Application of barley flavin in preparation of medicine for preventing or treating intestinal diseases or symptoms

Abstract

According to the application disclosed by the invention, a Caco-2 cell model verifies that the barley flavin has a remarkable protection effect on damage to the intestinal epithelial barrier caused by LPS induced inflammation, and a result shows that the barley flavin has a potential application value and can be used for preventing and treating diseases related to intestinal epithelial barrier dysfunction.

Description

Technical Field

[0001] The invention belongs to the technical field of natural compounds, and in particular relates to the use of barley xanthoside in preparing medicines for preventing or treating intestinal diseases or conditions. Background Art

[0002] The intestinal epithelial barrier (IEB) is an important barrier to maintain intestinal homeostasis, and its dysfunction is closely related to a variety of gastrointestinal diseases such as inflammatory bowel disease (IBD). Lipopolysaccharide (LPS) is a common inflammatory inducer that can damage the intestinal epithelial barrier function by destroying tight junction (TJ) proteins (such as occludin and ZO-1).

[0003] In recent years, plant-derived flavonoids have received extensive attention due to their anti-inflammatory, antioxidant and other biological activities. Lutonarin, as a natural flavonoid, also has good anti-inflammatory and antioxidant biological activities, but the symptoms of some inflammatory bowel diseases are more complicated, among which the intestinal epithelial barrier function is often damaged, causing intestinal epithelial barrier damage, and there are currently no reports that lutonarin plays a role in intestinal epithelial barrier damage associated with inflammatory bowel disease. Therefore, there is still an unmet need for the use of lutonarin in the prevention, treatment or relief of intestinal epithelial barrier damage associated with inflammatory bowel disease. Summary of the invention

[0004] In view of the defects of the prior art, the present invention verifies the molecular mechanism of the protective effect of hordein on the intestinal epithelial barrier in intestinal inflammation, and further provides the use of hordein in the preparation of drugs for preventing or treating intestinal diseases or disorders.

[0005] In one aspect, the present invention provides the use of hordenine in the preparation of a medicament for preventing or treating an intestinal disease or condition, wherein the intestinal disease or condition is intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction.

[0006] In one or more embodiments, the intestinal disease or disorder is intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction caused by intestinal inflammation or inflammatory bowel disease.

[0007] In one or more embodiments, the intestinal disease or disorder is accompanied by a symptom of decreased expression of tight junction proteins in intestinal cells.

[0008] In one or more embodiments, the intestinal disease or disorder is accompanied by symptoms of intestinal cell hyperpermeability.

[0009] In one or more embodiments, the intestinal disease or disorder is accompanied by abnormal expression of one or more genes selected from ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1, MATR3 and VAMP7.

[0010] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction, wherein the pharmaceutical composition comprises hordein and a pharmaceutically acceptable carrier.

[0011] In one or more embodiments, the concentration of hordein is 3 to 96 μM.

[0012] In one or more embodiments, the concentration of hordein is 6-12 μM.

[0013] Preferably, the concentration of hordein is 3 μM, 6 μM, 12 μM, 24 μM, 48 μM or 96 μM.

[0014] In one or more embodiments, the intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction is intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction caused by intestinal inflammation or inflammatory bowel disease.

[0015] Preferably, the intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction is accompanied by symptoms of decreased expression of tight junction proteins in intestinal cells.

[0016] Preferably, the intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction is accompanied by symptoms of excessive intestinal cell permeability.

[0017] In one or more embodiments, the intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction is accompanied by abnormal expression of one or more genes selected from ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1, MATR3 and VAMP7.

[0018] The present invention verifies the significant effect of barley xanthoside in preventing and treating intestinal epithelial barrier dysfunction, and its effects are specifically reflected in the following aspects:

[0019] 1. Hordeylin can inhibit the high cell permeability caused by LPS-induced inflammation, protect the low mRNA expression of tight junction proteins Occludin and ZO-1, and the high mRNA expression of inflammatory factors TLR4, IL-6, IL-1β and iNOS.

