Application of Atractylodes macrocephala polysaccharide in preventing and treating diarrhea caused by tyrosine kinase inhibitors

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to the application of Atractylodes macrocephala polysaccharide in preventing and treating diarrhea caused by tyrosine kinase inhibitors. Specifically, the present invention has found through research that Atractylodes macrocephala polysaccharide inhibits CFTR-mediated chloride ion secretion in cells after treatment with the tyrosine kinase inhibitor pyrotinib. A certain dose of Atractylodes macrocephala polysaccharide can alleviate diarrhea in rats caused by pyrotinib and significantly restore intestinal barrier function. Atractylodes macrocephala polysaccharide treatment improves the distribution of intestinal microorganisms and restores the increased levels of Clostridium difficile and Erysipelothrix caused by pyrotinib. In short, the present invention provides ideas for the specific mechanism of Atractylodes macrocephala polysaccharide in alleviating diarrhea caused by pyrotinib, provides insights into the complex interactions between Atractylodes macrocephala polysaccharide and intestinal microbiota, and provides a possible strategy for using Atractylodes macrocephala polysaccharide to treat diarrhea caused by pyrotinib, and therefore has good practical application value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to application of Atractylodes macrocephala polysaccharide in preventing and treating diarrhea caused by tyrosine kinase inhibitors. Background Art

[0002] Pyrotinib is a novel oral irreversible small molecule tyrosine kinase inhibitor of EGFR / HER2 / HER4, approved for the treatment of HER2-positive advanced breast cancer. Compared with other HER2-targeted drugs, pyrotinib has a stronger inhibitory effect on HER2-overexpressing tumors. Although pyrotinib has shown significant anti-tumor activity in clinical trials, its side effects, especially diarrhea, are one of the most common adverse reactions. Multiple studies have shown that more than 90% of patients report diarrhea symptoms, of which 30% to 40% experience severe diarrhea of ​​grade 3 or above. The mechanism of diarrhea caused by pyrotinib has not yet been elucidated, but it is generally believed to be related to EGFR inhibition, intestinal mucosal damage and intestinal flora imbalance. There is currently no specific drug for the treatment of diarrhea caused by pyrotinib. Montmorillonite powder and loperamide are used clinically to treat diarrhea caused by pyrotinib, but they can cause adverse reactions such as abdominal distension, abdominal pain and constipation, further reducing the quality of life of patients.

[0003] There is increasing evidence that traditional Chinese medicine can regulate intestinal microbiota and metabolites, and intestinal flora can metabolize traditional Chinese medicine components, thereby affecting the internal environment of the human body. Therefore, understanding the interaction between traditional Chinese medicine and microbiota is of great significance for understanding the pharmacological effects of traditional Chinese medicine in disease prognosis and treatment. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention provides the use of Atractylodes macrocephala polysaccharide in preventing and treating diarrhea caused by tyrosine kinase inhibitors. Specifically, the present invention found through research that Atractylodes macrocephala polysaccharide inhibits CFTR-mediated chloride ion secretion in cells after pyrotinib treatment. A certain dose of Atractylodes macrocephala polysaccharide can relieve diarrhea in rats caused by pyrotinib and significantly restore intestinal barrier function. Atractylodes macrocephala polysaccharide treatment improves the distribution of intestinal microorganisms and restores the increased levels of Clostridium difficile and Erysipelotrichi caused by pyrotinib. Metabolomics analysis suggests the AMPK signaling pathway. Atractylodes macrocephala polysaccharide activates LKB1 / AMPK and inhibits CFTR by inhibiting cAMP levels, thereby alleviating diarrhea caused by pyrotinib. Based on the above research results, the present invention is completed.

[0005] Specifically, the present invention relates to the following technical solutions:

[0006] The first aspect of the present invention provides the use of Atractylodes macrocephala polysaccharide in the preparation of a medicament for preventing and treating diarrhea caused by tyrosine kinase inhibitors.

[0007] Specifically, the prevention and treatment of diarrhea caused by tyrosine kinase inhibitors is specifically manifested in that while preventing and / or treating diarrhea caused by pyrotinib, the anti-tumor effect of pyrotinib is not affected.

[0008] More specifically, the prevention and treatment of diarrhea caused by Pyrotinib is manifested as:

[0009] (a1) inhibiting chloride ion secretion caused by pyrotinib;

[0010] (a2) Promote the recovery of intestinal barrier function damage caused by pyrotinib-induced diarrhea;

[0011] (a3) regulating the intestinal flora imbalance caused by pyrotinib;

[0012] (a4) Regulate the intestinal metabolite disorder caused by pyrotinib.

[0013] in,

[0014] The (a1) specifically includes: Atractylodes macrocephala polysaccharide inhibits the expression of chloride ion channel protein CFTR.

[0015] The (a3) ​​is specifically: Atractylodes macrocephala polysaccharide reduces the increase of Clostridium difficile and Erysipelothrix rhizogenes caused by pyrotinib.

[0016] The second aspect of the present invention provides the use of Atractylodes macrocephala polysaccharide combined with pyrotinib in the preparation of anti-tumor drugs.

[0017] Specifically, the anti-tumor drug specifically exhibits the following performances: exerting the anti-tumor effect of pyrotinib, and preventing and / or treating diarrhea side effects mediated by pyrotinib.

[0018] The mass ratio of the Atractylodes macrocephala polysaccharide to pyrotinib is 1-4:20; or, the mass molar ratio of the Atractylodes macrocephala polysaccharide to pyrotinib is 2.5-80:0.001-0.032, g / mmol.

[0019] The third aspect of the present invention provides an anti-tumor drug, the active ingredients of which at least include Atractylodes macrocephala polysaccharide and Pyrotinib.

