Application of bruscin compound in preparation of medicine for treating acute cerebral ischemic stroke

Abstract

The invention discloses an application of a bruscin compound in preparation of a medicine for treating acute cerebral ischemic stroke. The invention provides an application of a brusicin compound containing a quassin diterpene mother nucleus in preparation of a medicine for inhibiting PARthanatos, and further provides an application of brucein E in preparation of a medicine for treating cerebral apoplexy, a medicine for treating cerebral ischemia-reperfusion injury and a PARP-1 inhibitor. According to the application disclosed by the invention, the brucein E is screened and identified to become a potent inhibitor of PARthanaos due to inhibition of excessive activation of PARP-1 at a nanomolar concentration. The brucein E effectively prevents accumulation of PAR modified protein, reduces mitochondrial membrane potential collapse and inhibits AIF nuclear translocation. The brucein E can greatly reduce the infarct volume and denatured neurons, increases the microvascular density, has no adverse effect on cerebral blood flow, and shows the potential of single-time, delayed and low-dose central protection.

Description

Technical Field

[0001] This invention relates to a drug for treating acute ischemic stroke, and more particularly to the use of crotonin compounds in the preparation of drugs for treating acute ischemic stroke. Background Technology

[0002] Ischemic stroke, also known as cerebral ischemic stroke (CIS), is one of the leading causes of death. In 2021, there were 7.8 million new cases of ischemic stroke worldwide, accounting for more than 65% of all stroke cases. The pathophysiology of ischemic stroke is characterized by the loss of approximately 1.9 million neurons per minute after the event, resulting in irreversible brain tissue damage within six hours. Despite advances in treatment methods such as revascularization, more than 50% of patients still suffer from significant disability after intervention. While intravenous thrombolysis and mechanical thrombectomy can recanalize occluded vessels, their efficacy is limited by a narrow 4.5-hour treatment window, and reperfusion injury and hemorrhagic transformation pose significant challenges, resulting in less than 20% of patients actually benefiting.

[0003] A more pressing issue is the current lack of evidence-based, marketed neuroprotective drugs that improve prognosis. Since the 1970s, researchers have explored over 1,000 candidate molecules, including free radical scavengers like NXY-059, magnesium agents, calcium channel blockers, and excitotoxic inhibitors such as NA-1 and GABA modulators like S44819. While these candidates have shown encouraging results in animal models, they have all repeatedly failed in phase III clinical trials. The fundamental problem is that current technologies still cannot effectively target the "irreversible" links in the ischemic cascade.

[0004] In recent years, advancements in cell death biology have brought attention to PARthanatos, a cell death process mediated by the accumulation of poly(ADP-ribose) polymerases. Unlike classic cell death mechanisms such as apoptosis, necrosis, or pyroptosis, PARthanatos is initiated by the overactivation of poly(ADP-ribose) polymerase-1 (PARP-1) due to DNA single-strand breaks. This process manifests as PAR polymer accumulation, increased mitochondrial membrane permeability, and the translocation of apoptosis-inducing factor (AIF) to the nucleus, ultimately leading to widespread DNA breaks and energy depletion. Morphologically, PARthanatos is characterized by nuclear condensation without caspase activation. It occurs temporally before membrane rupture and spatially originates in the neuronal nucleus, thus being considered the "first wave" of cell death following ischemic events. Notably, once PARthanatos is initiated, it cannot be inhibited by other cell death pathway blockers, highlighting its crucial role in neuronal death after cerebral ischemia. Studies using transgenic mouse models have shown that knocking out PARP-1 or mutating the AIF nuclear localization sequence can significantly reduce infarct volume by 40%–60% without affecting cerebral blood flow, suggesting that targeting the PARthanatos pathway has potential therapeutic value. Unfortunately, currently used PARP inhibitors such as olaparib have significant systemic toxicity and poor central penetration, and have not yet been tested in the field of stroke.

[0005] Traditional Chinese medicine possesses unique advantages due to its multiple components, multiple targets, and low toxicity. Modern pharmacological studies have confirmed that compounds such as quercetin, resveratrol, silymarin, and flavonoids can cross the blood-brain barrier and indirectly inhibit PARP-1 enzyme activity and reduce PAR polymer levels at micromolar concentrations. However, these findings primarily focus on the effects on PAR modification, lacking comprehensive screening and evaluation of the PARthanatos signaling pathway; their sites of action, structure-activity relationships, in vivo efficacy windows, and safety profiles remain insufficiently elucidated.

[0006] Crotonin compounds are extracted from the dried, ripe fruit of Brucea javanica. Due to their strong cytotoxicity, they are used in the preparation of antitumor drugs. Current research findings (reference: Lan X, Wang Q, Liu Y, You Q, Wei W, Zhu C, Hai D, Cai Z, Yu J, Zhang J, Liu N. Isoliquiritigenin alleviates cerebral ischemia-reperfusion injury by reducing oxidative stress and ameliorating mitochondrial dysfunction via activating the Nrf2 pathway. Redox Biol. 2024 Nov;77:103406. doi: 10.1016 / j.redox.2024.103406. Epub 2024 Oct 22. PMID: 39454290; PMCID: PMC11546133.) show that crotonol, as an Nrf2 inhibitor, can reverse the mitochondrial protective properties and anti-apoptotic effects of isoliquiritigenin in vivo and in vitro, thus eliminating the potential neuroprotective effect of isoliquiritigenin against cerebral ischemia-reperfusion injury. Furthermore, numerous studies have demonstrated that crotonol is a drug that exacerbates stroke damage, through the mechanism of inhibiting the function of the antioxidant transcription factor Nrf2. Therefore, crotonol compounds not only lack significant neuroprotective effects but also have a negative impact on the treatment of ischemic brain injury due to their highly potent cytotoxicity. Based on these existing research findings, it can be inferred that crotonol compounds are not suitable as alternative drugs for the treatment of ischemic stroke or for inhibiting apoptosis. Summary of the Invention

[0007] Objectives of this invention: The first objective is to provide the application of crotonin compounds containing crotonin diterpenoid cores in the preparation of drugs that inhibit PARthanatos, thus solving the problem of how to inhibit PARthanatos. The second objective is to propose the application of crotonin E in the preparation of drugs for treating stroke, treating cerebral ischemia-reperfusion injury, and PARP-1 inhibitors, thus solving the problems of how to treat stroke, cerebral ischemia-reperfusion injury, and inhibit PARP-1 activity. The third objective is to propose a poly(ADP-ribose) polymerase 1 inhibitor containing crotonin E. The fourth objective is to propose a PARthanatos inhibitor containing crotonin E. The fifth objective is to propose a stroke treatment drug containing crotonin E.

