Use of rgds polypeptide in inhibiting neuronal ferroptosis after cerebral hemorrhage

Abstract

The application discloses application of an RGDS polypeptide in inhibition of neuron ferroptosis after cerebral hemorrhage. The application is a key time window of ferroptosis and iron overload 24 hours after cerebral hemorrhage, at which time the expression of fibronectin is significantly up-regulated, the expression / activity of glutathione peroxidase 4 (GPX4) is inhibited, and neuron ferroptosis is promoted; after intervention of the fibronectin activity inhibitor RGDS polypeptide, the inhibition on GPX4 can be removed, the expression level of GPX4 is restored, and iron deposition is reduced, so that neuron ferroptosis can be significantly inhibited. Meanwhile, the RGDS intervention can significantly improve short-term neurological function (corner test, modified Garcia score) and spatial memory ability (Y maze test) after cerebral hemorrhage. The application provides a new drug target and a clinical application scheme for treatment of cerebral hemorrhage, and has high application value.

Description

Technical Field

[0001] This invention relates to peptides and the field of peptides, and more particularly to the application of RGDS peptides in inhibiting neuronal ferroptosis after cerebral hemorrhage. Background Technology

[0002] Intracerebral hemorrhage (ICH) is a subtype of stroke with high mortality and disability rates, and currently effective clinical treatment strategies remain relatively limited. Its pathological mechanism involves a cascade of primary and secondary injuries, with progressive neuronal death in the peri-hematoma area being the core driving factor for neurological deterioration.

[0003] Ferroprelation—an iron-dependent regulated cell death characterized by increased lipid peroxidation and glutathione depletion—is a key mechanism of neuronal loss following intracerebral hemorrhage. Glutathione peroxidase 4 (GPX4), a crucial antioxidant enzyme combating ferroprelation, is exacerbated by decreased activity or expression. Excess free iron released from hematoma degradation initiates the Fenton reaction, producing cytotoxic reactive oxygen species (ROS), which in turn inhibit GPX4 activity, weaken cellular antioxidant capacity, and ultimately trigger neuronal ferroptosis, further aggravating brain injury. However, the specific molecular mechanisms connecting the pathological microenvironment of intracerebral hemorrhage with the initiation, amplification, and GPX4 regulation of neuronal ferroptosis remain unclear.

[0004] In existing technologies, protein expression analysis of the brain parenchyma surrounding the hematoma in a cerebral hemorrhage model revealed a significant upregulation of fibronectin expression. Fibronectin is a high-molecular-weight glycoprotein whose expression increases after tissue injury and participates in cell adhesion, migration, and inflammation regulation.

[0005] However, its specific role in the hematoma microenvironment after cerebral hemorrhage, particularly its role in regulating ferroptosis, remains unclear. Therefore, there is an urgent need to find drugs that can effectively regulate fibronectin and inhibit neuronal ferroptosis to improve the prognosis of patients with cerebral hemorrhage.

[0006] Therefore, this invention proposes the application of RGDS peptide in inhibiting neuronal ferroptosis after cerebral hemorrhage. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and to propose the application of RGDS peptide in inhibiting neuronal ferroptosis after cerebral hemorrhage.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides the application of RGDS peptides in the preparation of drugs that inhibit neuronal ferroptosis after cerebral hemorrhage.

[0009] Furthermore, the RGDS polypeptide inhibits neuronal ferroptosis by restoring the expression / activity of glutathione peroxidase 4 (GPX4) by inhibiting fibronectin function.

[0010] Furthermore, the RGDS peptide reduces iron overload by decreasing iron deposition after cerebral hemorrhage, thereby inhibiting neuronal ferroptosis.

[0011] This invention also provides the application of RGDS peptides in the preparation of drugs to improve neurological function after cerebral hemorrhage.

[0012] Furthermore, the improvement in neural function includes improving sensorimotor integration function and overall neural function.

[0013] Furthermore, the improved sensorimotor integration function was assessed using a turning angle test, and the overall neural function was assessed using a modified Garcia score.

[0014] This invention also provides the application of RGDS peptides in the preparation of drugs to improve spatial memory and cognitive function after cerebral hemorrhage.

[0015] Furthermore, the improvement in spatial memory and cognitive function was assessed through a Y-maze neo-arm experiment, which showed an increase in the number of times one entered a neo-arm and the time spent in a neo-arm.

