Bidens pilosa exosome as well as preparation method and application thereof in acute lung injury

Abstract

The invention provides a spanishneedles herb exosome as well as a preparation method and application thereof in acute lung injury, and belongs to the technical field of biological medicines. According to the preparation method of the spanishneedles herb exosome provided by the invention, the spanishneedles herb exosome can be extracted from a plant source through a simple method. In one embodiment of the invention, a lung injury model constructed by using LPS is used as an experimental animal to verify the therapeutic effect of the spanishneedles herb exosome. Results show that after the spanishneedles herb exosome is used for treatment, mouse lung tissue injury and inflammatory cell exudation can be relieved, and lung injury caused by LPS can be relieved; tNF-alpha and IL-1beta in lung lavage fluid of a mouse are remarkably reduced, and lung inflammation infiltration of the mouse can be relieved. Therefore, the spanishneedles herb exosome has the effect of treating the acute lung injury and can be used for preparing the medicine for treating the acute lung injury.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Bidens pilosa exosome, its preparation method, and its application in acute lung injury. Background Technology

[0002] Acute lung injury (ALI) is a severe respiratory disease with a high incidence and mortality rate worldwide. Its main clinical manifestations include pulmonary edema, inflammatory cell infiltration, and uncontrolled oxidative stress. Current treatment emphasizes symptomatic and supportive care after the onset of the disease, including mechanical ventilation, prophylactic or therapeutic antibiotics, and limiting fluid accumulation, as well as treatment of the initial injury or disease. However, even with treatment, ALI often leads to severe respiratory failure and mortality, with an in-hospital mortality rate as high as 38%-46%, and surviving patients frequently experience long-term physical, psychological, and / or cognitive impairment.

[0003] Intratracheal instillation of lipopolysaccharide (LPS) in mice is a common model for constructing atrial fibrillation (ALI). LPS is an outer membrane component of Gram-negative bacteria and can strongly activate the host's immune system. It triggers a series of signaling pathways by binding to pattern recognition receptors (such as TLR4) on the surface of host cells, inducing the release of inflammatory factors. These inflammatory factors (such as TNF-α, IL-6, and IL-1β) can directly cause lung tissue damage, thus mimicking the characteristic pathological process of ALI.

[0004] Currently, there are still no effective drugs for the treatment of ALI. It is crucial to develop new treatment options to prevent the development of ALI, restore alveolar function, reduce lung inflammation, improve prognosis, and reduce mortality. Summary of the Invention

[0005] The present invention aims to provide a Bidens pilosa exosome, its preparation method and its application in ALI, wherein the Bidens pilosa exosome can significantly alleviate ALI and reduce lung inflammation infiltration in mice, thereby improving their survival rate.

[0006] This invention provides a method for preparing Bidens pilosa exosomes, comprising the following steps: washing Bidens pilosa and then crushing it; mixing the crushed material with an enzymatic hydrolysate for enzymatic hydrolysis; filtering the obtained enzymatic hydrolysate and collecting the filtrate; The filtrate was subjected to gradient centrifugation in sequence, and the supernatant was collected. The supernatant was then filtered through a filter membrane, and the resulting filtrate contained the Bidens pilosa exosomes. The enzymatic hydrolysate includes cellulase and pectinase.

[0007] In a preferred embodiment of the present invention, the enzymatic hydrolysate contains cellulase at a mass of 2-4% of the mass of the pulverized material and pectinase at a mass of 1-2% of the mass of the pulverized material.

[0008] In a preferred embodiment of the present invention, the enzymatic hydrolysis is performed at a temperature of 4°C for a time of 10-14 hours.

[0009] In a preferred embodiment of the present invention, the gradient centrifugation includes centrifugation at 1000-3000g for 5-15 min and centrifugation at 8000-15000g for 20-40 min.

[0010] In a preferred embodiment of the present invention, the filtration includes sequential filtration through MCE (mixed cellulose ester, 1.5 / 0.8 / 0.45 / 0.22 μm) and PVDF (polyvinylidene fluoride, 0.45 μm) filter membranes.

[0011] In a preferred embodiment of the present invention, the step of extracting exosomes from the filtrate by ultracentrifugation is further included after filtration.

