Application of lilium lancifolium exosome in preparation of medicine for treating depression

Abstract

The invention provides an application of lilium lancifolium exosome in preparation of a medicine for treating depression. The lilium lancifolium exosome has a unique nano-structure, is better in biocompatibility, can accurately target pathological cells, penetrate a biological membrane and a blood brain barrier, reduces damage to normal cells, directly acts on a central nervous system, is less in dosage, is safer in clinical use, has many side effects when being used as an antidepressant drug, and is high in treatment resistance and recurrence risk, and the like. A brand-new efficient solution is provided, and existing treatment bottlenecks are expected to be broken through.

Description

Technical Field

[0001] This invention relates to the use of lily tigrinus exosomes in the preparation of medicaments for treating depression. Background Technology

[0002] Depression, as a neurological disorder, continues to rise in incidence. Clinically, it is often characterized by a persistently low interest in voluntary activities, slowed thinking, lethargy, and reduced speech and movement. Severe depression may involve suicidal thoughts or a sense of hopelessness. Recent studies have found that depression is also a systemic disease, associated with premature onset of early-onset diseases such as coronary heart disease, stroke, diabetes, and osteoporosis. Even without the influence of smoking, high blood pressure, obesity, or other known health risk factors, these diseases can shorten the life expectancy of patients with recurrent depression by 7–10 years. According to the World Health Organization, in 2019, an estimated 280 million people suffered from depression, 5% of whom were adults. Notably, the incidence of depression is higher in women than in men. Globally, more than 10% of pregnant and postpartum women experience depression, and 20% of the population in developed countries is affected. Furthermore, it is estimated that more than 75% of people with mental disorders in low- and middle-income countries do not receive treatment. Therefore, the World Health Organization has classified depression as the second leading cause of disability worldwide.

[0003] Currently, depression treatment relies on medication, psychotherapy, and physical therapy. Commonly used medications include selective serotonin reuptake inhibitors (SSRIs) and tricyclic antidepressants. However, these medications have significant limitations; approximately 30% of patients do not respond well to them, and long-term use can easily lead to drug tolerance, side effects, and addiction. Psychotherapy, such as cognitive behavioral therapy and interpersonal therapy, can alleviate symptoms, but requires a significant investment of time and energy from patients, and the effectiveness varies greatly from person to person, with some patients easily becoming frustrated. Physical therapy, such as electroconvulsive therapy (ECT), can alleviate severe depression, but due to its invasiveness and potential for cognitive impairment, patient acceptance is low. Therefore, the need for safe, effective, and low-side-effect antidepressants is urgent. Currently, fluoxetine is a commonly used selective serotonin reuptake inhibitor (SSRI) for treating depression. Common side effects in clinical use include: digestive system: including nausea, vomiting, diarrhea, indigestion, dry mouth, etc.; nervous system: may cause headaches, insomnia, dizziness, drowsiness, anxiety, agitation, etc.; sexual function problems: may include decreased libido, erectile dysfunction, delayed ejaculation, etc. Mood changes: In rare cases, it may cause mood swings, irritability, etc. More serious side effects include: Suicidal thoughts or behaviors: Especially in children, adolescents, and young adults, fluoxetine may increase the risk of suicidal thoughts or behaviors. Serotonin syndrome: This is a serious side effect characterized by high fever, sweating, rapid heart rate, confusion, muscle rigidity, etc. Allergic reactions: Rash, itching, difficulty breathing, etc., may occur. Seizures: Seizures may occur in a small number of patients. Hyponatremia: May cause polyuria, polydipsia, and hyponatremia.

[0004] Extracellular vesicles (EVs) are subcellular components produced by paracrine secretion and belong to the nanoscale particle category. Recent research hotspots in academia, such as exosomes, membrane microparticles (MPs), and microvesicles (MVs), all fall into this category. Exosomes typically have a particle size of 30–150 nm. Their lipid bimolecular structure carries a large number of bioactive substances such as proteins, lipids, and nucleic acids, enabling them to play important roles in cell communication, cell migration, and the occurrence and development of cancer.

