Application of traditional Chinese medicine composition in preparation of medicine for treating intestinal mucositis caused by 5-FU

Abstract

The invention belongs to the field of medicines, and particularly relates to application of a traditional Chinese medicine composition in preparation of a medicine for treating intestinal mucositis caused by 5-fluorouracil (5-FU). It is found for the first time that the traditional Chinese medicine composition has the functions of improving bile acid functions and restoring intestinal barriers and has the function of restoring expression of 5-FU key detoxification genes DPYD so as to achieve the effect of treating and chemotherapy of intestinal mucositis, and a new way is provided for treating 5-FU-caused intestinal mucositis.

Description

Technical Field

[0001] The invention belongs to the field of medicines, and particularly relates to application of a traditional Chinese medicine composition in preparing a medicine for treating intestinal mucositis caused by 5-FU. Background Art

[0002] Chemotherapy is one of the main treatments for malignant tumors. 5-FU is the basic chemotherapy regimen for colon cancer and other diseases. Diarrhea is the main dose-limiting side effect of 5-FU chemotherapy drugs, which greatly limits its clinical application. Although its mechanism of action is still unclear, improving chemotherapy-related intestinal mucositis can significantly improve the quality of life of patients and optimize the chemotherapy process.

[0003] Current studies have shown that the protein dihydropyrimidine dehydrogenase (DPYD) encoded by the DPYD gene is the initiator and rate-limiting factor of the 5-FU decomposition pathway. It catalyzes the conversion of 5-FU to 5,6-dihydrofluorouracil (DHFU), which is the first and most important step in 5-FU metabolism. When DPYD-deficient cancer patients receive 5-FU chemotherapy, the toxicity increases. Secondly, based on the results of clinical studies, the researchers conducted plasma metabolomics testing on patients with surgery and 5-FU treatment. The concentrations of glycochenodeoxycholic acid, glycodeoxycholic acid, glycocholic acid, glycolithocholic acid, glycoursodeoxycholic acid, taurochenodeoxycholic acid and taurocholic acid in the samples were relatively high, among which deoxycholic acid (DCA) and its analogs, hyodeoxycholic acid (HDCA) and glycodeoxycholic acid (GDCA) showed significantly higher levels in patients with diarrhea, while excess bile acid ( BAs can stimulate the secretion of fluid, mucus or sodium, increase intestinal motility, aggravate mucosal permeability and damage, and ultimately promote the occurrence of diarrhea. It was found that the combination of 5-FU and BAs would aggravate the level of apoptosis of HIEC (human intestinal epithelial cells). This suggests that bile acid metabolism disorders and decreased levels of DPYD are closely related to 5-FU-induced chemotherapy intestinal mucositis (CIM). Although it has been confirmed that bile acid metabolism disorders and decreased DPYD expression are the key mechanisms of 5-FU intestinal mucositis, there is still no effective solution to this problem in clinical practice.

[0004] Therefore, based on this, the technical solution of the present invention is proposed. Summary of the invention

[0005] In order to solve the problems existing in the prior art, the present invention provides an application of a traditional Chinese medicine composition in the preparation of a medicine for treating intestinal mucositis caused by 5-FU. The traditional Chinese medicine composition (DA BU PIDECOCTION, DBPD) comprises the following raw materials in parts by weight: 13-15 parts of ginseng, 8-10 parts of roasted licorice, 8-10 parts of atractylodes, 6-8 parts of ophiopogon, 4-5 parts of dried ginger, 4-5 parts of inula flower, and 4-5 parts of schisandra chinensis.

[0006] Preferably, the drug is a drug that reduces the expression level of IL-6 and TNF-α;

[0007] And / or, the drug is a drug that increases the expression level of IL-10.

[0008] Preferably, the drug is a drug that increases the expression level of Occludin, zo-1 protein;

[0009] And / or, the drug is a drug that increases the expression level of LGR5, Lyz1, and Muc2 proteins;

[0010] And / or, the drug is a drug that increases the expression level of Ki67.

