Application of loganin in preparation of medicine for treating cardiac traumatic diseases

Abstract

The invention provides application of loganin in preparation of drugs for treating cardiac traumatic diseases, and belongs to the technical field of drugs related to cardiac diseases. Researches on rat models with penetrable heart injury and operative heart injury find that loganin can promote myocardial cell regeneration after cardiac traumatic injury, action target proteins are ephrinB2 and EphB4, and cardiac structural repair is further realized through myocardial cell regeneration.

Description

Technical Field

[0001] This invention belongs to the field of drug technology related to heart diseases, and particularly relates to the application of loganin in the preparation of drugs for treating traumatic cardiac diseases. Background Technology

[0002] Cardiac injury is a syndrome of damage to the structure and function of the heart caused by various etiologies, including ischemic, traumatic, and inflammatory injuries. Traumatic cardiac injury is an acute and critical condition in cardiothoracic surgery, usually caused by trauma such as knives, gunshot wounds, surgery, or compression. It has a high mortality rate and can be divided into penetrating and non-penetrating cardiac injuries. The common cause of penetrating cardiac injuries is puncture wounds from sharp objects, while the common cause of non-penetrating injuries is compression or cutting of the heart by instruments during surgery. These injuries cause varying degrees of damage and necrosis of myocardial cells, ranging from asymptomatic minor abrasions to fatal cardiac rupture, depending on the severity and mechanism of injury. Its clinical manifestations are complex, often including hypotension, arrhythmias, and ventricular wall rupture. Therefore, timely promotion of myocardial regeneration and repair after injury plays a crucial role.

[0003] Existing research has disclosed studies on the use of loganin for myocardial ischemia (e.g., CN111166757A) and myocardial hypertrophy (e.g., CN113082041A). However, the core causes of myocardial ischemia and myocardial hypertrophy are vascular lesions, neuroendocrine disorders, or hemodynamic abnormalities. The core mechanism by which loganin is used for myocardial ischemia and myocardial hypertrophy is anti-inflammatory, inhibition of apoptosis, and regulation of inflammation-related signaling pathways. For example, CN113082041A clearly shows that it works by inhibiting the NF-κB / JAK2-STAT3 pathway and reducing inflammatory factors such as IL-1β. However, it has never been disclosed that loganin can be used to treat traumatic cardiac diseases. Summary of the Invention

[0004] This invention proposes a new use for loganin, specifically in the preparation of a medicament for treating traumatic cardiac diseases.

[0005] Existing research has only revealed that loganin can treat secondary cardiac injuries such as myocardial ischemia, myocardial hypertrophy, inflammation, and apoptosis. However, its nature, pathological mechanisms, and clinical treatment logic differ significantly from those of traumatic cardiac diseases. The core of cardiac trauma lies in primary injuries such as structural tears and hemorrhage, which often require surgical repair or structural repair.

[0006] This invention proposes the application of loganin in the preparation of drugs for treating traumatic cardiac diseases.

[0007] In this embodiment of the invention, loganin achieves the treatment of traumatic cardiac diseases by promoting the regeneration of cardiomyocytes after cardiac traumatic injury.

[0008] In a preferred embodiment of the present invention, the traumatic cardiac disease includes surgical cardiac injury, penetrating cardiac injury, or myocardial contusion caused by external force.

[0009] In a preferred embodiment of the present invention, the loganin promotes the regeneration of cardiomyocytes after traumatic cardiac injury, and the target proteins of the loganin are ephrin B2 and EphB4.

[0010] In this embodiment of the invention, loganin regulates the ephrinB2 / EphB4 signaling pathway by targeting and binding to ephrinB2 and EphB4, thereby promoting cardiomyocyte regeneration, inhibiting inflammatory response and cardiomyocyte apoptosis.

[0011] In a preferred embodiment of the present invention, the effective dose of loganin is 120 mg / kg to 240 mg / kg. For a given subject (e.g., a clinical patient), the effective daily intake of loganin is 120 mg to 240 mg.

[0012] In a preferred embodiment of the present invention, the dosage form of the drug includes at least one of oral preparations, injections, and topical preparations.

[0013] In a preferred embodiment of the present invention, the oral preparation is a tablet, capsule, granule, oral liquid, or drop; the injectable preparation is an intravenous injection, intramuscular injection, or subcutaneous injection. The topical preparation includes an ointment, gel, patch, or liniment.