[0005] Li Kun et al., Research Progress on Exosomes in the Diagnosis and Treatment of Depression, Chinese Journal of Psychiatry, May 2024, Vol. 57, No. 5, reported that exosomes are extracellular vesicles secreted by cells and containing various bioactive substances such as proteins, lipids, and nucleic acids. They participate in various physiological processes in the brain, including neurogenesis, synaptic plasticity, and intercellular communication, and are closely related to the occurrence and development of depression. Because exosomes can cross the blood-brain barrier and can be extracted from various peripheral fluids, exosomes from the central nervous system (i.e., animal or human sources) may help reveal the pathophysiology of brain-related diseases. Due to the different cell characteristics and bioactive substances carried by different exosomes, exosomes from different sources differ in efficacy. Different cell types of exosomes have unique roles in intercellular communication, tissue repair, and disease progression.

[0006] CN202411756649.6, Invention Title: Application of Lily Extracellular Vesicles in the Preparation of Tumor Therapeutic Drugs, discloses the application of lily extracellular vesicles in the preparation of tumor therapeutic drugs. The preparation method of the lily extracellular vesicles includes the following steps: S1, juicing lily bulb tissue and filtering to obtain lily bulb stock solution; S2, processing the lily bulb stock solution obtained in step S1 to obtain lily extracellular vesicle extract; S3, concentrating and purifying the lily extracellular vesicle extract obtained in step S2 to obtain lily extracellular vesicles; wherein, in step S2, the processing includes centrifugation and membrane filtration. The lily extracellular vesicles provided by this invention can effectively promote the polarization of M2 macrophages to M1 macrophages, exhibiting significant immunomodulatory capabilities, and have good biocompatibility, showing potential for the preparation of anticancer drugs. Furthermore, this application discloses extracellular vesicles extracted from Lanzhou lily.

[0007] Lilium lancifolium Thunb., belonging to the Liliaceae family, is used medicinally and as food for its bulbs. This plant is native to China, Japan, and Korea, and is widely distributed in East Asia. It possesses various biological activities, including anti-inflammatory, antidepressant, antioxidant, antibacterial, blood-activating, hypoxia-resistant, anti-fatigue, sedative, sleep-improving, anti-aging, antitussive, and acute toxicity-modifying effects, making it valuable as a raw material for health products and in clinical treatment. Zhang Ying et al., "Application and Research Status of Lily in Antidepressant Treatment," Medical Review, September 2016, Vol. 22, No. 17, published that lily-based compound preparations can also improve behavioral changes in depressed animals, increase the level of the hippocampal monoamine neurotransmitter serotonin, and downregulate the hyperactive hypothalamic-pituitary-adrenal (HPA) axis. The active ingredient in lily, lily saponins, also has the effect of increasing serotonin and dopamine levels, and decreasing adrenocortical hormone and cortisol levels.

[0008] Currently, there are no literature reports on the antidepressant effects of Lilium tigrinum exosomes. Summary of the Invention

[0009] The technical solution of this invention provides a new use for the exosomes of Lilium tigrinum.

[0010] This invention provides the use of lily tigrinus exosomes in the preparation of medicaments for treating depression.

[0011] Furthermore, the drug has neuroprotective effects.

[0012] The exosomes of *Lilium lancifolium* Thunb., a plant in the Liliaceae family, are prepared by differential centrifugation and sucrose density gradient centrifugation.

[0013] The exosomes have an average diameter of 50 nm and an average electrokinetic potential of -20.97 mV.

[0014] The method for preparing *Lilium tigrinum* exosomes includes the following steps:

[0015] a. Take fresh *Tilia lily* plants (Liliaceae family);

[0016] b. Differential centrifugation and density gradient centrifugation were used to extract exosomes from Lilium tigrinum.

[0017] The method for preparing *Lilium tigrinum* exosomes includes the following steps:

[0018] a. Take fresh bulbs of the Liliaceae family plant *Tilia lily*, wash and drain them;

[0019] b. Weigh out the drained Liliaceae lily plant, add phosphate-buffered saline (PBS) and place in a high-speed blender to chop at high speed;

[0020] c. Centrifuge sequentially at 4℃ using differential centrifugation: 1000g, 10min; 3000g, 20min; 10000g, 20min. Collect the supernatant after each centrifugation, discard the precipitate, and continue centrifugation.