[0011] Preferably, the drug is a drug that reduces BESP and increases the expression levels of UGT1A1, UGT1A9, TGR5, FXR, and DPYD;

[0012] And / or, the drug is a drug for alleviating gallbladder inflammation.

[0013] Preferably, the drug is a drug that increases the expression level of BCL-2;

[0014] And / or, the drug is a drug that reduces the expression level of Bax and Caspase-3.

[0015] Preferably, the drug is a drug that improves bile acid function and restores the intestinal barrier.

[0016] Preferably, the medicine is a preparation prepared by using the Chinese medicine composition as raw material and adding medically acceptable excipients.

[0017] Preferably, the preparations include but are not limited to oral preparations and injection preparations.

[0018] Preferably, the oral preparations include but are not limited to capsules, granules, tablets, powders, pills, and decoctions.

[0019] To facilitate understanding of the present invention, the compatibility mechanism of the Chinese medicine composition of the present invention is described:

[0020] Diarrhea after 5-FU chemotherapy is caused by "drug toxicity" in traditional Chinese medicine that damages the spleen and stomach. Although chemotherapy can expel evil and fight cancer, it can also harm the spleen and stomach, resulting in disorders of digestion and transportation, and a weak acquired foundation. On the one hand, damage to the spleen and stomach will lead to a lack of source for the production of qi and blood, the viscera will not be full, and the vital energy will become weaker. On the other hand, "there is no diarrhea without dampness". The spleen is responsible for the transportation and transformation of water. If the spleen is deficient, water and dampness will accumulate and cause diarrhea. Diarrhea will lead to the depletion of qi and body fluid over a long period of time, and it will develop into a symptom of qi and yin deficiency over time. Therefore, the treatment should focus on strengthening the spleen and removing dampness, invigorating qi and nourishing yin.

[0021] The Chinese medicine composition of the present invention comprises: ginseng is sweet in taste, can greatly invigorate vital energy, invigorate the spleen and benefit the lungs; roasted licorice is sweet in taste, can invigorate the middle qi, and the two medicines are used together to invigorate the spleen; dried ginger is pungent in taste, can warm the middle and dispel cold, can purge the spleen's body, and is matched with ginseng and roasted licorice, can transform yang with pungent and sweet, and help the spleen to transport and transform; white atractylodes is bitter in taste, can invigorate the spleen and benefit qi, dry dampness and promote diuresis, so it can help spleen transformation and regulate spleen deficiency; ophiopogon is sweet in taste, can invigorate lung yin, schisandra chinensis is sour in taste, can restrain the lung and produce fluid, and can help the spleen and lung together, and can transform yin with sour and sweet to prevent fluid damage; inula flower is salty in taste, can descend qi, transform phlegm and promote water circulation, and if the spleen is weak, water and dampness will accumulate and form a solid syndrome, and the solid syndrome will be purged, so inula flower is used to purge the spleen's body and lung, and the whole prescription has four uses, two bodies and one transformation, and has the functions of invigorating the spleen and dispelling dampness, invigorating qi and nourishing yin.

[0022] The beneficial effects of the present invention are:

[0023] The present invention provides an application of a traditional Chinese medicine composition in the preparation of a medicament for treating intestinal mucositis caused by 5-FU. The present invention firstly finds that the traditional Chinese medicine composition has the function of improving bile acid function and restoring the intestinal barrier, and has the function of restoring the expression of DPYD, a key detoxification gene of 5-FU, to play a role in treating chemotherapy intestinal mucositis, thus providing a new approach for treating intestinal mucositis caused by 5-FU. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 This is a data graph showing that DBPD alleviates 5-FU-induced intestinal mucositis in mice.

[0026] Figure 2 This is the data graph showing that DBPD restores 5-FU-induced intestinal barrier damage in mice.