[0020] 2. Hordei blue can inhibit the abnormal mRNA expression of nine key genes (ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1, MATR3 and VAMP7) related to inflammatory response and cell damage caused by LPS-induced inflammation.

[0021] The present invention thus provides the use of barley xanthoglobin in the preparation of drugs for preventing or treating intestinal diseases or conditions, and is expected to provide new ideas and methods for the clinical diagnosis, prevention and treatment of intestinal epithelial barrier dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the chemical structure of barley xanthoside.

[0023] Figure 2 The results of CCK-8 assay of the effect of barley xanthoside on the cell viability of Caco-2 cells. Figure 2 A is the 24h CCK-8 assay result of the effect of barley xanthoside on the cell viability of Caco-2 cells; Figure 2 B is the 48h CCK-8 assay result of the effect of hordein on the cell viability of Caco-2 cells.

[0024] Figure 3 These are the CCK-8 assay results of the effect of 48h LPS-induced inflammation on the cell viability of Caco-2 cells and the effect of hordein pretreatment on the cell viability of Caco-2 cells inhibited by LPS-induced inflammation.

[0025] Figure 4 These are the results of transepithelial electrical resistance measurement of cells in the LPS group and Lutonarin group.

[0026] Figure 5 These are the results of determination of intracellular FITC-dextran concentrations in the LPS group and the Lutonarin group.

[0027] Figure 6 is the mRNA expression level of tight junction proteins in the cells of LPS group and Lutonarin group.

[0028] Figure 7 These are immunofluorescence staining images for determining the expression levels of Occludin and ZO-1 in the cells of the LPS group and the Lutonarin group.

[0029] Figure 8 This is the result of quantitative analysis of the signal intensity of Occludin and ZO-1 and expression levels in the immunofluorescence staining of cells in the LPS group and Lutonarin group.

[0030] Fig. 9is the mRNA expression levels of TLR4, IL-6, IL-1β, and iNOS in the cells of the LPS group and Lutonarin group.

[0031] Fig.10 This is a high-throughput RNA sequencing analysis result of the effect of barley xanthophyllotoxin on gene expression in Caco-2 cells induced by LPS inflammation. Fig.10 A is the volcano plot of differentially expressed genes between cells in the LPS group and the control group (LPS_vs_Con); Fig.10 B is the volcano plot of differentially expressed genes between cells in the Lutonarin group and the LPS group (LU_vs_LPS); Fig.10 C is the Venn diagram of differentially expressed genes between cells in the LPS group and the control group (LPS_vs_Con); Fig.10 D is the Venn diagram of differentially expressed genes between cells in the Lutonarin group and the LPS group (LU_vs_LPS); Fig.10 E is the heat map of differentially expressed genes among the control group, LPS group, and Lutonarin group.

[0032] Fig.11 The results of GO and KEGG analysis of the effect of barley xanthophylls on the transcriptional response of Caco-2 cells induced by LPS inflammation. Fig.11 A is the GO annotation analysis results of differentially expressed genes; Fig.11 B is the changes of genes of interest in the GO entries of LPS_vs_Con and LU_vs_LPS; Fig.11 C is the GO enrichment analysis results of the target differentially expressed genes in the transcriptional response of LPS_vs_Con; Fig.11 D is the GO enrichment analysis results of the target differentially expressed genes in the transcriptional response of LU_vs_LPS; Fig.11 E is the KEGG enrichment analysis results in the transcriptional response of LPS_vs_Con; Fig.11 F is the result of KEGG enrichment analysis in the transcriptional response of LU_vs_LPS.

[0033] Fig.12 The qPCR results of the effects of LPS-induced inflammation and hordein pretreatment on the expression of ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1, MATR3 and VAMP7 genes, respectively.