[0021] Centrifuge the supernatant further: 4℃, 12000g, 90min, discard the supernatant, and suspend the precipitate in PBS;

[0022] d. Using density gradient centrifugation, the precipitate from step c is transferred to 15%, 30%, 45%, and 60% sucrose solutions and centrifuged in an ultracentrifuge at 4°C, 150,000g, for 90 min. The intermediate layer of the 30%–45% sucrose solution is collected to obtain exosome particles, which are then resuspended in sterile PBS and ultracentrifuged at 4°C, 150,000g, for 90 min. The centrifuged precipitate is then stored at -80°C.

[0023] The pharmaceutical preparations mentioned herein are oral or injectable preparations.

[0024] Furthermore, the oral preparations mentioned are sprays, capsules, tablets, granules, pills, or oral liquids.

[0025] Lilium tigrinum exosomes, as a type of plant-derived exosome, have attracted considerable attention from researchers in recent years. Plant exosomes possess characteristics and advantages comparable to animal exosomes, including carrying multiple bioactive molecules such as proteins, lipids, and nucleic acids; strong antioxidant and anti-inflammatory capabilities; low immunogenicity; natural origin and good biocompatibility; and nano-targeted delivery advantages. Compared to traditional drugs, Lilium tigrinum exosomes, due to their unique nanostructure, exhibit better biocompatibility, can precisely target diseased cells, penetrate biological membranes and the blood-brain barrier, reduce damage to normal cells, and act directly on the central nervous system. They require smaller doses, making clinical use safer. This provides a novel and highly effective solution to the problems of numerous side effects, treatment resistance, and high relapse risk associated with antidepressants, and holds promise for overcoming current treatment bottlenecks. Attached Figure Description

[0026] Figure 1 The process of extracting and purifying exosomes from Lilium tigrinum;

[0027] Figure 2 Morphology of exosome-like nanoparticles from Lilium tigrinum var. ...

[0028] Figure 3 Size distribution of exosomes from *Lilium tigrinum*;

[0029] Figure 4 Distribution of zeta potentials in exosomes of *Lilium tigrinum*;

[0030] Figure 5 Detection of the in vitro free radical scavenging capacity of exosomes from Lilium tigrinum;

[0031] Figure 6 Effects of Lilium tigrinum exosomes on body weight in depressed mice;

[0032] Figure 7 Percentage of people who prefer sweet soup;

[0033] Figure 8 Percentage of time the tail remained stationary during the suspension test;

[0034] Figure 9 Percentage of time spent in the mining area;

[0035] Figure 10 Percentage of horizontal movement frequency in the mine;

[0036] Figure 11 Mine field experiment trajectory diagram;

[0037] Figure 12 The effect of Lilium tigrinum exosomes on PC12 cell survival;

[0038] Figure 13 The effect of lipopolysaccharide on PC12 cell survival;

[0039] Figure 14 The protective effect of Lilium tigrinum exosomes against LPS-induced cellular oxidative stress. Detailed Implementation

[0040] Example 1: Extraction and characterization of exosomes from Lilium tigrinum according to the present invention

[0041] 1. Extraction and purification

[0042] Fresh *Tilia tigrinum* bulbs were selected, and moldy or rotten scales were removed. The bulbs were washed three times each with tap water and distilled water, drained, and then 0.5 kg of each bulb and an appropriate amount of phosphate-buffered saline (PBS) were placed in a high-speed blender and chopped for 3 minutes. The bulbs were then centrifuged sequentially at 4°C using differential centrifugation: 1000 g, 10 min; 3000 g, 20 min; 10000 g, 20 min. The supernatant was collected after each centrifugation, and the precipitate was discarded before further centrifugation. The supernatant was then centrifuged again at 4°C, 12000 g, 90 min. The supernatant was discarded, and the precipitate was resuspended in 1 mL of PBS. Samples were transferred to 15%, 30%, 45%, and 60% sucrose solutions using density gradient centrifugation and centrifuged in an ultracentrifuge at 4°C, 150,000g, for 90 min. The intermediate layer of the 30%–45% sucrose solution was collected to obtain exosome particles, which were then resuspended in sterile PBS and ultracentrifuged at 4°C, 150,000g, for 90 min. The centrifuged precipitate was then stored at -80°C.

