Weissella sinus ZMC030 and application of exopolysaccharide thereof in prevention and treatment of toxic and side effects caused by chemotherapeutic drugs

Abstract

The invention belongs to the technical field of biological medicines, and relates to application of Weissella sinus ZMC030 and exopolysaccharides thereof in prevention and treatment of toxic and side effects caused by chemotherapeutic drugs. The invention discovers that the Weissella cibaria ZMC030 exopolysaccharide can prevent, relieve or / and treat toxic and side effects caused by chemotherapeutic drugs for the first time; wherein the toxic and side effects of the heart comprise heart injury, myocardial cell apoptosis, cardiac fibrosis, heart tissue inflammation and the like; the intestinal toxic and side effects comprise intestinal injury, intestinal tissue structure disorder or / and intestinal mucosal barrier disorder, intestinal mucosal barrier permeability increase and intestinal flora disorder. Therefore, the Weissella cibaricola ZMC030 and the exopolysaccharide secreted by the Weissella cibaricola ZMC030 can be used for preparing products for preventing, relieving or / and treating cardiac toxic and side effects and intestinal toxic and side effects caused by chemotherapy drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of *Westernella esculenta* ZMC030 and its extracellular polysaccharides in preventing and treating the toxic side effects caused by chemotherapy drugs. Background Technology

[0002] Doxorubicin (Dox) is a widely used, broad-spectrum, and highly effective anthracycline chemotherapy drug in clinical practice, commonly used to treat various cancers such as breast cancer, ovarian cancer, prostate cancer, lung cancer, leukemia, liver cancer, and malignant lymphoma. However, while treating cancer, Dox can accumulate in other organs, causing serious toxic side effects, particularly dose-dependent and irreversible cardiotoxicity, ultimately leading to myocardial damage, arrhythmia, and heart failure. This greatly limits its clinical application, and Dox is listed as a drug with a risk of causing heart failure. Extensive basic research has explored ways to alleviate Dox's cardiotoxicity, achieving some progress. However, to date, effective prevention and treatment strategies are still lacking in clinical application. Currently, dexpromethazine is the only clinically approved cardioprotective agent against anthracycline toxicity. Dexpromethazine can prevent the reduction in resting left ventricular ejection fraction caused by anthracycline chemotherapy drugs, reducing the incidence of heart failure. However, dexpromethazine can exacerbate the myelosuppressive effect of Dox, affecting its antitumor efficacy and potentially causing secondary malignant tumors. Therefore, it is urgent to develop new treatment options and drugs with translational potential and value for Dox cardiotoxicity.

[0003] Besides causing cardiac damage, Dox also induces intestinal mucosal inflammation, disrupting intestinal barrier homeostasis, and intestinal damage precedes cardiac damage. Furthermore, Dox causes gut microbiota dysbiosis, leading to decreased gut microbiota diversity and an increase in pro-inflammatory bacteria, thereby exacerbating mucosal inflammation and increasing the amount of toxins entering the internal circulation. Studies have shown that Dox promotes increased TLR4 expression in macrophages, and the intestinal barrier disruption caused by Dox leads to LPS leakage into the circulation (hypoendotoxemia). Given that TLR4 is the receptor for LPS, this further enhances the sensitivity of macrophages to LPS, exacerbating cardiac inflammation and damage. All of these studies demonstrate that intestinal barrier damage is a crucial factor promoting the development and progression of cardiac injury.

[0004] Extracellular polysaccharides (EPS) are large carbohydrate molecules produced during the growth and metabolism of microorganisms. They are the main active substances for microbial physiological functions and possess natural advantages such as biodegradability, low or non-toxicity, and good biocompatibility. EPS can exert functions such as anti-inflammation, anti-oxidation, anti-tumor activity, and immune regulation. However, whether bacterial EPS can alleviate Dox cardiotoxicity remains unknown. *Weissella cibaria*, a type of lactic acid bacteria, is widely found in saliva, breast milk, the human gastrointestinal tract, and traditional fermented foods. *Weissella cibaria* produces high levels of EPS and is currently mainly used as a thickener, stabilizer, and gelling agent in food processing, cosmetics, and pharmaceuticals. However, no studies have reported the application of *Weissella cibaria* EPS in alleviating Dox toxicity. Summary of the Invention

[0005] In view of the problems and deficiencies in the existing technology, the purpose of this invention is to provide the application of Weissella esculenta ZMC030 and its extracellular polysaccharides in the prevention and treatment of toxic side effects caused by chemotherapy drugs.

[0006] To achieve the objectives of this invention, the technical solution adopted is as follows:

[0007] The first aspect of this invention provides the use of *Weissella cibaria*, a culture of *Weissella cibaria*, or extracellular polysaccharides secreted by *Weissella cibaria* in the preparation of products for preventing, alleviating, and / or treating the toxic side effects caused by chemotherapy drugs; wherein the *Weissella cibaria* is *Weissella cibaria* ZMC030, the accession number of which is CCTCC NO:M 20241261, the depository institution is the China Center for Type Culture Collection (CCTCC), located at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, China, China Center for Type Culture Collection, Wuhan University; the deposit date is June 17, 2024. This *Weissella cibaria* ZMC030 has been disclosed in patent application number 202411291801.8, entitled "Application of *Weissella cibaria* ZMC030 and its extracellular polysaccharides in the prevention and treatment of inflammatory bowel disease".

[0008] According to the above application, preferably, the toxic side effects caused by the chemotherapy drugs include cardiotoxic side effects and gastrointestinal side effects.

