CN112675159A - Application of L-malic acid in preparing medicine for preventing and treating liver ischemia reperfusion injury - Google Patents
Application of L-malic acid in preparing medicine for preventing and treating liver ischemia reperfusion injury Download PDFInfo
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Abstract
The invention discloses application of L-malic acid in preparing a medicament for preventing and treating liver ischemia-reperfusion injury, and particularly discloses application of L-malic acid as a unique active ingredient in preparing a medicament or a health-care product for preventing or treating liver ischemia-reperfusion injury. The invention is proved by mouse in vivo experiments that the L-malic acid can obviously reduce the increase of liver injury markers such as alanine aminotransferase, aspartate aminotransferase and lactate dehydrogenase in serum caused by liver ischemia-reperfusion, reduce the area of liver necrosis, inhibit liver tissue lipid peroxidation and cell iron death caused by ischemia-reperfusion, and further reduce liver injury. The invention provides a theoretical basis for treating diseases related to liver ischemia-reperfusion injury, and has clinical application value.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of L-malic acid in preparation of a medicine for preventing and treating liver ischemia-reperfusion injury.
Technical Field
Ischemia Reperfusion Injury (IRI) refers to the phenomenon that blood circulation is restored after a period of Ischemia of a tissue organ, and cell dysfunction and tissue structure damage are not reduced but aggravated. Ischemia reperfusion injury can occur in many vital organs including the heart, liver, lung, kidney, gastrointestinal tract, and the like.
Liver Ischemia Reperfusion Injury (HIRI) is a common complication in liver surgical operations such as hepatectomy and liver transplantation, and is one of the main reasons for poor surgical effect and liver transplantation failure. Severe HIRI can lead to massive death of hepatocytes, which in turn can lead to liver failure and even death of the patient. According to the existing research reports, the pathophysiological process has a complex occurrence mechanism, and a practical and effective method and a specific medicine aiming at HIRI are still lacking clinically at present; therefore, how to prevent and treat the liver ischemia-reperfusion injury becomes a great medical research direction.
Iron death (Ferroptosis) is a new type of programmed cell death pathway dependent on iron, and has obvious differences from apoptosis, necrosis and autophagy in aspects of morphology, biochemistry, genetics and the like. The occurrence of iron death is closely related to a variety of biochemical processes, including polyunsaturated fatty acid, iron and amino acid metabolism, and biosynthesis of glutathione, phospholipids, nicotinamide adenine dinucleotide phosphate and coenzyme Q10; specifically, the expression level of lipid peroxidation and the expression level of the marker gene Ptgs2 can be increased. Recent studies found that cellular iron death (Ferroptosis) is one of the important molecular mechanisms of ischemia reperfusion injury of liver. The use of specific inhibitors of iron death can effectively alleviate hepatic ischemia reperfusion injury in a mouse model, and therefore iron death is a therapeutic target with potential clinical application.
L-malic acid (L-Malate), also known as L-malic acid, is an important organic acid produced during the metabolic process of organisms. It plays a key role in the tricarboxylic acid cycle in which mitochondria generate energy substances ATP, is also an important component of a malic acid-aspartic acid shuttle system, and plays an important role in transferring reducing equivalents between cytoplasm and mitochondria. The L-malic acid is safe, non-toxic, harmless and edible, and has physiological functions of improving exercise capacity, resisting fatigue, protecting heart, improving memory, etc.
The Chinese patent application with the publication number of CN109966278A discloses the application of oxalyl malic acid in preparing the medicine for treating nerve cell injury; an academic paper, clinical application of sodium acetate malate ringer's solution in perioperative liver ischemia protection [ D ], discloses that the sodium acetate malate ringer's solution can relieve early liver injury after partial resection of the liver, reduce postoperative lactic acid level and protect liver function.
However, to date, no reports have been made of the role of L-malic acid as the only active ingredient in cell iron death and ischemia-reperfusion injury of the liver.
Disclosure of Invention
The invention aims to solve the technical problem of providing the application of L-malic acid as the only active ingredient in preparing the medicine or health care product for preventing and treating the liver ischemia reperfusion injury, and providing a foundation for the research and development of new medicines and innovative therapies.
