New application of gastrodin

Abstract

The invention provides novel application of gastrodin, and belongs to the technical field of biological medicine. By constructing an MCAO model of cerebral arterial thrombosis and detecting the influence of GAS on MCAO mouse and OGD-induced microglial cell ferroptosis, it is found that GAS alleviates the ferroptosis of cerebral arterial thrombosis, it is found through molecular docking and cell thermal displacement experiments that GAS is specifically bound with Alkbh5, the stability of protein is improved, nuclear translocation of Alkbh5 is promoted, and the GAS can be used for treating cerebral arterial thrombosis. Therefore, the methylation level of N6-methyladenosine (m6A) of a glutamic acid-cysteine ligase modified subunit (Gclm) is reduced, the expression of the Gclm is increased, the synthesis of glutathione is enhanced, and finally ferroptosis of microglial cells is inhibited. Therefore, the gastrodin can be used for preparing the medicine for treating the ferroptosis of the cerebral arterial thrombosis, and a new natural medicine source is provided for preventing and treating the ferroptosis of the cerebral arterial thrombosis.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a new use of gastrodin. Background Art

[0002] Stroke, also known as apoplexy or cerebrovascular accident, is an acute cerebrovascular disease. It is a group of diseases that cause brain tissue damage due to sudden rupture of brain blood vessels or blood vessel obstruction that prevents blood from flowing into the brain. The pathophysiology of ischemic brain damage involves the activation of a series of harmful signal cascades. Insufficient oxygen and glucose supply to brain tissue will lead to the immediate failure of energy-dependent ion pumps and channels, resulting in the release of excitatory neurotransmitters and inducing neuronal death. In the adult brain, neurons damaged by ischemia have limited or no ability to regenerate axons, resulting in long-term sensory, motor or cognitive disability. At present, recombinant tissue plasminogen activator (rt-PA) is still the only drug treatment for ischemic stroke patients, and surgical recanalization can also be performed in a few cases. However, although rapid reperfusion is necessary to restore brain metabolic activity, it is also accompanied by other related risks such as reperfusion injury and cerebral hemorrhage. Therefore, the development of new drugs to treat ischemic stroke and protect brain cells from ischemia and reperfusion injury is an important issue that needs to be solved urgently.

[0003] Gastrodin (C 13 H 18 GAS (GAS) is an effective monomer of the traditional Chinese medicinal material Gastrodia elata, and its chemical name is 4-hydroxymethylphenyl-β-D-pyrrolidone glucoside, also known as gastrodin. It has been found that gastrodin is an effective antioxidant and free radical scavenger, with anti-inflammatory, antioxidant, and free radical scavenging effects, and can effectively protect nerve cells from damage caused by oxidative stress. Our previous studies have found that GAS can effectively inhibit microglia-mediated apoptosis, inflammation, and oxidative stress after ischemic brain injury, but its specific mechanism of action on ferroptosis of neurons and microglia after stroke is still unclear. Therefore, revealing the new mechanism by which gastrodin regulates ferroptosis to exert neuroprotective effects is of great significance for the development of new drugs for ischemic stroke. Summary of the invention

[0004] The purpose of the present invention is to provide a new use of gastrodin in view of the above-mentioned deficiencies of the prior art. The study found that gastrodin did not change the expression level of Alkbh5, but promoted its nuclear localization, indicating that gastrodin regulates the subcellular distribution of Alkbh5, thereby affecting the downstream signaling pathway and promoting the m of Gclm. 6 A demethylation leads to increased Gclm expression and enhanced glutathione (GSH) synthesis, ultimately leading to the inhibition of microglial ferroptosis.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The first object of the present invention is to provide the use of gastrodin and pharmaceutically acceptable salts thereof in the preparation of ferroptosis inhibitors.

[0007] The second object of the present invention is to provide the use of gastrodin and pharmaceutically acceptable salts thereof in the preparation of ferroptosis drugs for treating ischemic stroke.

[0008] Furthermore, the gastrodin specifically binds to the protein Alkbh5.

[0009] Furthermore, the gastrodin promotes the nuclear translocation of the protein Alkbh5 from the cytoplasm to the nucleus, thereby reducing the N6-methyladenosine (m 6 A) Methylation levels.