[0020] The mass ratio of the Atractylodes macrocephala polysaccharide to pyrotinib is 1-4:20; or, the mass molar ratio of the Atractylodes macrocephala polysaccharide to pyrotinib is 2.5-80:0.001-0.032, g / mmol.

[0021] Specifically, the anti-tumor drug can not only effectively exert the anti-tumor effect of Pyrotinib, but also effectively alleviate the diarrhea side effect mediated by Pyrotinib.

[0022] The Atractylodes macrocephala polysaccharide in the present invention can be prepared by water extraction and alcohol precipitation. In a specific embodiment of the present invention, the specific preparation method of the Atractylodes macrocephala polysaccharide is as follows:

[0023] Add water to Atractylodes macrocephala for reflux extraction, filter and evaporate to concentrate, add ethanol for precipitation; let stand and then vacuum filter, wash the precipitate with anhydrous ethanol, acetone, petroleum ether and anhydrous ethanol in turn, evaporate the organic reagent to obtain crude Atractylodes macrocephala polysaccharide; purify the crude Atractylodes macrocephala polysaccharide to remove protein to obtain the Atractylodes macrocephala polysaccharide.

[0024] The specific method for purifying and removing protein may be the sevage method.

[0025] The mass ratio of Atractylodes macrocephala to water is 1:5-15, preferably 1:10.

[0026] The specific conditions of reflux extraction are: reflux extraction temperature is 80-100° C., reflux extraction time is 1-3 hours, and reflux extraction times are 2-4 times.

[0027] The preparation method further comprises dialyzing the Atractylodes macrocephala polysaccharide after the protein is removed, and the dialysis can be performed using a dialysis bag with a molecular weight cutoff of 5 KDa.

[0028] According to the present invention, the anti-tumor drug may further include at least one inactive drug ingredient.

[0029] The inactive ingredients of the medicine may be carriers, excipients, diluents, etc. commonly used in pharmacy, and are not specifically limited here.

[0030] The anti-tumor drug can be administered in a unit dosage form, specifically a liquid dosage form or a solid dosage form. Among them, the liquid dosage form can be a true solution, a colloid, a microparticle dosage form, an emulsion dosage form and a suspension dosage form, etc. The solid dosage form can be a tablet, a capsule, a dripping pill, a pill, a powder, an emulsion and a granule, etc.

[0031] In another specific embodiment of the present invention, the anti-tumor drug of the present invention can be administered to the body in a known manner. For example, it can be delivered systemically by vein. It can be optionally administered via intravenous, percutaneous, intranasal, oral, mucosal or other delivery methods. Such administration can be carried out via a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dose to be administered in the present invention can vary depending on a variety of factors to a great extent, such as target cells, biological types or their tissues, the general condition of the subject to be treated, the route of administration, the mode of administration, etc.

[0032] Beneficial technical effects of one or more of the above technical solutions:

[0033] The above technical solution has proved through experiments that Atractylodes macrocephala polysaccharide can effectively relieve diarrhea caused by the tyrosine kinase inhibitor pyrotinib. Atractylodes macrocephala polysaccharide regulates the AMPK signaling pathway, inhibits CFTR overexpression, restores intestinal mucosal damage and regulates intestinal homeostasis. The above technical solution provides ideas for the specific mechanism by which Atractylodes macrocephala polysaccharide relieves diarrhea caused by pyrotinib, provides insights into the complex interactions between Atractylodes macrocephala polysaccharide and intestinal microbiota, and provides a possible strategy for using Atractylodes macrocephala polysaccharide to treat diarrhea caused by pyrotinib, so it has good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The present invention shows that Atractylodes macrocephala polysaccharide inhibits CFTR-mediated chloride ion secretion in cells after pyrotinib treatment. (A) CCK-8 test was used to detect the effect of different concentrations of pyrotinib on the relative cell activity of T84 cells. (B) The dose-response curve of pyrotinib was drawn using GraphPad Prism. (C) CCK-8 test was used to detect the effect of different concentrations of Atractylodes macrocephala polysaccharide on the relative cell activity of T84 cells. (D) The dose-response curve of Atractylodes macrocephala polysaccharide was drawn using GraphPad Prism. (E) qRT-PCR was used to detect the expression of CFTR gene in each group of cells. (F) Western blot was used to detect the expression of CFTR protein in each group of cells. (G) The expression of CFTR protein in each group of cells was calculated based on the results of Western blot. (H) qRT-PCR was used to detect the expression of CFTR gene in CFTR-KD cells and CFTR-NC cells. (I) Western blot was used to detect the expression of CFTR protein in CFTR-KD cells and CFTR-NC cells. * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001; **** indicates P<0.0001; ns indicates P>0.05.

[0035] Figure 2 The following is the mechanism of Atractylodes macrocephala polysaccharide inhibiting the reduction of chloride ion secretion through CFTR after treatment with pyritinib in the embodiments of the present invention. (A) The expression of CFTR protein in CFTR-KD cells and CFTR-NC cells was calculated based on the results of Western blot. (B) MQAE was used to detect the chloride ion secretion of CFTR-KD cells and CFTR-NC cells in different drug treatment groups. (C) The relative fluorescence intensity of CFTR-KD cells and CFTR-NC cells in different drug treatment groups was calculated based on the MQAE results. One-way ANOVA was used for statistical significance. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates P > 0.05.

[0036] Figure 3The experimental design of the present invention is related to the effect of Atractylodes macrocephala polysaccharide on pyrotinib-induced diarrhea in rats. (A) Experimental design of Atractylodes macrocephala polysaccharide on pyrotinib-induced diarrhea rat model. (B) Fecal characteristics and diarrhea classification of rats. (C) Diarrhea rate of each group.

