Application of plantamajoside in treatment of ulcerative colitis

Abstract

The invention discloses application of plantamajoside in treatment of ulcerative colitis, and belongs to the field of medicine. The invention provides a new application of plantamajoside in a medicine for treating colitis, which can effectively relieve symptoms of ulcerative colitis and reduce pathological injury degree, inflammation and oxidative stress indexes; the expression of intestinal compact protein is recovered, and the intestinal barrier is protected; the intestinal flora composition is improved; and inflammation-related genes are effectively down-regulated.

Description

Technical Field

[0001] The invention relates to the field of medicine, and in particular to application of plantagogue glycoside in treating ulcerative colitis. Background Art

[0002] Plantamajoside (PMS) is the main active ingredient of Plantain, which has a wide range of pharmacological value and has a wide range of anti-oxidative stress, anti-inflammatory and anti-tumor effects. Current studies have found that although plantamajoside has a significant effect in anti-inflammatory aspects, there is no report on whether it has an effect on ulcerative colitis.

[0003] Ulcerative colitis (UC) is a chronic intestinal inflammatory disease characterized by recurrent or protracted inflammation of the intestinal mucosa. The lesions are located in the colorectum, and the incidence rate is increasing year by year worldwide. The main clinical manifestations of ulcerative colitis are bloody stools and diarrhea, which seriously affect the quality of life of patients. The pathogenesis of ulcerative colitis is still unclear, and its disease progression mainly includes impaired intestinal barrier function, intestinal mucosal inflammatory response, and intestinal flora disorders. Ulcerative colitis is difficult to cure clinically. Although the widespread use of biological drugs in the past decade has increased the clinical treatment remission rate of ulcerative colitis, the long-term cure rate has not increased significantly, and biological drugs are expensive, which has brought a heavy economic burden to social public health.

[0004] Therefore, research on drugs that are suitable for my country's national conditions and can effectively treat ulcerative colitis is of great significance to ulcerative colitis patients and national medical care. Summary of the invention

[0005] In view of the above-mentioned defects of the prior art, the present invention provides the use of plantago glycoside in the preparation of a drug for treating ulcerative colitis.

[0006] Furthermore, the psyllium glycoside increases the levels of intestinal barrier proteins ZO-1, Occludin, and Claudin-3.

[0007] Furthermore, the psyllium glycoside reduces the levels of inflammatory factors TNF-α, IL-1β and IL-6.

[0008] Furthermore, the psyllium glycoside improves the aggregation of colonic flora, so that the colonic flora maintains good diversity and uniformity.

[0009] Furthermore, the psyllium glycoside improves the abundance of bacterial flora.

[0010] Furthermore, the psyllium glycoside effectively up-regulates the CBS gene and inhibits NF-κB activation.

[0011] Compared with the prior art, the present invention has the following technical effects:

[0012] The present invention applies psyllium glycoside to a drug for treating ulcerative colitis. Experiments have shown that psyllium glycoside can effectively alleviate the symptoms of ulcerative colitis, reduce the degree of pathological damage, and reduce intestinal inflammation and oxidative stress indicators; it can restore the expression of intestinal tight proteins and protect the intestinal barrier; it can improve the composition of intestinal flora; it can effectively upregulate the CBS gene and inhibit NF-κB activation.

[0013] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is the result of the effect of PMS on the symptoms and pathological damage of colitis in UC mice in a specific embodiment of the present invention. Figure 1 A is the structure diagram of psyllium glycoside, Figure 1 B is a schematic diagram of animal experiment design. Figure 1 C is the weight change curve of mice. Figure 1 D is the change curve of DAI score of mice. Figure 1 E is a representative picture of mouse colon. Figure 1 F is the statistical result of mouse colon length, Figure 1 G is a representative microscopic image of H&E staining of colon tissues in each group. Figure 1 H is the pathological injury score of colon tissue in each group, Figure 1 I is a representative microscopic image of PAS staining of colon tissues in each group, Figure 1 J is the number of goblet cells in colon tissue of each group.