[0034] ( Fig.11English annotations in Chinese: transporter activity: transporter activity; antioxidant activity: antioxidant activity; extracellular region, extracellular region; cell junction, cell junction; membrane, membrane; extracellular region part, extracellular region part; membrane part, membrane part; response to stimulus: stress response; signaling: signal transduction; biological adhesion: biological adhesion; localization, localization; developmental process: developmental process; cellular process: cellular process; cell population proliferation: cell population proliferation; immune system process: immune system process; CP organization (cellular component organization): cellular component organization; negative regulation of glycolytic process: negative regulation of glycolytic process; negative regulation of type B pancreatic cell proliferation: negative regulation of type B pancreatic cell proliferation; intrinsic apoptotic signaling pathway: intrinsic apoptotic signaling pathway; acute inflammatory response: acute inflammatory response; defense response to symbiont: defense response to symbiont; defense response to virus: virus defense response; response to oxygen levels: response to oxygen levels; cellular response to stimulus: cellular stress response; negative regulation of small molecule metabolic process: negative regulation of small molecule metabolic process; positive regulation of oxidative phosphorylation: positive regulation of oxidative phosphorylation; acetyltransferase activator activity: acetyltransferase activator activity; response to stress: response to stress;regulation of female gonaddevelopment; chylomicron remodeling; defenseresponse; phospholipid efflux; chylomicron assembly; cellular response to oxygen levels; 14-3-3proteinbinding; negative regulation of carbohydrate metabolic process; negative regulation of DNA-binding transcription; tolerance induction negative regulation of glycolyticprocess; acute inflammatory response; intestinal absorption; carbohydrate import across plasma membrane; regulation of interieukin-3production; negative regulation of erythrocyte apoptotic process; regulation of erythrocyte apoptotic process process: regulation of erythrocyte apoptosis process; negative regulation of cardiac conduction: negative regulation of cardiac conduction; oxalate metabolic process: negative regulation of interleukin-3 production: negative regulation of interleukin-3 production; negative regulation of cell communication: negative regulation of cell communication; hexose import across plasma membrane: hexose import across plasma membrane; Cholesterol metabolism: cholesterol metabolism;Metabolism of xenobiotics by cytochrome P450;Bile secretion;Glyoxylate and dicarboxylate metabolism;Butanoate metabolism;Vitamin digestion and absorption;Valine, leucine and isoleucine degradation;MicroRNA in cancer;Arginine and proline metabolism;Other types of O-glycan biosynthesis;Carbohydrate digestion and absorption;Glycosylphosphatidylinositol(GPl);Glutathione metabolism;PPAR signaling pathway;Adipocytokine signaling pathway;Platinum drug resistance: platinum drug resistance; Mucintype O-glycan biosynthesis: mucintype O-glycan biosynthesis; Drug metabolism-cytochrome P450: cytochrome P450 metabolism of drugs; Arachidonic acid metabolism: arachidonic acid metabolism; Complement and coagulation cascades: complement and coagulation cascades; Glucagonsignaling pathway: glucagon signaling pathway; Neuroactive ligand-receptor interaction: neuroactive ligand-receptor interaction; Amyotrophic lateral sclerosis: amyotrophic lateral sclerosis; TGF-beta signaling pathway: TGF-beta signaling pathway; Serotonergic synapse: serotonergic synapse; HIF-1signaling pathway: HIF-1 signaling pathway;TNF signaling pathway: TNF signaling pathway; Tuberculosis: tuberculosis; Autophagy-animal: autophagy in animals; GABAergic synapse: GABAergic synapse; JAK-STAT signaling pathway: JAK-STAT signaling pathway; Herpes simplex virus 1 infection: Herpes simplex virus 1 infection; mTOR signaling pathway: mTOR signaling pathway; Pl3K-Aktsignaling pathway: Pl3K-Akt signaling pathway; Taste transduction: taste transduction; Transcriptional misregulation in cancer: transcriptional misregulation in cancer; IL-7signalingpathway: interleukin-7 signaling pathway); DETAILED DESCRIPTION

