Use of human plasma exosomes for preventing and treating brain injury related to ferroptosis after ich

Abstract

The application discloses a use of human plasma exosomes for preventing and treating brain injury related to iron death after intracerebral hemorrhage (ICH), wherein the human plasma exosomes are derived from plasma of healthy young people. The exosomes can reduce brain injury by inhibiting cell iron death, can significantly enhance behavior recovery of patients with ICH, and have no obvious side effects, cause no organ toxicity, and have good tolerance and safety.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the use of exosomes from the plasma of healthy young adults in the manufacture of drugs for the prevention and treatment of brain injury associated with ferroptosis following intracerebral hemorrhage (ICH). Background Technology

[0002] Intracerebral hemorrhage (ICH) is a life-threatening disease, accounting for 15%–20% of strokes, with most survivors suffering lifelong disability. The pathological basis for poor prognosis in ICH is primary and secondary brain injury (SBI), characterized by a complex series of pathological processes including oxidative stress, inflammation, and apoptosis. Ferropreservation, a newly discovered cell death process distinct from other cell death mechanisms, plays a crucial role in various brain diseases, including neurodegenerative diseases. It is characterized by the accumulation of reactive oxygen species (ROS) and lipid peroxidation. Particularly in ICH, ferrous iron produced from hematoma degradation contributes to the accumulation of ROS generated by the Fenton reaction, and ferrous iron also acts as a cofactor for lipid peroxidases. Therefore, ROS accumulation and iron deposition after ICH readily induce feropreservation. In fact, recent studies have also suggested that perihematoma feropreservation plays a vital role in post-ICH SBI. Therefore, strategies specifically inhibiting feropreservation after ICH hold great promise.

[0003] Exosomes are small vesicles of 50-150 nm in diameter derived from cells, capable of delivering nutrients and RNA sequences between different cell types. Studies have shown that exosomes derived from mesenchymal stem cells, neural stem cells, astrocytes, and microglia can protect nerve cells from various types of damage, including ICH, through different pathways. However, it remains unclear whether exosomes can protect nerve cells from ferroptosis.

[0004] Exosomes exist in circulation, and their production in plasma (or serum) is far greater than that of exosomes secreted by single cells. Previously, convalescent plasma therapy has been an attractive treatment strategy for infectious and immune diseases. Traditionally, plasma therapy is thought to provide patients with antibodies and nutrition. In recent years, blood-derived exosomes have been found to have various therapeutic effects, especially exosomes derived from the plasma of healthy young adults, which have been reported to show promising applications in the treatment of inflammation and COVID-19. However, the impact and potential of exosomes derived from the plasma of healthy young adults on ICH remain unclear. Therefore, the potential for researching and developing drugs using human plasma exosomes derived from young adults for the prevention and treatment of ICH has significant application and socioeconomic value. Summary of the Invention

[0005] The purpose of this invention is to provide the use of human plasma exosomes in the manufacture of drugs for the prevention and treatment of ferroptosis-related brain injury following intracerebral hemorrhage (ICH). These exosomes are derived from the plasma of healthy young adults and are therefore referred to herein as "human plasma exosomes".

[0006] The present invention provides the use of human plasma exosomes in the manufacture of drugs for the prevention and treatment of ferroptosis-related brain injury following cerebral hemorrhage, wherein the human plasma exosomes are derived from the plasma of healthy young adults.

[0007] Preferably, in the above-described uses of the present invention, the healthy young person refers to a young person aged 18-25 years who has no underlying diseases.

[0008] In the above-described uses of the present invention, the exosomes are extracted and separated from the plasma of young people, preferably from the plasma of young people aged 18-25 years without underlying diseases.

[0009] The present invention also provides a pharmaceutical composition for the prevention and treatment of ferroptosis-related brain injury following cerebral hemorrhage, comprising human plasma exosomes and pharmaceutical excipients, wherein the human plasma exosomes are derived from the plasma of healthy young adults.

[0010] Preferably, in the pharmaceutical composition of the present invention, the healthy young person refers to a young person aged 18-25 years without underlying diseases.

[0011] Preferably, the pharmaceutical composition of the present invention is in the form of an injection.

[0012] The aforementioned injection can be an injectable solution or a lyophilized powder for injection. Its excipients can be conventional injectable excipients used in the art, such as mannitol, lactose, water for injection, physiological saline, glucose, etc. It is prepared using conventional methods in the art, such as mixing extracted exosomes with excipients and water for injection, sterilizing, filling, and optionally lyophilizing.