[0016] The present invention also provides a method for inhibiting neuronal ferroptosis after cerebral hemorrhage, comprising administering an effective amount of RGDS peptide to the subject.

[0017] The present invention also provides a method for improving neurological function after cerebral hemorrhage, comprising administering an effective amount of RGDS peptide to the subject.

[0018] The present invention also provides a method for improving spatial memory and cognitive function after cerebral hemorrhage, comprising administering an effective amount of RGDS peptide to the subject.

[0019] Furthermore, the subject is preferably a mammal in the acute phase of cerebral hemorrhage (e.g., 24 hours after cerebral hemorrhage), and more preferably a mouse.

[0020] Furthermore, the RGDS polypeptide can be administered via intracranial injection, intraperitoneal injection, nasal administration, or other suitable routes of administration, and the specific dosage can be adjusted according to actual circumstances.

[0021] The beneficial effects of this invention are as follows: 1. This invention clarifies for the first time that RGDS peptides inhibit neuronal ferroptosis after cerebral hemorrhage by targeting fibronectin and restoring GPX4 function, thus providing a new molecular target for the treatment of cerebral hemorrhage. Attached Figure Description

[0022] Figure 1The images show the brain water content measurements taken at 6, 12, 24, 48, and 72 hours after ICH modeling in this embodiment of the invention. A. Brain water content measurement after cerebral hemorrhage; B. Free hemoglobin measurement after cerebral hemorrhage. Figure 2 The images shown are the results of Western blotting of proteins after cerebral hemorrhage in this embodiment of the invention. A. Representative Western blotting bands; B. Statistical histogram of the BA plot; C. Representative immunofluorescence image (scale bar = 50 μm). Figure 3 The images shown are from an embodiment of the present invention and illustrate the results of RGDS peptide inhibiting neuronal ferroptosis after cerebral hemorrhage. A. A representative Western blotting image; B. A statistical bar chart; C. A representative coronal section of brain tissue; D. A representative Prussian blue staining image (scale bar = 500 μm); E. A representative immunofluorescence double-labeled image, with green representing GPX4 and red representing NeuN (scale bar = 50 μm). Figure 4 The following are graphs showing the results of RGDS improving short-term spatial memory and cognitive function in mice after cerebral hemorrhage in this embodiment of the invention: A. Statistical histogram of modified Garcia score (n=8 per group); B. Statistical histogram of corner turning test (n=8 per group); C. Representative movement trajectory of mice in Y maze; D. Statistical histogram of the number of times mice entered the new arm (n=6 per group); E. Time spent by mice in the new arm (n=6 per group). Detailed Implementation

[0023] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0024] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "setting" should be interpreted broadly. For example, they can refer to a fixed connection or setting, a detachable connection or setting, or an integral connection or setting. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0025] Example 1: Preparation of experimental animals and cerebral hemorrhage models Six- to eight-week-old male C57BL / 6 mice were selected and sourced from the Experimental Animal Center of Chongqing Medical University. The mice were housed in the standardized animal facility of the Neuroscience Research Platform at Chongqing Medical University. The housing environment was strictly controlled with a 12-hour day-night cycle, allowing free access to food and water. The temperature was maintained at 22±2°C and the humidity at 50±10%. All experimental protocols were reviewed and approved by the Experimental Animal Ethics Committee of Chongqing Medical University (IACUC-CQMU-2025-11084), strictly adhering to the "Guidelines for Ethical Review of Experimental Animals" to minimize animal suffering and ensure the ethical and standardized nature of the experiments.

[0026] An ICH model was established using autologous blood injection into the basal ganglia. The specific steps were as follows: Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After anesthesia, the mice were fixed on a stereotaxic apparatus. The head was prepared and disinfected, the scalp was incised along the midline, the skull was dissected, and the anterior fontanelle was exposed. 20 μL of blood was drawn from the tail vein using a microsyringe. The syringe was fixed on the stereotaxic apparatus, and the coordinates of the basal ganglia region (0.2 mm anterior to the anterior fontanelle, 2.0 mm lateral to the midline, depth 3.5 mm) were determined using a mouse brain stereotaxic atlas. A small hole was drilled with a skull drill, and the microsyringe was slowly inserted to the target depth. Injection was performed at a rate of 1 μL / min, stopping for 2 minutes after injecting 10 μL, then continuing until completion. After injection, the injection was paused for 5 minutes, and the syringe was slowly withdrawn. The skull hole was sealed with bone wax, the scalp was sutured, and the incision was disinfected with iodine. Mice in the Sham group underwent only scalp incision, skull drilling, and microsyringe insertion (without autologous blood injection); the remaining procedures were the same as the ICH model group. After modeling, the mice were placed in a warm environment for resuscitation, and their mental state, diet and activity were closely observed. Mice that failed to model (those that showed severe bleeding, death or no neurological deficits) were removed.

