Application of plant-derived nanoscale vesicles in preparation of medicine for treating radiation-induced lung injury

Abstract

The invention provides application of plant-derived nanoscale vesicles in preparation of a medicine for treating radiation-induced lung injury. The plant-derived nanoscale vesicles are obtained by using aloe as a raw material through ultra-high-speed centrifugal purification. According to the invention, by separating and purifying the aloe-derived nano-scale vesicles, the aloe-derived nano-scale vesicles (AVNVs) are applied to protection of radiation-induced lung injury for the first time, and by establishing a cell model and an animal model of radiation-induced lung injury, it is determined that the AVNVs can inhibit lung cell oxidative stress, inflammation and DNA injury caused by radioactive rays from the cellular level; the traditional Chinese medicine composition can be used for regulating GSH, LDH and SOD levels in serum from the tissue level and reducing protein secretion in lung tissues, so that the lung inflammatory infiltration degree, the alveolar wall thickening degree and the tissue disorder degree are reduced, the effect of treating radiation-induced lung injury is achieved, and the traditional Chinese medicine composition has practical application and popularization values.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to the use of nanoscale vesicles of plant origin in the preparation of a medicament for treating radiation-induced lung injury. BACKGROUND

[0002] Radiation-induced lung injury (RILI) is a common and unavoidable complication in chest tumor radiotherapy. RILI is often accompanied by radiation pneumonia (RP) and radiation fibrosis, which occurs in about 5-20% of chest radiotherapy patients, which greatly limits the use of the maximum irradiation dose.

[0003] Given that late radiation fibrosis has irreversible damage, timely early intervention of RP is crucial. At present, amifostine is the only FDA-approved radio-protective drug, but its adverse reactions also limit its application. Therefore, it is imperative to develop new therapeutic drugs for intervention of RP. The core pathological mechanism of RILI is the vicious cycle of inflammatory response and oxidative stress: radiation directly induces reactive oxygen species (ROS) burst and cell damage, triggering pro-inflammatory pathways such as NF-κB, recruiting neutrophils and macrophages infiltration, further releasing ROS and inflammatory factors (such as TNF-α, IL-6, TGF-β), exacerbating lung tissue oxidative damage and driving fibrosis. This process is accompanied by the imbalance of the antioxidant system (such as the Nrf2 pathway), forming a continuous tissue destruction.

[0004] Given the limitations of traditional anti-inflammatory (such as glucocorticoids) and antioxidant therapy, plant-derived extracellular vesicles have become a potential intervention strategy due to their natural antioxidant components (such as polyphenols, flavonoids) and anti-inflammatory activity (regulating macrophage polarization, inhibiting TGF-β). Extracellular vesicles (EVs) are nanoscale particles (ranging from 30 to 150 nm) that can be released from any cell, including animal and plant cells. Due to the immunogenicity and clear resistance of immune cells, extracellular vesicles carry a variety of substances such as DNA, RNA, proteins and lipids, effectively delivering biomolecules and affecting cell behavior. Compared with mammalian-derived extracellular vesicles, plant-derived EVs have been shown by a variety of inventions to have lower immunological risks and fewer side effects. For example, lemon NVs, aloe NVs or cabbage NVs show anti-inflammatory properties, or ginger-derived nanoparticles (GiDNP) alleviate ulcerative colitis, oral (GiDNP) can reduce pro-inflammatory factors (IL-6, IL-1β and TNF-α), and citrus NVs inhibit cancer growth. A variety of inventions show that plant-derived EVs have repair potential, and the concerns associated with potential animal or human pathogens are reduced.

[0005] Aloe vera is a well-known natural remedy for treating skin rashes and burn wounds and has been widely invented in recent years. Aloe vera contains a variety of phenolic compounds, including cinnamic acid, chromone, anthracene and flavonoids, and the active components naturally carried have molecular complementarity with the host cell signaling pathways. Among them, aloe vera-derived nanoscale vesicles (AVNVs) also have good anti-inflammatory repair potential. After the invention of aloe vera-derived nanoscale vesicles (AVNVs) in the skin, they not only showed good cell compatibility with human skin cells, but also were internalized into HaCaT cells through membrane fusion, clathrin and caveolae-mediated endocytosis. Aloe vera-derived nanoscale vesicles (AVNVs) showed dose-dependent antioxidant activity and reduced intracellular ROS levels under high ROS environment. The invention found that aloe vera-derived nanoscale vesicles (AVNVs) can trigger wound healing and antioxidant defense mechanisms through Nrf2 activation.