[0014] In a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier or excipient, which includes diluents, excipients, binders, disintegrants, lubricants, etc. The diluents and excipients (such as starch and lactose) are used to increase volume for ease of production and administration. The binders (such as cellulose) are used to bind drug powders into granules or tablets. The disintegrants (such as sodium carboxymethyl starch) are used to rapidly disintegrate the drug in vivo, releasing the drug. The lubricants (such as magnesium stearate) are used to prevent powder from adhering to machinery, ensuring smooth production.

[0015] This invention has the following advantages:

[0016] The application of loganin proposed in this invention in the preparation of drugs for treating traumatic cardiac diseases has been found in rat models of penetrating cardiac injury and surgical cardiac injury. Loganin can promote the regeneration of cardiomyocytes after traumatic cardiac injury, with the target proteins being ephrinB2 and EphB4. Through cardiomyocyte regeneration, it can achieve structural repair of the heart. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 Ki67 was observed 7 days after the establishment of the rat penetrating cardiac injury model using loganin in Example 1 of this invention. + / cTnT + 、BrdU + / cTnT + The effect of cardiomyocyte count. Note: Data are expressed as mean ± standard error; n = 4, * P <0.05,** P <0.01, *** P <0.001 compared to the model group, scale bar = 20 micrometers; Figure 2 Ki67 of loganin in rats 7 days after the establishment of surgical cardiac injury model in Example 1 of this invention. + / cTnT + 、BrdU + / cTnT + The effect of cardiomyocyte count. Note: Data are expressed as mean ± standard error; n = 4, * P <0.05,** P <0.01, *** P <0.001 compared to the model group, scale bar = 20 micrometers; Figure 3 This invention relates to the effect of loganin in Example 1 on the expression levels of ephrinB2 and Eph4 proteins in heart tissue 7 days after the establishment of a rat penetrating heart injury model. Note: Data are expressed as mean ± standard error; n = 4, compared to the model group, * P <0.05,** P <0.01, *** P <0.001; Figure 4 This invention relates to the effect of loganin in Example 1 on the expression levels of ephrinB2 and EphB4 proteins in the heart tissue of a rat model of surgically induced cardiac injury 7 days after modeling. Note: Data are expressed as mean ± standard error; n = 4, compared to the model group, * P <0.05,** P <0.01, *** P <0.001. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0020] Example 1 Study on rat models of penetrating cardiac injury and surgical cardiac injury using loganin 1. Materials and Methods 1.1 Drugs The monomeric compound loganin, with a content greater than 98.5% detected by high performance liquid chromatography. Before use, it was dissolved in distilled water to form a drug solution with the required concentration for the experiment. The molecular structure is shown in Formula I.

[0021] Formula I.

[0022] 1.2 Experimental Animals SPF-grade male Sprague-Dawley rats, 7-8 weeks old, weighing 260-280 g. Purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., certificate number: SCXK (Beijing) 2016-0001. Raised conventionally, environmental temperature 24±1°C, humidity 55±5%, fasted for 12 h before surgery without water deprivation.

[0023] 1.3 Main Experimental Instruments Small animal ventilator (Harvard Instruments, USA); multi-channel physiological signal recorder (BIOPAC, USA); ultrasonic cell disruptor (Ningbo Xinzhi Technology Research Institute, China); bench-top micro freezing centrifuge (Beckman Coulter, USA); full wavelength microplate reader (Thermo Fisher, USA); fluorescence microscope (Nikon, Japan); cryostat (Leica, USA); Powerpac Basic electrophoresis apparatus (Bio-Rad, USA); chemiluminescent gel imaging system (Alpha, USA).

