Application of plasmalogen in preparation of medicine for treating intestinal vascular barrier injury

Abstract

The invention belongs to the technical field of medicine research, and particularly relates to application of plasmalogen in preparation of a medicine for treating intestinal vascular barrier injury. Tests prove that the plasmalogen can improve intestinal flora disorder induced by high fat diet and prevent intestinal bacteria from invading intestinal barriers, so that the integrity of intestinal vascular barriers is protected.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicine research, and particularly relates to application of an acetal phospholipid in preparation of a medicine for treating injury of a gut vascular barrier. BACKGROUND

[0002] In recent years, the incidence of diet-induced obesity and related diseases has increased sharply. Gut bacterial translocation-driven vascular barrier damage is a prerequisite for the development of high-fat diet-induced fatty liver and the like. Gut bacterial translocation refers to the process in which bacteria in the intestinal cavity and their products such as lipopolysaccharide pass through the intestinal barrier, enter the portal vein system through the intestinal blood vessels, and then invade the sterile normal tissues and organs. The gut vascular barrier GVB is one of the three intestinal barriers together with the mucosal barrier and the epithelial barrier, and is mainly composed of vascular endothelial cells. The GVB is the last line of defense against intestinal bacterial translocation, which can prevent the spread of bacteria to the liver and other organs and cause immune and metabolic disorders in extra-intestinal tissues. Therefore, improving the injury of the gut vascular barrier is one of the important targets for preventing and treating high-fat diet HFD-induced chronic metabolic diseases.

[0003] Existing studies have found that germ-free mice receiving fecal microbiota transplantation of HFD-fed mice will also have an increase in GVB permeability even if they are fed with low-fat feed. It can be seen that HFD-induced intestinal dysbiosis is the key to GVB damage; there may be a cooperative relationship between intestinal bacteria, for example, certain intestinal bacteria can destroy the intestinal epithelial barrier, and certain bacteria can further destroy the GVB. Intake of HFD will cause significant changes in the composition of the intestinal flora of mice, such as an increase in the ratio of Firmicutes / Bacteroidetes, promotion of the proliferation of LPS-producing bacteria Helicobacter and Desulfovibrionaceae, etc.; bioinformatics analysis results show that these bacteria are likely to be involved in the regulation of intestinal barrier function. In addition, after 30 days of HFD feeding, the spatial distribution of intestinal bacteria in the ileal intercrypt region of mice increased by 42.5%, and these bacteria are likely to be related to HFD-induced damage to the intestinal epithelial barrier. However, there is currently no report on HFD-induced GVB damage-related intestinal bacteria. In summary, it is very meaningful to study the intestinal bacteria related to HFD-induced GVB damage and prevent their proliferation and translocation through dietary intervention.

[0004] According to the difference of the connecting group at sn-1 position of glycerol skeleton, natural phospholipids are mainly divided into glycerophospholipids, acyl-alkyl phospholipids and ether phospholipids. Among them, acyl-alkyl phospholipids contain special olefinic ether bond at sn-1 position, so they may have more unique physiological functions. Acyl-alkyl phospholipids widely exist in human and animal bodies, and are important components of cell membrane structure. The digestion and absorption process of acyl-alkyl phospholipids from marine sources is the same as that of most phospholipid molecules. Phospholipase A2 in the intestine can catalyze the hydrolysis of ester bond at sn-2 position to produce free PUFA and hemolytic acyl-alkyl phospholipids, and the released PUFA can be rapidly absorbed by intestinal cells and maintained at a high level in the blood for a long time. In terms of biological activity, acyl-alkyl phospholipids from sea cucumbers and mussels can effectively improve the cognitive impairment of animals with Alzheimer's disease, and the related mechanisms include inhibition of oxidative stress, reduction of neuroinflammation and inhibition of neuron apoptosis. Acyl-alkyl phospholipids from sea cucumbers can also alleviate HFD-induced atherosclerosis in ApoE − / − mice by regulating liver bile acid metabolism, or alleviate hyperlipidemia in aging mice by promoting liver fatty acid beta oxidation and bile acid synthesis. In summary, most of the activities of acyl-alkyl phospholipids from marine sources reported so far are focused on regulating brain or liver function, and there is no related research on regulating intestinal barrier function. In fact, many natural phospholipid components have biological activity of repairing intestinal barrier damage. For example, DHA phospholipids from chickpea can regulate intestinal flora disorder, thereby improving the intestinal epithelial barrier function of HFD mice, but whether acyl-alkyl phospholipids can regulate intestinal flora disorder and repair intestinal vascular barrier damage needs further study. SUMMARY

[0005] Based on the problems existing in the prior art, the application provides an application of acyl-alkyl phospholipids in the preparation of a drug for treating intestinal vascular barrier damage.

