Sophora japonica exosomes and their use in preparing drugs for treating digestive tract mucosal barrier damage

Abstract

The present invention relates to the field of exosome preparation and application technology, and in particular to Sophora japonica exosomes and their use in the preparation of drugs for digestive tract mucosal barrier damage. The Sophora japonica exosomes have an average diameter of 100 to 500 nm, a surface membrane potential of ‑15 to ‑85 mV, and contain 51.7% fatty acyl groups, 17.7% glycerophospholipids, 7.9% glycerolipids, 7.4% sphingolipids, 6.9% sterol lipids, 5.9% pro-alcohol lipids and 2.5% polyketide compounds in the Sophora japonica exosomes by mass percentage.

Description

Technical Field

[0001] The present invention relates to the technical field of exosome preparation and application, and in particular to sophora japonica flower exosomes and use thereof in preparing a medicament for treating gastrointestinal mucosal barrier damage. Background Art

[0002] Exposure to high altitude hypoxia can cause a series of acute and chronic high altitude diseases, among which the incidence of high altitude gastrointestinal stress disorder is 21%-72% (the incidence increases with increasing altitude). At present, domestic and foreign research on high altitude hypoxia is still limited to organs such as the heart, brain, and lungs. There are few studies on damage to the digestive tract, and the research has not gone deep into the specific mechanism and systematic prevention and treatment level. Therefore, it is particularly important to focus on the mechanism of high altitude hypoxic gastrointestinal diseases and clarify the causes of high altitude gastrointestinal diseases.

[0003] The cells maintain homeostasis in hypoxic environment mainly relying on the regulation of hypoxia-inducible factor (HIF). HIF is composed of HIF-α subunits (HIF-1α and HIF-2α) and HIF-1β heterodimers, which regulate gene transcription by binding to hypoxia response elements (HRE) on specific gene promoters under hypoxic conditions. Under acute hypoxia, HIF-1α and HIF-2α expression increases to improve the adaptability of cells to hypoxic environment. However, if hypoxia persists, the continued high expression of HIF-1α and HIF-2α will lead to cell death. According to domestic and foreign research and the detection of cell death pattern markers in the present invention, it is determined that the digestive tract cell death pattern caused by the high expression of HIF-1α and HIF-2α during continuous hypoxia is ferroptosis. Ferroptosis is a programmed cell death characterized by lipid peroxidation, reactive oxygen species (ROS) accumulation and oxidative stress. Studies have found that ROS can mediate the stable expression of HIFs by promoting the oxidative inactivation of PHDs. Conversely, under hypoxic conditions, HIF-1α can also promote the increase of ROS levels. The nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) protein family can produce ROS. The arachidonic acid lipoxygenase (ALOX) family and cytochrome P450 oxidoreductase (POR, also known as CYPOR) mediate ferroptosis through the peroxidation of polyunsaturated fatty acids (PUFA).

[0004] Sophora japonica L.flower is a medicinal and edible plant. It mainly contains a variety of medicinal flavonoids, such as rutin, hyperoside and quercetin, which have various pharmacological effects such as antioxidant, antibacterial, antiviral and hemostatic effects. Plant-derived exosome-like nanoparticles (PDELNs) can play an immunomodulatory role, improve intestinal microbiota, interspecies exchange regeneration and antioxidant effects through the biomacromolecules (lipids, proteins and miRNAs) they contain. They have the advantages of low toxicity, low immunogenicity, high cellular uptake efficiency, high biocompatibility and stability, which makes PDELNs have great potential in clinical applications for the treatment of gastrointestinal diseases. For example, grape exosome nanoparticles induce the recovery of intestinal stem cells through the Wnt / β-catenin signaling pathway and protect mice from colitis caused by dextran sulfate sodium (DSS). Ginger-derived exosomal miRNA can shape the intestinal microbiota and has the potential to improve intestinal barrier function.

[0005] Sophora japonica flower belongs to the class of flowers that can be used as both medicine and food. It mainly contains a variety of medicinal flavonoid ingredients such as rutin, hyperoside, quercetin, etc. It has multiple pharmacological effects such as antioxidant, antibacterial, hemostatic and gastrointestinal protection. At present, there are no reports on the research of Sophora japonica flower exosomes. Summary of the invention

[0006] The first aspect of the present invention provides a Sophora japonica flower exosome, and the preparation method thereof comprises: soaking Sophora japonica flower in an ethanol solution under sterile conditions, then washing with distilled water to remove the ethanol, drying with sterile paper, adding an enzyme solution to the Sophora japonica flower, centrifuging the obtained mixture multiple times, filtering and collecting the supernatant, continuing to ultracentrifuge the supernatant multiple times, and resuspending the precipitate with NaCl to obtain Sophora japonica flower exosomes.

[0007] The sophora japonica flower of the present invention is derived from Robinia pseudoacacia L.flower in Latin.

[0008] In some embodiments, the average diameter of the Sophora japonica flower exosomes is 100 to 500 nm, and the surface membrane potential is -15 to -85 mV.

[0009] In some embodiments, by mass percentage, the Sophora japonica flower exosomes contain 51.72% fatty acyl groups, 17.73% glycerophospholipids, 7.88% glycerolipids, 7.39% sphingolipids, 6.90% sterol lipids, 5.91% pro-alcohol lipids and 2.46% polyketides.

[0010] The second aspect of the present invention provides the use of the above-mentioned Sophora japonica flower exosomes in the preparation of a drug for treating gastrointestinal mucosal barrier damage.

[0011] In some embodiments, the digestive tract mucosal barrier damage is digestive tract mucosal barrier damage caused by high altitude hypoxia.

[0012] The third aspect of the present invention provides a pharmaceutical preparation for treating gastrointestinal mucosal barrier damage, the pharmaceutical preparation comprising the above-mentioned Sophora japonica flower exosomes and optionally containing pharmaceutically acceptable excipients.

[0013] In some embodiments, the pharmaceutical preparation is any one of an injection, capsule, tablet, film, suppository, granule, oral solution, dripping pill, water pill, paste, powder, pill, granule, patch, aerosol, powder spray or powder injection.

[0014] In some embodiments, the administration of the pharmaceutical preparation includes any one of oral administration, injection administration, inhalation administration, sublingual administration, rectal administration or skin mucosal administration.

[0015] In some embodiments, the pharmaceutical preparation can reduce the levels of ROS, hypoxia-inducible factor HIF-α, and lipid peroxides that are elevated due to hypoxia in gastric and small intestinal mucosal cells.

[0016] In some embodiments, the pharmaceutical preparation is capable of inhibiting hypoxia-induced ferroptosis of gastric and small intestinal mucosal cells.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The animal experimental results of the present invention show that oral administration of Sophora japonica exosomes can reduce the level of HIF-α, inhibit cell ferroptosis, and alleviate hypoxia-induced gastric and small intestinal mucosal barrier dysfunction. Sophora japonica exosomes have the potential to become a treatment for gastric and small intestinal mucosal damage under hypoxic conditions.

[0019] 2. The animal experimental results of the present invention show that Sophora japonica exosomes can effectively alleviate hypoxia-induced ferroptosis of gastric and small intestinal cells in mice, and can also alleviate the microbial and metabolic disorders caused by hypoxia, indicating that Sophora japonica exosomes may become a therapy for gastrointestinal mucosal damage under hypoxic conditions.

[0020] 3. The results of cell experiments in the present invention show that Sophora japonica exosomes inhibit lipid peroxidation of gastric and small intestinal mucosal cells caused by hypoxia by inhibiting HIF-α-mediated NOX4 and ALOX5, thereby effectively alleviating cell ferroptosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an electron micrograph of the Sophora japonica exosomes prepared in Example 1.

[0022] Figure 2 This is a data graph of the average diameter of Sophora japonica exosomes prepared in Example 1.