[0009] According to the above application, preferably, the cardiotoxic side effects are at least one of the following:

[0010] (A1) Chemotherapy-induced cardiac damage;

[0011] (A2) Chemotherapy-induced cardiomyocyte apoptosis;

[0012] (A3) Chemotherapy-induced cardiac fibrosis;

[0013] (A4) Chemotherapy-induced inflammation of cardiac tissue;

[0014] (A5) Increased cardiac oxidative stress induced by chemotherapy drugs;

[0015] (A6) Chemotherapy drugs induce decreased activity of cardiac antioxidant enzymes;

[0016] (A7) Chemotherapy drugs induce an increase in lipopolysaccharide (LPS) levels in cardiac tissue.

[0017] According to the above application, preferably, the intestinal toxicity side effects are at least one of the following:

[0018] (B1) Chemotherapy-induced intestinal damage;

[0019] (B2) Chemotherapy-induced intestinal tissue structural disorder;

[0020] (B3) Chemotherapy drugs induce the proliferation of LPS-producing pro-inflammatory bacteria in the gut;

[0021] (B4) Chemotherapy-induced intestinal mucosal barrier disorder;

[0022] (B5) Chemotherapy-induced gut microbiota dysbiosis.

[0023] According to the above application, preferably, the chemotherapy drug is an anthracycline chemotherapy drug. More preferably, the anthracycline chemotherapy drug is DOX.

[0024] According to the above application, preferably, the culture of Weissella stolonifera includes a fermentation culture broth of Weissella stolonifera, a supernatant of the fermentation culture broth of Weissella stolonifera, and a dried powder of the fermentation culture broth of Weissella stolonifera.

[0025] According to the above application, preferably, the extracellular polysaccharide is obtained by fermenting and culturing the *Westernella esculenta* to obtain a fermentation broth, and then separating and purifying the fermentation broth.

[0026] According to the above application, preferably, the preparation method of the extracellular polysaccharide is as follows: *Westernella esculenta* is inoculated into a culture medium for fermentation to obtain a fermentation broth. The fermentation broth is then subjected to cell removal and protein removal treatment to obtain a supernatant. The supernatant is subjected to alcohol precipitation, and the precipitate is collected. The precipitate is then dialyzed to remove impurities and freeze-dried to obtain the extracellular polysaccharide. The extracellular polysaccharide is further purified by column chromatography using a DEAE-cellulose 52 column. The fraction containing the polysaccharide is dialyzed to remove impurities and freeze-dried to obtain purified extracellular polysaccharide.

[0027] According to the above application, preferably, the culture medium is MRS medium or mMRS medium (modified MRS medium); more preferably, the mMRS medium is composed of: diammonium citrate 5 g / L, sucrose 100 g / L, peptone 10 g / L, magnesium sulfate 0.2 g / L, Tween 80 1 ml / L, dipotassium hydrogen phosphate 2 g / L, beef extract 10 g / L, yeast extract 5 g / L, anhydrous sodium acetate 5 g / L, manganese sulfate 0.05 g / L, with the balance being water.

[0028] According to the above application, preferably, the fermentation culture temperature is 30℃~37℃; more preferably, the culture temperature is 30℃.

[0029] According to the above application, preferably, the dialysis is performed in distilled water, and the molecular weight cutoff for dialysis is 8000-14000 Da.

[0030] According to the above application, preferably, the alcohol precipitation treatment involves adding pre-cooled anhydrous ethanol to the supernatant, mixing it evenly, and then letting it stand at 4°C for 8 to 12 hours.

[0031] According to the above application, preferably, the specific operation of protein removal is as follows: trichloroacetic acid is added to the culture medium after removing the bacterial cells, mixed evenly to obtain a mixture, and the mixture is placed at 4°C for 8-12 hours, centrifuged, and the precipitate is removed. More preferably, the amount of trichloroacetic acid used is: the mass-volume percentage concentration of trichloroacetic acid in the mixture is 4%.

[0032] According to the above application, preferably, the specific operation for removing the bacterial cells is as follows: heating the fermentation broth at 100°C for 15 minutes, cooling to room temperature, and centrifuging to remove the bacterial cells. More preferably, the centrifugation conditions are: centrifuging at 12000g for 30 minutes at 4°C.

[0033] According to the above application, preferably, the crude extracellular polysaccharide lyophilized powder can be further purified by column chromatography using a DEAE-cellulose 52 column. During column chromatography purification, gradient elution is performed using deionized water or sodium chloride solutions (0.1M, 0.3M, and 0.5M) to obtain the polysaccharide-containing eluent. The collected eluent containing polysaccharides is then dialyzed with deionized water and freeze-dried to obtain the purified extracellular polysaccharide. More preferably, during column chromatography purification, 0.1M sodium chloride solution is used for elution.

[0034] According to the above applications, preferably, the product is a drug, food additive, or functional food.

[0035] Compared with the prior art, the positive and beneficial effects achieved by this invention are as follows:

[0036] (1) This invention is the first to discover that *Weissella cibaria* ZMC030 (W. cibaria ZMC030) and its secreted extracellular polysaccharides can reduce inflammation and oxidative stress levels in cardiac tissue, decrease cardiomyocyte apoptosis and fibrosis, and ultimately alleviate cardiac damage. Furthermore, it was found that *Weissella cibaria* ZMC030 and its secreted extracellular polysaccharides can alleviate chemotherapy-induced intestinal damage, intestinal tissue structural disorders, intestinal mucosal barrier disorders, inhibit the proliferation of LPS-producing pro-inflammatory bacteria in the intestine, and improve intestinal mucosal barrier permeability and intestinal flora imbalance. Therefore, *Weissella cibaria* ZMC030 and its secreted extracellular polysaccharides can be used to prepare products for the prevention, relief, and / or treatment of cardiotoxic and intestinal toxic side effects caused by chemotherapy drugs.