[0035] Example 1: Caco-2 cells and their grouping

[0036] The cells used in this example are human Caco-2 cells (human cloned colon adenocarcinoma cells, similar in structure and function to differentiated intestinal epithelial cells, from the Chinese Academy of Sciences Cell Bank, SCSP-5027); the culture medium is high-glucose Dulbecco's modified Eagle's medium (DMEM), the culture medium contains 10% fetal bovine serum (i.e., FBS, purchased from GIBCO) and 1% penicillin-streptomycin; the culture conditions are that the cells are cultured in a humidified environment at 37°C and 5% CO2. When the cells reach 80% to 90% confluence, they are digested with 0.25% trypsin-EDTA and passaged. Subsequently, the cells are cultured at 3×10 5 Cells / cm 2 The density of 100 μg / mL was inoculated into the top chamber of a polyethylene terephthalate (PET) membrane Transwell chamber with a pore size of 0.4 μm (Corning, USA, area of ​​0.33 cm 2 ) and cultured for 12 to 14 days to form a polarized monolayer cell model. During the culture period, the culture medium was replaced every 2 to 3 days.

[0037] To induce inflammation, after the cells form a polarized monolayer, 15 μg / mL lipopolysaccharide (LPS) can be added to the basolateral side of the Transwell chamber for induction, and different concentrations of Lutonarin (3-96 μmol / mL) can be added to the apical side for pretreatment. The structural formula of Lutonarin is as follows: Figure 1 shown.

[0038] Cell grouping: The cells were divided into three groups: control group (i.e., Ctrl or Control, no treatment), LPS group (incubated with 15 μg / mL LPS) and Lutonarin group (pretreated with 15 μg / mL LPS for 48 h and then co-incubated with 15 μg / mL LPS).

[0039] Example 2: Effects of Hordeoside and LPS on the cell viability of Caco-2 cells

[0040] Each group of Caco-2 cells was seeded in a 96-well plate at a density of 2×104 cells / well and cultured for 24 hours. Subsequently, the cells were grouped and treated with reference to the cells in Example 1, and the incubation or co-incubation time was 24 hours or 48 hours. Cell viability determination: After the incubation, the cell counting kit-8 (i.e., CCK-8 kit, purchased from Beyotime) was used to determine and evaluate the toxicity of barley xanthoglobin on Caco-2 cells and its inhibitory effect on the decrease in cell viability caused by LPS-induced inflammation, and the absorbance was measured at 450nm. All experiments were repeated three times. The measurement results are shown in the figure. Figure 2 and Figure 3 The results are shown as mean ± SD of three independent determinations. # indicates p < 0.05 compared with cells not treated with hordein. Data are shown as mean ± SD, * indicates p < 0.05 compared with the control group, n = 5.

[0041] Depend on Figure 2 It can be seen that, when incubated for 24 h or 48 h, hordein had no significant toxicity to Caco-2 cells within the concentration range of 3 to 96 μM. Figure 3 It can be seen that in the cell model with decreased cell viability 48 hours after LPS-induced inflammation, pretreatment with hordein significantly inhibited the decrease in cell viability, and the inhibitory effect showed a certain concentration dependence.

[0042] Example 3: Effects of Hordeoside and LPS on Barrier Integrity and Cell Permeability of Caco-2 Cells

[0043] Each group of Caco-2 cells was inoculated into a PET membrane Transwell chamber with a pore size of 0.4 μm at a density of 1×105 cells / well and cultured for 10 to 14 days to form a polarized monolayer of cells. Subsequently, the cells were grouped and treated with reference to the cells in Example 1, and the incubation or co-incubation time was 48 hours. In addition to setting up the control group, LPS group and Lutonarin group in Example 1 (pre-treated with barley glycosides for 48 hours and then adding 15 μg / mL LPS for co-incubation), a Lutonarin control group was also set up. The difference between this group of cells and the Lutonarin group was that LPS was not added for induction.