[0006] In a first aspect of the application, an application of acyl-alkyl phospholipids in the preparation of a drug for treating intestinal vascular barrier damage is provided, wherein the acyl-alkyl phospholipids are prepared according to the following steps: The mussels are extracted with a mixed solvent of ethanol and n-hexane as an extractant, the obtained extract is separated by silica gel column chromatography to separate phospholipids, and then thin layer chromatography is used to determine the component with the highest purity to obtain crude phospholipids; the volume ratio of ethanol to n-hexane is 2:1. Phosphatidylcholine in the crude phospholipids is converted to phosphatidylethanolamine by using phospholipase as a catalyst, the conversion product is eluted and separated by using a diol-based bonded silica gel column with a mixed solution of n-hexane and isopropanol and a mixed solution of isopropanol and water as elution solvents, and then the eluted components are analyzed by high performance liquid chromatography, and components with acyl-alkyl phospholipids having a purity greater than 90% are collected, thereby obtaining the acyl-alkyl phospholipids; the volume ratio of n-hexane to isopropanol is 1-7:1-3, and the volume ratio of isopropanol to water is 2-10:1.

[0007] As a preferred embodiment of the present application, the intestinal vascular barrier injury is induced by a high-fat diet.

[0008] As a preferred embodiment of the present application, the plasmalogen is used for preparing a medicine for increasing the abundance of intestinal Faecalibacterium population.

[0009] Further preferably, the plasmalogen is used for preparing a medicine for promoting the colonization of Faecalibacterium population in the intestine.

[0010] As a preferred embodiment of the present application, the plasmalogen is used for preparing a medicine for inhibiting the colonization of Escherichia-Shigella in the intestine.

[0011] As a preferred embodiment of the present application, the extraction condition is stirring at 40-55℃ for 85-95 min.

[0012] In the second aspect of the present application, a medicine for treating or preventing intestinal vascular barrier injury is provided, which is composed of the plasmalogen as the only active ingredient.

[0013] As a preferred embodiment of the present application, the medicine is composed of the plasmalogen and pharmaceutically acceptable excipients.

[0014] As a preferred embodiment of the present application, the medicine is prepared into an oral preparation or an injection preparation according to a pharmaceutically acceptable method. In the preparation, conventional excipients such as excipients, wetting agents, thickening agents, viscosity agents, flavoring agents, adjusting agents, coloring agents, suspending agents, suspensions, etc. can be added.

[0015] More preferably, the oral preparation is a granule, a tablet, a capsule, an oral liquid or other liquid preparations.

[0016] The present application has been proved by experiments that the mussel plasmalogen can improve the intestinal flora disorder induced by a high-fat diet, prevent intestinal bacteria from invading the intestinal barrier, and thus protect the integrity of the intestinal vascular barrier. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 is the animal grouping and treatment method (A) and the feed formula (B) of the experiment of exploring the mussel PlsEtn regulating HFD mouse GVB function.

[0018] Figure 2 is the mussel PlsEtn protecting the intestinal barrier integrity of HFD mice. (A) FITC-dextran penetration experiment design, (B) serum and fecal FITC-dextran level, (C) serum LPS level.

[0019] Figure 3Mussel PlsEtn reduced GVB permeability in HFD mice. (A) FITC-dextran permeation experiment design, (B) serum FITC-dextran level, (C) FITC-dextran fluorescence intensity expression in liver frozen sections, arrows in the figure point to FITC-dextran entering the colon tissue.