[0015] Figure 2 This is the result of the effect of PMS on the colon barrier integrity of UC mice in a specific embodiment of the present invention. Figure 2 A is a representative immunofluorescence image of Claudin-3 in colon tissue. Figure 2 B is the immunofluorescence image of Occlaudin in colon tissue. Figure 2 C is a representative immunofluorescence image of ZO-1 in colon tissue, Figure 2 D is the expression levels of ZO-1, Occlaudin and Claudin-3 in colon tissue detected by qPCR

[0016] Figure 3 This is the result of the effect of PMS on the inflammatory response of colon tissue in UC mice in a specific embodiment of the present invention. Figure 3 A is the result of fluorescence quantitative polymerase chain reaction experiment. Figure 3 B is the ELISA test result;

[0017] Figure 4 In a specific embodiment of the present invention, PMS affects the diversity of intestinal flora in UC mice. Figure 4 A is the relative distribution diagram of bacteria in each group. Figure 4 B is the species accumulation box plot, Figure 4 C is α diversity, which is the change results of Chao1, Shannon, Simpson and Pielou index of each group. Figure 4 D is β diversity, which are the results of principal coordinate analysis PcoA and NMSD analysis, respectively;

[0018] Figure 5 In a specific embodiment of the present invention, the effect of PMS on the intestinal flora of UC mice is shown. Figure 5 A is the relative abundance of intestinal flora at the phylum level, Figure 5 B is the relative abundance of intestinal flora at the genus level, Figure 5 C is the difference in relative abundance of specific bacteria, Figure 5 D is the LEfSe analysis of intestinal flora (LDA>3.5, P<0.05), Figure 5 E is the Tax4fun function annotation clustering heat map, Figure 5 F is the correlation analysis between intestinal flora and mouse colitis-related index, intestinal barrier proteins, and inflammatory factors

[0019] Figure 6 In a specific embodiment of the present invention, the results of transcriptomic analysis of the colon of UC mice treated with PMS are shown in FIG. Figure 6 A is the heat map of differentially expressed genes in each group. Figure 6 B is the volcano map of differentially expressed genes. Figure 6 C is the differential gene VEIN map, Figure 6 D is the GO analysis of differentially expressed genes, including molecular function (MF), biological process (BP) and cellular component (CC). Figure 6 E is the KEGG analysis of differentially expressed genes in each group. DETAILED DESCRIPTION

[0020] In order to make the purpose of the invention, technical scheme and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described in this specification are only for explaining the present invention, not for limiting the present invention. In the following embodiments, psyllium glucoside refers to CAS No. 104777-68-6, molecular formula C29H36O16, purchased from MCE Biotechnology Co., Ltd. (Shanghai, China) (PMS, purity ≥99%). The treatment in the following embodiments refers to delaying the occurrence of the disease or making it normal.

[0021] 1. Model building

[0022] C57BL / 6J male mice (6 weeks old, average body weight 16-20g) were selected and fed with standard food and pure water for 7 days of adaptive feeding. All mice were randomly divided into 5 groups, 5 mice in each group, namely control group, model group, PMS-L (12.5mg / kg) group, PMS-M (25mg / kg) group and PMS-H (50mg / kg) group. Except for the control group, all mice freely drank 2.5% DSS solution for 7 days to induce UC. The corresponding drugs were administered intragastrically with a volume of 0.1ml / 10g body weight for a total of 7 days. The body weight of the mice was recorded every day, and the mental state, hair color changes, fecal status and fecal bleeding of the mice during the experiment were observed. At the end of the experiment, the mice were killed by cervical dislocation. The internal organs were quickly removed, the colon tissue was completely removed, the cecal contents were collected, and the blood was collected.

[0023] 2. Detection experiment

[0024] 2.1 Disease Activity Index Evaluation:

[0025] During the experiment, the weight of mice was monitored daily, and the morphology of their feces and rectal bleeding were observed to check the occurrence of UC. Disease Activity Index (DAI) was used to assess the overall severity of the disease.

[0026] 2.2 Histopathological experiments

[0027] The colon tissue was fixed in 4% paraformaldehyde solution for 24 hours, embedded in paraffin blocks, and two slices with a thickness of 5 μm were prepared for hematoxylin and eosin (HE) and Alcian blue-periodic acid-Schiff (AB-PAS) staining. After dehydration and sealing, the slices were imaged with a digital microscope and the specific lesions were observed at appropriate magnifications. PAS staining was used to quantify the number of goblet cells, and HE staining was used to perform pathological scoring of tissue sections to determine the extent of lesions.

[0028] Histological scoring was performed for: (a) severity of inflammation (score 0-3: none, mild, moderate, severe), (b) extent of damage (score 0-3: none, mucosa, mucosa and submucosa, transmural), and (c) crypt damage (score 0-4: none, basal 1 / 3 damage, basal 2 / 3 damage, surface epithelium intact, loss of entire crypt and epithelium). The score for each parameter was multiplied by a factor reflecting the percentage of tissue involvement (×1, 0%-25%; ×2, 26%-50%; ×3, 51%-75%; ×4, 76%-100%), and the total score was obtained by adding these values.