[0037] Figure 4 The results of the present invention are analyzed for the effect of Atractylodes macrocephala polysaccharide on the relief of pyritinib-induced diarrhea in rats. (A) Diarrhea score of each group. Two-way ANOVA was used for statistical significance. (B) Diarrhea index of each group. One-way ANOVA was used for statistical significance. (C) Weight change of rats in each group. Two-way ANOVA was used for statistical significance. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates P > 0.05.

[0038] Figure 5 The results of the tissue level of the intestinal barrier function of rats with diarrhea caused by pyrotinib restored by Atractylodes macrocephala polysaccharide in the examples of the present invention. (A) H&E staining of rat small intestine, large intestine, kidney and spleen histological images. (B) Evaluation of the kidney index of rats in each group. (C) Evaluation of the spleen index of rats in each group. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates P > 0.05.

[0039] Figure 6 The results of the molecular level of the intestinal barrier function of rats with diarrhea caused by pyrotinib restored by Atractylodes macrocephala polysaccharide in the embodiments of the present invention. (A) Western blot was used to detect the expression of intestinal barrier-related proteins ZO-1, E-Cadherin and Occludin in the intestinal tissues of rats in each group. (B) The expression of ZO-1 protein in the intestinal tissues of rats in each group was calculated based on the results of Western blot. (C) The expression of E-Cadherin protein in the intestinal tissues of rats in each group was calculated based on the results of Western blot. (D) The expression of Occludin protein in the intestinal tissues of rats in each group was calculated based on the results of Western blot. Statistical significance was analyzed by one-way analysis of variance, * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates P > 0.05.

[0040] Figure 7 Alpha diversity analysis and PCoA analysis of the changes in the intestinal microbiota of rats by pyrotinib and Atractylodes macrocephala polysaccharide in the embodiments of the present invention. (A) Grouped box plot of the Alpha diversity index. The numbers under the diversity index labels are the P values ​​of the Kruskal-Wallis test. (B) Sample two-dimensional ordination diagram of the PCoA analysis.

[0041] Figure 8 The genus-species difference analysis of the changes in the intestinal microbiota of rats by pyrotinib and Atractylodes macrocephala polysaccharide in the examples of the present invention. (A) Venn diagram of ASV / OTU in each group. (B) Heat map of species composition at the genus level of species clustering. (C) LDA effect value bar chart of marker species.

[0042] Fig. 9 The intestinal metabolites of rats with diarrhea induced by pyrotinib regulated by Atractylodes macrocephala polysaccharide in the embodiments of the present invention. (A) Principal Component Analysis (PCA) results in positive ion mode. (B) Partial Least Squares Discrimination Analysis (PLS-DA) results in positive ion mode. (C) Principal Component Analysis results in negative ion mode. (D) Partial Least Squares Discrimination Analysis results in negative ion mode.

[0043] Fig.10 The results of molecular level-related detection of Atractylodes macrocephala polysaccharide in the present invention in alleviating diarrhea caused by pyrotinib through cAMP / LKB1 / AMPK / CFTR. (A) ELISA was used to detect the serum cAMP content of rats in each group. (B) qRT-PCR was used to detect the expression of LKB1 gene in the intestinal tissue of rats in each group. (C) qRT-PCR was used to detect the expression of AMPK gene in the intestinal tissue of rats in each group. (D) qRT-PCR was used to detect the expression of CFTR gene in the intestinal tissue of rats in each group. (E) Western blot was used to detect the expression of CFTR, AMPKα, P-AMPK, LKB1 and P-LKB1 proteins in the intestinal tissue of rats in each group. (F) The expression of CFTR protein in the intestinal tissue of rats in each group was calculated according to the results of Western blot. (G) The expression of AMPKα protein in the intestinal tissue of rats in each group was calculated according to the results of Western blot. (H) The expression of P-AMPK protein in the intestinal tissue of rats in each group was calculated according to the results of Western blot. (I) The expression level of LKB1 protein in the intestinal tissue of rats in each group was calculated based on the results of Western blot. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates P > 0.05.

[0044] Fig.11The results of immunohistochemical analysis of the effect of Atractylodes macrocephala polysaccharide on the alleviation of pyrotinib-induced diarrhea through cAMP / LKB1 / AMPK / CFTR in the embodiments of the present invention. (A) The expression of P-LKB1 protein in the intestinal tissue of each group of rats was calculated based on the results of Western blot. (B) The distribution of CFTR, P-AMPK and LKB1 in the intestinal tissue of each group of rats was determined by immunohistochemistry. (C) The expression of CFTR protein was calculated based on the results of immunohistochemistry. (D) The expression of P-AMPK protein was calculated based on the results of immunohistochemistry. (E) The expression of LKB1 protein was calculated based on the results of immunohistochemistry. One-way ANOVA was used for statistical significance. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001; **** indicates P < 0.0001; ns indicates P > 0.05.

[0045] Fig.12 1 is the dose-effect curve of pyrotinib and Atractylodes macrocephala polysaccharide in the embodiment of the present invention. DETAILED DESCRIPTION

[0046] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0047] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0048] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0049] Example

[0050] 1. Materials and Methods

[0051] 1.1 Cell culture

[0052] T84 cells were purchased from HyCyte (TCH-C354) and cultured in DMEM / F12 medium supplemented with 5% fetal bovine serum (FBS, Gibco, 10099141) and 1% penicillin and streptomycin at 37°C in a 5% CO2 incubator. Lenti-X293T cells were donated by Professor Wei Jian's team at Shandong University and cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS, Gibco, 10099141) and 1% penicillin and streptomycin at 37°C in a 5% CO2 incubator.

