Application of quercetin in improvement of intestinal barrier injury

Abstract

The invention belongs to the technical field of biological medicine, and particularly discloses application of quercetin in improvement of intestinal barrier injury. According to the application disclosed by the invention, the flavonoid quercetin is found to be possibly a potential medicine for improving the intestinal barrier injury of an HIV infected person for the first time, and the flavonoid quercetin has important clinical significance in finding an effective way for improving the intestinal barrier injury of the HIV infected person and reducing the disease risk of non-AIDS related complications in the population. According to the invention, the in-vitro established Caco-2 intestinal barrier model destroyed by the HIVgp120 protein is used for screening the flavonoid compound with a repairing effect for the first time, and the flavonoid compound quercetin is used for exploring whether the flavonoid compound quercetin can improve the intestinal barrier injury destroyed by the HIVgp120 protein or not.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to the application of quercetin in improving intestinal barrier damage. Background Art

[0002] The intestinal barrier is an important tissue structure that ensures the absorption of nutrients from the intestinal cavity and prevents harmful substances in the intestine (such as bacteria and toxins) from entering the body through the intestinal mucosa. However, the intestine is also the priority target and main site of replication of human immunodeficiency virus (HIV), which can cause CD4 + A large amount of T lymphocytes are consumed, leading to intestinal barrier dysfunction. HIV causes the intestinal barrier to be in a damaged state for a long time, the composition of the intestinal flora is dysregulated, the permeability is increased, and intestinal microorganisms and metabolites (such as peptidoglycan, lipoteichoic acid, lipopolysaccharide, etc.) are released into the blood in large quantities, which is called microbial translocation. The continuous high level of microbial translocation caused by impaired intestinal barrier function is an important cause of chronic inflammation and immune activation in AIDS patients. After entering the blood, microbial products can activate specific receptors and bind to these receptors to trigger signal cascade reactions, triggering a strong proinflammatory response, leading to the production of proinflammatory cytokines interleukin-1β (IL-1β), IL-6, tumor necrosis factor (TNF) and type I interferons (IFN). Proinflammatory cytokines such as IL-1β, IL-6 and TNF can induce vascular inflammation and tissue damage as well as the proliferation of local monocytes or macrophages and smooth muscle cells, leading to cardiovascular disease. In addition, they can promote the proliferation of precancerous and malignant cells, weaken the apoptosis of such cells, stimulate bone resorption, and thus lead to osteoporosis. Therefore, the risk of non-AIDS complications (such as cardiovascular disease, cognitive dysfunction, malignant tumors, and osteoporosis) in HIV-infected people is significantly higher than that in HIV-negative people. The occurrence of such complications is related to chronic inflammation and immune activation induced and maintained by microbial translocation, which is one of the main driving factors for the morbidity and mortality of AIDS patients. Even if HIV replication is completely suppressed by effective and highly effective antiretroviral (ART) treatment, the virus cannot be completely eliminated. HIV-infected people are in a state of intestinal barrier damage for a long time, and microbial translocation, immune activation, and chronic inflammation related to intestinal barrier damage continue to exist. Therefore, finding effective drugs to improve intestinal barrier damage and prevent microbial translocation in HIV-infected people is of great significance to reduce the risk of non-AIDS-related complications and improve the quality of life in this population. Summary of the invention

[0003] The purpose of the present invention is to provide the use of quercetin in improving intestinal barrier damage to solve the above technical problems.

[0004] One aspect of the present invention relates to the use of quercetin in improving intestinal barrier damage.

[0005] Preferably, the intestinal barrier damage is intestinal barrier damage in human immunodeficiency virus infected individuals.

[0006] Preferably, the intestinal barrier damage is a Caco-2 intestinal barrier model destroyed by HIVgp120 protein.

[0007] Preferably, quercetin can inhibit the increase in Caco-2 monolayer permeability caused by HIV gp120 protein.

[0008] Preferably, quercetin can improve the viability of Caco-2 cells and inhibit the apoptosis of Caco-2 cells caused by HIVgp120 protein.