[0020] Figure 4 Mussel PlsEtn reduced PV-1 expression in ileum and colon of HFD mice. (A) Representative results of ileum tissue PV-1 immunofluorescence staining, (B) statistical results of ileum tissue PV-1 mean fluorescence intensity MFI, (C) Representative results of colon tissue PV-1 immunofluorescence staining, (D) Statistical results of colon tissue PV-1 mean fluorescence intensity MFI.

[0021] Figure 5 Mussel PlsEtn regulated the spatial distribution of intestinal bacteria in ileum tissue of HFD mice.

[0022] Figure 6 Mussel PlsEtn regulated intestinal flora in ileum of HFD mice. (A) NMDS analysis results of intestinal flora in ileum contents, (B) community structure diagram at genus level, (C) significantly different bacteria between groups.

[0023] Figure 7 Mussel PlsEtn regulated intestinal flora in colon of HFD mice. (A) NMDS analysis results of intestinal flora in colon contents, (B) community structure diagram at genus level, (C) significantly different bacteria between groups.

[0024] Figure 8 Mussel PlsEtn regulated intestinal flora in ileum tissue of HFD mice. (A) Community structure diagram at genus level in ileum tissue, (B) Allobaculum Relative abundance.

[0025] Figure 9 Mussel PlsEtn regulated intestinal flora in colon tissue of HFD mice. (A) Community structure diagram at genus level in colon tissue, (B) Allobaculum Relative abundance.

[0026] Figure 10 Animal grouping and treatment method for exploring the effect of intestinal flora regulated by mussel PlsEtn on GVB.

[0027] Figure 11 Intestinal flora regulated by mussel PlsEtn reduced PV-1 expression in ileum of HFD mice. (A) Representative results of immunofluorescence staining, (B) statistical results of mean fluorescence intensity (MFI). DETAILED DESCRIPTION

[0028] For a better understanding of the above technical solutions, the exemplary embodiments of the present application will be described in more detail below with reference to the accompanying drawings.

[0029] Although the exemplary embodiments of the present application are shown in the drawings, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present application can be more clearly, thoroughly understood and the scope of the present application can be completely conveyed to those skilled in the art.

[0030] Example 1 Preparation and detection of mussel acetal phospholipid According to the different structures of phosphate groups, phospholipids can be further divided into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine, etc. Phosphatidylcholine is abbreviated as PC, phosphatidylethanolamine is abbreviated as PE, and phosphatidylserine is abbreviated as PS. At present, marine organisms used for extracting acetal phospholipids include scallops, mussels, sea squirts, sea cucumbers and squids. The acetal phospholipids extracted from the above marine organisms are all in the form of PE, i.e. acetal phosphatidylethanolamine, abbreviated as PlsEtn.

[0031] The present application takes purple mussels produced in Shandong Peninsula as raw materials, extracts mussel total lipids with anhydrous ethanol / n-hexane, separates phospholipids through silica gel column chromatography, uses phospholipase D to catalyze the enrichment reaction of PE, and finally separates common PE and PlsEtn by using diol-based bonded silica gel column chromatography. The purity of the acetal phospholipid sample is detected by high performance liquid chromatography-evaporative light scattering detector, the phospholipid structure is analyzed by high performance liquid chromatography-tandem mass spectrometry, and the fatty acid composition is analyzed by gas chromatography. The specific process is as follows:

[0032] 1. Preparation of acetal phospholipid The mussel total lipids are extracted with 9 volumes of ethanol-n-hexane at 50℃ for 90 min, and the volume ratio of ethanol-n-hexane is 2:1. Phospholipids are separated by silica gel column chromatography, and are eluted with chloroform, chloroform / methanol and methanol in turn, and the volume ratio of chloroform / methanol is 9:1. The component with the highest purity is determined by thin layer chromatography, and the crude phospholipid is obtained.

[0033] A reaction system containing phospholipase D and 0.3M ethanolamine is prepared, and is added to 2 volumes of crude phospholipid ethyl acetate solution, and the reaction is carried out at 30℃ for 2h to catalyze the conversion of PC to PE. Finally, diol-based bonded silica gel column is used, and n-hexane / isopropyl alcohol, isopropyl alcohol / water are used for elution, and the volume ratio of n-hexane / isopropyl alcohol is 7:3, and the volume ratio of isopropyl alcohol / water is 10:1. The phospholipid structure in each elution component is analyzed by HPLC method, the component with acetal phospholipid purity greater than 90% is collected, and after being combined, rotary evaporation is carried out for drying.

