Application of acanthus ilicifolius acid in repairing intestinal barrier

Abstract

The invention discloses application of acanthus ilicifolius acid in repairing intestinal barriers. The application refers to at least one of the following purposes: reducing the damage of goblet cells; the expression of mucin is increased; the expression of tight junction protein in an intestinal epithelial cell injury model caused by LPS is increased; the migration capability of intestinal epithelial cells is enhanced. The acanthus ilicifolius can also be used for preparing medicines for repairing intestinal barriers.

Description

Technical Field

[0001] The present invention relates to the field of new uses of cantharidic acid, and particularly to the use of cantharidic acid in repairing the intestinal barrier. Background Art

[0002] Ulcerative colitis is a chronic inflammatory disease of the colonic mucosa. Its onset begins in the rectum and usually extends proximally in a continuous manner through part or all of the colon. In recent years, the incidence of ulcerative colitis has been increasing. A significant disruption of the intestinal barrier function is a core pathological feature of ulcerative colitis. As the first line of defense for the intestine to contact the external environment and maintain the stability of the internal intestinal environment, the integrity of the intestinal barrier is essential. In patients with ulcerative colitis, the integrity of the intestinal barrier is impaired, leading to increased intestinal permeability, allowing intestinal microorganisms, toxins, and antigens to cross the epithelial layer, activating immune cells in the submucosa, and thus causing the occurrence and exacerbation of the inflammatory response. The disruption of such barrier function involves multiple complex mechanisms, including epithelial cell damage, abnormal expression of tight junction proteins, immune cell-mediated mucus layer function defects, and inflammatory responses, all of which are disruptions of the barrier function. Although the current clinical treatment options for ulcerative colitis are increasing, the main treatment methods for ulcerative colitis are currently drug treatment and surgical treatment; among them, drug treatment mainly focuses on anti-inflammatory and immunomodulatory effects, and commonly used drugs include aminosalicylates, glucocorticoids, immunosuppressants, biological agents, probiotics, and anti-infective drugs. However, due to problems such as high cost, unstable efficacy, and adverse reactions of the treatment drugs, it is easy to cause the recurrence of the patient's condition and the generation of drug resistance, thus limiting their clinical application. The treatment of ulcerative colitis with traditional Chinese medicine has significant effects, can reduce the inflammatory response, promote mucosal healing, and reduce recurrence, with few adverse reactions, and has the advantages of multi-target and multi-pathway regulation. As one of the main components of the traditional Chinese medicine Acanthus ilicifolius L., the pharmacological effects of cantharidic acid have not been developed, and its application and mechanism of action in ulcerative colitis have not been reported. Summary of the Invention

[0003] The purpose of the present invention is to provide an effect of cantharidic acid in repairing the intestinal barrier.

[0004] To achieve the above purpose, the present invention provides the use of cantharidic acid in repairing the intestinal barrier.

[0005] Furthermore, the above use refers to at least one of the following: Reducing the damage of goblet cells; Increasing the expression of mucin; Increasing the expression of tight junction proteins in the intestinal epithelial cell injury model caused by LPS; Enhancing the migration ability of intestinal epithelial cells.

[0006] The present invention also provides the use of acanthic acid in the preparation of a drug for repairing the intestinal barrier.

[0007] In terms of in vivo experiments, a mouse ulcerative colitis model induced by DSS was established and intervened by intragastric administration of acanthic acid. After sacrificing the mice, paraffin sections of colon tissues were taken for AB-PAS staining to observe the distribution of goblet cells and intestinal mucus in the colon tissues, so as to determine whether the intervention of acanthic acid can play a role in repairing the intestinal barrier in in vivo experiments.

[0008] In terms of in vitro experiments, an intestinal epithelial injury model was established by inducing IEC-6 cells with lipopolysaccharide, and acanthic acid was added for treatment. The expression levels of tight junction proteins and the migration of IEC-6 cells were detected by WB. In chronic inflammatory diseases such as inflammatory bowel disease (IBD), repairing the intestinal barrier is one of the important means to restore the body function. Therefore, the establishment of this model and the intervention of acanthic acid can further verify whether acanthic acid has the effect of repairing the intestinal barrier in in vitro experiments. Description of the Drawings

[0009] Figure 1 It is the experimental result diagram of Example 1.

