Application of 2, 6-DMHQ in resisting heart and liver ischemia-reperfusion injury

Abstract

The invention relates to the field of biological medicine, in particular to application of dimethylhydroquinone in improvement of heart and liver ischemia reperfusion related injuries. 2, 6-DMHQ can reduce ALT and AST in serum and improve hepatic necrosis; cK, CK / MB and LDH of serum myocardial zymogram can be reduced, and the area of cardiac infarction is reduced.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to application of dimethylhydroquinone in improving ischemia-reperfusion-related damage of the heart and liver. Background Art

[0002] Clinically, ischemia-reperfusion injury (IRI) refers to the phenomenon that after a period of insufficient blood supply (ischemia), when blood flow is restored (reperfusion), the damage to tissues or organs is aggravated. This type of injury is common in many clinical situations, such as myocardial infarction, liver and other organ transplantation, and microcirculation reperfusion after shock. Ischemia-reperfusion injury has a significant impact on the prognosis of the disease in clinical practice, especially in the fields of heart disease and liver disease. [1][2] The research on drugs against ischemia-reperfusion injury is an important branch of medicine. Currently, there is a lack of specific drugs for ischemia-reperfusion injury.

[0003] 2,6-Dimethylhydroquinone (2,6-DMHQ) has been reported to reduce ferric iron and is mainly involved in the redox reaction of iron. [3] The medical use of 2,6-DMHQ has not been reported yet.

[0004]

[0005] References:

[0006] 1. Xuexian Fang et al., Proceedings of the National Academy of Sciences of the United States of America, 2019Feb 12;116(7):2672-2680.

[0007] 2.Jose Pedro Friedmann Angeli et al., Nature Cell Biology, 2014Dec; 16(12):1180-91.

[0008] 3. Lelde Krumina et al., Environmental Science & Technology, 2017Aug 15;51(16):9053-9061.

[0009] Gry Lyngsie et al.,Scientific Reports,2018Jul 17;8(1):10834. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide application of 2,6-DMHQ in preventing ischemia-reperfusion injury of heart and liver.

[0011] In order to solve the above technical problems, the present invention provides the use of dimethylhydroquinone (2,6-DMHQ) in the preparation of anti-ischemia reperfusion injury drugs.

[0012] As an improvement of the application of the present invention: it is used to resist ischemia-reperfusion injury of heart and liver.

[0013] As a further improvement of the application of the present invention:

[0014] Can reduce serum ALT and AST, improve liver necrosis (reduce liver necrosis area);

[0015] It can reduce serum myocardial enzymes CK, CK / MB and LDH, and reduce the area of ​​myocardial infarction.

[0016] During the invention process, the mouse liver and heart ischemia-reperfusion injury models were introduced, and 2,6-DMHQ was injected intraperitoneally into the mice before surgery. It was found that 2,6-DMHQ could significantly improve liver and heart ischemia-reperfusion injuries. The dose required for the human body can be calculated according to the corresponding dose of the mouse. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The specific implementation modes of the present invention are further described in detail below with reference to the accompanying drawings.

[0018] Figure 1 2,6-DMHQ) significantly improved liver ischemia-reperfusion injury. ALT (A), AST (B), liver H&E staining (C); * represents p < 0.05, ** represents p < 0.01, the difference is statistically significant;

[0019] Sham represents sham operation experiment of wild-type mice, IR represents liver ischemia-reperfusion operation experiment, and IR+2,6-DMHQ represents liver ischemia-reperfusion experimental group intervened by 2,6-DMHQ.

[0020] Figure 2 Dimethylhydroquinone (2,6-DMHQ) significantly improved myocardial ischemia-reperfusion injury. CK (A), CK-MB (B), LDH (C), cardiac TTC staining (D), cardiac HE and Masson staining (E); * represents p < 0.05, ** represents p < 0.01, the difference is statistically significant.

[0021] IR represents the cardiac ischemia-reperfusion surgery experiment, and IR+2,6-DMHQ represents the cardiac ischemia-reperfusion experimental group intervened by 2,6-DMHQ. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0023] 1. Materials and Methods

[0024] 1.1 Experimental animals

[0025] 8-week-old or 12-week-old SPF-grade C57BL / 6 male mice (wild-type mice) were raised 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 raised in an SPF-grade animal room with a constant temperature (22±1℃) and a strict 12-h light-dark cycle. Gender- and age-matched control mice in the same cage were used for each experimental design. All animal experiments adhered to the welfare of experimental animals, minimized the number of experimental animals, reduced animal suffering, and strictly abided by experimental animal ethics and relevant operating regulations. They were randomly divided into groups according to body weight, with 5-10 mice in each group.

