Application of licochalcone D in preparation of medicine for treating spontaneous cerebral hemorrhage

Abstract

The invention discloses application of licochalcone D in preparation of a medicine for treating spontaneous cerebral hemorrhage, and belongs to the technical field of biological medicine. The invention provides application of licochalcone D in preparation of related drugs for treatment and prognosis improvement of spontaneous cerebral hemorrhage. Rat experiment results show that licochalcone D can relieve hematoma caused by cerebral hemorrhage, reduce death of nerve cells after hemorrhage and reduce the level of inflammatory factors; cell experiment results show that licochalcone D can be used as a ferroptosis inhibitor and a COX2 inhibitor to improve ischemia and hypoxia injury of nerve cells after spontaneous cerebral hemorrhage, and the effect of licochalcone D is superior to that of a common ferroptosis inhibitor Fer-1. It is reported for the first time that licochalcone D is used for treating spontaneous cerebral hemorrhage and improving secondary brain injury, a new scheme is provided for treatment of spontaneous cerebral hemorrhage, and the licochalcone D has good clinical medicine application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of licorice chalcone D in preparing medicine for treating spontaneous cerebral hemorrhage. Background Art

[0002] Spontaneous intracerebral hemorrhage (ICH) accounts for 10-15% of all strokes worldwide and has significant morbidity and mortality. It is estimated that the mortality rate is as high as 50% within 30 days of the onset of ICH. The sudden rupture of cerebral blood vessels forms a hematoma and causes mechanical damage to adjacent tissues, resulting in a sharp increase in intracranial pressure, which is considered to be primary brain injury. After ICH, the hematoma can lead to a rapid and sustained increase in intracranial reactivity, including inflammatory response, oxidative stress, neuronal apoptosis, mitochondrial dysfunction and blood-brain barrier (BBB) ​​disruption, leading to secondary neurological damage. However, in clinical practice, the treatment for primary brain injury after ICH is mainly early surgical removal of the hematoma, but there is a possibility of failure in surgical removal of blood clots in primary injury, and the clinical significance and survival advantage for patients may be small. Therefore, in recent decades, most experimental studies have focused on the mechanism of secondary injury caused by ICH in order to find new therapeutic targets for ICH, but have failed to achieve good results.

[0003] Ferroptosis is a newly defined form of programmed cell death that mediates a variety of pathological settings characterized by the presence of iron overload, retention and generation of lipid reactive oxygen species (L-ROS) accumulation, and numerous other transcriptional alterations. Recent reports have highlighted the effects of ferroptosis inhibition on short- and long-term neuroprotection during intracerebral hemorrhage. Although some progress has been made in the study of ferroptosis, many gaps remain.

[0004] Therefore, there is an urgent need to provide a raw material drug for treating cerebral hemorrhage and inhibiting ferroptosis. Summary of the invention

[0005] The purpose of the present invention is to provide an application of licorice chalcone D in the preparation of a drug for treating spontaneous cerebral hemorrhage, so as to solve the problems existing in the above-mentioned prior art. Licorice chalcone D can alleviate the symptoms of spontaneous cerebral hemorrhage and significantly improve the secondary damage of spontaneous cerebral hemorrhage.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of licorice chalcone D in preparing a medicine for treating and / or preventing spontaneous cerebral hemorrhage.

[0008] The invention provides an application of licorice chalcone D in preparing a medicine for improving secondary injury of spontaneous cerebral hemorrhage.

[0009] Preferably, the secondary injury is ischemic injury and / or hypoxic injury of nerve cells.

[0010] Preferably, the drug for treating and / or preventing spontaneous cerebral hemorrhage or the drug for improving secondary damage of cerebral hemorrhage further comprises a pharmaceutically acceptable excipient.

[0011] Preferably, the dosage form of the drug for treating and / or preventing spontaneous cerebral hemorrhage or the drug for improving secondary damage of cerebral hemorrhage is any one of capsules, tablets, pills, liquids, powders, granules and injections.

[0012] Preferably, the dosage of licoricechalcone D administered to the subject is 5-20 mg / kg.

[0013] The present invention also provides an application of licorice chalcone D in preparing a nerve cell ferroptosis inhibitor.

[0014] Preferably, the inhibitor of neuronal cell ferroptosis is an inhibitor for treating neuronal cell ferroptosis caused by spontaneous cerebral hemorrhage.

[0015] The present invention also provides an application of licorice chalcone D in the preparation of cyclooxygenase inhibitors.

[0016] Preferably, the licorice chalcone D inhibits cyclooxygenase by reducing the expression level of the cyclooxygenase and / or binding to the specific binding site of the cyclooxygenase.

