Pediococcus pentosaceus ncu-11 and its use in treating melanoma

Abstract

The application discloses pentosus NCU-11 and application thereof in treating melanoma, and relates to the technical field of biological medicine. The pentosus NCU-11 has been preserved in the China Center for Type Culture Collection on June 5, 2023, is located at Wuhan University in Wuhan, China, and has a preservation number of CCTCC NO: M2023898. The application further provides that the pentosus NCU-11 is used for preparing a drug for assisting in treating melanoma, a drug for relieving or reducing side effects caused by chemotherapy of melanoma, a melanoma chemotherapy sensitizer, and application of the pentosus NCU-11 combined with cisplatin and dacarbazine in preparing a drug for treating melanoma. The pentosus NCU-11 has good probiotic characteristics, and has a synergistic effect with cisplatin and dacarbazine, can enhance the effect of a chemotherapy drug in treating melanoma, and can reduce the chemotherapy toxicity of the chemotherapy drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a strain of Pediococcus pentosaceus NCU-11 and its application in the treatment of melanoma. Background Technology

[0002] Melanoma is a highly aggressive malignant tumor originating from melanocytes, characterized by rapid progression and significant metastatic potential, severely threatening patient survival. Combination chemotherapy regimens, such as cisplatin combined with dacarbazine, remain one of the treatment options for advanced melanoma. However, this regimen is accompanied by severe dose-limiting toxicities, including nephrotoxicity, hepatotoxicity, and myelosuppression, significantly impairing patients' quality of life and treatment tolerability. Although more effective systemic treatments are now available, the cisplatin (cis-dichlorodiammineplatinum)-dacarbazine (5-(3,3-dimethyl-1-triazine)imidazol-4-carboxamide) regimen remains an important second-line salvage therapy option for patients who do not benefit from these treatments. Therefore, developing adjuvant strategies that can enhance the efficacy of this regimen and reduce its toxicities is of clear clinical necessity.

[0003] In recent years, the role of gut microbiota in regulating the efficacy and toxicity of chemotherapy has received increasing attention. Clinical studies have shown that cisplatin-based chemotherapy can induce gut microbiota dysbiosis in patients with gastrointestinal tumors, and is closely related to chemotherapy-associated diarrhea. Furthermore, gut microbiota can influence chemotherapy efficacy through multiple mechanisms. For example, Bifidobacterium spp. (…) Bifidobacterium ) Species can activate CD8 + T cells and enhance anti-tumor immunity; Lactobacillus ( Lactobacillus Microbial microbiota can inhibit the proliferation of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), thereby reversing the immunosuppressive microenvironment. Microbial metabolites can participate in drug metabolism and repair the intestinal mucosal barrier by upregulating tight junction proteins ZO-1 and Occludin, thus reducing chemotherapy-induced gastrointestinal damage and systemic toxicity. However, the specific role of gut microbiota in the treatment of melanoma with cisplatin combined with dacarbazine remains to be elucidated.

[0004] As key biological agents regulating host microecology and immune function, probiotics can enhance the efficacy of chemotherapy drugs and host tolerance by reshaping the gut microbiota composition, improving the tumor immune microenvironment, and reducing chemotherapy-related toxicity. Pediococcus pentosaceus (… Pediococcus pentosaceus (This is a representative strain of the Pediococcus genus, and it has an adjuvant antitumor effect when administered as a supplement. In a breast cancer model, Pediococcus pentosaceus PP06 modulates the intestinal flora and promotes CD8...) +T-cell infiltration significantly enhanced the efficacy of doxorubicin immunochemotherapy. In colorectal cancer, conjugated linoleic acid derived from *Pediococcus pentosaceus* GS4 induced mitochondrial-dependent apoptosis by activating the PPARγ signaling pathway and inhibited colon cancer cell proliferation. Furthermore, *Pediococcus pentosaceus* PP34 alleviated 5-fluorouracil (5-FU)-induced intestinal damage by regulating gut microbiota and inhibiting oxidative stress, thus reducing inflammation. However, the antitumor and toxicity-reducing effects of *Pediococcus pentosaceus* in cisplatin-based combined treatment of melanoma have not been reported. Summary of the Invention

[0005] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a strain of Pediococcus pentosaceus NCU-11 and its application in the treatment of melanoma.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a strain of Pediococcus pentosaceus ( Pediococcus pentosaceus NCU-11, the Pediococcus pentosaceus NCU-11, was deposited on June 5, 2023, at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, with accession number CCTCC NO: M2023898.