[0023] Figure 3 This is a graph showing the surface membrane potential data of Sophora japonica exosomes prepared in Example 1.

[0024] Figure 4 This is a statistical chart of the percentage of each lipid component in the Sophora japonica exosomes prepared in Example 1.

[0025] Figure 5 This is a statistical diagram of the specific components of lipid subclasses in the Sophora japonica exosomes prepared in Example 1.

[0026] Figure 6 The effect of Sophora japonica exosomes on the hypoxic mouse model in Example 2, wherein: Figure 6 A is a schematic diagram of the mouse model and drug administration. Figure 6 B is the daily food consumption (g), Figure 6 C is the percentage of body weight change, Figure 6 D is the loose stool score, Figure 6 E is the bloody stool score, Figure 6 F is the disease activity index score.

[0027] Figure 7 These are cross-sectional views of the digestive tract and stomach of the hypoxic mouse in Example 2, wherein A is a cross-sectional view of the digestive tract and B is a cross-sectional view of the stomach.

[0028] Figure 8 These are the H&E staining results of the digestive tract of mice in Example 2, where A represents the esophagus, stomach, duodenum, jejunum, ileum, and colon, and B represents the pathological scores of the above digestive tract segments.

[0029] Fig. 9 This is a diagram of the in vivo tracing results of Sophora japonica exosomes in Example 2.

[0030] Fig.10 These are transmission electron micrographs of mouse stomach and small intestinal mucosal cells in Example 2, wherein A is an electron micrograph of stomach mucosal cells, and B is an electron micrograph of small intestinal mucosal cells.

[0031] Fig.11 This is a graph of FITC-dextran levels in mouse serum in Example 2.

[0032] Fig.12 These are the cytokine / chemokine level graphs of the gastric and small intestinal mucosa in Example 2, wherein A is the cytokine / chemokine level graph of the gastric mucosa, and B is the cytokine / chemokine level graph of the small intestinal mucosa.

[0033] Fig.13It is the Western blot of gastric and small intestinal mucosal tissues in Example 2, wherein A is the expression level of gastric mucosal hypoxia-inducible factor-1α, hypoxia-inducible factor-2α, glutathione peroxidase 4 and internal reference GAPDH, and B is the expression level of small intestinal mucosal hypoxia-inducible factor-1α, hypoxia-inducible factor-2α, glutathione peroxidase 4 and internal reference GAPDH.

[0034] Fig.14 It is a graph of the levels of gastric and small intestinal mucosal iron death markers in Example 2, wherein A is the level of gastric mucosal ferrous ion, reactive oxygen, 4-hydroxynonenal, and lipid peroxide, and B is the level of small intestinal mucosal ferrous ion, reactive oxygen, 4-hydroxynonenal, and lipid peroxide.

[0035] Fig.15 is the alpha diversity map of stomach and small intestine microorganisms in Example 2, wherein: Fig.15 A is the α diversity of gastric microorganisms, Fig.15 B is the α diversity of small intestinal microorganisms.

[0036] Fig.16 is the beta diversity map of stomach and small intestine microorganisms in Example 2, wherein: Fig.16 A is the β diversity of gastric microorganisms, Fig.16 B is the beta diversity of small intestinal microorganisms.

[0037] Fig.17 This is a diagram of the accumulation of stomach and small intestine microorganisms in Example 2, wherein: Fig.17 A and Fig.17 B is the accumulation diagram of gastric microorganisms (phylum, genus), Fig.17 C and Fig.17 D is the accumulation diagram of small intestinal microorganisms (phylum, genus).

[0038] Fig.18 These are the microorganisms with differences at the hilum and genus levels in Example 2, wherein A represents the microorganisms with differences at the hilum level, and B represents the microorganisms with differences at the genus level.

[0039] Fig.19 These are the microorganisms with differences in the phylum and genus levels of the small intestine in Example 2, wherein A represents the microorganisms with differences in the phylum level of the small intestine, and B represents the microorganisms with differences in the genus level of the small intestine.

[0040] Fig. 20 These are the differential metabolites of stomach and small intestine in Example 2, wherein A is the differential metabolite of stomach and B is the differential metabolite of small intestine.

[0041] Fig.21It is the combined analysis of stomach and small intestine microorganisms and metabolites in Example 2, wherein A is the combined analysis of differential microorganisms and differential metabolites at the gastric phylum level and the gastric genus level, and B is the combined analysis of differential microorganisms and differential metabolites at the small intestine phylum level and the small intestine genus level.

[0042] Fig. 22 The crystal violet staining results of cells in Example 3 (determining the relationship between hypoxia, hypoxia-inducible factor-α and ferroptosis), wherein A is the crystal violet staining of normal human gastric epithelial cells to detect surviving cells, and B is the crystal violet staining of normal human small intestinal epithelial cells to detect surviving cells.

[0043] Fig.23 The results of cell CCK-8 detection in Example 3 (determining the relationship between hypoxia, HIF-α and ferroptosis), wherein A is the CCK-8 detection of cell viability of normal human gastric epithelial cells, and B is the CCK-8 detection of cell viability of normal human small intestinal epithelial cells.

[0044] Fig.24 It is a diagram of the crystal violet staining results of cells in Example 3 (determining the effect of Sophora japonica exosomes on hypoxic death of gastric and small intestinal epithelial cells), wherein A is the crystal violet staining of normal human gastric epithelial cells to detect surviving cells, and B is the crystal violet staining of normal human small intestinal epithelial cells to detect surviving cells.

[0045] Fig.25 The results of cell CCK-8 detection in Example 3 show the effect of Sophora japonica exosomes on hypoxic death of gastric and small intestinal epithelial cells), wherein A is the CCK-8 detection of cell viability of normal human gastric epithelial cells, and B is the CCK-8 detection of cell viability of normal human small intestinal epithelial cells.

[0046] Fig.26 This is a result graph of the ferroptosis marker levels of NGEC cells in Example 3, wherein A is ferrous ions, B is reactive oxygen species, C is 4-hydroxynonenal, and D is malondialdehyde.

[0047] Fig. 27 This is a result graph of the levels of HIEC cell ferroptosis markers in Example 3, wherein A is ferrous ions, B is reactive oxygen species, C is 4-hydroxynonenal, and D is malondialdehyde.

[0048] Fig.28 These are transmission electron microscopy images of cells in Example 3, wherein A is the ferroptosis characteristics of NGEC detected by transmission electron microscopy, and B is the ferroptosis characteristics of HIEC detected by transmission electron microscopy.

[0049] Fig.29It is the Western blot of the cells in Example 3, wherein A is the expression of hypoxia-inducible factor-1α, hypoxia-inducible factor-2α, proline hydroxylase, nicotinamide adenine dinucleotide phosphate oxidase, arachidonic acid lipoxygenase, cytochrome P450 oxidoreductase and internal reference detected in NGEC, and B is the expression of hypoxia-inducible factor-1α, hypoxia-inducible factor-2α, proline hydroxylase, nicotinamide adenine dinucleotide phosphate oxidase, arachidonic acid lipoxygenase, cytochrome P450 oxidoreductase and internal reference detected in HIEC.

[0050] Fig.30 It is the Western blot of the cells in Example 3, wherein A is the detection of the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 and arachidonic acid lipoxygenase 5 after knocking down hypoxia-inducible factor-1α or hypoxia-inducible factor-2α in NGEC, and B is the detection of the expression of nicotinamide adenine dinucleotide phosphate oxidase 4 and arachidonic acid lipoxygenase 5 after knocking down hypoxia-inducible factor-1α or hypoxia-inducible factor-2α in HIEC.

