Use of parabacteroides johnsonii in the preparation of a medicament for alleviating toxic side effects induced by anthracycline chemotherapy

Abstract

The application relates to the technical field of new biological medicine uses, in particular to application of Bacteroides joyae in preparation of a medicine for relieving toxic side effects induced by anthracycline chemotherapy drugs. Related experiments show that the Bacteroides joyae can reduce the serum myocardial injury marker creatine kinase isoenzyme MB (CK-MB) and lactate dehydrogenase (LDH) levels of a doxorubicin-induced cardiotoxicity (DIC) model mouse, improve the left ventricular ejection fraction (LVEF) and left ventricular short axis shortening rate (LVFS), and effectively improve the cardiac dysfunction. Meanwhile, the Bacteroides joyae can up-regulate the expression of intestinal tight junction proteins and repair the intestinal barrier damage caused by doxorubicin chemotherapy. The above results show that the Bacteroides joyae has good application potential in reducing the toxic side effects induced by anthracycline chemotherapy drugs.

Description

Technical Field

[0001] This invention relates to the field of new applications in biomedicine, specifically to the use of *Pseudomonas johnsonii* in the preparation of drugs for alleviating the toxic side effects induced by anthracycline chemotherapy drugs. Background Technology

[0002] Cancer is a major global public health problem, seriously threatening human health. During cancer treatment, the toxic side effects of chemotherapy drugs severely impact patients' quality of life and long-term prognosis. Among these, doxorubicin-induced cardiotoxicity (DIC), induced by the anthracycline chemotherapy drug doxorubicin (DOX), is a challenging area of ​​research in cardio-oncology. DIC primarily manifests as decreased left ventricular ejection fraction, arrhythmias, myocardial contractile dysfunction, and dilated cardiomyopathy, and in severe cases, can lead to myocardial infarction and heart failure. Therefore, mitigating pathological myocardial damage is a crucial step in preventing and treating DIC.

[0003] Currently, clinical methods for preventing and treating disseminated intravascular coagulation (DIC) are very limited. Dexrazoxane is currently the only cardioprotective agent approved by the US Food and Drug Administration (FDA) to alleviate DIC, but its clinical application is significantly limited: on the one hand, dexrazoxane has strict indications and potential side effects such as immunosuppression, and is only recommended for advanced breast cancer patients receiving high-cumulative doses of doxorubicin; on the other hand, multiple clinical studies suggest that dexrazoxane may reduce the antitumor efficacy of anthracycline chemotherapy drugs, and the European Medicines Agency does not recommend its routine clinical use in all patients receiving anthracycline chemotherapy. These factors greatly restrict the application of dexrazoxane in clinical practice.

[0004] Therefore, there is an urgent need to develop new, safe, and effective targeted drugs for the prevention and treatment of DIC.

[0005] The inventors constructed an doxorubicin-induced mouse cardiotoxicity model and performed 16S rRNA sequencing on fecal samples from normal control and DIC model mice. The sequencing data identified 132 bacterial genera. Systematic analysis of these genera revealed nine significantly differentially expressed genera with an LDA threshold greater than 3: five in the normal control group and four in the model group. Subsequently, Spearman correlation analysis was performed on the abundance of these differentially expressed genera with mouse cardiac function indicators and myocardial injury markers. The key genera significantly associated with the pathological progression of DIC and the degree of myocardial injury were identified as *Pseudomonas parabens*. Parabacteroides ).

[0006] Parabacterium ( Parabacteroides ) are important members of the core gut microbiota of the human body, including Parabacterium johnsonii ( Parabacteroides johnsonii , P. johnsonii), Parabacterium dilatatum ( Parabacteroides distasonis , P. distasonis ), fecal parabacterium ( Parabacteroides merdae , P. merdae ) and Parabacterium goeringii ( Parabacteroides goldsteinii , P. goldsteinii Multiple strains, including *Parabacterium*, have been shown to have potential probiotic effects in various diseases such as obesity, inflammatory bowel disease, colorectal cancer, and atherosclerosis. Although international patent application WO2018112363A1 discloses that *Parabacterium goeringii* can be used to treat various cancers and in combination with chemotherapy drugs, its disclosure focuses on enhancing anti-tumor efficacy and does not address the mitigation of anthracycline-induced toxic side effects by *Parabacterium* strains.

[0007] Furthermore, to date, no other patent literature discloses the application of *Pseudomonas* strains in alleviating the toxic side effects (especially cardiotoxicity) induced by anthracycline chemotherapy drugs.

