Application of peppermint-derived exosomes in preparation of drugs for treating LPS-induced ARDS

Abstract

The application relates to application of a peppermint-derived exosome in preparation of a medicine for treating LPS-induced ARDS, and the peppermint-derived exosome is obtained through treatment such as soaking in a PBS solution, wall breaking, differential centrifugation, filtration, ultracentrifugation, sucrose density gradient centrifugation and the like. The peppermint-derived exosome in the application can not only reduce histological damage of lung tissue of LPS-induced ARDS mice, improve pulmonary edema, but also has an anti-inflammatory effect on the lung of the ARDS mice, so that the peppermint-derived exosome can be used for preparing a medicine for treating LPS-induced ARDS.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the application of peppermint-derived exosomes in the preparation of drugs for treating LPS-induced ARDS. Background Technology

[0002] Acute Respiratory Distress Syndrome (ARDS) is an acute and severe lung disease characterized by widespread alveolar damage and non-cardiogenic pulmonary edema, leading to severe oxygenation impairment and respiratory failure. The pathogenesis of ARDS involves complex pathophysiological processes, including damage to alveolar epithelial cells and capillary endothelial cells, excessive activation of the inflammatory response, and alveolar ventilation-perfusion mismatch, ultimately resulting in widespread inflammation and dysfunction of the lung tissue. Due to its high mortality rate and the challenges of clinical treatment, ARDS has become one of the most critical diseases requiring breakthroughs in critical care medicine. Although clinical understanding of ARDS is constantly deepening, early diagnosis and effective treatment still face many obstacles, particularly the lack of specific drug therapies targeting the pathological mechanisms beyond non-invasive oxygen therapy and mechanical ventilation management.

[0003] Current research on ARDS mainly focuses on the regulation of inflammatory responses, lung tissue protection mechanisms, and novel cell therapy strategies. Existing treatments are mostly supportive care, lacking effective drugs that target the specific molecular mechanisms of inflammation and cell damage, and research on the application of natural plants and their derivatives in ARDS treatment is relatively scarce.

[0004] Peppermint is a traditional medicinal plant that has been proven to have multiple pharmacological activities, including anti-inflammatory, analgesic, and antioxidant effects. However, its exosome components and functions have not yet been systematically elucidated. Summary of the Invention

[0005] The purpose of this invention is to provide the application of peppermint-derived exosomes in the preparation of drugs for treating LPS-induced ARDS, in order to solve the problem that supportive care is not effective in treating ARDS.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This application extracts exosomes with a particle size of 50-90 nm from peppermint. These peppermint-derived exosomes can alleviate the pathological changes in lung tissue and pulmonary edema in LPS (Lipopolysaccharide)-induced ARDS mice, and can also reduce the content of pro-inflammatory factors in bronchoalveolar lavage fluid (BALF). Therefore, they can be used to prepare drugs for treating LPS-induced ARDS.

[0007] In this application, peppermint-derived exosomes were centrifuged, resuspended in PBS (phosphate buffer saline), and sterilized to form a peppermint-derived exosome solution. The concentration of peppermint-derived exosomes in this solution was 7.97 × 10⁻⁶. 9 Particles / mL, protein concentration 4.7 mg / mL.

[0008] When peppermint-derived exosomes are used to prepare drugs for treating LPS-induced ARDS, the dosage of peppermint-derived exosome solution should not be less than 1.5 mg / kg based on mouse body weight.

[0009] The present invention has the following beneficial effects: (1) In this application, fresh peppermint is treated by soaking in PBS solution, breaking the cell wall, differential centrifugation, filtration, ultracentrifugation, and sucrose density gradient centrifugation to obtain exosomes with a particle size of 50-90nm and a "tea tray shape".