[0009] Preferably, quercetin can upregulate the expression of tight junction proteins in Caco-2 cells caused by HIVgp120 protein and improve the distribution of ZO-1, Occludin and Claudin-1, wherein the tight junction proteins are ZO-1, Occludin and Claudin-1.

[0010] Preferably, the concentration of quercetin is 1-64 μM.

[0011] The present invention discovers for the first time that the flavonoid compound quercetin may be a potential drug for improving intestinal barrier damage in HIV-infected individuals, which has important clinical significance for finding effective ways to improve intestinal barrier damage in human immunodeficiency virus-infected individuals and reduce the risk of non-AIDS-related complications in this population.

[0012] This invention is the first to use the Caco-2 intestinal barrier model destroyed by HIVgp120 protein established in vitro to screen flavonoids with repairing effects, and to explore whether the flavonoid quercetin can improve the intestinal barrier damage destroyed by HIVgp120 protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The cell morphology of Caco-2 cells was observed under a microscope.

[0014] Figure 2 To detect the TEER value of Caco-2 cells for 8-21 days.

[0015] Figure 3 The results are for alkaline phosphatase activity.

[0016] Figure 4 To detect the effect of HIV protein on the permeability of Caco-2 monolayer by TEER.

[0017] Figure 5 The effect of HIVgp120 protein on the tight junction genes ZO-1, Occludin, Claudin-1, Claudin-2, and Claudin-7 in Caco-2 cells.

[0018] Figure 6 The CCK-8 method was used to detect the effects of three flavonoid compounds on cytotoxicity after intervention in Caco-2 cells.

[0019] Figure 7 To screen the flavonoids with the greatest repair effect on HIV-related intestinal barrier damage by exploring the effects of three flavonoids on the expression of tight junction genes (ZO-1, occludin and claudin-1) in Caco-2 intestinal epithelial cells.

[0020] Figure 8 Effect of quercetin on the permeability of Caco-2 monolayer.

[0021] Fig. 9 Effects of different concentrations of quercetin on the viability of Caco-2 cells.

[0022] Fig.10 Flow cytometry was used to detect cell apoptosis.

[0023] Fig.11 The results of Western blot detection of the expression of tight junction proteins in cells.

[0024] Fig.12 To observe the localization and distribution of tight junction proteins using cell immunofluorescence experiments. DETAILED DESCRIPTION

[0025] The following is further described in detail through specific implementation methods:

[0026] 1. Establishment of Caco-2 Monolayer Model

[0027] To establish a Caco-2 intestinal epithelial cell model and evaluate the reliability and effectiveness of the Caco-2 model.

[0028] 1.1 Observation of Caco-2 cell monolayer compactness

[0029] Caco-2 cells (Chinese Academy of Sciences Cell Bank, SCSP-5027) were seeded in 6-well plates at a seeding density of 8,000 cells / well and cultured in an incubator at 37°C with 5% CO, with the medium changed every other day. Cells of different growth days were directly placed under an inverted microscope for observation.

[0030] like Figure 1As shown, observation of cell morphology under a microscope revealed that when Caco-2 cells grew for 3 days, the cells gradually became confluent.

[0031] When the cells grow for 1 week, the cells cover most of the area of ​​the well plate, and vacuoles will be generated during the growth process due to the death of a small number of cells.

[0032] In the second week of cell growth, the cells began to grow logarithmically, the cells were arranged gradually more closely, the cells were squeezed and became smaller, and the vacuoles were covered by new cells.

[0033] In the third week of cell growth, no vacuoles were observed in the wells, and the cells were tightly connected to form a dense monolayer.

[0034] 1.2 Measurement of transepithelial electrical resistance (TEER) of Caco-2 cell monolayers at different growth days

[0035] (1) Experimental methods

[0036] Caco-2 cells in the logarithmic growth phase were taken and 2×10 5 The cells were inoculated at a concentration of 100 / mL in a 12-well Transwell plate (membrane area 1.12 cm 2 , pore size 0.4μm). All volumes were kept constant, 0.5mL of cell suspension was added to the apical side (AP), and 1.5mL of normal culture medium was added to the basolateral side (BL). The medium was changed every 48h in the first week, and then the culture medium was changed every day. Before the test, the electrode was placed in a 75% ethanol solution for sterilization for 15min, then taken out and allowed to stand, air-dried, and then placed in a D-Hanks buffer (preheated to 37°C in advance) for 5min to equilibrate, and then the measurement could be started.