[0007] Preferably, the Pediococcus pentosaceus NCU-11 exhibits DPPH and hydroxyl radical scavenging rates of 88.24% and 96.94%, respectively; and the Pediococcus pentosaceus NCU-11 can survive at a bile salt concentration of 0.5% w / v and an environment with pH=1.0.

[0008] Secondly, the present invention provides the use of the aforementioned Pediococcus pentosaceus NCU-11 in the preparation of a medicament for adjuvant treatment of melanoma.

[0009] Thirdly, the present invention provides the use of the aforementioned Pediococcus pentosaceus NCU-11 in the preparation of a medicament for alleviating or reducing the side effects caused by chemotherapy for melanoma.

[0010] Preferably, the melanoma chemotherapy is performed using cisplatin and dacarbazine.

[0011] Fourthly, the present invention provides the application of the aforementioned Pediococcus pentosaceus NCU-11 in the preparation of melanoma chemotherapy sensitizers.

[0012] Preferably, the melanoma chemotherapy is performed using cisplatin and dacarbazine.

[0013] Chemotherapy sensitizers are substances that can increase the sensitivity of tumors to chemotherapeutic drugs and reverse or overcome drug resistance. Studies have found that the Pediococcus pentosaceus NCU-11 of this invention can enhance the efficacy of chemotherapeutic drugs cisplatin and dacarbazine in the treatment of melanoma and reduce the chemotherapeutic toxicity of cisplatin and dacarbazine. Therefore, it can be used to prepare melanoma chemotherapeutic agents.

[0014] Fifthly, the present invention provides the use of the aforementioned Pediococcus pentosaceus NCU-11 in combination with cisplatin and dacarbazine in the preparation of a medicament for treating melanoma.

[0015] Preferably, the Pediococcus pentosaceus NCU-11 can enhance the efficacy of cisplatin and dacarbazine in treating melanoma and reduce the chemotoxicity of cisplatin and dacarbazine.

[0016] Preferably, the drug has at least one of the following effects: a. Upregulates the expression levels of the pro-apoptotic factor Bax mRNA and protein; b. Downregulate the expression levels of the anti-apoptotic factor Bcl2 and / or the proliferation marker Survivin and / or the invasion-related protein MMP9 mRNA and protein; c. Reduce chemotherapy-induced intestinal inflammation; d. Restore intestinal morphology and / or restore intestinal peristalsis; e. Enhance the expression of tight junction proteins; f. Increase the abundance of Pediococcus and Heterobacterium in the gut; g. Restore the Firmicutes / Bacteroidetes ratio; h. Upregulates the levels of pro-inflammatory cytokines IL-2 and / or IFN-γ and / or TNF-α; i. Downregulate the levels of immunosuppressive factors IL-10 and / or TGF-β; j. Downregulate the protein expression levels of key immunosuppressive molecules iNOS and / or Arg1 in tumor tissues; k. Increase CD8 in tumor tissue + IFN-γ + The proportion of T cells.

[0017] In a sixth aspect, the present invention provides a melanoma chemotherapy sensitizer, wherein the active ingredient of the melanoma chemotherapy sensitizer includes the aforementioned Pediococcus pentosaceus NCU-11.

[0018] This invention has at least one of the following beneficial effects: 1. This invention screened a strain of Pediococcus pentosaceus NCU-11 from the feces of healthy adults. Through research, it was found that this strain has good probiotic properties. In terms of antioxidant capacity, Pediococcus pentosaceus NCU-11 exhibits excellent free radical scavenging activity, with DPPH and hydroxyl radical scavenging rates of 88.24% and 96.94%, respectively. It also has good acid resistance and can survive in an environment with pH=1.0.