[0010] (2) Peppermint-derived exosomes can not only reduce the histological damage of lung tissue in LPS-induced ARDS mice and improve pulmonary edema, but also have an anti-inflammatory effect on the lungs of ARDS mice. Therefore, peppermint-derived exosomes can be used to prepare drugs for treating LPS-induced ARDS. Attached Figure Description

[0011] Figure 1 Image of peppermint-derived exosomes obtained by transmission electron microscopy (TEM). Figure 2 This is a particle size distribution diagram of peppermint-derived exosomes; Figure 3 Figure showing the pathological effects of peppermint-derived exosomes on lung tissue in LPS-induced ARDS mice; Figure 4 Comparison of the effects of peppermint-derived exosomes on lung W / D in LPS-induced ARDS mice; Figure 5 Figure 1 shows the effect of peppermint-derived exosomes on pro-inflammatory cytokines in BALF of LPS-induced ARDS mice; Figure 2 shows the effect of peppermint-derived exosomes on IL-1β, and Figure 3 shows the effect of peppermint-derived exosomes on TNF-α. Figure 6 The diagram shows the effect of peppermint-derived exosomes on the differentiation of M1 and M2 macrophages in BALF. Among them, Figure A is the flow cytometry analysis of M1 macrophages, Figure B is the flow cytometry analysis of M2 macrophages, Figure C is a comparison of the percentage of M1 polarized cells in the total number of cells, and Figure D is a comparison of the percentage of M2 polarized cells in the total number of cells. Detailed Implementation

[0012] The technical solution of the present invention will be further explained and described below through specific embodiments.

[0013] Example 1 - Preparation of peppermint-derived exosomes This application provides a peppermint-derived exosome, the preparation method of which includes: S01: Fresh peppermint is soaked in PBS solution and then subjected to cell wall disruption to obtain peppermint juice.

[0014] Fresh mint was picked, washed, and dried, then soaked in PBS solution for 60 minutes. After soaking, the mint was processed using a high-speed blender to remove the cell walls and obtain mint juice.

[0015] S02: The peppermint juice was subjected to differential centrifugation, filtration, ultracentrifugation, and resuspending in PBS to obtain a crude extract.

[0016] Peppermint juice was added to centrifuge tubes and centrifuged sequentially at 2000×g for 10 min, 6000×g for 20 min, and 10000×g for 60 min at 4°C to remove precipitate. The supernatant obtained after centrifugation was filtered through a 0.45 μm filter and transferred to an ultracentrifuge tube. The tube was centrifuged at 100000×g for 70 min at 4°C. The supernatant was discarded, and the precipitate was resuspended in PBS to obtain the crude extract.

[0017] S03: After centrifugation of the crude extract using a sucrose density gradient, the strip precipitate containing 30-45% sucrose is collected, and after washing and centrifugation, peppermint-derived exosomes are obtained.

[0018] Sucrose solutions with concentrations of 8%, 15%, 30%, 45%, and 60% were added sequentially to ultracentrifuge tubes. The crude extract was slowly added above the sucrose solution, and the tubes were centrifuged using a sucrose density gradient at a centrifugation force of 100,000-120,000 × g for 60-120 min. The precipitate containing 30-45% sucrose was collected. The precipitate was mixed with an appropriate amount of PBS and centrifuged at 100,000 × g for 60 min to wash with sucrose. The precipitate was collected again, which is the peppermint-derived exosome. After resuspending in PBS, the solution was filtered through a 0.22 μm microporous membrane for sterilization to form an exosome solution. The solution was then aliquoted and stored at -80°C for later use.

[0019] Example 2 – Morphological and other detection of peppermint-derived exosomes 10 μL of peppermint-derived exosome solution was added to the front side of a copper grid. After precipitation for 1 minute, the liquid was absorbed with filter paper. Then, 10 μL of uranium acetate was added to the copper grid. After precipitation for 1 minute, the liquid was absorbed with filter paper. After drying at room temperature for 10 minutes, TEM was performed to obtain the attached... Figure 1 The peppermint-derived exosome suspension was diluted with ultrapure water to a particle concentration of 1×10⁻⁶. 7 / mL-1×10 9 / mL, the particle size distribution of peppermint-derived exosomes was detected using nanoparticle size tracer method, and the attached... Figure 2 .

[0020] From the appendix Figure 1 , 2 As can be seen, the exosomes derived from peppermint are "cafe-shaped", with a particle size of 50-90 nm and an average particle size of 66.4 nm, which is consistent with the distribution range of exosomes of 40-150 nm.