[0051] Fig.31 These are the in vitro tracing images of Sophora japonica exosomes in Example 3, wherein A is a image of Sophora japonica exosomes and NGECs engulfing Sophora japonica exosomes after 24 hours of culture, and B is a image of Sophora japonica exosomes and HIECs engulfing Sophora japonica exosomes after 24 hours of culture. DETAILED DESCRIPTION

[0052] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0053] Example 1

[0054] This example provides the extraction and identification of Sophora japonica exosomes, and the extraction method specifically includes:

[0055] Ultracentrifugation was used to isolate Sophora japonica exosomes. Sophora japonica was washed, dried, chopped and soaked in 75% alcohol for 1 minute under sterile conditions. The alcohol residue was removed with distilled water, and 20 mL of enzyme solution (4% cellulase, 2% pectinase and 0.6 mol / L mannitol, pH 5.8) was added after drying with sterile paper. The sample was then centrifuged (4°C, 2000×g, 30 minutes), and the supernatant was carefully transferred to a new centrifuge tube and centrifuged again (4°C, 10000×g, 45 minutes) to remove larger vesicles and dead cells. The supernatant was then collected by filtration with a 0.45μm filter and ultracentrifuged twice (4°C, 100000×g, 70 minutes). The precipitated exosomes were resuspended with 150μL of pre-cooled 1×NACL and then stored at -80°C.

[0056] The obtained Sophora japonica exosomes are identified, specifically including:

[0057] (1) Transmission electron microscopy observation of Sophora japonica exosomes

[0058] The exosomes stored at -80℃ were melted at room temperature, and then 5μL of the sample was added to the copper mesh for precipitation for 1min, and the floating liquid was removed with filter paper. Then 5μL of uranyl acetate was added to the copper mesh for precipitation for 1min, and the floating liquid was removed with filter paper. After drying at room temperature for several minutes, electron microscopy was performed at 100kV for imaging.

[0059] Electron micrographs see Figure 1 ,Depend on Figure 1 It can be seen that Sophora japonica exosomes are round and have a double-layer lipid membrane structure.

[0060] (2) Particle size analysis and potential measurement of Sophora japonica exosomes

[0061] The exosomes stored at -80°C were thawed in a 25°C water bath, then placed on ice, diluted with 1× NACL at a ratio of 1:10, and detected using a nanoparticle tracking analyzer.

[0062] See also Figure 2-3 The results showed that the average diameter of Sophora japonica exosomes was 176.3 nm and the surface membrane potential was -83.4 mV, which was consistent with the characteristics of exosomes, indicating that the extracted exosomes met their typical characteristics and had a high purity, which could be used for the next experiment.

[0063] (3 Analysis of lipid components of Sophora japonica exosomes

[0064] Sample extraction: Take 100 μL of Sophora japonica exosomes, grind with liquid nitrogen, add 3 times the volume of lysis buffer (80% methanol), vortex mix, grind with a tissue crusher (45Hz, 4min), ultrasonicate for 5min (ice water bath), stand at -20℃ for 30min, centrifuge at 14000g for 30min, take the supernatant and filter through a 0.45μm filter membrane and wait for machine detection. Take 20μL of the supernatant in the injection bottle for machine detection, and take an equal amount of supernatant to mix into the quality control sample for machine detection.

[0065] Mass spectrometry identification: Loading Pump flow rate 0.25mL / min, 20 minutes, mass spectrometry parameters see Table 1-2;

[0066] Table 1

[0067]

[0068] Table 2

[0069]

[0070] Mass spectrometry data processing: The mass spectrometry data were combined with the human metabolome database and the lipid map structure database (LIPID MAPS Structure Database, LMSD) to analyze the metabolite components of Sophora japonica exosomes.

[0071] The experimental results are shown in Figure 4-5 , indicating that Sophora japonica exosomes are rich in 51.7% fatty acyl groups, 17.7% glycerophospholipids, 7.9% glycerolipids, 7.4% sphingolipids, 6.9% sterol lipids, 5.9% pro-alcohol lipids and 2.5% polyketides. Among the fatty acyl groups, the content of fatty acids and conjugates is as high as 49%.

[0072] Example 2

[0073] Experimental animals: C57BL / 6J mice (male, 6 weeks) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were raised in the specific pathogen free (SPF) animal room of Tsinghua University at room temperature of 20-24°C, with a 12h / 12h day and night cycle, free access to food and water, and mice were allowed to adapt to the environment for one week before the experiment. The experimental protocol was approved by the Animal Care and Use Committee of Tsinghua University (Protocol No. 23-CZJ1).

[0074] This example provides a study on the inhibition of hypoxia-ferroptosis of gastric and small intestinal mucosal cells by Sophora japonica exosomes in mice, which specifically includes the following steps:

[0075] (1) Animal model construction

[0076] Mice were placed in a ventilated hypobaric oxygen chamber system to simulate an altitude of 5500 meters for 3 days to induce hypoxic gastrointestinal injury. The mice were randomly divided into five groups: ① normoxia + NaCl group, exposed to room air (21% O2) for 3 days, and gavaged with 100μL Nacl on the first day; ② hypoxia + NaCl group, placed in the hypoxic chamber for 3 days, and gavaged with 100μL NaCl on the first day; ③ hypoxia + low-dose Sophora japonica exosomes group, placed in the hypoxic chamber for 3 days, and gavaged with 100μL Sophora japonica exosomes (5×10 4 ④ Hypoxia + Sophora japonica exosomes medium-dose group, placed in the hypoxia chamber for 3 days, and gavage with 100 μL Sophora japonica exosomes (1×10 5 ⑤ Hypoxia + Sophora japonica exosomes high-dose group, placed in the hypoxia chamber for 3 days, and gavage with 100 μL Sophora japonica exosomes (5×10 5 The food intake, body weight, stool characteristics, and blood in stool of the mice were recorded every day, and the disease activity index was recorded. On the third day, the mice were euthanized and the entire gastrointestinal tract (including esophagus, stomach, duodenum, jejunum, ileum, and colon) was removed for H&E staining. Three H&E-stained specimens were taken from each group for histopathological scoring, and the gastric and small intestinal mucosa were collected for ROS detection.

[0077] The gavage method is as follows: fix the mouse with the left hand, hold the gavage device with the right hand (the gavage needle is installed and NACL or exosomes have been sucked), insert the gavage needle into the mouth from the corner of the mouse's mouth, press the mouse's head to make the mouth and esophagus in a straight line, gently turn the needle to stimulate the mouse to swallow, slowly insert the gavage needle into the esophagus along the posterior wall of the pharynx, until the front end of the gavage needle reaches the level of the diaphragm, slowly push the syringe to complete the operation, and aspirate NACL to clean the gavage needle before each gavage.

[0078] Disease activity index scoring criteria: percentage of mouse weight loss (0 points for unchanged weight, 1 point for a decrease of 1-5%, 2 points for a decrease of 5-10%, 3 points for a decrease of 10-15%, and 4 points for a decrease of more than 15%), stool viscosity (0 points for normal, 1 point for soft stools, 2 points for mucous stools, and 3 points for watery stools), and stool occult blood (0 points for normal, 1 point for light blue, 2 points for blue, 3 points for dark blue, and 4 points for overt bleeding).