[0034] It should be noted that the total mussel lipid is extracted at 40-55℃ for 85-95min, the volume ratio of n-hexane to isopropanol can be changed within 1-7:1-3, and the volume ratio of isopropanol to water can be changed within 2-10:1, and the acetal phospholipid in the present application can be obtained within the above range. The specific ratio in the above specific method is not a special limitation of the present application.

[0035] 2. Phospholipid structure analysis HPLC analysis was performed on an Agilent 1100 chromatograph using a 150 mm x 4.6 mm, 3 μm Cosmosil HILIC column. The mobile phase A was acetonitrile, and the mobile phase B was a solution containing 60 mM ammonium formate and pH 4.5, 0.1% formic acid solution. The flow rate was 0.6 mL / min. The gradient elution program was 5% B elution for 3 min, 30% B elution for 18 min, 50% B elution for 23 min and 5% B elution for 32 min.

[0036] MS analysis was performed on an AB Sciex 4000 QTRAP system equipped with an electrospray ion source. The primary mass spectrometry temperature was 500℃, the drying gas temperature was 350℃, the atomization gas was 30 psi, the capillary voltage was 4500V, and the scan range was m / z 400-1000. The secondary mass spectrometry conditions were unchanged, the collision energy was 30 eV, and the scan mode was sub-ion scan.

[0037] 3. Results It can be seen from the LC-MS / MS detection that the content of PE that can be identified in the prepared sample reaches 96.67%, of which PlsEtn accounts for 90.60%, as shown in Table 1. Analysis of the molecular species of phospholipids in the sample found that the content of 16:0p / 20:5 was the highest in PlsEtn, followed by 16:0p / 20:1, 18:1p / 22:6 and 18:1p / 20:0, etc., indicating that the sn-2 position of mussel PlsEtn molecules contains a large amount of MUFA and PUFA.

[0038] Table 1 Identification results of phospholipid molecular species of mussel PlsEtn sample Example 2 Effect of isolated mussel acetal phospholipids on HFD-induced GVB injury 1. Animal experiment design C57BL / 6J mice, male, 4 weeks old, adaptive feeding for one week. The animal experiment design is as follows Figure 1The mice were randomly divided into 4 groups, 10 in each group, namely low-fat diet group, high-fat diet group, low-fat diet + plasmenyl phospholipid group and high-fat diet + plasmenyl phospholipid group, and the low-fat diet group was marked as LFD, the high-fat diet group was marked as HFD, the low-fat diet + plasmenyl phospholipid group was marked as LFD + PlsEtn, and the high-fat diet + plasmenyl phospholipid group was marked as HFD + PlsEtn. The LFD and HFD used herein refer to refined feed with fat energy supply ratio of 10% and 45%, respectively. In addition, the mussels plasmenyl phospholipid was added to the LFD or HFD at an addition amount of 2% for intervention on the mice. The experiment lasted for 4 weeks, and after the experiment, all the mice were fasted for 12 h, anesthetized with ether, blood was taken from the left ventricle, and the mice were decapitated, and the ileum tissue and its contents were collected.

[0039] 2. Intestinal barrier integrity detection 2.1. 4KDa fluorescein isothiocyanate-dextran permeation experiment The mice were fasted for 4 h, and 200 μL of 80 mg / mL 4kDa FITC-dextran suspension was orally administered. After 4 h of gavage, the feces of each mouse were collected into a sterile tube. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital, and the mice were sacrificed after heart puncture. 50 mg of fecal samples were diluted with 200 μL of PBS, and the plasma was diluted with PBS at a ratio of 1:2. The fluorescence level of FITC in serum and fecal samples was analyzed using a fluorescence microplate reader.

[0040] 2.2. Analysis of serum LPS level The content of LPS in serum was detected by ELISA according to the method on the kit instruction, and the kit was from Shanghai Fanke Wei Biological Technology Co., Ltd.

