Application of curcumin in preparation of neuroprotective drug for inhibiting ferroptosis

Abstract

The invention discloses an application of curcumin in preparation of a neuroprotective drug for inhibiting ferroptosis, and relates to the technical field of medical application of natural drugs, curcumin is used as an active ingredient for preventing or treating nerve cell injury related to ferroptosis, and comprises an H2O2-induced oxidative stress model and a hemin-induced hemorrhagic stroke model; the optimal window of curcumin in a specific concentration range is defined, efficient and safe neuroprotection is achieved, the high-concentration toxicity risk is avoided, the medication accuracy is improved, curcumin can play a multi-target role, the active oxygen level and lipid peroxidation products in cells can be reduced in a concentration-dependent mode, the glutathione content is up-regulated, and the neuroprotective effect is achieved. According to the curcumin, the expression of GPX4 and xCT key proteins is enhanced, so that the ferroptosis process is synergistically inhibited, the neuroprotection theory of the curcumin is enriched, and a solid foundation is laid for clinical application of the curcumin in diseases such as Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the medical application technology field of natural drugs, and particularly relates to application of curcumin in preparation of a neuroprotective drug for inhibiting ferroptosis. BACKGROUND

[0002] Neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease and Huntington's disease, are global health challenges, and their pathogenesis is complex and closely related to the progressive loss of neurons. In recent years, ferroptosis, as a new type of cell death mode dependent on iron and driven by lipid peroxide, has been confirmed to be widely involved in the pathological process of these diseases. The characteristics of ferroptosis include glutathione depletion, inhibition of glutathione peroxidase 4 (GPX4) activity and accumulation of lipid peroxide, and finally leading to membrane structure damage. At present, the treatment methods are mainly targeted at symptom relief, and there is a lack of specific ferroptosis inhibitors.

[0003] At present, the treatment methods for neurodegenerative diseases are limited, mainly focused on symptom relief, and there is a lack of specific drugs targeting ferroptosis. Curcumin, as a natural polyphenolic compound, has been known to have antioxidant and anti-inflammatory properties, but its mechanism of action in ferroptosis-related neuroprotection is unclear, especially lacking precise concentration-dependent studies. The SH-SY5Y cell model induced by H2O2 (hydrogen peroxide) is a commonly used tool to simulate oxidative stress damage, and the SH-SY5Y cell model induced by hemin (iron porphyrin) can simulate the pathology of hemorrhagic stroke, but the inhibitory effect of curcumin on ferroptosis in the above models and the optimal concentration window have not been clearly defined, which restricts its drug development and application. Therefore, the present application proposes a kind of curcumin in preparation of neuroprotective drug for inhibiting ferroptosis to solve the problems existing in the prior art. SUMMARY

[0004] In view of the above problems, the present application aims to provide a kind of curcumin in preparation of neuroprotective drug for inhibiting ferroptosis, to provide a more solid theoretical basis for the application of curcumin in the prevention and treatment of neurodegenerative diseases mediated by ferroptosis pathway.

[0005] In order to achieve the purpose of the present application, the present application realizes the following technical scheme:

[0006] The application of curcumin in the preparation of neuroprotective drugs for inhibiting ferroptosis, the curcumin is used as an active ingredient for preventing or treating nerve cell damage related to ferroptosis, wherein ferroptosis is cell death caused by iron-dependent lipid peroxide accumulation.

[0007] Further improvement lies in that the administration concentration of the curcumin is 1.25-5 μM.

[0008] Further improvement lies in that the application is based on a ferroptosis induction model, and the ferroptosis induction model includes an H2O2 induction or hemin induction cell ferroptosis model, wherein the concentration of H2O2 is 200-500 μM, and the concentration of hemin is 50-200 μM.

[0009] Further improvement lies in that the neuroprotection is achieved by inhibiting intracellular reactive oxygen species (ROS) accumulation and lipid peroxidation (LPO), wherein curcumin can reduce the levels of ROS and LPO in a concentration-dependent manner.

[0010] Further improvement lies in that the neuroprotection is achieved by increasing intracellular glutathione (GSH) content and regulating iron ion homeostasis, wherein curcumin can increase the level of GSH and reduce intracellular iron ion accumulation.

[0011] Further improvement lies in that the neuroprotection is achieved by up-regulating the protein expression of glutathione peroxidase 4 (GPX4) and cysteine or glutamate antiporter system (xCT).

