Application of glyyrrhizin chalcone D in preparation of medicine for treating spontaneous cerebral hemorrhage

Abstract

The application discloses application of glycyrrhiza chalcone D in preparation of a drug for treating spontaneous cerebral hemorrhage and belongs to the technical field of biological medicine. The application provides application of glycyrrhiza chalcone D in preparation of a drug for treating and improving prognosis of spontaneous cerebral hemorrhage. Rat experiment results show that glycyrrhiza chalcone D can reduce hematoma of cerebral hemorrhage, reduce death of nerve cells after hemorrhage and reduce levels of inflammatory factors; cell experiment results show that glycyrrhiza chalcone D can be used as an inhibitor of ferroptosis and a COX2 inhibitor, improve ischemia and hypoxia injury of nerve cells after spontaneous cerebral hemorrhage and has a better effect than a commonly used ferroptosis inhibitor Fer-1. The application firstly reports that glycyrrhiza chalcone D is used for treating spontaneous cerebral hemorrhage and improving secondary brain injury, provides a new scheme for treatment of spontaneous cerebral hemorrhage and has a good clinical drug application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of glycyrrhizin D in the preparation of drugs for treating spontaneous intracerebral hemorrhage. Background Technology

[0002] Spontaneous intracerebral hemorrhage (ICH) accounts for 10-15% of all strokes worldwide, with significant morbidity and mortality. It is estimated that the mortality rate within 30 days of the onset of ICH can reach as high as 50%. The sudden rupture of a cerebral blood vessel, forming a hematoma and causing mechanical damage to adjacent tissues, leads to a rapid increase in intracranial pressure, which is considered primary brain injury. Following ICH, the hematoma can cause a rapid and sustained increase in intracranial reactivity, including pro-inflammatory responses, oxidative stress, neuronal apoptosis, mitochondrial dysfunction, and blood-brain barrier (BBB) ​​disruption, resulting in secondary neurological impairment. However, in clinical practice, the main treatment for primary brain injury following ICH is early surgical resection of the hematoma. However, there is a possibility of failure in surgically removing the hematoma from the primary injury, which may have limited clinical significance and survival advantage for patients. Therefore, in recent decades, most experimental research has focused on the mechanisms of secondary injury caused by ICH in order to find new therapeutic targets for ICH, but without satisfactory results.

[0003] Ferroptosis is a newly defined form of programmed cell death that mediates a variety of pathological environments, characterized by iron overload, retention and generation of lipid reactive oxygen species (L-ROS), and numerous other transcriptional alterations. Recent reports have highlighted the short- and long-term neuroprotective effects of ferroptosis suppression during intracerebral hemorrhage. Although some progress has been made in ferroptosis research, 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 this invention is to provide the application of glycyrrhizin D in the preparation of drugs for treating spontaneous intracerebral hemorrhage, so as to solve the problems existing in the prior art. Glycyrrhizin D can alleviate the symptoms of spontaneous intracerebral hemorrhage and significantly improve the secondary damage of spontaneous intracerebral hemorrhage.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the use of glycyrrhizin D in the preparation of medicaments for the treatment and / or prevention of spontaneous intracerebral hemorrhage.

[0008] This invention provides the application of glycyrrhizin D in the preparation of a drug for improving secondary damage in spontaneous cerebral hemorrhage.

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

[0010] Preferably, the medicament for treating and / or preventing spontaneous intracerebral hemorrhage or the medicament for improving secondary damage from intracerebral hemorrhage further includes pharmaceutically acceptable excipients.

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

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

[0013] This invention also provides the application of glycyrrhizin D in the preparation of neuronal ferroptosis inhibitors.

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

[0015] This invention also provides the application of glycyrrhizin D in the preparation of cyclooxygenase inhibitors.