[0037] (2) The *Westernella esculenta* ZMC030 of this invention was screened from the intestines of healthy infants. Its metabolite, extracellular polysaccharide, is also a natural component produced during the metabolism of lactic acid bacteria. From the perspective of clinical application, compared with other drugs for treating cardiac and intestinal toxicity caused by chemotherapy drugs, *Westernella esculenta* ZMC030 and its extracellular polysaccharide are safer and have important clinical application value and huge social and economic benefits. Attached Figure Description

[0038] Figure 1 The figure shows the results of polysaccharide content detection in the eluent obtained by separation and purification of crude polysaccharide from W. cibaria ZMC030 using a DEAE-cellulose 52 chromatography column;

[0039] Figure 2 The figure shows the results of detecting the effect of EPS-2 polysaccharide on the body weight of Dox-treated mice;

[0040] Figure 3 The figure shows the results of detecting the effect of EPS-2 polysaccharide on the heart-to-tibia ratio in Dox-treated mice;

[0041] Figure 4 The results of detecting the effect of EPS-2 polysaccharide on cardiac function in Dox-treated mice are shown in the figure; where A is the cardiac ultrasound result, B is the ejection fraction (EF%) result, and C is the short-axis contractility (FS%) result.

[0042] Figure 5 The results show the effects of EPS-2 polysaccharide on Dox-treated mouse cardiac injury markers; where A is the NT-proBNP result, B is the CKMB result, and C is the cTnT result.

[0043] Figure 6The graph shows the detection results of EPS-2 polysaccharide on inflammatory factors in Dox-treated mouse cardiac tissue; where A is the IFN-γ result, B is the IL-1β result, C is the IL-6 result, D is the TNF-α result, and EPS+DOX represents the EPS-2 / Dox group.

[0044] Figure 7 The results of detecting the effects of EPS-2 polysaccharide on oxidative stress in the heart of Dox-treated mice are shown in Figure 1; where A is the ROS result and B is the MDA result.

[0045] Figure 8 The figure shows the results of detecting the activity of antioxidant enzymes in the heart of mice treated with Dox using EPS-2 polysaccharide; where A represents the relative activity of SOD enzyme, B represents the relative activity of CAT enzyme, C represents the relative activity of GPX4 enzyme, and EPS / DOX represents the EPS-2 / Dox group;

[0046] Figure 9 The figure shows the effect of EPS-2 polysaccharide on the expression levels of Nrf2 and KEAP1 in the hearts of Dox-treated mice; where A represents the expression level of Nrf2 and B represents the expression level of KEAP1.

[0047] Figure 10 The figure shows the results of detecting the effect of EPS-2 polysaccharide on cardiac fibrosis in Dox-treated mice;

[0048] Figure 11 The figure shows the results of detecting the effect of EPS-2 polysaccharide on apoptosis of Dox-treated mouse cardiomyocytes;

[0049] Figure 12 The images show the results of detecting the effect of EPS-2 polysaccharide on the pathological results of the small intestine in Dox-treated mice; where A is the HE staining result of the jejunum and B is the staining result of the ileum.

[0050] Figure 13 The image shows the detection results of the effect of EPS-2 polysaccharide on Claudin-1, a tight junction protein in the small intestine of Dox-treated mice; where A is the immunofluorescence staining result of Claudin-1 in the jejunum and B is the immunofluorescence staining result of Claudin-1 in the ileum.

[0051] Figure 14 The figure shows the results of detecting the effects of EPS-2 polysaccharide on the gut microbiota of Dox-treated mice; where A is the result of changes at the Proteobacteria phylum level, B is the result of changes at the Escherichia-Shigella genus level, C is the result of the correlation between Escherichia-Shigella genus and cardiac function-related indicators, and D is the result of changes in Escherichia coli. EPS / DOX represents the EPS-2 / Dox group.

[0052] Figure 15 Figure 1 shows the results of detecting the effect of EPS-2 polysaccharide on the growth and proliferation of Escherichia coli; Figure A shows the result of EPS-2 polysaccharide inhibiting the growth of E. coli T1; Figure B shows the result of EPS-2 polysaccharide inhibiting the growth of E. coli BL21.

[0053] Figure 16 The figure shows the results of detecting the effect of EPS-2 polysaccharide on LPS content in serum and heart of Dox-treated mice; where A represents LPS content in serum; B represents LPS content in heart; and EPS / DOX represents the EPS-2 / Dox group.

[0054] Figure 17 The images show the effects of extracellular polysaccharides secreted by different *Westernella esculenta* species on the heart and small intestine of Dox-treated mice. A represents echocardiography results, B represents ejection fraction (EF%), C represents short-axis contraction rate (FS%), D represents HE staining results of mouse ileum, and D represents HE staining results of mouse jejunum. Detailed Implementation

[0055] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0056] The following detailed description is exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, components, and / or combinations thereof.

[0057] Unless otherwise specified, the experimental methods in the following examples all employ conventional techniques in this technical field or follow the conditions recommended by the manufacturer; reagents or instruments whose manufacturers are not specified are all commercially available products.

[0058] In addition, the *W. cibaria* described in the following examples is *W. cibaria* ZMC030, with accession number CCTCC NO:M 20241261, accession date June 17, 2024, and deposited at the China Center for Type Culture Collection (CCTCC). This *W. cibaria* ZMC030 has been disclosed in the patent application number 202411291801.8, entitled "Application of *W. cibaria* ZMC030 and its extracellular polysaccharides in the prevention and treatment of inflammatory bowel disease".