[0021] The protein concentration in the peppermint-derived exosome solution was detected using a BCA (Bicinchoninic Acid) protein quantification kit, and the protein concentration was found to be 4.7 mg / mL.

[0022] Example 3 – Effects of peppermint-derived exosomes on LPS-induced ARDS mice Six- to eight-week-old C57BL / 6 mice were randomly divided into five groups: blank control group, LPS group, LPS + 1.5 mg / kg menthol exosome group, LPS + 3 mg / kg menthol exosome group, and LPS + 2 mg / kg dexamethasone group.

[0023] The ARDS mouse model was established as follows: For five consecutive days prior to modeling, mice were administered medication according to their groupings. The LPS + peppermint exosome group received a tail vein injection of peppermint-derived exosomes, the LPS + 2 mg / kg dexamethasone group received a tail vein injection of dexamethasone, and the control group received a tail vein injection of PBS. One hour after the last administration on day 5, mice were anesthetized with isoflurane and stimulated with an intratracheal infusion of 200 μg / mL LPS. The control group received PBS. Modeling was then complete.

[0024] Sample processing: 24 hours after model establishment, mice were anesthetized and euthanized by cervical dislocation. After fixing the mice, the neck was cut to fully expose the trachea and lung tissue. A small incision was made in the trachea, and 1.5 mL of PBS was injected three times to flush the left lung of the mouse. BALF was collected, with a recovery rate exceeding 90%. The BALF was centrifuged at 4℃ and 2000 rpm for 10 min, and the BALF supernatant was collected and stored at -80℃ for later use.

[0025] The left lung was isolated, and the lung tissue was rinsed with PBS solution to remove bloodstains. Residual PBS was blotted dry with filter paper. The left lung was fixed in 4% paraformaldehyde for 24 hours, then removed and placed in a tissue embedding cassette. It was then dehydrated with ethanol solutions of different concentrations and embedded in paraffin. The embedded tissue sections were dewaxed sequentially in xylene, anhydrous ethanol, and ethanol, and stained with hematoxylin-eosin (HE) to prepare lung pathological specimens.

[0026] The right lung was isolated, and the lung tissue was rinsed with PBS solution to remove bloodstains. Residual PBS was blotted dry with filter paper, and the wet weight was measured. The lung was then placed in a 60°C incubator to dry for 48-72 hours. After drying, the lung was removed and weighed, and the wet-to-dry weight ratio (W / D) was calculated to assess the degree of pulmonary edema.

[0027] 1. Effects of peppermint-derived exosomes on lung tissue Lung specimens were prepared using hematoxylin and eosin (HE) staining for histopathological examination. The weight ratio of wet lung to dry lung was calculated to obtain the attached... Figure 3 Appendix Figure 4 .

[0028] From the appendix Figure 3 As can be seen, the lung structure of mice in the blank control group was intact and the alveoli were clearly visible. Mice in the LPS group showed significant alveolar wall thickening, pulmonary hemorrhage, and neutrophil infiltration in their lung tissue. The lung lesions in mice in the low- and high-dose menthol exosome groups were alleviated, indicating that menthol-derived exosomes can reduce alveolar wall thickening, improve pulmonary hemorrhage, and inhibit neutrophil infiltration.

[0029] From the appendix Figure 4 As can be seen, the W / D ratio of mice in the LPS group was significantly increased compared to the control group, indicating that LPS-induced pulmonary edema occurred in the mice. Compared to the LPS group, the W / D ratio of mice in the low- and high-dose menthol exosome groups was decreased.

[0030] This indicates that peppermint-derived exosomes can significantly reduce histological damage to lung tissue, improve pulmonary edema, and thus play a protective role in the lungs of ARDS mice.