[0008] Through experimental screening, the inventors confirmed that *Pseudomonas johnsonii* strains among the *Pseudomonas* genus strains were the most effective, and thus proposed this invention. Summary of the Invention

[0009] To alleviate the toxic side effects induced by anthracycline chemotherapy drugs, this invention provides *Pseudomonas johnsonii* (… Parabacteroides johnsonii Application of *Peribrobacter johnsonii* in the preparation of drugs to alleviate the toxic side effects induced by anthracycline chemotherapy. Related experiments showed that *Peribrobacter johnsonii* could reduce serum levels of creatine kinase isoenzyme MB (CK-MB) and lactate dehydrogenase (LDH), markers of myocardial injury, in mice with anthracycline-induced cardiotoxicity (DIC), and increase left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), effectively improving cardiac dysfunction. Simultaneously, *Peribrobacter johnsonii* could upregulate the expression of intestinal tight junction proteins, repairing intestinal barrier damage caused by doxorubicin chemotherapy. These results indicate that *Peribrobacter johnsonii* has good application potential in alleviating the toxic side effects induced by anthracycline chemotherapy.

[0010] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the use of *Pseudomonas johnsonii* in the preparation of a medicament for alleviating the toxic side effects induced by anthracycline chemotherapy drugs, wherein the toxic side effects are cardiotoxicity and / or intestinal barrier damage.

[0011] Specifically: The anthracycline chemotherapy drug mentioned is doxorubicin.

[0012] The cardiotoxicity refers to cardiac damage induced by anthracycline chemotherapy drugs, specifically including one or more of the following: myocardial fibrosis, cardiomyocyte apoptosis, myocardial contractile dysfunction, and heart failure.

[0013] The intestinal barrier damage refers to the disruption of tight junctions of the intestinal epithelium and damage to the colonic mucosal structure caused by anthracycline chemotherapy drugs.

[0014] The *Parabacteroides johnsonii* was a live bacterium.

[0015] The standard strain of *Pseudomonas johnsonii* is numbered as follows: P. johnsonii CCUG 54943T.

[0016] The *Pseudomonas johnsonii* species contain no less than 2 × 10⁻⁶ 8 Mice were administered CFU / mouse via gavage for 28 consecutive days.

[0017] The drug contains *Pseudomonas johnsonii* and a pharmaceutically acceptable carrier.

[0018] The drug is any one of a liquid preparation, a solid preparation, or a semi-solid preparation.

[0019] The beneficial effects of this invention are as follows: 1. This invention discovers that *Peribrobacter johnsonii* can alleviate the toxic side effects induced by anthracycline chemotherapy drugs and verifies its improving effect. Experimental results demonstrate that administration of *Peribrobacter johnsonii* significantly improves myocardial injury and cardiac dysfunction, restores cardiac contractile function in mice, and reduces myocardial tissue apoptosis and myocardial fibrosis. This invention also verifies that *Peribrobacter johnsonii* improves intestinal barrier damage caused by anthracycline chemotherapy drugs, confirming that it can repair intestinal barrier function by upregulating the expression of intestinal tight junction proteins and improving colonic mucosal structure. These findings provide a novel and effective approach for clinical intervention and drug selection for anthracycline chemotherapy drug-induced toxic side effects.

[0020] 2. Parabacterium johnsonii is a normal intestinal colonizing bacterium in humans. Based on the fact that the regimen of using Parabacterium johnsonii to alleviate myocardial damage and cardiac dysfunction induced by anthracycline chemotherapy drugs is simple to operate and highly feasible, drug compositions containing Parabacterium johnsonii are expected to be developed as drugs to alleviate the toxic side effects induced by anthracycline chemotherapy drugs.

[0021] 3. *Pseudomonas johnsonii* can simultaneously and significantly improve anthracycline-induced cardiac dysfunction, myocardial fibrosis, inhibit cardiomyocyte apoptosis, and improve intestinal barrier damage, thus having both cardioprotective and intestinal barrier repair effects.

[0022] This indicates that *Pseudomonas johnsonii* can be used as a safe and effective active ingredient in the preparation of formulations that alleviate cardiotoxicity and intestinal barrier damage induced by anthracycline chemotherapy drugs. It can effectively improve the damage to the body caused by chemotherapy side effects, make up for the deficiencies of existing prevention and treatment methods, and has good clinical translation and application value. Attached Figure Description