[0006] So far, there has been no report on aloe vera-derived nanoscale vesicles (AVNVs) for treating radiation-induced lung injury. SUMMARY

[0007] In view of the above technical problems in the prior art, the present application provides the use of plant-derived nanoscale vesicles in the preparation of a drug for treating radiation-induced lung injury. The use of plant-derived nanoscale vesicles in the preparation of a drug for treating radiation-induced lung injury solves the technical problem of poor drug effect of the prior art drug for treating radiation-induced lung injury.

[0008] The present application provides the use of plant-derived nanoscale vesicles in the preparation of a drug for treating radiation-induced lung injury.

[0009] Further, the plant-derived nanoscale vesicles are obtained by ultrahigh-speed centrifugation purification using aloe vera as raw material.

[0010] Further, the preparation method of the plant-derived nanoscale vesicles comprises the following steps: 1) Weigh aloe vera, wash it with tap water, remove the sticky gel of aloe vera, cut the aloe vera skin, mix it with PBS solution at a ratio of 1: (2~6) (w / w), and then crush it using a blender; 2) Treat with a pectinase solution with a mass percentage concentration of 0.05~0.2% for 1~3h, squeeze the juice through gauze, and centrifuge the liquid at 1000g, 2000g, 3000g and 10000g for 10min, take the supernatant, and filter it with 1.5μm, 0.7μm, 0.45μm and 0.22μm filter membranes in turn to obtain clear filtrate; 3) The filtrate of step 2) is transferred into an ultracentrifuge tube, and the centrifugation condition is set as 4℃, 80000~150000g centrifugation for 70min, and then the supernatant is discarded, and the precipitate is collected; 4) The precipitate of the ultracentrifugation is resuspended with PBS solution, and it is ensured that it is completely dissolved to obtain a uniform resuspension; 5) The resuspension of step 4) is added with a plant exosome purification reagent, and the centrifugation condition is set as 4℃, 80000~150000g centrifugation for 70min, and then the middle layer is taken, which is the exosome with high purity.

[0011] Further, the drug is a drug for reducing inflammatory infiltration of lung tissue caused by radiation.

[0012] Further, the drug is a drug for inhibiting the increase of LDH level in serum and / or the decrease of SOD and GSH content in lung tissue caused by radiation.

[0013] Further, the drug is a drug for inhibiting lung cell oxidative stress and DNA damage caused by radiation.

[0014] The application applies the aloe-derived nanoscale vesicles (AVNVs) to the protection of radiation-induced lung injury for the first time by separating and purifying the aloe-derived nanoscale vesicles (AVNVs), and determines that the AVNVs can inhibit the lung cell oxidative stress, inflammation and DNA damage caused by radiation at the cell level, regulate the GSH, LDH and SOD levels in serum at the tissue level, reduce the protein secretion in lung tissue, thereby reducing the inflammatory infiltration, alveolar wall thickening and tissue disorder degree of lung, and achieve the effect of treating the occurrence of radiation-induced lung injury, and has the value of practical application and popularization.

[0015] Compared with the prior art, the technical effect of the application is positive and obvious. The aloe-derived nanoscale vesicles (AVNVs) are successfully separated and purified, and the analysis results show that the aloe-derived nanoscale vesicles (AVNVs) can effectively regulate the oxidative stress of MLE-12 cells and RAW264.7 cells under irradiation induction in vitro, and then effectively reduce the oxidative stress and inflammation cascade reaction under acute radiation-induced lung injury, and effectively help the uptake and protection of tissue cells by nasal inhalation administration. The results show that the aloe-derived nanoscale vesicles (AVNVs) have potential effect on the treatment of radiation-induced lung injury after irradiation. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the extraction and characterization of aloe-derived nanoscale vesicles (AVNVs), wherein Figure 1 A is a schematic diagram of separation and preparation of AVNVs; Figure 1B is TEM images of AVNVs; Figure 1 C is NTA detection of AVNVs for particle size and distribution.