[0010] Furthermore, the gastrodin can inhibit OGD-induced microglial ferroptosis.

[0011] The third object of the present invention is to provide a ferroptosis inhibitor, the active ingredient of which includes gastrodin.

[0012] Furthermore, pharmaceutically acceptable excipients are also included.

[0013] The fourth object of the present invention is to provide a composition, the active ingredient of which includes gastrodin.

[0014] Furthermore, pharmaceutically acceptable excipients are also included.

[0015] The fifth object of the present invention is to provide a pharmaceutical composition for treating ferroptosis in ischemic stroke, the active ingredient of which includes gastrodin.

[0016] Furthermore, the effective dose of gastrodin is 100 mg / kg.

[0017] Furthermore, pharmaceutically acceptable excipients are also included.

[0018] The pharmaceutically acceptable salts of the above compounds refer to compounds having the same parent core structure as the compound itself, which can generate molecules with the same or similar activity as the original compound through hydrolysis in the body, achieving the same or similar therapeutic effects. Pharmaceutically acceptable salts of compounds can be synthesized from parent compounds by conventional chemical methods. Generally speaking, these salts can be prepared by reacting the free base and acid of the compound in water or an organic solvent or a mixture of the two; usually, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile are used.

[0019] Acid addition salts can be made by various acids (inorganic and organic acids). Examples of acid addition salts include salts made from acids, wherein the "inorganic acid" includes, but is not limited to, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, pyrosulfuric acid, phosphoric acid or nitric acid. The "organic acid" includes, but is not limited to, formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid, undecanoic acid, lauric acid, benzoic acid, phenylacetic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, naphthalenedisulfonic acid, nicotinic acid, pamoic acid , pectinic acid, persulfuric acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxyethanesulfonic acid, itaconic acid, sulfamic acid, trifluoromethanesulfonic acid, dodecylsulfuric acid, camphorsulfonic acid, citric acid, tartaric acid, stearic acid, lactic acid, oxalic acid, malonic acid, succinic acid, malic acid, adipic acid, alginic acid, maleic acid, fumaric acid, D-gluconic acid, mandelic acid, ascorbic acid, glucoheptanoic acid, glycerophosphoric acid, aspartic acid, sulfosalicylic acid, hemisulfuric acid or thiocyanic acid.

[0020] The term "effective amount" or "therapeutically effective amount" refers to an amount of the compound of the present invention sufficient to achieve the intended application (including but not limited to the treatment of diseases as defined below). The therapeutically effective amount may vary depending on the intended application (in vitro or in vivo), or the subject and disease condition to be treated, such as the weight and age of the subject, the severity of the disease condition, and the mode of administration, which can be easily determined by a person of ordinary skill in the art. The specific dosage will vary depending on the following factors: the specific compound selected, the dosage regimen relied on, whether it is administered in combination with other compounds, the timing of administration, the tissue to which it is administered, and the physical delivery system carried.

[0021] The term "excipient" refers to a pharmaceutically acceptable inert ingredient. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the drug, or promote the effective absorption of the active ingredient after the subject receives the composition. The pharmaceutical excipient can be an inert filler, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. The pharmaceutical excipient can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0022] The pharmaceutical composition of the present invention can be prepared according to the disclosed content using any method known to those skilled in the art, such as conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or freeze-drying processes.

[0023] The pharmaceutical composition of the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or delayed release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules, and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry preparations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories and lozenges.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention found that gastrodin specifically binds to the protein Alkbh5, increases its protein stability, and promotes the nuclear translocation of the protein Alkbh5.

[0026] (2) The present invention demonstrates that nuclear Alkbh5 reduces the N6-methyladenosine (m6-methyladenosine) of the glutamate-cysteine ​​ligase modifying subunit (Gclm). 6 A) Methylation level, leading to increased Gclm expression and enhanced glutathione (GSH) synthesis, ultimately leading to the inhibition of microglial ferroptosis.

