Application of Spexin active polypeptide in preparation of medicine for preventing and treating ischemia-reperfusion ventricular arrhythmia

Abstract

The invention relates to application of a Spexin active polypeptide in preparation of a medicine for preventing and treating ischemia-reperfusion ventricular arrhythmia, and belongs to the technical field of biological medicine manufacturing. The invention provides a new medicinal application of a Spexin active polypeptide, namely an application of the Spexin active polypeptide in preparation of a medicine for preventing and treating ischemia-reperfusion ventricular arrhythmia, and the amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ. Animal experiments prove that the Spexin active polypeptide can reduce the occurrence rate of ischemia-reperfusion mouse ventricular arrhythmia, improve the heart function of ischemia-reperfusion mice, reduce the cardiac infarction area and inhibit myocardial cell calcium overload caused by ischemia-reperfusion. The Spexin active polypeptide is applied to preparation of the medicine for preventing and treating ischemia-reperfusion arrhythmia, new clinical application of the Spexin active polypeptide is developed, and the Spexin active polypeptide has the advantages of biological multiple effects, high safety, small toxic and side effects and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical manufacturing, and particularly relates to the application of Spexin active polypeptide in the preparation of drugs for preventing and treating ischemia-reperfusion ventricular arrhythmia. Background Art

[0002] Reperfusion therapy after coronary artery occlusion is an important means to restore cardiac blood supply and salvage myocardium. However, reperfusion is usually accompanied by reperfusion injury, including problems such as cardiomyocyte death, increased myocardial infarction area, and fatal reperfusion ventricular arrhythmia. Among them, reperfusion ventricular fibrillation is considered to be an important cause of sudden death in patients during the ischemia-reperfusion period.

[0003] Reperfusion ventricular arrhythmia has a different occurrence mechanism from arrhythmias caused by atrial fibrillation, ventricular remodeling, or heart failure. Atrial fibrillation is mainly due to the loss of normal rhythm of atrial electrical activity, resulting in rapid and irregular fibrillation of the atrium, inability to effectively contract and relax, and thus affecting the normal pumping function of the heart. Arrhythmias related to ventricular remodeling are caused by changes in the structure and function of cardiomyocytes, myocardial interstitium, and cardiac blood vessels after the heart is damaged or under pressure. Arrhythmias related to heart failure are that when the heart fails, in order to maintain sufficient blood circulation, the heart may compensate by accelerating heart rate, increasing myocardial contractility, etc. This compensatory mechanism may cause changes in the electrophysiological properties of the heart, such as increased excitability of cardiomyocytes, changes in conduction velocity, etc., thus triggering arrhythmias.

[0004] Ventricular arrhythmia is an abnormal conduction originating from the ventricle, and the pacing point is in the ventricle rather than the atrium or the cardiac conduction system. Ventricular arrhythmia includes types such as ventricular premature beats, ventricular tachycardia, and ventricular flutter. Its occurrence mechanism is usually related to abnormal electrophysiological properties of ventricular myocytes, such as increased excitability of cardiomyocytes, changes in ion channel function, etc. During ischemia-reperfusion, the increased intracellular Na + rapidly activates Na + on the cell membrane and Ca 2+ exchanges, causing a large influx of extracellular Ca 2+ and forming calcium overload. Calcium overload affects multiple aspects such as the energy metabolism, cell membrane stability, and enzyme activity of cardiomyocytes, leading to changes in the electrophysiological properties of cardiomyocytes. These changes may include shortening of the action potential duration, changes in the refractory period, and disorders of ion channel function, etc., thereby increasing the susceptibility to ventricular arrhythmia.