[0027] Figure 3 This is the transcriptomic analysis data of ileum tissue of each group of mice.

[0028] Figure 4 This is a data map of key genes and mechanisms revealed by integrated transcriptome analysis.

[0029] Figure 5 It is a bioinformatics analysis of UGT1A1, UGT1A9, and FXR expression in gastric cancer and colorectal cancer and its correlation with prognosis.

[0030] Figure 6 This is a data graph showing that DBPD alleviates 5-FU-induced apoptosis and barrier destruction in human intestinal epithelial cells (HIEC).

[0031] Figure 7 This is the UGT1A1, UGT1A9 and FXR gene correlation analysis and Alpha fold3 prediction data chart. DETAILED DESCRIPTION

[0032] To make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention.

[0033] Example 1

[0034] This embodiment first provides a method for preparing a Chinese medicine composition (DBPD), which comprises the following steps:

[0035] According to a conventional method, 15 g of ginseng, 10 g of licorice root, 10 g of atractylodes macrocephala, 8 g of ophiopogon japonicus, 5 g of dried ginger, 5 g of inula flower, and 5 g of schisandra chinensis are mixed and prepared into a decoction.

[0036] Example 2

[0037] This example established a C57BL / 6 mouse chemotherapy intestinal mucositis model (such as Figure 1 A) was used to study the effect of DBPD on 5-FU-induced intestinal mucosal inflammation, wherein a total of 5 groups were set up, specifically:

[0038] (1) Control group;

[0039] (2) 5-FU intervention group (5-FU);

[0040] (3) low-dose DBPD intervention group (DBPD L), with a concentration of 3.75 g / kg;

[0041] (4) medium-dose DBPD intervention group (DBPD M), with a concentration of 7.5 g / kg;

[0042] (5) High-dose DBPD intervention group (DBPD H), with a concentration of 15 g / kg.

[0043] First, a chemotherapy-induced intestinal mucositis model was established in C57BL / 6 mice to examine the effect of DBPD on 5-FU-induced mucositis (e.g. Figure 1 Compared with the control group, the intestinal villi in the 5-FU group were significantly shortened and sparse, some epithelial cells were lost, and inflammatory cell infiltration was observed. DBPD treatment improved these manifestations, especially restoring the height of the intestinal crypts (e.g. Figure 1 B~ Figure 1 E). Since high-dose DBPD showed the most significant morphological recovery in 5-FU-induced intestinal damage, this group was selected for further analysis. The expression of inflammatory factors in small intestinal tissue was evaluated, and it was found that IL-6 and TNF-α were significantly increased, while IL-10 was significantly decreased in the 5-FU group. After DBPD treatment, decreased IL-6 and TNF-α levels were observed, while increased IL-10 levels (as shown in Figure 5E). Figure 1 F). These results indicate that DBPD can reduce 5-FU-induced morphological damage and inflammatory response in the small intestine of mice.

[0044] Example 3

[0045] To evaluate the effect of 5-FU on intestinal barrier integrity, the expression of barrier-related proteins and the abundance of LGR5+ stem cells, goblet cells, and Paneth cells were detected. Compared with the control group, 5-FU treatment resulted in a significant decrease in the expression of Occludin and Zo-1 proteins in the ileum tissue of mice. However, DBPD treatment significantly restored the expression of these proteins that were inhibited by 5-FU. Using Lyz1, Muc2 (PAS), and Lgr5 as specific markers for Paneth cells, goblet cells, and intestinal stem cells, respectively, 5-FU treatment significantly reduced the expression of Lyz1, Muc2 / PAS, and Lgr5 compared with the control group. In contrast, DBPD treatment significantly increased the expression of these proteins (e.g. Figure 2 A. Figure 2 B). Ki67 staining was used to evaluate intestinal proliferation; after DBPD treatment, Ki67 expression in the crypts was higher than that in the 5-FU group, indicating that DBPD promoted the proliferation of intestinal crypt cells (as shown in Figure 2A). Figure 2 C. Figure 2 D). Transmission electron microscopy showed structural changes in intestinal microvilli. Compared with the control group, the microvilli in the 5-FU group were significantly sparse and shortened, but these changes were partially reversed after DBPD treatment (see Figure 2). Figure 2 These results suggest that DBPD has a protective effect on the intestinal barrier, thereby alleviating the damage caused by 5-FU.