[0017] Figure 2 is the detection of AVNVs uptake by in vitro alveolar epithelial cells MLE-12, wherein Figure 2 A-2B is flow cytometry and fluorescence detection of AVNVs uptake by alveolar epithelial cells MLE-12; Figure 2 C-2D is the statistical chart of flow cytometry and fluorescence detection, respectively.

[0018] Figure 3 is the detection of AVNVs uptake by in vitro macrophage RAW264.7, wherein, wherein Figure 3 A-3B is flow cytometry and fluorescence detection of AVNVs uptake by macrophage RAW264.7; Figure 3 C-3D is the statistical chart of flow cytometry and fluorescence detection, respectively.

[0019] Figure 4 is the detection of AVNVs of aloe origin to reduce oxidative stress and inflammation induced by irradiation in alveolar epithelial cells MLE-12, wherein Figure 4 A-4B is immunofluorescence staining and statistical chart for detecting DNA damage in MLE-12 cells under 8Gy irradiation; Figure 4 C-4E is the detection of intracellular LDH, GSH and SOD levels in MLE-12 cells under 8Gy irradiation; Figure 4 F-4G is immunofluorescence staining and statistical chart for detecting pro-inflammatory factor IL-6 in MLE-12 cells under 8Gy irradiation.

[0020] Figure 5 is the detection of AVNVs of aloe origin to reduce oxidative stress and inflammation induced by irradiation in macrophage RAW264.7 cells, wherein 5A-5B is immunofluorescence staining and statistical chart for detecting DNA damage in RAW264.7 cells under 8Gy irradiation; Figure 5 C-5E is the detection of intracellular LDH, GSH and SOD levels in RAW264.7 cells under 8Gy irradiation; Figure 5 F-5I is immunofluorescence staining and statistical chart for detecting pro-inflammatory factors IL-6 and TNF-α in RAW264.7 cells under 8Gy irradiation.

[0021] Figure 6 is the detection of AVNVs of aloe origin to reduce oxidative damage, inflammation and lung tissue damage induced by irradiation in mice, wherein Figure 6 A is a schematic diagram of the animal irradiation model and detection sample; Figure 6B is the hematoxylin / eosin (HE) staining result of lung tissue of 20 Gy irradiation-induced mice after 14 days after injury; Figure 6 C-6D is the protein concentration and SOD activity detection result of alveolar lavage fluid of 20 Gy irradiation-induced mice after 14 days after injury; Figure 6 E-6F is the detection result of LDH, GSH, and SOD levels in serum of 20 Gy irradiation-induced mice after 14 days after injury.

[0022] Figure 7 is a preparation schematic of plant-derived nanoscale vesicles of the present application, and a therapeutic principle diagram for reducing inflammation and oxidative stress and preventing and treating radiation-induced lung injury. DETAILED DESCRIPTION

[0023] The aloe-derived nanoscale vesicles (AVNVs) are prepared according to the following method: Aloe vera (Kurazao Aloe Vera Base in Yunnan Province, China) is purchased, washed with tap water, and the sticky gel is removed. The aloe vera skin is chopped and mixed with PBS solution at a ratio of 1:3 w / w. Then, a commercial blender is used for pulverization, and a 0.1% pectinase (Jiawei Rixing Company) solution is used for treatment for 2 h. The juice is squeezed through gauze, and the liquid is gradually centrifuged at 1000g, 2000g, 3000g, and 10000g for 10 min. The supernatant is taken and filtered with 1.5μm, 0.7μm, 0.45μm, and 0.22μm filter membranes in sequence to obtain clear filtrate. The filtrate is transferred into an ultracentrifuge tube, and the centrifugation conditions are set as 4℃, 100000g for 70 min. The supernatant is discarded, and the precipitate is collected. The precipitate is resuspended with an appropriate amount of PBS solution to ensure complete dissolution, and a uniform resuspension is obtained. 2mL of VesPura plant exosome purification reagent (Primcyt, China) is added, and the centrifugation conditions are set as 4℃, 100000g for 70 min. The middle layer is taken, which is the exosome with high purity. The AVNVs are stored at -70℃ until further use.

