Application of lily polysaccharide in the preparation of drugs for treating inflammatory bowel disease

Abstract

The present invention provides the application of lily polysaccharide in the preparation of drugs for treating inflammatory bowel disease, and relates to the field of new drug uses. Lily polysaccharide can improve inflammatory bowel disease by regulating the differentiation of CD4+ T cells, and has broad application prospects in the development of new targeted drugs.

Description

Technical Field

[0001] The present invention relates to the field of new uses of drugs, and particularly to the application of lily polysaccharide in the preparation of drugs for treating inflammatory bowel disease. Background Art

[0002] Inflammatory bowel disease (IBD) is a group of digestive system diseases with a continuously high incidence globally in recent years. Due to the complex pathogenesis of IBD, which is difficult to cure, protracted and recurrent, and accompanied by various complications, it poses a huge challenge to global public health security. Currently, three classic drugs, namely aminosalicylate derivatives, glucocorticoids, and immunosuppressants, are mainly used to treat IBD. Although these drugs can relieve the symptoms of IBD, they all have deficiencies such as easy recurrence after drug withdrawal and low patient compliance with long-term use. Understanding the pathogenesis of IBD is crucial for seeking new alternative treatment strategies. IBD is a complex inflammatory process in which multiple immune cells are activated. Currently, it is considered that the key to its pathogenesis is the imbalance in the differentiation of CD4 + T cells. As one of the CD4 + T cell subsets, Th17 plays a leading role in the pro-inflammatory effect of IBD, that is, intestinal inflammation is caused by pro-inflammatory cytokines. Under the stimulation of Th17 cells, the inflamed intestine further secretes cytokines such as IL-17, GM-CSF, IFN-γ, TNF-α, IL-1β, and IL-22, which in turn trigger and exacerbate the inflammatory process on the mucosal surface. Treg cells are another subset of CD4 + T cells, which participate in the host immune response by secreting cytokines such as IL-4 and IL-10 to maintain the body's immune balance. It has been found that the deletion of Treg cells will exacerbate the disease in ulcerative colitis mice. Treg cells inhibit other Th cells by secreting inhibitory cytokines, lysing cells, interrupting cell metabolism, and regulating the function and maturation of dendritic cells. Therefore, regulating the Th17 / Treg cell balance is expected to become a new target for treating IBD.

[0003] Lilium davidii var. willmottiae belongs to the genus Lilium of the family Liliaceae and is a variant of Lilium davidii. Lilium davidii var. willmottiae has excellent quality and is the only agricultural product with a national geographical indication among the edible lilies in China. Due to its long cultivation history, high nutritional value and medicinal value, it has become a leader in the characteristic industries of Lanzhou. The bulb polysaccharides of lilies (Lilium davidii var. willmottiae polysaccharides, LDP) are rich, which are important active ingredients that exert effects in Lilium davidii var. willmottiae. Many studies have shown that lily polysaccharides have physiological activities such as regulating immunity, anti-fatigue, antioxidant, hypoglycemic, and anti-cancer effects. Zhao Zhimeng et al. (2020) reported that lily polysaccharides can regulate intestinal flora imbalance by fostering beneficial bacteria and inhibiting the content of harmful bacteria in the intestines of mice, but the effects of lily polysaccharides on inflammatory bowel disease have not been reported yet. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides the application of lily polysaccharides in the preparation of drugs for treating inflammatory bowel disease, providing a new use for lily polysaccharides.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the application of lily polysaccharides in the preparation of drugs for treating inflammatory bowel disease.

[0007] The present invention also provides the application of lily polysaccharides in the preparation of drugs for regulating the differentiation of CD4 + T cells.

[0008] Preferably, the lily polysaccharides inhibit the differentiation of Th1 cells in CD4 + T cells.

[0009] Preferably, the lily polysaccharides inhibit the secretion of the inflammatory factor IFNγ by Th1 cells.

[0010] Preferably, the lily polysaccharides inhibit the differentiation of Th17 cells in CD4 + T cells.

[0011] Preferably, the lily polysaccharides inhibit the secretion of IL-17 by Th17 cells.

[0012] Preferably, the lily polysaccharides increase the differentiation of Treg cells in CD4 + T cells.

[0013] Preferably, the lily polysaccharides increase the secretion of the cytokine IL-10 by Treg cells.

[0014] The beneficial effects of the present invention are:

[0015] Lily polysaccharide can improve inflammatory bowel disease by regulating CD4 + T cell differentiation, and has broad application prospects in the development of new targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments.