[0046] Example 4

[0047] This example further performed transcriptomic analysis on the ileum tissues of each group of mice. Principal coordinate analysis (PCoA) based on the unweighted UniFrac index showed that there were significant differences in PC1 (74%) and PC2 (10%) between the control group and the 5-FU group (e.g. Figure 3 The cluster heat map showed that the gene expression after DBPD treatment was more consistent with the control group compared with the 5-FU group (as shown in Figure 2A). Figure 3The volcano plot of differentially expressed genes (DEGs) showed that there were 3350 up-regulated genes and 2470 down-regulated genes after 5-FU intervention compared with the control group (as shown in Figure 2A). Figure 3 C), and compared with the 5-FU group, there were 463 up-regulated and 427 down-regulated DEGs after DBPD treatment (as shown in Figure 3 D). The intersection of the above differentially expressed genes showed 772 DEGs in the three groups (as shown in Figure 3 E).

[0048] Example 5

[0049] KEGG and GO analyses were performed on 772 DEGs. KEGG analysis highlighted the enrichment of pathways related to bile secretion, protein digestion and absorption, histidine metabolism, arachidonic acid metabolism, and CYP-mediated drug metabolism (e.g. Figure 4 GO analysis showed that DBPD regulates cellular components such as the extracellular region and the cell apical plasma membrane, biological processes including responses to external stimuli, small molecule metabolic processes and lipid metabolic processes, related molecular functions and transport protein activities and receptor regulatory activities (such as Figure 4 B).

[0050] The frequency of 772 gene lineages was analyzed using the Wordcloud method, and high occurrence rates of Ugts, Cyps, and Slcs gene families were found (e.g. Figure 4 C). Ugts are essential for BA metabolism and enterohepatic circulation, affecting cholestatic diarrhea. In clinical studies involving CPT11, decreased Ugts expression was associated with increased intestinal mucositis. The bladder primarily metabolizes various drugs to mitigate toxicity, while the Slcs family is involved in the transport of small molecules, amino acids, and lipids, indicating potential involvement in DBPD and 5-FU transport, and studies have shown that cells with low expression can increase resistance to 5-FU.

[0051] These results suggest that regulation of BA and drug metabolism is the main mechanism by which DBPD alleviates 5-FU-induced intestinal mucositis. Gene set enrichment analysis (GSEA) showed that genes related to the BA metabolic pathway were suppressed in the 5-FU group, while DBPD significantly enhanced BA metabolism and regulated core genes controlling BA secretion (such as Figure 4 D. Figure 4 E). Further analysis of the cluster heat map analysis of BA-related genes and key detoxification enzymes showed that the key receptors FXR (NR1H4), TGR5 (GPBAR1) and detoxification enzymes UGT1A1, UGT1A9 and key enzyme DPYD were reduced in the 5-FU group. After DBPD treatment, the levels of the above genes were increased, and the bile flow control pump BSEP (ABCB11) was increased under the action of 5-FU and decreased after DBPD treatment (as shown in Figure 5). Figure 4 F). These results indicate that DBPD regulates multiple genes involved in BA metabolism and small molecule metabolism, enhances biological functions, and increases the level of the key detoxification enzyme DPYD, thereby alleviating 5-FU-induced intestinal mucositis. Based on the above, the present invention believes that DBPD may be the mechanism of action for alleviating 5-FU-induced intestinal mucositis by regulating bile acid metabolism and DPYD expression.