[0023] Figure 1 In Example 1 of this invention, 16S rRNA sequencing, Spearman correlation analysis, and species-level qPCR were performed on feces from mice in the normal control group and the DIC model group. Figure 1 A is the LDA score graph of differential bacterial genera among groups obtained by LEfSe analysis; Figure 1 B is a Spearman correlation diagram showing the differential bacterial abundance and mouse cardiac function indicators (LVEF, LVFS) and myocardial injury markers (LDH, CK-MB); Figure 1 C represents the species-specific qPCR detection results of the genus *Pseudomonas*, with the horizontal axis representing different groups and the vertical axis representing relative abundance. Figure 2 This is a graph showing the detection results of *Peribrobacter johnsonii* improving cardiac function in a mouse model of anthracycline-induced cardiotoxicity in Example 2 of this invention. Figure 2 A represents the cardiac ultrasound images of each group; Figure 2 B represents the effect on the left ventricular ejection fraction in mice, with grouping as the x-axis and left ventricular ejection fraction as the y-axis. Figure 2 C represents the effect on the shortening rate of the left ventricular fractional axis in mice, with grouping as the x-axis and the shortening rate of the left ventricular fractional axis as the y-axis. Figure 2 D and 2E are the statistical results of the levels of CK-MB and LDH, markers of myocardial injury in mouse serum, measured by a fully automated biochemical analyzer. Figures 3-5 The figures show the detection results of *Peribrobacter johnsonii* improving myocardial structural damage and inhibiting cardiomyocyte apoptosis in a mouse model of anthracycline-induced cardiotoxicity in Example 3 of this invention. Figure 3 Representative images of mouse myocardial tissue stained with hematoxylin-eosin (HE); Figure 4 A is a representative image of Masson staining in mouse myocardial tissue; Figure 4 B represents the quantitative statistical results of Masson staining for fibrosis, with experimental groups on the x-axis and the percentage of myocardial fibrosis area on the y-axis. Figure 5 A is a representative image of TUNEL fluorescence staining in myocardial tissue; Figure 5 B represents the statistical results of cardiomyocyte apoptosis rate, with experimental grouping as the x-axis and cardiomyocyte apoptosis rate as the y-axis. Figure 6 , Figure 7 This is a graph showing the detection results of *Pseudomonas johnsonii* remodeling the damaged intestinal barrier and alleviating anthracycline-induced intestinal barrier damage in Example 4 of this invention. Figure 6 A is a representative image of mouse colon tissue stained with HE; Figure 6 B is a statistical graph showing the serum lipopolysaccharide (LPS) levels in mice measured using an ELISA research kit; Figure 7 Figure A is a Western blot (WB) representation of the colonic tight junction proteins Occludin and ZO-1. Figure 7 B and 7C are the quantitative statistical results of the relative expression levels of Occludin and ZO-1 proteins, respectively. The experimental group is the x-axis and the relative expression level of Occludin / ZO-1 protein is the y-axis. In the above figure, Ctrl represents the normal control group, DOX represents the model group, and PJ ( Parabacteroidesjohnsonii ) is the doxorubicin + Parabacterium johnsonii group, HPJ (Heat-killed) Parabacteroidesjohnsonii The group consisted of doxorubicin plus heat-inactivated Pleurobacterium johnsonii. Detailed Implementation

[0024] The following detailed experimental examples systematically explain the core technical concepts and in vivo pharmacodynamic effects of this invention, enabling those skilled in the art to fully understand its research and development objectives, technical features, and application value. Experimental procedures not otherwise specified in this application all adopt standard experimental protocols commonly used in the field; the required bacterial strains, reagents, and consumables can all be obtained through routine commercial procurement channels. The exemplary embodiments listed in this invention are for illustrative purposes only and do not constitute a limitation on the scope of protection of this invention.

[0025] Experimental reagents Doxorubicin hydrochloride (DOX), purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number S17092, specification 100 mg / vial; Fecal genomic DNA extraction kit, purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number DP328-02, specification 50 tests / kit; Parabacterium johnsonii ( Parabacteroides johnsonii abbreviation P . johnsonii The strain was purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd., with the product number BNCC364594. This strain originated from the Microbial Collection Center of the University of Gothenburg, Sweden, and is the same strain as CCUG 54943T. The heat-killed Parabacteroides johnsonii bacterial suspension is prepared by heating the viable bacterial suspension of Parabacteroides johnsonii (CCUG 54943T) in a 100°C water bath for 15 minutes for heat inactivation, and the suspension is reserved after no viable bacterial growth is verified by plate coating; Columbia blood agar plate, purchased from Beijing Beina Chuanglian Biotechnology Co., Ltd., with the product number BNCC352241; Anaerobic gas generating sachet, purchased from Mitsubishi MGC Corporation of Japan, with the product number C-1; Anaerobic indicator, purchased from Mitsubishi MGC Corporation of Japan, with the product number C-22; Phosphate-Buffered Saline (PBS, pH 7.4), purchased from Gibco Company. Loosen the cap of the sterilized and cooled sterile PBS, and let it stand for equilibration for 12 in an anaerobic environment to 24 hours, so that the dissolved oxygen inside the liquid diffuses and releases into the gas phase to achieve pre-reduction, and tighten the cap before use; Mouse lipopolysaccharide (LPS) ELISA research kit (96T), purchased from Jiangsu Kete Biotechnology Co., Ltd., with the product number KT2631-A.