[0053] 1.2 Preparation of Atractylodes macrocephala polysaccharide

[0054] After washing, the Atractylodes macrocephala was dried and crushed into powder. 10 times the amount of double distilled water was added and refluxed at 90°C for 2 hours. The extraction was repeated 3 times. The filtrates were combined and concentrated by rotary evaporation to 1 / 4 of the combined filtrate volume to obtain a concentrate. 4 times the volume of ethanol was added to the concentrate for precipitation. After standing at 4°C for 24 hours, vacuum filtration was performed to obtain crude Atractylodes macrocephala polysaccharide. The crude Atractylodes macrocephala polysaccharide was dissolved in distilled water and mixed with Sevage reagent at a volume ratio of 1:3 for 25 minutes, centrifuged at 4000r / min for 10 minutes, and repeated several times until there was no protein precipitate. The supernatant was transferred to a dialysis bag (molecular weight cutoff 5 KDa) and dialyzed in double distilled water for 24 hours. The water was changed at 2 hours, 6 hours and 8 hours during the dialysis process. After the dialysis was completed, it was freeze-dried to obtain Atractylodes macrocephala polysaccharide.

[0055] 1.3 CCK-8 test

[0056] T84 cells were seeded in 96-well culture plates (1×10 4 / well), incubate overnight at 37°C in a 5% CO2 incubator. Add drugs to the cells inoculated the day before according to the set drug concentration gradient, and incubate at 37°C in a 5% CO2 incubator for 24 hours. Wash the treated cells twice with PBS and add 100ul DMEM / F12 complete medium. Add 10 μl CCK-8 solution to each well of the 96-well plate to be tested, and incubate at 37°C in a 5% CO2 incubator for 2 hours. Use an enzyme reader (Multiskan SkyHigh) to measure the OD value of each experimental well at 450nm to detect the cell activity of each group.

[0057] 1.4 Plasmid construction, transfection and transduction

[0058] Construct a CFTR knockdown (CFTR-KD) plasmid. The constructed plasmid was transformed into competent cells, evenly spread on a plate containing ampicillin, and then inverted and placed in a 37°C incubator overnight. The next day, colonies were selected from the plate for monoclonal culture, and the colony PCR identification bands were sent for sequencing. After correct sequencing, plasmid extraction was performed. Lenti-X293T cells were successfully transfected with the constructed CFTR knockdown plasmid and its empty control (CFTR-NC) plasmid, and viral supernatants were collected at 48 hours and 72 hours. The collected viral supernatant was used for transduction to successfully construct CFTR knockdown T84 cells. In addition, in order to obtain stable CFTR knockdown T84 cells, puromycin was used to select cells.

[0059] 1.5 RNA extraction and quantitative reverse transcription polymerase chain reaction (qRT-PCR)

[0060] Total RNA was extracted from cells and tissues using Trizol reagent, and the quality of the extracted RNA was tested using NanoDrop One. Genomic DNA contamination was removed and cDNA was obtained by reverse transcription. The qRT-PCR reaction system was prepared, the reaction program was set, and the fluorescence quantitative PCR instrument (QuantStudio 1) was used for analysis. The original Ct value was obtained based on the standard reaction curve and the result data, and 2 -△△Ct The primer sequences of each gene are shown in Table 1.

[0061] Table 1 Primer sequences

[0062]

[0063] 1.6 Western blotting

[0064] Proteins were extracted from fresh cells and frozen tissues, and protease inhibitors and phosphatase inhibitors were added. The samples were separated by 7.5% and 10% SDS-PAGE and transferred to PVDF membranes. The primary antibody was incubated overnight on a shaker at 4°C, and the immune reaction area was detected by enhanced chemiluminescence (ECL) detection reagent after incubation with the corresponding secondary antibody. The images were taken using a chemiluminescence imaging system (Tanon5200). Image J was used to analyze the grayscale of western blot images and quantify them.

[0065] The primary antibodies used for western blot included LKB1 antibody (Abclonal, A2122), P-LKB1 antibody (AbwaysTechnology, AY8180), AMPKα antibody (Santa Cruz Biotechnology, sc-74461), P-AMPK antibody (Abways Technology, CY5608), CFTR antibody (Santa Cruz Biotechnology, sc-376683), ZO-1 antibody (Affinity Biosciences, AF5145), E-Cadherin antibody (Abclonal, A20798), Occludin antibody (Abways Technology, CY5997), and β-actin antibody (Abways Technology, AB0035).

[0066] 1.7 Chloride ion fluorescent probe

[0067] Cell culture was performed according to the grouping, and the drug treatment was added after the cell morphology in the culture dish was stable and the growth was uniform. Culture was carried out in a 37°C, 5% CO2 incubator for 24 hours. After the cells in the culture dish were washed once with buffer, MQAE reagent (MedChemExpress, 162558-52-3) was added and cultured in a 37°C, 5% CO2 incubator for 50 minutes. After washing the cells in the culture dish 3 to 5 times with buffer, the images were collected using an intelligent fluorescence microscopy system (KEYENCE, BZ-X810). The collected images were analyzed for fluorescence quantification using Image J.

[0068] 1.8 Construction of animal models

[0069] Forty female SPF Wistar rats (Vital River) aged 4 to 6 weeks were purchased. After one week of adaptive feeding, they were randomly divided into 8 groups, with 5 rats in each group, namely the control group (equal amount of normal saline), the pyrotinib group (80 mg / kg / day), the low, medium and high dose groups of Atractylodes macrocephala polysaccharide (4, 8, 16 mg / kg / day), and the low, medium and high dose groups of pyrotinib + Atractylodes macrocephala polysaccharide. Rats were gavaged with pyrotinib (Hengrui Pharma, 6124230401cs) and Atractylodes macrocephala polysaccharide for 10 days. The low, medium and high dose groups of pyrotinib + Atractylodes macrocephala polysaccharide were given by gavage 30 minutes after the gavage of Atractylodes macrocephala polysaccharide. The rats were weighed and recorded before gavage every day, and the general condition and diarrhea of ​​the rats were observed and recorded. The rats were kept in an SPF animal breeding room with a temperature of 22±1℃ and a relative humidity of 50±1%. After 10 days, the rats were euthanized and tissue samples, including blood, intestine, kidney, and spleen, were collected. All experimental procedures on rats complied with the protocols of the Animal Ethics Committee of the Provincial Hospital Affiliated to Shandong First Medical University.