[0008] Technical solution: This invention provides the application of crotonin compounds containing quassinolide nuclei in the preparation of drugs that inhibit PARthanatos.

[0009] Preferably, the crotonin compounds include at least one of crotonin E, Pasakbumin B, 13,21-dihydropasakbumin, crotonin D, and crotonin lactone A.

[0010] Preferably, the PARthanatos are PARthanatos caused by ischemic stroke.

[0011] The second aspect of this invention discloses the use of crocin E in any of the following: (1) Preparation of drugs for the treatment of stroke; (2) Preparation of drugs for the treatment of cerebral ischemia-reperfusion injury; (3) Preparation of poly(ADP-ribose) polymerase 1 inhibitor; (4) Preparation of drugs to prevent the accumulation of PAR-modified proteins; (5) To prepare drugs that slow down the collapse of mitochondrial membrane potential; (6) Prepare drugs that inhibit AIF nuclear translocation.

[0012] Preferably, the stroke is an acute ischemic stroke.

[0013] In some embodiments, PAR-modified protein accumulation, mitochondrial membrane potential collapse, and AIF nuclear translocation events all occur in brain tissue following ischemic stroke.

[0014] A third aspect of the present invention provides a poly(ADP-ribose) polymerase 1 inhibitor containing crocin E.

[0015] A fourth aspect of the present invention provides a PARthanatos inhibitor containing crotonin E.

[0016] The fifth aspect of this invention provides a stroke treatment drug containing crotonin E.

[0017] Preferably, the concentration of crocin E in the aforementioned drug or inhibitor is at least 800 nM.

[0018] More preferably, the concentration of crocin E in the drug or inhibitor is 1-30 μM.

[0019] In some embodiments, the aforementioned drug or inhibitor further contains pharmaceutically acceptable excipients.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: This invention utilizes an MNNG-induced cellular PARthanatos model to screen and identify crocin E as a potent inhibitor of PARthanatos at nanomolar concentrations by inhibiting PARP-1 overactivation. Crocin E effectively prevents the accumulation of PAR-modified proteins, alleviates mitochondrial membrane potential collapse, and inhibits AIF nuclear translocation. Molecular docking, surface plasmon resonance (SPR), and thermal stability experiments demonstrate that crocin E binds to the NAD+ of PARP-1 via five hydrogen bonds. + The catalytic pocket interaction exhibits a fast binding and slow dissociation kinetic profile. In a mouse model of permanent distal middle cerebral artery occlusion, a single administration of 10 mg / kg was administered 4.5 hours after occlusion. -1 Brucea javanica extract E reduced infarct volume and degenerated neurons by 70%, increased microvascular density, and had no adverse effects on cerebral blood flow. These findings suggest that Brucea javanica extract E is a novel first-in-class candidate molecule for stroke neuroprotection, demonstrating the potential for single-dose, delayed, low-dose central protective effects. Attached Figure Description

[0021] Figure 1 The screening and identification process for candidate molecules that can effectively inhibit PARthanatos; Figure 2 The effects of different quassinolide diterpenoid compounds on the inhibition of PARthanatos; Figure 3 The experimental results show that crotonin E inhibits PARthanatos in nerve cells in a dose-dependent manner. Figure 4 Cytotoxicity of Yadanziolid A, Brusatol, Bruceine E and ola pretreatment and its effect on MNNG-induced cytotoxicity; Figure 5 The experimental results show that crocin E inhibits PARP1 activation in the PARthanatos process; Figure 6 The results show that crotonin E does not affect PARP1 protein expression, but inhibits DNA damage repair by suppressing PARP1 activity. Figure 7 The results of the analysis on the interaction between crotonin E and PARP1; Figure 8 Experimental results showing that pretreatment with crotonin E can prevent MNNG-induced changes in mitochondrial membrane potential and AIF nuclear translocation. Figure 9The results show that crocin E significantly inhibited neuronal death in a mouse model of permanent cerebral ischemia; Figure 10 To investigate the effect of crocin E on PARthanatos-related proteins in a mouse model of permanent cerebral ischemia. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0023] Example 1: Candidate molecules with potent inhibitory effects on PARthanatos were identified by screening a library of traditional Chinese medicine monomer compounds. The method is as follows: (1) Experimental materials: Bruceine E (CAS: 21586-90-3, purity: 99.88%, molecular weight: 412.43 Da, chemical formula: C) 20 H 28 O9), 13,21-dihydrothionone (CAS: 129587-06-0, purity: 98.01%, molecular weight: 410.42, chemical formula: C 20 H 26 O9), Brucea javanica extract D (CAS: 21499-66-1, purity: 99.99%, molecular weight: 410.42 Da, chemical formula: C 20 H 26 O9) and Pasakbumin B (CAS: 138809-10-6, purity: 99.93%, molecular weight: 424.4 Da, chemical formula: C 20 H 24 O 10 Purchased from TargetMol. Olaparib (OLA; CAS: 763113-22-0, purity: 99.98%, molecular weight: 434.46, chemical formula: C 24 H 23 FN4O3) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG; CAS: 70-25-7, purity: 99.97%, molecular weight: 147.09, chemical formula: C2H5N5O3) were purchased from MedChemExpress (MCE). Brusatol (CAS: 14907-98-3, purity: 99.73%, molecular weight: 520.53, chemical formula: C) was also purchased. 26 H 32 O 11 ); Yadanziolitholide A (CAS: 95258-14-3, purity: 99.42%, molecular weight: 426.41, chemical formula: C 20 H26 O 10 ).