[0026] Example 1: Correlation analysis between DIC model mice and Parabacteroides johnsonii 1. Experimental animals and grouping 1.1 Experimental animals: A total of 12 SPF-grade healthy male C57BL / 6J mice aged 6-8 weeks, weighing 20-25 g, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK (Beijing) 2023-0011).

[0027] 1.2 Feeding conditions: SPF-grade Animal Experiment Center of Beijing University of Chinese Medicine. All animals had free access to standard feed and drinking water, the feeding environment was kept at a constant temperature, and the day-night light-dark cycle was set to 12 h light / 12 h dark. All animal operation procedures in this study were approved by the Institutional Animal Ethics Committee, and the relevant specifications for laboratory animal ethics were strictly followed (Ethics Review No.: BUCM20251031-002).

[0028] 1.3 Grouping: After 1 week of adaptive feeding, the mice were randomly divided into 2 groups (normal control group and model group), with 6 mice in each group. After the mice were fixed, tail vein injection was performed. The injection dose of doxorubicin (1 mg / mL) was 5 mg / kg, once every 7 days for 4 consecutive weeks (cumulative dose of 20 mg / kg). The Ctrl group was synchronously injected with an equal volume of normal saline. After the last administration, the mice were kept for another 4 weeks, and then the mice were uniformly sampled for subsequent detection of various indicators.

[0029] 2. Detection indicators: 2.1 16S rRNA sequencing: Fecal samples were collected from normal control and model mice. Total microbial DNA was extracted from the feces. PCR amplification of the V4 variable region of the bacterial 16S rRNA gene was performed using primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). The PCR amplification products were purified with magnetic beads and used to construct sequencing libraries. High-throughput sequencing was performed using NovaSeq 6000 or DNBSEQ-G99 sequencing platforms. After quality control of the sequencing data, subsequent microbiome bioinformatics analysis was conducted.

[0030] 2.2 Correlation analysis: Based on the genus-level microbial abundance matrix obtained by 16S rRNA sequencing, Spearman correlation analysis was performed on the differential bacterial genus abundance and cardiac function-related indicators LVEF, LVFS, LDH, and CK-MB to assess the relationship between gut microbiota and cardiac injury phenotype.

[0031] 2.3 qPCR detection: Species-specific qPCR primers were used to detect the target *Pseudomonas* spp. ( Parabacteroides The relative abundance of the following four key bacterial species was quantitatively verified, including... P. goldsteinii , P. distasonis , P. merdae and P. johnsonii To verify the changes in the relative abundance of bacterial species.

[0032] 3. Statistical Analysis GraphPad Prism 9.0 software was used for statistical processing of experimental data. Measurement data are expressed as mean ± standard error (Mean ± SEM). Unpaired t-tests were used to compare the DOX and Ctrl groups. P A value <0.05 indicates a statistically significant difference.

[0033] 4. Experimental Results: 4.1 After 16S rRNA sequencing, 132 bacterial genera were identified in the feces of mice in the normal control group and the model group. LEfSe analysis was used to screen for differentially enriched genera between the two groups (LDA score > 3), ultimately yielding 9 differentially enriched genera. For example... Figure 1 As shown in Figure A, the differentially enriched bacterial genera in the normal control group include: *Corydalis* spp. (g_ Colidextribacter ), Parabacterium (g_ Parabacteroides ), *Femtobacter* spp. (g_ Faecalibaculum ), genus *Gastromycetes* (g_ Alistipes ) and Lactobacillus spp. (g_ Ligilactobacillus The differentially enriched bacterial genera in the model group included: Lactobacillus (g_ Lactobacillus ), genus *Rikiensis* (g_ RikenellaBifidobacterium spp. (g_) Bifidobacterium ) and anaerobic clumps (g_ Anaerotruncus ).

[0034] 4.2 As shown in Tables 1-1, 1-2 and Figure 1 As shown in Figure B, Spearman correlation analysis was performed on the differentially enriched bacterial genera and cardiac function indicators and myocardial injury markers. The results showed: g_ Bifidobacterium and g_ Lactobacillus The relative abundance of LVEF and LVFS was significantly negatively correlated with LDH and CK-MB, and significantly positively correlated with LDH and CK-MB. g_ Rikenella The relative abundance of LVEF and LVFS was significantly negatively correlated with LDH and CK-MB, but not significantly correlated with LDH and CK-MB. g_ Parabacteroides The relative abundance of LVEF and LVFS was significantly positively correlated with LDH and CK-MB, and significantly negatively correlated with LDH and CK-MB. g_ Alistipes The relative abundance of CK-MB was significantly negatively correlated only with LVEF, LVFS, and LDH, but not significantly correlated with LVEF, LVFS, and LDH. g_ Anaerotruncus g_ Ligilactobacillus g_ Faecalibaculum and g_ Colidextribacter No significant correlation was observed with the above indicators.