[0012] Further improvement lies in that the neuroprotective drug is used for treating or preventing neurodegenerative diseases selected from the group consisting of Alzheimer's disease, Parkinson's disease and Huntington's disease.

[0013] Further improvement lies in that the drug contains curcumin as the only active ingredient or a pharmaceutical composition combined with a pharmaceutically acceptable carrier, and the pharmaceutically acceptable carrier includes a solvent, an excipient or a stabilizer.

[0014] Further improvement lies in that the hemin-induced model is specifically a hemorrhagic stroke cell model, wherein hemin induces ferroptosis by degrading to generate iron ions.

[0015] Further improvement lies in that the inhibition of ferroptosis includes regulating intracellular iron ion homeostasis, wherein curcumin affects the total iron and ferrous ion content.

[0016] Further improvement lies in that curcumin promotes ferroptosis at a concentration of 10 μM, and the neuroprotective drug is configured to avoid a concentration exceeding 5 μM.

[0017] The beneficial effects of the present application are: the present application realizes efficient and safe neuroprotection by determining the optimal window of curcumin in the concentration range of 1.25-5 muM, avoids the toxicity risk of high concentration (such as 10 muM), improves the drug precision, and curcumin can play a multi-target role: can reduce the intracellular reactive oxygen species level and lipid peroxidation product in a concentration-dependent manner, up-regulate the glutathione content, and enhance the expression of GPX4 and xCT key proteins, thereby synergistically inhibiting the ferroptosis process, in addition, the present application verifies the effect by using the SH-SY5Y cell model induced by H2O2, and verifies the bidirectional concentration effect and multi-target mechanism of curcumin in the hemin model, ensures the repeatability and reliability of the experiment, and provides a standardized platform for drug screening. These mechanisms not only enrich the neuroprotective theory of curcumin, but also lay a solid foundation for its clinical application in Alzheimer's disease and other diseases, and have significant conversion potential and market prospect. BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below.

[0019] Figure 1 is a chemical structure diagram of curcumin in the embodiments of the present application;

[0020] Figure 2 is a diagram showing the influence of different concentrations of H2O2 on cell survival rate in the embodiment 1 of the present application;

[0021] Figure 3 is a diagram showing the influence of different concentrations of H2O2 on cell morphology under an inverted microscope in the embodiment 2 of the present application;

[0022] Figure 4 is a diagram showing the influence of different concentrations of H2O2 on cell morphology under a fluorescence microscope in the embodiment 2 of the present application;

[0023] Figure 5 is a diagram showing the influence of different concentrations of curcumin on the level of reactive oxygen species in cells in the embodiment 3 of the present application;

[0024] Figure 6 is a diagram showing the influence of different concentrations of curcumin on the level of LPO in the embodiment 4 of the present application;

[0025] Figure 7 is a diagram showing the OD value of different concentrations of GSSG in the embodiment 5 of the present application;

[0026] Figure 8 is a diagram showing the OD value of different concentrations of total glutathione in the embodiment 5 of the present application;

[0027] Figure 9 is a schematic diagram of the influence of different concentrations of curcumin on GSH concentration in the embodiment 5 of the present application;

[0028] Figure 10 is a schematic diagram of the influence of curcumin on the expression of GPX4 and xCT in H2O2-induced SHSY-5Y cells in the embodiment 6 of the present application;

[0029] Figure 11 is a schematic diagram of the influence of hemin on the proliferation of SH-SY5Y cells detected by MTT method in the embodiment 8 of the present application;

[0030] Figure 12 is a schematic diagram of the influence of different concentrations of hemin on the morphology of SH-SY5Y cells observed under an inverted microscope in the embodiment 8 of the present application;

[0031] Figure 13 is a schematic diagram of the influence of Cur on the proliferation of SH-SY5Y cells under hemin damage detected by MTT method in the embodiment 9 of the present application;

[0032] Figure 14 is a schematic diagram of the influence of curcumin on the relative content change of reactive oxygen species (ROS) at different concentrations in the embodiment 9 of the present application;

[0033] Figure 15 is a schematic diagram of the influence of curcumin on hemin-induced GPX4 (glutathione peroxidase 4) protein oxidative damage in the embodiment 9 of the present application. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0035] Curcumin is one of the most effective nutritional antioxidants against free radical-related diseases. Curcumin appears as an orange-yellow crystalline powder, which is an acidic polyphenol substance. Its chemical structure is unique, consisting of an unsaturated aliphatic and aromatic group as the main chain. In terms of solubility, it is soluble in ethanol, propylene glycol, glacial acetic acid and alkaline solution, but not in water. It is yellow in acidic and neutral conditions, and reddish brown in alkaline conditions. It is sensitive to light, heat and Fe 3+ , but has good stability and is not easy to fade. Based on its unique color change characteristics, curcumin is often used as a natural coloring agent for meat products, and can play an important role as an acid-base indicator in chemical experiments.