[0012] The present invention also provides Bidens pilosa exosomes prepared using the above preparation method.

[0013] The present invention also provides the application of the above-mentioned Bidens pilosa exosomes in the preparation of a drug for treating acute lung injury.

[0014] In a preferred embodiment of the present invention, the amount of Bidens pilosa exosomes used is not less than 25 μg / mouse.

[0015] The present invention also provides a medicament for treating acute lung injury, comprising the above-mentioned Bidens pilosa exosomes and pharmaceutically acceptable excipients.

[0016] In a preferred embodiment of the present invention, the dosage form of the drug includes at least one of the following: spray, solution, gelatin capsule, soft capsule, lozenge, chewing gum, and dry powder preparation.

[0017] In a preferred embodiment of the present invention, the drug further includes probiotics.

[0018] Compared with the prior art, this application has the following beneficial effects: This invention provides a method for preparing Bidens pilosa exosomes, which can be extracted from plant sources using a simple method. In one embodiment of this invention, a lung injury model constructed using LPS was used as the experimental animal to verify the therapeutic effect of the Bidens pilosa exosomes. The results showed that after treatment with Bidens pilosa exosomes, lung tissue damage and inflammatory cell exudation in mice were reduced, and LPS-induced lung injury was alleviated; TNF-α and IL-1β in the bronchoalveolar lavage fluid of mice were significantly reduced, which could alleviate inflammatory infiltration in the lungs of mice. Therefore, the Bidens pilosa exosomes of this invention have a therapeutic effect on acute lung injury and can be used to prepare drugs for treating acute lung injury. Attached Figure Description

[0019] Figure 1 This is a particle size distribution diagram of Bidens pilosa exosomes; Figure 2 Electron micrograph of Bidens pilosa exosomes; Figure 3 Standard curve for the determination of exosome proteins from Bidens pilosa; Figure 4 Figure 1 shows the results of Bidens pilosa exosomes alleviating LPS-induced lung injury. A: HE staining results; B: Lung injury score in mice; WT: Wild-type mice; LPS: Mice treated with LPS via airway infusion; LPS+EV-0.25: Mice treated with 0.25 μg / μL Bidens pilosa exosomes via airway infusion after LPS infusion; LPS+EV-0.5: Mice treated with 0.5 μg / μL Bidens pilosa exosomes via airway infusion after LPS infusion; **** P <0.0001; Figure 5 Figure 1 shows the results of using Bidens pilosa exosomes to reduce LPS-induced inflammatory factor infiltration. In the figure, A represents the concentration of IL-6 in bronchoalveolar lavage fluid (BALF); B represents the concentration of TNF-α in BALF; C represents the concentration of IL-1β in BALF; and D represents the concentration of IL-1α in BALF. P <0.01; **** P <0.0001. Detailed Implementation

[0020] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0021] This invention provides a method for preparing Bidens pilosa exosomes, comprising the following steps: washing Bidens pilosa and then crushing it; mixing the crushed material with an enzymatic hydrolysate for enzymatic hydrolysis; filtering the enzymatic hydrolysate and collecting the filtrate; The filtrate was subjected to gradient centrifugation in sequence, and the supernatant was collected. The supernatant was then filtered through a filter membrane, and the filtrate contained the Bidens pilosa exosomes. The enzymatic hydrolysate includes cellulase and pectinase.

[0022] This invention uses fresh Bidens pilosa as raw material, which is washed and then crushed. The washing process includes sequential washing with clean water and then with pure water, followed by draining and crushing. The crushing is a physical process, such as using a blunt-press juicing method in one embodiment.

[0023] In this invention, pulverized Bidens pilosa and enzymatic hydrolysate are mixed and enzymatically hydrolyzed overnight (10-14 h) at 4°C. The enzymatic hydrolysate includes cellulase and pectinase. In the enzymatic hydrolysate, the mass of cellulase is 2-4% of the mass of the pulverized material, and the mass of pectinase is 1-2% of the mass of the pulverized material.