[0005] Spexin bioactive polypeptide is a novel bioactive peptide hormone that can be used to prevent ventricular remodeling, enhance myocardial contractility, and treat atrial fibrillation. However, whether Spexin bioactive polypeptide can be used to prevent and treat ischemia-reperfusion ventricular arrhythmia has not been reported. The invention patent "Application of Spexin Bioactive Polypeptide in the Preparation of Drugs for Enhancing Myocardial Contractility" with the authorization number CN117815360B discloses that Spexin bioactive polypeptide can enhance myocardial cell contractility by increasing the calcium transient in myocardial cells, and solve the problem of calcium homeostasis imbalance and decreased myocardial contractility in heart failure. Increasing the calcium transient in myocardial cells means that the concentration of free calcium ions in myocardial cells suddenly increases, and calcium ions bind to troponin C, triggering the allosteric change of myosin, thereby enhancing myocardial contractility. Since there are significant differences in the structure, function, and electrophysiology of myocardial cells during heart failure and reperfusion injury, it is impossible to determine whether Spexin bioactive polypeptide can be used to prevent and treat ischemia-reperfusion ventricular arrhythmia. Summary of the Invention

[0006] The present invention provides a new pharmaceutical use of Spexin bioactive polypeptide, that is, the application of Spexin bioactive polypeptide in the preparation of drugs for preventing and treating ischemia-reperfusion ventricular arrhythmia.

[0007] Technical solution of the present invention:

[0008] The application of Spexin bioactive polypeptide in the preparation of drugs for preventing and treating ischemia-reperfusion ventricular arrhythmia, and the amino acid sequence of the Spexin bioactive polypeptide is NWTPQAMLYLKGAQ.

[0009] Further, the drug uses Spexin bioactive polypeptide as the sole active ingredient or one of the active ingredients.

[0010] Further, the content of Spexin bioactive polypeptide in the drug is 0.1wt% - 99wt%.

[0011] Further, the drug also includes pharmaceutically acceptable excipients and / or carriers.

[0012] Further, the drug can reduce the incidence and duration of ventricular arrhythmia after ischemia-reperfusion.

[0013] Further, the drug can improve the damaged heart function after ischemia-reperfusion.

[0014] Further, the drug can reduce the increase in myocardial infarction area after ischemia-reperfusion.

[0015] Further, the drug can inhibit the calcium overload of myocardial cells caused by ischemia-reperfusion.

[0016] Advantages of the present invention:

[0017] The present invention has confirmed through animal experiments that Spexin active polypeptide can reduce the incidence of ventricular arrhythmia in mice with ischemia-reperfusion, improve the cardiac function of mice with ischemia-reperfusion, reduce the area of cardiac infarction, inhibit the calcium overload of cardiomyocytes caused by ischemia-reperfusion, and thus reduce the occurrence of arrhythmia after ischemia-reperfusion. The present invention applies Spexin active polypeptide to the preparation of drugs for preventing and treating ischemia-reperfusion arrhythmia, develops its new clinical uses, has the characteristics of biological pleiotropy, high safety, and low toxicity and side effects, is expected to become a new and effective means for the treatment of ischemia-reperfusion arrhythmia, and has important translational significance. Description of the Drawings

[0018] Figure 1 Electrocardiograms of mice in the Sham group, IR group, and IR+SPX group in Example 1;

[0019] Figure 2 Comparison chart of the incidence of ventricular arrhythmia in mice in the Sham group, IR group, and IR+SPX group in Example 1;

[0020] Figure 3 Comparison chart of the duration of ventricular arrhythmia in mice in the Sham group, IR group, and IR+SPX group in Example 1;

[0021] Figure 4 Echocardiogram images of four groups of mice in Example 2;

[0022] Figure 5 Comparison chart of left ventricular EF (ejection fraction) of four groups of mice in Example 2;

[0023] Figure 6 Comparison chart of left ventricular FS (short-axis shortening rate) of four groups of mice in Example 2;

[0024] Figure 7 Comparison photos of the TTC staining results of heart sections of four groups of mice in Example 3;

[0025] Figure 8 Comparison chart of the myocardial infarction area of the hearts of four groups of mice in Example 3;

[0026] Figure 9 Comparison chart of WB of L-type calcium channel protein Cav1.2 in heart tissue proteins of four groups of mice in Example 4;

[0027] Figure 10 Comparison chart of the expression levels of L-type calcium channel protein Cav1.2 in heart tissue proteins of four groups of mice in Example 4;