The technical scheme provided by the invention for solving the technical problems is as follows:
the application of L-malic acid as the only active component in preparing the medicine or health product for preventing or treating liver ischemia-reperfusion injury.
The L-malic acid is applied to the preparation of medicines or health products for preventing or treating the increase of liver injury markers caused by liver ischemia-reperfusion.
The L-malic acid is used as the only active ingredient in the preparation of drugs or health products for preventing or treating the increase of liver injury markers caused by liver ischemia-reperfusion.
The liver injury marker is alanine aminotransferase, aspartate aminotransferase or lactate dehydrogenase.
The L-malic acid is applied to the preparation of medicines or health products for preventing or treating lipid peroxidation caused by liver ischemia-reperfusion.
The L-malic acid is used as the only active component in the preparation of drugs or health products for preventing or treating lipid peroxidation caused by liver ischemia-reperfusion.
The L-malic acid is applied to the preparation of medicines or health products for preventing or treating liver cell iron death caused by liver ischemia-reperfusion.
The L-malic acid is used as the only active component in the preparation of the medicine or health care product for preventing or treating the liver cell iron death caused by liver ischemia reperfusion.
The invention has the following beneficial effects:
the invention discloses that L-malic acid as the only active ingredient has a protective effect on liver ischemia-reperfusion injury for the first time, and can be used for preparing a medicament or a health-care product for preventing or treating the liver ischemia-reperfusion injury. Experiments prove that the L-malic acid treatment can obviously reduce serum liver injury markers and liver pathological changes of mice after receiving liver ischemia reperfusion operations, inhibit the occurrence of liver iron death, and has wide clinical application value.
The research result provides a theoretical basis for the L-malic acid to improve the liver ischemia reperfusion injury, and particularly provides a basis for the research and development of related medicaments or health care products.
Drawings
FIG. 1 is a graph comparing the content change of serum liver injury markers in mice in different experimental groups, wherein, the graph A is the content comparison of serum alanine aminotransferase, the graph B is the content comparison of aspartate aminotransferase, and the graph C is the content comparison of lactate dehydrogenase.
FIG. 2 shows the effect of L-malic acid on pathological changes after ischemia-reperfusion surgery of mouse liver.
FIG. 3 is a graph showing the comparison of the change of the iron death marker gene and the malondialdehyde content in lipid peroxidation products in different experimental groups, wherein, the graph A shows the comparison of the content of the liver iron death marker gene Ptgs2 mRNA, and the graph B shows the comparison of the content of the malondialdehyde in lipid peroxidation products.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Example 1 animal experiments: application of L-malic acid in preparation of medicine for preventing and treating liver ischemia reperfusion injury
1. Materials and methods
1.1 Experimental animals
SPF grade C57BL/6 male mice at 6 weeks of age were purchased from Jiangsu Jiejiaokang Biotech, Inc. and bred in SPF environment. After 2 weeks of acclimation feeding, standard feed AIN-76A (Research Diets, Inc) was randomly assigned to each group of 8-10 animals by body weight.
1.2 Experimental drugs
L-malic acid was purchased from Sigma-Aldrich (cat. 02288) and had a purity of 99% or higher, and was dissolved in physiological saline.
1.3 construction of mouse liver ischemia-reperfusion model
Mice were anesthetized with isoflurane (3% concentration for anesthesia induction, 1.5% concentration for anesthesia maintenance) and fixed on a 37 ℃ thermostated platform, depilated routinely, sterilized with alcohol cotton balls. The skin and muscle are cut once along the median line of the abdomen, the falciform ligament and the left and right triangular ligaments around the liver are separated, the blood vessel of the hepatic portal part is fully exposed, and the portal vein, the hepatic artery and the common bile duct are clamped by a miniature nondestructive vascular clamp (the color of the hepatic lobe is changed from bright red to dark). At the beginning of the protocol, the incision was covered with warm saline gauze. After 1 hour of ischemia, the vascular clamps are released, and the blood flow of the liver lobes is restored and the abdomen is closed layer by layer. After 6 hours of reperfusion, mice were sacrificed and liver tissue and blood samples were left.