[0044] Barrier integrity measurement: TEER values ​​were measured using an ohmmeter with electrodes (purchased from Millipore, model ESR-2). The results were expressed in Ω×cm 2 The resistance value of the filter membrane is deducted. Figure 4 shown.

[0045] Cell permeability assay: 1 mg / mL FITC-labeled dextran (i.e., FITC-dextran or fluorescein isothiocyanate-dextran, molecular weight 4 kDa) was added to the apical chamber, and the basolateral culture medium was collected after 30 minutes. The fluorescence intensity of the cells was measured using a fluorescence microplate reader (purchased from Tecan, model Infinite 200Pro) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. The assay results are shown in Figure 2. Figure 5 shown.

[0046] Depend on Figure 4 and Figure 5 It can be seen that LPS-induced inflammation significantly reduced the transepithelial electrical resistance (TEER) value of Caco-2 monolayer cells and increased the FITC-dextran content in the Caco-2 monolayer cells. Both results indicate that the cell permeability increased, that is, the barrier function was impaired, while pretreatment with hordein alone did not significantly affect the TEER value of Caco-2 monolayer cells or the content of FITC-dextran in the cells. Pretreatment with hordein can significantly inhibit the decrease in TEER value and the increase in FITC-dextran content in the cells caused by LPS-induced inflammation, indicating that pretreatment with hordein can significantly inhibit the destructive effect of LPS-induced inflammation on the barrier.

[0047] Example 4: Hordeyolate inhibits changes in tight junction protein expression levels caused by LPS-induced inflammation

[0048] Detection of mRNA expression levels:

[0049] Referring to the cell grouping in Example 1, Caco-2 cells of the control group, LPS group and Lutonarin group (pretreated with 12 μM hordenine for 48 h and then added with 15 μg / mL LPS for 48 h) were collected, total RNA was extracted and reverse transcribed into cDNA, and qPCR was performed on the LightCycler480 system (purchased from Roche) using SYBR Green fluorescent dye to detect the mRNA expression levels of tight junction proteins Occludin and ZO-1. All experiments were repeated three times.

[0050] Test results such as Figure 6As shown, it can be seen that LPS-induced inflammation caused a significant decrease in the mRNA expression levels of Occludin and ZO-1, and pretreatment with barley xanthoglobin in advance could significantly inhibit the decrease in the expression of the two caused by LPS-induced inflammation, and make the expression levels of the two close to that of the control group.

[0051] Immunofluorescence staining:

[0052] Caco-2 cells were seeded on coverslips and treated with LPS or Lutonarin+LPS for 24 hours, then washed with PBS and fixed with 4% paraformaldehyde for 20 minutes. Subsequently, the cells were permeabilized with a blocking solution containing 0.1% Triton X-100 and 2% BSA, and antibodies against Occludin (purchased from Abcam, Catalog No. AB216327, dilution ratio 1:400 (v / v)) and ZO-1 (purchased from Abcam, AB221547, dilution ratio 1:200 (v / v)) were added and incubated overnight at 4°C. The next day, fluorescently labeled secondary antibodies (purchased from Invitrogen, dilution ratio 1:200 (v / v)) were added, and cell nuclei were counterstained with DAPI, images were taken using a laser confocal microscope (Leica TCS-SP8), and the signal intensity in the images was quantitatively analyzed using ImageJ software.

[0053] The immunofluorescence staining results were as follows Figure 7 The quantitative analysis results of signal intensity are shown in Figure 8 As shown in Figure 2, LPS-induced inflammation led to the mRNA expression of Occludin and ZO-1 ( Figure 7 The levels of the two proteins (corresponding orange-red) were significantly decreased, and pretreatment with hordein in advance could significantly inhibit the decrease in the expression of the two proteins caused by LPS-induced inflammation, and make the expression levels of the two proteins close to that of the control group.

