CN115282142B - Application of luteolin in preparation of medicines for treating gastric precancerous diseases - Google Patents

Abstract

The invention verifies that luteolin (Lut) can obviously improve the gastric mucosa epithelial cell state after bile acid intervention, and down regulate the expression of intestinal epithelial metaplasia (IM) specific molecules CDX2, MUC2 and KLF4 for the first time. In vivo experiments prove that the Lut can prevent and treat a mouse model of Spasmolytic Polypeptide Expression Metaplasia (SPEM) induced by tamoxifen, remarkably improve the mental state and activity change of the mouse, remarkably improve the gastric mucosa morphology and gastric tissue pathology of the mouse, and down regulate the expression of a SPEM marker TFF 2. The used effective dose has no toxic or side effect on normal gastric mucosa epithelial cells and other organs, and verifies two administration modes of injection and oral administration, so that Lut becomes a safe and effective new means for preventing and treating gastric precancerous diseases (IM and SPEM) and brings a new thought for reversing and blocking gastric precancerous diseases.

Description

Application of luteolin in preparation of medicines for treating gastric precancerous diseases

Technical Field

The invention relates to the technical field of novel application of medicines, in particular to application of a small molecular compound Luteolin (Lut) in preparing medicines for treating gastric precancerous diseases.

Background

Gastric Cancer (GC) is the fifth most common cancer, and is also the third leading cause of cancer death worldwide. Gastric cancer is well known as a mucosal cascade of changes: superficial gastritis, chronic atrophic gastritis (Chronic atrophic gastritis, CAG), intestinal metaplasia (Intestinal metaplasia, IM), intraepithelial neoplasia, gastric cancer. Of these, two pre-neoplastic events that are considered critical for final malignant transformation are critical for the occurrence of gastric cancer. IM is the main precursor for gastric changes in intestinal gastric cancer, and increases risk of intestinal GC exponentially; furthermore, the initiation of spasmolytic polypeptide expression metaplasia (Spasmolytic polypeptide expressing metaplasia, SPEM) that occurs with wall cell loss, neck mucosal cell proliferation, and reprogramming of mature host cells that secrete acids is considered a potential cellular source for IM, intraepithelial neoplasia, and ultimately adenocarcinoma. At present, no effective therapeutic drug aiming at IM and SPEM exists, and eradication of helicobacter pylori has positive effect on gastric precancerous diseases, but blocking and reversing effects of helicobacter pylori still need to be further studied. Although the traditional Chinese medicine formula can improve symptoms of patients with IM or SPEM and prevent further development, the related prescription has excessive medicine flavor and large dosage, needs to be taken for a long time, aggravates the economic burden of the patients, has complex prescription components, has unknown active ingredients and has difficult elucidation of toxic and side effects. Therefore, the monomer medicine with high efficiency, economy and no toxic or side effect is actively explored from the traditional Chinese medicine prescription, and is a real problem faced clinically.

Luteolin (Lut) is a flavonoid compound widely existing in natural plants such as honeysuckle, herba schizonepetae, brussels sprouts, sweet peppers, peanuts and the like, has obvious anti-inflammatory and anticancer properties, and can resist various human malignant tumors such as lung cancer, breast cancer, glioblastoma, prostate cancer, colon cancer and pancreatic cancer; the partial absorption of UVA and UVB radiation to resist ultraviolet radiation reduces undesirable photo-biological effects; in addition, it has antioxidant and anti-inflammatory activities on keratinocytes and fibroblasts, and various immune cells (such as macrophages, mast cells, neutrophils, dendritic cells, and T cells); can improve cognitive decline in patients suffering from neurodegenerative diseases, brain trauma and cerebral apoplexy, and enhance neuroprotection. In the aspect of the digestive system, lut inhibits proliferation of gastric cancer cells induced by oxaliplatin at a small dose by inducing G2/M cell cycle arrest and apoptosis, and can also induce apoptosis and autophagy of HCT116 colon cancer cells through a p53 dependent pathway. It can act by inhibiting the pro-inflammatory mediators IL-1 beta, IL-6, IL-8, IL-17, IL-22, TNF-alpha, etc., and modulating a variety of signaling pathways. However, the prevention and treatment effects of Lut in pre-gastric cancer diseases, especially IM and SPEM, have not been studied.