[0024] EXPERIMENTAL METHODS

[0025] Radiation cell injury model group: lung alveolar epithelial cells (MLE-12) and macrophages (RAW264.7) are cultured, and when the cell fusion degree reaches 70%-80%, an 8Gy dose of radiation is performed by a biological X-ray irradiator (RadSource Company, USA). Radiation-induced mouse lung injury model group: C57BL / 6 mice are given a single dose of 20Gy X-ray radiation to the chest by a biological X-ray irradiator (RadSource Company, USA).

[0026] Cell experiment: divided into normal group, simple aloe-derived nanoscale vesicles (AVNVs), simple irradiation group, irradiation + aloe-derived nanoscale vesicles (AVNVs) group (AVNVs prepared according to Example 1 were diluted with physiological saline, and the concentration was 100 ug / mL). Normal group: no treatment was given to the cells; simple aloe-derived nanoscale vesicles (AVNVs): cells were co-incubated with AVNVs for 24 h, and the concentration was 100 ug / mL; simple irradiation group: irradiated by a biological X-ray irradiator (RadSource company, USA) at a dose of 8 Gy; irradiation + aloe-derived nanoscale vesicles (AVNVs) group: AVNVs were used to pretreat cells at a concentration of 100 ug / mL, and then irradiated by a biological X-ray irradiator (RadSource company, USA) at a dose of 8 Gy; Animal experiment (8 C57BL / 6N mice per group): divided into normal group, simple irradiation group, aloe-derived nanoscale vesicles (AVNVs) group (the solution of Example 1 was diluted with physiological saline, and the administration dose concentration was 5 mg / kg per mouse). Normal group: 50 uL of physiological saline was nose-dropped to the mice every day; simple irradiation group: after 20 Gy X-ray single irradiation, 50 uL of physiological saline was nose-dropped for 3 consecutive days; aloe-derived nanoscale vesicles (AVNVs) group: after 20 Gy X-ray single irradiation, 50 uL of aloe-derived nanoscale vesicles (AVNVs) was nose-dropped for 3 consecutive days.

[0027] 1.3.1 Characterization of aloe-derived nanoscale vesicles (AVNVs) The protein concentration in the aloe-derived nanoscale vesicles (AVNVs) sample was measured using the BCA kit (Bi Yun Tian, China) according to the manufacturer's instructions. Transmission electron microscopy observation of aloe-derived nanoscale vesicles (AVNVs): 10 uL of plant nanoscale vesicles were taken out, 10 uL of the sample was dropped onto a copper mesh and precipitated for 1 min, the supernatant was absorbed with filter paper, 10 uL of uranyl acetate (Zhongjing Keyi) was dropped onto the copper mesh and precipitated for 1 min, the supernatant was absorbed with filter paper, and the copper mesh was dried at room temperature for several minutes. Electron microscopy (Hitachi) detection imaging was performed at 80 kv to obtain transmission electron microscopy imaging results. Particle size analysis of aloe-derived nanoscale vesicles (AVNVs): the frozen sample was thawed in a 25°C water bath, placed on ice, and the plant exosome sample was diluted with 1 × PBS. NTA detection was performed directly using a nanoparticle tracking analyzer (PARTICLE METRIX).

[0028] 0.5 μM DiD far-red fluorescent probe (C1039, Biyun Tian) was used to stain the nanoscale vesicles (AVNVs) from aloe vera. When the cell confluence reached 70-80%, the nanoscale vesicles (AVNVs) (100 ug / mL) were incubated in the cell culture box (USA, Thermo) for 0 h, 1 h, 2 h, and 4 h, respectively. The cell uptake amount was detected by flow cytometry and fluorescence microscopy.

[0029] Cell slides were placed in a 24-well plate, the cells were digested and counted using a cell counting plate, and then seeded in a 24-well plate at a density of 2x10^5 cells per well. The cells were evenly distributed by shaking the plate up and down and left and right. The cells were cultured in an incubator for 24 hours overnight. The pretreated cells were irradiated with 8 Gy X-rays. After 24 hours, 0.5 mL of paraformaldehyde was added to each well for fixation. After fixation, the cells were washed with PBS for 3x5 min, then 0.5 mL of fine permeation solution was added to each well and incubated at 37°C for 10 min, and then washed with PBS for 3x5 min. Subsequently, the cells were blocked with 2% BSA at room temperature for 2 h, and then incubated with antibody γ-H2AX (1:200; CST) at 4°C overnight. After washing with PBS for 3x5 min, the secondary antibody was incubated for 2 h. The cell nuclei were stained with DAPI. After washing with PBS for 3x5 min, 10 μL of anti-fluorescence quencher was added to the glass slide, and the slide was placed on the glass slide. The slide was observed under a upright fluorescence microscope (Germany Carl Zeiss Company).