Grouping experiments:
after 2 weeks of standard dietary equilibration, 6-week-old C57BL/6 male mice were randomly assigned to 4 groups, the first and second groups were sham (only anesthesia and laparotomy, and only liver tissue was isolated by traction), and the third and fourth groups were subjected to liver ischemia reperfusion (1 hour of ischemia, 6 hours of reperfusion).
In which the second and fourth groups were intraperitoneally injected with L-malic acid (10 mg/kg body weight) 2 hours before ischemia and 10 hours before reperfusion, and the first and third groups were administered with the same dose of physiological saline at the same time point.
Mice were dissected after completion of reperfusion, serum and liver tissue were collected, and serum liver injury markers, liver pathological changes, and iron death levels were examined.
1.4 detection of serum liver injury markers
After the collected blood was left at room temperature for 2 hours, the serum was collected by centrifugation at 3000g for 10 minutes. The contents of alanine Aminotransferase (ALT), aspartate Aminotransferase (AST) and Lactate Dehydrogenase (LDH) in serum were measured by a Roche COAS INTEGRA 800 full-automatic biochemical analyzer, and the results are shown in FIG. 1.
As shown in fig. 1, compared with the sham operation group, the serum contents of the three enzymes in the ischemia reperfusion group are significantly increased, which indicates that the liver injury modeling is successful; the treatment of the L-malic acid has no influence on a sham operation group, which indicates that the L-malic acid is safe and harmless; the L-malic acid treatment can obviously reduce the content of the enzymes in the mice subjected to liver ischemia reperfusion, and the protective effect of the L-malic acid treatment on the liver function in the pathological state is suggested.
1.5 liver Paraffin section HE staining
(1) Fixing liver tissue in 10% formaldehyde solution for 24 hr, dewatering, transparentizing, soaking in wax and embedding;
(2) slicing wax block, baking in oven at 65 deg.C for 2 hr to obtain slices with thickness of 5 μm, and storing at room temperature;
(3) soaking in hematoxylin dye solution for 5 min, washing with deionized water to remove loose color, differentiating with 1% hydrochloric acid alcohol for 2-5 s, and washing with deionized water for 10 min;
(4) soaking the eosin dye solution for 3 minutes, and washing away the flooding color by deionized water;
(5) gradient ethanol dehydration, xylene transparency, and neutral gum sealing;
(6) and (4) observing under an optical microscope.
The results are shown in fig. 2, and the L-malic acid treatment can obviously relieve pathological injuries such as hepatocyte necrosis, vacuolation-like degeneration, hepatic sinus congestion and the like caused by liver ischemia-reperfusion.
1.6 RNA extraction and fluorescent quantitation real-time PCR
Trizol (Life technologies) method for extracting RNA from cells and tissues, the specific operation is performed according to the instruction. RNA purity (OD) was determined on a Nanodrop 1000Spectrophotometer260/OD2801.9-2.1) and RNA concentration(ng/l), adjust the RNA concentration to 1. mu.g/l.
2.0. mu.g of RNA was treated with DNase (Promega), and reverse transcribed with M-MLV reverse transcriptase (Promega) and oligo (dT)18primer (Takara Bio Inc.). The fluorescence quantitative Real-Time (Real-Time PCR) detection of mRNA level in CFX96 Real-Time System (Bio-Rad) for gene expression of each related gene is carried out in a detection volume of 10ul, the iQ SYBR Green Supermix (Bio-Rad) is adopted as a reagent, and the primer sequence is as follows:
mouse Gapdh primers:
forward direction: ATCATCCCTGCATCCACT
And (3) reversing: ATCCACGACGGACACATT
Mouse Ptgs2 primer:
forward direction: CTGCGCCTTTTCAAGGATGG
And (3) reversing: GGGGATACACCTCTCCACCA
The results are shown in fig. 3A, where L-malic acid treatment was able to significantly fallback the iron death marker gene elevated due to liver ischemia reperfusion injury.