Disclosure of Invention

The invention aims to provide a novel application of a small molecular compound luteolin (Lut), in particular to an application for preventing and treating gastrointestinal epithelialization (IM) and Spasmolytic Polypeptide Expression Metaplasia (SPEM), and provides a novel approach for blocking or reversing gastric precancerous diseases.

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

the experimental results show that Lut can inhibit chenodeoxycholic acid (Chenodeoxycholic acid, CDCA) induced gastrointestinal epithelial metaplasia in vitro, improve the growth state of cells and lower the index of intestinal epithelial metaplasia. In vivo experiments prove that the Lut can prevent and treat the formation of SPEM, has obvious effects on injection and oral administration, has no toxic or side effect on other organs, and prompts that the Lut can be used as a new means for preventing and treating the intervention of IM and SPEM medicaments.

In a first aspect of the invention, there is provided the use of Lut in the manufacture of a medicament for the prevention and/or treatment of IM.

In one embodiment, the IM is induced by bile acids.

In a second aspect of the present invention, there is provided a use of Lut in the manufacture of a medicament for preventing and/or treating SPEM.

In one embodiment, the SPEM is induced by tamoxifen.

In one embodiment, the Lut administration mode described above is both injection and oral administration.

In one embodiment, in the medicament, lut is the sole active ingredient.

In one embodiment, the Lut is mixed with other active ingredients in the medicament.

In one embodiment, the Lut is used at a concentration of 10 μm or less.

Compared with the prior art, the invention has the following remarkable improvements:

the invention verifies that the Lut can obviously improve the state of gastric mucosa epithelial cells after bile acid intervention, and down regulate the expression of intestinal epithelial metaplasia specific molecules CDX2, MUC2 and KLF4 for the first time. In vivo experiments prove that the Lut can prevent and treat a SPEM mouse model induced by tamoxifen, remarkably improve the mental state and activity change of the mouse, remarkably improve the gastric mucosa morphology and gastric tissue pathology of the mouse, and down regulate the expression of a SPEM marker TFF 2. The used effective dose has no toxic or side effect on normal gastric mucosa epithelial cells and other organs, and verifies two administration modes of injection and oral administration, so that Lut becomes a safe and effective new means for preventing and treating gastric precancerous diseases (IM and SPEM) and brings a new thought for reversing and blocking gastric precancerous diseases.

Drawings

The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate the invention and together with the embodiments of the invention, serve to explain the invention. In the drawings:

FIG. 1.Lut molecular architecture diagram.

Figure 2. Influence of lut on normal gastric mucosal epithelial cell viability. Altogether, 6 groups of luts with different concentrations are respectively: cell viability was examined at 3 time points of 0. Mu.M, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M, 0h, 24h, 48 h.

FIG. 3. Influence of lut on the growth status of bile acid-induced intestinal metaplastic cell model. The Lut concentration was 10. Mu.M and the cell model was intervened for 24h.

FIG. 4. Influence of lut on bile acid induced intestinal epithelial metaplastic cell model specific molecules CDX2, KLF4 at mRNA and protein levels.

FIG. 5. Influence of lut on the level of mRNA and protein of the bile acid induced intestinal epithelial metaplastic cell model specific molecule MUC 2.

FIG. 6. Influence of lut on stomach tissue morphology in a SPEM mouse model. The study was designed with 2 modes of administration (20 mg/kg for injection, 40mg/kg for oral administration).

FIG. 7. Influence of lut on gastric mucosal pathology in a SPEM mouse model.

FIG. 8. Influence of lut on SPEM mouse model marker TFF2 expression.

FIG. 9. Influence of lut on pathological states of other organs of a SPEM mouse model. Organs include liver, heart, spleen, kidney, large intestine, small intestine.

FIG. 10 shows the principal component analysis results of three groups of samples. D: normal group; c: a model group; l: lut treatment group.