[0029] 2.3 Immunofluorescence experiments

[0030] Colon tissue sections were baked, dewaxed in xylene, dehydrated in 100%, 95%, 85% and 75% ethanol, heated in citrate buffer (pH 6.0) by microwave, inactivated endogenous enzymes by 3% H2O2, added serum and allowed to stand at room temperature for 20 minutes, dried and added with primary antibody (Occludin 1:100, ZO-11:100, Claudin-3 1:100), and the control group was added with an equal amount of PBS at 4°C overnight, washed with PBS, protected from light, and excess liquid was blotted, fluorescent secondary antibody was added, incubated at 37°C for 1 hour, washed with PBS, added with DAPI and incubated in the dark for 5 minutes to stain the nucleus, washed with PBS to remove excess DAPI, sealed with sealing medium, and observed under a fluorescence microscope.

[0031] 2.4 Fluorescence quantitative polymerase chain reaction (RT-qPCR) experiment:

[0032] Total RNA from colon tissue was extracted using RNA Simple Total RNA Kit and quantitatively detected using a microplate reader. It was then converted into cDNA using FastKingRTKit (containing gDNase). A 20 μL reaction system was prepared according to the instructions of the SuperRealPreMixPlus (SYBRGreen) kit, and RealTimePCR analysis was performed. β-actin was used as the internal reference gene, and the relative expression of the gene to be tested was calculated using the 2-ΔΔCt algorithm.

[0033] 2.5ELISA experiment:

[0034] Mouse blood was collected in a centrifuge tube, placed at room temperature for 2 h, and centrifuged at low temperature and high speed. The serum was separated into a new centrifuge tube and stored at -80 °C. The content of IL-1β, IL-6, and TNF-α was detected according to the ELISA kit specifications (Protein Detection Group, Wuhan, China). The test samples were added to the microplate containing antibodies in sequence, and then the HRP-labeled antibody was combined with the test sample to form an antibody-antigen-enzyme-labeled antibody complex. The plate was washed and stained with the substrate TMB, and the absorbance was measured at 450 nm.

[0035] 2.616srRNA sequencing experiment:

[0036] The feces of mice in the control group, model group, and PMS-H group were sequenced for 16SrRNA gene. Cecal contents were collected after dissection, microbial DNA was extracted, and the hypervariable regions (such as V3-V4 regions) of the 16SrRNA gene were amplified by specific primers. After purification, the amplified products were connected to the sequencing adapters to construct libraries, and sequenced on high-throughput sequencing platforms (such as Illumina MiSeq). After quality control, splicing, and de-redundancy, the obtained raw data were clustered into operational taxonomic units (OTUs) or amplicon sequence variants (ASVs), and species annotation was performed by database comparison. The α diversity index was further calculated to evaluate species richness and evenness, and the β diversity analysis was performed to explore the differences between communities. The relative abundance of bacterial communities at the phylum and genus levels was analyzed, and the representative bacterial communities of each group were analyzed by LEfSe, and the potential functions and metabolic pathways of microorganisms were inferred by combining functional prediction tools (such as TAX4FUN). Spearman analyzed the correlation between intestinal flora and enteritis-related indexes, inflammatory factors, and intestinal barrier proteins.

[0037] 2.7 Colon tissue RNA sequencing experiment:

[0038] Three mice were randomly selected from each of the control group, model group, and PMS-H group, and total RNA from colon tissue was extracted. Total RNA was extracted from colon tissue samples, and mRNA was enriched after rRNA removal and reverse transcribed into cDNA, followed by construction of a sequencing library. The library was sequenced on a high-throughput sequencing platform (such as Illumina NovaSeq) to generate a large amount of raw data. The sequencing data were quality controlled, aligned, and normalized by bioinformatics analysis to identify differentially expressed genes (DEGs). Functional enrichment and pathway analysis were further performed in combination with the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

[0039] 2.8 Statistical analysis:

[0040] Statistical analysis was performed using Prism 9.0 (GraphPad Software, CA). For multiple comparisons, one-way analysis of variance was performed, followed by Tukey's comparison test. When the data were not normally distributed, the Kruskal-Wallis test was used to determine the significant differences between different groups. All experimental data are expressed as mean ± SEM. P < 0.05 was considered statistically significant.

[0041] 3. Experimental results

[0042] Effects of PMS on symptoms and histological damage in UC mice

[0043] We conducted animal experiments to evaluate PMS (chemical formula Figure 1A) Therapeutic effect in DSS-induced UC mouse model ( Figure 1 B) Monitor the weight of mice every day during the experiment and record the weight changes. Figure 1 As shown in C, the mice in the model group showed obvious weight loss, but the body weight of the mice given PMS (12.5, 25 mg / kg and 50 mg / kg) increased significantly.