[0028] Figure 11Schematic diagram of QT interval (ventricular electrical contraction time) of wild mice and SPX knockout mice in Example 5;

[0029] Figure 12 Comparison chart of QT intervals of wild mice and SPX knockout mice in Example 5;

[0030] Figure 13 Electrocardiogram of wild mice and SPX knockout mice in Example 5;

[0031] Figure 14 Comparison chart of the incidence of ventricular arrhythmia in wild mice and SPX knockout mice in Example 5;

[0032] Figure 15 Comparison chart of the duration of ventricular arrhythmia in wild mice and SPX knockout mice in Example 5. Detailed implementation manners

[0033] The technical solutions of the present invention will be further described below in conjunction with the embodiments, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention shall be covered by the protection scope of the present invention. For the process equipment or devices not specifically noted in the following embodiments, conventional equipment or devices in the art are used. Unless otherwise specified, the raw materials used in the embodiments of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the embodiments of the present invention are all conventional means well known to those skilled in the art.

[0034] Example 1

[0035] This example investigated the effect of Spexin active polypeptide on reducing the incidence of ventricular arrhythmia in ischemia-reperfusion mice.

[0036] The Spexin active polypeptide used in this example was purchased from Shangcheng Qiangyao Biotechnology Co., Ltd. In the study, the Spexin active polypeptide was prepared into a concentration of 1 mg / ml with physiological saline and intraperitoneally injected at 50 mg / kg / d.

[0037] I. The methods for modeling, grouping, and administering the test mice in the example are as follows:

[0038] 1. Experimental animals

[0039] 8-week-old C57 mice were purchased from Beijing Biotechnology Co., Ltd. All animals were housed in a specific pathogen-free facility, with the temperature controlled at (20 - 25°C) and the humidity at (40 - 70%), a 12-hour light cycle, and free access to standard laboratory food and tap water.

[0040] 2. Grouping method:

[0041] After 1 week of adaptive feeding, all mice were randomly divided into four groups:

[0042] Sham group (control group), SPX group (control + Spexin bioactive polypeptide treatment group), IR group (ischemia-reperfusion group), and IR + SPX group (ischemia-reperfusion + Spexin bioactive polypeptide treatment group).

[0043] 3. Administration method:

[0044] Mice in the SPX group and the IR + SPX group were intraperitoneally injected with Spexin bioactive polypeptide (50 mg / kg / day) for three consecutive days. Subsequently, the IR group and the IR + SPX group were subjected to ischemia-reperfusion treatment, and relevant data were collected 2 days after reperfusion to evaluate the effect of Spexin bioactive polypeptide in reducing the occurrence of ventricular arrhythmia caused by ischemia-reperfusion injury.

[0045] 4. Ischemia-reperfusion surgery

[0046] C57 mice were weighed and anesthetized by intraperitoneal injection of pentobarbital (50 mg / kg). The mice were intubated and mechanically ventilated using a respirator (KW-100, KEW BASIS, China). Thoracotomy was performed on the mice in the IR group and the IR + SPX group. The left anterior descending coronary artery was observed and ligated proximally using 6-0 silk sutures. A PE tube was placed between the left coronary artery and the 6-0 silk suture to minimize coronary artery trauma caused by occlusion and promote reperfusion. The LAD was completely occluded for 50 min; subsequently, reperfusion was initiated by removing the 6-0 suture. During reperfusion, the color of the heart gradually returned, and the electrocardiogram was continuously monitored to observe the occurrence of arrhythmia. Finally, the intercostal muscles and skin layer were gradually sutured with sutures. After the surgery, the mice were placed on a heating table to wake up.

[0047] II. Evaluation method

[0048] 1. Cardiac electrophysiological detection

[0049] Two days after the ischemia-reperfusion surgery, C57 mice were weighed and anesthetized by intraperitoneal injection of pentobarbital (50 mg / kg). Surface electrocardiogram electrodes were installed on the anesthetized mice. The electrodes were placed in specific areas of the chest, connected to the electrocardiogram device, and monitoring was initiated to record the basic heart rhythm of the mice. Pay attention to the changes in heart rhythm and whether the waveforms of ventricular tachycardia (VT) or ventricular fibrillation (VF) appear. The number of animals with VT / VF in each group was counted, and the incidence of ventricular arrhythmia was calculated.