[0059] Example 1: Obtaining extracellular polysaccharides from W. cibaria ZMC030

[0060] Thaw W. cibaria ZMC030 cryovials in a 37°C water bath. Using a sterile inoculating loop, take 1-2 drops of bacterial suspension and inoculate using the three-section streak method. Incubate at 37°C for 48 hours until single colonies are observed. Use a micropipette to pick up the obtained single colonies and inoculate them into 5 ml of liquid MRS medium. Incubate at 37°C for 24 hours until the suspension becomes turbid. Inoculate the bacterial suspension at a 1% (v / v) inoculation rate into mMRS liquid medium and incubate at 30°C for 48 hours to obtain the fermentation broth. Boil the fermentation broth in a 100°C water bath for 15 minutes to inactivate bacteria and enzymes, then cool to room temperature. Centrifuge the boiled broth at 12000g at 4°C for 30 minutes using a high-speed refrigerated centrifuge, discarding the bacterial cells and other precipitates to obtain the supernatant. Add 80% (m / v) trichloroacetic acid (TCA) to the supernatant until the final concentration of TCA is 4% (m / v) to remove proteins. After standing at 4°C for 12 hours, centrifuge at 4°C and 12000g for 30 minutes. Collect the supernatant. Repeat this step 2-3 times to remove as much protein as possible. Pre-cool anhydrous ethanol at 4°C. Add three times the volume (v / v) of cold anhydrous ethanol to the supernatant after removing proteins and other impurities. Let stand at 4°C for 12 hours (to precipitate extracellular polysaccharides). Centrifuge at 4°C and 12000g for 30 minutes to obtain the precipitate, which is the crude extracellular polysaccharide. Dissolve the crude extracellular polysaccharide precipitate in pure water and repeat the ethanol precipitation step 2-3 times. After alcohol precipitation, the extracellular polysaccharides obtained were reconstituted and then dialyzed in distilled water at 4°C (dialysis molecular weight cutoff of 8000–14000 Da) for 72 hours to remove impurities, with the water changed every 8 hours. The dialyzed polysaccharide solution was then freeze-dried under vacuum to obtain crude extracellular polysaccharide lyophilized powder, which was then weighed. Weighing revealed that after static cultivation at 30°C for 48 hours, the yield of crude extracellular polysaccharides in the fermentation broth of *W. cibaria* ZMC030 was 24.2 g / L, indicating a high extracellular polysaccharide yield in strain ZMC030.

[0061] The crude extracellular polysaccharide lyophilized powder was prepared into a 10 mg / mL crude polysaccharide aqueous solution, which was then loaded into a DEAE-cellulose 52 chromatography column (2.6 x 30 cm). Elution was performed with deionized water and sodium chloride solutions (0.1 M, 0.3 M, and 0.5 M), and the corresponding eluates (10 mL / tube) were collected. The sugar content in the eluates was determined using the phenol-sulfuric acid method. The experimental results are as follows: Figure 1 As shown.

[0062] Depend on Figure 1 It was found that the crude polysaccharide of *W. cibaria* ZMC030, after separation and purification by DEAE-cellulose 52 chromatography, showed elution peaks in deionized water and 0.1M sodium chloride eluent, respectively, and were named 030-EPS-1 and 030-EPS-2 (hereinafter referred to as EPS-2). 030-EPS-1 and EPS-2 were dialyzed against deionized water (dialysis cutoff of 8000-14000 Da), freeze-dried, and their yields were calculated. The polysaccharide yields in 030-EPS-1 and EPS-2 were found to be 8.6% and 53.2%, respectively. Given the low polysaccharide yield in the 030-EPS-1 eluent, the polysaccharide in the EPS-2 eluent (hereinafter referred to as EPS-2 polysaccharide) was selected for subsequent functional identification.

[0063] Example 2: Efficacy study of W. cibaria ZMC030 extracellular polysaccharide EPS-2 in preventing and treating DOX-induced cardiotoxicity.

[0064] 1. Grouping of experimental animals and construction of a mouse model of chronic myocardial toxicity

[0065] Six- to eight-week-old SPF-grade female C57BL / 6J mice (purchased from Beijing Spaford Laboratory Animal Co., Ltd.) were randomly divided into three groups: NC group, Dox group, and EPS-2 / Dox group.

[0066] The treatment methods for mice in the NC group, Dox group, and EPS-2 / Dox group were as follows:

[0067] Mice in the Dox group were administered saline via gavage (gavage volume was 10 μL of mouse body weight), with the saline volume being 10 μL of mouse body weight, administered every other day. After one week of continuous gavage intervention, Dox was injected via tail vein (injection method: 5 mg / kg of Dox was injected once a week for a total of 4 injections, with a cumulative Dox dose of 20 mg / kg) to establish a mouse model of chronic myocardial toxicity.

[0068] Mice in the EPS-2 / Dox group were administered the EPS-2 polysaccharide solution obtained in Example 1 by gavage (gavage volume was 10 μL of mouse body weight) every other day. The EPS-2 polysaccharide solution was prepared using physiological saline as a solvent and the concentration of the EPS-2 polysaccharide solution was 10 mg / ml. After one week of continuous gavage intervention, Dox was injected via tail vein (injection method: 5 mg / kg of Dox was injected once a week for a total of 4 injections, with a cumulative Dox dose of 20 mg / kg) to establish a mouse model of chronic myocardial toxicity.

[0069] All mice in the NC group were administered saline by gavage (gavage volume was 10 μL of mouse body weight), with the saline volume being 10 μL of mouse body weight. The gavage was performed every other day for one week, followed by a tail vein injection of the same dose of sterile saline.

[0070] The date of the first Dox injection in mice was recorded as Day 0. After the last Dox injection, the mice were fed for another 2 weeks, at which point the experiment ended (the experimental endpoint). Throughout the experiment, the mice's weight was monitored. At the experimental endpoint, echocardiography was performed on mice in each group, and then serum, heart, small intestine, and small intestine contents were collected for relevant experimental analysis.

[0071] 2. Effect of EPS-2 on body weight of Dox-treated mice

[0072] Starting from day 0, the weight of mice in each group was recorded every other day until the end of the experiment. Statistical results regarding mouse weight are provided as follows: Figure 2 As shown.