[0010] Figure 2 It is the diagram showing the effect of acanthic acid at different concentrations on the proliferation activity of IEC-6 cells in Example 2.

[0011] Figure 3 It is the diagram showing the detection of the expression level of tight junction proteins in IEC-6 cells in Example 2.

[0012] Figure 4 It is the diagram showing the migration result of IEC-6 cells detected by scratch experiment in Example 2.

[0013] Figure 5 It is the statistical diagram of the migration result of IEC-6 cells detected by scratch experiment in Example 2. Detailed Embodiments

[0014] The embodiments of the present invention are described in detail below. The examples of the embodiments are shown in the drawings, wherein the same or similar reference numerals represent the same or similar elements or elements with the same or similar functions from beginning to end. The embodiments described below by referring to the drawings are exemplary and are intended to explain the present invention, but should not be construed as a limitation to the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the art or according to the product specifications. For the reagents or instruments not specified for the manufacturers, they are all conventional products that can be obtained through commercial purchase.

[0015] Acanthic acid: It is the tannin compound in a tannin compound, its extraction method and application, namely CN113402573B.

[0016] Example 1: In Vivo Experiment of Acanthospermum hispidum DC. Acid A mouse ulcerative colitis model induced by DSS was established and intervened by intragastric administration of Acanthospermum hispidum DC. acid. After sacrificing the mice, paraffin sections of colon tissues were taken for AB-PAS staining. The mucus layer of UC patients will be significantly affected, with a decrease in the number of goblet cells and a decline in mucus secretion ability, resulting in a thinning or even disappearance of the mucus layer, which makes it easier for bacteria to directly contact epithelial cells. By observing the distribution of goblet cells and intestinal mucus in colon tissues through AB-PAS staining, to determine whether the intervention of Acanthospermum hispidum DC. acid can play a role in repairing the intestinal barrier in in vivo experiments.

[0017] Animal experiment procedures were carried out in accordance with Chinese national guidelines and approved by the Animal Ethics Committee of Xiamen University.

[0018] Thirty SPF-grade C57BL / 6 male mice (weighing 21 - 22 g) were selected. After 7 days of pre-breeding, they were randomly divided into 5 groups: control group, DSS model group, DSS + 25 mg / kg Acanthospermum hispidum DC. acid group (i.e., DSS + IaA(25 mg / kg)), DSS + 50 mg / kg Acanthospermum hispidum DC. acid group (i.e., DSS + IaA(50 mg / kg)), DSS + 100 mg / kg SASP group (i.e., DSS + SASP(100 mg / kg)); 6 mice in each group. DSS refers to dextran sulfate sodium, with a molecular weight of 36,000 - 50,000; IaA refers to Acanthospermum hispidum DC. acid; SASP refers to the positive drug sulfasalazine.

[0019] Except for the control group, the mice in the other groups freely drank water containing 2.5% DSS from day 0 to day 7. The DSS + 25 mg / kg Acanthospermum hispidum DC. acid group, DSS + 50 mg / kg Acanthospermum hispidum DC. acid group, and DSS + 100 mg / kg SASP group were given intragastric administration from day 3 to day 7, once a day. The control group mice freely drank normal water from day 0 to day 7.

[0020] The mice were sacrificed on the 8th day for the next experiment.

[0021] The experimental results are shown in Figure 1 . The staining results showed that the number of goblet cells in the DSS model group decreased and the mucin in the mucus layer was almost invisible, indicating that the intestinal barrier was severely damaged; the Acanthospermum hispidum DC. acid administration group significantly improved this situation, the damage of goblet cells was significantly reduced, and the expression of mucin also increased significantly. This shows that Acanthospermum hispidum DC. acid can effectively relieve and repair the damage of the intestinal barrier in UC mice.