[0024] SH-SY5Y cells (RRID: CVCL_0019) and PC-9 cells (RRID: CVCL_B260) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All compounds were derived from MCE (Shanghai, China) or TargetMol.

[0025] (2) Cell culture: Adherent cells were maintained at 37°C and 5% CO2 in 10cm culture dishes (Corning 430167) in complete medium (high glucose DMEM, VivaCell, China; 10% FBS, VivaCell; 1% penicillin-streptomycin, Biosharp, China). When the cell density exceeded 90%, PBS, trypsin, and medium were preheated to room temperature; the old medium was discarded, PBS was used for washing, trypsin was added, and the cells were allowed to become rounded but not detached; the trypsin was discarded, fresh medium was added to stop digestion, a single-cell suspension was prepared, transferred to a new dish, medium was added, and incubation continued. Cells from passages 3-5 with 80% confluence were used in the experiment. For cryopreservation, the medium was discarded, the cells were washed with PBS and trypsin, centrifuged at 1000 rpm for 10 min, the supernatant was discarded, and the cells were resuspended in cryopreservation buffer (10% DMSO + 90% FBS), 1 mL per tube, incubated overnight at -80°C, and then transferred to liquid nitrogen. During resuscitation, rapidly thaw the medium in a 37°C water bath for 1–3 min, transfer it to a centrifuge tube, add fresh culture medium, centrifuge at 1000 rpm for 10 min, discard the supernatant, resuspend the medium in fresh medium, inoculate it into a 10 cm dish, bring the volume to 10 mL, and incubate at 37°C with 5% CO2. Observe and change the medium or subculture after 24 h.

[0026] (3) MNNG-induced PARthanatos model: Cells were evenly seeded into 12-well plates and allowed to reach 80% confluence after 24 hours. MNNG (60 μM, 15 min) was added to induce PARthanatos. The compound was added 24 hours before MNNG treatment; after MNNG exposure, the medium was replaced with fresh medium containing the compound; morphology was assessed under a microscope after 24 hours.

[0027] (4) Experimental methods CCK-8 Experiment: Cells were spaced at 8 × 10⁸ cells per well. 3 Cells were seeded in 96-well plates (triple replicates). After treatment, 10 μL of CCK-8 reagent (Beyotime, China) was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance was read at 450 nm. Cell viability was calculated using the following formula: Cell viability (%) = [(Experimental group mean – Blank mean) / (Control group mean – Blank mean)] × 100%.

[0028] Protein extraction: Pre-cool PBS and trypsin to room temperature. Discard the culture medium, wash with PBS, digest with trypsin until cells become rounded, discard the trypsin, add PBS, centrifuge at 1000 rpm for 5 min at 4°C, discard the supernatant, add RIPA lysis buffer containing a 1× protease inhibitor cocktail, vortex for 15-20 s every 5 min for a total of 6 times, centrifuge at 12000 rpm for 15 min at 4°C, collect the supernatant, quantify the protein, add 4× loading buffer at a 4:1 ratio, and boil at 99°C for 10 min.

[0029] Sytox staining: Discard the culture medium, wash with PBS, add culture medium containing SytoxGreen and Hoechst 33342, incubate at 37°C and 5% CO2 in the dark for 30 min, wash twice with PBS, and image with a fluorescence microscope.

[0030] Cellular immunofluorescence: Discard the culture medium, wash the fixed cells with PBS for 3 min × 3, permeabilize with 0.3% Triton X-100 PBT for 30 min, block for 30 min, incubate with primary antibody at 4°C overnight, wash with PBS for 5 min × 3, incubate with secondary antibody in the dark for 2 h, wash with PBS for 5 min × 3, and mount with anti-quenching agent.

[0031] Molecular docking: Preliminary molecular docking predictions were performed as previously described. The protein crystal structure was obtained from the RCSB protein database, and the 3D structure of crotonin E was obtained from the PubChem database. Crotonin E was used as the ligand, and the target was the acceptor. Semi-flexible docking of crotonin E and PARP1 protein was performed using AutoDockVina 1.1.2 software: 3D conformation of crotonin E (mol: 21586-90-3), PARP1 protein (PDB code: 7KK2). Binding site selection criteria: The binding region was determined using the GridBox module of AutoDockTools; Docking box size criteria: The docking box was dynamically adjusted to ensure coverage of all potential binding conformations. All water molecules and heteroatoms were removed from the acceptor using PyMOL 2.4 and AutoDockVina 1.1.2, followed by the addition of charge and hydrogen atoms. Ligand-acceptor interactions were predicted using AutoDockTools; lower binding energies indicated stronger affinity. Two-dimensional interaction information was generated and analyzed using LigPlot+ software.

[0032] Cell thermal migration experiment: Cells at 80% confluence in a 10 cm culture dish were treated with a compound or DMSO for 24 h. The cell pellet was collected, resuspended in PBS containing 1× protease inhibitor, aliquoted into 8-tube strips (100 μL per tube), heated in a PCR instrument with a temperature gradient, and subjected to three freeze-thaw cycles at 37°C and liquid nitrogen. The cells were then centrifuged at 12000 rpm for 30 min at 4°C. The supernatant was used for Western blot analysis.

[0033] Western blot: An equal amount of protein (50 μg) was loaded onto an SDS-PAGE gel (Wansheng Haotian, Shanghai), transferred to a 0.22 μm PVDF membrane, blocked with 5% skim milk for 2 h or with protein-free blocking buffer for 1 h, incubated with primary antibody overnight at 4°C, washed with PBST for 10 min × 3, incubated with HRP-labeled secondary antibody at room temperature for 2 h, washed with PBST for 10 min × 3, developed with ECL reagent (A:B=1:1), acquired using a BIO-RED imaging system, and the ImageJ quantitative bands were corrected with GAPDH.