[0073] Depend on Figure 2 It was observed that, starting from the first Dox injection, the Dox group mice showed a decreasing body weight trend throughout the experiment compared to the NC group, while the EPS-2 / Dox group mice showed a significant increase in body weight compared to the Dox group. This indicates that EPS-2 polysaccharide intervention can alleviate the body weight loss induced by DOX treatment in mice.

[0074] 3. Effect of EPS-2 polysaccharide on the heart-to-tibia ratio in Dox-treated mice

[0075] After each group of mice was sacrificed, the weight of the mouse heart and the length of the tibia were measured, and the heart-to-tibia ratio was calculated. The results are as follows: Figure 3 As shown.

[0076] Depend on Figure 3 It was found that, compared with the NC group, the heart-to-tibia ratio of mice in the Dox group was significantly reduced; the heart-to-tibia ratio of mice in the EPS-2 / Dox group was significantly increased compared with the Dox group. This indicates that EPS-2 polysaccharide can significantly increase the heart-to-tibia ratio of Dox-treated mice.

[0077] 4. Effects of EPS-2 polysaccharide on cardiac function in Dox-treated mice

[0078] At the end of the experiment, echocardiography was performed on the cardiac function of mice in each group using a Vevo 3100 small animal ultrasound imaging system (visualsonic, Canada). The probe was 40 MHz with a resolution of 30 μm. The specific procedure for echocardiography was as follows: mice were first anesthetized with 1-1.5% isoflurane and placed in a supine position. Chest hair was removed with depilatory cream, and a suitable amount of coupling gel was applied. The ultrasound probe was placed at the level of the left anterior pectoral papilla muscle. Two-dimensional and m-type images were obtained from the parasternal short- and long-axis views. Heart rate (400-500 beats / min) and respiratory rate were monitored throughout the experiment. Data analysis was performed using Vevo Lab, analyzing the short-axis contraction percentage (FS%) and ejection fraction (EF%) to assess cardiac function. Results are as follows: Figure 4 As shown.

[0079] Depend on Figure 4 It was found that, compared with the NC group, the fractional shortening rate (FS%) and ejection fraction (EF%) of mice in the Dox group were significantly reduced; while the fractional shortening rate (FS%) and ejection fraction (EF%) of mice in the EPS-2 / Dox group were significantly increased compared with the Dox group. This indicates that EPS-2 polysaccharide intervention can significantly improve cardiac function in mice.

[0080] 5. Effects of EPS-2 polysaccharide on Dox-treated mouse cardiac injury markers

[0081] Blood was collected from the orbital venous plexus of mice in each group. After collection, the blood was allowed to stand at room temperature for 2 hours, centrifuged at 4000 rpm for 15 min, and the supernatant was collected to obtain serum. The levels of cTnT (manufacturer: Elabscience, catalog number E-EL-M1801), NT-proBNP (manufacturer: Elabscience, catalog number E-EL-M0834), and CKMB (manufacturer: Elabscience, catalog number E-EL-M0355) in the serum were detected and analyzed. The results are as follows: Figure 5 As shown.

[0082] Depend on Figure 5 It was found that Dox treatment increased the levels of cardiac injury markers CKMB, cTnT, and NT-proBNP in serum, while EPS-2 polysaccharide intervention significantly reduced the levels of CKMB, cTnT, and NT-proBNP.

[0083] The above results indicate that EPS-2 polysaccharide can effectively alleviate Dox-induced cardiac damage.

[0084] 6. Effects of EPS-2 polysaccharide on cardiac inflammatory factors in Dox-treated mice

[0085] At the experimental endpoint, mice in each group were sacrificed, and their heart tissue was collected. The heart tissue was cut into small pieces and added to pre-cooled PBS containing PMSF. After being processed using a cryo-homogenizer, the tissue was centrifuged at 12000×g for 15 min, and the supernatant was collected to obtain heart tissue lysate. The levels of inflammatory factors IL-1β (manufacturer: Elabscience, catalog number E-EL-M0037), IFN-γ (manufacturer: Elabscience, catalog number E-EL-M0048), IL-6 (manufacturer: Elabscience, catalog number E-EL-M0044), and TNF-α (manufacturer: Elabscience, catalog number E-EL-M3063) in the heart tissue lysate were detected. The results are as follows: Figure 6 As shown.

[0086] Depend on Figure 6 It was found that, compared with the NC group, Dox treatment caused an increase in the levels of pro-inflammatory factors IFN-γ, IL-1β, IL-6, and TNF-α in cardiac tissue, while EPS-2 polysaccharide intervention reversed this phenomenon. This result indicates that EPS-2 polysaccharide can reduce the Dox-induced inflammatory response in cardiac tissue.

[0087] 7. Effects of EPS-2 polysaccharide on oxidative stress in the heart of Dox-treated mice

[0088] At the experimental endpoint, after the mice in each group were sacrificed, heart tissue was collected. A portion of the heart tissue was cut into small pieces and added to pre-cooled PBS containing PMSF. After cryogenic homogenization, the tissue was centrifuged at 12000×g for 15 min, and the supernatant was collected to obtain heart tissue lysate. Changes in oxidative stress-related indicators ROS (manufacturer: Elabscience, catalog number E-BC-K138-F) and MDA (manufacturer: Elabscience, catalog number E-EL-0060) were detected. The results are as follows: Figure 7 As shown.

[0089] Depend on Figure 7 It was found that Dox treatment significantly increased the levels of ROS and MDA in cardiac tissue, while EPS-2 polysaccharide intervention decreased the levels of ROS and MDA in the heart. This indicates that EPS-2 polysaccharide can reduce the level of oxidative stress in the hearts of Dox-treated mice, thereby alleviating cardiac damage in mice.