[0036] Curcumin in this embodiment is obtained by commercial purchase, and its chemical structure is as shown in Figure 1 which is used as an active ingredient for preventing or treating nerve cell injury associated with ferroptosis, wherein ferroptosis is cell death caused by iron-dependent lipid peroxide accumulation.

[0037] Example 1

[0038] Referring to Figure 2 , this embodiment evaluates the effect of curcumin on the survival rate of H2O2-induced SH-SY5Y cells, and determines the optimal concentration window for the neuroprotective effect of curcumin, and the specific steps are as follows:

[0039] S1, cell culture and grouping: SH-SY5Y cells were seeded in a 96-well plate (3000 cells per well), and cultured using MEM / F12 complete medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody). The experimental groups include:

[0040] Blank control group (only medium)

[0041] Model group (300 μM of H2O2)

[0042] Curcumin protection group (1.25, 2.5, 5, 10 μM of curcumin pretreatment for 4-6 h, and then 300 μM of H2O2 was added)

[0043] Solvent control group (equal volume of curcumin solvent, such as DMSO, which does not affect cell activity at the final concentration).

[0044] S2, MTT detection: After 24 h of treatment, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation was continued at 37°C for 4 h. The culture medium in the wells was carefully aspirated, 150 μL of DMSO was added, and the plate was placed on a constant temperature microplate shaker at low speed for 10 min to dissolve the formazan crystals.

[0045] S3, data acquisition and analysis: The optical density (OD value) of each well was measured at 570 nm using a full-wave enzyme marker. Cell survival rate (%) = [(OD experimental group-OD blank group) / (OD control group-OD blank group)] x 100%. The data are expressed as mean ± standard deviation, and single factor analysis of variance (ANOVA) is used for statistical software, LSD-t test is used for comparison between groups, and P<0.05 is considered to have statistical significance.

[0046] The results are as shown in Figure 2As shown, compared with the blank control group, the cell survival rate of the 300 mM H2O2 model group was significantly reduced (P < 0.01). The cell survival rate of the protection group pretreated with 1.25, 2.5, 5 mM curcumin showed a concentration-dependent increase, and the protection effect was most significant at 5 mM, and the survival rate was restored to near the control group level. However, when the concentration of curcumin increased to 10 mM, the cell survival rate decreased significantly, even lower than the model group, indicating that too high concentration of curcumin itself had a toxic effect on cells. This result clearly shows that curcumin has a clear protective effect on H2O2-induced cell damage in the concentration range of 1.25-5 mM.

[0047] This example determines the safe and effective concentration range of curcumin for neuroprotection, providing a key basis for subsequent mechanism research and drug dosage design.

[0048] Example 2

[0049] Referring to Figure 3 , Figure 4 , this example establishes a stable and reliable H2O2-induced SH-SY5Y cell ferroptosis model to provide a platform for drug screening. The specific steps are as follows:

[0050] S1, concentration gradient screening: set different concentrations of H2O2 (0, 25, 50, 100, 200, 300, 400, 500 mM) to treat SH-SY5Y cells for 24 h.

[0051] S2, multi-index evaluation model:

[0052] Cell morphology observation: observe cell morphology changes such as cell shrinkage, rounding, and shedding by inverted optical microscope.

[0053] Cell survival rate detection: MTT method was used to detect cell proliferation activity.

[0054] Cell apoptosis / death detection: Hoechst 33258 fluorescence staining was used. After washing with PBS, Hoechst 33258 staining solution (final concentration 5 mg / mL) was added, and incubated for 15 min in the dark. After washing with PBS, observe under fluorescence microscope (excitation wavelength 350 nm, emission wavelength 460 nm). Normal cell nuclei show uniform light blue fluorescence, while apoptotic or dead cell nuclei show dense bright blue fluorescence or fragmented.