[0034] JC-1 staining: JC-1 reagent (ThermoFisher, China) should be equilibrated to room temperature 2–3 hours before the experiment. Positive control wells should be treated with CCCP for 10 min, followed by incubation with JC-1 in the dark for 20 min, washed twice with PBS, and imaged with constant exposure parameters; the remaining experimental wells should be treated in the same way.

[0035] (5) Test results: To identify drugs that could mitigate the effects of MNNG, a library of traditional Chinese medicine monomeric compounds was screened under the conditions of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG, 60 μM, 15 min). In the initial screening stage, five compounds in the library showed survival rates comparable to the classic PARP1 inhibitor olaparib (OLA). In the secondary screening, these compounds consistently exhibited a normalized survival rate of 70% after MNNG exposure (e.g., ...). Figure 1 (As shown in the AD diagram). Figure 1 Figure A shows a schematic diagram of compound library screening, Figure B shows a schematic diagram of drug screening process, Figure C shows the initial screening results, and Figure D shows the secondary screening verification results. Figure 1 In Figures C and D, the CCK-8 cell viability assays show the protective effect of the compound after MNNG treatment; the dashed line represents the cell viability of the MNNG-treated control group.

[0036] The verification results showed that bruceine E (BE), a crotonin compound, had a significant protective effect against MNNG toxicity and had the highest survival rate, so it was selected for further research.

[0037] Pasakbumin E is a diterpenoid belonging to the Simaroubaceae family. To elucidate the structure-activity relationship of BE in inhibiting PARthanatos, three other Simaroubaceae diterpenoids with structures similar to BE were evaluated: Pasakbumin B, 13,21-dihydropanthenone, and pasakbumin D. Their chemical structures are shown in [reference needed]. Figure 2 Figure A shows that all natural products possess a highly conserved C-20 skeleton, with a core of three six-membered rings and one lactone ring; their common structural feature is C8-CH2-OC. 13 The oxygen bridge spans between C8 and C13 of the C ring and contains α,β-unsaturated ketones, lactone rings, and multiple hydroxyl groups, collectively forming the key pharmacophore. Experimental setup: PC9 cells were pretreated with Pasakbumin B, BE, 13,21-dihydrocaproic acid, and crotonin D (10 μM) for 24 h, followed by MNNG exposure for 15 min to induce parthanatos, and then incubated for another 24 h after medium replacement.

[0038] The results are as follows Figure 2 and Figure 3 As shown, Figure 2Figure A shows the molecular structures of crocin E, Pasakbumin B, 13,21-dihydropassone, and crocin D; Figure B shows a microscopic image of cells treated with crocin E, Pasakbumin B, 13,21-dihydropassone, and crocin D for 24 h, followed by treatment with MNNG for 15 min and further culture for 24 h (scale bar 25 μm); Figure C shows cell viability measured by CCK-8 assay after the above drug treatments (data expressed as mean ± SD, n=3) (P < 0.01 vs. DMSO; nsP > 0.05 vs. DMSO; ****P < 0.0001 vs. DMSO; ####P < 0.0001 vs. MNNG); Figure D shows typical images of cells treated with 1, 5, 10, 20, 30, 40, and 50 μM for 24 h, and images of cells treated with 1... Image 1 shows microscopic images of cells treated with 5, 10, 20, and 30 μM for 24 h, followed by the addition of 60 μM MNNG for 15 min and further culture in fresh BE-containing medium for 24 h (scale bar 25 μm). Image 2 shows typical images of cell death detected by Sytox staining after 18 h of MNNG treatment (scale bar 25 μm). Image 3 shows three-dimensional images of cell death detected by Sytox green fluorescence. Image 4 shows cell viability detected by CCK-8 assay after 24 h of treatment with 1, 5, 10, 20, 30, 40, and 50 μM MNNG (mean ± SD, n=3) (nsP>0.05 vs. DMSO; P<0.01 vs. DMSO; P<0.001; P<0.0001 vs. DMSO). Image 5 shows cells treated with 25, 50, 100, 150, and 200 μM for 24 h, followed by the addition of 60 μM MNNG. MNNG treatment for 15 min followed by culturing in fresh BE medium for 24 h was performed. Viability was assessed using CCK-8 assay, data are mean ± SD (n=3) (P ≤ 0.01 vs. DMSO; *P < 0.0001 vs. DMSO); Figure I shows the percentage of Sytox-positive cells after MNNG treatment, data are mean ± SD (n=3) (**P < 0.0001 vs. DMSO; ####P < 0.0001 vs. MNNG); Figure J shows the EC50 of BE. 50 Fitted curve, EC 50 =798.3nM (GraphPadPrism10.1.1).

[0039] Figure 3Figure A shows representative phase-contrast micrographs of SH-SY5Y cells pretreated with crocin E (10 μM, 24 h), followed by MNNG (60 μM, 15 min) treatment and further cultured for 24 h (scale bar 25 μm); Figure B shows quantitative data of CCK-8 activity (mean ± SD, n=3; *P<0.001 vs DMSO; ####P<0.0001 vs MNNG); Figure C shows the Western blot time progression of total PARP-1 protein 12 h and 24 h after BE treatment; Figure D shows the density metric (PARP-1 / GAPDH) confirming no significant change in total PARP-1 expression. Significant changes (nsP>0.05 vs. DMSO); Figure E shows PC9 cells treated with 400nM–900nM crotonin E for 24 h, followed by 60 μM NNG for 15 min, and then replaced with fresh medium containing crotonin E. Cell viability was measured by CCK-8 after 24 h, and the data are mean ± SD (n=3) (**P<0.0001 vs. DMSO); Figure F shows microscopic images of PC9 cells treated with 400nM–900nM crotonin E for 24 h, followed by 60 μM NNG for 15 min, and then replaced with fresh medium containing BE for 24 h. Scale bar: 25 μm.

[0040] Microscopic morphological observation (results are shown in...) Figure 2 Figure B) and CCK-8 activity assay (results are shown in Figure B). Figure 2 Figure C shows that all four compounds significantly inhibited MNNG-induced PARthanatos, with BE showing the strongest protective effect, followed by Pasakbumin B, suggesting that the quassinolide diterpenoid nucleus is the structural basis for inhibiting PARthanatos.