[0017] The present invention discloses the following technical effects:

[0018] The present invention provides the use of licorice chalcone D in the preparation of relevant drugs for treating and improving the prognosis of spontaneous cerebral hemorrhage. The results of rat experiments show that licorice chalcone D can reduce the hematoma of cerebral hemorrhage, reduce the death of nerve cells after hemorrhage and reduce the level of inflammatory factors; the results of cell experiments show that licorice chalcone D can be used as a ferroptosis inhibitor and COX2 inhibitor to improve the ischemic hypoxic injury of nerve cells after spontaneous cerebral hemorrhage, and the effect is better than the commonly used ferroptosis inhibitor Fer-1. The present invention reports for the first time that licorice chalcone D is used to treat spontaneous cerebral hemorrhage and improve secondary brain damage, providing a new solution for the treatment of spontaneous cerebral hemorrhage, and has good prospects for clinical drug application. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 This is the observation picture of brain tissue in the ICH animal model after treatment with licorice chalcone D;

[0021] Figure 2 The TUNEL staining observation and fluorescence intensity statistical chart in the ICH animal model after treatment with licorice chalcone D; * indicates P < 0.05, ** indicates P < 0.01;

[0022] Figure 3 The electrophoresis results of neuronal cell death proteins P53, BCL2 and BAX in the ICH animal model after treatment with licorice chalcone D;

[0023] Figure 4 This is a statistical chart of the expression of inflammatory factor IL-6 in the ICH animal model after treatment with licorice chalcone D; ** indicates P < 0.01;

[0024] Figure 5 Statistical graph of catalase (CAT) activity in ICH animal model after treatment with licorice chalcone D; * indicates P < 0.05, ** indicates P < 0.01;

[0025] Figure 6 Statistical graph of catalase (CAT) activity in the ICH model of PC12 cells after treatment with licorice chalcone D; ** indicates P < 0.01;

[0026] Figure 7 This is a statistical chart of the expression of inflammatory factor IL-6 in the ICH model of PC12 cells after treatment with licorice chalcone D; ** indicates P < 0.01;

[0027] Figure 8 Statistical graph of catalase (CAT) activity in SH-SY5Y cell ICH model after treatment with licorice chalcone D; ** indicates P < 0.01;

[0028] Fig. 9 Statistical diagram of the expression of inflammatory factor IL-6 in the SH-SY5Y cell ICH model after treatment with licorice chalcone D; * indicates P < 0.05, ** indicates P < 0.01;

[0029] Fig.10 This is the prediction result diagram of the binding site between Licorice Chalcone D and protein COX2;

[0030] Fig.11 This is the prediction result diagram of the binding site and binding mode of Licorice Chalcone D to protein COX2;

[0031] Fig.12 The immunohistochemical results of COX2 protein and the statistical results of cells expressing COX2 protein in the ICH animal model after treatment with licorice chalcone D; * indicates P < 0.05, ** indicates P < 0.01;

[0032] Fig.13 Statistical diagram of the ratio of reduced glutathione to oxidized glutathione in the ICH animal model after treatment with licorice chalcone D; * indicates P < 0.05, ** indicates P < 0.01;

[0033] Fig.14 Statistical diagram of the ratio of reduced glutathione to oxidized glutathione in the ICH model of PC12 cells after treatment with licorice chalcone D; ** indicates P < 0.01;

[0034] Fig.15 Statistical diagram of the ratio of reduced glutathione to oxidized glutathione in the SH-SY5Y cell ICH model after treatment with licorice chalcone D; ** indicates P < 0.01;

[0035] Fig.16 The electrophoresis results of GPX4 protein and COX2 protein in the ICH model of PC12 cells after treatment with licorice chalcone D;

[0036] Fig.17 This is the electrophoresis result of GPX4 protein and COX2 protein in SH-SY5Y cell ICH model after treatment with licorice chalcone D. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Cyclooxygenases (COX) are a group of heme-containing enzymes that catalyze the rate-limiting conversion of arachidonic acid (AA) to biologically active prostaglandins (PGs) by adding molecular oxygen. There are two major isoforms of COX enzymes (i.e., COX1 and COX2), which differ in their structure. In the human brain, COX1 is preferentially expressed in microglia as a clean enzyme constitutively expressed in all tissues, while COX2 is usually expressed in cortical neurons at low basal levels under normal conditions, but is induced by stimuli such as fever, infection, inflammation, hypertension, and growth factors and excessive neuronal cell activity, and is widely considered to be the main proinflammatory mediator. Acute neurological injury (including encephalitis and ischemia-reperfusion, etc.) can rapidly and strongly induce COX2 in the brain.

[0043] Since COX2 is specifically highly expressed in acute neurological injury and is involved in hypoxia-induced responses, small molecule compounds targeting this molecule may be able to inhibit secondary damage after cerebral hemorrhage and open up a new strategy for the treatment of spontaneous cerebral hemorrhage.