[0055] The results of inverted microscope observation (as shown in Figure 3 ) showed that with the increase of H2O2 concentration, the cell density decreased, and the morphology gradually changed from spindle or polygonal to round, shrinkage, and even shedding. The results of Hoechst 33258 staining (as shown in Figure 4indicated that there were less apoptotic cells under low concentration of H2O2 (e.g. 100 μM); when the concentration increased to 300 μM, a large number of cells showed karyopyknosis and fragmentation, presenting bright blue fluorescence; and when the concentration was 500 μM, the cell damage was extremely serious. In combination with the MTT results (as shown in Table 1), the H2O2 treatment at 300 μM could reduce the cell survival rate to a moderate damage level (e.g. 80-90%) and stably induce the morphological changes characteristic of ferroptosis, and thus 300 μM was selected as the optimal modeling concentration. Figure 2

[0056] In this embodiment, a H2O2-induced SH-SY5Y cell ferroptosis model was successfully constructed, which has the advantages of good repeatability and obvious characteristics, and is suitable for in vitro screening of neuroprotective drugs.

[0057] Example 3

[0058] Referring to Table 1, Figure 5 , in this embodiment, the ability of curcumin to remove ROS accumulation in H2O2-induced SH-SY5Y cells was explored, and the specific steps were as follows:

[0059] S1, cell treatment and grouping: same as in Example 1.

[0060] S2, loading of DCFH-DA fluorescent probe: after the cell treatment was completed, the culture medium was discarded, and the cells were gently washed twice with PBS. DCFH-DA probe working solution (final concentration 10 μM) diluted with serum-free medium at a ratio of 1:1000 was added, and the cells were incubated at 37°C in the dark for 20 min.

[0061] S3, fluorescence detection:

[0062] Fluorescence microscope observation: the cells were washed with PBS three times to remove the probe that did not enter the cells, and then immediately observed and photographed under a fluorescence microscope (excitation wavelength 488 nm, emission wavelength 525 nm). The green fluorescence intensity represented the level of intracellular ROS.

[0063] Fluorescence microplate reader quantification: after the cells were digested and resuspended, the cell suspension was inoculated into a black 96-well plate at 180 μL per well, and the fluorescence intensity values of each well were detected using a fluorescence microplate reader at the same wavelength.

[0064] The results are shown in Table 2. Figure 5 Compared with the control group, the green fluorescence intensity of the H2O2 model group cells was significantly enhanced, indicating that ROS accumulated in large amounts. After pre-treatment with 1.25-5 μM curcumin, the fluorescence intensity showed a concentration-dependent decrease, and especially at 5 μM, the fluorescence intensity was close to the normal level. However, the fluorescence intensity in the 10 μM curcumin group did not decrease significantly and even increased. The quantitative results of the fluorescence microplate reader (as shown in Table 2) also showed that the fluorescence intensity of the H2O2 model group was significantly higher than that of the control group, and the fluorescence intensity of the 1.25-5 μM curcumin groups was significantly lower than that of the H2O2 model group, especially at 5 μM, the fluorescence intensity was close to the normal level. However, the fluorescence intensity in the 10 μM curcumin group did not decrease significantly and even increased. Figure 5 ​The trend shown in the image is consistent with that of the microscopic spectral data, providing objective data support. This indicates that an appropriate concentration of curcumin can effectively remove excess ROS induced by H2O2.

[0065] This embodiment elucidates one of the important mechanisms of curcumin's neuroprotective effect from an antioxidant perspective: inhibiting the initiating factors of ferroptosis by reducing oxidative stress levels.

[0066] Example 4

[0067] See Figure 6 This embodiment verifies the core aspect of whether curcumin can inhibit ferroptosis, namely, curcumin's regulation of lipid peroxidation (LPO) levels. The specific steps are as follows:

[0068] S1. Cell treatment and grouping: Same as in Example 1.

[0069] S2. Lipid peroxide detection: A commercial lipid peroxide detection kit (colorimetric method) was used. Cells were collected, washed with pre-cooled PBS, and lysed on ice with extraction buffer. Cells were then sonicated on ice (20% power, 3s sonication, 7s interval, repeated 30 times). The cells were centrifuged at 8000×g for 10min at 4℃, and the supernatant was collected.

[0070] S3. Colorimetric Reaction: Take an appropriate amount of supernatant and add Reagent 1 (detection buffer, e.g., 300 μL) and Reagent 2 (chromogenic agent, e.g., 100 μL) sequentially according to the volume ratio recommended in the kit instructions. Mix thoroughly and heat in a 95°C water bath for 40 min (to decompose lipid peroxides and produce colored substances). After cooling under running water, centrifuge at 3000×g for 10 min. Add 200 μL of supernatant to a 96-well plate and measure the OD value at 535 nm using a microplate reader.