[0035] The above results indicate that among the bacterial genera that were significantly enriched in the normal control group, g_ Parabacteroides The relative abundance of the bacteria was significantly positively correlated with cardiac function indicators (LVEF, LVFS) and significantly negatively correlated with myocardial injury markers (LDH, CK-MB), suggesting a protective association between Parabacterium and doxorubicin-induced cardiotoxicity. Supplementing with Parabacterium strains may be a potential strategy to alleviate DIC.

[0036] Table 1-1. Spearman correlation coefficient (r) and P-value between differential bacterial genus abundance and cardiac function

[0037] Note: Compared to the Ctrl group, the DOX group... P <0.05, P <0.01, P <0.001 Table 1-2. Spearman correlation coefficients (r) and p-values ​​between differential bacterial genus abundance and myocardial injury markers.

[0038] Note: Compared to the Ctrl group, the DOX group... P <0.05, P <0.01, P <0.001 4.3 As shown in Table 2 and Figure 1 As shown in Figure C, qPCR was used to quantitatively validate the major species of the genus *Pseudomonas* at the species level. The results showed that, compared with the normal control group, P. goldsteinii and P. merdae Adjust the model group. P. distasonis It shows a downward trend but has not reached a significant level. P >0.05), P. johnsonii Significantly downregulated in the model group ( P <0.01).

[0039] Based on the aforementioned correlation analysis results, P. johnsonii Decreased levels of *Pseudomonas johnsonii* were closely associated with impaired cardiac function and elevated markers of myocardial injury, suggesting the need for supplementation. P. johnsonii This could be a potential strategy to improve the toxic side effects induced by anthracycline chemotherapy drugs.

[0040] Table 2. Relative abundance of four parabacterial bacteria detected by qPCR

[0041] Note: Compared to the Ctrl group, the DOX group... P <0.05, P <0.01, P <0.001 Example 2: Detection of *Pseudomonas johnsonii* improving cardiac dysfunction in a mouse model of anthracycline-induced cardiotoxicity. 1. Laboratory animals and grouping 1.1 Experimental animals: 24 healthy male SPF-grade C57BL / 6J mice aged 6-8 weeks, weighing 20-25 g, provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No.: SCXK (Beijing) 2021-0006).

[0042] 1.2 Feeding conditions: Same as in Example 1 (Ethics Review No.: BUCM20260304-001).

[0043] 1.3 Grouping: After 1 week of adaptive feeding, the mice were randomly divided into 4 groups, with 6 mice in each group. The specific grouping and operation methods are detailed in Table 3.

[0044] Table 3. Experimental grouping and operation methods

[0045] After the mice were fixed, tail intravenous injection was performed. The injection dose of doxorubicin (1 mg / mL) was 5 mg / kg, once every 7 days for 4 consecutive weeks (cumulative dose of 20 mg / kg). The Ctrl group was synchronously injected with an equal volume of normal saline. 7 days after the last tail intravenous injection, intragastric administration was started, with an intragastric volume of 200 μL per mouse, once a day for 28 consecutive days: In the Ctrl group and the DOX group, an equal volume of sterile anaerobic PBS was intragastrically administered daily; In the PJ group, the bacterial suspension of live Parabacteroides johnsonii was intragastrically administered at a dose of 2×10 per day 8 CFU per mouse for 28 consecutive days of intragastric administration; In the HPJ group, the bacterial suspension of heat-inactivated Parabacteroides johnsonii was intragastrically administered at a dose of 2×10 per day 8 CFU per mouse for 28 consecutive days of intragastric administration; After the entire intragastric administration cycle, samples were collected uniformly for subsequent detection of various indicators.

[0046] 2. Detection indicators: 2.1 Cardiac LVEF and LVFS: The Vevo 2100 small animal cardiac ultrasound imaging system (VisualSonics, USA) was used to perform cardiac ultrasound detection on the mice after the end of administration. After the mice were anesthetized with isoflurane, M-mode ultrasound images were collected to detect LVEF and LVFS of mice in each group, so as to evaluate myocardial systolic function; 2.2 Serum myocardial injury markers CK-MB and LDH: Peripheral blood was collected through the abdominal aorta. After the serum was separated by standing, the TBA-120FR automatic biochemical analyzer (Toshiba, Japan) was used to determine the levels of CK-MB and LDH.

[0047] 3. Statistical analysis GraphPad Prism 9.0 software was used for statistical processing of experimental data. Measurement data are expressed as mean ± standard error (Mean ± SEM). One-way ANOVA was used for comparisons among multiple groups. P A value <0.05 indicates a statistically significant difference.