[0050] 2. Statistical analysis

[0051] Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Software, Inc, La Jolla, CA). Continuous variables were expressed as mean ± standard error of the mean (SEM). Categorical variables were expressed as numbers and percentages. Comparisons between two groups were evaluated by Student's non-paired t-test or Wilcoxon (Mann-Whitney U) test, and normality test was performed using Shapiro-Wilk. Variables over two groups were analyzed by one-way ANOVA, followed by Tukey test. Chi-square test was used for categorical variables. Univariate and multivariate Logistic regression were used for clinical correlation analysis. P < 0.05 indicated a statistically significant difference between the two groups.

[0052] The results of electrophysiological detection of mouse hearts were as Figure 1 , Figure 2 and Figure 3 shown. Compared with the control group, the incidences of ventricular arrhythmias such as ventricular tachycardia and ventricular fibrillation in the IR group were significantly increased, while the IR + SPX group could significantly reduce the occurrence of ventricular arrhythmias after ischemia-reperfusion. This indicated that Spexin bioactive polypeptide treatment significantly reduced the incidence and duration of ventricular arrhythmias after ischemia-reperfusion (P < 0.0001).

[0053] Example 2

[0054] This example investigated the effect of Spexin bioactive polypeptide on improving the impaired cardiac function after ischemia-reperfusion.

[0055] On the basis of the modeling and treatment in Example 1, echocardiography was further performed on four groups of mice. The specific examination method was as follows:

[0056] Two days after the ischemia-reperfusion surgery of the mice, they were anesthetized with 2% isoflurane. The chest hair on the left side of the mice was fully removed using a razor, and a small amount of coupling agent was applied to the prepared skin area for transthoracic echocardiography. Images were obtained using a high-frequency ultrasound system, and in vivo cardiac structure changes and cardiac function were detected using echocardiography. In addition, two-dimensional guided m-mode images of the left ventricle were recorded on the parasternal short-axis view at the level of the papillary muscle. Left ventricular EF and FS were calculated based on the m-mode images.

[0057] The echocardiography detection results of the four groups of mice were as Figure 4 , Figure 5 and Figure 6As shown, compared with the control group, the left ventricular EF and FS of the mice in the IR group decreased after ischemia-reperfusion, indicating that ischemia-reperfusion caused impaired cardiac systolic function. Compared with the IR group, the left ventricular EF and FS of the mice in the IR+SPX group were significantly increased, indicating that the Spexin active polypeptide has a potential protective effect in improving the cardiac function of mice with ischemia-reperfusion.

[0058] Example 3

[0059] This example investigated the effect of the Spexin active polypeptide in reducing the increase in myocardial infarction area after ischemia-reperfusion.

[0060] The size of the myocardial infarction area is one of the main determinants of the risk of ventricular arrhythmia. The larger the infarction area, the higher the incidence of ventricular arrhythmia. Based on the modeling and treatment in Example 1, this example investigated the myocardial infarction area of four groups of mice by TTC staining (2,3,5-triphenyltetrazolium chloride staining method). The specific method is as follows:

[0061] After sacrificing the animals by cervical dislocation, the heart was quickly removed. The myocardial tissue was placed in a mouse section mold, and 2-mm continuous sections were obtained with a surgical blade. Then, the sections were immersed in the TTC dye solution and incubated at 37 °C for 30 minutes for sufficient staining. The heart sections were taken out and washed with PBS solution for 3 - 5 min, and the stained sections were photographed.

[0062] The TTC staining results of the heart sections of the four groups of mice and the comparison results of the myocardial infarction area are as Figure 7 and Figure 8 shown. Compared with the control group, the myocardial infarction area of the mice in the IR group was significantly increased, indicating that reperfusion injury led to the death of mouse myocardial cells, thereby increasing the myocardial infarction area. Compared with the IR group, the increase in the myocardial infarction area in the IR+SPX group was significantly improved, indicating that the Spexin active polypeptide can indirectly reduce the incidence of ischemia-reperfusion ventricular arrhythmia by reducing the myocardial infarction area.