[0070] 1.9 Hematoxylin and eosin (H&E) staining

[0071] The tissue was fixed, dehydrated, and embedded in paraffin. After sectioning, the slides were placed in a 60°C oven overnight. After dewaxing, they were stained with hematoxylin solution, then separated and reversed, and finally stained with eosin solution. The sections were dehydrated in different concentrations of alcohol and xylene in turn, and finally sealed with neutral gum. The sections were scanned and images were collected, and the staining intensity was analyzed and quantified using Image J.

[0072] 1.10 Enzyme-linked immunosorbent assay

[0073] The cAMP level in the serum of rats in each group was measured and calculated using the cyclic adenosine monophosphate (cAMP) enzyme-linked immunosorbent assay kit (Elabscience, E-EL-0056) purchased from the company according to the product instructions. All standards and samples were tested in duplicate.

[0074] 1.11 Immunohistochemical staining and quantitative analysis

[0075] The tissue was fixed, dehydrated, and embedded in paraffin. After sectioning, the slides were placed in a 60°C oven overnight. After dewaxing, immersion in water, and antigen retrieval, immunohistochemical staining was performed using CFTR antibodies (Santa Cruz Biotechnology, sc-376683), P-AMPK (AbwaysTechnology, CY5608), and LKB1 antibodies (Abclonal, A2122). The sections were scanned and images were collected, and the staining intensity was analyzed and quantified using Image J.

[0076] 1.12 Statistical analysis

[0077] All data are expressed as mean ± standard deviation. One-way analysis of variance was used for comparison among multiple groups. Two-way analysis of variance was used for repeated measurement data. All statistical analyses were performed using GraphPad Prism 9.0 software. P values ​​< 0.05 were considered statistically significant.

[0078] 2. Results

[0079] 2.1 Atractylodes macrocephala polysaccharide inhibits CFTR-mediated chloride secretion in cells treated with pyrotinib

[0080] In order to explore the effects of pyrotinib and Atractylodes macrocephala polysaccharide on the chloride channel protein CFTR, we set up a drug concentration gradient to intervene in T84 cells. The CCK-8 test was used to detect the cell activity after drug intervention, and the relative cell activity was calculated. The dose-response curve was drawn with the logarithm of the drug concentration as the horizontal axis and the relative cell activity as the vertical axis to calculate the half-inhibitory concentration (IC 50 ) is 0.01428mM, and the IC 50 27.98mg / ml( Figure 1 A to D in the middle). Combining the effects of the two drugs on cell activity, we finally chose 0.08mM dose of pyrotinib and 10mg / ml dose of Atractylodes macrocephala polysaccharide to treat T84 cells. A control group, a pyrotinib group, an Atractylodes macrocephala polysaccharide group, and a pyrotinib + Atractylodes macrocephala polysaccharide group were set up. Cells were collected 24 hours after drug intervention, and RNA and protein were extracted. The results of qRT-PCR showed that pyrotinib significantly increased CFTR gene expression at the transcriptional level (P<0.0001), and Atractylodes macrocephala polysaccharide could inhibit this process (P<0.01) ( Figure 1 Middle E). Western blot results showed that pyrotinib increased CFTR protein expression at the protein level (P<0.05), and Atractylodes macrocephala polysaccharide restored protein expression to normal levels (P<0.05) ( Figure 1 F and G).

[0081] To elucidate the effect of pyrotinib on chloride secretion of CFTR, we constructed stable CFTR knockdown (CFTR-KD) cells and negative control (CFTR-NC) cells in T84 cell line using CRISPR / Cas9 plasmid. Knockdown of CFTR gene resulted in decreased transcription level and protein expression, which were detected by qRT-PCR and Western blot, respectively ( Figure 1 In H and I, Figure 2Middle A). CFTR-KD cells and CFTR-NC cells were set up with a control group, a pyrotinib group, a white atractylodes polysaccharide group, and a pyrotinib + white atractylodes polysaccharide group, respectively. After 24 hours of treatment, the MQAE test was used to evaluate the chloride ion secretion. The results showed that pyrotinib promoted the chloride ion secretion in cells (P<0.0001), and white atractylodes polysaccharide inhibited the abnormal chloride ion secretion caused by pyrotinib (P<0.0001). The chloride ion secretion in CFTR knockdown cells treated with pyrotinib was significantly reduced compared with that in the negative control cells (P<0.0001), confirming that pyrotinib promotes chloride ion secretion by activating CFTR. In CFTR knockdown cells, the intervention of white atractylodes polysaccharide could not alleviate the chloride ion secretion caused by pyrotinib (P>0.05), verifying that white atractylodes polysaccharide reduces chloride ion secretion through CFTR ( Figure 2 B and C).