FIG. 11 shows a volcanic diagram of a differentially expressed gene. Group1: normal group; group2: a model group; group3: lut treatment group. In the figure, the abscissa is log2FoldChange value, the ordinate is-log 10padj or-log 10pvalue, and the blue dotted line represents the threshold line of the differential gene screening criteria.

FIG. 12 differential expressed gene cluster heat map. D: normal group; c: a model group; l: lut treatment group. The abscissa is the sample name, the ordinate is the normalized value of the differential gene FPKM, the redder the color, the higher the expression quantity, the greener the expression quantity, and the lower the expression quantity. The thermal map also adds the chromosome to which each gene belongs, the length of the gene and the biological type of the gene.

FIG. 13A differential gene GO enrichment analysis scatter plot. The abscissa in the graph is the ratio of the difference base factor annotated to GO Term to the total number of difference genes, and the ordinate is GO Term.

FIG. 14 shows a differential gene KEGG enrichment scatter plot. The ratio of the differential base factor annotated to the KEGG pathway to the total number of differential genes is plotted on the abscissa and the KEGG pathway is plotted on the ordinate.

FIG. 15 shows the result of differential gene RT-qPCR verification.

FIG. 16 NCTD inhibits proliferation of gastric cancer cell lines AGS, SGC 7901 and normal gastric mucosal epithelial cells GES-1.

FIG. 17 NCTD structural formula and expression of an index of gastrointestinal epithelial metaplasia induced by an intervention bile acid.

FIG. 18 shows the structural formula of Esculetin, the inhibition of GES-1 proliferation and the intervention in bile acid-induced expression of the index of gastrointestinal epithelial metaplasia.

FIG. 19A schematic of the inhibition of GES-1 proliferation by forsononetin.

Figure 20.Formononetin structural formula and expression of the gastrointestinal epithelialization indicators MUC2 and KLF4 induced by bile acid.

Detailed Description

The preferred embodiments of the present invention will be described below with reference to the accompanying drawings, it being understood that the preferred embodiments described herein are for illustration and explanation of the present invention only, and are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments used in the following examples are laboratory conventional instruments unless otherwise specified; the experimental materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

Example 1 in vitro experiments

Effect of lut on normal gastric mucosal epithelial cell survival

To determine the maximum non-toxic concentration of Lut on normal gastric mucosal epithelial cells we selected the normal gastric mucosal epithelial cell line GES-1 (stored in the subject group laboratory, 1640 medium) as the subject. The GES-1 cell lines were treated with 6 groups of different concentrations (0, 10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M, 50. Mu.M) of Lut (structure shown in FIG. 1, purchased from Med Chem Express Co.) for 0h, 24h, 48h, respectively, and the CCK8 assay was used to measure cell proliferation, and cell viability was calculated. As shown in FIG. 2, the cell viability was not different from group to group before dosing (0 h), and the intervention of the concentration of Lut 10. Mu.M for 24h and 48h had no effect on the cell viability, while the other concentrations had inhibitory effect on the cells, indicating that the concentration of Lut 10. Mu.M had no toxic or side effect on normal cells.

Effect of lut on bile acid-induced intestinal metaplastic cell model growth status and specific molecule expression

According to the results of the CCK-8 assay, the concentration of Lut 10. Mu.M did not affect cell proliferation. Therefore, after GES-1 cells were treated at a CDCA concentration of 150. Mu.M, a 10. Mu.M concentration of Lut was added for intervention for 24 hours, and the growth state of each group of cells was observed under the mirror, as compared with the case of the synchronous normal GES-1 cells.

As shown in FIG. 3, the cells in the normal group (DMSO) are good in growth state, regular in cell morphology, fusiform, orderly in arrangement, scattered in growth, poor in growth state of cells in the model group (CDCA), long and slender in trilateral, polygonal or spindle shape, accompanied by pseudopodia formation, cell polarity loss and arrangement disorder, and the cells in the drug intervention group (CDCA+lut) are recovered to a large extent.

Subsequently, we detected the expression of cell enteronitization specific molecules CDX2, KLF4, MUC2 at mRNA and protein levels using RT-qPCR, western Blot, and immunofluorescence techniques. The results are shown in FIGS. 4-5, and the CDX2, KLF4 and MUC2 were significantly down-regulated at both mRNA and protein levels after the cell was subjected to Lut 10. Mu.M intervention, and the differences were statistically significant.

