Use of rhubarb-derived exosome-like nanovesicles in preparation of a medicament for treating acute respiratory distress syndrome caused by severe acute pancreatitis

Abstract

This invention discloses the application of rhubarb-derived exosome-like nanovesicles in the preparation of a drug for treating acute respiratory distress syndrome (ARDS) induced by severe acute pancreatitis, belonging to the field of nanomedicine technology. This invention is the first to propose the application of rhubarb-derived exosome-like nanovesicles in the preparation of a drug for treating ARDS induced by severe acute pancreatitis, providing a new method for treating ARDS and expanding the application of rhubarb-derived exosome-like nanovesicles in the pharmaceutical field. This invention is also the first to propose using rhubarb-derived exosome-like nanovesicles as the active ingredient in a pharmaceutical composition for treating ARDS induced by severe acute pancreatitis.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, specifically relating to the application of rhubarb-derived exosome-like nanovesicles in the preparation of a drug for treating acute respiratory distress syndrome caused by severe acute pancreatitis. Background Technology

[0002] Severe acute pancreatitis (SAP) is a common acute abdominal condition characterized by abnormal activation of pancreatic enzymes leading to autodigestion, edema, hemorrhage, and necrosis of pancreatic tissue, resulting in a high mortality rate. Patients typically present with acute upper abdominal pain, nausea, vomiting, and fever. Approximately one-third of SAP patients progress to acute lung injury or even acute respiratory distress syndrome (ARDS). The mechanisms of SAP-induced ARDS are complex, primarily involving uncontrolled inflammatory responses mediated by inflammatory cells in the lungs, disrupting the alveolar-capillary barrier and leading to diffuse alveolar damage and refractory hypoxemia. Although progress has been made in the etiological study of SAP-related ARDS, the pathological mechanisms remain largely unknown.

[0003] The treatment of SAP-induced ARDS remains extremely challenging. On the one hand, SAP is complex and progresses rapidly, and the risk of SAP-induced ARDS differs from the progression of the disease, increasing the complexity of treatment. On the other hand, there are limitations in respiratory support techniques. Although mechanical ventilation is an important means of treating ARDS, its application to these patients often faces many problems. While extracorporeal membrane oxygenation (ECMO) technology has made some progress, the equipment required is specialized, the technology is technically demanding, and the cost is high, limiting its widespread application.

[0004] Plant exosome-like nanovesicles (PELNs) are nanovesicles that are released into the extracellular matrix after multivesicles in plant cells fuse with the cell membrane and invaginate. They possess advantages such as abundant resources, low immunogenicity and organ toxicity, and carry a large amount of proteins, lipids, nucleic acids, and plant-specific secondary metabolites such as flavonoids and polyphenols, which are antioxidant and anti-inflammatory small molecules. They show potential in drug delivery and disease treatment. Multiple preclinical studies have reported that the main active components of rhubarb, emodin and rhein, have significant efficacy in treating SAP-induced ARDS.

[0005] No studies have yet shown that rhubarb-derived exosome-like nanovesicles (RDEVs) have been applied to SAP-induced ARDS. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide an application of rhubarb-derived exosome-like nanovesicles (Rheum palmatum L.) in the preparation of a drug for treating acute respiratory distress syndrome caused by severe acute pancreatitis, providing a new method for treating acute respiratory distress syndrome caused by severe acute pancreatitis and expanding the application of rhubarb-derived exosome-like nanovesicles in the pharmaceutical field.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides the application of rhubarb-derived exosome-like nanovesicles in the preparation of a medicament for treating acute respiratory distress syndrome caused by severe acute pancreatitis.

[0009] Based on the above technical solution, the effective concentration of the rhubarb-derived exosome-like nanovesicles is further 0.8-1.2 mg / mL.

[0010] In a second aspect, the present invention provides a pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis, wherein the active substance comprises rhubarb-derived exosome-like nanovesicles.

[0011] Based on the above technical solution, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0012] Based on the above technical solution, the pharmaceutical composition is further described as an oral formulation or an injectable formulation.

[0013] Based on the above technical solution, the rhubarb-derived exosome-like nanovesicles are further prepared using the following method: Fresh rhubarb rhizome tissue was broken up, water was added for solid-liquid separation, and the supernatant was obtained after at least 3 centrifugations. The supernatant was filtered through a 0.22 μm filter membrane and centrifuged to obtain the precipitate. The precipitate was resuspended in PBS to obtain rhubarb-derived exosome-like nanovesicles.