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

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

[0018] This invention provides the application of glycyrrhizin chalcone D in the preparation of drugs for the treatment and prognosis improvement of spontaneous intracerebral hemorrhage. Rat experiments showed that glycyrrhizin chalcone D can reduce hematoma in intracerebral hemorrhage, decrease post-hemorrhagic neuronal cell death, and lower inflammatory factor levels. Cell experiments showed that glycyrrhizin chalcone D can act as a ferroptosis inhibitor and COX2 inhibitor, improving ischemic and hypoxic damage to neuronal cells after spontaneous intracerebral hemorrhage, with better effects than the commonly used ferroptosis inhibitor Fer-1. This invention is the first to report the use of glycyrrhizin chalcone D in the treatment of spontaneous intracerebral hemorrhage and the improvement of secondary brain injury, providing a new treatment option for spontaneous intracerebral hemorrhage and showing promising clinical application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0020] Figure 1 Image showing brain tissue in an ICH animal model treated with glycyrrhizin D;

[0021] Figure 2 TUNEL staining and fluorescence intensity statistics in an ICH animal model treated with glycyrrhizin D; * indicates P<0.05, ** indicates P<0.01;

[0022] Figure 3 Electrophoresis results of neuronal cell death proteins P53, BCL2, and BAX in an ICH animal model treated with glycyrrhizin D;

[0023] Figure 4 Statistical graph showing the expression level of inflammatory cytokine IL-6 in an ICH animal model after treatment with glycyrrhizin chalcone D; ** indicates P<0.01;

[0024] Figure 5 Statistical graph of catalase (CAT) activity in ICH animal models treated with glycyrrhizin D; * indicates P<0.05, ** indicates P<0.01;

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

[0026] Figure 7 Statistical graph of IL-6 expression level in PC12 cells in ICH model after treatment with glycyrrhizin chalcone D; ** indicates P<0.01;

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

[0028] Figure 9 Statistical graph showing the expression level of IL-6, an inflammatory factor, in SH-SY5Y cells in an ICH model after treatment with glycyrrhizin chalcone D; * indicates P<0.05, ** indicates P<0.01;

[0029] Figure 10 The diagram shows the predicted binding sites of glycyrrhizin D and protein COX2.

[0030] Figure 11 The diagram shows the predicted binding sites and binding modes of glycyrrhizin D with protein COX2.

[0031] Figure 12 Figure 1 shows the immunohistochemical results of COX2 protein and the statistical results of COX2 protein expression cells in an ICH animal model treated with glycyrrhizin D; * indicates P<0.05, ** indicates P<0.01;

[0032] Figure 13 Statistical graph showing the ratio of reduced glutathione to oxidized glutathione in an ICH animal model treated with glycyrrhizin chalcone D; * indicates P<0.05, ** indicates P<0.01;

[0033] Figure 14 Statistical graph of the ratio of reduced glutathione to oxidized glutathione in PC12 cells in an ICH model after treatment with glycyrrhizin chalcone D; ** indicates P<0.01;

[0034] Figure 15 Statistical graph showing the ratio of reduced glutathione to oxidized glutathione in SH-SY5Y cells in an ICH model after treatment with glycyrrhizin chalcone D; ** indicates P<0.01;

[0035] Figure 16 Electrophoresis results of GPX4 and COX2 proteins in PC12 cells ICH model after treatment with glycyrrhizin D;

[0036] Figure 17 Electrophoresis results of GPX4 and COX2 proteins in SH-SY5Y cells ICH model after treatment with glycyrrhizin D. Detailed Implementation

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

[0038] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0040] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0041] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are 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 (pg) by the addition of molecular oxygen. COX enzymes have two main isomers (COX1 and COX2) with different structures. In the human brain, COX1, as a cleansing enzyme expressed in all tissues, is preferentially expressed in microglia, while COX2 is normally expressed at a low basal level in cortical neurons. However, it is widely considered a major pro-inflammatory mediator induced by stimuli such as fever, infection, inflammation, hypertension, growth factors, and excessive neuronal activity. Acute neurological injury (including encephalitis and ischemia-reperfusion) 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 response, small molecule compounds targeting this molecule may be able to inhibit secondary damage caused by cerebral hemorrhage, opening up a new strategy for the treatment of spontaneous cerebral hemorrhage.