Example 2 in vivo experiments

C57BL/6 mice (SPF grade, male, 6-8 weeks old, supplied by the university of beijing department of medicine laboratory department of zoology) were randomly divided into 4 groups, normal, SPEM model, lut treatment (injection), and Lut treatment (oral), 6 each, and Lut treatment groups were given Lut interventions in both intraperitoneal (20 mg/kg, 1 time daily) and oral (40 mg/kg, 1 time daily) modes, for 7 consecutive days, after which, in addition to the normal group, the other three groups induced SPEM mice model (5 mg/20g, 1 time daily, 3 days of continuous intraperitoneal injection) with Tamoxifen (Tamoxifen, TMX), and gastric tissues were sacrificed for HE staining, and the expression of TFF2 was detected for each group of mice by immunohistochemistry while liver, heart, spleen, kidney, large intestine, and small intestine tissues were left to complete HE staining.

The visual observation of the gastric mucosa morphology shows that the gastric mucosa thickness is uniform after normal mice are in groups, the mucosa is smooth and glossy, and the mucosa folds are arranged regularly; after the mice are in the model group, the gastric mucosa is thinned, the local mucosa is whitish, the glossiness is poor, and the mucosa fold arrangement is irregular; the color, glossiness and fold arrangement of the gastric mucosa of the mice in the Lut treatment group (injection and oral administration) are all recovered. Pathological and immunohistochemical results showed that the model group had decreased gastric glandular wall cells, which exhibited pseudo-pyloroadenogenesis, increased expression of TFF2 at the middle-bottom of gastric glandular glands, whereas the Lut treatment group (injection and oral) had increased numbers of wall cells, no pseudo-pyloroadenogenesis was seen in the gastric body, and no significant expression of TFF2 at the middle-bottom of gastric glandular glands, as shown in FIGS. 7-8. The HE results of other organs are shown in figure 9, and liver, heart, spleen, kidney, large intestine and small intestine tissue pathology is not abnormal, which indicates that the Lut administration dose has no toxic or side effect on other organs.

EXAMPLE 3Lut mechanism of action study

To fully prove the curative effect of the Lut, we explored the action mechanism of the Lut, and after treating the GES-1 cells with CDCA at 150 mu M concentration, adding 10 mu M concentration of the Lut to intervene for 24 hours, comparing the synchronous normal GES-1 cells, namely grouping into a normal group (DMSO), a model group (CDCA) and a treatment group (CDCA+lut), screening differential genes by a transcriptome sequencing technology, and carrying out GO function enrichment analysis and KEGG channel enrichment analysis. As a result, as shown in fig. 10 to 14, the inter-group samples are dispersed, the intra-group samples are polymerized, and three groups of common differential genes are enriched to transport of bile acid and bile salt, intracellular transport, aldehyde ketal reductase (NADP) activity, retinoid metabolic process, cell differentiation, etc., the mechanism of which may be involved in glycolipid metabolism, central carbon metabolism of cancer, jak-STAT signaling pathway, etc. As shown in FIG. 15, the results of the RT-qPCR verification of key molecules show that IFNL1, IL12RB2 and Tyk2 molecules in the Jak-STAT signal channel are obviously down-regulated after the Lut intervention, and the molecules PAPA 2 related to cell proliferation, the molecules AKR1C1 related to cell differentiation and the bile acid transmembrane transporter SLCO2B1 are also obviously down-regulated after the Lut intervention, so that one of the acting mechanisms of the Lut is possibly to influence the bile acid transmembrane transport activity and regulate the cell growth and differentiation through the IFNL1/IL12RB2/Tyk2 pathway.

The experimental results show that Lut can inhibit the gastrointestinal epithelial metaplasia induced by CDCA in vitro, improve the growth state of cells and lower the index of intestinal epithelial metaplasia. In vivo experiments prove that the Lut can prevent and treat the formation of SPEM, has good injection and oral administration effects, has no toxic or side effect on other organs, and one of the action mechanisms of the Lut in preventing and treating gastric precancerous diseases probably influences the trans-membrane transport activity of bile acid through IFNL1/IL12RB2/Tyk2 pathway so as to regulate and control cell growth and differentiation. The Lut is suggested to be a new means for preventing and treating the intervention of IM and SPEM medicines.