[0024] The present invention filters the enzymatic hydrolysis products. For example, in one embodiment, all the enzymatic hydrolysates are placed in a 200-mesh nylon filter bag, and the filtrate is collected under the action of a jack squeezer. The filtrate is then transferred to a centrifuge bottle and centrifuged at 2000g and 4°C for 10 min. The supernatant is carefully transferred to a clean centrifuge bottle and centrifuged at 10000g and 4°C for 30 min, and the supernatant is collected.

[0025] The supernatant was passed sequentially through MCE (1.5 / 0.8 / 0.45 / 0.22 μm) and PVDF (0.45 μm) filter membranes under positive pressure, and the filtrate was collected. The filtrate contained the Bidens pilosa exosomes. The filtrate of this invention is a sample for extracting Bidens pilosa exosomes and can be frozen and stored at -80°C.

[0026] After obtaining the filtrate, this invention also includes an extraction step, such as rapidly thawing the frozen filtrate at 37°C and then transferring it to a new centrifuge tube for extraction using ultracentrifugation. Specifically, the ultracentrifugation method includes the following steps: centrifuging at 10000g and 4°C for 30 min to remove larger vesicles; collecting the supernatant, filtering it through a 0.45 μm filter membrane, and collecting the filtrate; transferring the filtrate to a new centrifuge tube, selecting an ultracentrifuge rotor, and centrifuging at 4°C and 100000g for 90 min; removing the supernatant, resuspending the filtrate in 10 mL of pre-cooled 1×PBS, selecting an ultracentrifuge rotor, and centrifuging again at 4°C and 100000g for 90 min; removing the supernatant to obtain purified exosomes.

[0027] The present invention also provides Bidens pilosa exosomes prepared using the above preparation method.

[0028] The present invention also provides the application of the above-mentioned Bidens pilosa exosomes in the preparation of a drug for treating acute lung injury.

[0029] The amount of Bidens pilosa exosomes described in this invention is not less than 25 μg / mouse.

[0030] The present invention also provides a medicament for treating acute lung injury, comprising the above-mentioned Bidens pilosa exosomes and pharmaceutically acceptable excipients.

[0031] The dosage forms of the drugs described in this invention include at least one of the following: spray, solution, gelatin capsules, soft capsules, lozenges, chewing gum, and dry powder preparations. The drugs described in this invention may also include probiotics.

[0032] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a Bidens pilosa exosome, its preparation method, and its application in acute lung injury, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0033] Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0034] Example 1 1. Preparation of Bidens pilosa exocytic fluid samples: Wash fresh Bidens pilosa with clean water and purified water, drain, and extract juice by blunt pressing; prepare enzymatic hydrolysate: 4% cellulase, 2% pectinase; mix the crushed Bidens pilosa with the enzymatic hydrolysate and incubate overnight at 4°C; put the enzymatic hydrolysate into a 200-mesh nylon filter bag and collect the filtrate under the action of a jack; transfer the filtrate to a centrifuge bottle and centrifuge at 2000g, 4°C for 10min; carefully transfer the supernatant to a clean centrifuge bottle and centrifuge at 10000g, 4°C for 30min, and collect the supernatant; under positive pressure, pass the supernatant through MCE (1.5 / 0.8 / 0.45 / 0.22μm) and PVDF (0.45μm) filter membranes in sequence, collect the filtrate, and freeze at -80°C.

[0035] 2. The method for obtaining exosomes from Bidens pilosa was ultracentrifugation: Samples were thawed at 37℃ using a medium-speed centrifugation method; the sample was transferred to a new centrifuge tube and centrifuged at 2000g, 4℃ for 10 min; the supernatant was carefully transferred to a new centrifuge tube and centrifuged at 10000g, 4℃ for 30 min to remove larger vesicles; the supernatant was collected and filtered through a 0.45μm filter membrane, and the filtrate was collected; the filtrate was transferred to a new centrifuge tube, and centrifuged at 4℃, 100000g for 90 min using an ultracentrifuge rotor; the supernatant was removed, and the sample was resuspended in 10mL of pre-cooled 1×PBS, and then centrifuged again at 4℃, 100000g for 90 min using an ultracentrifuge rotor; the supernatant was removed, and the sample was resuspended in pre-cooled 1×PBS, 20μL was used for electron microscopy, 10μL for particle size analysis, and 10μL for protein extraction; the remaining exosomes were stored at -80℃.