[0044] The present invention relates to Licochalcone D, CAS No. 144506-15-0, with the molecular formula C. 21 H 22 O5, its chemical structure is shown below:

[0045]

[0046] The Fer-1 of this invention is Ferrostatin-1, CAS number 347174-05-4, which is a specific inhibitor of ferroptosis.

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

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

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

[0050] The PC12 and SH-SY5Y cell lines were cultured as follows: The cells were seeded in DMEM (InVitrogen) medium containing 10% fetal bovine serum and cultured in a 37°C, 5% CO2 incubator. The cells were cultured without any drugs for two weeks before the experiment.

[0051] The method 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 glycyrrhizin D in a rat model of spontaneous intracerebral hemorrhage (ICH)

[0054] An autologous intracerebral hemorrhage (ICH) rat model was established by inducing autologous blood. Two hours prior to modeling, rats were pretreated with glycyrrhizin D via tail vein injection at doses of 5 mg / kg and 20 mg / kg. HE staining was used to observe the hematoma formation 24 hours after glycyrrhizin D injection. Microscopic observation revealed extensive loss and sparse neuronal cells, interstitial edema, and significant erythrocyte infiltration at the ICH hematoma site. With the intervention of glycyrrhizin D, a reduction in the ICH hematoma area was observed. Figure 1 ).

[0055] The level of neuronal cell death was observed by TUNEL staining, and the effect of glycyrrhizin chalcone D on secondary brain injury was further investigated. The results showed that the quantitative TUNEL fluorescence level decreased with the intervention of glycyrrhizin chalcone D. Figure 2Further 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 glycyrrhizin D, further demonstrating that glycyrrhizin D can alleviate ICH cell death. Figure 3 The expression level of the inflammatory cytokine IL-6 mRNA in brain tissue was detected, and the results showed that glycyrrhizin chalcone D can reduce the production of the inflammatory cytokine IL-6. Figure 4 ), and quantitative detection of catalase (CAT) revealed that glycyrrhizin D can improve oxidative stress levels after cerebral hemorrhage. Figure 5 ).

[0056] 2. Glycyrrhizin chalcone D improves oxidative stress and inflammatory factor levels in neurons after ICH.

[0057] Ischemia-hypoxia models were induced in PC12 and SH-SY5Y cells using CoCl2 (400 μg / mL, MCE Co., China, 7791-13-1). Patients were treated with glycyrrhizin D and Fer-1 for 24 hours (Fer-1 concentration 1 μM); pretreatment lasted 2 hours. The concentration of glycyrrhizin D in PC12 cells was 5 μM, and in SH-SY5Y cells it was 10 μM.

[0058] Detection of CAT catalase activity in cell models ( Figure 6 , Figure 8 The expression level of IL-6 mRNA in cells was detected, indicating that glycyrrhizin chalcone D can reduce the production of IL-6. Figure 7 , Figure 9 These findings indicate that glycyrrhizin D can improve secondary damage caused by ICH.

[0059] 3. Glycyrrhizin can act as a COX2 inhibitor to suppress ferroptosis in ICH.