[0041] To assess the safety of BE, cells were treated for 24 h with different concentration gradients (1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM). Morphological observation (see results) Figure 2 (Figure D) and CCK-8 cell viability assay (results are shown in Figure D) Figure 2 (Figure G) confirmed that the cytotoxicity of BE was positively correlated with drug concentration, exhibiting a typical dose-dependent toxicity response. To define the effective protective concentration range, cells were pretreated with BE at concentrations of 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 5 μM, 10 μM, 20 μM, and 30 μM for 24 h before establishing an MNNG injury model. Morphological assessment ( Figure 2 Chinese D diagram and Figure 3 (Figure F) and CCK-8 activity assay ( Figure 2 H-diagram and Figure 3The figure (E) shows that low to medium concentrations of BE (1-10 μM) provide significant protection; however, as the concentration increases, the inherent toxicity also increases, leading to a parabolic decline in the protective effect. The half-maximal effective concentration (EC50) is also mentioned. 50 The value was determined to be 798.3 nM (see...). Figure 2 (J figure).

[0042] Meanwhile, in SH-SY5Y human neurons, BE was also shown to effectively block MNNG-induced PARthanatos process (e.g. Figure 3 (As shown in Figures A and B). Therefore, 10 μM was chosen as the effective drug concentration. SytoxGreen staining was used to analyze cell death and survival (…). Figure 2 Figures E, F, and I show that BE pretreatment significantly inhibited MNNG-induced cell death, confirming its potent anti-PARthanatos activity and making it the most effective natural PARthanatos inhibitor.

[0043] Further investigation was conducted to examine the effects of pretreatment with Yadanziolide A, Brusatol, Bruceine E, and ola on MNNG-induced cytotoxicity. Methods: PC9 cells were pretreated for 24 hours with the specified compounds: Yadanziolide A (YA, 1 ​​µM), Brusatol (BR, 25 nM and 50 nM), Bruceine E (BE, 10 µM), and ola (50 µM). Subsequently, they were treated with 85 µM MNNG for 17 minutes. After MNNG treatment, the cells were cultured in fresh medium for another 24 hours. Representative bright-field microscopy images were captured at this time, and cell viability was assessed using the CCK-8 assay. Data are presented as mean ± standard deviation of at least three independent experiments. Results are as follows: Figure 4 As shown. Figure 4 The results showed that crotonol not only exhibited strong cytotoxicity but also failed to alleviate MNNG-induced cytotoxicity. While yadanziolid A showed weaker cytotoxicity, its effect in alleviating MNNG-induced cytotoxicity was significantly less than that of the BE group.

[0044] Early characteristics of PARthanatos include upregulation of PARP1 activation and increased PAR modification following DNA damage. To elucidate the effect of BE on PARylation after MNNG exposure, PC9 cells were pretreated with BE or the positive control olaparib (OLA) for 24 h, followed by exposure to MNNG for 15 min to induce PARthanatos; cells were collected 1 h after DNA damage, and total protein was extracted for analysis. Results are as follows... Figure 5 As shown, Figure 5Figures A and B show the results of immunoblotting and quantitative analysis of PAR levels in PC9 cells after 24 h of BE pretreatment followed by MNNG (60 μM, 15 min) and 30 min of cell collection. The results show that BE significantly inhibited MNNG-induced PAR overactivation, with an effect comparable to that of the PARP-1 inhibitor OLA.

[0045] Figure 5 Figures C-E show the results of further validation of cellular-level immunofluorescence colocalization. Figure C shows the quantitative analysis results of PAR fluorescence intensity in Figure D, corrected with DAPI (P<0.0001 vs. MNNG). Figure D shows the confocal image of PAR obtained by immunofluorescence staining after PC9 cells were pretreated with BE for 24 h and treated with MNNG for 15 min, with a scale bar of 25 μm and a magnified scale bar of 5 μm. Figure E shows the three-dimensional analysis image of PAR immunofluorescence changes in Figure D. Figure 5 Figures C-E show that BE effectively antagonizes MNNG-induced PARP1 hyperactivation and subsequent increase in PARylation, similar to the established PARP1 inhibitor OLA. Given BE's strong inhibition of MNNG-induced PARylation, it is speculated that BE may be a novel PARP-1 inhibitor.

[0046] Initial Western blot analysis showed no change in total PARP-1 protein levels in cells treated with BE or OLA (see [link to analysis]). Figure 3 (Figures C and D). Subsequently, immunofluorescence images were used to dynamically assess the subcellular localization and fluorescence intensity of PARP-1 at different time points after MNNG treatment. The results are shown in Figure C and Figure D. Figure 6 As shown in Figures A and B. Figure 6 Image A shows PC9 cells pretreated with BE for 24 h, then treated with MNNG for 15 min, and immunofluorescence stained with PARP1; confocal image, scale bar 25 μm, magnified image scale bar 5 μm. Figure 6 Figure B in the middle section shows the quantitative analysis results of PARP1 fluorescence intensity in Figure A, corrected with DAPI (****P<0.0001 vs. DMSO, ####P<0.0001 vs. MNNG). Figure 6Figures A and B show that after BE or OLA pretreatment, PARP-1 protein changed from a diffuse nuclear distribution to aggregation at DNA damage sites. This phenomenon is consistent with the understanding that PARP1 binds to damage sites, undergoes conformational changes, anchors DNA, and enhances catalytic activity to mediate its own and its substrate's parylation. After parylation, PARP dissociates from DNA and changes to an inactive conformation; conversely, PARP1 inhibitors retain PARP1 at the damage site, preventing its release. To determine whether BE has a similar effect to OLA, its influence on MNNG-induced DNA damage repair was investigated. Time-series immunofluorescence assays were used to dynamically monitor the changes in the DNA damage marker γ-H2AX at 1, 6, and 18 h after damage. The results are shown below. Figure 6 Figures C and D are shown in the image. Figure C is a confocal image of PC9 cells after BE pretreatment for 24 h, MNNG treatment for 15 min, and immunofluorescence staining for γ-H2AX. The scale bar is 25 μm, and the scale bar of the magnified image is 5 μm. Figure D is the quantitative analysis result of γ-H2AX fluorescence intensity in Figure C, corrected with DAPI (nsP>0.05 vs. DMSO, **P<0.01 vs. MNNG, nsP>0.05 vs. MNNG). Figure 6 Figures C and D show that BE treatment did not significantly reduce the number of γ-H2AX focal points or fluorescence intensity, confirming that BE, similar to OLA, inhibits the repair of MNNG-induced DNA damage by suppressing PARP1 activity, thus confirming that BE is a PARP1 inhibitor. Figure 7 The results of the interaction analysis between BE and PARP1 are shown in Figure A, which shows the representative structure after BE and PARP1 molecular docking; Figure B shows the interaction diagram of BE and PARP1 amino acid residues generated by LigPlot+; Figure C shows the Western blot detection of PARP1 thermostability after PC9 cells were pretreated with 10 μMBE or DMSO for 24 h (n=3); Figure D shows the ImageJ quantitative results of the band intensity in Figure C (n=3) (**P<0.01BEvs.DMSO, ****P<0.0001BEvs.DMSO); Figure E shows the KD value of BE and PARP1 protein determined by SPR.