[0041] 3. GVB permeability detection 3.1. 70KDa FITC-dextran permeation experiment As Figure 3 After fasting for 15 h, the mice were subjected to midline laparotomy under isoflurane anesthesia, and the distal ileum was taken and ligated with a 2 cm intestinal loop. 8 mg of 70kD AFITC-dextran was dissolved in 200 μL of saline, and injected into the intestinal loop. 1 h after injection, the mice were sacrificed and serum and liver were harvested. The fluorescence level of FITC in serum was analyzed using a fluorescence microplate reader. The liver was quickly frozen with liquid nitrogen to make 5 μm frozen sections, which were stained with DAPI and observed under a fluorescence microscope to express and take photos of the fluorescence intensity of FITC-dextran.

[0042] 3.2. PV-1 protein expression analysis Intestinal tissues were taken and frozen sections were prepared for immunofluorescence staining. After treatment with blocking solution at 37°C for 2 h, CD34 and PV-1 antibodies were added and incubated at 4°C overnight. Immunofluorescence secondary antibodies were added and incubated at 37°C for 30 min in the dark. Anti-fluorescence quenching agent was mounted and observed under a microscope.

[0043] 4. Results 4.1. Intestinal barrier integrity detection The intestinal barrier integrity was detected by 4KDa FITC-dextran tracing method. The results showed that the FITC-dextran level in serum of HFD group mice was 1.86±0.47 μg / μL, which was about 1.71 times of that of LF group. After supplementation of mussel PlsEtn, the FITC-dextran level was significantly decreased to 1.57±0.34 μg / μL. In addition, the serum LPS level detection results also showed that the level of intestinal flora product LPS entering the blood circulation of HFP group mice was significantly lower than that of HFD group, as shown in Figure 2 . In summary, the intestinal barrier integrity of mice was significantly improved after intervention of mussel PlsEtn.

[0044] 4.2. GVB permeability detection Under normal circumstances, macromolecules above 70KDa cannot pass through GVB, but in the case of damaged GVB, they can enter the portal vein through the intestinal blood vessels and then enter the liver and other extra-intestinal tissues. As shown in Figure 3 B, after injection of 70KDa FITC-dextran into the ileal loop for 1 h, the serum FITC-dextran level of HFD group was significantly higher than that of LFD group, indicating that the GVB of mice fed with 60% HFD for 16 weeks was significantly damaged. The serum FITC-dextran level of HFP group was significantly lower than that of HFD group, but there was no significant difference between the serum FITC-dextran level of LFP group and that of LFD group. The expression of FITC-dextran fluorescence intensity in liver is shown in Figure 3 C. Intervention of mussel PlsEtn can prevent FITC-dextran from entering the extra-intestinal tissues of HFD mice, as indicated by the arrows in the figure.

[0045] In addition, the results of immunofluorescence staining are shown in Figure 4 . In the figure, CD34 is a marker for vascular endothelial cells. Intervention of mussel PlsEtn can significantly reduce the expression level of PV-1 protein in the ileum and colon of HFD mice, further indicating that it can improve the increase of GVB permeability induced by HFD.

[0046] Example 3 Effect of isolated mussel acetal phospholipid on HFD-induced GVB damage-related intestinal bacteria 1. Detection of spatial distribution of intestinal bacteria The ileum section in Example 2 was taken for FISH analysis. The section was treated with lysozyme at 37℃ for 10 min, and then hybridization buffer containing fluorescent probes was added, and incubated at 42℃ overnight. The corresponding oligonucleotide probes were designed according to the sequences in Table 2. The intestinal cells were fluorescently stained with DAPI, and observed under a microscope.

[0047] Table 2 In situ fluorescence hybridization probe sequence 2, Intestinal flora composition detection The ileum and colon tissues and their contents of the mice in Example 2 were taken, and the total bacterial DNA was extracted by using QIAamp DNA extraction kit, and the DNA concentration and purity were detected by Nanodrop. The DNA extracted from the tissues was amplified by using 16S rRNA primer pair V4 region, and the DNA extracted from the contents was amplified by using 16S rRNA primer pair V3-V4 region. The amplification conditions are shown in Table 3. After purification, the product was subjected to library construction, and then sequencing on the PacBio SMRT platform.