[0063] Example 4

[0064] This example investigated the inhibitory effect of the Spexin active polypeptide on myocardial cell calcium overload after ischemia-reperfusion.

[0065] Based on the modeling and treatment in Example 1, this example collected mouse heart tissue and extracted proteins, and investigated the expression level of the L-type calcium channel protein Cav1.2 by Western blot experiment. The specific method is as follows:

[0066] 1. Collection of mouse heart tissue

[0067] After the ultrasonic detection, collect the mouse heart tissue, and pay attention to maintaining anesthesia. After opening the chest, take out the mouse heart, remove the excess connective tissue, place it in pre-cooled physiological saline to remove the residual blood, quickly freeze it in liquid nitrogen, and then store it at -80 °C.

[0068] 2. Protein extraction from mouse heart tissue

[0069] Take out the frozen heart tissue, place it on ice to thaw, weigh 10 mg of ventricular tissue and put it into a 1.5 ml EP tube, add 1 ml of PBS solution, cut the tissue into pieces and centrifuge at a speed of 10,000 revolutions per minute for 5 minutes, then pour out the supernatant. The lysis buffer is prepared according to RIPA:PMSP = 100:1, add 100 μl of lysis buffer to each tube, grind the ventricular tissue thoroughly, and lyse it on ice for 20 minutes, vortexing once every 5 minutes to ensure complete lysis of the tissue. Set the centrifugation parameters and centrifuge for 15 minutes: 4 °C, 13,500 revolutions per minute. Take the supernatant to obtain the protein of mouse heart tissue.

[0070] 3. Western blot experiment

[0071] (1) Preparation of experimental reagents

[0072] 1) Prepare 10% SDS-PAGE separating gel - Table 1

[0073] Reagent 15 ml system (10%) Deionized water 4ml 30% Acr - Bis (29:1) 5ml 1 mol / L Tris, pH = 8.8 5.7ml 10% SDS 150 μl 10% Ammonium persulfate 150 μl TEMED 6 μl

[0074] 2) Prepare 5% SDS-PAGE stacking gel - Table 2

[0075] Reagent 6 ml system (5%) Deionized water 4.1ml 30% Acr - Bis (29:1) 1ml 1 mol / L Tris, pH = 6.8 0.75ml 10% SDS 60 μl 10% Ammonium persulfate 60 μl TEMED 6 μl

[0076] 3) Prepare electrophoresis buffer - Table 3

[0077]

[0078]

[0079] 4) Prepare electrotransfer buffer - Table 4

[0080] Reagent 1 L system Tris 3.03g Glycine 14.4g Methanol 200ml Deionized water 800ml

[0081] 5) Prepare TBST solution - Table 5

[0082]

[0083] 6) Prepare 5% blocking solution - Table 6

[0084] Reagent 20 ml system TBST solution 20ml Non - fat milk powder 1g

[0085] 7) Prepare BSA antibody dilution buffer - Table 7

[0086] Reagent 20 ml system TBST solution 20ml BSA 1g

[0087] (2) Experimental procedures

[0088] 1) Leak detection: Rinse the impurities remaining on the glass plate with tap water, and then rinse it again with deionized water. Assemble the glass plate and the rubber rack correctly and place them on a horizontal experimental table. Fill the concave groove formed by the glass plate with deionized water.

[0089] 2) Prepare the separating gel: Discard the liquid in the groove and blot the residual liquid with filter paper. Select the appropriate concentration of the separating gel according to the molecular weight of the target protein to be detected. Add the reagents according to the corresponding ratio, mix well, and then add them evenly from one corner of the groove at a constant speed. Do not generate bubbles. Add about 4.5 ml of separating gel into each groove, and then slowly inject 1 ml of absolute ethanol from one corner of the groove and let it stand for 20 minutes.