[0027] Example 2: Brain water content measurement Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After anesthesia, the mice were decapitated and the brains were removed. The meninges and blood vessels were quickly dissected, and the surface moisture of the brain tissue was blotted dry with filter paper. The wet weight of the brain tissue was immediately measured using an electronic balance (accuracy 0.1 mg). The brain tissue was then placed in a 65°C constant temperature oven and baked for 72 hours until the brain tissue weight stabilized. The dry weight of the brain tissue was measured again. The brain water content of each group of mice was calculated using the formula: Brain water content (%) = [(wet weight - dry weight) / wet weight] × 100%.

[0028] Example 3: Determination of Free Hemoglobin After deep anesthesia, mice were perfused cardiacally with 0.9% saline via the apex of the heart. Following cervical dislocation, the brain was rapidly removed, and the olfactory bulb and cerebellum were discarded. The remaining brain tissue was weighed and homogenized in ice-cold 0.9% saline using an ultrasonic homogenizer. The homogenate was centrifuged, and the supernatant was collected. A microhemoglobin assay kit (A071-1-1, Nanjing Jiancheng) was used, with the chromogenic reagent prepared strictly according to the manufacturer's instructions. For each test, 150 μL of supernatant was mixed with 2.5 mL of chromogenic reagent (blank: 150 μL distilled water plus 2.5 mL of chromogenic reagent). After thorough vortexing, the reaction mixture was incubated at 37°C for 20 minutes, and the absorbance at 510 nm was recorded.

[0029] Example 4: Protein Immunoblotting Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After anesthesia, 0.9% saline was perfused through the apex of the heart until the liver turned pale. The mice were then euthanized by cervical dislocation, and brain tissue from the right cerebral hemisphere was rapidly extracted. The tissue was placed in pre-chilled RIPA lysis buffer (containing protease and phosphatase inhibitors), sonicated on ice, and homogenized for 30 minutes. It was then centrifuged at 12000 rpm for 30 minutes at 4°C, and the supernatant was collected as the total protein extract. Protein concentration was determined using a BCA protein quantification kit, and all sample protein concentrations were standardized to a uniform level. 30 μg of standardized protein sample was added to 5xSDS loading buffer, denatured at 95°C for 5 minutes, cooled to room temperature, and loaded onto a 10%–15% SDS-PAGE gel. Electrophoresis was performed at constant voltage (80V for the stacking gel and 120V for the separating gel) until the bromophenol blue indicator reached the bottom of the gel. After electrophoresis, the proteins in the gel were transferred to an activated PVDF membrane using a wet transfer method. After transfer, the PVDF membrane was placed in 5% skim milk and blocked on a shaker at room temperature for 2 hours. After blocking, it was washed three times with PBST buffer, 5 minutes each time. Primary antibody was then added, and the membrane was incubated overnight at 4°C on a shaker. The next day, the PVDF membrane was washed three times with PBST buffer, 5 minutes each time. HRP-conjugated secondary antibody was added, and the membrane was incubated on a shaker at room temperature for 1 hour. It was then washed three more times with PBST buffer, 5 minutes each time. After washing, the PVDF membrane was placed in ECL chemiluminescence solution, and protein bands were imaged using a gel imaging system with chemiluminescence. ImageJ software was used for quantitative analysis of the band grayscale values.