[0030] According to the manufacturer's instructions, the cell samples / serum treated for 24 hours were collected. After washing the cells twice with 2 mL of PBS in each sample, 1 mL of reagent was added to resuspend the cells, ultrasonic treatment was performed (200 W, ultrasonic for 3 s, stop for 10 s, repeat for 30 times), centrifugation was performed at 8000 g for 10 min, the supernatant was collected, and commercially available kits (Nanjing Jiancheng, Nanjing, China) and ultraviolet-visible spectrophotometer were used to detect the biochemical indexes of superoxide dismutase (SOD), glutathione (GSH), and lactate dehydrogenase (LDH).

[0031] All mice were anesthetized with sodium pentobarbital at a dose of 40 mg / kg, fixed in a supine position on a dissection board, and the skin was disinfected with 75% ethanol. The trachea was incised: the muscles were separated to expose the trachea. A blunt needle was inserted into the trachea to a depth of about 5 mm, and silk was used to fix it to prevent slipping. Pre-cooled PBS was slowly injected using a 1 mL syringe (0.8 mL each time, a total of 3 times, a total of 2.4 mL). After each injection, the liquid was gently withdrawn and collected into an EP tube. It was immediately placed on ice, centrifuged at 4°C (300g, 10 min), and the supernatant was separated (-80°C for storage) and the cell pellet was separated. The supernatant was used to quantitatively detect the protein content of BALF using a BCA protein assay kit (Biyun Tian, China).

[0032] Figure 1 A shows the isolation and purification process of the present application, Figure 1 B Transmission electron microscopy (TEM) imaging shows that the resulting AVNVs present a typical exosome-like cup-shaped morphology, with intact capsular structure and no visible impurities, Figure 1 C Nanoparticle tracking analysis (NTA) further quantifies the particle size distribution, showing that the average hydrodynamic diameter of AVNVs is 176 nm, which is within the standard size range of exosomes (30-200 nm), and the concentration is 2.3*10^11 Particles / mL, indicating that we successfully extracted and purified high-concentration AVNVs.

[0033] Figure 2 A-2B shows that alpine nanocapsule (AVNVs) labeled with DiD on the lung alveolar epithelial cells MLE-12 is successfully and efficiently taken up, according to Figure 2 C-2D shows that the uptake of AVNVs by MLE-12 cells presents a significant time-dependent manner: after 1h of 100 μg / mL AVNVs treatment, cells generally take up AVNVs, and with time accumulation, the endocytosis dose increases in cells (* P<0.5, ** P<0.01, *** P<0.001).

[0034] Figure 3 A-3B shows that alpine nanocapsule (AVNVs) labeled with DiD on the macrophage RAW 264.7 cells is successfully and efficiently taken up, according to Figure 3 C-3D shows that the uptake of AVNVs by MLE-12 cells presents a significant time-dependent manner: after 1h of 100 μg / mL AVNVs treatment, cells generally take up AVNVs, and with 2h of incubation, the endocytosis dose reaches a peak in cells (* P<0.05, ** P<0.01, *** P<0.001).

[0035] Figure 4 A-4B shows that under the basis of non-toxicity of alpine nanocapsule (AVNVs) to MLE-12, after 8Gy X-ray irradiation, the IR group DNA damage is significantly aggravated, while AVNVs effectively alleviate the DNA damage induced by X-ray, which is reduced to about 75% of the original damage (* P<0.05, ** P<0.01, *** P<0.001). Figure 4C-4E shows that AVNVs effectively regulate the oxidative stress induced by irradiation in MLE-12 cells, making the SOD activity recover to 40±5.3% of the normal level (vs. IR group 32.1±6.8%, P<0.05), the GSH content increase 2 times (15.7±1.9 vs. IR group 7.8±1.2 μmol / g prot, P<0.001), and the LDH release amount decrease 81% (P<0.001). Figure 4 F-4G shows that AVNVs effectively reduce the secretion of inflammatory factor IL-6, and thus reduce the inflammatory response.