[0044] Licochalcone D of the present invention has a CAS number of 144506-15-0 and a molecular formula of C 21 H 22 O 5 , the chemical structure is shown below:

[0045]

[0046] The Fer-1 of the present invention is Ferrostatin-1, with CAS No. 347174-05-4, and is a specific ferroptosis inhibitor.

[0047] The cell lines of the present invention are all used to simulate neuronal cell models, and the specific information is as follows:

[0048] PC12 cell line: PC12 cell line was purchased from ATCC, which is a rat pheochromocytoma cell line.

[0049] SH-SY5Y cell line: purchased from ATCC, which is a human neuroblastoma cell line.

[0050] The PC12 and SH-SY5Y cell lines were cultured in DMEM (InVitrogen) containing 10% calf serum and placed at 37°C with 5% CO. 2 The cells were routinely cultured in an incubator without drugs for two weeks before the experiment.

[0051] The method of the present invention for establishing an autologous cerebral hemorrhage rat model by inducing autologous blood has been disclosed in the literature Wang Yuelong et al. "Simvastatin accelerates hematoma resolution after intracerebral hemorrhage in a PPARγ-dependent manner." Neuropharmacology vol. 128 (2018): 244-254.

[0052] Example 1

[0053] 1. Drug intervention of licoricechalcone D in spontaneous intracerebral hemorrhage (ICH) rat model

[0054] The rat model of autologous cerebral hemorrhage was established by induction of autologous blood. Licorice chalcone D was injected into the rat tail vein 2 hours before modeling for pretreatment. The doses were 5 mg / kg and 20 mg / kg, respectively. HE staining was used to observe the hematoma of cerebral hemorrhage 24 hours after injection of licorice chalcone D. Microscopic observation results showed that a large area of ​​nerve cells disappeared and became sparse in the ICH hematoma, with interstitial edema and obvious red blood cell infiltration. With the intervention of licorice chalcone D, the ICH hematoma area was observed to decrease ( Figure 1 ).

[0055] The level of neuronal cell death was observed by TUNEL staining, and the effect of licorice chalcone D on secondary brain injury was further observed. The results showed that the TUNEL fluorescence quantitative level decreased with the intervention of licorice chalcone D ( Figure 2). Further observation of brain tissue at the protein level showed that the expression levels of P53, BAX, and Bcl-2 proteins gradually decreased with the intervention of licorice chalcone D, which once again proved that licorice chalcone D can alleviate ICH cell death ( Figure 3 ). The expression level of inflammatory factor IL-6 mRNA in brain tissue was detected. The results showed that licorice chalcone D can reduce the production of inflammatory factor IL-6 ( Figure 4 ) and detected the quantitative expression of catalase (CAT) and found that licorice chalcone D can improve the oxidative stress level after cerebral hemorrhage ( Figure 5 ).

[0056] 2. Licorice chalcone D improves oxidative stress and inflammatory factor levels in neuronal cells after ICH

[0057] By CoCl 2 (400μg / mL, MCE Co., China, 7791-13-1) was used to induce ischemia-hypoxia model in PC12 and SH-SY5Y cells. Licorice chalcone D and Fer-1 were used for 24 hours of treatment, respectively, and the concentration of Fer-1 was 1μM; pretreatment was 2h; the concentration of Licorice chalcone D in PC12 cell treatment was 5μM, and the concentration of Licorice chalcone D in SH-SY5Y cell treatment was 10μM.

[0058] Detection of CAT catalase activity in cell models ( Figure 6 , Figure 8 ), and the expression level of IL-6 mRNA in the cells was detected, indicating that licorice chalcone D can reduce the production of inflammatory factor IL-6 ( Figure 7 , Fig. 9 ), all of which indicate that licorice chalcone D can improve the secondary injury of ICH.

[0059] 3. Licorice chalcone can act as a COX2 inhibitor to inhibit ferroptosis in ICH