[0048] The 16S rRNA primer sequence used for extracting DNA from the tissues is as follows: 515F: 5'-GTGYCAGCMGCCGCGGTAA-3', SEQ ID NO. 4; 806R: 5'-GGACTACNVGGGTWTCTAAT-3', SEQ ID NO. 5.

[0049] The 16S rRNA primer sequence used for extracting DNA from the contents is as follows: 341F: 5'-CCTAYGGGRBGCASCAG-3', SEQ ID NO. 6; 806R: 5'-GGACTACNNGGGTATCTAAT-3', SEQ ID NO. 7; Table 3 16S rRNA analysis amplification conditions Low-quality sequencing data was removed on the QIIME platform, OTU clustering was performed, representative sequences were selected, and taxonomic information in the Silva database was aligned to obtain species annotation information. The R language ANCOMBC package was used to analyze the significantly different species between groups, and the vegan package was used to analyze the correlation between the abundance of different species and the GVB damage index.

[0050] 3, Results 3.1, Intestinal bacterial spatial distribution detection The effect of mussel PlsEtn on the total intestinal bacteria was observed by FISH analysis. Compared with the LFD group, the number of intestinal bacteria entering the ileum tissue of the HFD group was significantly increased, while the number of total bacteria in the ileum tissue of the HFP group was significantly lower than that of the HFD group, as shown in Figure 5 .

[0051] 3.2, Intestinal flora composition detection NMDS analysis was performed to evaluate the overall differences in intestinal flora in the ileum and colon of mice in each group, and the results are shown in Figure 6 A and Figure 7 A. After feeding mussel PlsEtn, the intestinal flora structure of the HFP group of mice was obviously different from that of the HFD group. Taxonomic results at the genus level showed that there were obvious differences in the intestinal flora structure in the ileum and colon contents of mice in each group, as shown in Figure 6 B and Figure 7 B. As shown in Figure 6 C and Figure 7 C, t-test was used to screen the intestinal flora significantly regulated by mussel PlsEtn, and it was found that compared with the HFD group, the abundance of Allobaculum Fecalibacterium was significantly increased in the ileum and colon contents of the HFP group.

[0052] In addition, from the detection results of the intestinal flora composition in the ileum and colon tissues, it can be seen that the supplementation of mussel PlsEtn can effectively promote Allobaculum to colonize in the intestinal mucosa of HFD mice, and inhibit the colonization of Escherichia- Shigella , as shown in Figure 8 and Figure 9 . Escherichia-Shigella is a pathogenic bacterium with high infection pathogenicity under inflammatory conditions, and this bacterium may be related to the damage of GVB induced by HFD. Although the overgrowth of this bacterium can also be observed in the LFP group, it is possible that Akkermansia and Ligilactobacillus and other beneficial bacteria also proliferate significantly, effectively maintaining the intestinal homeostasis of mice in this group. Importantly, mussel PlsEtn may promote the growth of Allobaculum , increase the production of beneficial metabolites such as short-chain fatty acids, and maintain the integrity of GVB 。

[0053] Example 4 Effect of intestinal flora regulated by mussel acetal phospholipid on HFD-induced GVB damage 1. Animal experiment design C57BL / 6J mice, male, 4 weeks old, were adaptively fed for one week. The mice were randomly divided into 4 groups, including: LFD recipient group, HFD recipient group, LFD+PlsEtn recipient group and HFD+PlsEtn recipient group, 10 mice in each group. Freshly prepared compound broad-spectrum antibiotics containing 40 mg / kg ampicillin, 40 mg / kg metronidazole, 20 mg / kg vancomycin and 40 mg / kg neomycin were added to the drinking water of each group of mice, and the antibiotic treatment was carried out for 1 week to construct a pseudo-sterile mouse model. During the feeding period, the mice in each group ingested 10% LFD, and the intestinal flora was cleared for 7 days. The feces of mice without antibiotic treatment and treated with antibiotics for 7 days were collected, and the weight of the feces was recorded. The fecal bacterial DNA was extracted using a fecal genomic DNA extraction kit, and qPCR analysis of total bacteria in the feces was performed to determine the modeling effect. The specific process is shown in Figure 10

[0054] Fresh feces of the 4 groups of mice in Example 2 were taken, added to sterile phosphate buffer, and homogenized for 5 min, then centrifuged at 800 g for 3 min to take the supernatant. The fecal bacteria suspension was administered to the above-mentioned 4 groups of mice by gavage, once a week, for 16 weeks of continuous gavage to perform fecal bacteria transplantation. At the end of the experiment, all mice were anesthetized and sacrificed, and the ileum tissue and its contents were collected.