[0048] 4. Experimental Results: 4.1 Cardiac echocardiography was used to detect LVEF and LVFS in each group of mice to evaluate myocardial contractile function. The results are shown in Table 4. Figure 2 AC.

[0049] Table 4. Echocardiographic results of mice in each group (Mean ± SEM, n = 6)

[0050] Note: Compared to the DOX group. P <0.05, P <0.01, P <0.001 Figure 2 A shows representative echocardiographic images from each group. Compared to the Ctrl group, DOX processing leads to significant left ventricular enlargement, a flattened ventricular wall motion curve, and reduced amplitude. In contrast to the DOX group, the live *Parabacterium johnsonii* administration group showed significant left ventricular chamber shrinkage, restored ventricular wall motion amplitude, and a systolic curve approaching the Ctrl level, suggesting that *Parabacterium johnsonii* effectively reversed DOX-induced cardiac dysfunction. The HPJ group was similar to the DOX group, indicating that the protective effect of this bacterium depends on its viability.

[0051] Table 4 Figure 2 Results B and 2C showed that, compared with the Ctrl group, the LVEF and LVFS levels of mice in the DOX group were significantly decreased, suggesting that doxorubicin induced cardiac dysfunction in mice; compared with the DOX group and the HPJ group, the LVEF and LVFS of mice in the live Bacteroides johnsonii administration group were significantly restored.

[0052] The results show that live Pleurobacterium johnsonii can significantly alleviate doxorubicin-induced cardiac dysfunction and restore myocardial contractility.

[0053] 4.2 Peripheral blood was collected via the abdominal aorta. After serum separation by allowing it to stand, serum levels of myocardial injury markers CK-MB and LDH were measured using a fully automated biochemical analyzer. The results are shown in Table 5. Figure 2 D and 2E.

[0054] Table 5. Results of serum myocardial injury marker detection in mice of each group (Mean ± SEM, n = 6)

[0055] Note: Compared to the DOX group. P <0.05, P <0.01, P <0.001 Table 5 Figure 2 Results D and 2E showed that serum CK-MB and LDH levels were significantly elevated in the DOX group; after administration of live Pleurobacterium johnsonii, the levels of both myocardial injury markers were significantly downregulated, while there was no significant difference between the HPJ group and the DOX group.

[0056] The results confirm that live *Pseudomonas johnsonii* can alleviate myocardial damage induced by anthracycline chemotherapy drugs and improve the decline in cardiac function in model mice.

[0057] Example 3: *Pseudomonas johnsonii* improves myocardial injury and inhibits cardiomyocyte apoptosis in an anthracycline-induced cardiotoxicity model mice. 1. Experimental animals and grouping: Same as in Example 2.

[0058] 2. Experimental method: Same as Example 2.

[0059] 3. Detection indicators: 3.1 Mouse heart tissue samples from each group in Example 2 were taken, fixed with 4% paraformaldehyde, and prepared into paraffin sections for HE staining and Masson staining, respectively. Wherein: The HE staining procedure is as follows: hematoxylin staining of cell nuclei, eosin staining of cytoplasm, gradient dehydration, clearing treatment, and mounting with neutral resin. Finally, myocardial tissue morphology images are observed and acquired using an optical microscope.

[0060] Masson staining followed by dewaxing and rehydration of sections, stepwise staining with reagents, differentiation with acetic acid, gradient dehydration, xylene clearing, and mounting. The myocardial tissue structure and degree of fibrosis were then observed under a microscope.

[0061] 3.2 The apoptosis level of cardiomyocytes in each group was detected by TUNEL fluorescence staining.

[0062] After sample dewaxing, proteinase K antigen retrieval, and equilibration buffer treatment, apoptosis reaction solution was added and incubated in the dark. DAPI was used for nuclear counterstaining. After mounting with anti-fluorescence quencher, the samples were observed and photographed under a fluorescence microscope, and the apoptosis rate of cardiomyocytes in each group was counted.

[0063] 4. Experimental Results: 4.1 The results of HE and Masson staining of mouse heart tissue are shown in Table 6. Figure 3 and Figure 4 .

[0064] Table 6. Masson staining results of mice in each group (Mean ± SEM, n = 6)

[0065] Note: Compared to the DOX group. P <0.05, P <0.01, P <0.001 Figure 3 These are representative images of hematoxylin-eosin (HE) staining of mouse myocardial tissue. In the Ctrl group, the myocardial cells are neatly arranged and dense, with regular nuclei morphology, and no obvious inflammatory cell infiltration is observed in the interstitium. In the DOX group, the myocardial tissue shows significant pathological damage, characterized by significantly disordered and loose arrangement of myocardial fibers, accompanied by a large number of inflammatory cell infiltrations. In contrast, the PJ group shows significantly improved myocardial cell morphology, with a more regular arrangement, significantly reduced inflammatory cell infiltration, and good restoration of myocardial structural integrity. The HPJ group is basically the same as the DOX group, with no significant improvement.