[0090] 8. Effects of EPS-2 polysaccharide on the activity of antioxidant enzymes in the heart of Dox-treated mice

[0091] At the experimental endpoint, after the mice in each group were sacrificed, heart tissue was collected. A portion of the heart tissue was cut into small pieces and added to pre-cooled PBS. After cryogenic homogenization, the tissue was centrifuged at 12000×g for 15 min, and the supernatant was collected to obtain heart tissue lysate. The activities of antioxidant enzymes SOD (manufacturer: Elabscience, catalog number E-BC-K019-M), CAT (manufacturer: Elabscience, catalog number E-BC-K031-M), and GPX4 (manufacturer: Elabscience, catalog number E-BC-K883-M) in the heart tissue lysate were detected. The results are as follows: Figure 8 As shown.

[0092] Depend on Figure 8 It was found that Dox treatment significantly reduced the activities of SOD, CAT, and GPX4 antioxidant enzymes in cardiac tissue, while EPS-2 polysaccharide intervention increased the activities of SOD, CAT, and GPX4 antioxidant enzymes. This indicates that EPS-2 polysaccharide can enhance the antioxidant capacity of the heart in Dox-treated mice.

[0093] 9. Effects of EPS-2 polysaccharide on the expression levels of Nrf2 and KEAP1 in the hearts of Dox-treated mice

[0094] At the end of the experiment, the heart tissue of mice in each group was perfused with pre-cooled sterile saline to flush out residual blood. After removing the heart, the circumferentially sectioned heart tissue was immediately fixed in 4% paraformaldehyde. The paraformaldehyde-fixed heart tissue was then prepared into paraffin blocks and sectioned for Nrf2 and KEAP1 immunofluorescence detection. Results are as follows: Figure 9 As shown.

[0095] Depend on Figure 9 It was found that Dox significantly reduced the expression level of Nrf2 and increased the expression level of KEAP1 in cardiac tissue. However, EPS-2 polysaccharide intervention increased the expression level of Nrf2 and decreased the expression level of KEAP1 in cardiac tissue. This indicates that EPS-2 polysaccharide affects the antioxidant capacity of mouse heart by regulating the Nrf2 / KEAP1 signaling pathway.

[0096] 10. Effects of EPS-2 polysaccharide on cardiac fibrosis in Dox-treated mice

[0097] At the end of the experiment, the heart tissue of each group of mice was perfused with pre-cooled sterile saline to flush out residual blood. After removing the heart, the circumferentially sectioned heart tissue was immediately fixed in 4% paraformaldehyde. The paraformaldehyde-fixed heart tissue was then prepared into paraffin blocks, sectioned, and stained with Masson-trichrome. The results are as follows: Figure 10 As shown.

[0098] Depend on Figure 10 It is known that Dox caused cardiac fibrosis in mice, while EPS-2 polysaccharide intervention could significantly reduce the level of cardiac fibrosis.

[0099] 11. Effects of EPS-2 polysaccharide on Dox-treated mouse cardiomyocyte apoptosis

[0100] At the end of the experiment, the heart tissue of each group of mice was perfused with pre-cooled sterile saline to flush out residual blood. After removing the heart, the circumferentially sectioned heart tissue was immediately fixed in 4% paraformaldehyde. The paraformaldehyde-fixed heart tissue was then prepared into paraffin blocks, sectioned, and stained with TUNEL. The results are as follows: Figure 11 As shown.

[0101] Depend on Figure 11 The results showed that the TUNEL positivity rate of Dox-treated mouse cardiomyocytes increased, indicating that Dox induced cardiomyocyte apoptosis. The TUNEL positivity rate of EPS-2 polysaccharide-treated mouse cardiomyocytes decreased, indicating that EPS-2 polysaccharide can effectively alleviate Dox-induced apoptosis in mouse cardiomyocytes.

[0102] 12. Effects of EPS-2 polysaccharide on pathological results of small intestine in Dox-treated mice

[0103] At the end of the experiment, small intestinal tissue from each group of mice was collected, and the contents were washed with pre-cooled sterile saline. Ileum and jejunum tissues, approximately 0.5 cm in length, were fixed in 4% paraformaldehyde. The paraformaldehyde-fixed ileum and jejunum tissues were prepared into paraffin blocks, sectioned, and then stained with hematoxylin and eosin (HE). The results are as follows: Figure 12 As shown.

[0104] Depend on Figure 12 It can be seen that the small intestinal tissue structure of Dox group mice was disordered and the villi were shortened, indicating that Dox caused damage to the small intestinal structure; after EPS-2 polysaccharide intervention, the small intestinal villi of mice grew significantly and the structure tended to be normal, indicating that EPS-2 polysaccharide can effectively alleviate Dox-induced small intestinal structural disorder in mice.

[0105] 13. Effects of EPS-2 polysaccharide on Dox-treated mouse small intestinal barrier tight junction protein

[0106] At the end of the experiment, small intestinal tissue from mice in each group was collected, and the contents were washed with pre-cooled sterile saline. Ileal and jejunal tissue sections of approximately 0.5 cm were fixed in 4% paraformaldehyde. Paraformaldehyde-fixed ileal and jejunal tissues were prepared into paraffin blocks, sectioned, and then subjected to immunofluorescence staining to analyze the effect of EPS-2 polysaccharide on Claudin-1, a tight junction protein in the small intestinal barrier of Dox-treated mice. Results are as follows: Figure 13 As shown.

[0107] Depend on Figure 13It was found that the expression of the tight junction protein Claudin-1 was reduced and its structural distribution was disordered in the Dox group mice, indicating that Dox affected the expression of Claudin-1 in epithelial cells, causing the mucosal barrier to be disordered. EPS-2 polysaccharide intervention increased the expression of Claudin-1 in the small intestinal epithelial cells of Dox-treated mice and improved its distribution, indicating that EPS-2 polysaccharide can protect the homeostasis of the small intestinal mucosal barrier.

