Use of 2,6-DMHQ in combatting cardiac and hepatic ischemia-reperfusion injuries

Abstract

The use of 2,6-DMHQ in ischemia-reperfusion-related injuries, preferably against cardiac and hepatic ischemia-reperfusion injuries. 2,6-DMHQ can reduce serum ALT and AST and ameliorate liver necrosis. 2,6-DMHQ can also reduce the cardiac enzyme profile, including CK, CK / MB and LDH, and reduce myocardial infarct size.

Description

Application of 2,6-DMHQ in the treatment of cardiac and hepatic ischemia-reperfusion injury Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the application of dimethylhydroquinone in improving ischemia-reperfusion-related injury of the heart and liver. Background Technology

[0002] In clinical practice, ischemia-reperfusion injury (IRI) refers to the phenomenon where, after a period of insufficient blood supply (ischemia), the damage to tissues or organs worsens upon restoration of blood flow (reperfusion). This injury is common in various clinical situations, such as myocardial infarction, organ transplantation (e.g., liver), and microcirculation recanalization after shock. ISI has a significant impact on disease prognosis, particularly in the fields of cardiology and liver disease. [1][2] Drug research to combat ischemia-reperfusion injury is an important branch of medicine. Currently, there are no specific drugs for ischemia-reperfusion injury.

[0003] 2,6-Dimethylhydroquinone (2,6-Dimethylhydroquinone, or 2,6-DMHQ) has been reported to reduce ferric iron and mainly participates in iron redox reactions. [3] There are currently no reports on the pharmaceutical applications of 2,6-DMHQ.

[0004] References:

[0005] 1. Xuexian Fang et al., Proceedings of the National Academy of Sciences of the United States of America, 2019Feb 12;116(7):2672-2680.

[0006] 2.Jose Pedro Friedmann Angeli et al., Nature Cell Biology, 2014Dec; 16(12):1180-91.

[0007] 3. Lelde Krumina et al., Environmental Science & Technology, 2017Aug 15; 51(16): 9053-9061. Gry Lyngsie et al., Scientific Reports, 2018Jul 17; 8(1): 10834. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides the application of dimethylhydroquinone (2,6-DMHQ) in the preparation of drugs for treating ischemia-reperfusion injury.

[0009] In particular, the application of 2,6-DMHQ in the treatment of cardiac and hepatic ischemia-reperfusion injury.

[0010] 2,6-DMHQ can reduce serum ALT and AST, improve liver necrosis, reduce serum myocardial enzyme levels CK, CK / MB and LDH, and reduce the area of ​​myocardial infarction.

[0011] In addition, the present invention also provides the application of 2,6-DMHQ in the preparation of drugs that improve cardiac ejection function.

[0012] 2,6-DMHQ is used in the preparation of drugs to improve lipid peroxidation levels and iron accumulation.

[0013] During the invention process, mouse models of liver and heart ischemia-reperfusion injury were introduced. 2,6-DMHQ was injected into the mice intraperitoneally before surgery, and it was found that 2,6-DMHQ could significantly improve liver and heart ischemia-reperfusion injury.

[0014] The experimental results also proved that:

[0015] Serum ALT and AST levels in the experimental group (IR+2,6-DMHQ) were significantly lower than those in the experimental group (IR);

[0016] The area of ​​liver necrosis in the experimental group (IR+2,6-DMHQ) was reduced compared to that in the experimental group (IR);

[0017] The serum myocardial enzyme levels of the experimental group (IR+2,6-DMHQ) were significantly lower than those of the experimental group (IR).

[0018] The left ventricular ejection fraction in the experimental group (IR+2,6-DMHQ) was significantly higher than that in the experimental group (IR), indicating that the cardiac function of the mice in the experimental group (IR+2,6-DMHQ) was significantly improved.

[0019] The experimental group (IR+2,6-DMHQ) had a smaller infarct area compared to the experimental group (IR).