[0055] 2. GVB permeability detection The ileum tissue was taken to make frozen sections, and the expression level of PV-1 was detected by immunofluorescence staining, according to the method described in Example 2.

[0056] 3. Results The results of immunofluorescence staining are shown in Figure 11 Figure 11 In the above-mentioned examples, CD34 is a marker for vascular endothelial cells. The intestinal flora regulated by mussel PlsEtn can significantly reduce the expression level of PV-1 protein in the ileum of HFD mice, indicating that the intestinal flora plays a key role in the improvement of HFD-induced GVB permeability by mussel PlsEtn.

[0057] Although the embodiments of the present application have been shown and described above, it should be understood that the above-mentioned embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can modify, modify, replace and modify the above-mentioned embodiments within the scope of the present application.​​

Claims

1. The application of phospholipid acetal in the preparation of drugs for treating intestinal vascular barrier damage, characterized in that, The acetal phospholipid was prepared according to the following steps: Mussels were extracted using a mixed solvent of ethanol and n-hexane. The resulting extract was subjected to silica gel column chromatography to separate phospholipids, and then thin-layer chromatography was used to determine the component with the highest purity to obtain crude phospholipids. The volume ratio of ethanol to n-hexane was 2:

1. Phosphatidylcholine in the crude phospholipid was converted to phosphatidylethanolamine using phospholipase catalysis. The conversion products were eluted sequentially using a mixture of n-hexane and isopropanol, and then a mixture of isopropanol and water as elution solvents, using a diol-bonded silica column. The eluted fractions were then analyzed by high performance liquid chromatography. The fractions with a purity greater than 90% of acetal phospholipids were collected. The volume ratio of n-hexane to isopropanol was 1~7:1~3, and the volume ratio of isopropanol to water was 2~10:

1.

2. The use of the acetal phospholipid according to claim 1 in the preparation of a medicament for treating intestinal vascular barrier damage, characterized in that, The intestinal vascular barrier damage was induced by a high-fat diet.

3. The use of the acetal phospholipid according to claim 1 in the preparation of a medicament for treating intestinal vascular barrier damage, characterized in that, The acetal phospholipid is used to prepare drugs that increase the abundance of the genus *Ischemicum* in the intestine.

4. The use of the acetal phospholipid according to claim 3 in the preparation of a medicament for treating intestinal vascular barrier damage, characterized in that, The acetal phospholipid is used to prepare a drug that promotes the colonization of *Isobacterium fecalith* in the intestine.

5. The use of the acetal phospholipid according to claim 1 in the preparation of a medicament for treating intestinal vascular barrier damage, characterized in that, The acetal phospholipid is used to prepare a drug that inhibits the colonization of Shigella spp. in the intestines of Escherichia coli.

6. The use of the acetal phospholipid according to claim 1 in the preparation of a medicament for treating intestinal vascular barrier damage, characterized in that, The extraction conditions were: stirring at 40℃~55℃ for 85min~95min.

7. A drug for treating or preventing damage to the intestinal vascular barrier, characterized in that, It uses the acetal phospholipid described in claim 1 as the sole active ingredient.

8. The medicament for treating or preventing intestinal vascular barrier damage according to claim 7, characterized in that, The drug is composed of the acetal phospholipid and pharmaceutically acceptable excipients.

9. The medicament for treating or preventing intestinal vascular barrier damage according to claim 8, characterized in that, The drug is an oral or injectable formulation.

10. The medicament for treating or preventing intestinal vascular barrier damage according to claim 9, characterized in that, The oral preparation is a granule, tablet, capsule, or oral liquid.

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