[0054] Example 5: Hordeyolate inhibits changes in the expression levels of inflammatory markers caused by LPS-induced inflammation

[0055] Referring to the cell grouping in Example 1, Caco-2 cells of the control group, LPS group and Lutonarin group (pretreated with 12 μM barley glycosides for 48 h and then incubated with 15 μg / mL LPS) were collected, total RNA was extracted and reverse transcribed into cDNA, and qPCR was performed on the LightCycler480 system using SYBR Green fluorescent dye to detect the mRNA expression levels of inflammatory markers TLR4, IL-6, IL-1β (IL-1B) and iNOS. All experiments were repeated three times, and the results are expressed as the mean ± standard deviation (mean ± SD) of three independent experiments. * indicates p < 0.05, ** indicates p < 0.01.

[0056] qPCR test results Fig. 9 As shown, LPS-induced inflammation led to a significant increase in the mRNA expression levels of inflammatory markers TLR4, IL-6, IL-1β and iNOS, while pretreatment with hordenine in advance could significantly inhibit the increase in the mRNA expression levels of these inflammatory markers caused by LPS-induced inflammation, among which the expression levels of IL-6 and iNOS in the Lutonarin group were even lower than those in the control group.

[0057] Example 6: Transcriptomic analysis of the effect of hordein on protecting intestinal epithelial cell barrier damage caused by LPS-induced inflammation

[0058] Referring to the cell grouping in Example 1, Caco-2 cells of the control group, LPS group (lipopolysaccharide group) and Lutonarin group (hordeum glycoside group) were collected respectively, and total RNA was extracted and reverse transcribed and library constructed. High-throughput sequencing was performed using the IlluminaNovaSeq6000 platform to generate 2×150bp double-end read length data. HISAT2 was used to align to the reference genome, StringTie was used for transcriptome assembly, and RSEM was used to calculate gene expression levels (TPM). Differentially expressed genes (DEGs) analysis was completed using DESeq2, and the screening criteria were p value <0.05 and fold change>1.5.

[0059] The effects of barley xanthophylls on gene expression in Caco-2 cells induced by LPS inflammation were analyzed by high-throughput RNA sequencing (RNA-seq). Fig.10 As shown, a total of 105 differentially expressed genes (DEGs) were identified in the LPS group compared with the control group, of which 34 genes were upregulated and 71 genes were downregulated ( Fig.10 A); A total of 101 differentially expressed genes were identified in the Lutonarin group compared with the LPS group, of which 61 genes were upregulated and 40 genes were downregulated ( Fig.10 B). Further analysis revealed that hordenine pretreatment reversed the expression patterns of 17 key genes, 15 of which were upregulated in the LPS group but downregulated in the hordenine pretreatment group ( Fig.10 C), 2 genes were down-regulated in the LPS group and up-regulated in the barley glycoside pretreatment group ( Fig.10 D), these genes may be key regulators of hordein's ability to protect intestinal epithelial barrier function. Fig.10 The heat map of E shows the relevant differentially expressed genes among the control group, LPS group, and Lutonarin group.

[0060] Example 7: GO analysis and KEGG analysis of hordenine protecting intestinal epithelial cell barrier damage caused by LPS-induced inflammation

[0061] Based on the high-throughput RNA sequencing data of Example 6, significantly enriched biological process and signaling pathway related genes were identified by GO and KEGG enrichment analysis.

[0062] GO functional enrichment analysis showed that the differentially expressed genes between the LPS group and the control group were significantly enriched in biological processes such as inflammatory response, intestinal epithelial barrier function, cell apoptosis and cell stress response ( Fig.11 A~C), while the differentially expressed genes between the Lutonarin group and the LPS group were significantly enriched in biological processes such as metabolic regulation, immune regulation, cell protection and cell-cell interaction ( Fig.11 A, B and D).

[0063] KEGG pathway analysis showed ( Fig.11 E, F), the differentially expressed genes between the LPS group and the control group were mainly enriched in inflammation and metabolism-related pathways such as complement and coagulation cascades, arachidonic acid metabolism, etc. ( Fig.11 E), while the differentially expressed genes between the Lutonarin group and the LPS group were significantly enriched in inflammatory and immune regulatory pathways such as IL-17, TNF, JAK-STAT, and TGF-beta, as well as cytoprotective pathways such as mTOR and PI3K-Akt ( Fig.11 F).