[0020] Compared with the experimental group (IR), the experimental group (IR+2,6-DMHQ) showed significantly lower levels of 4-HNE, Perls' staining of the heart, and non-heme iron in the heart, and a significantly upregulated GSH / GSSG ratio. This indicates that the lipid peroxidation level and iron accumulation in the heart of mice in the experimental group (IR+2,6-DMHQ) were significantly improved, meaning that 2,6-DMHQ can significantly improve the ferroptosis phenotype of I / R injury. Attached Figure Description

[0021] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0022] Figure 1 shows that 2,6-DMHQ significantly improved hepatic ischemia-reperfusion injury. ALT (A), AST (B), and liver H&E staining (C); * indicates p < 0.05, ** indicates p < 0.01, and the difference is statistically significant;

[0023] Sham represents wild-type mice undergoing sham surgery, IR represents liver ischemia-reperfusion surgery, and IR+2,6-DMHQ represents the liver ischemia-reperfusion experimental group treated with 2,6-DMHQ.

[0024] Figure 2 shows that dimethylhydroquinone (2,6-DMHQ) significantly improved myocardial ischemia-reperfusion injury. CK (A), CK-MB (B), LDH (C), echocardiography and left ventricular ejection fraction (EF) (D), cardiac TTC staining (E), cardiac HE and Masson staining (F), cardiac 4-HNE and Prussian blue staining (G), cardiac non-heme iron level (H), and cardiac reducing to oxidized glutathione (GSH / GSSG) ratio (I); * indicates p < 0.05, ** indicates p < 0.01, indicating statistically significant differences.

[0025] IR represents the experimental group undergoing cardiac ischemia-reperfusion surgery, and IR+2,6-DMHQ represents the experimental group undergoing cardiac ischemia-reperfusion intervention with 2,6-DMHQ. Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0027] 1. Materials and Methods

[0028] 1.1 Laboratory Animals

[0029] Eight- or twelve-week-old SPF-grade male C57BL / 6 mice (wild-type mice) were housed in an SPF environment. All mice were fed a standard AIN-76A diet (Research Diets) with an iron content of 50 mg / kg. All mice were housed in an SPF-grade animal facility at a constant temperature (22±1℃) with a strict 12-hour light-dark cycle. Each experimental design used sex- and age-matched control mice in the same cage. All animal experiments adhered to strict animal welfare principles, minimizing the number of animals and reducing animal suffering, and strictly followed animal ethics and related operating procedures. Mice were randomly assigned to groups of 5-10 mice based on body weight.

[0030] 1.2 Drugs and Treatment

[0031] The monomeric compound 2,6-DMHQ was purchased from MCE and dissolved in physiological saline containing 5% dimethyl sulfoxide (DMSO) to obtain a 2,6-DMHQ injection solution with a concentration of 2 mg / mL. Mice were treated with 2,6-DMHQ at a dose of 20 mg / kg body weight.

[0032] Experiment 1: Liver ischemia-reperfusion injury

[0033] Eight-week-old male wild-type C57BL / 6 mice were randomly divided into three groups (n=10 per group) under standard diet. The first group was the sham intervention group (Sham), the second group was the liver ischemia-reperfusion surgery experimental group (IR), and the third group was the liver ischemia-reperfusion experimental group with 2,6-DMHQ intervention (IR+2,6-DMHQ).

[0034] The liver ischemia-reperfusion injury experimental group (IR+2,6-DMHQ) was treated with 2,6-DMHQ intervention: 2,6-DMHQ injection was administered intraperitoneally once daily for 10 days prior to the liver ischemia-reperfusion injury experiment, with the final injection given 1 hour before surgery. Six hours post-surgery, orbital blood samples were collected for serum biochemical analysis. Twenty-four hours later, ischemic liver lobes from sacrificed mice were fixed in 4% formaldehyde fixative and then embedded for sectioning and HE staining.

[0035] The surgical treatment for liver ischemia-reperfusion injury is as follows:

[0036] a) Administer pet analgesics to mice via gavage;

[0037] b) Place the mouse's mouth and nose over the mouth of the isoflurane anesthesia mask. After the mouse is anesthetized, shave the hair in the liver area of ​​its abdomen.

[0038] c) Record the weight; then fix the mouse to the operating table with medical tape and disinfect the surgical area with alcohol;

[0039] d) Use surgical scissors to make a small incision (less than 1.5cm) in the abdomen horizontally, and use a vascular clamp to clamp the branch of the portal vein triple canal in the large hepatic lobe, causing ischemia in the left large hepatic lobe. The darkening of the ischemic lobe is an indication that the ischemia was successful.