[0043] 2. Characterization

[0044] 2.1 Cryo-transmission electron microscopy observation of exosome morphology

[0045] Add an equal volume of 3% paraformaldehyde (PFA) to the exosome suspension, mix thoroughly by pipetting, and transfer 10 μL to a sterile petri dish. Incubate the sample with a copper mesh for 20 min, wash with PBS, and fix with 1% glutaraldehyde for 5 min. After fixation, wash five times with distilled water and stain with 3% phosphotungstic acid negative staining solution for 10 min. Add a small amount of staining solution to 1 mL of 2% methylcellulose solution in a 1 mL EP tube, mix well, and centrifuge at high speed for 5 min to obtain the coating solution. Place the stained sample on the surface of the copper mesh and place it on ice for 10 min. Remove the copper mesh, absorb the liquid with filter paper, and then dry it under infrared light with sterile filter paper. Adjust the electron microscope and take pictures.

[0046] 2.2 Determination of particle size and potential of exosomes from Lilium tigrinum

[0047] The sample, resuspended in 1 mL of PBS solution, was diluted 50 times and filtered through a 0.22 μm filter membrane. 2 mL of the diluted solution was slowly injected into the sample cell using a syringe. The particle size and potential of the exosomes were measured using a Zeta potential and particle size analyzer. The instrument was calibrated using polystyrene microspheres (100 nm).

[0048] 2.3 Determination of the in vitro free radical scavenging activity of exosomes from Lilium tigrinum

[0049] 1 mg of DPPH (394.32 g / mol) reagent was dissolved in 12.68 mL of anhydrous methanol to obtain a 0.2 mM solution, which was incubated at room temperature in the dark for 2 hours. 20 μL of different concentrations of *Lilium tigrinum* exosomes were mixed with 220 μL of DPPH methanol solution, thoroughly mixed, and incubated at room temperature in the dark for 30 minutes. The mixture was then transferred to a 96-well plate, and the absorbance was read at 515 nm using a microplate reader. The ability of different concentrations of *Lilium tigrinum* exosomes to scavenge peroxide free radicals in vitro was expressed as the DPPH free radical scavenging rate, calculated using the following formula. Each concentration was measured three times.

[0050]

[0051] 3. Results

[0052] Lilium tigrinum exosomes were separated from the sap of Lilium tigrinum using differential centrifugation and sucrose density gradient centrifugation, such as... Figure 1 As shown. Combined transmission electron microscopy imaging and nanoparticle tracking analysis revealed that the exosomes of *Lilium tigrinum* are membrane-closed vesicles. Figure 2 The structure is irregular in shape and has an average diameter of 50 nm. Figure 3 Its average electromotive force is -20.97mV. Figure 4 Therefore, *Lilium tigrinum* exosomes resuspended in PBS solution at room temperature are unstable. Furthermore, in vitro free radical scavenging activity assays show that *Lilium tigrinum* exosomes possess good free radical scavenging capabilities. Figure 5 ).

[0053] The following pharmacodynamic tests demonstrate the beneficial effects of this invention.

[0054] Example 1: Study on the antidepressant effect of Lilium tigrinum exosomes on a mouse model of depression.

[0055] 1. Laboratory animals

[0056] Male wild-type C57BL / 6 mice (24 - 26 g, 8 - 10 weeks old) were purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd., with the license number: SCXK (Su) 2023 - 0009. All experimental mice were fed with sufficient food and water under the conditions of room temperature 22 ± 2°C, humidity 55 ± 60%, and natural lighting. After 7 days of adaptive feeding, they were randomly grouped for experiments. All animal experiments in this project were approved by the Animal Experiment Ethics Committee of Lanzhou University, and the experimental operations were in line with the "3R" principle and the relevant regulations on laboratory animal protection, animal welfare, and animal ethics.