[0037] TEER values ​​were measured by Millicell ERS-2 voltage resistance meter to monitor cell monolayer integrity. 200Ω·cm 2 It is the basic value for forming a cell monolayer. Select Caco-2 cells on days 8, 10, 12, 14, 16, 19, 20, and 21, discard the old culture medium of the cell plate, add 1.5 mL and 2.6 mL of 37°C preheated D-Hanks buffer to the AP side and BL side of the cells, respectively, and place in the incubator for equilibrium for 15-30 minutes. Insert the balanced test electrodes vertically into each well in turn, with the short end immersed in the AP side and the long end immersed in the BL side. Start reading after the resistance value stabilizes, and record the resistance value after stabilization. Measure each well three times, and the average of the three sets of data is the resistance value of the well. The formula for calculating the transmembrane resistance value is as follows:

[0038] TEER (Ω·cm 2 ) = [TEER sample well (Ω) - TEER blank well (Ω)] × area (cm2 );

[0039] Among them, the TEER sample well is the transmembrane resistance value of the well containing cells; the TEER blank well is the transmembrane resistance value of the well without cells; the membrane area of ​​the 12-well Transwell culture plate used in the experiment is 1.12cm 2 .

[0040] (2) Experimental results

[0041] The transmembrane resistance value of the cell monolayer model reflects the tightness and integrity of the cell model to a certain extent. The larger the resistance value, the tighter the connection between cells. Figure 2 As shown in Figure 2, the TEER value of Caco-2 cells from 8 to 21 days showed that the longer the cell culture time, the greater the TEER value, and it tended to stabilize at 400Ω·cm from 19 to 21 days. 2 This indicates that Caco-2 cells have formed a tight cell monolayer membrane, which meets the needs of subsequent experiments.

[0042] 1.3 Alkaline phosphatase activity of Caco-2 cells at different growth days

[0043] (1) Experimental methods

[0044] During the formation of the monolayer, Caco-2 cells differentiate alkaline phosphatase, which is a brush border marker enzyme. Determination of alkaline phosphatase activity can reflect the biochemical characteristics and cell polarity of cells. According to the alkaline phosphatase assay kit method, 30 μL of culture medium was taken from the AP side and BL side of the Transwell cell plate, and 50 μL of buffer and matrix solution were added to the 96-well ELISA plate in turn. After incubation at 37°C for 15 minutes, 150 μL of color developer was added, and the absorbance was measured at 520 nm after gentle mixing. Alkaline phosphatase activity AP / BL greater than 5 is used as an indicator that cell growth has formed a polar distribution. The calculation formula for alkaline phosphatase activity AP / BL is:

[0045] Alkaline phosphatase activity AP / BL = (OD AP -OD 空白 ) / (OD BL -OD 空白 );

[0046] Where: OD AP and OD BL The absorbance values ​​of alkaline phosphatase in the culture medium of AP layer and BL layer were measured respectively; OD 空白 It is the absorbance value when 5 μL distilled water is used as blank control.

[0047] (2) Experimental results

[0048] like Figure 3As shown in the figure, the alkaline phosphatase activity results showed that with the increase of cell culture days, the enzyme activity of alkaline phosphatase on the apical side (AP) of Caco-2 cells increased significantly, while the enzyme activity on the basolateral side (BL) first increased and then decreased. The enzyme activity ratio of the AP side to the BL side increased from 2.29±0.22 to 5.6±0.13. The results showed that most of the alkaline phosphatase had accumulated on the AP side of the cells, and the cell polarization was formed, which can be used for subsequent experimental studies.

[0049] 2. Establishment of HIV-related intestinal barrier damage model

[0050] By detecting the effects of HIV gp120, Tat and Nef proteins on Caco-2 monolayer permeability and tight junction gene expression, the most effective HIV protein for intestinal barrier damage can be screened. Human immunodeficiency virus (HIV) is an RNA virus with a genome length of about 9.8kb. It is composed of structural genes, regulatory genes and auxiliary genes, and can encode structural proteins such as P24, P41, gp120, gp140 and regulatory proteins such as Tat, Nef, and rev. gp120: HIV capsid protein structure, is a specific glycoprotein present on the HIV viral envelope, forming the outer spikes of the envelope, is the site where the virus body binds to the CD4 molecule on the surface of the host cell, and mediates the fusion of the virus and the host cell.