[0066] Figure 4 Image A shows a representative Masson staining image of mouse myocardial tissue, where no obvious interstitial fibrosis was observed in the Ctrl group. In the DOX group, blue collagen fiber deposition in the myocardial interstitium was significantly increased, appearing in sheet-like or cord-like patterns around necrotic myocardium, indicating that DOX induced significant myocardial fibrotic remodeling. In contrast, blue collagen deposition in the myocardial interstitium was significantly reduced in the PJ group, and the degree of fibrosis was significantly alleviated; its collagen distribution pattern was similar to that of the Ctrl group. There was no significant difference in myocardial pathological changes between the HPJ group and the DOX group.

[0067] Table 6 and Figure 4 The quantitative results of group B further corroborated the above histological observations: compared with the Ctrl group, the percentage of fibrosis area in the DOX group was significantly increased, confirming that doxorubicin successfully induced myocardial injury. Compared with the DOX group, the PJ group showed a significant reduction in fibrosis area, indicating that *P. johnsonii* has a certain cardioprotective effect. Under the experimental conditions, no significant protective effect was observed in *P. johnsonii* after heat inactivation treatment, suggesting that the survival status of the strain plays an important role in the efficacy of the present invention.

[0068] The results showed that live *Pseudomonas johnsonii* could effectively alleviate doxorubicin-induced myocardial tissue structural disorder, inhibit inflammatory cell infiltration, and reduce myocardial pathological damage, and this protective effect depended on the survival status of the strain.

[0069] 4.2 The results of TUNEL staining of mouse heart tissue are shown in Table 7. Figure 5 .

[0070] Table 7. TUNEL staining results of mice in each group (Mean ± SEM, n = 6)

[0071] Note: Compared to the DOX group. P <0.05, P <0.01, P <0.001 Table 7 Figure 5 The results of the AB study showed that the apoptosis rate of cardiomyocytes in the DOX group was significantly higher than that in the Ctrl group, confirming that doxorubicin can significantly induce cardiomyocyte apoptosis; the apoptosis rate of cardiomyocytes in the PJ group was significantly lower than that in the DOX group, while there was no significant difference between the HPJ inactivated bacteria group and the DOX group.

[0072] The results suggest that live *Pseudomonas johnsonii* can significantly inhibit anthracycline-induced cardiomyocyte apoptosis and reduce cardiomyocyte damage, and this protective effect depends on the survival status of the strain.

[0073] Example 4: *Pseudomonas johnsonii* improves the damaged intestinal barrier and reduces anthracycline-induced intestinal barrier damage. 1. Experimental animals and grouping: Same as in Example 2.

[0074] 2. The experimental method is the same as in Example 2.

[0075] 3. Detection indicators: To investigate the regulatory role of *Peribrobacter johnsonii* on the toxic side effects, especially intestinal barrier damage, induced by anthracycline chemotherapy drugs, the effects of *Peribrobacter johnsonii* on improving doxorubicin-induced intestinal barrier damage were evaluated through colonic histopathological staining, serum LPS measurement, and intestinal tight junction protein expression detection. 3.1 Pathological staining of mouse colon tissue: The colon tissue of each group of mice in Example 2 was fixed with 4% paraformaldehyde, embedded in paraffin, and prepared by sectioning. After routine staining, dehydration, clearing, and mounting, the morphology of the colonic mucosa, crypt structure and inflammatory infiltration were observed under an optical microscope.

[0076] 3.2 Measurement of mouse serum LPS: Peripheral blood was collected via the abdominal aorta, and after the serum was separated by standing, the serum LPS level was measured using a mouse LPS ELISA research kit.

[0077] 3.3 Detection of intestinal tight junction protein expression: Western blot was used to detect the expression level of intestinal tight junction proteins in colon tissue. Total protein was extracted from colon tissue, and after protein quantification, electrophoretic separation, membrane transfer, and blocking, the tissue was incubated with Occludin, ZO-1 specific primary antibody, and corresponding secondary antibody, respectively. Protein bands were obtained by imaging. GAPDH was used as an internal control to correct the gray value of the bands, and the relative expression level of intestinal tight junction proteins in each group was quantitatively analyzed to evaluate the integrity of the intestinal barrier.

[0078] 4. Experimental Results: 4.1 HE staining results of mouse colon tissue are shown in [the table below]. Figure 6 A. In the DOX group, the colonic mucosal crypts of mice showed disordered and irregular arrangement, impaired mucosal integrity, and inflammatory cell infiltration, suggesting that doxorubicin-induced colonic pathological damage. In contrast, the PJ group showed intact colonic mucosal structure, regular crypt arrangement, and significantly reduced inflammatory infiltration, indicating that *Peribrobacter johnsonii* can effectively improve colonic tissue damage induced by anthracycline chemotherapy drugs.