[0082] 2.2 Atractylodes macrocephala polysaccharide alleviates pyrotinib-induced diarrhea in rats

[0083] To investigate the alleviating effect of Atractylodes macrocephala polysaccharide on pyrotinib-induced diarrhea, we intragastrically administered Atractylodes macrocephala polysaccharide to Wistar rats treated with 80 mg / kg of pyrotinib at doses of 4 mg / kg (low dose), 8 mg / kg (medium dose), and 16 mg / kg (high dose) ( Figure 3 Diarrhea is graded according to the characteristics of rat feces: dry and hard granular feces are grade 0 diarrhea, formed soft feces are grade 1 diarrhea, porridge-like feces are grade 2 diarrhea, and watery feces are grade 3 diarrhea ( Figure 3 Rats in the control group and the low, medium, and high dose Atractylodes polysaccharide groups did not develop diarrhea. Some rats in the pyrotinib group developed diarrhea on the second day, all developed diarrhea on the fifth day, and the diarrhea reached grade 2-3 on the tenth day. The time of diarrhea in the pyrotinib + Atractylodes polysaccharide group was delayed, and the degree of diarrhea was reduced. Among them, some rats in the pyrotinib + Atractylodes polysaccharide (medium dose) group developed diarrhea on the sixth day, all developed diarrhea on the eighth day, and the degree of diarrhea was significantly reduced, and the diarrhea reached grade 1-2 on the tenth day ( Figure 3 (C). The diarrhea score was calculated based on the diarrhea rate. Neither the control group nor the Atractylodes polysaccharide group had diarrhea, and the diarrhea score was 0. Compared with the control group, the diarrhea score in the pyrotinib group increased on the third day (P<0.01). As the experiment progressed, the diarrhea score continued to increase, reaching the highest score in the pyrotinib group on the tenth day (P<0.0001). Compared with the pyrotinib group, the diarrhea score in the pyrotinib + Atractylodes polysaccharide group decreased to varying degrees, among which the pyrotinib + Atractylodes polysaccharide (medium dose) group was the most significant (P<0.0001) ( Figure 4 Therefore, Atractylodes macrocephala polysaccharide has a relieving effect in the pyrotinib-induced diarrhea rat model, and 8 mg / kg (medium dose) of Atractylodes macrocephala polysaccharide has the most significant therapeutic effect. This dose group of rats was used in subsequent studies.

[0084] Next, we calculated the diarrhea index. Neither the control group nor the Atractylodes macrocephala polysaccharide group had diarrhea, and the diarrhea index was 0. Compared with the control group, the diarrhea index in the pyrotinib group was significantly increased (P<0.0001). Compared with the pyrotinib group, the diarrhea index in the pyrotinib + Atractylodes macrocephala polysaccharide group was significantly reduced (P<0.0001) ( Figure 4 Middle B). The body weight of rats in each group was measured every 2 days, and the changes in body weight of rats in each group were statistically obtained. The body weight of rats in the control group and the Atractylodes macrocephala polysaccharide group increased steadily, and there was no significant difference between the two groups (P>0.05). The body weight of rats in the pyrotinib group decreased overall, which was significantly different from the control group (P<0.0001). The body weight of rats in the pyrotinib + Atractylodes macrocephala polysaccharide group gradually stabilized after a steady increase, which was significantly different from the pyrotinib group (P<0.001) ( Figure 4 Middle C).

[0085] 2.3 Atractylodes macrocephala polysaccharide restores intestinal barrier function in rats with pyrotinib-induced diarrhea

[0086] In order to evaluate the effects of pyrotinib and Atractylodes macrocephala polysaccharide on various organs of rats, we performed hematoxylin and eosin (H&E) staining on the small intestine, large intestine, kidney and spleen of rats. The results showed that the small intestinal mucosa of rats in the pyrotinib group was significantly damaged, the small intestinal villi were significantly shortened, and some villi fell off. In contrast, the Atractylodes macrocephala polysaccharide group showed a significant repair effect. The length of the small intestinal villi of rats was significantly restored, the villi structure tended to be normal, and the integrity of the small intestinal villi was restored. There was no significant difference in the structure of the large intestine of rats in each group ( Figure 5 In addition, we also evaluated the effects of the drug on the kidney and spleen. H&E staining showed that the pathological changes in the rats in the pyrotinib group were not obvious, and there was no difference in the renal index of the rats in each group (P>0.05) ( Figure 5 Middle B). H&E staining showed that the proportion of spleen white pulp in the rats in the Atractylodes macrocephala polysaccharide group increased, and the spleen index of the rats in the Atractylodes macrocephala polysaccharide group was significantly increased compared with the rats in the control group and the pyrotinib group (P<0.01) ( Figure 5 These results indicate that the dose of Atractylodes macrocephala polysaccharide (8 mg / kg) used in the experiment is within the controllable toxicity range and has potential application value in preventing and treating intestinal barrier function damage caused by pyrotinib-induced diarrhea.

[0087] Consistent with this, Western blot showed that the expression of tight junction proteins in rats treated with pyrotinib was reduced, including ZO-1 (a key tight junction protein, as a marker of intestinal barrier integrity), E-Cadherin (a cell adhesion molecule in the plasma membrane), and Occludin (a tight junction protein that acts as a biological barrier to prevent the passage of harmful substances). Western blot quantitative analysis results showed that pyrotinib significantly reduced the levels of ZO-1, E-Cadherin and Occludin (P < 0.05), and Atractylodes macrocephala polysaccharide could significantly restore this change (P < 0.01) ( Figure 6 These findings suggest that Atractylodes macrocephala polysaccharides may contribute to the restoration of intestinal barrier function.

[0088] 2.4 Pyrotinib and Atractylodes macrocephala polysaccharide alter the intestinal microbiota of rats

[0089] To determine how pyrotinib and Atractylodes macrocephala polysaccharide affect the composition of the intestinal microbiota, we performed 16S rRNA sequencing on fecal samples from four groups of rat models. In order to comprehensively evaluate the alpha diversity of the intestinal microbial community, Chao1 and Observed species indices were used to characterize richness, Shannon and Simpson indices were used to characterize diversity, Faith's PD index was used to characterize evolution-based diversity, Pielou's evenness index was used to characterize uniformity, and Good's coverage index was used to characterize coverage. The results showed that the intestinal microbiota of rats in each group was high in richness, rich in diversity, high in coverage, and evenly distributed ( Figure 7 Middle A). There was no significant difference in the Alpha diversity of the intestinal flora composition between different groups. Principal coordinates analysis (PCoA) based on Jaccard and further PERMANOVA analysis showed that the samples within each group were similar. There was no significant difference in the colony composition between the pyrotinib group and the Atractylodes macrocephala polysaccharide group and the control group (P>0.05). There was a significant difference in the flora composition between the control group and the pyrotinib group and the pyrotinib + Atractylodes macrocephala polysaccharide group (P<0.05). The Beta diversity of the intestinal flora suggests that pyrotinib and Atractylodes macrocephala polysaccharide can jointly change the composition of the intestinal flora ( Figure 7 Middle B).