[0051] 2.1 HIV gp120 protein significantly reduces Caco-2 monolayer permeability

[0052] (1) Experimental methods

[0053] 2×10 5 0.5 mL of Caco-2 cell suspension was inoculated into a Transwell culture chamber (membrane area 1.12 cm 2 After 21 days of culture, the TEER value was measured using a Millicell-ERS2 volt-ohmmeter, and the TEER value > 400 Ω·cm was selected. 2 The cells in the chamber were used for subsequent experiments. After treating Caco-2 cell monolayers with gp120 protein (50, 100 ng / ml), Tat protein (1, 2 μg / ml) and Nef protein (0.5, 1 μg / ml) for 24 hours, the TEER value was measured to observe the permeability of the three HIV proteins to the Caco-2 monolayer.

[0054] (2) Experimental results

[0055] The effect of HIV protein on Caco-2 monolayer permeability was detected by TEER to screen out the most effective HIV protein for intestinal barrier damage. Figure 4A shows that compared with the NC group, 100 ng / ml gp120 protein significantly reduced the Caco-2 monolayer TEER value by about 40% from 6h to 48h, and the effect was most significant at 24h; Figure 4 B and Figure 4 As shown in Figure C, HIVTat and Nef proteins had little effect on the permeability of Caco-2 monolayer, only reducing the TEER value of Caco-2 monolayer by 26% and 32% at 24h and 12h, respectively. Therefore, 100ng / ml gp120 protein was used to intervene in Caco-2 cell monolayer for 24h as the model group of the present invention.

[0056] 2.2 HIV gp120 protein dose-dependently reduces the expression of tight junction genes in Caco-2 cell monolayers

[0057] (1) Experimental methods

[0058] Syber Green RT-qPCR was used to detect the expression of tight junction genes in Caco-2 cells exposed to HIV gp120 protein (1, 25, 100 ng / ml) for 24 hours and compared with unexposed control Caco-2 cells. The tight junction genes detected were ZO-1, Occludin, Claudin-1, Claudin-2, and Claudin-7.

[0059] 12-well plate with 2×10 5 / ml Caco-2 cells were inoculated, and when the cells adhered to the wall and grew to about 80%, total RNA was extracted by adsorption column method, and cDNA was obtained by reverse transcription. RT-qPCR detection was performed with GAPDH as the internal reference. The program was: 95℃10min, 95℃10s, 60℃1min, 45 cycles. RT-qPCR results were analyzed by relative quantitative method 2 -△△ Ct was used to analyze the data.

[0060] (2) Experimental results

[0061] The effect of HIV gp120 protein on tight junction genes in Caco-2 cell monolayers was detected by RT-qPCR. Figure 5 The results showed that compared with the control group, 100 ng / ml gp120 protein had the most significant effect, which could significantly downregulate the mRNA expression levels of Caco-2 cell tight junction genes ZO-1, Occludin and Claudin-1 (0.71±0.07, P<0.001; 0.67±0.02, P<0.001; 0.46±0.01, P<0.001, respectively).

[0062] At present, there are studies on the effect of HIV gp120 protein on intestinal lamina propria T cells and T84 colorectal cancer lung metastasis cell line, but there are no studies on the effect of HIV gp120 protein on Caco-2 cell monolayer model. Caco-2 cell monolayer model is a human cloned colon adenocarcinoma cell, which has a structure and function similar to differentiated small intestinal epithelial cells, has structures such as microvilli, and contains enzyme systems related to small intestinal brush border epithelium. It is a typical model used to simulate intestinal structure and function. The present invention uses Caco-2 cell monolayer model for the first time to study the effect of HIV gp120 protein on intestinal barrier damage.