[0040] e) During ischemia, cover the abdomen with sterile gauze and moisten it with saline.

[0041] f) After 1 hour, remove the vascular clamp. If the liver color returns to red, the reperfusion is considered successful.

[0042] g) Carefully suture the opening to prevent the wound from reopening; weigh and record the weight;

[0043] h) Subcutaneously administer 400 μL of physiological saline to the neck of mice;

[0044] i) After the mouse regains consciousness, return it to its cage; 24 hours later, sacrifice the mouse and harvest the surgical liver lobe and other tissues.

[0045] During the surgery, the operating table was heated to maintain the mice's body temperature.

[0046] In the liver ischemia-reperfusion surgery experimental group (IR), "2,6-DMHQ injection" was replaced with "physiological saline with a volume ratio of 5% dimethyl sulfoxide DMSO", while the volume remained the same, and the rest was the same as the experimental group (IR+2,6-DMHQ).

[0047] The sham intervention group (Sham) only underwent laparotomy without surgical intervention; that is, step d) was simply to open a small incision in the abdomen horizontally with surgical scissors and correspondingly cancel step f), and the incision was sutured 1 hour after the laparotomy; the rest was the same as the experimental group (IR+2,6-DMHQ).

[0048] Experiment 2: Myocardial Ischemia-Reperfusion Injury

[0049] Twelve-week-old male wild-type C57BL / 6 mice were randomly divided into two groups (n=4-5 per group) under standard diet. The first group was the myocardial ischemia-reperfusion surgery experimental group (IR), and the second group was the myocardial ischemia-reperfusion experimental group with 2,6-DMHQ intervention (IR+2,6-DMHQ).

[0050] For the 2,6-DMHQ-interventional group of myocardial ischemia-reperfusion injury (IR+2,6-DMHQ), the 2,6-DMHQ injection was administered intraperitoneally once one day before the myocardial ischemia-reperfusion injury experiment, followed by myocardial ischemia-reperfusion injury surgery. Mice were sacrificed 24 hours after the operation for testing.

[0051] The experimental group (IR) changed "2,6-DMHQ injection" to "physiological saline with a volume ratio of 5% dimethyl sulfoxide DMSO", while keeping the volume unchanged, and the rest was the same as the experimental group (IR+2,6-DMHQ).

[0052] illustrate:

[0053] The surgical treatment for myocardial ischemia-reperfusion injury is as follows:

[0054] Each experimental mouse was weighed. Mice were anesthetized by intraperitoneal injection of 0.16 ml / 25 g of 1% sodium pentobarbital (prepared with physiological saline). After being fixed in a dorsal position on a board, the chest area was disinfected and prepared, and then disinfected again. A 20G intravenous catheter was used for endotracheal intubation, and the mice were connected to a small animal ventilator for assisted breathing (parameters: respiratory rate 100 breaths / min, tidal volume 1.0, respiratory ratio 1:1). A thoracotomy was performed at the 3rd and 4th intercostal spaces along the left sternal border. Blunt dissection was used, and the heart was fully exposed using a thoracotomy device. The left anterior descending coronary artery (LAD) below the left atrial appendage was ligated with 7-0 surgical sutures, secured with a slipknot; the heart surface immediately turned white. Blood and debris in the pleural cavity were cleared, and the pleural cavity was closed but not sutured immediately. A gauze soaked in physiological saline was placed over the wound, and the ventilator was continuously ventilated to maintain the LAD ligation for 45 minutes. After 45 minutes of ischemia, the heart was fully exposed again using a thoracotomy apparatus. The slipknots of the ligatures were loosened to allow reperfusion of the mouse's coronary arteries. At this point, the heart was observed to change from white to slightly pink. The muscle and skin layers were sutured layer by layer. The ventilator was removed, and the mouse was transferred to an electric blanket to keep warm and recover until it was fully awake before being placed back in its cage.

[0055] Twenty-four hours after cardiac surgery, mice were anesthetized with 2% isoflurane. After successful anesthesia, the mice were fixed in a supine position on the worktable, and anesthesia was maintained. Ultrasound coupling gel was applied to the left anterior chest wall of the mice, and the ultrasound probe was manually adjusted to locate the papillary muscles in short-axis mode. The corresponding data were recorded. After the examination, the mice were removed, the coupling gel was dried, and they were placed in cages to await awakening. The results were processed using the ultrasound equipment's accompanying data analysis software. Left ventricular ejection fraction (EF%) was measured by echocardiography.