1.7 determination of Malondialdehyde (MDA) in liver tissue
Malondialdehyde (MDA) is a product of lipid oxidation in living organisms, and the content of MDA can be used for judging the level of lipid oxidation in cells, MDA generated by degradation of peroxidized lipids can be condensed with thiobarbituric acid to generate a red product, and a maximum absorption peak is formed at 532 nm. Therefore, the detection of the MDA content can reflect the lipid peroxidation degree in the organism, and can indirectly indicate the iron death degree of the cells. The invention adopts a Biyuntian Biotechnology (Beyotime Biotechnology) lipid oxidation (MDA) detection kit (S0131), and the operations are carried out according to the instruction:
(1) homogenizing and cracking the tissue by PBS;
(2) centrifuging at 1, 600g for 10 minutes, and taking the supernatant for subsequent measurement;
(3) measuring the protein concentration so as to calculate the MDA content in the tissue or the cell by unit protein weight;
(4) weighing a proper amount of TBA, and preparing TBA storage solution with the concentration of 0.37% by using TBA preparation solution;
(5) preparing MDA detection working solution;
(6) diluting a proper amount of standard substance with distilled water to 1, 2, 5, 10, 20 and 50 mu M for subsequent standard curve preparation;
(7) adding 0.1ml PBS into a centrifuge tube as a blank control, adding 0.1ml of the standard substance with different concentrations for preparing a standard curve, adding 0.1ml sample for determination, and then adding 0.2ml MDA detection working solution;
(8) after mixing uniformly, heating for 15 minutes at 100 ℃ or in a boiling water bath;
(9) cooling to room temperature, and centrifuging at 1,000 g for 10 minutes at room temperature;
(10) 200. mu.l of the supernatant was added to a 96-well plate, followed by measurement of absorbance at 532nm with a microplate reader.
The results are shown in fig. 3B, where ischemia reperfusion injury can significantly increase MDA levels in mouse liver, suggesting the occurrence of lipid peroxidation and iron death; after the L-malic acid is used for treatment, MDA (increased MDA) of the mouse liver after injury is obviously inhibited, which shows that the L-malic acid can inhibit the iron death process which takes lipid peroxidation as a key molecular mechanism.
1.8 statistical methods
All data are expressed as mean ± standard error (mean ± SD) and statistically analyzed using GraphPad Prism (version 6.0, GraphPad software; san diego, CA, USA) statistical software. P < 0.05 represents that the difference is significant and has statistical significance.
2. Results of the experiment
The results show that L-malic acid treatment significantly inhibited the serum liver injury markers significantly upregulated by surgery (fig. 1) and the pathological changes in liver (fig. 2) compared to the ischemia-reperfusion group alone, demonstrating its protective effect on liver function. At the same time, the level of iron death significantly activated by ischemia reperfusion treatment was also significantly inhibited by L-malate (fig. 3).
And (4) conclusion: based on the experiments, the application of L-malic acid to inhibit iron death is a novel therapy for preventing and treating liver ischemia reperfusion.
Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (8)
- The application of L-malic acid as the only active component in preparing the medicine or health product for preventing or treating liver ischemia-reperfusion injury.
- Application of L-malic acid in preparation of medicines or health products for preventing or treating liver injury marker elevation caused by liver ischemia-reperfusion.
- The application of the L-malic acid as the only active ingredient in preparing the medicine or the health care product for preventing or treating the increase of the liver injury marker caused by the liver ischemia-reperfusion.
- 4. The use according to claim 2 or 3, wherein the marker of liver injury is alanine aminotransferase, aspartate aminotransferase or lactate dehydrogenase.
- The application of the L-malic acid in preparing the medicine or health-care product for preventing or treating lipid peroxidation caused by liver ischemia-reperfusion.
- The application of the L-malic acid as the only active ingredient in preparing the medicine or health care product for preventing or treating lipid peroxidation caused by liver ischemia reperfusion.
- Application of L-malic acid in preparation of medicines or health products for preventing or treating liver cell iron death caused by liver ischemia-reperfusion.
- The application of the L-malic acid as the only active ingredient in preparing the medicine or health care product for preventing or treating the liver cell iron death caused by liver ischemia reperfusion.
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