[0047] Molecular docking analysis showed that the optimal conformational binding energy was 7.48 kcal / mol (see) Figure 7 (Figures A and B). Crotonin E is anchored to NAD+ of PARP-1 via multiple hydrogen bonds and hydrophobic interactions. +Catalytic pockets: BEO3 (C17) is bonded to PARP1Tyr896N; BEO6 (C10) is bonded to PARP1His862NE2; BEO7 (C7) is bonded to PARP1Asp770OD1 and Asn868OD1; BEO6 (C14) is bonded to PARP1Ser864OG and Asn868OD1; BEO9 (C18) is bonded to PARP1Tyr907O. Figure 7 The results of the middle B diagram show that BE occupies NAD. + The PARP1 catalytic pocket forms five hydrogen bonds at all four subsites (nicotinamide, ribose, pyrophosphate, and adenine), stabilizing the pocket's D ring through bonding with Tyr907, thus rendering the PARP1 catalytic pocket an inactive "dead space" and achieving nanomolar-level high-efficiency inhibition. Thermostability experiments of PC9 cell lysates pre-incubated with BE or DMSO for 24 h confirmed that BE binds to PARP1 and enhances its thermostability (see...). Figure 7 (Figures C and D in the image). Human PARP1 protein was immobilized on a CM5 chip using a Biacore T200 microarray, and binding affinity was measured. The results are shown below. Figure 7 As shown in Figure E, the results indicate that the interaction between BE and PARP1 follows a fast binding / slow dissociation pattern, with affinity increasing with increasing BE concentration. The equilibrium dissociation constant is 8.43 × 10⁻⁶. -6 M.

[0048] To further elucidate the molecular mechanism by which BE inhibits MNNG-induced PARthanatos, the JC-1 fluorescent probe was used to assess changes in mitochondrial membrane potential (ΔΨm) 18 h after DNA damage. Furthermore, immunofluorescence confocal microscopy was used to detect nuclear translocation of AIF 18 h after MNNG treatment. The results are as follows: Figure 8 As shown, Figure 8 Figure A shows a typical image of PC9 cells treated with WS for 24 h, then treated with MNNG for 15 min and cultured for another 18 h, stained with JC-1 (scale bar 25 μm); Figure B shows a representative confocal image with DAPI as the nuclear label, showing the effect of BE on AIF translocation 18 h after MNNG treatment (scale bar 25 μm); Figure C shows the fluorescence analysis results of AIF nuclear translocation induced by MNNG in Figure B.

[0049] Figure 8 The results in Figure A show that MNNG exposure led to a significant collapse of ΔΨm, while BE intervention significantly alleviated this effect. Figure 8 Figures B and C show that BE effectively blocks the translocation of AIF from mitochondria to the nucleus. In summary, BE exerts its anti-PARthanatos effect by maintaining mitochondrial functional integrity and blocking AIF-mediated nuclear signaling.

[0050] Example 2: Detection of the efficacy of crotonin E in an animal stroke model, the method is as follows: (1) Experimental method: Construction of animal stroke models: Male C57BL / 6J mice (7-8 weeks old) were purchased from Henan Skobes Biotechnology Co., Ltd. All procedures complied with ARRIVE guidelines, national regulations, and were approved by the Medical Ethics Committee of Wannan Medical College (WNMC-AWE-2025489). Mice were anesthetized with 1.5% isoflurane, and a 0.5 cm incision was made between the ear and eye. The fascia and muscles were bluntly dissected to expose the distal middle cerebral artery. The skull was thinned to translucent using a micro-drill (avoiding penetration), the artery was electrocoagulated and occluded, the incision was sutured, and the mice were kept warm until fully awake.

[0051] Brain slice immunofluorescence: Brain slices were selected, washed with PBS, permeabilized with 0.3% Triton X-100 PBT for 1.5 h (37°C), microwave antigen retrieval (high heat for 2 min, low heat for 14 min, sodium citrate buffer), naturally cooled, blocked at 37°C for 1 h, incubated with primary antibody at 4°C overnight, washed with PBT for 7 min × 4, incubated with fluorescent secondary antibody in the dark for 2 h, washed with PBT for 7 min × 4, and mounted with anti-quenching agent.

[0052] TTC staining: Preheat TTC solution (0.5g TTC dissolved in 50mL PBS) to 40°C. 24 hours after occlusion, brain tissue was harvested, rinsed with physiological saline, and rapidly frozen in a brain matrix at -80°C until sclerosis. 1mm thick sections were cut, incubated with CTTC at 40°C for 7 minutes, flipped, and incubated for another 4 minutes. The sections were then fixed overnight in 4% paraformaldehyde at 4°C and photographed the next day. Normal tissue appeared reddish-pink, while infarcted tissue remained white.