[0013] The present invention relates to the use of human plasma exosomes, wherein the exosomes can be taken up by neurons and microglia, and can reduce ferroptosis after ICH. This can prevent brain damage and accelerate behavioral recovery in ICH patients.

[0014] The present invention relates to the use of human plasma exosomes derived from healthy young adults, which can effectively reduce ferroptosis in mice after ICH and promote behavioral recovery in ICH patients. Attached Figure Description

[0015] Figure 1This is a characterization diagram of plasma exosomes from healthy young adults. In this diagram, A is a transmission electron microscopy image of plasma exosomes from young adults, B is the Zeta potential of plasma exosomes from young adults, C is the expression of exosome markers CD63, CD9, CD81 and TSG101 in plasma exosomes from young adults, and D is the particle size distribution of plasma exosomes from young adults.

[0016] Figure 2 This is a schematic diagram of the ICH autologous blood injection model and lateral ventricle injection. In this diagram, A shows the location of the ICH autologous blood injection model and lateral ventricle injection in the coronal position of the mouse brain atlas, and B shows the schematic diagram of the mouse ICH autologous blood injection model and the actual location of the lateral ventricle injection.

[0017] Figure 3 This is a distribution and uptake map of human plasma exosomes in mouse brains after ICH (n=3). In this map, A shows the in vivo distribution of DiD-labeled exosomes in the brains of sham-operated mice and ICH mice as shown by confocal imaging, and B shows the uptake of DiD-labeled exosomes by neurons and microglia in vivo, but not by astrocytes as shown by confocal imaging.

[0018] Figure 4 This is a dose-response curve of exosomes derived from plasma of young and elderly individuals in the behavioral recovery of ICH mice;

[0019] Figure 5 This is a diagram showing the effect of plasma exosomes in young people on reducing brain damage and ferroptosis after ICH.

[0020] Figure 6 This is a diagram showing the effect of plasma exosomes in young adults on antagonizing ICH-induced ferroptosis through the SLC7A11 / GPX4 pathway. Detailed Implementation

[0021] The following examples are provided to further understand and illustrate the essence of the present invention, but do not limit the scope of the present invention in any way.

[0022] In this embodiment, the experimental animals C57BL / 6J were purchased from Hunan Enswell Experimental Animal Technology Co., Ltd. Plasma from healthy young adults was collected with the approval of the Ethics Committee of Southwest Hospital, Army Medical University.

[0023] Example 1: Extraction of human plasma exosomes

[0024] Methods for extracting exosomes from plasma:

[0025] Peripheral blood was collected from healthy young adults and the elderly aged 18-25 using EDTA anticoagulant collection tubes and allowed to stand for 2 hours.

[0026] Centrifuge at 2000g for 15 minutes using a refrigerated high-speed centrifuge at 4℃, and collect the upper plasma layer.

[0027] Centrifuge again at 2000g for 15 minutes using a refrigerated high-speed centrifuge at 4℃. Take the upper plasma layer, aliquot it, and store it in a -80℃ refrigerator.

[0028] Remove the aliquoted frozen plasma and thaw it in a water bath at 25–37°C. Once thawed, place it on ice.

[0029] Centrifuge at 2000g for 10 minutes using a refrigerated high-speed centrifuge at 4℃, and collect the supernatant.

[0030] Centrifuge at 12000g for 30 minutes using a refrigerated high-speed centrifuge at 4℃, and collect the supernatant.

[0031] Plasma was filtered through a 0.22 μm microporous membrane and then slowly poured into a dedicated ultracentrifuge tube. The solution was then diluted with filtered sterile PBS. The mixture was centrifuged at 120,000 g for 120 minutes at 4°C using a refrigerated ultracentrifuge. After centrifugation, the supernatant was slowly aspirated, and the precipitate was resuspended in 100 μL of sterile PBS to collect the exosomes.

[0032] Exosomes were identified using transmission electron microscopy (TEM), particle size analysis (NTA), zeta potential measurement, and Western blot analysis of exosome markers. Results are shown in [Figure number missing]. Figure 1 .

[0033] Example 2: Labeling plasma exosomes in healthy young adults

[0034] Fluorescently labeled exosomes were synthesized using a DiD kit. DiD dye was added to the exosome resuspension, and the mixture was incubated for 10 min to stain the exosomes. The dye was then removed by centrifugation at 100,000 g for 2 h at 4 °C. After centrifugation for 2 h, the precipitate was washed with PBS to obtain the dye-stained exosomes.