[0024] 1.4 Main Experimental Reagents Rabbit anti-Ki67 primary antibody (Abcam, USA); mouse anti-BrdU primary antibody (Roche, USA); mouse anti-Cardiac Troponin T (cTnT) primary antibody (Abcam, USA); rabbit anti-Cardiac Troponin T (cTnT) primary antibody (Abcam, USA); rabbit anti-ephrinB2 primary antibody (Cell Signaling Technology, USA); mouse anti-EphB4 primary antibody (Abcam, USA); mouse anti-GAPDH primary antibody (Solebio Technology, Beijing); donkey anti-mouse IgG (H+L) secondary antibody AlexaFluor 488 (Thermo Fisher Scientific, USA); donkey anti-mouse IgG (H+L) secondary antibody AlexaFluor 594 (Thermo Fisher Scientific, USA); donkey anti-rabbit IgG (H+L) secondary antibody AlexaFluor 488 (Thermo Fisher Scientific, USA); donkey anti-rabbit IgG (H+L) secondary antibody AlexaFluor 594 (Thermo Fisher Scientific, USA); horseradish enzyme-labeled goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); Horseradish enzyme-labeled goat anti-mouse IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.); ECL chemiluminescence detection kit (Beijing Botes Biotechnology Co., Ltd.).

[0025] 1.5 Experimental Methods 1.5.1 Preparation of a rat model of penetrating cardiac injury After weighing, rats were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg). The rats were then fixed in a supine position on the operating table, endotracheally intubated, and connected to a small animal ventilator with a respiratory ratio of 2:1, a tidal volume of 2.5 mL / 100g, and a respiratory rate of 50 breaths / min. Once the air pressure stabilized, the rats were adjusted to a right lateral decubitus position. The skin was prepared and disinfected, exposing the sternal skin. A 2-3 cm transverse incision was made at the point of most pronounced pulsation, through the left 4th and 5th intercostal spaces, exposing the heart. The heart was gently stabilized with forceps (avoiding excessive compression). A pre-selected 14G intravenous catheter cannula was held and quickly and forcefully inserted perpendicularly to the ventricular wall into the left ventricle to a depth of approximately 3-4 mm (ensuring complete penetration of the ventricular wall), and immediately withdrawn. The insertion site was approximately 3-4 mm above the apex of the left ventricle on the anterior wall, in an area with few visible blood vessels. Note: The puncture and withdrawal actions should be completed within 1 second. Hemostasis was then achieved by applying temporary pressure with a finger, followed by mattress closure of the cardiac wound using 6-0 Prolene sutures with padding. Once the cardiac wound was confirmed closed and no active bleeding was observed, the pleural cavity was flushed with warm saline. The intercostal muscles, chest wall muscles, and skin were sutured layer by layer. Before suturing the skin, a three-way stopcock was connected to a syringe to carefully aspirate any small amount of gas or blood that might have accumulated in the pleural cavity to restore negative pressure. After the rats resumed spontaneous breathing, the endotracheal tube was removed, and the rats were returned to their cages after regaining consciousness. Rats in the sham-operated group underwent the same thoracotomy, but without the insertion of a wounding needle. Three hours post-operation, pre-prepared and preheated BrdU solution was injected intraperitoneally at a dose of 50 mg / kg once daily for seven consecutive days.

[0026] 1.5.2 Preparation of a rat model of surgical cardiac injury After weighing, rats were anesthetized via intraperitoneal injection of 2% sodium pentobarbital (50 mg / kg). The rats were then fixed in a supine position on the operating table, endotracheally intubated, and connected to a small animal ventilator with a respiratory ratio of 2:1, a tidal volume of 2.5 mL / 100g, and a respiratory rate of 50 breaths / min. Once the air pressure stabilized, the rats were positioned to the right lateral decubitus position. The skin was prepared and disinfected, exposing the sternal skin. A 2-3 cm transverse incision was made at the point of most pronounced pulsation, through the left 4th and 5th intercostal spaces, exposing the heart. A 2-3 mm superficial incision was gently made in the avascular area of ​​the left ventricular anterior wall using a 15-gauge round blade, only puncturing the epicardium and a small amount of myocardium, without penetrating the ventricular wall. A linear bleeding band was observed postoperatively, but no large amount of blood flowed out; care was taken to avoid the coronary arteries. After the injury was completed, the heart bleeding was carefully observed, and hemostasis was achieved by applying gentle pressure with a cotton swab for a few seconds. After confirming no active bleeding, the intercostal muscles and chest wall muscles were sutured layer by layer. Using a syringe connected to a three-way stopcock, carefully aspirate any small amount of gas or blood that may have accumulated in the thoracic cavity to restore negative pressure, and suture the skin incision. Disconnect the ventilator and allow the rat to breathe spontaneously. Once its breathing is stable and strong, remove the endotracheal tube and place the rat alone in a warm, clean cage for resuscitation, continuing to observe until it is fully awake. Rats in the sham-operated group also underwent open-chest surgery, but without any surgical incisions. Three hours post-surgery, they were intraperitoneally injected with pre-prepared, preheated BrdU solution at a dose of 50 mg / kg, once daily for 7 consecutive days.