[0079] Scoring criteria for esophageal and gastric histopathology: mucosal injury (normal: 0 points, epithelial detachment: 1 point, superficial lamina propria or 1 / 3 epithelial detachment: 2 points, middle lamina propria or 2 / 3 epithelial detachment: 3 points, lower lamina propria or more than 2 / 3 epithelial detachment: 4 points), inflammatory cell infiltration (normal: 0 points, lamina propria infiltration: 1 point, muscularis mucosa infiltration: 2 points, extensive muscularis mucosa infiltration with edema: 3 points, submucosal infiltration: 4 points)

[0080] Scoring criteria for intestinal histopathology: epithelial cell damage (normal: 0 points, mild goblet cell loss: 1 point, extensive goblet cell loss: 2 points, mild crypt loss and extensive goblet cell loss: 3 points, extensive crypt loss: 4 points), inflammatory cell infiltration (normal: 0 points, infiltration around the crypt base: 1 point, infiltration of the muscularis mucosa: 2 points, extensive infiltration of the muscularis mucosa with edema: 3 points, infiltration of the submucosal layer: 4 points), bleeding (normal: 0 points, bleeding area less than or equal to 10%: 1 point, bleeding area of ​​11%-20%: 2 points, 21%-30%: 3 points, and greater than 30%: 4 points)

[0081] Results Figure 6-7 , Figure 6 The results showed that compared with the normoxic control group, hypoxia caused the mice to eat less, have bloody stools, loose stools and lose weight. The three doses of Sophora japonica exosomes gavage could significantly improve these symptoms, among which the high dose of Sophora japonica exosomes (5×10 5 Therefore, a high dose of Sophora japonica exosomes (5×10 5 Particles / 100 μL NaCl) were used for subsequent experiments. *P<0.05, **P<0.01, ***P<0.001. Figure 7 The results showed that compared with the normoxia + NaCl group, the entire digestive tract and gastric mucosa of mice in the hypoxia + NaCl group were congested and red, but they were significantly restored after treatment with Sophora japonica exosomes.

[0082] (2) H&E staining steps

[0083] Fresh mouse gastrointestinal tissue was fixed with 4% paraformaldehyde fixative for more than 24 hours. The fixed specimens were trimmed and then dehydrated step by step, embedded in paraffin, and the wax blocks were cut into 4 μm thick slides, and then the prepared sections were placed in a section box for storage.

[0084] The prepared sections were dewaxed and hydrated, then placed in hematoxylin for staining for 3-5 minutes, rinsed with distilled water, dried, and then placed in eosin for counterstaining. After transparent dehydration, the sections were sealed with neutral resin.

[0085] Results Figure 8 , Figure 8 The results showed that hypoxia can cause serious damage to the gastric and small intestinal mucosal barriers, including loss, shedding and death of gastrointestinal mucosal cells, as well as smaller villi, shallower crypts and thinner small intestinal mucosa. Taking Sophora japonica exosomes can improve this mucosal damage. It is worth noting that hypoxia did not cause damage to the esophagus and colon. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0086] (3) In vivo tracing of Sophora japonica exosomes

[0087] The mice were shaved the night before, and the remaining short floating hair was removed with shaving cream, and then wiped clean with saline or NaCl. The mice were gavaged with DiR-labeled Sophora japonica exosomes at a single dose of 5×10 5 Particles / 100μL NaCl. In vivo imaging was performed using the Odyssey CLx imaging system at 0, 6, 16, and 24 hours after administration. The specific operation method is: place the mouse in a pre-anesthetized transparent chamber, and when the animal is in an anesthetized state, take the animal out of the pre-anesthetized transparent box, put the animal into the imaging box, close the box door, perform in vivo imaging, and observe the in vivo distribution of Sophora japonica exosomes.

[0088] Results Fig. 9 , Fig. 9 It was shown that strong fluorescence signals were found in the entire gastrointestinal tract of mice after 6 hours, and the fluorescence signals gradually weakened after 16 hours.

[0089] (4) Transmission electron microscopy observation

[0090] After the mice were euthanized, the stomach and small intestine were quickly removed and cut into 1 mm 3 Size, fixed with 2.5% glutaraldehyde, while cells were fixed with 2.5% glutaraldehyde after 2 hours of medium removal. Tissues and cells were then fixed with osmium tetroxide, dehydrated in alcohol gradients, infiltrated with acetone and epoxy resin, embedded in epoxy resin, cut into 80-100 nm ultrathin sections with an ultramicrotome, stained with 2% saturated aqueous uranyl acetate and lead citrate, and observed under a transmission electron microscope.

[0091] Results Fig.10 , Fig.10 It showed that hypoxia caused the mitochondria of the gastric and small intestinal mucosal cells of mice to shrink, increase membrane density, and lose mitochondrial cristae, which is one of the morphological characteristics of ferroptosis. Oral administration of Sophora japonica exosomes can alleviate this phenomenon.

[0092] (5) In vivo gastrointestinal permeability test

[0093] Mice were fasted for 4 hours before oral gavage. Fluorescein isothiocyanate isomer (FITC)-dextran was orally administered at a concentration of 0.6 mg / g body weight. Serum was collected four hours later and the fluorescence intensity was measured by fluorescence measurement.

[0094] Results Fig.11 , Fig.11The results showed that hypoxia caused an increase in serum FITC-dextran levels, while Sophora japonica exosomes could significantly reduce serum FITC-dextran levels, indicating that Sophora japonica exosomes could significantly improve the gastrointestinal mucosal barrier damage caused by hypoxia. ***P<0.001,***P<0.001.

[0095] (6) Multiplex ELISA test, comprising the following steps:

[0096] S1. Tissue protein extraction: The protein lysis buffer formula is shown in Table 3; the extraction method is as follows: collect about 1 cm of gastric and small intestinal mucosal tissue in a 1.5 mL EP tube, add 200 μL of protein lysis buffer, cut the tissue on ice and grind it with a homogenizer, place it on ice, vortex once every 10 minutes, vortex three times and lyse it on ice for 30 minutes, then centrifuge at 12000 rpm and 4°C for 20 minutes, and transfer the supernatant to a new 1.5 mL EP tube.

[0097] Table 3

[0098]

[0099]

[0100] S2. Organize the test, including the following steps:

[0101] 1. Add 200 μL of washing solution to each well of the plate, seal the plate and shake on a rocking plate at room temperature (20-25°C) for 10 minutes.

[0102] 2. Pour off the wash solution, invert the plate and tap it on an absorbent towel several times to remove any residue from all wells.

[0103] 3. Add 25 μL of standard or control to the corresponding wells, and add 0 pg / mL of standard to the blank group as background.

[0104] 4. Add 25 μL of assay buffer to the sample wells.

[0105] 5. Add 25 μL of the appropriate matrix solution to the background, standard, and control wells.

[0106] 6. Add 25 μL of sample to the corresponding wells.

[0107] 7. Add 25 μL of mixed beads to each well and vortex the bottle to mix.

[0108] 8. Seal the plate, wrap it with tin foil and incubate it on a rocking plate at 2-8°C overnight.

[0109] 9. Remove the samples from the wells and wash the plate twice according to the instructions in section 1-2.

[0110] 10. Add 25 μL of detection antibody to each well (Note: Warm the antibody to room temperature before adding).

[0111] 11. Seal the plate, wrap it with tin foil and incubate it on a shaker at room temperature (20-25°C) for 1 hour.

[0112] 12. Add 25 μL of streptavidin-phycoerythrin to each well containing detection antibody.

[0113] 13. Seal the plate, wrap it with tin foil and incubate it on a shaker at room temperature (20-25°C) for 30 minutes.

[0114] 14. Remove the samples from the wells and wash the plate twice according to the instructions in section 1-2.

[0115] 15. Add 150 μL of Sheath Fluid PLUS to all wells, add beads back in and shake on a rocking plate for 5 minutes.

[0116] 16. Read the data using INTELLIFLEX software and calculate the cytokine / chemokine concentration in the sample using the sample fitting curve.

[0117] The information of standard products is shown in Table 4.

[0118] Table 4

[0119]

[0120]

[0121] Results Fig.12 , Fig.12 The results showed that after treatment with Sophora japonica exosomes, the levels of eotaxin, granulocyte macrophage colony-stimulating factor, interleukin-1α, interleukin-3, interleukin-6, interleukin-7, interleukin-9, interleukin-12p40, leukemia inhibitory factor, interferon-γ-induced protein 10, macrophage colony-stimulating factor, interferon-induced monocyte factor, T cell-specific chemokine, and vascular endothelial growth factor, which were downregulated in the gastric mucosa due to hypoxia, were restored, and the levels of eotaxin, interleukin-9, interferon-γ-induced protein 10, macrophage inflammatory protein 2, interferon-induced monocyte factor, T cell-specific chemokine, and vascular endothelial growth factor, which were downregulated in the small intestinal mucosa due to hypoxia, were restored.