[0090] To study the similarities and differences between different groups, a Venn diagram was used to perform community analysis ( Figure 8 In order to further compare the differences in species composition between samples and show the distribution trend of species abundance in each group, the abundance data of the top 50 intestinal microorganisms in terms of average abundance were used to draw a cluster heat map ( Figure 8(B). LEfSe analysis was used to perform differential analysis at all taxonomic levels simultaneously to find marker species between groups. The intestinal species composition of the control group and the pyrotinib group was significantly different. Prevotellaceae / Prevotella, Desulfovibrionales / Desulfovibrionaceae / Desulfovibrio, and Deltaproteobacteria were the marker species of the control group, while Clostridiaceae, Clostridium, Erysipelotrichales / Erysipelotrichaceae, and Erysipelotrichi were the marker species of the pyrotinib group ( Figure 8 These results suggest that pyrotinib may cause intestinal flora imbalance and diarrhea by increasing the levels of Clostridium difficile and Erysipelothrix. By counting the abundance of secondary functional pathways in the KEGG Pathway Database, we found that changes in the flora were closely related to pathways related to metabolism, gene information processing, and cell processes.

[0091] 2.5 Atractylodes macrocephala polysaccharide regulates intestinal metabolites in rats with pyrotinib-induced diarrhea

[0092] Given that metabolites secreted by the intestinal microbiota play an important role in regulating health and disease, we then performed metabolite analysis on the fecal samples of the four groups of rats. Based on principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) in positive and negative ion modes, the metabolomes of the fecal samples of the four groups of rats were separated ( Fig. 9 A to D in the figure). Bidirectional clustering of samples and differential metabolites revealed significant changes in multiple metabolites in the feces of rats in the control group, pyrotinib group, Atractylodes macrocephala polysaccharide group, and pyrotinib + Atractylodes macrocephala polysaccharide group. KEGG pathway enrichment analysis of differential metabolites showed that drug effects affected tumor carbon center metabolism, mineral absorption, AMPK signaling pathway, and aldosterone synthesis and secretion.

[0093] 2.6 Atractylodes macrocephala polysaccharide alleviates pyrotinib-induced diarrhea via cAMP / LKB1 / AMPK / CFTR

[0094] KEGG pathway enrichment analysis of differential metabolites suggested that Atractylodes macrocephala polysaccharide may alleviate pyrotinib-induced diarrhea through the AMPK signaling pathway. In order to gain a deeper understanding of its mechanism of action, we used ELISA to detect the cAMP content in the serum of each group of rats, used qRT-PCR to detect the expression of LKB1, AMPKα and CFTR in the intestinal tissue of each group of rats, and used Western blot to detect the expression of LKB1, P-LKB1, AMPKα, P-AMPK and CFTR in the intestinal tissue of each group of rats. We observed that pyrotinib significantly increased the cAMP content in rat serum (P < 0.01), and Atractylodes macrocephala polysaccharide reversed this change (P < 0.05) ( Fig.10 Middle A). qRT-PCR results showed that pyrotinib significantly reduced LKB1 gene expression (P < 0.0001) and increased CFTR gene expression (P < 0.0001), and Atractylodes macrocephala polysaccharide could restore these changes to varying degrees ( Fig.10 There was no significant difference in the transcription level of AMPK (P>0.05) ( Fig.10 C), we think it may be a change in phosphorylation level. Western blot results showed that pyrotinib reduced LKB1 protein expression in rat intestinal tissue (P < 0.0001), reduced P-AMPK protein expression (P < 0.001), and increased CFTR protein expression (P < 0.0001), while Atractylodes macrocephala polysaccharide could restore it to a level close to that of the control group ( Fig.10 Middle E to I, Fig.11 A). It can be seen that Atractylodes macrocephala polysaccharide relieves pyrotinib-induced diarrhea by reducing cAMP content, activating the LKB1 / P-AMPK signaling pathway, and reducing CFTR protein expression. Consistent with this, immunohistochemical staining and its quantitative analysis showed that Atractylodes macrocephala polysaccharide could reverse the decreased LKB1 expression, decreased P-AMPK expression, and increased CFTR expression caused by pyrotinib ( Fig.11 Collectively, these data suggest that the effect of Atractylodes macrocephala polysaccharide on pyrotinib-induced diarrhea is mediated by the cAMP / LKB1 / AMPK / CFTR signaling pathway.

[0095] 2.7 Atractylodes macrocephala polysaccharide does not affect the anti-tumor efficacy of pyrotinib

[0096] To explore the drug safety of Atractylodes macrocephala polysaccharide, we used CCK-8 to measure and calculate the cell death rate, and used CompuSyn software to obtain the dose-effect curve of Atractylodes macrocephala polysaccharide and pyrotinib, and obtained the combination index (CI) of the actual experimental point. The results showed that the combined use of Atractylodes macrocephala polysaccharide did not affect the anti-tumor effect of pyrotinib, and the CI suggested a synergistic or additive effect between the two drugs ( Fig.12 and Table 2).

[0097] Table 2 Combination index of drugs

[0098]

[0099] CI value: <0.1 very strong synergistic effect; 0.1-0.3 strong synergistic effect; 0.3-0.7 synergistic effect; 0.7-0.85 moderate synergistic effect; 0.85-0.9 mild synergistic effect; 0.9-1.1 additive effect; 1.1-1.2 mild antagonistic effect; 1.2-1.45 moderate antagonistic effect; 1.45-3.3 antagonistic effect; 3.3-10 strong antagonistic effect; >10 very strong antagonistic effect.