[0071] The results are as follows Figure 6 As shown, the OD value of the H2O2 model group was significantly higher than that of the control group, indicating an increased level of intracellular lipid peroxidation. The OD values ​​of the 1.25–5 μM curcumin pretreatment groups decreased with increasing concentration, indicating that curcumin can inhibit H2O2-induced lipid peroxidation in a concentration-dependent manner. When the curcumin concentration was 10 μM, the OD value rebounded, suggesting a weakened protective effect or a pro-oxidative effect. This result is consistent with the ROS detection results.

[0072] This embodiment demonstrates that curcumin can inhibit lipid peroxidation, a key biochemical event in ferroptosis, providing solid evidence for its anti-ferroptosis effect.

[0073] Example 5

[0074] See Figure 7 , Figure 8 , Figure 9The embodiment studies the influence of curcumin on the key antioxidant system in cells, i.e., glutathione system, and the specific steps are as follows:

[0075] S1, cell treatment and sample preparation: same as embodiment 1.

[0076] After cell digestion, PBS washing, centrifugal collection of cell precipitate, and accurate weighing, lysis is performed according to the proportion of 30 μL protein removal reagent M solution per 10 mg cell precipitate, repeated freezing and thawing in liquid nitrogen twice, 4°C standing for 5 min, 10000 x g, 4°C centrifugation for 10 min, and the supernatant is taken for determination.

[0077] S2, total glutathione (T-GSH) and oxidized glutathione (GSSG) determination: glutathione detection kit is used. First, the standard curve of T-GSH and GSSG is prepared by using standard products (as shown in Figure 7 , Figure 8 GSSG standard product refers to the pure product of oxidized glutathione (Glutathione Oxidized); T-GSH standard product refers to the pure product of reduced glutathione (GSH)). When GSSG is determined, the sample needs to be treated with GSH scavenger to remove GSH, and then the total glutathione determination method is used. The content of reduced glutathione (GSH) is calculated by the formula: GSH = T-GSH - 2 x GSSG.

[0078] The standard curve is linear (R 2 > 0.99), and the results are shown in Figure 9 The GSH content in the H2O2 model group cells is significantly lower than that in the control group. After being pretreated with 1.25-5 μM curcumin, the GSH content increases in a concentration-dependent manner, and reaches a peak at 5 μM. The GSH content in the 10 μM curcumin treatment group decreases. This shows that appropriate concentration of curcumin can maintain or increase the GSH level in cells and enhance the antioxidant capacity of cells.

[0079] GSH is an important cofactor of GPX4 enzyme, and its sufficient content is the key to inhibit lipid peroxidation and resist ferroptosis. The results reveal the mechanism of curcumin from the perspective of restoring endogenous antioxidant reserves.

[0080] Embodiment 6

[0081] Referring to Figure 10 , the embodiment verifies whether curcumin plays a protective role by regulating the ferroptosis core proteins GPX4 and xCT at the protein level, and the specific steps are as follows:

[0082] S1, Protein sample preparation: After cell treatment, the pre-cooled RIPA lysis buffer (containing PMSF and protease inhibitors) was used to lyse for 30 min on ice. 4°C, 12000xg centrifugation for 30 min, take the supernatant.

[0083] S2, BCA method protein quantification: strictly according to the BCA kit instructions, according to the standard curve to calculate the sample protein concentration. Adjust the same protein concentration of each sample, mixed with 5x loading buffer, 100℃ boiling 5min to denature the protein.

[0084] S3, Western Blot:

[0085] Electrophoresis: preparation of 10% separation gel and 5% concentrated gel (formula as shown in Table 1, Table 2). Equal amount of protein was loaded into each well, 80V constant voltage electrophoresis to the concentrated gel, and 120V constant voltage electrophoresis to the bottom of the separation gel.

[0086] Membrane transfer: wet transfer method, 66mA constant current transfer membrane for 2h, the protein was transferred to PVDF membrane.

[0087] Blocking and hybridization: blocking with 5% skim milk at room temperature for 1h. Add GPX4 and xCT primary antibody, 4°C shaking incubation overnight. After TBST washing, add the corresponding secondary antibody, incubate at room temperature for 1h.