[0022] Example 2: In Vitro Experiment of Acanthospermum hispidum DC. Acid In vitro experiments, an intestinal epithelial injury model was established by lipopolysaccharide-induced IEC-6 cells, and acanthic acid was added for treatment. Western blot was used to detect the expression levels of tight junction proteins and the migration of IEC-6 cells. In chronic inflammatory diseases such as inflammatory bowel disease (IBD), repairing the intestinal barrier is one of the important means to restore the body's functions. The migration ability of intestinal epithelial cells is a key indicator to measure intestinal health, mucosal repair and barrier function. Its migration ability directly affects the repair efficiency after intestinal mucosal injury. The stronger the migration ability, the faster the repair speed, which helps to maintain the integrity of the intestinal barrier. If the migration ability is weakened, it may lead to delayed mucosal repair, increased intestinal permeability, making it easier for pathogens and toxins to invade the body, causing inflammation or infection. The effect of acanthic acid on the migration ability of IEC-6 cells was evaluated by scratch assay. Therefore, the establishment of this model and the intervention of acanthic acid can further verify whether acanthic acid has the effect of repairing the intestinal barrier in in vitro experiments.

[0023] The concentration range in which acanthic acid has no effect on the proliferation of mouse macrophage IEC-6 cells was obtained by CCK-8 method ( Figure 2 ).

[0024] Cells were seeded into 96-well plates at a density of approximately 7000 cells per well. To prevent evaporation of the culture medium, 200 μL of PBS was added to each well around the wells where the cells were cultured. After the cells adhered, the medium containing different concentrations of acanthic acid was added to the wells. Each concentration (0, 0.5, 1, 1.5, 2, 2.5 mM respectively) was set with 3 replicates, and the cells were cultured for another 24 hours. Then, the medium containing the drug was aspirated, and the medium containing CCK8 reagent was added to each well and incubated in the incubator for 2 hours. Finally, the absorbance (OD value) of each well was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the relative cell viability was calculated accordingly. Figure 2 The results showed that there was no significant effect on the proliferation activity of IEC-6 cells when the concentration of acanthic acid was 0.5, 1, 1.5, 2, 2.5 mM.

[0025] An appropriate concentration was selected from this concentration range, acanthic acid was co-cultured with IEC-6, and an in vitro intestinal epithelial injury model was constructed using lipopolysaccharide to study the effect of repairing the intestinal barrier in vitro.

[0026] A blank control group, M1 group (i.e., LPS group), M1 + Acanthus ilicifolius acid (0.5 mM) group (i.e., LPS + IaA 0.5), M1 + Acanthus ilicifolius acid (1 mM) group (i.e., LPS + IaA 1), M1 + Acanthus ilicifolius acid (1.5 mM) group (i.e., LPS + IaA 1.5), and M1 + Acanthus ilicifolius acid (2 mM) group (i.e., LPS + IaA 2) were set up. First, IEC-6 cells were seeded into six-well plates. After the cells adhered to the wall, except for the blank control group, lipopolysaccharide at 5 mg / mL was added to the remaining groups. After 24 hours, the cells were collected in the drug groups (i.e., LPS group, LPS + IaA 0.5 group, LPS + IaA 1 group, LPS + IaA 1.5 group, LPS + IaA 2 group), and proteins were extracted to detect the expression levels of tight junction proteins in IEC-6 cells ( Figure 3 ). The WB results showed that after LPS induction, the expressions of ZO-1 and Occludin proteins were downregulated; after treatment with different concentrations of Acanthus ilicifolius acid, the expressions of these two tight junction proteins showed an upward trend. This indicates that Acanthus ilicifolius acid can increase the expression of tight junction proteins in the IEC-6 cell injury model caused by LPS.

[0027] The above experiment was repeated, and a scratch assay was carried out to detect the migration of IEC-6 cells ( Figure 4-5 ). Compared with the blank control group, the migration ability of IEC-6 cells in the LPS group was significantly decreased, while treatment with Acanthus ilicifolius acid significantly improved this phenomenon. Further statistical analysis of the migration distance found that Acanthus ilicifolius acid at a concentration of 1 mM had the most significant effect on promoting the migration of IEC-6 cells. In summary, Acanthus ilicifolius acid can effectively enhance the migration ability of intestinal epithelial cells IEC-6.

[0028] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and purposes of the present invention.

Claims

1. Acanthic acid has the use of repairing the intestinal barrier.

2. The use according to claim 1, wherein The said use refers to at least one of the following: Reducing the damage of goblet cells; Increasing the expression of mucin; Increasing the expression of tight junction proteins in the intestinal epithelial cell injury model caused by LPS; Enhancing the migration ability of intestinal epithelial cells.

3. The use of acanthic acid for preparing drugs for repairing the intestinal barrier.

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