[0100] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of Atractylodes macrocephala polysaccharide in the preparation of drugs for preventing and treating diarrhea caused by pyrotinib; The preparation method of the Atractylodes macrocephala polysaccharide is as follows: the Atractylodes macrocephala is washed, dried and crushed into powder, 10 times the amount of double distilled water is added, and the mixture is refluxed and extracted at 90° C. for 2 hours, extracted 3 times, the filtrates are combined, and concentrated by rotary evaporation to 1 / 4 of the volume of the combined filtrate to obtain a concentrated solution, and 4 times the volume of ethanol is added to the concentrated solution for precipitation treatment; after standing at 4° C. for 24 hours, vacuum filtration is performed to obtain crude Atractylodes macrocephala polysaccharide; the crude Atractylodes macrocephala polysaccharide is dissolved in distilled water and mixed with Sevage reagent at a volume ratio of 1:3 for 25 minutes, centrifuged at 4000 r / min for 10 minutes, and repeated multiple times until there is no protein precipitate; the supernatant is transferred to a dialysis bag and dialyzed in double distilled water for 24 hours, the molecular weight cutoff of the dialysis bag is 5 KDa, and the water is changed at 2 hours, 6 hours and 8 hours during the dialysis process; after the dialysis is completed, freeze-drying is performed to obtain Atractylodes macrocephala polysaccharide.

2. The use according to claim 1, characterized in that: The method for preventing and treating diarrhea caused by pyrotinib is specifically characterized in that while preventing and / or treating diarrhea caused by pyrotinib, the anti-tumor effect of pyrotinib is not affected.

3. The use according to claim 1, characterized in that: The prevention and treatment of diarrhea caused by Pyrotinib is manifested as: (a1) inhibiting chloride ion secretion caused by pyrotinib; (a2) Promote the recovery of intestinal barrier function damage caused by pyrotinib-induced diarrhea; (a3) regulating the intestinal flora imbalance caused by pyrotinib; (a4) Regulate the intestinal metabolite disorder caused by pyrotinib.

4. The use according to claim 3, characterized in that: The (a1) specifically includes: Atractylodes macrocephala polysaccharide inhibits the expression of chloride ion channel protein CFTR.

5. The use according to claim 3, characterized in that: The (a3) ​​is specifically: Atractylodes macrocephala polysaccharide reduces the increase of Clostridium difficile and Erysipelothrix rhizogenes caused by pyrotinib.

6. Application of Atractylodes macrocephala polysaccharide combined with pyrotinib in the preparation of anti-tumor drugs; the dosage ratio of Atractylodes macrocephala polysaccharide to pyrotinib is 2.5 mg / ml to 0.001 mM; The preparation method of the Atractylodes macrocephala polysaccharide is as follows: the Atractylodes macrocephala is washed, dried and crushed into powder, 10 times the amount of double distilled water is added, and the mixture is refluxed and extracted at 90° C. for 2 hours, extracted 3 times, the filtrates are combined, and concentrated by rotary evaporation to 1 / 4 of the volume of the combined filtrate to obtain a concentrated solution, and 4 times the volume of ethanol is added to the concentrated solution for precipitation treatment; after standing at 4° C. for 24 hours, vacuum filtration is performed to obtain crude Atractylodes macrocephala polysaccharide; the crude Atractylodes macrocephala polysaccharide is dissolved in distilled water and mixed with Sevage reagent at a volume ratio of 1:3 for 25 minutes, centrifuged at 4000 r / min for 10 minutes, and repeated multiple times until there is no protein precipitate; the supernatant is transferred to a dialysis bag and dialyzed in double distilled water for 24 hours, the molecular weight cutoff of the dialysis bag is 5 KDa, and the water is changed at 2 hours, 6 hours and 8 hours during the dialysis process; after the dialysis is completed, freeze-drying is performed to obtain Atractylodes macrocephala polysaccharide.

7. The use according to claim 6, characterized in that The anti-tumor drug specifically exhibits the anti-tumor effect of pyrotinib and prevents and / or treats diarrhea side effects mediated by pyrotinib.

8. An anti-tumor drug, characterized in that: The active ingredients of the anti-tumor drug are Atractylodes macrocephala polysaccharide and pyrotinib; The dosage ratio of Atractylodes macrocephala polysaccharide to Pyrotinib is 2.5 mg / ml to 0.001 mM; The preparation method of the Atractylodes macrocephala polysaccharide is as follows: the Atractylodes macrocephala is washed, dried and crushed into powder, 10 times the amount of double distilled water is added, and the mixture is refluxed and extracted at 90° C. for 2 hours, extracted 3 times, the filtrates are combined, and concentrated by rotary evaporation to 1 / 4 of the volume of the combined filtrate to obtain a concentrated solution, and 4 times the volume of ethanol is added to the concentrated solution for precipitation treatment; after standing at 4° C. for 24 hours, vacuum filtration is performed to obtain crude Atractylodes macrocephala polysaccharide; the crude Atractylodes macrocephala polysaccharide is dissolved in distilled water and mixed with Sevage reagent at a volume ratio of 1:3 for 25 minutes, centrifuged at 4000 r / min for 10 minutes, and repeated multiple times until there is no protein precipitate; the supernatant is transferred to a dialysis bag and dialyzed in double distilled water for 24 hours, the molecular weight cutoff of the dialysis bag is 5 KDa, and the water is changed at 2 hours, 6 hours and 8 hours during the dialysis process; after the dialysis is completed, freeze-drying is performed to obtain Atractylodes macrocephala polysaccharide.

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