[0108] 14. Effects of EPS-2 polysaccharide on gut microbiota in Dox-treated mice

[0109] At the end of the experiment, feces from each group of mice were collected and rapidly frozen in liquid nitrogen. The feces were then sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for gut microbiota sequencing, and the results were analyzed. The results are as follows: Figure 14 As shown.

[0110] Depend on Figure 14 As shown in Figure A, the level of the Proteobacteria phylum in the intestine of Dox group mice was significantly increased, while EPS-2 polysaccharide intervention reduced the abundance of the Proteobacteria phylum; Figure 14 As shown in Figure B, the levels of pro-inflammatory bacteria *Escherichia-Shigella* (belonging to the phylum *Proteobacteria*) producing lipopolysaccharides (LPS) were significantly increased in the intestines of Dox group mice, while EPS-2 polysaccharide intervention decreased the levels of *Escherichia-Shigella*. Figure 14 As shown in C, the genus *Escherichia-Shigella* is negatively correlated with cardiac function. (From...) Figure 14 The results showed that the level of *Escherichia coli* belonging to the *Escherichia-Shigella* group increased in the intestines of Dox-treated mice, while EPS-2 polysaccharide intervention reduced its level. These results indicate that EPS-2 polysaccharide can reduce the increase in LPS-producing pro-inflammatory bacteria levels in the mouse intestines induced by Dox.

[0111] 15. Effects of EPS-2 polysaccharide on the growth and proliferation of Escherichia coli

[0112] Escherichia coli was cultured to the logarithmic growth phase, and the bacteria were collected and washed twice with sterile physiological saline. After resuspending in physiological saline, the bacteria were aliquoted and incubated with different concentrations of EPS-2 polysaccharide for 3 hours. The E. coli was then serially diluted, and the diluted solutions were plated onto LB agar plates and incubated overnight at 37°C until single colonies appeared. The effect of EPS-2 polysaccharide on the growth and proliferation of E. coli was observed. Results Figure 15 As shown.

[0113] Depend on Figure 15It can be seen that EPS-2 polysaccharide can significantly inhibit the growth of two different E. coli strains, indicating that EPS-2 polysaccharide can inhibit the growth and proliferation of LPS-producing pro-inflammatory bacteria E. coli, which may be the key reason for its reduction of Escherichia-Shigella levels in the intestines of Dox-treated mice.

[0114] 16. Effects of EPS-2 polysaccharide on LPS levels in serum and heart of Dox-treated mice

[0115] At the end of the experiment, blood was collected from the orbital venous plexus of mice in each group. After collection, the blood was allowed to stand at room temperature for 2 hours, centrifuged at 4000 rpm for 15 min, and the supernatant was collected. The LPS content in the serum was measured. Simultaneously, a portion of mouse heart tissue was taken and added to pre-chilled PBS containing PMSF. After cryogenic homogenization, the tissue was centrifuged at 12000×g for 15 min, and the supernatant was collected to obtain heart tissue lysate. The LPS content in the heart tissue was measured, and the results are as follows: Figure 16 As shown.

[0116] LPS is an important indicator of intestinal mucosal barrier permeability. Figure 16 The results showed that Dox induced increased LPS levels in the blood and heart, indicating that Dox treatment induced increased permeability of the intestinal mucosal barrier in mice. EPS-2 polysaccharide intervention significantly reduced LPS levels in the blood and heart, alleviating hypoendotoxemia in mice. These results demonstrate that EPS-2 polysaccharide can effectively alleviate Dox-induced increased intestinal mucosal barrier permeability and protect the function of the small intestinal mucosal barrier.

[0117] Example 3: Comparison of the efficacy of extracellular polysaccharides from *Westernella esculenta* ZMC030 with other *Westernella esculenta* extracellular polysaccharides in alleviating the toxic side effects of chemotherapy drugs.

[0118] Extracellular polysaccharides from different lactic acid bacteria often exhibit different functions. To further demonstrate the functional differences among different polysaccharides, the efficacy of EPS-2 polysaccharide secreted by *Westernella esculenta* ZMC030 and extracellular polysaccharide secreted by *Westernella esculenta* D-2 (for details on the preparation of *Westernella esculenta* D-2 extracellular polysaccharide, please refer to the invention patent or article with patent number ZL 202310908858.7, DOI:10.1038 / s41598-023-47943-7) (denoted as D-2-EPS polysaccharide) was compared.

[0119] (1) Grouping of experimental animals and construction of a mouse chronic myocardial toxicity model

[0120] Six- to eight-week-old SPF-grade female C57BL / 6J mice (purchased from Beijing Spaford Laboratory Animal Co., Ltd.) were randomly divided into three groups: NC group, Dox group, EPS-2 / Dox group, and D-2-EPS / Dox group.

[0121] The treatment methods for NC group, Dox group, EPS-2 / Dox group and D-2-EPS / Dox mice were as follows:

[0122] All mice in the NC group were administered saline by gavage (gavage volume was 10 μL of mouse body weight), with the saline volume being 10 μL of mouse body weight. The gavage was performed every other day for one week, followed by a tail vein injection of the same dose of sterile saline.

[0123] Mice in the Dox group were administered saline via gavage (gavage volume was 10 μL of mouse body weight), with the saline volume being 10 μL of mouse body weight, administered every other day. After one week of continuous gavage intervention, Dox was injected via tail vein (injection method: 5 mg / kg of Dox was injected once a week for a total of 4 injections, with a cumulative Dox dose of 20 mg / kg) to establish a mouse model of chronic myocardial toxicity.