[0122] (7) Western blot detection, including the following steps:

[0123] S1. Tissue protein extraction

[0124] Extract stomach and small intestinal tissue proteins according to the method described in step (6).

[0125] S2. BCA method to detect tissue protein concentration

[0126] According to the BCA kit, the working solution was prepared with a ratio of 50:1 between solution A and solution B. Take 10uL of 5ng / mL BSA and dilute it with 90μL NaCl to prepare 100μL of 0.5mg / ml BSA solution. Prepare a 96-well plate and add 180μL BCA working solution to the standard wells and sample wells respectively; add 0.5mg / mL BCA solution 0, 1, 2, 4, 8, 16, 20μL and deionized water 20, 19, 18, 16, 12, 4, 0μL to the standard wells in sequence, add 18μL of deionized water and 2μL of protein sample to the sample wells, mix well and incubate in a 37℃ incubator for 30min. Use a spectrophotometer to record the optical density value of each well at 562nm, draw a standard curve with the optical density value of the standard well as the ordinate and the protein concentration as the abscissa, and calculate the protein concentration of the sample wells based on this standard curve.

[0127] S3. Protein concentration normalization

[0128] The sample protein concentration was adjusted to be consistent according to the calculation results. Taking the lowest concentration as the standard, the remaining samples were added with appropriate amounts of protein lysis buffer to make up, and then protein loading buffer was added to prepare the sample solution, which was mixed and stored in a -80℃ refrigerator.

[0129] S4.Western blot

[0130] S401. Preparation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0131] Align the two glass plates and place them on the clamping plate, then install them on the fixed frame. Prepare the lower glue mixture according to the reagent instructions: take one piece of glue as an example, the lower glue mixture contains 5 mL of liquid A and liquid B, add 100 μL of ammonium persulfate (APS), mix thoroughly, add the lower glue mixture to the glass plate sandwich, and then gently add 2 mL of anhydrous ethanol along the plate wall to press the glue. After the lower glue solidifies (about 40 minutes), pour out the anhydrous ethanol and absorb the residual anhydrous ethanol with absorbent paper. Prepare the upper glue: take one piece of glue as an example, the upper glue mixture contains 1 mL of liquid A and liquid B, add 20 μL of APS, mix thoroughly, add the upper glue mixture to the glass plate sandwich, and then insert a comb that has been cleaned and dried in advance. After the upper layer of gel solidifies (about 20 minutes), remove the glass plate, wipe it dry and insert it into the electrophoresis tank, add 1× electrophoresis solution (10× electrophoresis solution prepared with deionized water), then take out the comb and wait for sample loading.

[0132] S402. Electrophoresis

[0133] Add 2μL protein Maker and 18μL protein loading buffer to the Maker wells, and 20μL protein sample (previously boiled and cooled in a metal bath) to the sample wells, leaving the holes at both ends. After loading, connect the electrophoresis tank and power supply, 80V for the upper gel, 30-40min, 120V for the lower gel, 80-100min.

[0134] S403. Transfer

[0135] Add 4℃ pre-cooled transfer solution (10× transfer solution, methanol and deionized water in a ratio of 1:2:7) to the transfer tank and operation tank, place the transfer clip, sponge pad, and filter paper (from bottom to top) in the operation tank, gently pry open the glass plate along the gap with the cutting board, cut the gel according to the molecular weight of the protein Maker, place the cut gel strip on the filter paper of the black transfer clip, take out the pre-cut nitrocellulose filter membrane (Nitrocellulose filter membrane, NC membrane) and stick it on the gel strip, close the sponge pad and use a roller brush to gently drive out bubbles, close the transfer clip and transfer to the transfer tank. Transfer the membrane at 4℃, 100V, and 100min constant voltage (taking 10% SDS-PAGE as an example, the specific duration is adjusted according to the molecular weight).

[0136] S404. Ponceau Staining

[0137] Use tweezers to take out the NC membrane and place it in the incubation box. Add the pre-prepared Ponceau red dye solution and place it on a shaker. After 5 minutes, discard the Ponceau red dye solution and add distilled water to rinse repeatedly to reduce the background color.

[0138] S405. Film cutting

[0139] The membrane was cut according to the molecular weight of the target protein, the cut NC membrane was placed in an incubation box, and 1×TBST was used to wash away the residual Ponceau red dye.

[0140] S406. Closed

[0141] Add 5% skim milk (prepared in 1×TBST) prepared in advance and block at room temperature for 2 h.

[0142] S407. Incubate with primary antibody

[0143] After blocking, carefully discard the milk. Add 1×TBST (10×TBST and deionized water) and rinse on a shaker for 10 minutes and discard. Repeat three times and add the pre-prepared primary antibody incubation solution (5% skim milk) and incubate on a shaker at 4°C overnight.

[0144] S408. Incubate with secondary antibody

[0145] After the primary antibody incubation is completed, carefully recover the primary antibody incubation solution and store it in a -20℃ refrigerator. Add 1×TBST and rinse on a shaker for 10 minutes and discard. Repeat three times and select the corresponding secondary antibody according to the species of the primary antibody. Prepare the secondary antibody incubation solution (the ratio of secondary antibody to 1×TBST is 1:10000), mix thoroughly and add to the corresponding incubation box, and incubate at room temperature for 2 hours.

[0146] S409. Chemiluminescence imaging

[0147] After the secondary antibody incubation is completed, discard it, add 1×TBST, place it on a shaker to rinse for 10 minutes and discard it, repeat three times for development. Place the membrane on the imaging system and drop the prepared enhanced chemiluminescence (ECL) developer (prepared in dark, A solution and B solution are 1:1), start exposure and save the image.

[0148] Results Fig.13 , Fig.13 It was shown that Sophora japonica exosomes could significantly reduce the level of HIF-1α elevated by hypoxia in the gastric and small intestinal mucosa, but did not affect the protein expression of HIF-2α and GPX4.

[0149] (8) Detection of ferroptosis markers

[0150] The gastric and small intestinal tissue proteins were extracted according to the method described in step (6). The levels of ferrous ions, reactive oxygen species, 4-hydroxynonenal, and lipid peroxides in the gastric mucosa and small intestinal mucosa were analyzed according to the reagent instructions.

[0151] Results Fig.14 , the figure shows that Sophora japonica exosomes can reduce the levels of reactive oxygen species, 4-hydroxynonenal and lipid peroxides induced by hypoxia in the gastric and small intestinal mucosa, while hypoxia and Sophora japonica exosomes treatment have no effect on the levels of ferrous ions in the gastric and small intestinal mucosa. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0152] (9) 16S rRNA sequencing

[0153] After mice were euthanized, the stomach and small intestine contents were immediately isolated, frozen in liquid nitrogen and transferred to -80°C for subsequent DNA extraction and microbial analysis.

[0154] The primers were amplified with the primer (5'-ACTCCTACGGGAGGCAGCA-3') and the reverse primer (5'-GGACTACHVGGGTWTCTAAT-3'). The 16S rRNA gene sequences were analyzed using the QIIME platform script. By clustering the effective sequence similarity, sequences with 97% or more similarity were extracted as operational taxonomic units (OTUs). The OTU sequences were systematically classified using the Ribosomal Database Project classifier with reference to the Silva database.