[0036] 3. Exosome assay 3.1 Exosome particle size analysis Take 10 μL of exosomes and dilute it to 30 μL. After passing the instrument performance test with a standard, the exosome sample can be loaded. Note that serial dilution is necessary to avoid clogging the injection needle. Once the sample is analyzed, the particle size and concentration information of the exosomes detected by the instrument can be obtained. The average particle size and concentration information of exosomes are shown in Table 1, and a schematic diagram of particle size and concentration is shown in [reference needed]. Figure 1 .

[0037] Table 1. Average particle size and concentration information of exosomes

[0038] 3.2 Electron microscopy results of exosomes 20 μL of exocrine fluid was used for electron microscopy. The specific results are as follows: Figure 2 As shown, cells and exosomes coexist.

[0039] 3.3 Protein extraction and concentration determination from exosome samples The specific steps for determining exosome protein concentration using BCA are as follows: Exosomes are lysed at 37℃ using a medium-speed lysis method, and 5× RIPA lysis buffer is quickly added; after mixing, lysis is performed on ice for 30 min, with constant mixing during lysis; a standard sample for protein concentration determination using the BCA method is prepared, and 20 μL of the diluted sample is added to the BCA mixture and mixed; the sample is incubated at 37℃ for 30 min, and the absorbance is measured and recorded at OD562 nm using a microplate reader; the protein concentration of the sample is calculated based on the standard curve. The standard curve is shown below. Figure 3 The protein concentrations are shown in Table 2. Before dilution, the concentration of exosome protein obtained was 3.180317 μg / μL.

[0040] Table 2 Protein concentrations in exosome samples

[0041] 3.4 Exosome sterility test Take 5 mL of LB medium, add 10 μL of exosomes, and incubate on a shaker at 37℃ for 24 h. Observe the bacterial contamination. The results show that there are no contaminating bacteria in the exosome samples.

[0042] 4. Experimental verification 4.1 Animal Model Thirty-two 8-week-old male C57BL / 6 mice were selected and divided into four groups.

[0043] Group 1: Wild-type blank control group (WT); Group 2: LPS infusion via airway; Group 3: Intratracheal instillation of LPS and nasal drops of 0.25 μg / μL Bidens pilosa exosomes (LPS + EV 0.25); Group 4: LPS inhalation via airway and nasal drops of 0.5 μg / μL Bidens pilosa exosomes (LPS + EV0.5).

[0044] like Figure 4 As shown in Figure A, the LPS group successfully induced an ALI model through airway infusion of LPS (2 mg / kg). Exosomes were dissolved in PBS buffer to prepare exosome solutions with concentrations of 0.25 μg / μL and 0.5 μg / μL. At 2 hours, 2 days, 4 days, and 6 days after LPS-induced ALI, 100 μL of the prepared exosomes (0.25 μg / μL) were administered via nasal drops to the LPS+EV0.25 group (total 25 μg), and 100 μL of the exosomes (0.5 μg / μL) were administered to the LPS+EV0.5 group (total 50 μg). Samples were collected on day 7.

[0045] 4.2 Hematoxylin-eosin staining (HE staining) Right upper lung tissue from mice was fixed in 4% paraformaldehyde solution for 24 hours and then embedded in paraffin. The embedded lung tissue was cut into 3 μm thick paraffin sections for subsequent staining. HE staining was performed according to standard histological staining protocols. See below for detailed results. Figure 4 .

[0046] Results are expressed as (X±S). Statistical analysis was performed using Graphpad Prism 9.0 to compare parameters between two groups, and one-way ANOVA was used for statistical analysis among multiple groups. P A value <0.05 indicates a significant difference between the two groups.

[0047] HE staining results are as follows Figure 4 As shown in Figure A, compared with the control group mice, the alveolar walls of the LPS group mice were significantly damaged after airway administration of LPS, with a large number of inflammatory cells and erythrocytes exuded, resulting in severe damage to the lung tissue. However, treatment with Bidens pilosa exosome nasal drops reduced lung tissue damage and inflammatory cell exudation in the mice. These results indicate that Bidens pilosa exosome nasal drops can significantly alleviate LPS-induced lung damage in mice.