[0060] The docking score of licorice chalcone D with rat COX2 was -8.36 kcal / mol. The smaller the docking score, the better the binding affinity of the ligand to the receptor. The predicted binding mode of licorice chalcone D with rat COX2 was elucidated. Licorice chalcone D formed a suitable spatial complementary relationship with the binding site of rat COX2. Conventional hydrogen bonds, carbon-hydrogen bonds, hydrophobic interactions and van der Waals (VDW) interactions were formed between licorice chalcone D and rat COX2. The oxygen atom of licorice chalcone D formed two conventional hydrogen bond interactions with Ser516 and Arg106 residues of rat COX2. The oxygen atom of licorice chalcone D formed three carbon-hydrogen bond interactions with Ser339 and Ser516 residues of rat COX2. In rat COX2, the oxygen atom of licorice chalcone D formed a pi-donor hydrogen bond interaction with the residue of Tyr341. The carbon atoms of Licoricealcone D form alkyl-alkyl hydrophobic interactions with residues Val335, Ile331, Leu345, Leu517, Met99, Val102, and Leu103 in rat COX2. Licoricealcone D forms amide-pi stacking hydrophobic interactions with residues Gly512 and Ala513 of rat COX2. Licoricealcone D forms pi-alkyl hydrophobic interactions with residues Leu338 and Ala513 in rat COX2. Licoricealcone D forms VDW interactions with residues Val509, Leu370, Met508, Phe504, Trp373, Phe367, Tyr371, and Tyr334 in rat COX2. These interactions mainly contribute to the binding ability of Licoricealcone D to rat COX2 ( Fig.10 and Fig.11 ).

[0061] According to the method in "1. Drug intervention of licorice chalcone D in spontaneous intracerebral hemorrhage (ICH) rat model", the autologous intracerebral hemorrhage rat model was established by autologous blood induction. Licorice chalcone D was injected into the tail vein of the rats 2 hours before modeling for pretreatment. The doses were 5 mg / kg and 20 mg / kg, respectively. The expression of COX2 was observed by immunohistochemistry. We found that with the intervention of licorice chalcone D, the expression of COX2 was observed to be reduced ( Fig.12 ). The ratio of reduced glutathione to oxidized glutathione (GSH / GSSG) was observed in brain tissue to observe the metabolic level of reduced glutathione ( Fig.13 ), which indicated that licorice chalcone D could inhibit COX2 expression in ICH animal models and improve ICH ferroptosis.

[0062] According to the method in “2. Licorice chalcone D as a method to improve the oxidative stress and inflammatory factor levels of neuronal cells after ICH”, CoCl 2Ischemia-hypoxia models were induced in PC12 and SH-SY5Y cells and treated. Common indicators of ferroptosis were detected in cell models, including the detection of GSH / GSSG ratio and the observation of reduced glutathione metabolism level ( Fig.14 , Fig.15 ), detection of COX2 and GPX4 ferroptosis regulatory proteins ( Fig.16 , Fig.17 ), and the experiments were performed using the ferroptosis inhibitor Fer-1 as a control, which plays a key role in ferroptosis after ICH.

[0063] The experimental results showed that in the animal model of spontaneous cerebral hemorrhage, the GSH / GSSG ratio was increased and the expression of COX2 was reduced. In the ischemic hypoxia injury after spontaneous cerebral hemorrhage, the GSH / GSSG ratio and the expression level of GPX4 protein decreased, and the COX2 protein level increased. With the intervention of licorice chalcone D and Fer-1, the GSH / GSSG ratio and the expression level of GPX4 rebounded, and the COX2 protein level decreased; and the pharmacological effect of licorice chalcone D was more obvious than that of Fer-1. The above results show that licorice chalcone D can participate in the process of neuronal cell ferroptosis in secondary brain injury of ICH as an inhibitor of ferroptosis.

[0064] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A use of licorice chalcone D in the preparation of a medicament for treating and / or preventing spontaneous cerebral hemorrhage.

2. Use of licorice chalcone D in the preparation of a drug for improving secondary damage of spontaneous cerebral hemorrhage.

3. The use according to claim 2, characterized in that The secondary injury is ischemic injury and / or hypoxic injury.

4. The use according to any one of claims 1 to 3, characterized in that: The drug for treating and / or preventing spontaneous cerebral hemorrhage or the drug for improving secondary damage of cerebral hemorrhage also includes pharmaceutically acceptable excipients.

5. The use according to any one of claims 1 to 3, characterized in that: The dosage form of the drug for treating and / or preventing spontaneous cerebral hemorrhage or the drug for improving secondary damage of cerebral hemorrhage is any one of capsules, tablets, pills, liquids, powders, granules and injections.

6. The use according to any one of claims 1 to 3, characterized in that: The dosage of Licoricechalcone D administered to the subject is 5-20 mg / kg.

7. An application of licorice chalcone D in the preparation of a neuronal cell ferroptosis inhibitor.

8. The use according to claim 6, characterized in that The neuronal cell ferroptosis inhibitor is an inhibitor for treating neuronal cell ferroptosis caused by spontaneous cerebral hemorrhage.

9. Use of licorice chalcone D in the preparation of cyclooxygenase inhibitors.

10. The use according to claim 9, characterized in that The licorice chalcone D inhibits cyclooxygenase by reducing the expression amount of the cyclooxygenase and / or binding to the specific binding site of the cyclooxygenase.

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