[0027] (3) The present invention found that gastrodin can improve ferroptosis in ischemic stroke models. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the molecular docking diagram of gastrodin and Alkbh5;

[0029] Figure 2 This is a gel image of protein Alkbh5 detected by Western blots after cells were treated with CETSA technology;

[0030] Figure 3This is a statistical graph of protein levels detected by Western blots after cells were treated with CETSA technology;

[0031] Figure 4 The figure shows the results of Western blotting after microglia were treated with GAS at concentrations of 0.17mM (GAS-L) and 0.34mM (GAS-H) and the nucleus and cytoplasm were separated;

[0032] Figure 5 The results of the protein Alkbh5 levels in the nucleus and cytoplasm of microglia after treatment with gastrodin, where a is the nucleus; b is the cytoplasm;

[0033] Figure 6 This is the RT-qPCR result of microglia after being treated with OGD and GAS;

[0034] Figure 7 This is the result of MeRIP-qPCR of microglia after being treated with OGD and GAS;

[0035] Figure 8 This is a diagram of the laser speckle imaging system of the MCAO model;

[0036] Fig. 9 Transmission electron microscopy images of mitochondria in the MCAO group showed mitochondrial shrinkage, increased membrane density, and reduced cristae;

[0037] Fig.10 This is the MDA measurement result graph one day after surgery;

[0038] Fig.11 This is the MDA measurement result 3 days after surgery;

[0039] Fig.12 This is the result of GSH measurement one day after surgery;

[0040] Fig.13 This is the result of GSH measurement 3 days after surgery;

[0041] Fig.14 This is a graph showing the results of Western blotting to detect the levels of ferroptosis-related factors. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention clearer, embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0043] Example 1

[0044] Study on the specific binding of gastrodin to Alkbh5.

[0045] Molecular docking was performed using AutoDock software, and it was found that gastrodin specifically binds to Alkbh5. Its binding energy Affinity = -7.2 kcal / mol. When Affinity > -4 kcal / mol, the binding force is extremely weak or considered non-binding; when -7 kcal / mol < Affinity ≤ -4 kcal / mol, the binding force is medium; when Affinity ≤ -7 kcal / mol, the binding force is strong; the lower the binding energy, the stronger the binding force, as Figure 1 shown.

[0046] Example 2

[0047] Study on the nuclear translocation of protein Alkbh5 promoted by gastrodin.

[0048] Cell culture and grouping treatment: The OGD-induced BV2 microglial cells were divided into: 0.1% DMSO control group (DMSO), 0.17 μM GAS treatment group (GAS-L), and 0.34 μM GAS treatment group (GAS-H). DMSO or GAS was pre-treated for 1 h, and then the medium was changed to serum-free and sugar-free medium, and cultured in an anaerobic chamber at 37 °C, 5% CO2, and 95% N2 for 2 h to construct an OGD model.

[0049] Cell thermal shift assay (CETSA): A method for studying drug targets based on the principle of ligand-induced changes in the thermodynamic stability of target proteins.

[0050] Specifically, the cells were heated at different temperatures. The higher-order structure of non-drug-bound proteins would change at this temperature, resulting in heat denaturation and loss of protein molecular function; the structure of the drug-bound target protein becomes stable and is not easily denatured at the same temperature. Specific method: The cells of the control group (DMSO) and the drug-administered group (GAS) were heated to different gradient temperatures, namely 60 °C, 62 °C, 64 °C, 66 °C, and 68 °C. After cooling, the cells were lysed with cell lysis buffer, and then the samples were ground and centrifuged to remove insoluble precipitates. The supernatant of the soluble protein was taken, and protein loading buffer was added and reacted at 95 °C for 5 min. Finally, Western blotting was performed for detection.

[0051] As Figure 2 and Figure 3As shown, after the cells were treated with CETSA technology, Western blotting analysis was performed. As the temperature increased, the protein degradation rate in the GAS-treated group slowed down, indicating that GAS specifically bound to Alkbh5 and enhanced its protein stability.