[0027] 1.5.3 Animal grouping and administration Rats that successfully developed the model were randomly divided into 5 groups using a random number table method: sham-operated group, model group, low-dose loganin group (60 mg / kg), medium-dose loganin group (120 mg / kg), and high-dose loganin group (240 mg / kg). Loganin was dissolved in distilled water and administered 3 hours after modeling, once daily by gavage. The sham-operated group and the model group were given the same amount of distilled water.

[0028] 1.5.4 Immunofluorescence staining of cardiac tissue Seven days after rat modeling, heart samples were perfused and collected, then embedded in OCT embedding medium, rapidly frozen in liquid nitrogen, and stored at -80°C. 6μm frozen sections were excised. Tissue sections were permeated with 0.1% Triton X-100 in PBS for 30 min. BrdU-stained sections were treated with 2N hydrochloric acid and incubated in a 37°C water bath for 30 min. After removing the hydrochloric acid, the sections were washed three times with PBS, then blocked with 5% donkey serum at room temperature for 2 h, followed by overnight incubation with primary antibody at 4°C, and then incubated with Alexa Fluor-488 or Alexa Fluor-594-labeled secondary antibody. Cell nuclei were stained with mounting medium containing DAPI. Fluorescence signals were observed using a Nikon fluorescence microscope. Positively stained cardiomyocytes in each section were quantified using Image-Pro Plus software.

[0029] 1.5.5 Western blot detection Left ventricular tissues from rats in each group were collected, minced, and lysed with RIPA lysis buffer at a tissue weight ratio of 1:9. After thorough homogenization, sonication, and centrifugation, the supernatant was collected and protein quantification was performed using a BCA protein quantification kit. Proteins were separated by SDS-PAGE, transferred to a membrane, and blocked. Primary antibodies ephrinB2 (1:1000), EphB4 (1:1000), and GAPDH (1:1000) were added, and the membrane was incubated overnight at 4 °C. Horseradish peroxidase-labeled secondary antibody (1:2000) was then incubated at room temperature for 2 h, followed by imaging. ImageJ software was used to analyze the grayscale values ​​of protein bands, and the relative expression levels of ephrinB2 and EphB4 proteins were calculated using GAPDH as an internal control.

[0030] 1.5.6 Data Analysis Experimental data were analyzed using SPSS 22.0 statistical software, and results are expressed as Mean ± SD. One-way analysis of variance (ANOVA) was used to compare sample means between groups. P <0.05 indicates statistical significance.

[0031] 2. Experimental Results 2.1 Strychnine-induced penetrating cardiac injury model in rats: Ki67 7 days after modeling + / cTnT + 、BrdU + / cTnT + Effect of cardiomyocyte count To observe the effect of loganin on cardiomyocyte proliferation in rats with penetrating heart injury, we first performed Ki67 / cTnT immunofluorescence staining on heart tissue sections from rats 7 days after penetrating heart injury modeling. Ki67 protein is expressed only during the active phases of the cell cycle (G1, S, G2, M phases) and not during the quiescent phase (G0 phase). By detecting the expression of Ki67 protein in tissue samples, we can determine whether cells are in a proliferative state. Figure 1 (Figure A in the diagram). The results are as follows: Figure 1 Figure B shows that, compared with the sham-operated group, the number of Ki67-positive cardiomyocytes in the model group showed an increasing trend, but the difference was not significant; compared with the model group, the high-dose loganin group (240 mg / kg) ( P The number of Ki67-positive cardiomyocytes with a value <0.001 was significantly increased. This suggests that loganin can promote cardiomyocyte proliferation after penetrating cardiac injury in rats. To further verify its role in promoting cardiomyocyte proliferation, we performed BrdU / cTnT immunofluorescence staining on visceral tissue sections. BrdU (5-bromo-2'-deoxyuridine) is a thymine nucleoside analog that competitively substitutes thymine into the newly synthesized DNA strand of S-phase cells, thereby marking cells in a proliferating state. Figure 1 (Figure A in the diagram). The results are as follows: Figure 1 Figure C shows that, compared with the sham-operated group, the number of BrdU-positive cardiomyocytes in the model group showed an increasing trend, but the difference was not significant; compared with the model group, the number of BrdU-positive cardiomyocytes in the medium-dose loganin group (120 mg / kg) was significantly higher. P <0.05) and the high-dose group of loganin (240 mg / kg) ( P The number of BrdU-positive cardiomyocytes (<0.001) was significantly increased. These results suggest that loganin can promote cardiomyocyte proliferation and achieve myocardial regeneration in rats after penetrating cardiac injury.