[0014] Based on the above technical solution, further, in the three centrifugations, the first centrifugation conditions are 1000-1500 g for 20 min, the second centrifugation conditions are 3000-5000 g for 20 min, and the third centrifugation conditions are 12000-15000 g for 60 min.

[0015] Based on the above technical solution, the centrifugation conditions for obtaining the precipitate are 100,000 g - 110,000 g for 120 min.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention is the first to propose the application of rhubarb-derived exosome-like nanovesicles in the preparation of drugs for treating acute respiratory distress syndrome caused by severe acute pancreatitis, providing a new method for treating acute respiratory distress syndrome caused by severe acute pancreatitis and expanding the application of rhubarb-derived exosome-like nanovesicles in the pharmaceutical field.

[0017] 2. This invention is the first to propose using rhubarb-derived exosome-like nanovesicles as the active ingredient in a pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis. Attached Figure Description

[0018] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0019] Figure 1 Figure 1 shows the preparation and characterization results of rhubarb-derived PELNs in Example 1 of this invention: A is the TEM image of RDEVs structure, and B is the NTA image of RDEVs particle size. Figure 2 This is a graph showing the component analysis results of rhubarb-derived RDEVs from Example 1 of the present invention; Figure 3 The following figures show the effects of rhubarb-derived RDEVs on LPS-induced RAW264.7 in Example 3 of this invention: A shows that rhubarb-derived RDEVs significantly inhibited lipopolysaccharide-induced inflammatory factor TNF-α; B shows that rhubarb-derived RDEVs significantly inhibited lipopolysaccharide-induced inflammatory factor IL-6; and C shows that rhubarb-derived RDEVs significantly inhibited lipopolysaccharide-induced inflammatory factor IL-1β. Figure 4 This is a graph showing the colonization results of rhubarb-derived RDEVs in rat pancreas and lung tissue in Example 2 of the present invention; Figure 5 The following figures show the effects of rhubarb-derived RDEVs on the morphology of pancreatic tissue in SAP rats according to Example 2 of this invention: A is the result of HE staining observation of pancreatic tissue pathological changes, B is the result of pancreatic tissue pathological scoring in each group, and C is the result of rat serum amylase activity. Figure 6 The following figures show the effects of rhubarb-derived RDEVs on the morphology of lung tissue in SAP rats in Example 2 of this invention: A is the result of HE staining observation of lung tissue pathological changes, and B is the result of lung tissue pathological scores for each group. Figure 7 The following graphs show the effects of rhubarb-derived RDEVs on serum parameters in SAP rats in Example 2 of this invention: A, B, and C are the results of ALT, AST, and CREA levels in rat serum; D, E, and F are the results of inflammatory factors TNF-α, IL-6, and IL-1β levels in rat serum. Figure 8 The figure shows the effect of rhubarb-derived RDEVs on the protein expression level of BAX / Bcl-2 in lung tissue of SAP rats in Example 2 of this invention. Detailed Implementation

[0020] The present invention will be described in detail below with reference to embodiments. However, the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention. When μM is the concentration unit below, it specifically refers to μmol / L.

[0021] Example 1 In this embodiment, extracellular vesicles derived from rhubarb roots (hereinafter referred to as rhubarb-derived RDEVs or RDEVs) were prepared, and the obtained rhubarb-derived RDEVs were characterized and analyzed.

[0022] The preparation process of rhubarb-derived RDEVs is as follows: Fresh rhubarb rhizome tissue was broken up, and tap water was added for solid-liquid separation. After at least three low-speed centrifugations, the supernatant was obtained. The supernatant was filtered through a 0.22 μm filter membrane and then subjected to ultracentrifugation to obtain the precipitate. The precipitate was resuspended in PBS to obtain rhubarb-derived exosome-like nanovesicles. The low-speed centrifugation conditions were: first centrifugation at 1000-1500 g for 20 min; second centrifugation at 3000-5000 g for 20 min; and third centrifugation at 12000-15000 g for 60 min. The ultracentrifugation conditions were: 100000-110000 g for 120 min.

[0023] The characterization analysis of rhubarb-derived RDEVs is as follows: 1. Nanoparticle tracking analysis (NTA).

[0024] Using the Malvern's nanosight ns 300 instrument, the power supply and computer software connections were confirmed to be normal. The sample cell was cleaned and installed on the laser module. The extracted rhubarb-derived RDEVs were placed on an ice box. 20 μL of RDEVs sample was taken out and diluted 50 times with 980 μL PBS. The diluted solution was loaded onto the sample using a 1 mL syringe. The focus was adjusted until the particle image was clear, and then data acquisition and analysis were performed.