[0053] Fluoro-JadeC staining: Brain slices were rinsed with PBS, dehydrated for 5 min with 80% ethanol + 150 μL A solution (FJC kit), 2 min with 70% ethanol, 2 min with distilled water, incubated for 10 min with 350 μL distilled water + 150 μL B solution, 2 min with distilled water, incubated for 10 min in the dark with 350 μL distilled water + 150 μL C solution + 150 μL DPI, 1 min with distilled water × 3, dried at 55°C for 5 min before imaging.

[0054] SPR method: Experiments were conducted at 25°C on a BIAcore T200 using a CM5 sensor chip. Ligands were immobilized after EDC / NHS activation, blocked with ethanolamine, and kinetics were monitored over multiple cycles. The regeneration solution was 10 mM glycine-HCl, pH 2.0. Data were fitted to a 1:1 Langmuir model using Biacore Insight Evaluation software and exported to Origin7 for plotting. The PARP1 protein was a human sf9 expression His-tagged protein (MCE, catalog number HY-P74652).

[0055] Experimental Units and Group Design: The experimental unit was an individual mouse. The neuroprotective effect was evaluated by comparing the model group and the drug group. TTC staining was used to determine the 24-hour infarct volume (%). The experiment included a model group and a drug group, with n=3 in each group, for a total of 24 mice; n=3 is the minimum scientific number required to obtain a reliable variance estimate according to the 3R principle (replacement, reduction, optimization).

[0056] Randomization and Blinding: Six pdMCAO mice were randomly divided into two groups (n=3 in each group). To reduce environmental bias, cage sizes were balanced across treatment groups. A double-blind approach was implemented: sampling personnel performed group concealment, while the model preparation personnel were blinded during the procedure.

[0057] Statistical analysis: All experiments were repeated at least three times. Data are expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups, and t-tests were used for comparisons between two groups. P < 0.05 was considered statistically significant.

[0058] (2) Experimental results Figure 9 The experimental results show that electrocoagulation (BE) effectively inhibited neuronal loss in a mouse model of permanent cerebral ischemia. Figure A is a flowchart of the electrocoagulation ischemic stroke model; Figure B shows representative cerebral infarction images stained with TTC 4.5 h after BE administration and 24 h after ischemia; Figure C is a statistical chart of TTC staining: percentage of ischemic area to hemispheric area (n=3) (P<0.001 vs. Model); Figure D shows FJC staining images 24 h after BE administration and 4.5 h after ischemia (blue fluorescence indicates nuclei, green fluorescence indicates FJC-positive degenerated neurons; scale bar, 100 μm); Figure E shows the statistical analysis results of the FJC-positive neuron ratio (n=3) (P<0.001 vs. Model). Figure 9 As shown in Figure A, this invention establishes a permanent ischemia model in mice by electrocoagulation occlusion of the distal middle cerebral artery to systematically evaluate the protective effect of BE against cerebral ischemia-reperfusion injury. In the treatment group, a single intraperitoneal injection of BE 10 mg / kg was administered 4.5 h after occlusion; the infarct area was assessed 24 h after occlusion using 2,3,5-triphenyltetrazolium (TTC) staining (results are shown in Figure A). Figure 9(As shown in Figures B and C), the results showed that the infarct volume in the BE treatment group was significantly smaller than that in the model group, indicating strong protection in the acute phase. To assess the impact of ischemic injury on cortical neurons, Fluoro-Jade C (FJC) staining was used to identify degenerated neurons, and the results are as follows: Figure 7 As shown in Figures D and E, ischemic injury led to a significant increase in FJC-positive cells, while neuronal death in the ischemic area was significantly reduced in the BE-treated group, indicating a significant neuroprotective effect.

[0059] Figure 10 The results show the effects of BE on the expression of PARthanatos-related proteins in a mouse model of permanent cerebral ischemia. Figure 10 Figure A shows the protein extraction sites and immunofluorescence detection sites in mouse brain slices; Figure B shows the CD31 immunofluorescence detection in frozen sections of the coronary brain 24 hours after ischemia (scale bar 50 μm); Figure C shows the statistical analysis results of the number of CD31-positive microvessels in frozen sections 24 hours after ischemia (n=3) (P<0.001 vs. Model); Figure D shows the AIF immunofluorescence detection in frozen sections of the coronary brain 24 hours after ischemia (scale bar 50 μm); Figure E shows the AIF translocation immunofluorescence analysis in frozen sections 24 hours after ischemia; Figure F shows the statistical analysis results of the number of AIF nuclear translocation events in frozen sections 24 hours after ischemia (n=3). (**P<0.0001 vs. Model); Figure G is a confocal image obtained from γ-H2AX immunofluorescence detection of frozen sections of the coronary brain 24 h after ischemia, scale bar 50 μm; Figure H is a three-dimensional analysis image of γ-H2AX immunofluorescence in Figure G; Figure I is the statistical analysis result of γ-H2AX fluorescence intensity in Figure G, average fluorescence intensity of γ-H2AX / average fluorescence intensity of DAPI (n=3) (nsp>0.05 vs. Model); Figure J is a confocal image obtained from PARP1 immunofluorescence detection of frozen sections of the coronary brain 24 h after perfusion for brain harvesting, scale bar 50 μm; Figure K shows the three-dimensional immunofluorescence analysis image of PARP1 in Figure J; Figure L shows the statistical analysis results of PARP1 fluorescence intensity in Figure J, with the average fluorescence intensity of PARP1 / average fluorescence intensity of DAPI (n=3) (*P<0.0001 vs. Model). Figure M shows the confocal image obtained from the PAR immunofluorescence detection of frozen sections of the coronary brain 24 hours after perfusion following ischemia, with a scale bar of 50 μm; Figure N shows the three-dimensional immunofluorescence analysis image of PAR mentioned above; Figure O shows the statistical analysis results of PAR fluorescence intensity in Figure M (n=3, **P<0.0001 vs. Model).