[0032] 2.2 Strychnine-induced effect on Ki67 in rats 7 days after surgical cardiac injury model establishment + / cTnT + 、BrdU + / cTnT + Effect of cardiomyocyte count Similarly, to observe the effect of loganin on cardiomyocyte proliferation in rats with surgically induced cardiac injury, we first performed Ki67 / cTnT immunofluorescence staining on heart tissue sections from rats 7 days after the surgical cardiac injury model was established, and observed the number of Ki67-positive cardiomyocytes. Figure 2 (Figure A). The results are as follows: Figure 2 Figure B shows that, compared with the sham-operated group, the number of Ki67-positive cardiomyocytes in the model group showed an increasing trend, but the difference was not significant; compared with the model group, the high-dose loganin group (240 mg / kg) (P The number of Ki67-positive cardiomyocytes with a value <0.01 was significantly increased. Further BrdU / cTnT immunofluorescence staining was performed on cardiac tissue sections to observe the number of BrdU-positive cardiomyocytes in the heart. Figure 2 (Figure A). The results are as follows: Figure 2 The C-plot shows that, compared with the sham-operated group, the number of BrdU-positive cardiomyocytes in the model group showed an increasing trend, but the difference was not statistically significant; compared with the model group, the number of BrdU-positive cardiomyocytes in the medium-dose loganin group (120 mg / kg) was significantly higher. P <0.05) and the high-dose group of loganin (240 mg / kg) ( P The number of BrdU-positive cardiomyocytes (<0.01) was significantly increased. These results suggest that loganin can promote cardiomyocyte proliferation and achieve myocardial regeneration after surgical cardiac injury in rats.

[0033] 2.3 Regulatory effect of loganin on ephrin B2 and Eph B4 proteins in rats 7 days after the establishment of a penetrating heart injury model Western blot analysis was used to detect the protein expression of myocardial ephrin B2 and EphB4, such as... Figure 3 As shown, compared with the sham-operated group, the protein expression levels of ephrinB2 and EphB4 in the model group showed an increasing trend, but the difference was not significant; compared with the model group, the protein expression levels of ephrinB2 and EphB4 in the medium-dose and high-dose loganin administration groups were significantly increased. P <0.05, P <0.01, P <0.001).

[0034] 2.4 Regulatory effect of loganin on ephrin B2 and Eph B4 proteins in rats 7 days after the establishment of a surgical cardiac injury model Western blot analysis was used to detect the protein expression of myocardial ephrin B2 and EphB4, such as... Figure 4 As shown, compared with the sham-operated group, the protein expression levels of ephrinB2 and EphB4 in the model group showed an increasing trend, but the difference was not significant; compared with the model group, the protein expression level of ephrinB2 in the high-dose loganin administration group was significantly increased ( P <0.01, the protein expression level of EphB4 was significantly increased in the medium- and high-dose groups of loganin ( P <0.05, P <0.001).

[0035] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Use of loganin in the preparation of a medicament for treating a traumatic heart disease.

2. Use according to claim 1, characterized in that, The traumatic heart disease includes a surgical heart injury, a penetrating heart injury or a myocardial contusion caused by an external force.

3. Use according to claim 1, characterized in that, The target protein of the loganin is ephrinB2 and EphB4.

4. Use according to claim 1, characterized in that, The effective dose of the loganin is 120 mg / kg ~ 240 mg / kg.

5. The use according to claim 1, characterized in that, The dosage form of the medicament includes an oral preparation, an injection or a topical preparation.

6. The use according to claim 5, wherein at least one of the following conditions is met: (1) the oral preparation includes a tablet, a capsule, a granule, an oral liquid or a dripping pill; (2) the injection includes an intravenous injection, an intramuscular injection or a subcutaneous injection; (3) the topical preparation includes an ointment, a gel, a patch or a liniment. ​

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