[0044] The DAI (Disease Activity Index) score was obtained based on the weight change, feces morphology and rectal bleeding of each group of mice to evaluate the overall disease severity. The DAI score is as follows: Figure 1 As shown in D, through Figure 1 As shown in D, the DAI score of the model group increased. By comparison, the DAI score of mice given PMS (12.5, 25 mg / kg and 50 mg / kg) was significantly reduced.

[0045] The colon length of each group of mice was measured. Figure 1 As shown in EF, it can be seen that the administration of PMS (12.5, 25 mg / kg and 50 mg / kg) improved the colon shortening induced by DSS in a dose-dependent manner.

[0046] Colon tissues were fixed in 4% paraformaldehyde solution for 24 hours. 5 μm sections were stained with hematoxylin-eosin (HE) and alcian blue-periodic acid-Schiff (AB-PAS). Figure 1 GH) showed that the mucosal layer of the model group was incomplete, the glands were irregular, and there were more inflammatory cells infiltrating. The PMS group had more complete tissue, regular arrangement of glands, and fewer infiltrating inflammatory cells, which significantly reversed colon tissue damage and improved the histopathological score of colon inflammation in a dose-dependent manner. PAS staining was used to evaluate the number of goblet cells. The results of PAS staining are shown in Figure 1 As shown in IG, compared with the control group, the number of goblet cells in the model group was reduced and the degree of epithelial cell apoptosis was severe, but it was improved in the PMS group in a dose-dependent manner.

[0047] Effects of PMS on intestinal mucosal barrier damage in UC mice

[0048] Intestinal barrier destruction is a key factor in the progression of UC, and tight junction proteins are the main factors in maintaining the intestinal epithelial barrier. The recovery of intestinal tight junction proteins is closely related to the healing of the intestinal mucosa of UC patients and is a key indicator of the efficacy of drug treatment. In this example, immunofluorescence analysis of mouse colon tissue sections was used to detect intestinal epithelial tight junction proteins Occludin, Claudin-3, and ZO-1. The results are as follows: Figure 2As shown, compared with the control group, the fluorescence intensity of ZO-1, Occludin and Claudin-3 in the model group was weakened, but the fluorescence intensity of each PMS group was restored, indicating that PMS can alleviate intestinal barrier damage in UC mice by enhancing the expression of tight junction proteins.

[0049] Effects of PMS on inflammation in UC mice

[0050] The number of inflammatory factors and barrier proteins in colon tissue was detected by RT-qPCR. The verification factors included IL-1β, IL-6, and TNF-α, and the barrier proteins included Claudin-3, ZO-1, and Occludin to verify the inflammatory effect of PMS on UC mice. The RT-qPCR results are shown in Figure 3 As shown in A, compared with the control group, the levels of TNF-α, IL-1β and IL-6 in the colon of the model group mice were significantly increased, indicating that PMS can significantly reduce the levels of TNF-α, IL-1β and IL-6 in a dose-dependent manner. The ELISA experiment was used to detect the levels of IL-6, IL-1β and TNF-α in serum and cell supernatant. The ELISA results are shown in Figure 3 As shown in B, compared with the control group mice, the levels of serum TNF-α, IL-1β and IL-6 in the model group mice were significantly increased, and PMS could significantly reduce the levels of TNF-α, IL-1β and IL-6 in a dose-dependent manner.

[0051] 3.4 Microbial diversity analysis

[0052] The relative distribution of bacterial taxonomic operational units (OTUs) between groups is shown in the Venn diagram. Figure 4 As shown in A, there were 430 common OTUs and 300, 262, and 182 unique OTUs in the control group, model group, and PMS-H group, respectively. Figure 4 The species accumulation box plot shown in B shows that the number of OTUs increased significantly with the increase in sequencing volume, but the curve gradually flattened with the increase in sample size. Compared with the control group, the Chao1, Shannon, Simpson and Pielou indexes in the model group decreased significantly. Figure 4 C shows that the PMS-H group can significantly restore the Shannon, Simpson and Pielou indices, indicating that PMS can improve the diversity of the microbiota of UC mice and solve the problem of decreased uniformity. β-diversity is used to show the composition and aggregation of the colonic flora of samples in each group. Figure 4 The principal coordinate analysis PcoA results and NMSD analysis results shown in D show that the individual distances between samples in each group are relatively close, while there is a clear separation between different groups. The closer the distance on the coordinate graph, the higher the similarity, indicating that there are indeed differences in the intestinal flora between different groups.