[0155] Results Figure 15-19 ,Depend on Figure 15-16 The results showed that Sophora japonica exosomes could improve the decrease of gastric and small intestinal α diversity and gastric β diversity caused by hypoxia, but had no significant improvement on small intestinal β diversity; Figure 17-19 The results showed that at the phylum level of the stomach, hypoxia caused a significant decrease in the abundance of Actinobacteria and Bacteroidetes, and there was no significant change in the abundance of Actinobacteria and Bacteroidetes after treatment with Sophora japonica exosomes. At the phylum level of the small intestine, the abundance of Actinobacteria and Proteobacteria was significantly reduced under hypoxia, and there was no significant change in the abundance of Actinobacteria and Proteobacteria after treatment with Sophora japonica exosomes. The abundance of the genus Rhodobacteraceae_unclassified in the Proteobacteria phylum was significantly reduced under hypoxia, while its abundance was partially restored after treatment with Sophora japonica exosomes. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0156] (10) Metabolomics analysis of gastrointestinal contents, comprising the following steps:

[0157] S1. Metabolite extraction method

[0158] Weigh an appropriate amount of sample into a 2 mL centrifuge tube, add 600 μL of methanol (containing 2-chloro-L-phenylalanine (4 ppm)), and vortex for 30 seconds; then add steel beads and put it into a tissue grinder, grind at 50 Hz for 120 seconds; ultrasonicate at room temperature for 10 minutes, then centrifuge (12000 rpm, 4°C) for 10 minutes, take the supernatant and filter it through a 0.22 μm membrane, and add the filtrate to the detection bottle for LC-MS detection.

[0159] S2. Chromatographic conditions

[0160] The analysis was performed using an ultra-high performance liquid chromatography system and ACQUITY HSS T3 (2.1×100 mm, 1.8 μm) column, flow rate of 0.3 mL / min, column temperature of 40°C, injection volume of 2 μL. Positive ion mode, mobile phase of 0.1% formic acid acetonitrile (B2) and 0.1% formic acid water (A2), gradient elution program: 0-1 min, 8% B2; 1-8 min, 8%-98% B2; 8-10 min, 98% B2; 10-10.1 min, 98%-8% B2; 10.1-12 min, 8% B2. In negative ion mode, the mobile phase was acetonitrile (B3) and 5 mM ammonium formate water (A3), and the gradient elution program was: 0-1 min, 8% B3; 1-8 min, 8%-98% B3; 8-10 min, 98% B3; 10-10.1 min, 98%-8% B3; 10.1-12 min, 8% B3.

[0161] S3. Mass spectrometry conditions

[0162] The mass spectrometer was used for detection, and the electrospray ionization source (ESI) was used to collect data in positive and negative ion modes. The positive ion spray voltage was 3.50 kV, the negative ion spray voltage was -2.50 kV, the sheath gas was 40 arb, and the auxiliary gas was 10 arb. The capillary temperature was 325 ° C, and the first-level full scan was performed at a resolution of 60000. The first-level ion scan range was m / z100-1000, and the second-level fragmentation was performed by high-energy collision induced dissociation (HCD), the collision energy was 30%, and the second-level resolution was 15000. The data was scanned four times in each cycle, and dynamic exclusion was used to remove unnecessary information.

[0163] Results Fig. 20As shown in the figure, Sophora japonica exosomes partially restored the imbalance of 3,4-dihydroxymandelaldehyde, γ-L-Glutamyl-D-alanine, octadec-9-ene-1,18-dioic-acid, 3,5-diiodo-L-tyrosine and palmitoyl-L-carnitine caused by hypoxia in the stomach, and partially restored the imbalance of (1S,2R,4S)-(-)-bornyl acetate, deoxyadenosine, guanosine, nervonic acid and xanthoxicacid caused by hypoxia in the small intestine. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0164] (11) Combined analysis of microbiome and metabolomics

[0165] The differential microbiota of mouse stomach and small intestine contents were identified by 16S rRNA, and the differential metabolites were identified by LC-MS. The correlation analysis of differential microbiota and metabolites was performed using R software package.

[0166] Results Fig.21, as shown in the figure, γ-L-Glutamyl-D-alanine was significantly negatively correlated with Bacteroidetes, γ-L-Glutamyl-D-alanine and octadec-9-ene-1,18-dioic-acid were significantly negatively correlated with Actinobacteria, while 3,5-Diiodo-L-tyrosine and palmitoyl-L-carnitine were significantly positively correlated with Actinobacteria in the stomach. At the genus level of the stomach, 3,5-Diiodo-L-tyrosine was significantly positively correlated with Chitinophagaceae_unclassified of the Bacteroidetes, while γ-L-Glutamyl-D-alanine was significantly negatively correlated with Chitinophagaceae_unclassified of the Bacteroidetes.

[0167] At the phylum level in the small intestine, deoxyadenosine and guanosine were significantly positively correlated with Actinobacteria, and neuronic acid was significantly positively correlated with Proteobacteria. At the genus level, neuronic acid and guanosine were significantly positively correlated with Sulfitobacter and Octadecabacter of Proteobacteria, respectively. In addition, deoxyadenosine and guanosine, as well as deoxyadenosine, guanosine, (1S,2R,4S)-(-)-bornyl acetate, and xanthoxic acid were significantly positively correlated with Nocardioides and Rhodobacteraceae_unclassified of Proteobacteria, respectively. *P<0.05, **P<0.01, ***P<0.001.

[0168] (12) Statistical analysis

[0169] The research data were analyzed using GraphPad Prism 9.5 software. The differences between the two groups were compared using t-tests, and the research data of three or more groups were analyzed using one-way analysis of variance (Analysis of variance, ANOVA). The difference was statistically significant when p < 0.05, and the data were expressed as mean ± standard error. express.

[0170] Example 3

[0171] Human normal gastric epithelial cell line (NGEC) cells and human normal small intestinal epithelial cell line (HIEC) cells were purchased from Shenzhen Haodi Huatuo Biotechnology Co., Ltd.

[0172] This example provides a study on the inhibition of hypoxia-ferroptosis of gastric and small intestinal cells by Sophora japonica exosomes in vitro, which specifically includes the following steps:

[0173] (1) NGEC and HIEC cell culture

[0174] S1. Cell Recovery

[0175] The frozen NGEC cells and HIEC cells were thawed in a 37°C water bath, and then 1 mL of Dulbecco's modified eagle medium (DMEM) medium (containing 10% fetal bovine serum, 100 IU / mL penicillin G and 100 μg / mL streptomycin sulfate) was added to the cryotubes. The tubes were centrifuged at 1000 rpm for 3 min, and the supernatant was removed by a negative pressure aspirator. The prepared 1 mL of DMEM medium was added again and mixed by repeated blowing. An appropriate amount of cell suspension was transferred to a cell culture dish to which the culture medium had been added in advance. The culture dish was gently shaken in an "8" shape to evenly inoculate the cells in the culture dish, and then the culture dish was transferred to a CO2 constant temperature incubator for culture.

[0176] S2. Cell culture and passaging

[0177] The morphology of NGEC cells and HIEC cells was observed under a microscope every day, and the medium was changed every 24 or 48 hours according to the cell condition. The culture medium was sucked out using a negative pressure aspirator, and NaCl buffer was gently added along the side wall of the culture dish to wash away the non-adherent cells and impurities. The NaCl buffer was sucked out using a negative pressure aspirator again, and then the culture medium was added and transferred to a CO2 constant temperature incubator.