[0052] Nuclear and cytoplasmic separation: Use Biyuntian's nuclear protein and cytoplasmic protein extraction kit to separate the nuclear and cytoplasmic of cells. Specifically: Collect the cell pellet that has grown in the T75 culture flask, add 200ul of cytoplasmic protein extraction reagent A with PMSF, vortex at the highest speed for 5 seconds, and ice bath for 10-15min. Add 10μl of cytoplasmic protein extraction reagent B. Vortex at the highest speed for 5 seconds, and ice bath for 1min. Vortex at the highest speed for 5 seconds, and centrifuge at 12,000-16,000g at 4℃ for 5min. Immediately aspirate the supernatant into a pre-cooled plastic tube, which is the extracted cytoplasmic protein. For the precipitate, completely aspirate the remaining supernatant and add 50μl of nuclear protein extraction reagent with PMSF. Vortex at the highest speed for 15-30 seconds to completely suspend and disperse the cell pellet. Then put it back in the ice bath, and vortex at high speed for 15-30 seconds every 1-2min for a total of 30min. Centrifuge at 12,000-16,000g for 10 min at 4℃. Immediately transfer the supernatant to a pre-cooled plastic tube, which is the extracted nuclear protein for Western blotting detection.

[0053] Western blotting experiment: Add lysis buffer mixture (containing 1×RIPA lysis buffer, protease inhibitors and phosphatase inhibitors) to the collected cells and tissue samples, place at 4℃ for 10min, centrifuge at 12000rpm for 15min, collect supernatant, and determine protein concentration according to BCA kit. Protein denaturation, electrophoresis, transfer, block with 5% skim milk for 2h, primary antibody at 4℃ overnight, wash 3 times with TBST, 15min each time, incubate with corresponding secondary antibody at room temperature for 1h, wash 3 times with TBST, 15min each time, add chemiluminescent (ECL) solution on the membrane, detect bands with chemiluminescent imager, analyze gray value of each band with ImageJ software, and repeat the experiment 3 times.

[0054] like Figure 4 and Figure 5 As shown, after microglia were treated with GAS at concentrations of 0.17 mM (GAS-L) and 0.34 mM (GAS-H), Western blotting results after nucleus-cytoplasm separation showed that compared with the control DMSO group, the expression of Alkbh5 in the nucleus increased and the expression of Alkbh5 in the cytoplasm decreased after GAS treatment.

[0055] Example 3

[0056] Gastrodin reduces the N6-methyladenosine (m 6 A) Methylation levels.

[0057] RT-qPCR experiment: Total mRNA was extracted from cells using TRIpure Reagent and reverse transcribed into cDNA using a reverse transcription kit. RT-qPCR system was prepared according to the kit instructions for amplification. mRNA levels were measured using 2 -ΔΔCT The primers for glutamate-cysteine ​​ligase modification subunit (Gclm) were 5'-CAGTTGACATGGCATGCTC-3' and 5'-ACTCCATCTTCAATCGGAGG-3'. The primers for β-actin were 5'-GCTATGTTGCTCTAGACTTCG-3 and 5'-GGATTCCATACCCAAGAAGG-3'.

[0058] like Figure 6 As shown, after microglia were treated with OGD and GAS, RT-qPCR results showed that compared with the Control group, the expression of Gclm in the OGD group decreased, and the expression of Gclm increased after GAS intervention.

[0059] MeRIP-qPCR experiment: using methylated RNA immunoprecipitation kit to evaluate Gclm m 6 A. Methylation level. Total RNA was extracted from BV-2 cells and fragmented into approximately 300 nt fragments. 50 μL RNA was mixed with 4 μg m 6 Antibody A was incubated at 4°C for 4 hours, and then protein A / G magnetic beads were added and mixed and incubated for 1 hour at 4°C. The primers used were 5'-GGAACCTGCTCAACTG-3' and 5'-GGTGCATTCCAAAACATCTG-3'.

[0060] like Figure 7 As shown in the figure, after microglia were treated with OGD and GAS, the MeRIP-qPCR results showed that compared with the Control group, the m value of Gclm in the OGD group was 6 A level increased significantly, and m of Gclm increased significantly after GAS intervention. 6 A levels decreased.

[0061] Example 4

[0062] Study on the inhibition of ferroptosis by gastrodin in ischemic stroke.

[0063] MCAO model construction: C57BL / 6 male mice, weighing 20±2 g, were purchased from Henan Scobes Biotechnology Co., Ltd. All animal experiments were approved by the Animal Research Ethics Committee of Kunming Medical University (approval number: kmmu20231361). According to the description of previous studies, the middle cerebral artery occlusion (MCAO) technique was used to simulate the ischemic stroke model. In brief, mice were anesthetized with 4% isoflurane, and the common carotid artery and internal carotid artery were exposed. Then the nylon monofilament line was carefully inserted into the internal carotid artery and advanced to occlude the middle cerebral artery. The sham operation group followed the same steps except that the monofilament was not inserted. The laser speckle contrast imaging system was then used to detect cerebral blood flow and evaluate the success rate of the MCAO model.

