles; Melt Curve Stage: 95℃ (15s)-60℃ (1min)-95℃ (15s). After the PCR is completed, the results are exported and the expression level of the target gene is 2 ΔCT (ΔCT=CT 内参 -CT 目的基因 ).

[0079] 1.7 Western Blotting

[0080] RIPA buffer (P0013K, Beyotime) was used to extract cell and atrial tissue proteins, and the concentration of protein samples was determined using a BCA kit (P0010S, Beyotime). SDS-PAGE (PG112, Yazyme) gel separation, membrane transfer and blocking were performed, and then primary and secondary antibodies were incubated to detect the expression levels of related proteins, including immunoblotting to detect inflammatory signaling pathway NLRP3 antibodies (1:800; 19771-1-AP, Wuhan Tri-Index), p-p65 (1:500; 310013, Zhengneng Biotechnology), p65 (1:1000; #4764, CST), oxidative stress signaling pathway NOX2 (1:1000; 19013-1-AP, Wuhan Tri-Index), NOX4 (1:1000; 14347-1-AP, Wuhan Tri-Index), fibrosis signaling pathway p-SMAD2 (1:1000; ab280888, Abcam), SMAD2 (1:1000; 12570-1-AP, Wuhan Tri-Index), ion channel-related protein K V1.5 (1:800; 21659-1-AP, Wuhan Tri-Index).

[0081] 1.8 Statistical analysis

[0082] All data were analyzed using GraphPad Prism 9.0 software and expressed as mean ± SD. The Shapiro-Wilk and Levene's tests were used to test the normality and homogeneity of variance. For data with normal distribution and homogeneity of variance, Student's t-test was used for analysis of variance between two groups, and one-way ANOVA or two-way ANOVA (bonferroni correction) was used for differences between multiple groups. For data that did not obey normal distribution or had unequal variance, the nonparametric Mann-Whitney U test was used. p<0.05 was considered statistically significant.

[0083] 2 Experimental results

[0084] 2.1 6RK73 pretreatment in mice improves the occurrence of atrial fibrillation induced by hypertension factor (Ang II) After Ang II perfusion for 3 weeks, the mice were subjected to high-frequency programmed electrical stimulation to induce atrial fibrillation, and the electrophysiological recorder collected the electrocardiogram of atrial fibrillation in the mice. Figure 1 As shown, the incidence and duration of atrial fibrillation in wild-type mice infused with Ang II (model group) were significantly higher than those in the control group, while the incidence and duration of atrial fibrillation in mice pretreated with 6RK73 infused with Ang II (treatment group) were significantly reduced compared with the control group. This indicates that 6RK73 pretreatment can improve the occurrence of atrial fibrillation induced by Ang II (n=9-12, *: p<0.05; **: p<0.01).

[0085] 2.2 6RK73 pretreatment in mice improves Ang II-induced atrial dilatation in mice

[0086] Echocardiography was used to detect changes in the atrial structure of mice. Figure 2 As shown, the atrial diameter of mice perfused with Ang II was significantly increased compared with that of the control group, but 6RK73 pretreatment significantly alleviated the increase in atrial diameter induced by Ang II perfusion (n=6, **: p<0.01).

[0087] 2.3 6RK73 pretreatment in mice improves Ang II-induced atrial fibrosis in mice

[0088] Masson staining and real-time quantitative PCR were used to detect atrial fibrosis in mice. Figure 3 and Figure 4As shown in the figure, after Ang II perfusion, the area of ​​atrial interstitial fibrosis increased significantly, and the mRNA expression levels of type I collagen and type III collagen (Collagen I and Collagen III) also increased significantly; and the above changes were significantly alleviated in Ang II perfused mice pretreated with 6RK73 (n=6, *: p<0.05; **: p<0.01), indicating that 6RK73 pretreatment improved Ang II-induced fibrosis.