[0056] After the mice regained consciousness, they were anesthetized with pentobarbital (70 mg / kg). The thoracic cavity was opened with surgical scissors, and the skin of the thoracic cavity was fixed with hemostatic forceps to expose the heart. The needle of a 1 mL syringe was quickly inserted into the right ventricle, the needle position was adjusted, and blood was slowly and steadily drawn. The serum was separated by centrifugation at 1.0XG for 5 minutes, and 100 μL was aliquoted into 1.5 EP tubes for subsequent serum biochemical analysis (Siemens blood biochemistry analyzer). The main results included creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH). Fresh heart tissue was frozen at -80℃ for 15 min, sectioned, and subjected to TTC staining. The ischemic border region of the heart was fixed in 4% formaldehyde fixative and embedded, sectioned, and stained with HE, Masson's stain, 4-HNE immunohistochemical staining, and Perls' staining. A portion of the heart border region tissue was used for non-heme iron detection and the detection of reduced and oxidized glutathione (GSH / GSSG).

[0057] 1.3 Statistical Methods

[0058] All data are expressed as mean ± standard error (mean ± SEM). Statistical analysis was performed using GraphPad Prism (version 8.0, GraphPad software; San Diego, CA, USA) statistical software. * indicates p < 0.05, ** indicates p < 0.01, and the difference is statistically significant.

[0059] 2. Results

[0060] The results of Experiment 1 are shown in Figure 1;

[0061] As shown in Figures A and B, the serum ALT and AST levels in the experimental group (IR+2,6-DMHQ) were significantly lower than those in the experimental group (IR).

[0062] As shown in Figure 1C, the area of ​​liver necrosis in the experimental group (IR+2,6-DMHQ) was reduced compared to that in the experimental group (IR), indicating improved liver necrosis-like pathology.

[0063] In summary, 2,6-DMHQ improves hepatic ischemia-reperfusion injury.

[0064] The results of Experiment 2 are shown in Figure 2;

[0065] As shown in Figures 2A, 2B, and 2C, the serum myocardial enzyme levels of CK, CK / MB, and LDH in the experimental group (IR+2,6-DMHQ) were significantly lower than those in the experimental group (IR). As shown in Figure 2D, the left ventricular ejection fraction (EF) in the experimental group (IR+2,6-DMHQ) was significantly higher than that in the experimental group (IR), indicating that the cardiac function of the mice in the experimental group (IR+2,6-DMHQ) was significantly improved.

[0066] As shown in Figures E and F, the experimental group (IR+2,6-DMHQ) had a reduction in cardiac inflammation and infarct area compared to the experimental group (IR).

[0067] As shown in G, H, and I of Figure 2, the experimental group (IR+2,6-DMHQ) showed a significant decrease in 4-HNE, Perls' staining of the heart, and non-heme iron in the heart compared to the experimental group (IR). The GSH / GSSG ratio was significantly upregulated, indicating that the lipid peroxidation level and iron accumulation in the heart of the experimental group (IR+2,6-DMHQ) mice were significantly improved. That is, 2,6-DMHQ can significantly improve the ferroptosis phenotype of I / R injury.

[0068] In summary, 2,6-DMHQ improves myocardial ischemia-reperfusion injury.

[0069] Therefore, it can be concluded that dimethylhydroquinone (2,6-DMHQ) is a novel drug for the preparation of drugs to combat liver and heart ischemia-reperfusion injury.

[0070] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. 2,6-DMHQ in the preparation of drugs for treating ischemia-reperfusion injury.

2. The application according to claim 1, characterized in that: To combat ischemia-reperfusion injury of the heart and liver.

3. The application according to claim 2, characterized in that: 2,6-DMHQ can reduce serum ALT and AST and improve liver necrosis; 2,6-DMHQ can reduce serum myocardial enzyme levels CK, CK / MB, and LDH, and decrease the area of ​​myocardial infarction. 4.2, Application of 6-DMHQ in the preparation of drugs to improve cardiac ejection function. 5.2,6-DMHQ is used in the preparation of drugs to improve lipid peroxidation levels and iron accumulation.

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