[0030] Example 5: Immunofluorescence labeling Mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (50 mg / kg). After anesthesia, 0.9% saline and 4% paraformaldehyde were perfused sequentially through the apex of the heart. After perfusion, the mice were euthanized by cervical dislocation, the cerebellum was removed, and the intact brain tissue was extracted and fixed overnight in 4% PFA solution at 4°C. After fixation, the tissue was sent to Seville for paraffin embedding. 5 μm thick brain slices were cut using a paraffin microtome, mounted on poly-L-lysine-coated slides, and air-dried at room temperature. The slides were baked at 65℃ for 2 hours, dewaxed twice with xylene for 20 minutes each time, and then hydrated in a gradient of 100%, 95%, 85%, and 75% ethanol, respectively. Antigen retrieval was then performed using sodium citrate for 15 minutes. After cooling to room temperature, 0.5% Triton X-100 permeabilization buffer was added, and the slides were incubated at room temperature for 10 minutes. The slides were washed three times with PBS for 5 minutes each time. 5% BSA blocking buffer was added, and the slides were blocked at room temperature for 1 hour. The blocking buffer was discarded, and primary antibody was added. The slides were then placed in a humidified chamber and incubated overnight at 4℃. The next day, the humidified chamber was removed, and the slides were warmed to room temperature for 30 minutes. The slides were washed three times with PBS buffer for 5 minutes each time to remove unbound primary antibody. Diluted fluorescent secondary antibody was added and incubated at room temperature in the dark for 1 hour. The slides were washed three times with PBS for 5 minutes each time. DAPI was added to mount the slides, and images were taken under a fluorescence microscope. Quantitative fluorescence intensity analysis was performed using ImageJ software.

[0031] Example 6: Corner Experiment Twenty-four hours after a brain hemorrhage, mice underwent a corner-turning test to assess sensorimotor integration. The experimental setup consisted of two gray acrylic panels (30cm × 20cm × 1cm) connected at a 30° angle, with a 5mm slit at the top. Before the experiment, mice were placed in a quiet experimental room for acclimatization for 10 minutes. A researcher unaware of the experimental groupings then gently pushed the mouse into the angle between the two panels until both whiskers touched the panel walls. Each mouse underwent 10 consecutive trials with intervals ≥30 seconds, and the number of right turns was recorded. The formula used was: Right Turn Percentage = (Number of Right Turns / Number of Effective Trials) × 100%. A higher right turn percentage indicated a more severe sensorimotor integration deficit. At least eight mice were tested in each group, and the data were independently analyzed by two researchers unaware of the groupings.

[0032] Example 7: Improved Garcia Score Twenty-four hours after cerebral hemorrhage, mice were assessed using a modified Garcia score to evaluate overall neurological function. Before the experiment, mice were transferred to a quiet behavioral testing room and allowed 10 minutes to acclimatize. Two researchers, unaware of the experimental group assignments, jointly evaluated the mice. The score comprised six items: spontaneous movement, body symmetry, forelimb extension, cage grasping / climbing ability, bilateral body tactile responses, and bilateral whisker tactile responses. Each item was scored from 0 to 3 points, for a total score of 0 to 18 points. Higher scores indicated better neurological recovery. At least eight mice were tested in each group, and the average score from the two evaluators was used as the final score to ensure the objectivity of the results.

[0033] Example 8; Prussian blue staining Paraffin sections were dewaxed and rehydrated as described above, then stained with Prussian blue (G1428, Solarbio) strictly according to the manufacturer's instructions. The kit was equilibrated at room temperature (25-30°C) for 20 minutes, preheated to 37°C in a humidified chamber, and freshly prepared Perls working solution (1:1) was used to completely cover the sections and incubated at 37°C for 20 minutes. The slides were then gently rinsed three times with distilled water (10 seconds each time), covered with the incubation solution, and returned to the 37°C chamber for 10-20 minutes. After washing three times in 1×PBS for 60 seconds, the enhancing working solution (C1:C2:1xPBS=1:1:18) was layered onto the tissue and incubated again at 37°C for 10-20 minutes. When counterstaining was required, the sections were treated three times. A rapid immersion in 1xPBS for 5 seconds, staining for 3-5 minutes, a brief rinse, and then immersion in distilled water for 10 minutes were performed. Finally, the slides were dehydrated with graded ethanol, cleared with xylene, and sealed with neutral resin.