[0057] 2. Animal grouping, model establishment, and drug administration

[0058] After 7 days of adaptive feeding, the mice were randomly divided into a normal group, a blank group, a model group, and a positive drug group. Except for the normal group being fed normally, the other mice were subjected to 6 - week chronic mild stress (CMS) stimulation. The stressors included wet cage for 24 h, co - housing for 24 h, empty cage for 24 h, fasting for 12 h, water deprivation for 12 h, restraint for 7 h, ice bath for 5 min, and circadian rhythm reversal for 24 h. Drug administration started in the 7th week, with a drug administration cycle of 2 weeks. The administration method was intraperitoneal injection. The normal group and the model group: 200 μL of normal saline daily; the positive drug group: 5 mg / kg, 200 μL of fluoxetine daily; the treatment group: 4 mg / kg of Lilium lancifolium exosomes once every 3 days.

[0059] Compared with current first - line antidepressant drugs, such as selective serotonin reuptake inhibitors like sertraline (the general dose is 50 - 200 mg per day, calculated as 0.714 mg / kg for a 70 - kg body weight), fluvoxamine (the commonly used dose is 100 - 300 mg per day, calculated as 1.429 mg / kg for a 70 - kg body weight), etc., Lilium lancifolium exosomes have a lower effective dose (the converted human - equivalent dose is approximately 0.43 mg / kg), which is more beneficial for reducing the risk of drug accumulation, optimizing the treatment plan, and enhancing treatment safety and tolerance.

[0060] 3. Behavioral experiments

[0061] 3.1 Sucrose preference test

[0062] The sucrose preference test (SPT) is used to evaluate anhedonia, a key depressive symptom. Mice were pre - treated with two bottles of 1% sucrose solution 24 h before the experiment. On the experimental day, the mice were housed individually and fasted and water - deprived for 24 h. Then, they were provided with 1% sucrose solution and water, and allowed to freely consume one of the liquids. The consumption of water and 1% sucrose solution was measured within 24 h, and they were exchanged every 2 h to control for side bias.

[0063] 3.2 Open - field test

[0064] The open field test (OFT) evaluates spontaneous activity, anxiety, and exploratory abilities in mice. The test is conducted in a 50cm × 50cm × 50cm area with a 10cm × 10cm central square as the central zone. Each mouse is placed in the center and allowed free exploration for 5 minutes. An automated tracking system records the mice's movements. Motor activity is assessed by measuring the total distance walked, while the time spent in the zone and the frequency of horizontal movements represent anxiety-like behaviors.

[0065] 3.3 Tail Suspension Experiment

[0066] The tail suspension test was performed 24 hours after the last drug administration. The mouse tail was attached approximately 1 cm from the tip to the suspension box in an inverted position using medical tape, with the mouse's head about 15 cm above the experimental platform. The time the mouse remained still (time when it stopped struggling or was inactive) was recorded over 6 minutes using a camera. The first 2 minutes were considered adaptation time and were not recorded; only the time the mouse remained still from 3 to 6 minutes was recorded (in seconds).

[0067] 4. Results

[0068] 4.1 Effects of Lilium tigrinum exosomes on mouse body weight

[0069] Therefore, the body weight of the mice was monitored during the experimental period, and the results were as follows: Figure 6 As shown, during the modeling period, except for the blank group, the body weight of other mice decreased to varying degrees. During the treatment period, compared with the model group, the body weight of mice in both the positive group and the treatment group increased, and ultimately there was no significant difference compared with the saline group, indicating that the exosomes of *Tilia tiglium* have relatively few toxic side effects and have an ameliorative effect on changes in mouse body weight.

[0070] 4.2 Sugar water preference and tail suspension experiment

[0071] The results of the sugar solution monitoring in this experiment are as follows: Figure 7 As shown, both the positive control group and the treatment group showed significant differences compared to the model group. Tail suspension is an important experiment for assessing depression-like symptoms; the results of the antidepressant effect of *Lilium tigrinum* exosomes on depression-like mice are as follows... Figure 8 As shown, compared with the blank control group, the immobility time of mice in the model group was significantly increased, while there was no significant difference between the positive group and the treatment group, indicating that Lilium tigrinum exosomes and fluoxetine have a significant ameliorative effect on depressive-like behavior.