[0090] 3) Prepare the stacking gel: Discard and blot the liquid on the separating gel. Add the reagents according to the stacking gel preparation ratio, mix well, and then add them evenly from one corner of the groove at a constant speed to ensure no bubbles are generated. Add about 1.5 ml of stacking gel into each groove.

[0091] 4) Insert the comb: Immediately insert the comb corresponding to the glass plate above the stacking gel after the stacking gel is injected. Gently insert one end of the comb from one corner of the groove to remove the bubbles in the stacking gel. Let it stand for 15 minutes after the comb is inserted and there are no bubbles in the stacking gel.

[0092] 5) Remove the comb: Take down the rubber rack and put it into the electrophoresis tank. Inject the electrophoresis buffer from the inner tank until the whole electrophoresis tank is filled with the electrophoresis buffer. Slowly and evenly remove the comb. If the lanes are skewed, adjust the pores with a syringe needle. Then place the electrophoresis tank in a 4°C refrigerator for standby or carry out subsequent experiments.

[0093] 6) Loading: Take out the denatured protein sample from the -20°C refrigerator, heat it at 100°C for 10 minutes in a metal bath again, then place it on ice to cool, centrifuge at low speed after shaking for 30 seconds, mix the protein with a 10 μl pipette, and then take the corresponding volume of the sample and add it into the lanes. Load the samples from left to right in sequence, and add 3 μl of protein marker to each of the two lanes at both ends.

[0094] 7) Electrophoresis: Connect the connection wires correctly to the electrophoresis tank and the power supply according to the principle of "red to red, black to black". Carry out electrophoresis at a constant voltage of 80 V. When the protein marker electrophoreses to about 1 cm from the separating gel, adjust the voltage to 120 V, and stop electrophoresis when the bromophenol blue reaches the bottom of the separating gel.

[0095] 8) Membrane transfer: Take out the glass plate in the electrophoresis tank and place it in the electrotransfer solution. Cut a PVDF membrane, activate it with methanol for 1 minute, and equilibrate it in the electrotransfer solution for several minutes. Place the electrotransfer clamp in the electrotransfer solution with the black side down, cover it with a sponge and filter paper, cut off the corners of the gel and then transfer it to the filter paper, cover it with the PVDF membrane, use a roller to expel the air bubbles between the membrane and the gel, then cover it with filter paper and sponge, and then close the white side of the electrotransfer clamp and fasten the electrotransfer clamp. Place the electrotransfer clamp into the electrotransfer tank according to the principle of "red to red, black to black", fill it with electrotransfer solution, connect the power cord, adjust the current to 300 mA, and perform constant current electrotransfer for 2 hours.

[0096] 9) Membrane cutting: After the electrotransfer is completed, disconnect the power supply, take out the PVDF membrane, activate it with methanol for 1 minute, place it on the filter paper to dry, then stain the membrane with Ponceau S staining solution for 1 minute, wash the membrane twice with deionized water, and use filter paper to absorb the residual liquid on the membrane, and cut and mark the target band.

[0097] 10) Blocking: Activate the target band with methanol for 1 minute, wash it once with TBST buffer, and wash it at 120 rpm for 5 minutes each time. Add 3 ml of blocking solution to each compartment of the antibody box, and slowly shake and block the band at 60 rpm at room temperature for 2 hours.

[0098] 11) Incubate with primary antibody: Take out the band in the blocking solution, wash it once with TBST, add 3 ml of primary antibody solution to each compartment, put in the corresponding band, shake slowly at 60 rpm at room temperature for 20 minutes, and then place it in the refrigerator at 4 °C overnight.

[0099] 12) Incubate with secondary antibody: Recover the primary antibody, wash the membrane three times with TBST, and incubate with the secondary antibody for 1 hour.

[0100] 13) Wash the membrane: Discard the secondary antibody, wash the membrane three times with TBST, and wash it at 120 rpm for 10 minutes each time.

[0101] 14) Imaging: Mix the ECL developing working solution A and B in a ratio of 1:1 evenly, incubate the band in the dark for 3 minutes, then develop and image with Bio-Rad, and calculate the gray value with Image J.