[0060] The docking score of glycyrrhizin D with rat COX2 was -8.36 kcal / mol; a smaller docking score indicates a better affinity between the ligand and the receptor. The predicted binding mode of glycyrrhizin D with rat COX2 was elucidated. The binding sites of glycyrrhizin D and rat COX2 exhibited suitable spatial complementarity. Conventional hydrogen bonds, C-H bonds, hydrophobic interactions, and van der Waals (VDW) interactions formed between glycyrrhizin D and rat COX2. The oxygen atom of glycyrrhizin D formed two conventional hydrogen bonds with the Ser516 and Arg106 residues of rat COX2. The oxygen atom of glycyrrhizin D formed three C-H bonds with the Ser339 and Ser516 residues of rat COX2. In rat COX2, the oxygen atom of glycyrrhizin D formed a pi-donor hydrogen bond with the Tyr341 residue. The carbon atoms of glycyrrhizin D form alkyl-alkyl hydrophobic interactions with residues in rat COX2, specifically Val335, Ile331, Leu345, Leu517, Met99, Val102, and Leu103. Glycyrrhizin D also forms amide-pi-stacking hydrophobic interactions with Gly512 and Ala513 residues in rat COX2. Furthermore, glycyrrhizin D forms pi-alkyl hydrophobic interactions with Leu338 and Ala513 residues in rat COX2. Finally, glycyrrhizin D forms VDW interactions with residues in rat COX2, specifically Val509, Leu370, Met508, Phe504, Trp373, Phe367, Tyr371, and Tyr334. These interactions primarily contribute to the binding affinity of glycyrrhizin D to rat COX2. Figure 10 and Figure 11 ).

[0061] Following the method described in "1. Drug Intervention of Glycyrrhizin Chalcone D in a Rat Model of Spontaneous Intracerebral Hemorrhage (ICH)," an autologous intracerebral hemorrhage rat model was established by inducing autologous blood. Two hours before modeling, rats were pretreated with glycyrrhizin chalcone D injected via the tail vein at doses of 5 mg / kg and 20 mg / kg. Immunohistochemical observation of COX2 expression revealed a decrease in COX2 expression with the intervention of glycyrrhizin chalcone D. Figure 12 The ratio of reduced glutathione to oxidized glutathione (GSH / GSSG) was observed in brain tissue to assess the metabolic level of reduced glutathione. Figure 13 This indicates that glycyrrhizin D can inhibit COX2 expression in an ICH animal model and improve ferroptosis in ICH.

[0062] Following the method described in "2. Glycyrrhizin-Chalone D as an Aid to Improving Oxidative Stress and Inflammatory Factor Levels in Neuronal Cells After ICH", ischemia-hypoxia models were induced in PC12 and SH-SY5Y cells using CoCl2, followed by treatment. Commonly used indicators of ferroptosis were detected in the cell models, including the GSH / GSSG ratio to observe the level of reduced glutathione metabolism. Figure 14 , Figure 15 ), and detect COX2 and GPX4 ferroptosis regulatory proteins ( Figure 16 , Figure 17 The study used the ferroptosis inhibitor Fer-1 as a control, and these drugs play a key role in ferroptosis after cerebral hemorrhage.

[0063] Experimental results showed that in an animal model of spontaneous intracerebral hemorrhage (ICH), the GSH / GSSG ratio was reduced, and COX2 expression was decreased. In ischemic-hypoxic injury following ICH, the GSH / GSSG ratio and GPX4 protein expression levels decreased, while COX2 protein levels increased. With the intervention of glycyrrhizin D and Fer-1, the GSH / GSSG ratio and GPX4 expression levels rebounded, while COX2 protein levels decreased; moreover, glycyrrhizin D showed a more significant effect than Fer-1. These results indicate that glycyrrhizin D can act as an inhibitor of ferroptosis in the neuronal ferroptosis process secondary to ICH.

[0064] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of glycyrrhizin D in the preparation of drugs for the treatment and / or prevention of spontaneous intracerebral hemorrhage.

2. Application of glycyrrhizin D in the preparation of drugs to improve secondary damage in spontaneous intracerebral hemorrhage.

3. The application as described in claim 2, characterized in that, The secondary injury is ischemic injury and / or hypoxic injury.

4. The application as described in any one of claims 1-3, characterized in that, The medicine for treating and / or preventing spontaneous intracerebral hemorrhage or the medicine for improving secondary damage from spontaneous intracerebral hemorrhage also includes pharmaceutically acceptable excipients.

5. The application as described in any one of claims 1-3, characterized in that, The dosage form of the drug for treating and / or preventing spontaneous intracerebral hemorrhage or the drug for improving secondary damage caused by spontaneous intracerebral hemorrhage is any one of capsules, tablets, pills, liquids, powders, and granules.

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