[0072] 4.3 Mine Field Experiment

[0073] The open field experiment, by recording the activity trajectory, walking distance, and horizontal exploration of mice over a certain period of time, can directly reflect their spontaneous activity level and disease or normal state. Therefore, this experiment used the open field experiment to investigate the effect of *Lilium tigrinum* exosomes on the spontaneous activity of CMS-stimulated mice. The results are as follows: Figure 9 , Figure 10 and Figure 11As shown, the results indicated that the number of horizontal movements and the time spent in the treatment area were significantly different in the treatment group compared with the model group, suggesting that *Lilium tigrinum* exosomes and fluoxetine affected the spontaneous activity of CMS-stimulated mice and improved their depressive-like state.

[0074] Experimental Example 2: Effect of Lilium tigrinum exosomes on LPS-induced PC12 cells

[0075] PC12 cells are a cell line derived from rat adrenal medullary pheochromocytoma. They possess neuroendocrine cell characteristics and are commonly used in neurobiological research. In depression research, LPS is used to induce oxidative stress in PC12 cells, thereby affecting the viability of nerve cells and contributing to the formation of depressive symptoms.

[0076] 1. Experimental cells

[0077] PC12 cells (undifferentiated) were purchased from Xiamen Yimo Biotechnology Co., Ltd.

[0078] 2. Cell Culture

[0079] PC12 cells were cultured in RPMI-1640 complete medium containing 10% horse serum (HS), 5% fetal bovine serum (FBS), and 1% penicillin-streptomycin, and were placed in a constant temperature incubator at 37°C and 5% CO2.

[0080] When the cells have grown to about 85%, digest them with 0.25% trypsin, repeatedly pipette them into centrifuge tubes, centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the cells in complete culture medium for passage and use in subsequent experiments, and resuspend the remaining cells in cryopreservation solution and store them in liquid nitrogen.

[0081] 3. CCK-8 assay for cell viability

[0082] Effects of Lilium tigrinum exosomes on PC12 cell survival

[0083] The effect of different concentrations of *Lilium tigrinum* exosomes on cell survival was detected using a CCK-8 assay. Log-phase PC12 cells were selected and stored at 5.0 × 10⁶ cells per well. 3A certain number of cells were seeded into 96-well plates and incubated overnight in a cell culture incubator. After cell adhesion, different concentrations of *Lilium tigrinum* exosomes were added, and the cells were cultured for different time periods. Then, CCK-8 reagent and complete culture medium were mixed in an appropriate ratio, and 100 μL of the mixture was added to each well of the 96-well plate. The plate was gently shaken to ensure the liquid evenly covered the bottom of the wells without generating air bubbles. The plate was returned to the incubator and cultured for another 2 hours. The absorbance of each well was measured at 450 nm using a microplate reader. The cell viability calculation formula is as follows:

[0084]

[0085] Effects of different concentrations of LPS on cell survival

[0086] Lipopolysaccharide (LPS) was used to simulate an in vitro inflammatory environment, and the survival of PC12 cells treated with different concentrations of LPS was detected to determine the LPS induction concentration. Cell plating was performed as described above. After cell adhesion, cells were treated with different concentrations of LPS and cultured for 12 hours. Afterward, cells were incubated with CCK-8 solution at 37°C for 2 hours, and the absorbance at 450 nm was measured. Cell viability was calculated using the same formula as above.

[0087] Interventional effect of Lilium tigrinum exosomes on LPS-induced oxidative stress in PC12 cells

[0088] Cells were stimulated with LPS for 12 hours, and then treated with the optimal concentration of *Lilium tigrinum* exosomes obtained above for 48 hours. Fluoxetine was used as a positive control. Cell proliferation activity was detected by CCK-8 assay, using the same method as above.