[0025] See Figure 1As shown in Figure A; NTA results showed that the average particle size of rhubarb-derived RDEVs was 168 nm, consistent with the typical size range (30-150 nm) of exosome-like nanovesicles. The particle size distribution exhibited a single peak at 118 nm, and the particle concentration was 1.72 × 10⁹ particles / mL. Dynamic observation revealed that the nanovesicles were spherical, well-dispersed, and exhibited active Brownian motion. See Figure 1 As shown in B. In summary, the NTA results indicate that rhubarb-derived RDEVs exhibit typical exosome-like nanovesicle characteristics, providing fundamental data support for further experiments.

[0026] 2. Transmission electron microscope (TEM).

[0027] 20 μL of rhubarb-derived RDEVs sample was dropped onto a copper grid and allowed to adsorb for 1 minute. It was then negatively stained with phosphotungstic acid solution for 30 seconds, dried at room temperature for several minutes, and subjected to TEM imaging at an accelerating voltage of 80 kV to observe the structure of rhubarb-derived RDEVs.

[0028] TEM results showed that rhubarb-derived RDEVs exhibited saucer-like single or elliptical double vesicle structures with clearly defined membranes, slightly smaller than 100 nm. The vesicle outlines were clear, edges were intact, and there was no obvious damage or deformation. Random measurements revealed that the particle size was mainly distributed between 80-150 nm, and uneven electron density was observed within some vesicles. These results indicate that rhubarb-derived RDEVs possess the typical morphological characteristics of exosome-like nanovesicles.

[0029] 3. Mass spectrometry (LC / MS) Take 600 μL of rhubarb-derived RDEVs sample, add 400 μL of pure methanol, and vortex for 10 seconds to mix. Take 200 μL of the above mixture, add 200 μL of 40% methanol aqueous solution, vortex for 10 seconds, centrifuge at 6000 g for 15 min, and collect the supernatant for later use. Gradient elution separation is performed using an ultra-high performance liquid chromatography (UHPLC) system with a specific column, and primary and secondary spectra of the sample are acquired using a Q-Exactive HFX mass spectrometer. Accurately pipette 6 μL of rhubarb-derived RDEVs solution for LC-MS analysis, repeat the injection 5 times, and normalize the data.

[0030] Based on the base peak chromatograms (BPCs) of the traditional Chinese medicine (TCM), peak shapes and secondary chromatograms were examined for high abundance. The positive and negative ion peaks were then numbered sequentially, resulting in the labeling of 31 peaks from the TCM. Corresponding compound information was then identified. Mass spectrometry analysis showed that RDEVs derived from rhubarb contained known active ingredients such as emodin, emodin-8-glucoside, and rhein. (See...) Figure 2 As shown.

[0031] Example 2 This embodiment provides the therapeutic effect of rhubarb-derived RDEVs on a rat model of acute respiratory distress syndrome (ARDS) induced by severe acute pancreatitis (SAP).

[0032] 1. Model building and grouping.

[0033] Forty SPF-grade male SD rats were randomly divided into four groups (n=10) using a randomized controlled design: CON group, SAP-ALI group, low-dose rhubarb-derived RDEVs group, and high-dose rhubarb-derived RDEVs group. The CON group consisted of healthy SPF-grade male SD rats that received no treatment. The other groups used a rat model of acute pancreatitis-related acute lung injury (APLEI) induced by retrograde injection of 5% sodium taurocholate. The low-dose rhubarb-derived RDEVs group received a tail vein injection of 0.06 mg / 200 g of RDEVs two hours after modeling, and the high-dose rhubarb-derived RDEVs group received a tail vein injection of 0.12 mg / 200 g of RDEVs 12 hours after modeling. Rats' body weight was recorded during the experiment, and blood, pancreatic tissue, and lung tissue samples were collected 24 hours after modeling. Serum amylase activity and inflammatory factor levels were measured, and pathological changes in the pancreas and lung tissue were observed using H&E staining.

[0034] 2. H&E staining Fresh pancreatic and lung tissues were fixed by immersion in paraformaldehyde solution, embedded in paraffin, sectioned, dewaxed and dehydrated, and then stained with hematoxylin and eosin (H&E). Pathological changes in the pancreatic and lung tissues were then observed using an optical microscope.