[0035] Example 3: Construction of the ICH Mouse Model

[0036] The laboratory animal C57BL / 6J was purchased from Hunan Silek Jingda Laboratory Animal Technology Co., Ltd., and was approved by the Ethics Committee of Southwest Hospital of Army Medical University.

[0037] The autologous blood injection method is implemented as follows:

[0038] Mice were first anesthetized with a 2% isoflurane / air mixture and fixed on a stereotaxic apparatus. The anterior and posterior fontanelles were then exposed, and a hole was drilled 0.8 mm anterior to the anterior fontanelle and 2 mm lateral to the midline. The mouse tail was disinfected with 75% alcohol, and 25 μL of autologous blood was collected from the mouse tail using a microsyringe and slowly injected into the basal ganglia at a rate of 2 μL / min to a depth of 3.5 mm.

[0039] For mice in the sham-operated group (Sham group), the same procedure was performed, but the same volume of saline was injected. During the operation, a constant-temperature heating plate was used to maintain the mice's body temperature at 37℃±0.3℃ until they awoke. The procedure was performed by skilled researchers to minimize pain or discomfort during the experiment. Post-operatively, the mice had free access to food and water under a constant photoperiod (12-hour light / dark cycle). The location and successful model establishment of the ICH mouse model can be found in [link to documentation]. Figure 2 .

[0040] Example 4: Distribution and absorption of human plasma exosomes in mouse brain after ICH

[0041] DiD-labeled exosomes derived from the blood of healthy young adults were injected into ICH mice. The injection site was located in the lateral ventricle of the hemisphere of the hemorrhage. Injections were administered once daily for the first three days after ICH. On the third day, brain samples were collected to observe the distribution of exosomes. Results are shown below. Figure 3 .

[0042] Specific injection procedures:

[0043] (1) Collect exosomes, first use BCA kit to determine the concentration, accurately determine the volume of exosomes, and store the exosomes at 80℃.

[0044] (2) Exosomes were thawed at room temperature and 20 μg, 40 μg and 60 μg of exosomes were injected into the lateral ventricle of the hemisphere of the hemorrhage site in ICH mice, while the control group was injected with the same volume of sterile PBS.

[0045] (3) Mice were injected once a day for the first three days after ICH, and then injected again on the seventh day, for a total of 4 injections.

[0046] Example 5: Comparison of plasma exosome activity between healthy young adults and the elderly

[0047] After extracting exosomes from the plasma of healthy young adults and elderly individuals, the exosomes were injected into ICH mice according to the experimental method described in Example 4. The behavioral characteristics of the ICH group and the exosome injection group (hereinafter referred to as the EXO group) were assessed at days 1, 3, 7, 14, and 28, including open field, beam-walking, and modified Garcia score. Results are shown below. Figure 4 . Figure 4This is a dose-response curve of exosomes derived from the plasma of young and elderly individuals in the behavioral recovery of ICH mice, n=6. In the figure, **P<0.01, ***P<0.001 represent ICH vs. Sham; #P<0.05 represents ICH+EXO vs. ICH. Figures A and C show the motor function performance of ICH mice 1, 3, 7, 14, and 28 days after injection of 20 μg, 40 μg, and 60 μg of exosomes from young human plasma into the lateral ventricle, respectively. Figure A shows the results of the open field test, which records the total distance the mice move within a specified time; the longer the distance, the better. Comparing 20μg and 60μg doses, mice injected with 40μg of exosomes performed best in the open field test, covering the farthest distance at day 28. Figure B shows the Modified Garcia score results for each group of mice; higher scores are better in this experiment. On day 28, compared to the 20μg dose, mice injected with 40μg and 60μg doses had higher scores. Figure C shows the Beam walking test, recording the number of times the contralateral limb slipped while walking on a beam. This test reflects the fine motor skills of mice; fewer slips indicate better coordination. On day 28, compared to the 20μg dose, mice injected with 40μg and 60μg doses had fewer limb slips while walking on the beam. In summary, both 40μg and 60μg doses are optimal, with 40μg showing the best performance in all aspects. Therefore, 40μg of human plasma exosomes was chosen as the dose for subsequent experiments. Figures D and E show the motor function performance of ICH mice at 1, 3, 7, 14, and 28 days after 40 μg of elderly plasma exosomes was injected into the lateral ventricle. Figure D (open field test), Figure E (behavioral scoring), and Figure F (beam walking) all showed no significant difference in behavioral performance between ICH mice injected with elderly plasma exosomes and the ICH group mice. This comparative experiment revealed that plasma exosomes from young adults can promote the recovery of motor function in ICH mice, while plasma exosomes from elderly adults have no effect. The results are surprising: plasma exosomes from elderly adults have no therapeutic effect on ICH ferroptosis-related brain injury, while plasma exosomes from young adults, especially those aged 20-25, show therapeutic efficacy, particularly at doses of 40-60 μg, which show a significant therapeutic effect on ICH ferroptosis-related brain injury.