[0063] 3. Screening of flavonoid compounds with repairing effects on HIV-related intestinal barrier damage model

[0064] 3.1 Selection of quercetin drug concentration

[0065] (1) Experimental methods

[0066] Prepare three flavonoid solutions: ① Dissolve 0.61 mg of quercetin powder completely with 2 ml of DMSO to make a 1 mM storage solution, wrap it with aluminum foil after aliquoting, and store it at -80 ° C for a long time. Dilute it with DMSO solution to a working concentration of 1-64 μM before use. ② Dissolve 0.93 mg of hyperoside powder completely with 2 ml of DMSO to make a 1 mM storage solution, wrap it with aluminum foil after aliquoting, and store it at -80 ° C for a long time. Dilute it with DMSO solution to a working concentration of 1-64 μM before use. ③ Dissolve 0.89 mg of quercetin powder completely with 2 ml of DMSO to make a 1 mM storage solution, and store it at -80 ° C for a long time after aliquoting. Dilute it with DMSO solution to a working concentration of 1-64 μM before use.

[0067] The CCK-8 method was used to detect the effects of flavonoid compounds quercetin, hyperoside and baicalin on cell viability. 4 Caco-2 cells were inoculated per well. After the cells were completely attached, the culture medium was used as the blank group, the cells cultured in the culture medium were used as the control group, and the cells cultured in the culture medium with three flavonoid compound solutions (concentrations were 1-64 μM, preferably 1, 2, 4, 8, 16, 32, and 64 μM in the experiment) were used as the sample group. After incubation for 24 hours, 10 μL of CCK-8 solution was added to each well, incubated for 1 hour in a 5% CO2, 37°C environment, and the absorbance of each well measured at 450 nm was collected. The cell viability calculation method is as follows: Cell viability (%) = [(sample)-(blank)] / [(control)-(blank)]×100.

[0068] (2) Experimental results

[0069] like Figure 6 As shown, the flavonoid compound quercetin (quercetin, Figure 6 A), hyperoside Figure 6 B) baicalin Figure 6 C) Effects on cell viability after 24 hours of intervention on Caco-2 cells. The results showed that 2 μM of the three drugs had no significant effect on the cells. Therefore, the concentration of the flavonoid drugs in the present invention is 2 μM as the working concentration.

[0070] 3.2 Screening of flavonoid compounds that have a repairing effect on the Caco-2 intestinal barrier model damage caused by HIV gp120 protein

[0071] (1) Experimental methods

[0072] In order to screen for flavonoid compounds that can repair the intestinal barrier damage caused by HIV gp120 protein, 2×10 5 / ml inoculated Caco-2 cells, and when the cells adhered to the wall and grew to about 80%, the Caco-2 cells were divided into 6 groups, negative control group (cell culture medium), positive control group (10μg / ml LPS), gp120 model group (100ng / mL gp120), and three groups of flavonoid compounds quercetin (Quercetin), hyperoside (Hyperoside) and baicalin (Baicalin), all at a concentration of 2μM. After Caco-2 cells were stimulated with different factors for 24h, RT-qPCR was used to detect the expression of tight junction genes (ZO-1, occludin and claudin-1) in Caco-2 intestinal epithelial cells. The method is the same as 2.2.

[0073] (2) Experimental results

[0074] The results are as follows Figure 7 The results showed that the flavonoid compounds quercetin, hyperoside, and baicalin can prevent the decrease in the expression of tight junction genes in Caco-2 cell monolayers caused by gp120, and quercetin (concentration 2 μM) has the most significant effect. Therefore, the present invention selects quercetin as a representative drug for repairing the damage of the Caco-2 intestinal barrier model caused by HIV gp120 protein.

[0075] 3.3 Quercetin inhibits the increase in Caco-2 monolayer permeability induced by HIV gp120 protein

[0076] (1) Experimental methods

[0077] In order to investigate the effect of quercetin on the permeability of Caco-2 monolayer induced by HIV gp120 protein, Caco-2 cells were divided into 4 groups: negative control group (cell culture medium), positive control group (10 μg / ml LPS), gp120 model group (100 ng / mL gp120) and treatment group (2 μM Quercetin + 100 ng / mL gp120). After Caco-2 cells were stimulated by different factors for 6 h, 12 h, and 24 h, the cell TEER values ​​were measured using the same method as in 2.1.