[0052] In this example, the transcriptomic analysis data were verified using the ileum tissues of each group of mice. Compared with the 5-FU group, DBPD significantly increased the mRNA levels of bile acid metabolism-related factors UGT1A1, UGT1A9, TGR5, and FXR in the intestinal tissues of CIM mice (e.g. Figure 4 G). ELISA was used to detect the concentrations of DCA and LCA in the ileum of mice. Compared with the control group, 5-FU significantly increased the concentrations of DCA and LCA, while DBPD decreased the concentrations of DCA and LCA in the ileum (as shown in Figure 2A). Figure 4 H). We observed for the first time that DBPD can increase the level of DPYD, a key detoxification enzyme induced by 5-FU. The gallbladder is a key organ for bile concentration and storage, and can also be damaged by excessive BAs. Gallbladder morphological examination showed that the gallbladder wall in the 5-FU group was significantly thickened compared with the control group, with extensive inflammatory cell infiltration in the mucosa and muscle layer, accompanied by calcification areas. Intervention after DBPD significantly alleviated this inflammatory response (e.g. Figure 4 I). The changes caused by 5-FU and BAs may damage not only the intestinal mucosa but also the gallbladder. This damage reduces lipid digestion capacity and is associated with the reduced food intake observed in chemotherapy patients. In summary, DBPD may improve 5-FU-induced intestinal mucosal inflammation by regulating BA metabolism and the detoxification enzyme DPYD.

[0053] Example 6

[0054] In the current NCCN and CSCO guidelines, first-line chemotherapy drugs for patients with gastric cancer and colorectal cancer include 5-FU derivatives, such as fluorouracil injection and capecitabine. Patients receiving neoadjuvant chemotherapy received capecitabine treatment 4-6 cycles before surgery. In order to explore the effects of UGT1A1, UGT1A9 and FXR, key genes in bile acid metabolism, on patients with gastrointestinal tumors, the GSE17538 chip in the TCGA database was analyzed. The pan-cancer analysis results showed that the expression levels of UGT1A1, UGT1A9 and FXR in gastrointestinal tumor tissues were significantly lower than those in normal tissues (such as Figure 5 A. Figure 5 B. Figure 5 C). Tumor mutation burden (TMB) analysis showed that UGT1A1 and FXR have high mutation potential in colorectal cancer, gastric cancer, and cholangiocarcinoma (as shown in Figure 2A). Figure 5 D). Survival analysis showed that patients with elevated expression of UGT1A1, UGT1A9, and FXR had a significantly lower OS in gastrointestinal cancers and CRCs (Figure ​Figure4D). Figure 5 E) and median survival (as shown Figure 5 These results suggest that UGT1A1, UGT1A9, and FXR may serve as a promising potential prognostic prediction and therapeutic target for gastrointestinal tumors.

[0055] Example 7

[0056] The optimal intervention concentration of DBPD serum was determined by HIEC method. Serum concentrations of 2.5%, 5% and 10% significantly promoted HIEC cell proliferation, among which 10% concentration had the most significant effect on HIEC cell proliferation (e.g. Figure 6 After HIEC cells were treated with 80 μM 5-FU for 48 h, the effects of DBPD-containing serum on 5-FU-induced apoptosis and barrier proteins were examined. Flow cytometric analysis showed that 5-FU increased the apoptosis of HIEC cells compared with the control group, while DBPD intervention alleviated the apoptosis of HIEC cells (as shown in Figure 2A). Figure 6 B. Figure 6 C). Apoptosis-related protein analysis showed that after 5-FU treatment, the anti-apoptotic protein BCL-2 decreased, and the pro-apoptotic factors Bax and Caspase-3 increased. DBPD treatment led to increased BCL-2 expression, while Bax and Caspase-3 levels decreased. Intestinal barrier function assessment showed that 5-FU treatment reduced the mRNA and protein expression of barrier proteins Occludin and ZO-1 in HIEC cells (as shown in Figure 5C). Figure 6 D. Figure 6 E. Figure 6 F). However, DBPD treatment enhanced the expression of Occludin and ZO-1, suggesting improved intestinal barrier integrity and reduced HIEC cell apoptosis. Further analysis showed that 5-FU decreased the mRNA and protein levels of UGT1A1, UGT1A9, FXR, TGR5, and DPYD compared with the control group. On the other hand, DBPD treatment increased the expression of UGT1A1, UGT1A9, FXR, DPYD, and TGR5 (Fig. Figure 6 G. Figure 6 H. Figure 6 I). These results suggest that DBPD regulates the metabolism of BA in intestinal epithelial cells through the UGT1A1 / TGR5 / FXR pathway and increases the expression of the key detoxification enzyme DPYD, thereby alleviating 5-FU-induced intestinal cell apoptosis.