[0026] 1.2 Drugs and treatment

[0027] The compound monomer 2,6-DMHQ was purchased from MCE and dissolved in a physiological saline solution containing 5% dimethyl sulfoxide (DMSO) by volume to obtain a 2,6-DMHQ injection solution with a 20 mg / mL 2,6-DMHQ concentration. Mice were intervened with 2,6-DMHQ at a dose of 10 mg / kg body weight.

[0028] Experiment 1: Hepatic ischemia-reperfusion injury

[0029] Eight-week-old C57BL / 6 male wild-type mice were randomly divided into three groups (n=10 in each group) under standard diet. The first group was the sham operation 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 intervened by 2,6-DMHQ (IR+2,6-DMHQ).

[0030] 2,6-DMHQ intervention liver ischemia reperfusion experimental group (IR+2,6-DMHQ): The drug 2,6-DMHQ injection was intraperitoneally injected once a day before the liver ischemia reperfusion injury experiment for 10 days, and the last injection was injected 1 hour before the operation, and then the liver ischemia reperfusion injury surgery was performed. Six hours after the operation, blood was collected from the orbit for serum biochemical testing. After 24 hours, the mice were sacrificed and the ischemic lobe of the liver was fixed in 4% formaldehyde fixative and embedded in sections and HE staining experiments.

[0031] The surgical treatment of liver ischemia-reperfusion injury is as follows:

[0032] a) PET analgesics were administered to mice by gavage;

[0033] b) Place the mouse's mouth and nose toward the mouth of the isoflurane anesthesia mask, and after the mouse is anesthetized, shave the liver area of ​​the abdomen;

[0034] c) Record the body weight; then fix the mouse on the operating table with medical tape and disinfect the operating area with alcohol;

[0035] d) Use surgical scissors to make a small incision (less than 1.5 cm) in the abdomen transversely, and clamp the branch of the portal vein triad of the major hepatic lobe with a vascular clamp to cause ischemia of the left major hepatic lobe. The ischemic liver lobe will darken, indicating successful ischemia.

[0036] e) During the ischemic period, the abdomen was covered with sterile gauze and moistened with saline;

[0037] f) After 1 hour, remove the vascular clamp and observe that the liver has returned to red, indicating that reperfusion is successful;

[0038] g) Carefully suture the opening to avoid wound dehiscence; weigh and record the body weight;

[0039] h) 400 μL of normal saline was added subcutaneously in the neck of the mouse;

[0040] i) After the mouse wakes up, put it back into the cage; after 24 hours, sacrifice the mouse and remove the surgical liver lobe and other tissues.

[0041] During mouse surgery, the operating table was equipped with a constant temperature heating plate to maintain the mouse body temperature.

[0042] In the liver ischemia-reperfusion surgery experimental group (IR), the "2,6-DMHQ injection" was changed to "normal saline with 5% dimethyl sulfoxide (DMSO) by volume", the volume dosage remained unchanged, and the rest was the same as the experimental group (IR+2,6-DMHQ);

[0043] The sham intervention group (Sham) only underwent laparotomy without surgical treatment; that is, step d) was only to open the abdomen horizontally with surgical scissors, and step f) was cancelled accordingly, and the opening was sutured 1 hour after laparotomy; the rest was the same as the experimental group (IR+2,6-DMHQ).

[0044] Experiment 2: Cardiac ischemia-reperfusion injury

[0045] Twelve-week-old C57BL / 6 male wild-type mice were randomly divided into two groups (4-5 mice in each group) under standard diet. The first group was the cardiac ischemia-reperfusion surgery experimental group (IR), and the second group was the cardiac ischemia-reperfusion experimental group intervened by 2,6-DMHQ (IR+2,6-DMHQ).

[0046] For the cardiac ischemia-reperfusion experimental group intervened by 2,6-DMHQ (IR+2,6-DMHQ), the drug 2,6-DMHQ injection was intraperitoneally injected once one day before the cardiac ischemia-reperfusion injury experiment and continued for 10 days. The last injection was injected one hour before the operation. Then, the cardiac ischemia-reperfusion injury surgery was performed and the mice were sacrificed 24 hours after the operation for sample collection and testing.

[0047] In the experimental group (IR), the "2,6-DMHQ injection" was changed to "physiological saline with 5% dimethyl sulfoxide (DMSO) by volume", and the volume dosage remained unchanged, and the rest was the same as the experimental group (IR+2,6-DMHQ).