[0178] Observe under the microscope and use a negative pressure aspirator to remove the culture medium after the cells grow to 80%. Gently add NaCl buffer along the side wall of the culture dish to wash away the non-adherent cells and impurities. Use a negative pressure aspirator again to remove the NaCl buffer, add 1mL of trypsin and transfer to a CO2 constant temperature incubator to digest the cells. Take out the culture dish after 1-3 minutes and observe under the microscope. If the cells no longer adhere to the wall and become smaller and round, add culture medium to terminate digestion. Tilt the culture dish appropriately, remove the liquid in the culture dish and blow gently repeatedly until the cells are completely detached. Transfer the liquid in the culture dish to a 15mL centrifuge tube, centrifuge at 1000rpm for 3min, use a negative pressure aspirator to remove the supernatant, add 1mL of culture medium and blow gently repeatedly to make a cell suspension, then transfer the cell suspension to the cell culture dish added with culture medium in advance according to the experimental plan in an appropriate proportion, gently shake the culture dish in an "8" shape to evenly inoculate the cells in the culture dish, and then transfer the culture dish to a CO2 constant temperature incubator for culture.

[0179] (2) Construction of hypoxic cell model

[0180] NGEC cells and HIEC cells were placed in a 12-well cell culture plate and cultured in a pre-set hypoxic cell culture chamber (37°C, 1% O2, 5% CO2) to construct a hypoxic cell model. After 24 hours of hypoxia, different concentrations of Sophora japonica exosomes (5×10 3 Particles / mL, 2.5×10 4 Particles / mL and 5×10 4 particles / mL) or NaCl buffer, and the cells were collected after hypoxia treatment for 24 h for crystal violet staining, CCK-8 detection, Western blot, ferroptosis marker detection, transmission electron microscopy analysis, and lipid metabolomics.

[0181] The experimental results are shown in Figure 24-25 As shown in the figure, the three concentrations of Sophora japonica exosomes can significantly reduce the hypoxia-induced ferroptosis of NGEC cells and HIEC cells. In addition, the low concentration of Sophora japonica exosomes (5×10 3 The treatment effect was the best, so low concentration of Sophora japonica exosomes were selected for subsequent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0182] (3) Gene overexpression

[0183] NGEC cells and HIEC cells were cultured in 12-well cell culture plates, and cells were transfected after the cells grew to 80%. NGEC cells and HIEC cells were transiently transfected with HIF-1α and HIF-2α using plasmids pCMV-HIF-1α (containing the full-length wild-type cDNA of hHIF-1α) and pCMV-HIF-2α (containing the full-length wild-type cDNA of hHIF-2α), and the cells were cultured under hypoxia for 24 hours after 24 hours. After replacing the cell culture medium, dimethyl sulfoxide (DMSO), ferroptosis inhibitor Ferrostatin-1 (Fer-1) or ferroptosis inhibitor Liproxstatin-1 (Lip-1) were added to the culture plates and treated under hypoxia for 24 hours, and the cells were collected for crystal violet staining and CCK-8 detection.

[0184] (4) Gene knockdown

[0185] NGEC cells and HIEC cells were cultured in 12-well cell culture plates, and cells were transfected after the cells grew to 80%. NGEC cells and HIEC cells were transiently transfected with siRNA (si control, si hypoxia-inducible factor-1α, si hypoxia-inducible factor-2α) to knock down hypoxia-inducible factor-1α or hypoxia-inducible factor-2α, and cells were cultured under hypoxic conditions for 48 hours after 24 hours, and cell extracts were collected for Western blot detection.

[0186] (5) Crystal violet staining

[0187] Add 1 mL of 4% paraformaldehyde to the cell culture plate for fixation for 1 h, rinse with NaCl three times, add 1 mL of crystal violet stain to each well for staining, rinse slowly with NaCl buffer after 30 minutes to remove excess crystal violet, and take pictures of each well after drying using a scanner.

[0188] (6) CCK-8 assay

[0189] After NGEC and HIEC cells were treated with hypoxia for 24 h, Sophora japonica exosomes (5×10 3Particles / mL) or NaCl, and the cells were collected after 24 hours of hypoxia treatment again, and the cell suspension was prepared after trypsin digestion, centrifugation, and resuspension and inoculated into a 96-well plate (100 μL / well), and placed in a CO2 constant temperature incubator for culture. After 24 hours, 10 μL of CCK-8 solution was added to each well, and bubbles were avoided during the process. Then the 96 wells were placed in a CO2 constant temperature incubator and incubated in the dark for 2 hours, and the absorbance was measured at 450nm using an ELISA reader. Cell survival rate = [(OD value of the experimental group-OD value of the blank group) / (OD value of the control group-OD value of the blank group)] × 100%.

[0190] Crystal violet staining and CCK-8 results are shown in Figure 22-23 As shown in the figure, in NGEC cells and HIEC cells, overexpression of hypoxia-inducible factor-1α or hypoxia-inducible factor-2α will aggravate hypoxia-induced cell death, while ferroptosis inhibitors Fer-1 and Lip-1 can significantly reduce cell death induced by overexpression of hypoxia-inducible factor-1α or hypoxia-inducible factor-2α under hypoxic conditions. The above experimental results show that the mode of hypoxia-induced cell death is mainly ferroptosis. *P<0.05, ****P<0.0001.

[0191] (7) Detection of cell ferroptosis markers

[0192] S1. Cell protein extraction

[0193] After NGEC and HIEC cells were treated with hypoxia for 24 h, Sophora japonica exosomes (5×10 3 Particles / mL) or NaCl, and then collect cells for ferroptosis marker detection after hypoxia treatment for 24 hours. The preparation of protein lysis buffer refers to Chapter 2. Use a negative pressure aspirator to suck out the supernatant in the cell culture dish, slowly add 1mL NaCl along the side wall of the culture dish to rinse, suck out NaCl with a negative pressure aspirator, add 1mL NaCl again, scrape the cells with a sterile cell scraper, use a 1mL pipette to suck out NaCl in the culture dish to elute the cells and collect them in a 1.5mL EP tube, add 200μL protein lysis buffer and place on ice, vortex once every 10min, vortex 3 times and lyse on ice for 30min, then centrifuge at 12000rpm, 4℃ for 20min, and transfer the supernatant to a new 1.5mL EP tube.

[0194] S2. Detection of ferroptosis markers

[0195] The levels of ferrous ions, reactive oxygen species, 4-hydroxynonenal, and lipid peroxides in NGEC cells and HIEC cells were analyzed according to the reagent instructions.

[0196] Results Figure 26-27As shown in the figure, hypoxia significantly increased the levels of ferrous ions, reactive oxygen species, 4-hydroxynonenal, and malondialdehyde in NGEC and HIEC cells. 2+ In addition, the levels of ferrous ions, reactive oxygen species, and 4-hydroxynonenal were all reduced. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0197] (8) Transmission electron microscopy observation, including the following steps:

[0198] 1. Collection and fixation: Suspend cells / bacteria, collect by centrifugation, discard the culture medium, add 2.5% glutaraldehyde, use a toothpick to break up the cell clumps, and store at 4 degrees. For adherent cells, first pour out the culture medium, add 2.5% glutaraldehyde, fix at 4 degrees for 15 minutes, scrape with a cell scraper, collect by centrifugation (retain the fixative), and store at 4 degrees. Fix for at least 4 hours.

[0199] 2. Osmium acid fixation: The cells fixed with glutaraldehyde were rinsed three times with 0.1 M phosphate buffer (pH 7.4) for 15 minutes each time, fixed with 1% osmium acid × 0.1 M phosphate buffer (pH 7.4) at room temperature (20°C) for 2 hours, and then rinsed three times with 0.1 M phosphate buffer (pH 7.2) for 15 minutes each time.

[0200] 3. Dehydration: Dehydrate the cells with 30%, 50%, 70%, 80%, 85%, 90%, and 100% (twice) alcohol in an ascending gradient, each time for 15-20 minutes (the dehydration time can be appropriately extended for samples with high water content and thick cell membrane).

[0201] 4. Penetration: The penetrants are acetone: epoxy resin (2:1), acetone: epoxy resin (1:1), and epoxy resin, in a 37-degree incubator, and each penetration lasts 8-12 hours.

[0202] 5. Embedding: Place the infiltrated sample into the embedding plate, add embedding agent epoxy resin, and polymerize in a 60-degree incubator for 48 hours.