[0017] Figure 1 is the experimental grouping and dosing regimen;

[0018] Figure 2 is the change in body weight of mice in different experimental groups;

[0019] Figure 3 is the change in mortality of mice in different experimental groups;

[0020] Figure 4 is the DAI score of mice in different experimental groups (n = 8; *, P < 0.05; **, P < 0.01; ***, P < 0.001, mean ± S.E.M.);

[0021] Figure 5 is the effect of lily polysaccharide on the differentiation ratios of Th17, Th1, Th2 and Treg. The left and right respectively represent the percentage statistics of IL-17, Foxp3, IFNγ and IL-4 positive cells in the spleen and CNS;

[0022] Figure 6 is the effect of lily polysaccharide on the cytokine secretion of Th17, Th1, Th2 and Treg cells; the left and right respectively represent the expression of cytokines IFNγ, IL-17, IL-4 and IL-10 in the supernatant of splenocytes and CNS cells detected by ELISA;

[0023] Figure 7 is the effect of lily polysaccharide on the mRNA expression levels of specific transcription factors of Th17, Th1, Th2 and Treg cells. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides the application of lily polysaccharide in the preparation of drugs for treating inflammatory bowel disease. In the present invention, the lily polysaccharide is obtained by extraction using the water immersion and alcohol precipitation method. Take fresh lily bulbs, break them into slices, wash them, dry the moisture at a constant temperature of 60 °C, crush them and pass through a 60-mesh sieve to obtain lily powder. Take 2 g of lily powder, and the material-liquid ratio is 1:20 g·mL -1, extract with 75°C hot water for 1.5 h. Use the Sevage method to remove proteins, precipitate polysaccharides with absolute ethanol, and freeze-dry to obtain lily polysaccharides. Use gas chromatography-mass spectrometry (GC-MS) to determine the residues of organic substances (chloroform, n-butanol, and ethanol) in the polysaccharide sample and evaluate the safety of the polysaccharide. The present invention does not particularly limit the dosage form and preparation method of the drug. Those skilled in the art can select a pharmaceutically acceptable dosage form of lily polysaccharides according to routine and prepare it using a conventional preparation method.

[0025] The present invention also provides the use of lily polysaccharides in the preparation of drugs for regulating CD4 + T cell differentiation. The present invention does not particularly limit the dosage form and preparation method of the drug. Those skilled in the art can select a pharmaceutically acceptable dosage form of lily polysaccharides according to routine and prepare it using a conventional preparation method. In the present invention, the lily polysaccharides preferably inhibit the differentiation of Th1 cells in CD4 + T cells, and the lily polysaccharides preferably inhibit the secretion of the inflammatory factor IFNγ by Th1 cells. In the present invention, the lily polysaccharides preferably inhibit the differentiation of Th17 cells in CD4 + T cells, and the lily polysaccharides preferably inhibit the secretion of IL-17 by Th17 cells. In the present invention, the lily polysaccharides preferably increase the differentiation of Treg cells in CD4 + T cells, and the lily polysaccharides preferably increase the secretion of the cytokine IL-10 by Treg cells.

[0026] To further illustrate the present invention, the following describes the present invention in detail with reference to examples, but they should not be construed as limiting the protection scope of the present invention.

[0027] Example 1

[0028] (1) Therapeutic effect of LDP on the murine model of inflammatory bowel disease

[0029] ① Experimental animals

[0030] Use C57BL / 6 mice (wild type), male, 8 - 10 weeks old, weighing 18 - 22 g. Throughout the adaptation and study period, all animals were maintained on a 12-hour light-dark cycle (21 ± 2°C, relative humidity of 45 ± 10%) under specific pathogen-free conditions and had free access to food and water. All animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health of the United States and were approved by the Experimental Animal Ethics Committee of Northwest Minzu University.

[0031] ② Establishment of the IBD model and therapeutic effect of lily polysaccharides

[0032] Forty-eight experimental mice were adaptively fed for one week and randomly divided into 6 groups, with 8 mice in each group: blank control group, model group, and three low, medium, and high dose groups of lily polysaccharide LDP (100, 200, 300 mg / kg).

[0033] The blank control group freely drank sterile water and was intragastrically administered sterile water every day. The remaining groups freely drank 3% DSS solution for 7 days to establish an IBD mouse model. Meanwhile, the model group was intragastrically administered an equal volume of sterile water, and the treatment groups were intragastrically administered low, medium, and high dose lily polysaccharide solutions at 100, 200, and 300 mg / kg for 14 days. The morbidity of the mice was checked every day and the body weight changes were recorded. The pathological features of each mouse were scored, including fecal consistency, bloody stools, and body weight. The individual scores were combined to generate the DAI calculated for each mouse every day. After the experiment, the colon morphology and length, and pathological histological changes of each group of mice were compared to clarify the therapeutic effect of LDP on IBD mice.