[0035] H&E staining results showed that the pancreatic structure in the CON group was clear and intact; compared with the CON group, the pancreatic acinar structure of the SAP group rats was damaged, with some showing liquefactive necrosis accompanied by inflammatory cell infiltration and acinar cell edema; after administration of RDEVs, the pathological damage to the pancreatic tissue of rats was reduced, see Figure 5 As shown in A; the pathology score is decreased. See Figure 5 As shown in B. Compared with the CON group, the serum amylase activity in the SAP group was significantly increased. Treatment with rhubarb-derived RDEVs reduced serum amylase activity in SAP rats, as shown in Figure B. Figure 5 As shown in C.

[0036] H&E staining results showed that the lung tissue structure of the CON group was clear and intact; compared with the CON group, the lung tissue of the SAP group rats showed alveolar wall thickening, fusion, and inflammatory cell infiltration; after administration of rhubarb-derived RDEVs, the pathological damage in the lung tissue of rats was reduced, as shown in [the following text is missing]. Figure 6 As shown in A; the pathology score is decreased, see... Figure 6As shown in B.

[0037] 3. Immunofluorescence staining To trace the distribution of rhubarb-derived RDEVs in pancreatic and lung tissues, PKH67 dye was used for labeling. PKH67 was diluted 10-fold with diluent C according to the manufacturer's instructions to prepare a 100 μM working solution. The rhubarb-derived RDEVs were vortexed with the dye working solution for 1 minute, incubated for 10 minutes to obtain a final concentration of 5 μM PKH67. An appropriate amount of PBS was then added and mixed thoroughly. The RDEVs were re-extracted by ultracentrifugation to remove free dye. The precipitate was resuspended in an appropriate amount of PBS to obtain PKH67-labeled RDEVs. 200 μL of the precipitate was injected into rats via the tail vein. After collection, the fluorescence signals in the pancreatic and lung tissues were observed using an inverted fluorescence microscope.

[0038] To visualize the in vivo tissue distribution of rhubarb-derived RDEVs, PKH67-RDEVs were prepared and administered to rats. Results are as follows: Figure 4 The results showed that PKH67-RDEVs could be detected in pancreatic and lung tissues, suggesting that RDEVs can be delivered to pancreatic and lung tissues.

[0039] 4. Serum marker testing Blood was collected from the abdominal aorta and allowed to stand for 2 hours. The blood was then centrifuged at 4°C, 3000 rpm for 10 minutes to obtain serum. Following the kit instructions, the absorbance of the standard wells, assay wells, and blank wells was measured using a multi-mode microplate reader to determine serum ALT, AST, CREA, and amylase activities. The expression levels of serum inflammatory cytokines TNF-α, IL-6, and IL-1β were also determined using enzyme-linked immunosorbent assay (ELISA), following the kit instructions.

[0040] Lung tissues from rats in each group were homogenized with RIPA lysis buffer containing the protease inhibitor, centrifuged at 12,000 rpm for 15 min to obtain the supernatant, and the concentration was determined by the BCA method and normalized to 6 μg / μL. 48 μg of protein samples were subjected to 12% SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes using the wet transfer method, blocked with 5% skim milk, and then incubated overnight at 4°C with rabbit anti-rat Bax (1:1000), Bcl-2 (1:1000), and β-actin (1:50000) primary antibodies, respectively. The next day, they were incubated with HRP-labeled secondary antibody (1:8000). After ECL chemiluminescence development, images were acquired using a chemiluminescence imaging system, and the band gray values ​​were analyzed using ImageJ software. One-way ANOVA was performed after β-actin correction.

[0041] The results are as follows Figure 7As shown, RDEVs treatment can alleviate SAP-induced pathological tissue damage in rats. Compared with the CON group, the SAP group showed significantly increased levels of ALT, AST, and CREA, while treatment with rhubarb-derived RDEVs significantly reduced ALT, AST, and CREA levels (see Figures A and B). Serum TNF-α, IL-6, and IL-1β levels in rats were detected using kits. Compared with the CON group, the SAP group showed significantly increased levels of inflammatory factors. Different doses of RDEVs effectively reduced the levels of inflammatory factors. The 0.12 mg / 200 g dose showed better anti-inflammatory efficacy. See [link to relevant documentation]. Figure 7 As shown in DF.