[0088] Development: ECL chemiluminescence reagent treatment film, exposure and development in chemiluminescence gel imager. With β-actin or GAPDH as internal reference, the relative expression of target protein was analyzed.

[0089] The results are shown in Figure 10 Compared with the control group, the expression levels of GPX4 and xCT proteins in the H2O2 model group were significantly down-regulated. While 1.25-5μM curcumin pretreatment could reverse this down-regulation trend in a concentration-dependent manner, and the effect was most significant at 5μM. The up-regulation of 10μM curcumin on protein expression was weakened or disappeared. GPX4 is a key enzyme for degrading lipid peroxide, and xCT is a key transporter for taking cystine to synthesize GSH, and the up-regulation of both constitutes the core molecular mechanism of curcumin inhibiting ferroptosis.

[0090] This example further explains the molecular target of curcumin against ferroptosis from the protein expression level, making the mechanism more clear and perfect.

[0091] Table 110% separation adhesive preparation Table 2-310%SeparationAdhesive Preparation

[0092]

[0093] Table 2-45% Concentrated Adhesive Preparation

[0094]

[0095]

[0096] Example 7

[0097] This embodiment designs a neuroprotective drug composition containing curcumin, and the specific steps are as follows:

[0098] Active ingredient: curcumin, the effective concentration range of which in the drug composition is determined based on in vitro experiments to be 1.25 μM to 5 μM. In actual preparations, dose conversion needs to be made according to the administration route (such as oral administration, injection) and bioavailability.

[0099] Dosage form design:

[0100] Oral preparation: curcumin is mixed with a pharmaceutically acceptable carrier, such as a filler (microcrystalline cellulose, starch), a disintegrant (cross-linked polyvinylpyrrolidone), and a lubricant (magnesium stearate), to form tablets or capsules. To improve bioavailability, solid dispersions, cyclodextrin inclusion, or nanocrystal technology is used.

[0101] Injection: curcumin is dissolved in a suitable solvent (such as sterile PBS containing a small amount of ethanol and polysorbate 80), filtered to remove bacteria, and then divided into ampoules. The clarity and stability of the preparation need to be ensured.

[0102] Composition application: the drug composition can be used for the prevention and / or treatment of neurodegenerative diseases mediated by the ferroptosis pathway, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, etc. Its mechanism of action is to inhibit the ferroptosis process in nerve cells, which is manifested by reducing ROS, inhibiting LPO, increasing GSH levels, up-regulating GPX4 and xCT protein expression, and regulating iron homeostasis.

[0103] The above embodiment system systematically and in-depthly verifies that curcumin, within the concentration range of 1.25-5 μM, inhibits H2O2-induced SH-SY5Y cell ferroptosis through multi-target and multi-pathway, thereby exerting a neuroprotective effect.

[0104] Example 8

[0105] Reference Figure 11 , Figure 12 This embodiment establishes a stable and reliable hemin-induced SH-SY5Y cell ferroptosis model to simulate the pathology of hemorrhagic stroke, and the specific steps are as follows:

[0106] Cell culture: Human neuroblastoma SH-SY5Y cells were seeded in 96-well plates (density 5 x 10 3 cells / well) and cultured in 50% MEM / F12 medium (containing 10% fetal bovine serum) at 37°C in a 5% CO2 incubator for 24 h to a density of 70-80%.

[0107] Model induction: A hemin concentration gradient (0 μM, 25 μM, 50 μM, 100 μM, 200 μM, 400 μM, 500 μM) was set up, and 50 μL of hemin solution was added to each well for 24 h. The untreated group was used as a control, and the same volume of DMSO was added to the solvent group.

[0108] Survival rate detection: The cell survival rate was determined by MTT method. The culture medium was discarded, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubated for 4 h. The formazan was dissolved with DMSO, and the OD value was measured at 570 nm by a microplate reader. Survival rate (%) = (OD experimental group / OD control group) x 100%.

[0109] Morphological observation: The morphological changes of the cells were recorded by taking pictures under an inverted microscope.

[0110] The results are shown in Figure 11 , and the cell survival rate decreased to about 40% (IC 50 of 80.3 μM), which was determined as the optimal modeling concentration.

[0111] The morphological results (as shown in Figures 3-4 ) showed that the cells in the 100 μM hemin treatment group were shriveled and ruptured, showing the characteristics of ferroptosis.