[0034] (2) Detection of the regulation of lily polysaccharide on CD4 + T cell differentiation in vivo and in vitro

[0035] ① Preparation of CD4 + T cells

[0036] Peripheral blood, colon tissue, and spleen samples of mice in the control group, DSS group, and the LDP treatment group with the best intervention effect were collected to prepare PBMC, colonic mucosal lamina propria, and spleen single cell suspensions. According to the instructions of the Easysep Mouse CD4 + T Cell Isolation Kit, CD4 T cells were prepared. +

[0037] ② In vitro differentiation of Th1 / Th2 / Th17 / Treg cells

[0038] Before the cell differentiation experiment, anti-mouse CD3ε was used to pre-coat a 48-well plate and placed at 4°C overnight or incubated at 37°C for 2 h. CD4 +When plating T cells, add anti-mouse CD28 antibody for activation. For Th1 cell differentiation, add IL-12, IL-2, and anti-mouse IL-4; for Th2 cell differentiation, add IL-2, IL-4, anti-mouse IL-IFNγ, anti-mouse IL-12; for Th17 cell differentiation, add TGF-β1, IL-6, IL-23, IL-1β, anti-mouse IL-4, and anti-IFNγ; for Treg cell differentiation, add IL-2 and TGF-β1. CD4 + T cells were cultured in a 37°C, 5% CO2 incubator for 2 - 3 days under the polarization conditions of Th1, Th2, Th17, and Treg cells. After removing anti-mouse CD3ε / CD28 (changing plates, with other cytokines unchanged), culture for another 2 - 3 days, and then detect the differentiation of various subtype cells by flow cytometry.

[0039] (3) Effects of lily polysaccharide on the expression of related factors in CD4 + T cell subtype cells

[0040] According to the instructions of the kit, perform enzyme-linked immunosorbent assay (ELISA) to detect the concentrations of cytokines IFN-γ, IL-17, IL-4, and IL-10. Use the Trizol method to extract the RNA of the cell suspension and detect the total RNA concentration on a NanoDrop micro-spectrophotometer; use the BiomarkerScriptⅡ 1st Strand cDNA Synthesis Kit to remove genomic DNA and reverse transcribe the RNA, and use real-time quantitative PCR (qRT-PCR) to detect the expression levels of the specific transcription factors T-bet of Th1 cells, RORγt of Th17 cells, Foxp3 of Treg cells, and GATA3 mRNA of Th2 cells in CD4 + T cells of IBD mice. Clarify the effects of LDP on the mRNA expression of subtype-specific transcription factors in CD4 + T cells of IBD mice.

[0041] Results:

[0042] Intervene in DSS-induced colitis with refined LDP solutions at different concentrations (high dose 300 mg / kg, medium dose 200 mg / kg, low dose 100 mg / kg), and set a blank control at the same time to evaluate the therapeutic effect of LDP on DSS-induced colitis. The results showed that after treatment with LDP, the body weight of the mice ( Figure 2 ), mortality rate ( Figure 3) and DAI score ( Figure 4 ) were alleviated to varying degrees.

[0043] Flow cytometry was used to detect the proportion of each subtype of cells in CD4 + T cells of IBD mice 14 days after immunization. The results showed that, whether in the periphery (spleen) or in the CNS, lily polysaccharide (treatment administration) significantly inhibited the differentiation ratios of Th1 (IFNγ) and Th17 (IL-17) cells in CD4 + T cells of IBD mice (P<0.001 and P<0.01), increased the differentiation ratio of Treg (Foxp3) cells (P<0.001), while having no obvious effect on Th2 (IL-2) cells (P>0.05).

[0044] ELISA was used to detect the secretion of specific cytokines of each subtype of CD4 + T cells of IBD mice. Consistent with the flow cytometry results, whether in the spleen or in the CNS, lily polysaccharide (treatment administration) significantly inhibited the secretion of the Th1 cell-specific inflammatory factor IFNγ and the Th17 cell-specific inflammatory factor IL-17 (P<0.01, P<0.001), increased the secretion of the Treg cell-specific cytokine IL-10 (P<0.05), while having no obvious effect on the secretion of the Th2 cell-specific cytokine IL-2 (P>0.05).

[0045] qPCR was used to detect the mRNA expression levels of subtype-specific transcription factors of CD4 + T cells of IBD mice. Consistent with the above-mentioned flow cytometry and ELISA results, in the spleen cells of IBD mice, after administration of lily polysaccharide, the mRNA expression levels of the Th1 cell-specific transcription factor T-bet and the Th17 cell-specific transcription factor RORγt were significantly inhibited (P<0.01, P<0.001), and the mRNA expression level of the Treg cell-specific transcription factor Foxp3 was increased (P<0.05), but it did not affect the Th2 cell-specific transcription.

[0046] In summary, lily polysaccharide can improve inflammatory bowel disease by regulating the differentiation of CD4 + T cells and has broad application prospects in the development of new targeted drugs.

[0047] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments according to this embodiment without creative efforts, and these embodiments all belong to the protection scope of the present invention.

Claims

1. Use of lily polysaccharide in the preparation of a drug for treating inflammatory bowel disease, characterized in that, The lily polysaccharide is prepared by a water immersion and alcohol extraction method. The steps of the water immersion and alcohol extraction method are as follows: Take fresh lily bulbs, break them into slices, wash them, dry the moisture at a constant temperature of 60 °C, crush them and pass through a 60-mesh sieve to obtain lily powder; Take 2 g of lily powder, and the material-liquid ratio is 1:20 g·mL -1 , extract with hot water at 75 °C for 1.5 h; Use the Sevage method to remove proteins, precipitate the polysaccharide with absolute ethanol, and freeze-dry to obtain the lily polysaccharide.

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