[0078] The permeability of Caco-2 cell monolayer epithelium was reflected by measuring the flux of FITC-dextran with a molecular weight of 4 kDa. After detecting the TEER value of Caco-2 cells stimulated by different factors for 24 hours, 100 μL of 1 mg / ml FITC-dextran was added to the top chamber and incubated at 37°C for 30 minutes. 100 μl of culture medium was taken from the bottom chamber of the transwell chamber and added to a 96-well plate. The fluorescence absorbance with an excitation wavelength of 480 nm and an emission wavelength of 520 nm was detected by a microplate reader.

[0079] (2) Experimental results

[0080] like Figure 8 The results showed that 2 μM quercetin could increase the TEER of Caco-2 cells caused by HIV gp120 protein at 6h, 12h, and 24h, and reduce the flux of FITC-dextran at 24h. Therefore, quercetin can inhibit the increase of Caco-2 monolayer permeability caused by HIV gp120 protein.

[0081] 4. Quercetin can inhibit the cytotoxicity caused by HIVgp120 protein

[0082] (1) Experimental methods

[0083] In order to explore whether quercetin can prevent the decrease in cell viability caused by HIV gp120 protein, Caco-2 cell monolayers were treated with 100 ng / ml gp120 protein and 1-16 μM quercetin for 24 h, and the absorbance at 450 nm was detected. The method was the same as 3.1.

[0084] (2) Experimental results

[0085] like Fig. 9 The results showed that compared with the Caco-2 cell monolayer treated with 100 ng / ml gp120 protein for 24 hours, quercetin at concentrations of 1-16 μM significantly increased the viability of Caco-2 cells.

[0086] 5. Quercetin can inhibit the apoptosis of Caco-2 cells induced by HIVgp120 protein

[0087] (1) Experimental methods

[0088] In order to explore the effect of quercetin on the apoptosis of Caco-2 cells induced by HIV gp120 protein, flow cytometry was used to detect cell apoptosis. 5 / ml Caco-2 cells were inoculated, and when the cells grew to about 80% of the attachment, the Caco-2 cells were divided into 4 groups, negative control group (cell culture medium), positive control group (10μg / ml LPS), gp120 model group (100ng / mL gp120) and treatment group (2μM Quercetin+100ng / mL gp120). After Caco-2 cells were stimulated with different factors for 24h, the state of cell apoptosis was displayed by AnnexinV / PI dual fluorescent dye. After the cells were damaged according to step 2.3.5, they were washed with pre-cooled PBS buffer and collected by centrifugation. 5μL AnnexinV-FITC and 10μlPI were added in sequence, gently mixed, and incubated in the dark for 5min at room temperature for flow analysis. The experimental data were analyzed and processed using FlowJo 10.8.1.

[0089] (2) Experimental results

[0090] like Fig.10 The results showed that compared with the NC group, HIV gp120 protein could cause 29.7% apoptosis of Caco-2 cells, while quercetin could restore the apoptosis of Caco-2 cells to the original level, that is, inhibiting the apoptosis caused by HIV gp120 protein.

[0091] 6. Quercetin can upregulate the expression of tight junction proteins in Caco-2 cells induced by HIVgp120 protein

[0092] (1) Experimental methods

[0093] In order to investigate the effect of quercetin on the expression of tight junction proteins (ZO-1, Occludin and Claudin-1) in Caco-2 cells induced by HIV gp120 protein, 5 × 10 5 / ml Caco-2 cells were inoculated, and when the cells grew to about 80% of the attachment, the Caco-2 cells were divided into 4 groups, negative control group (cell culture medium), positive control group (10μg / ml LPS), gp120 model group (100ng / mL gp120) and treatment group (2μM Quercetin+100ng / mL gp120). After Caco-2 cells were stimulated with different factors for 48h, the expression of tight junction proteins in cells was detected by Western blot. After Caco-2 cells were treated, cells were lysed in an ice bath, protein was quantified by BCA method, and then electrophoresed by SDS-PAG. After electrophoresis, the protein was transferred to a nitrocellulose membrane, blocked with 5% skim milk powder for 1 hour, incubated with the corresponding protein antibody at 4°C overnight, and then incubated with HRP-labeled secondary antibody at room temperature for 1 hour. The ECL chemical substrate luminescence working solution was prepared, and the bands were placed in the dark box of a gel imager for chemiluminescence. Image J software was used to quantify the intensity of the protein bands for detection, and the ratio of the target protein to the internal reference protein was used as the relative expression level of the target protein.