[0064] Drug treatment: The GAS group was intraperitoneally injected with 100 mg / kg GAS 2 hours before surgery, 6 hours and 12 hours after surgery. The brain was removed under isoflurane inhalation anesthesia 1 day and 3 days after surgery, and the ischemic penumbra tissue was collected for subsequent testing.

[0065] like Figure 8 As shown in the figure, the laser speckle contrast imaging system showed that the blood flow on the ischemic side of the MCAO group was significantly reduced compared with the sham operation group, indicating that the MCAO model was successfully established.

[0066] like Fig. 9 As shown, transmission electron microscopy showed that the mitochondria in the MCAO group became smaller and condensed, the mitochondrial cristae decreased, and the membrane density increased. The mitochondrial morphology was improved after GAS treatment.

[0067] Malondialdehyde (MDA) and glutathione (GSH) level detection experiment: Brain tissue was collected, and the MDA level was measured using the MDA kit of Beyotime. GSH was detected using the glutathione detection kit of Solebao. The specific steps refer to the kit instructions.

[0068] The results of MDA determination were as follows Figures 10-11 The GSH assay results are shown in Figures 12-13 As shown in the figure, at 1 day and 3 days, the MDA level in the GAS group was lower than that in the MCAO group, and the GSH level was significantly higher than that in the MCAO group.

[0069] Western blotting experiment: The steps were as described above. Changes in ferroptosis-related factors acyl-CoA synthase (Acsl4), prostaglandin endoperoxide synthase 2 (Ptgs2), solute carrier family 7 member 11 (Slc7a11), spermidine / spermine N1-acetyltransferase 1 (Sat1), and glutathione peroxidase (Gpx4) were detected.

[0070] Western blotting results Fig.14As shown, the protein expression levels of Acsl4, Ptgs2 and Sat1 in the GAS group were significantly lower than those in the MCAO group at 1 day and 3 days, while the levels of Gpx4 and Slc7a11 were significantly increased compared with the MCAO group. This proves that gastrodin can inhibit ferroptosis in ischemic stroke.

[0071] For matters not mentioned above, the prior art applies.

[0072] Although some specific embodiments of the present invention have been described in detail through examples, those skilled in the art should understand that the above examples are for illustration only and are not intended to limit the scope of the present invention. Those skilled in the art to which the present invention belongs may make modifications or supplements to the described specific embodiments or replace them in a similar manner, but they will not deviate from the direction of the present invention or exceed the scope defined by the attached claims. Those skilled in the art should understand that any modifications, equivalent substitutions, improvements, etc. made to the above implementations based on the technical essence of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of gastrodin and its pharmaceutically acceptable salts in the preparation of ferroptosis inhibitors.

2. Use of gastrodin and its pharmaceutically acceptable salts in the preparation of ferroptosis drugs for the treatment of ischemic stroke.

3. The use according to claim 1 or 2, characterized in that The gastrodin specifically binds to the protein Alkbh5.

4. The use according to claim 1 or 2, characterized in that The gastrodin promotes the nuclear translocation of the protein Alkbh5 from the cytoplasm to the cell nucleus, thereby reducing the N6-methyladenosine m 6 The methylation level of A.

5. The use according to claim 1 or 2, characterized in that The gastrodin can inhibit OGD-induced microglial ferroptosis.

6. A ferroptosis inhibitor according to claim 1, characterized in that Its active ingredients include gastrodin.

7. The ferroptosis inhibitor according to claim 6, characterized in that Pharmaceutically acceptable excipients are also included.

8. A pharmaceutical composition for treating ferroptosis in ischemic stroke, characterized in that: Its active ingredients include gastrodin.

9. The pharmaceutical composition according to claim 8, characterized in that The effective dose of gastrodin is 100 mg / kg.

10. The pharmaceutical composition according to claim 8, characterized in that Pharmaceutically acceptable excipients are also included.

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