[0203] 6. Sectioning: Ultra-thin sectioning machine 80-100nm.

[0204] 7. Double staining: Uranium-lead double staining (2% saturated aqueous solution of uranyl acetate, lead citrate, staining at room temperature for 15 minutes, sections dried at room temperature overnight, and observed under an electron microscope.

[0205] Results Fig.28 The figure shows that hypoxia causes the mitochondria of NGEC cells and HIEC cells to shrink, decrease in number, increase in membrane density, and lack of mitochondrial cristae, which is one of the morphological characteristics of ferroptosis. This once again verifies that the mode of cell death caused by hypoxia is mainly ferroptosis, and this phenomenon can be alleviated after treatment with Sophora japonica exosomes.

[0206] (9) Western blot detection, including the following steps:

[0207] S1. Cell protein extraction

[0208] The preparation of protein lysis buffer refers to Chapter 2. Use a negative pressure aspirator to suck out the supernatant in the cell culture dish, slowly add 1 mL of NaCl along the side wall of the culture dish to rinse, suck out the NaCl with a negative pressure aspirator, add 1 mL of NaCl again, use a sterile cell scraper to scrape the cells, use a 1 mL pipette to suck out the NaCl in the culture dish to elute the cells and collect them in a 1.5 mL EP tube, add 200 μL of protein lysis buffer and place on ice, vortex once every 10 minutes, vortex 3 times and lyse on ice for 30 minutes, then centrifuge at 12000 rpm and 4°C for 20 minutes, and transfer the supernatant to a new 1.5 mL EP tube.

[0209] S2. The method for detecting tissue protein concentration by BCA method refers to Example 2.

[0210] S3. The method for normalizing protein concentration is as described in Example 2.

[0211] S4. The Western blot method refers to Example 2.

[0212] Results Fig.29 , as shown in the figure, Sophora japonica exosomes reduced the levels of hypoxia-inducible factor-1α, hypoxia-inducible factor-2α, nicotinamide adenine dinucleotide phosphate oxidase 4, and arachidonic acid lipoxygenase 5 in NGEC cells and HIEC cells, which were elevated due to hypoxia, but did not affect the protein expression of proline hydroxylase 1, proline hydroxylase 2, proline hydroxylase 3, cytochrome P450 oxidoreductase, nicotinamide adenine dinucleotide phosphate oxidase 1, and nicotinamide adenine dinucleotide phosphate oxidase 2.

[0213] Results Fig.30 As shown in the figure, after knocking down the expression of hypoxia-inducible factor-1α and hypoxia-inducible factor-2α in hypoxic NGEC cells and HIEC cells, the levels of nicotinamide adenine dinucleotide phosphate oxidase 4 and arachidonic acid lipoxygenase 5 also decreased.

[0214] (10) Sophora japonica exosome tracing, comprising the following steps:

[0215] S1. Cell protein extraction and concentration determination

[0216] Rapidly thaw exosomes at 37°C and quickly add 5× RIPA lysis buffer. Mix well and lyse on ice for 30 minutes. Prepare standard samples for protein concentration measurement by BCA method, and take 20μL of diluted samples and add them to BCA mixed solution and mix well. Incubate at 37°C for 30 minutes, and detect and record the absorbance value at OD562nm on the microplate reader. Finally, calculate the protein concentration of the sample to be tested based on the standard curve.

[0217] S2. Exosome tracing

[0218] S3. Exosome staining (protected from light throughout the process)

[0219] Prepare the dye according to the instructions: 1μLPKH67 dye + 9μLDiulent C. Add PKH26 dye to the exosomes (control group with NaCl), mix well, and incubate at room temperature in the dark for 10 minutes. Add 1mL NaCl, OptimaMAX-XP+ rotor TLA120.2, 100000g, centrifuge for 17 minutes, discard the supernatant and resuspend with sterile NaCl, repeat the steps and wash three times with sterile NaCl. Resuspend with 100μL sterile NaCl (keep in the dark), take 10μL to extract protein, and measure the protein concentration (BCA method).

[0220] S4. Co-incubation of stained exosomes with cells

[0221] According to the proposed protein concentration, the amount of Sophora japonica exosomes was calculated (1×10 4 The cells were allowed to climb onto the slides, and after the cells adhered to the wall, the stained exosomes were added to the cells and incubated in a cell culture incubator for 24 h.

[0222] S5. Nuclear staining and microscopy

[0223] The cell slides were removed and washed three times with NaCl. 4% paraformaldehyde was added for fixation at room temperature for 30 min, and then washed three times with NaCl. After nuclear staining for 10 min, the slides were sealed and then examined under a confocal microscope.

[0224] Results Fig.31 , as shown in the figure, Sophora japonica exosomes can be phagocytosed by NGEC cells and HIEC cells.

[0225] (12) Statistical analysis

[0226] The research data were analyzed using GraphPad Prism 9.5 software. The differences between the two groups were compared using t-test, and the research data of three or more groups were compared using single-factor ANOVA. The difference was statistically significant when p < 0.05, and the data were expressed as mean ± standard error. express.

[0227] The results of animal experiments showed that digestive tract damage under hypoxic conditions is mainly concentrated in the stomach and small intestine, manifested as mucosal damage. Sophora japonica exosomes can alleviate hypoxia-induced gastric and small intestinal mucosal damage in mice, restore cytokines downregulated by hypoxia, and reduce the levels of hypoxia-inducible factor-1α, reactive oxygen species, and lipid peroxides, which can improve hypoxia-induced gastric and small intestinal microbial and metabolic disorders to a certain extent. In vitro experiments showed that Sophora japonica exosomes inhibit hypoxia-induced lipid peroxidation of NGEC and HIEC by inhibiting hypoxia-inducible factor-α-mediated nicotinamide adenine dinucleotide phosphate oxidase 4 and arachidonic acid lipoxygenase 5, thereby effectively alleviating cell ferroptosis.

[0228] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of Sophora japonica exosomes in the preparation of a drug for treating gastrointestinal mucosal barrier damage, the preparation method of the Sophora japonica exosomes comprising: Sophora japonica flowers are soaked in an ethanol solution under sterile conditions, and then washed with distilled water to remove the ethanol. After being dried with sterile paper, an enzyme solution is added to the Sophora japonica flowers. The obtained mixture is centrifuged multiple times, and the supernatant is collected by filtration. The supernatant is further subjected to multiple ultracentrifugation, and the precipitation is resuspended with NaCl to obtain Sophora japonica flower exosomes. The enzyme solution contains 4% cellulase, 2% pectinase and 0.6 mol / L mannitol, and the pH value is 5.8; The Latin name of the sophora japonica flower is Robiniaseudoacacia L.flower.

2. The use according to claim 1, characterized in that The average diameter of the Sophora japonica flower exosomes is 100 to 500 nm, and the surface membrane potential is -15 to -85 mV.

3. The use according to claim 1, characterized in that By mass percentage, the Sophora japonica flower exosomes contain 51.7% fatty acyl groups, 17.7% glycerophospholipids, 7.9% glycerolipids, 7.4% sphingolipids, 6.9% sterol lipids, 5.9% pro-alcohol lipids and 2.5% polyketide compounds.

4. The use according to claim 1, characterized in that The digestive tract mucosal barrier damage is digestive tract mucosal barrier damage caused by plateau hypoxia.

5. The use according to claim 1, characterized in that: The medicine also contains pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that The medicine is any one of injection, capsule, tablet, film, suppository, oral solution, dripping pill, water pill, ointment, powder, granule, patch, aerosol, powder spray or powder injection.

7. The use according to claim 5, characterized in that The administration method of the drug includes any one of oral administration, injection administration, inhalation administration, sublingual administration, rectal administration or skin mucosal administration.

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