[0064] In summary, the results of GO analysis and KEGG analysis showed that barley xanthoside alleviated the inflammatory response and cell damage in LPS-induced inflammation by regulating specific inflammatory signaling pathways and cell protection mechanisms.

[0065] Example 8: Hordeoside inhibits changes in expression levels of key differentially expressed genes caused by LPS-induced inflammation

[0066] Based on the high-throughput RNA sequencing data of Example 6 and the inflammatory response and cell damage-related pathways determined in Example 7, 9 key differentially expressed genes (ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1, MATR3 and VAMP7) were determined.

[0067] Referring to the cell grouping in Example 1, Caco-2 cells in the control group, LPS group and Lutonarin group were collected, total RNA was extracted and reverse transcribed into cDNA, and qPCR was performed on the LightCycler480 system using SYBR Green fluorescent dye to detect the mRNA expression levels of 9 key differentially expressed genes. All experiments were repeated three times.

[0068] Test results such as Fig.12As shown, it was shown that barley glycosides pretreatment largely inhibited and reversed the changes in the expression levels of key differentially expressed genes caused by LPS-induced inflammation. Specifically, barley glycosides pretreatment upregulated the expression of ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1 and MATR3, and downregulated the expression of VAMP7. It can be seen that the results of the aforementioned RNA sequencing, GO analysis and KEGG analysis were largely verified, and barley glycosides could protect the intestinal epithelial cell barrier from the inflammatory response and cell damage-related gene expression disorders caused by LPS-induced inflammation.

[0069] In summary, the present invention verifies the significant protective effect of barley glycosides on the damage of intestinal epithelial barrier caused by LPS-induced inflammation through the Caco-2 cell model, which is specifically manifested as: restoring cell viability, reducing the cytotoxicity of LPS-induced inflammation, improving barrier integrity, restoring TEER value and cell permeability; upregulating the expression of tight junction proteins Occludin and ZO-1; regulating the expression of inflammation-related genes, and significantly affecting the signal pathways related to inflammation and barrier function. These results show that barley glycosides have potential application value and can be used to prevent and treat diseases related to intestinal epithelial barrier dysfunction.

Claims

1. Use of barley xanthoside in the preparation of a medicament for preventing or treating intestinal diseases or conditions, characterized in that: The intestinal disease or disorder is intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction.

2. The use according to claim 1, characterized in that The intestinal disease or disorder is intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction caused by intestinal inflammation or inflammatory bowel disease.

3. The use according to claim 1, characterized in that The intestinal disease or disorder is accompanied by symptoms of decreased expression of tight junction proteins in intestinal cells.

4. The use according to claim 1, characterized in that The intestinal disease or disorder is accompanied by symptoms of intestinal cell hyperpermeability.

5. The use according to claim 1, characterized in that The intestinal disease or disorder is accompanied by abnormal expression of one or more genes selected from ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1, MATR3 and VAMP7.

6. A pharmaceutical composition for preventing or treating intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction, characterized in that: The pharmaceutical composition comprises hordein and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that The concentration of the hordein is 3-96 μM / ml.

8. The pharmaceutical composition according to claim 7, characterized in that The concentration of the hordein is 6-12 μM / ml.

9. The pharmaceutical composition according to any one of claims 6, characterized in that The intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction is intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction caused by intestinal inflammation or inflammatory bowel disease.

10. The pharmaceutical composition according to claim 6, characterized in that The intestinal epithelial barrier damage or intestinal epithelial barrier dysfunction is accompanied by abnormal expression of one or more genes selected from ASCL2, DDIT4, HOGA1, HTR1D, IFI6, INHBE, NUPR1, MATR3 and VAMP7.

Images