[0102] The WB results and the comparison results of the expression levels of the L-type calcium channel protein Cav1.2 in the heart tissue proteins of the four groups of mice are as Figure 9 and Figure 10As shown, compared with the IR group, the expression level of the voltage-dependent L-type calcium channel protein Cav1.2 in the mice of the IR+SPX group was significantly reduced, indicating that the Spexin active polypeptide could significantly decrease the level of protein Cav1.2. As one of the main channels for calcium ions to enter cells, the reduction in the expression level of protein Cav1.2 will directly lead to a decrease in calcium ion influx. To a certain extent, this will alleviate the calcium overload in cardiomyocytes caused by excessive calcium ion influx. By alleviating the calcium overload in cardiomyocytes, the normal electrophysiological properties of cardiomyocytes can be restored, including the normal formation and propagation of action potentials, and the stability of the repolarization process. This helps to reduce ventricular arrhythmias triggered by abnormal electrophysiological properties.

[0103] Example 5

[0104] In this example, by constructing Spexin active polypeptide-encoding gene SPX knockout mice, the effect of SPX on ventricular arrhythmias during ischemia-reperfusion in mice was investigated.

[0105] The gene knockout mice were constructed by Cyagen Biosciences (Guangzhou) Co., Ltd. The Spexin gene knockout (SPX-KO) mouse strain was generated by CRISPR / Cas9-mediated genome engineering technology. Targeting the 2nd-5th exons of the Spexin gene, Cas9 mRNA and single-guide RNA (sgRNA) synthesized by in vitro transcription were microinjected into fertilized eggs to achieve the knockout of the Spexin gene. The F0 generation founder mice were genotyped by PCR and DNA sequencing, and positive mice were screened and bred to the F1 generation. The F1 generation mice were further verified by PCR genotyping and DNA sequencing. Genotyping was performed using tail genomic DNA. The knockout primers were forward 5'-GACAGGGTCGGAACATGAAGG-3' and reverse 5'-ACACACTTGTTAGCTGGGTGC-3'; the wild-type primers were forward 5'-GACAGGGTCGGAACATGAAGG-3' and reverse 5'-GTTCTCTGCATTACCTGTGGAG-3'. All experimental mice were 8-10 weeks old and were compared with wild-type or homozygous control mice in the same litter.

[0106] The comparison results of the QT intervals between wild mice and SPX knockout mice are as Figure 11 and Figure 12 shown. It can be seen through comparison that after the Spexin gene was knocked out, the QT interval increased significantly.

[0107] The comparison results of the incidence and duration of ventricular arrhythmias between wild mice and SPX knockout mice are as Figure 13 , Figure 14 and Figure 15As shown, Spexin gene knockout increased the incidence and duration of ventricular arrhythmia in mice (P < 0.0001).

Claims

1. Application of Spexin active polypeptide in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The amino acid sequence of the Spexin active polypeptide is NWTPQAMLYLKGAQ.

2. The use of the Spexin active polypeptide according to claim 1 in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The drug uses Spexin active polypeptide as the only active ingredient or one of the active ingredients.

3. Use of the Spexin active polypeptide according to claim 1 or 2 in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The content of Spexin active polypeptide in the medicine is 0.1wt% to 99wt%.

4. The use of the Spexin active polypeptide according to claim 3 in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The drug also includes pharmaceutically acceptable excipients and / or carriers.

5. The use of the Spexin active polypeptide according to claim 4 in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The drug can reduce the incidence and duration of ventricular arrhythmias after ischemia-reperfusion.

6. The use of the Spexin active polypeptide according to claim 4 in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The drug can improve impaired cardiac function after ischemia-reperfusion.

7. The use of the Spexin active polypeptide according to claim 4 in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The drug can reduce the increase in myocardial infarction area after ischemia-reperfusion.

8. The use of the Spexin active polypeptide according to claim 4 in the preparation of a drug for preventing and treating ischemia-reperfusion ventricular arrhythmia, characterized in that: The drug can inhibit calcium overload of myocardial cells caused by ischemia-reperfusion.

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