[0060] Take brain slices from the same anatomical location ( Figure 10The analysis (as shown by the arrow in Figure A) was conducted to further investigate the changes in PARP1 and PARthanatos-related proteins in a permanent ischemia model after BE treatment. Brain tissue was perfused 24 hours after occlusion, and immunofluorescence analysis showed that the expression of the endothelial marker CD31 in the BE group was significantly higher than that in the Vehicle ischemia group (see Figure A). Figure 10 (Figures B and C in the image) This result suggests that BE effectively inhibits cerebral microvascular endothelial cell death, which is beneficial for subsequent vascular recanalization or collateral reconstruction and promotes the restoration of blood flow in the ischemic area. Confocal imaging showed that the AIF in the ischemic area of ​​the Vehicle group had translocated to the nucleus, and BE treatment inhibited AIF nuclear input, confirming its blocking of AIF-mediated PARthanatos (see Figure B and C in the image). Figure 10 (Figures E and F). The DNA double-strand break marker γ-H2AX remained at high levels in both groups (see Figure E). Figure 10 (Figure G-I) indicates that BE did not alleviate neuronal DNA double-strand breaks; however, the fluorescence intensity of poly-ADP-ribose (PAR) in the damaged area decreased after BE treatment (see Figure G-I). Figure 10 (M-O diagram), PARP1 intensity decreases and accumulates in the lesion focus (see M-O diagram). Figure 10 (Figures J-L) These experimental results are consistent with in vitro data. In summary, BE penetrates the blood-brain barrier and inhibits PARthanatos at the lesion site: it inhibits PARP1 activity in the early post-ischemic period, reduces protein PARylation, and prevents AIF nuclear translocation, thereby blocking PARthanatos, promoting endothelial and neuronal survival, and providing multi-faceted protection for subsequent neurological function recovery.

[0061] Evidence-based neuroprotective agents for ischemic stroke remain lacking, primarily due to the absence of established targets such as calcium channels, NMDA receptors, and free radicals, which are often located in the "middle and lower" of the ischemic cascade. By the time drugs reach the lesion, cell death has often already crossed the "irreversible" threshold. Once PARthanatos is activated, other inhibitors of cell death pathways become ineffective. Therefore, discovering new PARthanatos inhibitors may be key to achieving neuroprotection in ischemic stroke. Clinically, PARP inhibitors such as olaparib and veriparib have not been adopted for stroke treatment due to insufficient central penetration and hematological toxicity. This invention bypasses the "skeletal bottleneck" of synthetic chemistry and, for the first time, identifies crotonin E (BE), a tetracyclic triterpenoid lactone, as a PARP1 inhibitor using a traditional Chinese medicine monomer library. Its skeleton is completely different from that of known PARP inhibitors (nicotinamide, naphthidone, quinoxalone, tricyclic heterocyclic compounds, and benzoxazinone). BE is a 412 Da tetracyclic triterpenoid-β-lactone-α,β-unsaturated ketone-polyhydroxy cage structure. Molecular docking shows that it occupies NAD through seven hydrogen bonds. + Subsite binding energy 7.48 kcalmol -¹, SPR showed fast binding / slow dissociation, KD≈8.4μM, and a dissociation phase half-life>30min, approaching the timescale of covalent inhibitors while remaining reversible, suggesting long-lasting protection with a single dose. In vitro, in PC9 and SH-SY5Y cells, BE inhibited MNNG-induced PAR accumulation, mitochondrial membrane potential collapse, and Sytox-positive cell death, with an EC50 of 798nM, a therapeutic window of 1-10μM, and toxicity only appearing at >30μM. In a mouse model of permanent distal middle cerebral artery occlusion, a single intraperitoneal injection of 10mg / kg (4.5h post-ischemia) reduced infarct volume by 40% and FJC-positive degenerated neurons by 50%, without adverse effects on cerebral blood flow, and CD31... + Increased microvascular density suggests preservation of vascular endothelium, establishing a "tissue reserve" for subsequent recanalization or collateral circulation. Compared to the PSD-95 interfering peptide nerinetide, which requires recanalization to be effective, and the NMDA antagonist aptiganel, which has cardiovascular side effects, BE does not require recanalization, has no sedation or cardiovascular side effects, and is suitable for use in combination with thrombolysis / thrombectomy.

[0062] This invention is the first to introduce the natural product quassin into the stroke intervention stage, proposing a patentable and transferable paradigm that "neuroprotection must be moved forward to the irreversible node." Subsequent systematic research will be conducted on structural optimization, treatment time window expansion, immune safety, and clinical biomarkers, in order to address the current lack of evidence-based neuroprotective agents for ischemic stroke and provide a general technology platform for other DNA damage-related neurodegenerative diseases such as Parkinson's disease and neonatal hypoxic-ischemic encephalopathy.

Claims

1. Application of crotonin compounds containing quassinolide nuclei in the preparation of drugs that inhibit PARthanatos.

2. The application according to claim 1, characterized in that, The crotonin class of compounds includes at least one of crotonin E, Pasakbumin B, 13,21-dihydrocaproic acid ketone, crotonin D, and crotonin lactone A.

3. The application according to claim 1, characterized in that, The PARthanatos referred to are PARthanatos caused by ischemic stroke.

4. Application of crotonin E in any of the following: (1) Preparation of drugs for the treatment of stroke; (2) Preparation of drugs for the treatment of cerebral ischemia-reperfusion injury; (3) Preparation of poly(ADP-ribose) polymerase 1 inhibitor; (4) Preparation of drugs to prevent the accumulation of PAR-modified proteins; (5) To prepare drugs that slow down the collapse of mitochondrial membrane potential; (6) Prepare drugs that inhibit AIF nuclear translocation.

5. The application according to claim 4, characterized in that, The stroke mentioned is an acute ischemic stroke.

6. A poly(ADP-ribose) polymerase 1 inhibitor containing crocin E.

7. A PARthanatos inhibitor containing crocin E.

8. A stroke treatment drug containing crotonin E.

9. The drug or inhibitor according to any one of claims 6-8, characterized in that, The concentration of crotonin E is at least 800 nM.

10. The drug or inhibitor according to claim 9, characterized in that, The concentration of crotonin E is 1-30 μM.

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