[0079] 4.2 Peripheral blood was collected via the abdominal aorta. After serum separation by allowing the blood to stand, serum LPS levels were measured using a mouse LPS ELISA research kit. The results are shown in Table 8. Figure 6 B.

[0080] Table 8. Serum LPS detection results of mice in each group (Mean ± SEM, n = 6)

[0081] Note: Compared to the DOX group. P <0.05, P <0.01, P <0.001 Table 8 and Figure 6 Results B showed that serum LPS levels were significantly elevated in the DOX group; LPS levels were significantly downregulated after administration of live Parabacterium johnsonii, while there was no significant difference between the HPJ group and the DOX group.

[0082] The results showed that live *Pseudomonas johnsonii* bacteria could effectively repair DOX-induced intestinal barrier damage.

[0083] 4.3 Figure 7 A shows representative bands of the tight junction proteins Occludin and ZO-1 in mouse colon tissue via Western blot. The results show that DOX significantly downregulated the expression levels of Occludin and ZO-1 proteins in colon tissue, indicating disruption of the intestinal barrier structure. After administration of *P. johnsonii*, the expression levels of these tight junction proteins in the colon tissue of PJ group mice significantly increased, indicating effective improvement of the intestinal barrier structure. These results confirm that *P. johnsonii* can alleviate anthracycline-induced intestinal barrier dysfunction by upregulating the expression of intestinal tight junction proteins, improving colonic pathological damage, and that this protective effect depends on the survival status of the strain.

[0084] Table 9 and Figure 7 B and 7C are the quantitative statistical results of the relative expression levels of Occludin and ZO-1 proteins in mouse colon tissue.

[0085] Table 9. Statistical results of Western blot analysis of colon tissue from mice in each group (Mean ± SEM, n = 6)

[0086] Note: Compared to the DOX group. P <0.05, P <0.01, P <0.001 Based on the results of the above embodiments, it can be concluded that *Pseudomonas johnsonii* can significantly improve anthracycline-induced cardiotoxicity and intestinal barrier damage, while also exhibiting cardioprotective and intestinal barrier repair effects. It can be used to alleviate the two types of toxic side effects caused by anthracycline chemotherapy drugs.

[0087] This indicates that live *Pseudomonas johnsonii* can be used as a safe and effective active ingredient in the preparation of formulations that alleviate cardiotoxicity and intestinal barrier damage induced by anthracycline chemotherapy drugs. It can effectively improve the damage to the body caused by chemotherapy side effects, make up for the deficiencies of existing prevention and treatment methods, and has good clinical translation and application value.

[0088] It should be noted that the above description is only a preferred exemplary embodiment of the present invention, and not all feasible embodiments. Based on the embodiments of the present invention, various embodiments derived by those skilled in the art based on the disclosure of the present invention without the need for inventive experimentation are all equivalent embodiments of the present invention. The scope of protection claimed by the present invention should be determined by the claims. The description and drawings can be used to interpret the claims, but should not be construed as limiting the scope of the claims.

Claims

1. *Pseudomonas johnsonii* ( Parabacteroides johnsonii Its application in the preparation of drugs for alleviating the toxic side effects induced by anthracycline chemotherapy drugs, characterized in that, The toxic side effects are cardiotoxicity and / or intestinal barrier damage.

2. The application according to claim 1, characterized in that, The anthracycline chemotherapy drug mentioned is doxorubicin.

3. The application according to claim 1, characterized in that, The cardiotoxicity refers to cardiac damage induced by anthracycline chemotherapy drugs, specifically including one or more of the following: myocardial fibrosis, cardiomyocyte apoptosis, myocardial contractile dysfunction, and heart failure.

4. The application according to claim 1, characterized in that, The intestinal barrier damage refers to the disruption of tight junctions of the intestinal epithelium and damage to the colonic mucosal structure caused by anthracycline chemotherapy drugs.

5. The application according to claim 1, characterized in that, The *Parabacteroides johnsonii* was a live bacterium.

6. The application according to claim 1, characterized in that, The standard strain of *Pseudomonas johnsonii* is numbered as follows: P. johnsonii CCUG 54943T.

7. The application according to claim 1, characterized in that, The *Pseudomonas johnsonii* species contain no less than 2 × 10⁻⁶ 8 Mice were administered CFU / mouse via gavage for 28 consecutive days.

8. The application according to claim 1, characterized in that, The drug contains *Pseudomonas johnsonii* and a pharmaceutically acceptable carrier.

9. The application according to claim 1, characterized in that, The drug is any one of a liquid preparation, a solid preparation, or a semi-solid preparation.

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