[0048] Lung injury score results as follows Figure 4As shown in Figure B, Bidens pilosa exosomes significantly reduced lung injury scores. The lung injury scoring principle was as follows: 10 fields of view were randomly selected from each mouse's HE slides under low magnification (×100). Pathological features were as follows: (I) pulmonary edema; (II) alveolar and interstitial inflammation; (III) alveolar and interstitial congestion; (IV) atelectasis and hyaline membrane formation. Each feature was scored from 0 to 4, with 0 points for absence or lesion extent <25% (1 point), 25%-50% (2 points), 50%-75% (3 points), and >75% (4 points), accumulating to a total histological score. The score for each parameter was averaged across ten images. Therefore, Figure 4 The results show that Bidens pilosa exosomes can alleviate LPS-induced lung damage.

[0049] 4.3 Determination of the levels of inflammatory factors IL-6, TNF-α, IL-1α and IL-1β in bronchoalveolar lavage fluid The left lung of mice was lavaged with 500 μL of PBS buffer to obtain approximately 400 μL of bronchoalveolar lavage fluid. The supernatant was obtained after centrifugation at 1000g for 5 min. The levels of IL-6, TNF-α, IL-1α, and IL-1β in the bronchoalveolar lavage fluid were detected using ELISA.

[0050] The results are as follows Figure 5 As shown, intranasal administration of Bidens pilosa exosomes reduced pulmonary inflammatory infiltration in mice after LPS injury. Specifically, intranasal administration of Bidens pilosa exosomes significantly reduced IL-6 levels in the bronchoalveolar lavage fluid of mice. Figure 5 (A); After nasal administration of Bidens pilosa exosomes, TNF-α in the bronchoalveolar lavage fluid of mice was significantly reduced ( Figure 5 (B) After nasal administration of Bidens pilosa exosomes, IL-1α levels in the bronchoalveolar lavage fluid of mice were significantly reduced ( Figure 5 (C). Following nasal administration of Bidens pilosa exosomes, IL-1β levels in the bronchoalveolar lavage fluid of mice were significantly reduced (C). Figure 5 (D). All of the above indicate that, following LPS injury, nasal administration of Bidens pilosa exosomes can reduce inflammatory infiltration in the lungs of mice.

[0051] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing Bidens pilosa exosomes, characterized in that, Includes the following steps: After washing the Bidens pilosa, it was crushed and pulverized. The pulverized material was mixed with an enzymatic hydrolysate for enzymatic hydrolysis. The resulting enzymatic hydrolysate was filtered, and the filtrate was collected. The filtrate was subjected to gradient centrifugation in sequence, and the supernatant was collected. The supernatant was then filtered through a filter membrane in sequence, and the resulting filtrate contained the Bidens pilosa exosomes. The enzymatic hydrolysate includes cellulase and pectinase.

2. The preparation method according to claim 1, characterized in that, In the enzymatic hydrolysate, the mass of the cellulase is 2-4% of the mass of the pulverized material, and the mass of the pectinase is 1-2% of the mass of the pulverized material.

3. The preparation method according to claim 1 or 2, characterized in that, The enzymatic hydrolysis was performed at a temperature of 4°C for 10-14 hours.

4. The preparation method according to claim 1, characterized in that, The gradient centrifugation includes centrifugation at 1000~3000g for 5~15min and centrifugation at 8000~15000g for 20~40min. And / or, the filtration includes sequential filtration through an MCE and a PVDF membrane.

5. The preparation method according to claim 1, characterized in that, The filtration process also includes a step of extracting exosomes from the filtrate using ultracentrifugation.

6. Bidens pilosa exosomes prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the Bidens pilosa exosomes according to claim 6 in the preparation of a medicament for treating acute lung injury.

8. A drug for treating acute lung injury, characterized in that, It includes the Bidens pilosa exosomes as described in claim 5 and pharmaceutically acceptable excipients.

9. The medicament according to claim 8, characterized in that, The dosage form of the drug includes at least one of the following: spray, solution, gelatin capsule, soft capsule, lozenge, chewing gum, and dry powder preparation.

10. The medicament according to claim 8, characterized in that, The drug also includes probiotics.

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