[0112] This example successfully established a hemin-induced hemorrhagic stroke cell model, providing a standardized platform for drug screening.

[0113] Example 9

[0114] Referring to Figure 13 , Figure 14 , Figure 15 , this example used a kit and Western blot method to verify the effect of curcumin on inhibiting ferroptosis in the hemin model, and detected key indicators (ROS, LPO, iron ions, GPX4 / xCT). The specific steps are as follows:

[0115] 1. Cell treatment: SH-SY5Y cells were seeded in 6-well plates (density 1 x 10 6 cells / well), and the groups were as follows:

[0116] Control group: no treatment;

[0117] Model group: 100 μM hemin treatment for 24 h;

[0118] Experimental group: 1.25 μM, 2.5 μM, 5 μM, 10 μM curcumin pretreatment for 6 h, then hemin treatment;

[0119] 2. Index detection:

[0120] ROS level: DCFH-DA fluorescent probe method. Cells were incubated with 10 μM DCFH-DA for 20 min, observed by fluorescence microscope (488 / 525 nm), and quantified by microplate reader;

[0121] LPO and iron ion: Commercial kit colorimetric method. After cell lysis, OD values at 535 nm (LPO) and 593 nm (iron ion) were measured, and the contents were calculated;

[0122] GPX4 / xCT protein: Western blot method. Cell lysis, BCA method for protein quantification, SDS-PAGE electrophoresis, membrane transfer, primary antibody (GPX4 / xCT) incubation overnight, and secondary antibody development.

[0123] The results are shown in Figure 13 and Figure 14 It is shown that curcumin at a concentration of 1.25-5 μM reduces ROS and iron ion levels in a concentration-dependent manner (2.5 μM is the best), and the indicators rebound at 10 μM.

[0124] Figures 3-14 It is shown that curcumin at 2.5 μM up-regulates GPX4 / xCT expression and inhibits ferroptosis.

[0125] Therefore, it can be seen that curcumin at 1.25-5 μM inhibits hemin-induced ferroptosis through multiple targets, and the optimal concentration is 2.5 μM.

[0126] The above only describes the preferred embodiments of the present application and does not limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. Use of curcumin in the manufacture of a neuroprotective agent for inhibiting ferroptosis, characterized in that: The curcumin is used as an active ingredient for preventing or treating nerve cell damage associated with ferroptosis.

2. The use of curcumin according to claim 1 for the preparation of a neuroprotective drug for inhibiting ferroptosis, characterized in that: The curcumin is administered at a concentration of 1.25-5 μM.

3. The use of curcumin in the preparation of a neuroprotective drug for inhibiting ferroptosis according to claim 1, characterized in that: The application is based on a ferroptosis induction model, which includes a H2O2 induction or hemin induction cell ferroptosis model, wherein the concentration of H2O2 is 200-500 μM, and the concentration of hemin is 50-200 μM.

4. The use of curcumin for the preparation of a neuroprotective drug for inhibiting ferroptosis according to claim 3, characterized in that: The hemin induction cell ferroptosis model is specifically a cell model of hemorrhagic stroke, wherein hemin induces ferroptosis by generating iron ions through degradation.

5. The use of curcumin according to claim 1 for the preparation of a neuroprotective drug for inhibiting ferroptosis, characterized in that: The neuroprotection is achieved by inhibiting the accumulation of reactive oxygen species and lipid peroxidation in cells.

6. The use of curcumin according to claim 1 for the preparation of a neuroprotective drug for inhibiting ferroptosis, characterized in that: The neuroprotection is achieved by increasing the content of glutathione in cells and regulating iron ion homeostasis.

7. The use of curcumin according to claim 1 for the preparation of a neuroprotective drug for inhibiting ferroptosis, characterized in that: The neuroprotection is achieved by up-regulating the protein expression of glutathione peroxidase 4, cystine or glutamate antiporter system.

8. The use of curcumin according to claim 1 for the preparation of a neuroprotective drug for inhibiting ferroptosis, characterized in that: The neuroprotective drug is used for treating or preventing neurodegenerative diseases selected from the group consisting of Alzheimer's disease, Parkinson's disease and Huntington's disease.

9. The use of curcumin according to claim 1 for the preparation of a neuroprotective drug for inhibiting ferroptosis, characterized in that: The neuroprotective drug comprises curcumin as the only active ingredient or a pharmaceutical composition combined with a pharmaceutically acceptable carrier, which includes a solvent, an excipient or a stabilizer.

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