[0042] 5. Western Blot Lung tissues from rats in each group were homogenized with RIPA lysis buffer containing the protease inhibitor, centrifuged at 12,000 rpm for 15 min to obtain the supernatant, and the concentration was determined by the BCA method and normalized to 6 μg / μL. 48 μg of protein samples were subjected to 12% SDS-PAGE electrophoresis. Proteins were transferred to PVDF membranes using the wet transfer method, blocked with 5% skim milk, and then incubated overnight at 4°C with rabbit anti-rat Bax (1:1000), Bcl-2 (1:1000), and β-actin (1:50000) primary antibodies, respectively. The next day, they were incubated with HRP-labeled secondary antibody (1:8000). After ECL chemiluminescence development, images were acquired using a chemiluminescence imaging system, and the band gray values ​​were analyzed using ImageJ software. One-way ANOVA was performed after β-actin correction.

[0043] To investigate the repair effect of rhubarb-derived RDEVs on lung tissue in SAP-ALI rats, the protein-related expression of Bax and Bcl-2 was measured. Compared with the CON group, Bax expression was significantly increased and Bcl-2 expression was inhibited in the SAP group; the opposite was true in the rhubarb-derived RDEVs group. (See...) Figure 8 As shown.

[0044] Example 3 This embodiment evaluates the in vitro anti-inflammatory activity of RDEVs.

[0045] 1. Effects of different concentrations of rhubarb-derived RDEVs on the viability of RAW264.7 cells.

[0046] The mouse mononuclear-macrophage cell line RAW264.7 was selected. Cells were placed in culture flasks containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin DMEM and cultured in a 37°C, 5% CO2 incubator. Cells were passaged when the cell density reached 80%-90% confluence. RAW264.7 macrophages were then seeded in 96-well plates (1×10⁻⁶ cells / well). 4Cells were collected per well and, after cell adhesion, incubated with different concentrations of rhubarb-derived RDEVs (0, 0.156, 0.312, 0.625, 1.25, 2.5, and 5 μg / mL) for 24 hours. 10 μL of CCK8 reagent was added to each well. After further incubation for 30 minutes to 1 hour, the absorbance at 450 nm was measured using a microplate reader. The results showed that RDEVs had no significant effect on the survival rate of RAW264.7 macrophages in the range of 0–0.625 μg / mL, but cell survival rate decreased significantly after the concentration exceeded 0.625 μg / mL (P<0.05).

[0047] 2. RT-qPCR Log-phase RAW264.7 macrophages were seeded at a rate of 1×10⁶ cells / well in 6-well plates. After cell attachment, the cells were co-incubated with different concentrations of rhubarb-derived RDEVs (0, 0.156, 0.312, and 0.625 μg / mL) for 24 hours. LPS (1 μg / mL) was used as a positive control. Cell pellets were collected. Total RNA was extracted using the Trizol method and reverse transcribed into cDNA. An RT-qPCR system was established according to the manufacturer's instructions to detect the expression levels of inflammatory factors and internal reference genes.

[0048] RT-qPCR results showed that rhubarb-derived RDEVs significantly inhibited the upregulation of lipopolysaccharide-induced mRNA expression of inflammatory cytokines TNF-α, IL-6, and IL-1β. (See attached image) Figure 3 As shown in AC.

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a rhubarb-derived exosome-like nanovesicle in the preparation of a drug for treating acute respiratory distress syndrome caused by severe acute pancreatitis.

2. The application according to claim 1, characterized in that, The effective concentration of the rhubarb-derived exosome-like nanovesicles is 0.8-1.2 mg / mL.

3. A pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis, characterized in that, The active substances include rhubarb-derived exosome-like nanovesicles.

4. The pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis according to claim 3, characterized in that, The pharmaceutical composition includes pharmaceutically acceptable excipients.

5. The pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis according to claim 3, characterized in that, The pharmaceutical composition is an oral or injectable formulation.

6. The pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis according to claim 3, characterized in that, The rhubarb-derived exosome-like nanovesicles were prepared using the following method: Fresh rhubarb rhizome tissue was broken up, water was added for solid-liquid separation, and the supernatant was obtained after at least 3 centrifugations. The supernatant was filtered through a 0.22 μm filter membrane and centrifuged to obtain the precipitate. The precipitate was resuspended in PBS to obtain rhubarb-derived exosome-like nanovesicles.

7. A pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis according to claim 6, characterized in that, In the three centrifugations, the first centrifugation conditions were 1000-1500 g for 20 min, the second centrifugation conditions were 3000-5000 g for 20 min, and the third centrifugation conditions were 12000-15000 g for 60 min.

8. A pharmaceutical composition for treating acute respiratory distress syndrome caused by severe acute pancreatitis according to claim 6, characterized in that, The centrifugation conditions for obtaining the precipitate were 100,000 g - 110,000 g for 120 min.

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