[0034] Example 9: Y-maze experiment Seven days after ICH, a Y-maze test was conducted to assess the spatial and short-term memory of mice. After a 10-minute acclimatization period, the test was conducted in a quiet, dimly lit room, with the experimenter blinded to the group task. The gray PVC Y-maze consisted of three identical arms (30 cm long × 8 cm wide × 15 cm high) arranged at a 120° angle, named the neoplasm arm, the initiating arm, and the other arms. During the sampling phase, the neoplasm arm was covered by a removable gray partition. Each mouse was placed at the end of the initiating arm and allowed free exploration of the initiating arm and the other arms for 5 minutes. Afterward, the animals were returned to their cages, and all arms were wiped with 75% ethanol and dried to eliminate odor cues. The partition was removed after a 2-hour test interval. The testing phase then began, with the mice again placed in the initiating arm and given 8 minutes of free access to all three arms. An overhead camera connected to Y-maze software (SMART-2000, San Diego Instruments) automatically recorded the data and time for each arm. The key metrics are the number of times a new arm is entered and the cumulative duration spent in the new arm.

[0035] Example 10: RGDS Peptide Intervention Experiment After the cerebral hemorrhage model was successfully established, RGDS peptide intervention was immediately administered (nasal administration, 5 mg / kg per mouse). The above-mentioned indicators were measured at different time points after intervention (e.g., 24 hours, 7 days).

[0036] result The 24-hour window following a brain hemorrhage is a critical time window for ferrodeogenesis and iron overload: brain water content reaches its highest level within 24 hours after a brain hemorrhage, and the accumulation of free hemoglobin also reaches its maximum within 24 hours.

[0037] Increased fibronectin expression after cerebral hemorrhage: Western blotting and immunofluorescence results showed that fibronectin expression was significantly upregulated 24 hours after cerebral hemorrhage, and it was more widely distributed in the brain parenchyma around the hematoma.

[0038] Inhibition of fibronectin improves neuronal ferroptosis: RGDS intervention can significantly reduce the area of ​​hemorrhage foci, restore GPX4 protein expression, reduce the area of ​​iron deposition, and increase the proportion of GPX4 positive neurons, thereby inhibiting neuronal ferroptosis.

[0039] RGDS intervention improves short-term neurological function and spatial memory: RGDS intervention can significantly improve modified Garcia scores, reduce the percentage of right turns in the corner test, increase the number of entries and dwell time in the novel arm in the Y maze, and improve neurological and cognitive functions.

[0040] Example 11: Data Analysis Data are expressed as mean ± SEM of joint independent experiments. For comparisons between two groups, unpaired two-tailed Student's t-tests were used to analyze datasets that met the assumptions of normality and homogeneity of variance. Multiple group comparisons were performed using one-way or two-way ANOVA, with Bonferroni correction applied to post-hoc comparisons where appropriate. A p-value less than 0.05 was defined as statistically significant, and results not meeting this threshold were labeled "ns". All statistical analyses were performed using GraphPad Prism software.

[0041] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. Application of RGDS peptides in the preparation of drugs that inhibit neuronal ferroptosis after cerebral hemorrhage.

2. The application according to claim 1, characterized in that, The RGDS polypeptide inhibits neuronal ferroptosis by suppressing fibronectin function and restoring the expression / activity of glutathione peroxidase 4GPX4.

3. The application according to claim 1 or 2, characterized in that, The RGDS peptide reduces iron overload by decreasing iron deposition after cerebral hemorrhage, thereby inhibiting neuronal ferroptosis.

4. The application according to claim 1, characterized in that, The cerebral hemorrhage is a basal ganglia hemorrhage, preferably an autologous blood injection-induced cerebral hemorrhage model.

5. The application according to claim 1, characterized in that, The drug is administered 1 hour after cerebral hemorrhage, which is a critical time window for ferroptosis and iron overload following cerebral hemorrhage.

6. Application of RGDS peptides in the preparation of drugs to improve neurological function after cerebral hemorrhage.

7. The application according to claim 6, characterized in that, The improvement in neurological function includes improved sensorimotor integration, which is assessed by a turning test and is manifested as a reduction in the percentage of right turns.

8. The application according to claim 6, characterized in that, The improvement in neurological function includes the improvement of overall neurological function, which is assessed by a modified Garcia score and is characterized by an increase in score.

9. Application of RGDS peptides in the preparation of drugs to improve spatial memory and cognitive function after cerebral hemorrhage.

10. The application according to claim 9, characterized in that, The improved spatial memory and cognitive function were assessed using the Y-maze neo-arm experiment, which showed an increase in the number of times one entered a neo-arm and the time spent in a neo-arm.

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