[0048] Therefore, the following further experiment selected 40 μg of plasma exosomes from young people as experimental material.

[0049] On day 7, the brains of mice injected with exosomes derived from the plasma of healthy young adults were collected to detect the following indicators:

[0050] (1) Observe brain injury and neuronal damage after cerebral hemorrhage by HE staining and Nissl staining;

[0051] (2) The expression of GPX4, a marker of ferroptosis around the hematoma after ICH, was observed by immunofluorescence staining;

[0052] (3) Transmission electron microscopy was used to examine the morphology of mitochondria around the mouse hematoma. The results are shown in […]. Figure 5 ;

[0053] (4) The expression changes of the ferroptosis markers GPX4 and SLC7A11 were detected by western blot.

[0054] (5) The expression of ferroptosis markers GPX4 and SLC7A11 mRNA was detected by qPCR;

[0055] (6) The expression levels of MDA and GSH were detected, and the results are shown in [the table below]. Figure 6 .

[0056] Figure 5 This figure shows the effect of plasma exosomes on reducing brain damage and ferroptosis after ICH in young adults. In the figure, n=3, **P<0.01, ***P<0.001 represent ICH vs. Sham; #P<0.05, ##P<0.01 represent ICH+EXO vs. ICH. Figure A shows the gross brain damage observed by HE staining in the Sham, ICH, and ICH+EXO groups; B shows representative Nissl staining micrographs of different groups; C shows the quantitative analysis of Nissl-positive neurons in the experiment; D shows representative immunofluorescence images of GPX4 around the hematoma in different groups; E shows the quantitative analysis of GPX4 staining; and F shows transmission electron microscopy images showing mitochondrial changes in different groups. The results indicate that the ICH+EXO group showed significant differences compared to the ICH and ICH control groups in mitochondrial changes, GPX4 expression density around the hematoma, brain damage, and positive neuron damage, and was comparable to or similar to Sham, demonstrating significant therapeutic efficacy with statistical significance.

[0057] Figure 6This figure shows the effect of plasma exosomes in young adults on antagonizing ferroptosis induced by ICH via the SLC7A11 / GPX4 pathway; n=6, **P<0.01 indicates ICH+EXO vs. ICH. Figure A shows Western blot analysis of SLC7A11 and GPX4 around hematomas in different groups, with β-actin and β-tubulin serving as internal controls for Western blot hybridization (SLC7A11 and GPX4); B and C show quantitative analysis of GPX4 and SLC7A11; D shows quantitative analysis of MDA around hematomas in each group; E shows quantitative analysis of GSH around hematomas in different groups; F and G show the expression levels of SLC7A11 and GPX4 mRNA around hematomas in different groups detected by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that, compared with the ICH group, the ICH+EXO group exhibited significant advantages in perihematoma SLC7A11 and GPX4 expression, β-actin and β-tubulin control, GPX4 and SLC7A11 quantification, and streptokinase response. In some aspects, it was at least comparable to, and even better than, the Sham group. This demonstrates the statistically significant role and efficacy of plasma exosomes in combating ICH-induced ferroptosis in young adults.

[0058] Based on the above experimental results, it is suggested that plasma exosomes from young adults can be used to treat brain injury associated with ferroptosis after ICH, especially with animal doses of 40-60 μg showing particularly good efficacy.

Claims

1. The use of human plasma exosomes in the manufacture of a medicament for the prevention and treatment of ferroptosis-related brain injury following cerebral hemorrhage, characterized in that: The human plasma exosomes are derived from the plasma of healthy young adults aged 18-25 years.

2. The use as described in claim 1, wherein the exosomes are extracted and separated from the plasma of young people.

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