[0057] Example 8

[0058] We analyzed the correlation between UGT1A1, UGT1A9 and FXR genes in gastric cancer and adjacent tissues, colon cancer and adjacent tissues, and jejunal tissues using the gene correlation analysis module in the GEPIA2 online database. The results showed that there was a significant positive correlation between UGT1A1 (R = 0.63), UGT1A9 (R = 0.58) and FXR, respectively (see Table 2). Figure 7 A).

[0059] In addition, we used Alpha Fold3 to confirm that there is a strong interaction between UGT1A1, UGT1A9 and FXR (e.g. Figure 7 B). Based on the TM-score, the ipTM score of UGT1A1 and FXR was 0.47, and the pTM score was 0.59. A total of 12 hydrogen bonds were found between UGT1A1 and FXR that may be combined (such as Figure 7 C). The ipTM score of UGT1A9 and FXR was 0.62, and the pTM score was 0.63. A total of 10 hydrogen bonds were found between UGT1A9 and FXR (e.g. Figure 7 The above results demonstrate that the interaction between UGT1A1, UGT1A9 and FXR is strong.

[0060] In summary, UGT1A1 and UGT1A9 have the possibility of regulating bile acid metabolism by interacting with FXR.

[0061] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. Use of a Chinese medicine composition in the preparation of a medicament for treating intestinal mucositis caused by 5-FU, characterized in that: The Chinese medicine composition comprises the following raw materials in parts by weight: 13-15 parts of ginseng, 8-10 parts of licorice root, 8-10 parts of atractylodes macrocephala, 6-8 parts of ophiopogon japonicus, 4-5 parts of dried ginger, 4-5 parts of inula flower, and 4-5 parts of schisandra chinensis.

2. The use according to claim 1, characterized in that: The drug is a drug that reduces the expression levels of IL-6 and TNF-α; And / or, the drug is a drug that increases the expression level of IL-10.

3. The use according to claim 1, characterized in that: The drug is a drug for increasing the expression level of Occludin and zo-1 proteins; And / or, the drug is a drug that increases the expression level of LGR5, Lyz1, and Muc2 proteins; And / or, the drug is a drug that increases the expression level of Ki67.

4. The use according to claim 1, characterized in that: The drug is a drug that reduces BESP and increases the expression levels of UGT1A1, UGT1A9, TGR5, FXR, and DPYD; And / or, the drug is a drug for alleviating gallbladder inflammation.

5. The use according to claim 1, characterized in that: The drug is a drug that increases the expression level of BCL-2; And / or, the drug is a drug that reduces the expression level of Bax and Caspase-3.

6. The use according to claim 1, characterized in that: The medicine is a medicine for improving bile acid function and restoring intestinal barrier.

7. The use according to any one of claims 1 to 6, characterized in that: The medicine is a preparation prepared by taking the Chinese medicine composition as raw material and adding medically acceptable auxiliary materials.

8. The use according to any one of claim 7, characterized in that: The preparation is an oral preparation or an injection preparation.

9. The use according to any one of claim 8, characterized in that: The oral preparation is a capsule, granule, tablet, powder, pill or decoction.

Images