[0053] Effect of PMS on the intestinal flora structure of UC mice

[0054] like Figure 5 As shown in A, at the phylum level, compared with the control group, the abundance of Firmicutes and Proteobacteria in the intestinal tract of mice in the model group increased, while that of Bacteroidetes and Verrucomicrobia decreased. Intake of PMS increased the relative abundance of Bacteroidetes and Verrucomicrobia, and decreased the relative abundance of Firmicutes and Proteobacteria. Figure 5 As shown in BC, at the genus level, compared with the control group, the relative abundance of Turicibacter in the model group mice increased significantly, and the abundance of [Eubacterium]_xylanophilum_group decreased significantly. The PMS group significantly increased the abundance of [Eubacterium]_xylanophilum_group and significantly decreased the abundance of Turicibacter. Figure 5 As shown in D, linear discriminant analysis (LDA) effect size (LEfSe) analyzed the microbial characteristics. Ligilactobacillus, Akkermansia, and [Eubacterium]_xylanophilum_group were the dominant flora in the PMS-H group, and Turicibacter and Romboutsia were the dominant flora in the model group, which was consistent with the results of intestinal microbial composition analysis. Figure 5 As shown in E, the predicted results of TAX4FUN analysis showed that signal transduction, biodegradation and metabolism, cell movement, and metabolism were improved in the model group mice, while these functions decreased in the PMS-H group; folding, classification and degradation, transport and catabolism, vitamin metabolism, lipid metabolism, and amino acid metabolism were significantly improved in the model group mice, but these functions were improved in the PMS-H group. In order to determine the relationship between changes in intestinal flora and UC. Spearman was used to analyze the correlation between intestinal flora and enteritis-related indexes, inflammatory factors, and barrier proteins. The results are shown in Figure 2. Figure 5 As shown in F, Turicibacter was positively correlated with TNF-α, IL-1β, IL-6, and DAI, and negatively correlated with mouse weight, colon length, ZO-1, and Occludin. [Eubacterium]_xylanophilum_group was positively correlated with mouse weight, colon length, ZO-1, Claudin-3, and Occludin, and negatively correlated with DAI, IL-1β, and IL-6.

[0055] 3.6 Transcriptome analysis of PMS-treated UC mice

[0056] The CBS gene is closely related to UC. The inhibition of CBS expression leads to a decrease in endogenous H2S, which aggravates inflammation and mucosal damage in the small colon. Leukocyte migration, cytokines, and NF-κB activation play a key role in the progression of ulcerative colitis. Figure 6 The heat map of differentially expressed genes shown in A shows that the distance between the samples in each of the three groups is relatively close, while there are obvious differences between the groups. Figure 6 As shown in BC, there are 385 DEGs that meet the model group > control group and PMS-H group < model group, and 338 DEGs that meet the model group < control group and PMS-H group > model group. Compared with the control group, the expression of CBS gene in the model group decreased significantly, while the expression of CBS gene in the PMS-H group increased most significantly. GO enrichment analysis was performed on the above DEGs, and the results are as follows Figure 6 As shown in D, the most enriched pathways are mainly related to leukocyte migration and cytokine activity. KEGG enrichment analysis was performed on the above DEGs, and the results are shown in Figure 6 As shown in E, the NF-κB signaling pathway was significantly enriched. The above results indicate that the anti-inflammatory mechanism of PMS can promote the upregulation of CBS and inhibit the NF-κB signaling pathway.

[0057] In summary, psyllium can effectively alleviate the symptoms of ulcerative colitis, reduce the degree of pathological damage, inflammation and oxidative stress indicators; restore the expression of intestinal tight proteins and protect the intestinal barrier; improve the composition of intestinal flora; effectively upregulate the CBS gene and inhibit the activation of the NF-κB signaling pathway.

[0058] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Application of plantago glycoside in the preparation of drugs for the treatment of ulcerative colitis.

2. The use according to claim 1, characterized in that: The psyllium glycoside increases the levels of intestinal barrier proteins ZO-1, Occludin and Claudin-3.

3. The use according to claim 1, characterized in that: The plantagogue glycoside reduces the levels of inflammatory factors TNF-α, IL-1β and IL-6.

4. The use according to claim 1, characterized in that: The psyllium glucoside improves the aggregation of colonic flora, so that the colonic flora maintains good diversity and uniformity.

5. The use according to claim 1, characterized in that: The psyllium glycoside improves the abundance of bacterial flora.

6. The use according to claim 1, characterized in that: The psyllium glycoside up-regulates the CBS gene and inhibits NF-κB activation.

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