[0089] 2.4 6RK73 pretreatment in mice improves Ang II-induced atrial inflammatory response

[0090] Immunofluorescence staining and real-time quantitative PCR were used to detect the inflammatory response of the mouse atria. Figure 5 As shown in the figure, compared with the healthy control group and the normal drug group, the macrophage marker CD68 in the atrial tissue of mice in the model group (after Ang II perfusion) increased significantly, while the macrophage infiltration in the atrial tissue of mice in the treatment group (Ang II perfusion 6RK73 pretreatment) was significantly improved compared with the model group (n=6, *: p<0.05; **: p<0.01). Figure 6 As shown, compared with the healthy control group and the normal drug group, the mRNA expression levels of interleukin-1β and interleukin-6 (IL-1β and IL-6) in the atrial tissue of the mice in the model group were significantly increased, while the mRNA expression levels of interleukin-1β and interleukin-6 in the atrial tissue of the mice in the treatment group were significantly lower than those in the model group (n=6), indicating that 6RK73 pretreatment improves Ang II-induced inflammatory response.

[0091] 2.5 Pretreatment of mice with 6RK73 improves Ang II-induced atrial oxidative stress in mice

[0092] The results are as follows Figure 7 As shown in the figure, compared with the healthy control group and the normal drug group, the level of reactive oxygen species (i.e., the fluorescence intensity of the DHE probe) in the atrial tissue of the mice in the model group increased significantly, while the level of reactive oxygen species in the atrial tissue of the mice in the treatment group was significantly improved compared with the model group (n=6, *: p<0.05; **: p<0.01). Figure 8 As shown, compared with the healthy control group and the normal drug group, the mRNA expression levels of NADPH oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4) in the atrial tissue of the mice in the model group were significantly increased, while the mRNA expression levels of NADPH oxidase 2 (NOX2) and NADPH oxidase 4 (NOX4) in the atrial tissue of the mice in the treatment group were significantly lower than those in the model group (n=6). This suggests that 6RK73 pretreatment improves Ang II-induced atrial oxidative stress.

[0093] 2.6 6RK73 pretreatment in mice improves Ang II-induced activation of atrial ion channels, inflammatory response, oxidative stress, and fibrosis signaling pathways

[0094] Immunoblotting was used to detect a series of signaling pathways, including the expression of ion channel-related proteins, inflammatory response, oxidative stress, and fibrosis-related proteins, to explore the signaling pathways through which 6RK73 pretreatment affects the occurrence of atrial fibrillation. Fig. 9 As shown in A, 6RK73 pretreatment alleviated the ion channel-related protein K induced by Ang II V 1.5 expression increased (n = 6). NADPH oxidase 2 (NOX2) in the NADPH subtype is the main source of reactive oxygen species in the heart. Fig. 9 As shown in B, 6RK73 pretreatment improved the increase of NOX2 induced by Ang II (n=6). Fig. 9 As shown in C and 9D, compared with the healthy control group and the normal drug group, the atrial tissue NF-κB p-p65 / NF-κB p65 ( Fig. 9 The levels of NLRP3 and p-SMAD2 / SMAD2 proteins in the atrial tissue of the treated mice were significantly increased compared with those in the model group (n=6). The above results suggest that 6RK73 pretreatment improves the activation of Ang II-induced atrial ion channels, inflammatory response, oxidative stress and fibrosis signaling pathways.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. 6 Use of RK73 in the preparation of a drug for preventing or treating atrial fibrillation.

2. The use according to claim 1, characterized in that The atrial fibrillation is angiotensin Ang II-induced atrial fibrillation.

3. The use according to claim 1 or 2, characterized in that The medicament comprises an effective amount of 6RK73.

4. The use according to claim 3, characterized in that The effective amount is 0.1-1 mg / kg, administered at least twice a week.

5. The use according to claim 3, characterized in that: The drug is prepared from 6RK73 and at least one pharmaceutical carrier and / or at least one pharmaceutical excipient via a general pharmaceutical method.

6. The use according to claim 4, characterized in that The pharmaceutical excipients include at least one of a diluent, a binder, a disintegrant, a lubricant and a flavoring agent.

7. The use according to claim 4, characterized in that The dosage form of the drug includes tablets, capsules, granules, injections or sprays.

8. The use according to claim 4, characterized in that The drug administration method includes oral or parenteral administration.

9. A pharmaceutical composition for preventing or treating atrial fibrillation, characterized in that: The invention comprises an effective amount of 6RK73, at least one pharmaceutically acceptable carrier and / or at least one pharmaceutically acceptable excipient.

10. The pharmaceutical composition according to claim 9, characterized in that The effective amount of 6RK73 is 0.1-1 mg / kg, administered at least twice a week.

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