[0048] illustrate:

[0049] The surgical treatment of cardiac ischemia-reperfusion injury is as follows:

[0050] The experimental mice were weighed one by one, and 0.16ml / 25g of 1% sodium pentobarbital prepared in normal saline was injected intraperitoneally to anesthetize the mice. After being fixed on the mouse board in dorsal position, the chest area was disinfected and prepared for skin preparation, and then disinfected again after skin preparation. The mice were intubated with a 20G intravenous indwelling needle, and connected to a small animal ventilator for assisted breathing (parameter settings: respiratory rate 100 times / min, tidal volume 1.0, respiratory ratio 1:1). The thoracotomy was performed at the 3rd and 4th intercostal spaces on the left side of the mouse sternum, blunt separation, and the heart was fully exposed using a thoracotomy device. At the left anterior descending coronary artery (LAD) under the left atrial appendage, a 7-0 surgical suture was used to ligate and tie a slipknot, and the surface of the heart was immediately whitened. The blood stains in the chest cavity were removed, the chest cavity was closed but not sutured temporarily, and the gauze soaked in normal saline was covered on the wound surface, and the ventilator was continuously ventilated to maintain the LAD ligation state for 45min. After 45 minutes of ischemia, the heart was fully exposed using a chest opener again, and the ligature was untied to allow the coronary artery of the mouse heart to be reperfused. At this time, the heart was seen to turn slightly pink from white. The muscle layer and cortex were sutured layer by layer, and the ventilator was removed and the mouse was transferred to an electric blanket for warmth and recovery until it was fully awake and then placed in a cage.

[0051] 24 hours after cardiac surgery, mice were anesthetized with pentobarbital (70 mg / kg), the chest was cut open with surgical scissors, and the chest skin was fixed with hemostats to expose the heart. The needle tip of a 1 mL syringe was quickly inserted into the right ventricle, the position of the needle was adjusted, blood was slowly and steadily drawn, and the serum was separated by centrifugation at 1.0XG for 5 minutes. 100 μL was dispensed into 1.5EP tubes for subsequent serum biochemical tests (blood biochemical analyzer Siemens); mainly including alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatine kinase (CK), creatine kinase isoenzyme (CK-MB), and lactate dehydrogenase (LDH). Fresh heart tissue was frozen in a -80℃ refrigerator for 15 minutes, then sliced ​​and subjected to TTC staining experiments. The ischemic part of the heart was fixed in 4% formaldehyde fixative and embedded and sliced, HE stained, and Masson's experiment was performed.

[0052] 1.3 Statistical methods

[0053] All data were expressed as mean ± SEM, and statistical analysis was performed using GraphPad Prism (version 8.0, GraphPad software; San Diego, CA, USA). * represents p < 0.05, ** represents p < 0.01, and the difference is statistically significant.

[0054] 2. Results

[0055] The results of Experiment 1 are as follows Figure 1 as stated;

[0056] according to Figure 1 From A and B, we can see that the serum ALT and AST levels of the experimental group (IR+2,6-DMHQ) were significantly lower than those of the experimental group (IR);

[0057] according to Figure 1 From C, we can see that: the liver necrosis area of ​​the experimental group (IR+2,6-DMHQ) was reduced compared with the experimental group (IR), that is, the liver necrosis-like pathology was improved;

[0058] In conclusion, 2,6-DMHQ improves hepatic ischemia-reperfusion injury.

[0059] The results of Experiment 2 are as follows Figure 2 as stated;

[0060] according to Figure 2 A, B and C show that the levels of serum myocardial enzymes CK, CK / MB and LDH in the experimental group (IR+2,6-DMHQ) were significantly lower than those in the experimental group (IR);

[0061] according to Figure 2From D and E, we can see that the experimental group (IR+2,6-DMHQ) reduced the area of ​​myocardial infarction compared with the experimental group (IR).

[0062] In conclusion, 2,6-DMHQ improves cardiac ischemia-reperfusion injury.

[0063] Therefore, it can be concluded that dimethylhydroquinone (2,6-DMHQ) is a new type of drug for preparing anti-ischemia-reperfusion injury of liver and heart.

[0064] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of 2,6-DMHQ in the preparation of drugs against ischemia-reperfusion injury.

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

3. The use according to claim 2, characterized in that: Can reduce serum ALT and AST and improve liver necrosis; It can reduce serum myocardial enzymes CK, CK / MB and LDH, and reduce the area of ​​myocardial infarction.

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