[0036] Figure 5 A-5B shows that AVNVs effectively reduce the DNA damage induced by X-ray irradiation, to about 55% of the original damage (P<0.5, P<0.01, P<0.001). Figure 5 C-5E shows that AVNVs effectively regulate the oxidative stress induced by irradiation in 264.7 cells, making the SOD activity recover to 48±5.3% of the normal level (vs. IR group 22.1±3.8%, P<0.001), the GSH content increase 1.31 times (11.7±1.9 vs. 7.8±2.2 μmol / g prot, P<0.05), and the LDH release amount decrease 81% (P<0.001). Figure 5 F-5I shows that AVNVs effectively reduce the secretion of inflammatory factors IL-6 and TNF-α induced by irradiation in 264.7 cells, and thus reduce the inflammatory response.

[0037] Figure 6 A shows that we establish a C57BL / 6N mouse whole chest single 20 Gy irradiation model, and give AVNVs (5 mg / kg, once a day) through nasal inhalation for continuous intervention for 3 days. Figure 6 B histopathological analysis shows that AVNVs intervention group effectively relieves the tissue damage induced by irradiation: inflammatory infiltration, alveolar wall thickening and tissue disorder degree, while the alveolar structure integrity remains good. Figure 6 C-6D shows that the protein concentration in the bronchoalveolar lavage fluid of the AVNVs intervention group is significantly reduced, and the SOD activity recovers to 76.2% of the normal level (vs. IR group 42.6%, P<0.01). Figure 6 E-6G shows that AVNVs effectively reduce the oxidative stress in the serum of mice after irradiation.

[0038] In summary, the present application determines that aloe nano vesicles (AVNVs) can inhibit oxidative stress, inflammation and DNA damage caused by radiation at the cellular level by establishing a cell model and an animal model of radiation-induced lung injury; regulate the levels of SOD, LDH and GSH in the serum, and reduce protein secretion in the alveolar lavage fluid at the animal level, thereby reducing lung inflammatory infiltration, alveolar wall thickening and the degree of tissue disorder, achieving the effect of treating the occurrence of radiation-induced lung injury, and having practical application and promotion value.

Claims

1. Use of plant-derived nanoscale vesicles in the preparation of drugs for treating radiation-induced lung injury.

2. The use as described in claim 1, characterized in that, The plant-derived nano-sized vesicles were obtained by purifying aloe vera through ultra-high-speed centrifugation.

3. The use as described in claim 1, characterized in that, The method for preparing plant-derived nanoscale vesicles includes the following steps: 1) Weigh out aloe vera, wash it with tap water to remove the sticky gel, chop the aloe vera peel, mix it with PBS solution at a ratio of 1:(2~6) (w / w), and then crush it with a blender; 2) Treat with a pectinase solution with a mass percentage concentration of 0.05~0.2% for 1~3 hours, squeeze the juice through gauze, and centrifuge the liquid stepwise at 1000g, 2000g, 3000g and 10000g for 10 minutes. Take the supernatant and filter it through filter membranes of 1.5μm, 0.7μm, 0.45μm and 0.22μm respectively to obtain a clear filtrate. 3) Transfer the filtrate from step 2) into an ultracentrifuge tube, set the centrifugation conditions to 4°C, centrifuge at 80,000~150,000g for 70 minutes, discard the supernatant and collect the precipitate; 4) Resuspend the exosome precipitate after separation in PBS solution to ensure complete dissolution and obtain a homogeneous resuspension; 5) Add plant exosome purification reagent to the resuspension in step 4), set the centrifugation conditions to 4℃, centrifuge at 80000~150000g for 70min, and take the middle layer, which is the high-purity exosome.

4. The use as described in claim 1, characterized in that, The drug is used to reduce inflammatory infiltration of lung tissue caused by radiation.

5. The use as described in claim 1, characterized in that, The drug is used to inhibit the increase in serum LDH levels and / or the decrease in SOD and GSH levels in lung tissue caused by radiation.

6. The use as described in claim 1, characterized in that, The drug is used to inhibit radiation-induced oxidative stress in lung cells and DNA damage.

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