[0094] (2) Experimental results

[0095] like Fig.11 The results showed that compared with the control group, HIV gp120 protein could reduce the expression of tight junction proteins (ZO-1, Occludin and Claudin-1) in Caco-2 cells (0.51±0.16, P<0.01; 0.55±0.09, P<0.05; 0.27±0.09, P<0.001), while quercetin could restore the expression of tight junction proteins in Caco-2 cells (0.94±0.31, P<0.05; 1.03±0.18, P<0.01; 0.81±0.21, P<0.01).

[0096] 7. Quercetin can improve the distribution of tight junction proteins in Caco-2 cells destroyed by HIV gp120 protein

[0097] (1) Experimental methods

[0098] In order to investigate the effect of quercetin on the localization and distribution of tight junction proteins (ZO-1, Occludin and Claudin-1) in Caco-2 cells induced by HIV gp120 protein, confocal culture dishes (glass bottom diameter 20 mm) were used with 5 × 10 5 / ml Caco-2 cells were inoculated, and when the cells adhered to the wall and grew to about 80%, the Caco-2 cells were divided into 4 groups, negative control group (cell culture medium), positive control group (10μg / ml LPS), gp120 model group (100ng / mL gp120) and treatment group (2μM Quercetin + 100ng / mL gp120). After Caco-2 cells were stimulated by different factors for 48h, the localization and distribution of tight junction proteins in cells were detected by immunofluorescence staining. Caco-2 cells were fully fused and cultured on confocal culture dishes, and after various experimental conditions were treated, the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 20min at room temperature, and then blocked with 3% bovine serum albumin at 37℃ for 1h. Caco-2 cells were incubated with rabbit anti-ZO-1, occludin and claudin-1 antibodies at 4℃ overnight. The secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (1:500) was incubated for 1.5h in the dark at room temperature. Then, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) at room temperature for 5 min, and observed under a fluorescence microscope.

[0099] (3) Experimental results

[0100] In order to visualize the expression of tight junction proteins, cell immunofluorescence experiments were used to visually display the location and distribution of tight junction proteins. Green fluorescence represents the content of tight junction proteins, and blue fluorescence is the staining of the cell nucleus to locate tight junction proteins. Fig.12 The results showed that the fluorescence intensity of tight junction proteins ZO-1, Occludin and Claudin-1 in the control group was strong. The distribution of ZO-1, Occludin and Claudin-1 in cells treated with gp120 protein changed significantly, the rearrangement was significantly weakened, the staining intensity decreased, and the tight junction structure of cells was destroyed. Quercetin further improved the distribution of ZO-1, Occludin and Claudin-1.

[0101] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection determined by the claims.

Claims

1. Application of quercetin in improving intestinal barrier damage.

2. The use according to claim 1, characterized in that: The intestinal barrier damage is intestinal barrier damage in human immunodeficiency virus infected people.

3. The use according to claim 1, characterized in that: The intestinal barrier damage is a Caco-2 intestinal barrier model destroyed by HIVgp120 protein.

4. The use according to claim 3, characterized in that: Quercetin can inhibit the increase in Caco-2 monolayer permeability caused by HIV gp120 protein.

5. The use according to claim 3, characterized in that: Quercetin can enhance the viability of Caco-2 cells and inhibit the apoptosis of Caco-2 cells induced by HIVgp120 protein.

6. The use according to claim 3, characterized in that: Quercetin can upregulate the expression of tight junction proteins in Caco-2 cells caused by HIVgp120 protein and improve the distribution of ZO-1, Occludin and Claudin-1, wherein the tight junction proteins are ZO-1, Occludin and Claudin-1.

7. The use according to claim 1, characterized in that: The concentration of quercetin ranged from 1 to 64 μM.

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