Example 4 comparative experiments

In order to explore whether the same drugs can be used for the treatment of gastric cancer and pre-gastric cancer diseases, we further selected 3 compounds for treating gastric cancer to perform the following experimental verification (all the following experiments are performed at concentrations where the drugs do not have great damage to normal gastric mucosal epithelial cells):

1. norcantharidin (Norcanthoridin, NCTD)

Norcantharidin (NCTD) is well documented in the pharmaceutical description that the medicament is suitable for esophageal cancer, gastric and cardiac cancer and the like. Our experiments also demonstrate that NCTD can significantly inhibit proliferation of gastric cancer cell lines AGS and SGC 7901, as shown in fig. 16A, 16B. Subsequently, we examined the proliferation of normal gastric mucosal epithelial cells (GES-1) inhibited by NCTD and found less damaging concentrations of 10 μm, as shown in fig. 16C, which is the maximum effective non-toxic concentration. We selected this concentration and performed an intervention for bile acid-induced gastrointestinal epithelialization according to the methods and experimental conditions described previously.

The results showed that the intestinal epithelial metaplasia indices CDX2, MUC2, KLF4 were not decreased at mRNA levels (fig. 17B, 17C, 17D) nor were MUC2 expression down-regulated at protein levels after NCTD intervention in bile acid-induced gastrointestinal metaplasia (fig. 17E). It can be stated that NCTD does not inhibit bile acid-induced gastrointestinal epithelialization.

2. Esculin (Esculetin)

The literature reports that esculin (esciletin) has an antitumor effect on gastric cancer. We therefore used Esculetin to intervene in GES-1, and found the maximum non-toxic concentration based on CCK-8 results, i.e., 50. Mu.M (FIG. 18A), and therefore later selected this concentration and interfered with bile acid-induced gastrointestinal metaplasia according to the methods and experimental conditions described above.

The results showed that after the esciletin intervenes in bile acid-induced gastrointestinal metaplasia, the key indicators of intestinal metaplasia CDX2, MUC2 were not down-regulated at mRNA levels, only KLF4 was down-regulated, as shown in fig. 18B, indicating that esciletin did not inhibit bile acid-induced gastrointestinal metaplasia.

3. Formononetin (Formononetin)

The literature reports that formononetin has antitumor effect on gastric cancer and also has therapeutic effect on gastric ulcer. We used Formonenetin to intervene in GES-1 and found the maximum non-toxic concentration, i.e., 160. Mu.M, based on CCK-8 results, which was then selected and used to intervene in bile acid-induced gastrointestinal epithelialization according to the methods and experimental conditions described above.

The results showed that after forstronetin intervention in bile acid-induced gastrointestinal metaplasia, intestinal metaplasia index MUC2 was not down-regulated at protein level nor was KLF4 down-regulated at mRNA level (fig. 20), indicating that forstronetin was not able to inhibit bile acid-induced gastrointestinal metaplasia.

It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.

Claims (8)

1. Application of luteolin in preparing medicine for preventing and/or treating gastrointestinal epithelial metaplasia is provided.

2. The use of claim 1, wherein the gastrointestinal epithelial metaplasia is caused by bile acid stimulation.

3. Application of luteolin in preparing medicine for preventing and/or treating spasmolytic polypeptide expression metaplasia is provided.

4. The use according to claim 3, wherein the spasmolytic polypeptide expresses a metaplasia inducing agent that is tamoxifen.

5. The use according to claim 1 or 3, wherein luteolin is administered by injection or orally.

6. Use according to claim 1 or 3, characterized in that in the medicament luteolin is the only active ingredient.

7. Use according to claim 1 or 3, characterized in that in the medicament luteolin is mixed with other active ingredients.

8. The use according to claim 1 or 3, characterized in that luteolin is used in a concentration of 10 μm or less.

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