[0124] Mice in the EPS-2 / Dox group were administered the EPS-2 polysaccharide solution prepared in Example 1 by gavage (gavage volume was 10 μL of mouse body weight) every other day. The EPS-2 polysaccharide solution was prepared using physiological saline as a solvent and the concentration of the EPS-2 polysaccharide solution was 10 mg / ml. After one week of continuous gavage intervention, Dox was injected via tail vein (injection method: 5 mg / kg of Dox was injected once a week for a total of 4 injections, with a cumulative Dox dose of 20 mg / kg) to establish a mouse model of chronic myocardial toxicity.

[0125] Mice in the D-2-EPS / Dox group were administered D-2-EPS polysaccharide (extracellular polysaccharide secreted by *Westernella diffusa* D-2) solution by gavage (gavage volume was 10 μL * mouse body weight) every other day. The D-2-EPS polysaccharide solution was prepared using physiological saline as a solvent, and the concentration of the D-2-EPS solution was 10 mg / ml. After one week of continuous gavage intervention, Dox was injected via tail vein (injection method: 5 mg / kg of Dox was injected once a week for a total of 4 injections, with a cumulative Dox dose of 20 mg / kg) to establish a mouse model of chronic myocardial toxicity.

[0126] The date of the first Dox injection in mice was recorded as Day 0. After the last Dox injection, the mice were fed for another 2 weeks, and the experiment ended (the experimental endpoint).

[0127] At the end of the experiment, echocardiography was performed on the cardiac function of mice in each group using a Vevo 3100 small animal ultrasound imaging system (visualsonic, Canada). Then, ileum and jejunum tissues from the mice were fixed in 4% paraformaldehyde. Paraformaldehyde-fixed ileum and jejunum tissues were prepared into paraffin blocks, sectioned, and stained with hematoxylin and eosin (HE) to assess the degree of damage to the two types of intestines. The results are as follows: Figure 17As shown.

[0128] Depend on Figure 17 As shown in A, B, and C, although both are extracellular polysaccharides of Weissella taurida, the extracellular polysaccharide EPS-2 of Weissella taurida ZMC030 can effectively alleviate Dox-induced cardiac damage, while the extracellular polysaccharide D-2-EPS secreted by Weissella taurida D-2 cannot alleviate Dox-induced cardiac damage.

[0129] Depend on Figure 17 As shown in sections D and E, the extracellular polysaccharide D-2-EPS secreted by *Westernella esculenta* D-2 cannot alleviate Dox-induced intestinal damage, while the extracellular polysaccharide EPS-2 secreted by *Westernella esculenta* ZMC030 can effectively alleviate Dox-induced intestinal damage. This indicates that different strains of the same bacteria produce extracellular polysaccharides with different functions, and not all *Westernella esculenta* extracellular polysaccharides have the effect of alleviating Dox toxicity.

[0130] Finally, it should be noted that the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any other way. Any person skilled in the art may use the above technical content as inspiration to make changes or modifications. These are equivalent embodiments with variations. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the technical concept of the present invention still fall within the protection scope of the claims of the present invention.

Claims

1. The use of *Westernophora stolonifer*, its culture, or its secreted extracellular polysaccharides in the preparation of products for the prevention, relief, and / or treatment of toxic side effects caused by chemotherapy drugs; wherein, The *Westernophora stolonifer* mentioned is *Westernophora stolonifer* ZMC030, and the preservation number of *Westernophora stolonifer* ZMC030 is CCTCC NO: M 20241261.

2. The application according to claim 1, characterized in that, The toxic side effects caused by the chemotherapy drugs include cardiotoxicity and gastrointestinal toxicity.

3. The application according to claim 2, characterized in that, The cardiotoxic side effects are at least one of the following: (A1) Chemotherapy-induced cardiac damage; (A2) Chemotherapy-induced cardiomyocyte apoptosis; (A3) Chemotherapy-induced cardiac fibrosis; (A4) Chemotherapy-induced inflammation of cardiac tissue; (A5) Increased cardiac oxidative stress induced by chemotherapy drugs; (A6) Chemotherapy drugs induce decreased activity of cardiac antioxidant enzymes; (A7) Chemotherapy drugs induce an increase in lipopolysaccharide levels in cardiac tissue.

4. The application according to claim 2, characterized in that, The intestinal toxicity side effects are at least one of the following: (B1) Chemotherapy-induced intestinal damage; (B2) Chemotherapy-induced intestinal tissue structural disorder; (B3) Chemotherapy-induced proliferation of LPS-producing pro-inflammatory bacteria in the gut; (B4) Chemotherapy-induced intestinal mucosal barrier disturbance; (B5) Chemotherapy-induced gut microbiota dysbiosis.

5. The application according to any one of claims 1-4, characterized in that, The chemotherapy drug is an anthracycline chemotherapy drug.

6. The application according to claim 1, characterized in that, The culture of *Westernella stolonifera* includes fermentation broth of *Westernella stolonifera*, supernatant of fermentation broth of *Westernella stolonifera*, and dried powder of fermentation broth of *Westernella stolonifera*.

7. The application according to claim 1, characterized in that, The extracellular polysaccharide is obtained by fermenting and culturing the *Westernella esculenta* to obtain a fermentation broth, and then separating and purifying the fermentation broth.

8. The application according to claim 7, characterized in that, The method for preparing the extracellular polysaccharide is as follows: the *Westernella esculenta* is inoculated into a culture medium for fermentation to obtain a fermentation broth. The fermentation broth is then subjected to cell removal and protein removal treatment to obtain a supernatant. The supernatant is subjected to alcohol precipitation treatment, and the precipitate is collected. The precipitate is then subjected to dialysis to remove impurities and freeze-dried to obtain the extracellular polysaccharide.

9. The application according to claim 8, characterized in that, The culture medium is MRS medium or mMRS medium; the fermentation culture temperature is 30℃~37℃; the dialysis molecular weight cutoff is 8000~14000 Da.

10. The application according to claim 1, characterized in that, The products mentioned are pharmaceuticals, food additives, and functional foods.

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