[0031] 2. Effects of peppermint-derived exosomes on cytokines The concentrations of cytokines IL-1β and TNF-α in BALF supernatant were determined by enzyme-linked immunosorbent assay (ELISA). Figure 5 From the appendix Figure 5As can be seen, compared with the control group, LPS stimulation significantly upregulated the pro-inflammatory cytokines TNF-α and IL-1β in BALF, while the LPS + 1.5 mg / kg peppermint exosome group, LPS + 3 mg / kg peppermint exosome group, and LPS + 2 mg / kg dexamethasone group significantly inhibited the production of TNF-α and IL-1β in the BALF of LPS-damaged mice. This indicates the anti-inflammatory effect of peppermint-derived exosomes on the lungs of ARDS mice.

[0032] 3. Effects of peppermint-derived exosomes on macrophages Macrophages are a key component of the immune system, capable of engulfing pathogens, clearing abnormal cells, and transmitting antigen information. In addition, macrophages secrete cytokines to regulate immune responses, but excessive activation can damage tissues. Based on this, this application's embodiments performed macrophage polarization detection on M1 and M2 macrophages in BALF supernatant to obtain... Figure 6 CD86 is a surface marker of M1 macrophages, and CD206 is a surface marker of M2 macrophages.

[0033] From the appendix Figure 6 It is evident that CD86 expression was low in the blank group and significantly increased in the LPS group. Furthermore, compared to the LPS group, CD86 expression was lower in the LPS+1.5 mg / kg menthol exosome group, the LPS+3 mg / kg menthol exosome group, and the LPS+2 mg / kg dexamethasone group, especially the LPS+3 mg / kg menthol exosome group. This indicates that LPS promotes macrophage polarization towards the M1 type, while menthol-derived exosomes inhibit macrophage polarization towards the M1 type in a dose-dependent manner.

[0034] In addition, CD206 expression was low in the blank group and the LPS group; meanwhile, compared with the LPS group, CD206 expression was significantly increased in the LPS+1.5mg / kg peppermint exosome group, LPS+3mg / kg peppermint exosome group, and LPS+2mg / kg dexamethasone group, and increased with increasing dose. These results may reveal the potential mechanism of peppermint-derived exosomes in anti-inflammatory effects.

[0035] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of peppermint-derived exosomes in the preparation of drugs for treating LPS-induced ARDS, among which, The method for preparing the peppermint-derived exosomes includes: Fresh peppermint was soaked in PBS solution and then subjected to cell wall disruption to obtain peppermint juice. The peppermint juice was subjected to differential centrifugation, filtration, ultracentrifugation, and resuspended in PBS to obtain a crude extract. After centrifugation of the crude extract using a sucrose density gradient, the precipitate with a sucrose layer of 30-45% was collected. After washing, centrifugation, resuspension, and sterilization, a peppermint-derived exosome solution was obtained.

2. The application according to claim 1, characterized in that, The peppermint-derived exosomes were resuspended in phosphate buffer and sterilized to form a peppermint-derived exosome solution, the concentration of peppermint-derived exosomes in the solution being 7.97 × 10⁻⁶. 9 Particles / mL, protein concentration 4.7 mg / mL.

3. The application according to claim 2, characterized in that, The dosage of the peppermint-derived exosome solution is not less than 1.5 mg / kg based on mouse body weight.

4. The application according to claim 1, characterized in that, The particle size of the peppermint-derived exosomes is 50-90 nm.

5. The application according to claim 1, characterized in that, The differential centrifugation was performed at 4°C, with centrifugation at 2000×g for 10 min, 6000×g for 20 min, and 10000×g for 60 min in sequence.

6. The application according to claim 1, characterized in that, The filter pore size is 0.45μm.

7. The application according to claim 1, characterized in that, The ultracentrifugation was performed at a temperature of 4°C, a centrifugal force of 100,000 × g, and a time of 70 min.

8. The application according to claim 1, characterized in that, The sucrose density gradient centrifugation is as follows: Add sucrose solutions of concentrations of 8%, 15%, 30%, 45%, and 60% sequentially to ultracentrifuge tubes. Slowly add the crude extract above the sucrose solution and centrifuge at 100,000-120,000 × g for 60-120 min.

9. The application according to claim 3, characterized in that, The peppermint-derived exosomes were resuspended in PBS and filtered through a 0.22 μm microporous membrane for sterilization.

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