[0089] 4. Results

[0090] 4.1 Effects of Lilium tigrinum exosomes and LPS on PC12 cell survival

[0091] The effect of different concentrations of *Lilium tigrinum* exosomes on PC12 cell survival was evaluated using the CCK-8 assay. Cells were treated with *Lilium tigrinum* exosomes at concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 μg / mL for 48 h, and cell survival was then assessed. The results showed that, after 48 h of treatment, within the concentration range of 0.625–20 μg / mL, there was no significant difference in the effect of *Lilium tigrinum* exosomes on PC12 cell survival compared to the control group, suggesting that *Lilium tigrinum* exosomes may have low neurotoxicity. Figure 12As shown. PC12 cells were treated with 0.2–25 μg / mL LPS for 12 hours. Low concentrations (0.2, 1 μg / mL) of LPS resulted in PC12 cell survival rates of 93.08% and 92.21%, respectively. When the LPS concentration reached 5 μg / mL, the PC12 cell survival rate was 84.34%. Figure 13 As shown.

[0092] 4.2 Protective effect of different concentrations of *Lilium tigrinum* exosomes against LPS-induced cellular oxidative stress

[0093] Oxidative stress is closely related to the pathogenesis of depression. Therefore, LPS was used to induce oxidative stress in cells to cause cell damage, so as to simulate the pathogenesis of depression in the brain and explore the neuroprotective effect of Lilium tigrinum exosomes.

[0094] PC12 cells were stimulated with LPS (5 μg / mL) for 12 hours, followed by treatment with 0.625 μg / mL *Lilium tigrinum* exosomes and fluoxetine (3.85 μg / mL) for 48 hours. Absorbance values ​​were measured and cell viability was calculated. Figure 14 As shown in the figure, treatment of PC12 cells with 0.625 and 2.5 μg / mL *Lilium tigrinum* exosomes significantly increased cell survival compared to the LPS-induced group, and was superior to the positive control drug fluoxetine group. The results suggest that LPS stimulation causes oxidative stress damage and significantly reduces cell survival. In this case, the fluoxetine group effectively protected nerve cells, and different concentrations of *Lilium tigrinum* exosomes also showed significant protective effects on nerve cells.

Claims

1. The use of Lilium tigrinum exosomes in the preparation of drugs for treating depression.

2. The use according to claim 1, characterized in that: The drug described has neuroprotective effects.

3. The use according to claim 1 or 2, characterized in that: The exosomes of *Lilium lancifolium* Thunb., a plant in the Liliaceae family, were prepared by differential centrifugation and sucrose density gradient centrifugation.

4. The use according to claim 1 or 2, characterized in that: The exosomes have an average diameter of 50 nm and an average electromotive force of -20.97 mV.

5. The use according to any one of claims 1-4, characterized in that: The method for preparing *Lilium tigrinum* exosomes includes the following steps: a. Take fresh *Tilia lily* plants (Liliaceae family); b. Differential centrifugation and density gradient centrifugation were used to extract exosomes from Lilium tigrinum.

6. The use according to claim 5, characterized in that: The method for preparing *Lilium tigrinum* exosomes includes the following steps: a. Take fresh bulbs of the Liliaceae family plant *Tilia lily*, wash and drain them; b. Weigh out the drained Liliaceae lily plant, add phosphate-buffered saline (PBS) and place in a high-speed blender to chop at high speed; c. Centrifuge sequentially at 4℃ using differential centrifugation: 1000g, 10min; 3000g, 20min; 10000g, 20min. Collect the supernatant after each centrifugation, discard the precipitate, and continue centrifugation. Centrifuge the supernatant further: 4℃, 12000g, 90min, discard the supernatant, and suspend the precipitate in PBS; d. Using density gradient centrifugation, the precipitate from step c is transferred to 15%, 30%, 45%, and 60% sucrose solutions and centrifuged in an ultracentrifuge at 4°C, 150,000g, for 90 min. The intermediate layer of the 30%–45% sucrose solution is collected to obtain exosome particles, which are then resuspended in sterile PBS and ultracentrifuged at 4°C, 150,000g, for 90 min. The centrifuged precipitate is then stored at -80°C.

7. The use according to any one of claims 1-4, characterized in that: The pharmaceutical preparation is an oral or injectable preparation.

8. The use according to claim 7, characterized in that: The oral preparations mentioned are sprays, capsules, tablets, granules, pills, and oral liquids.

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