[0019] 2. Further research in this invention revealed that *Pediococcus pentosaceus* NCU-11 has a synergistic effect with cisplatin and dacarbazine, enhancing the efficacy of anti-melanoma treatment while reducing toxicity. Specifically, the combination therapy remodels the gut microbiota and the tumor immune microenvironment, synergistically enhancing the efficacy of chemotherapy. Mechanistically, it inhibits PMN-MDSCs recruitment and Treg amplification while enhancing CD8. + T cell and TH1 responses largely explain the chemosensitizing effect of *Pediococcus pentosaceus*. In a mouse melanoma model, this combined strategy showed the potential to inhibit tumor growth and reduce chemotherapy-related toxicities. Attached Figure Description

[0020] Figure 1 This study evaluated the growth kinetics, antioxidant capacity, and gastrointestinal tolerance of six candidate probiotic strains in vitro. Figure A shows the growth curves of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Pediococcus pentosaceus* NCU-11, *Lactobacillus salivarius*, *Bifidobacterium longum*, and *Leuconostoc mesenteroides*, plotted using optical density (OD) measurements. Figure B shows DPPH scavenging rate. Figure C shows hydroxyl radical scavenging rate. Figure D shows ferrous ion chelating capacity. Figure E shows superoxide anion scavenging rate. Figure F shows total reducing power measurement. Figure G shows bile salt tolerance tests at 0%, 0.1%, 0.3%, and 0.5% bile salt concentrations. Figure H shows acid tolerance tests at pH 7.0, pH 5.0, pH 3.0, and pH 1.0. All data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA. * p< 0.05 ， ** p <0.01 ， *** p <0.001, **** p <0.0001 indicates a statistically significant difference.

[0021] Figure 2This study describes the effects of Pediococcus pentosaceus NCU-11 on sensitizing melanoma by inducing apoptosis and inhibiting invasion. Figure A shows the timeline and treatment regimen of the animal experiments. Figure B shows representative tumor images of each treatment group at the experimental endpoint. Figure C compares the final tumor volumes between groups. Figure D shows the histological evaluation of tumor tissue sections by H&E staining (original magnification × 200; scale bar = 100 µm). Figures E–F show the histological evaluation of tumor tissue sections by TUNEL staining (original magnification × 200; scale bar = 100 µm). Figures G–H show the evaluation of proliferation activity by Ki67 immunohistochemical (IHC) staining; the second row of G shows magnified views of selected areas (original magnification × 200 and × 400; scale bars 100 µm and 50 µm, respectively). Figures I–L show the expression of key genes related to apoptosis, proliferation, and invasion analyzed by qRT-PCR. In the figure, M–Q represent the corresponding changes in the expression of apoptosis, proliferation, and invasion-related proteins as verified by Western blot. Experimental groups: MP (melanoma model group), HP (intraperitoneal injection of cisplatin / dacarbazine after modeling), and MWP (intraperitoneal injection of cisplatin / dacarbazine combined with Pediococcus pentosaceus gavage after modeling). All data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way repeated measures ANOVA followed by Tukey's post-hoc test. * p< 0.05 ， ** p <0.01 ， *** p <0.001, **** p <0.0001.

[0022] Figure 3This study aimed to alleviate chemotherapy-induced systemic toxicity and maintain intestinal barrier integrity in *Pediococcus pentosaceus*. Figures A-E represent complete blood cell counts (white blood cells, neutrophils, platelets, erythrocytes, and red blood cells) in each group of experimental animals. Figures F-H represent the measurement results of ALT, AST, and Cr levels in each group of experimental animals. Figures I-J represent representative images and quantitative statistical results of colon length. Figure K represents gastrointestinal motility function testing. Figure L represents the histological assessment of colon tissue by H&E staining (original magnification × 200; scale bar = 100 µm). Figures M-P represent the immunofluorescence staining analysis of tight junction proteins ZO-1 and Occludin in mouse colon tissue (scale bar = 100 μm). All data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way repeated measures ANOVA followed by Tukey's post-hoc test. ns indicates no statistical difference. p >0.05), * p< 0.05 ， ** p <0.01 ， *** p <0.001, **** p <0.0001.

[0023] Figure 4 This study aimed to restore gut microbiota diversity and reshape taxonomic distribution in chemotherapy-bearing tumor mice using *Pediococcus pentosaceus*. In the figures, A represents the Chao1 index, reflecting gut microbiota species richness; B represents the Shannon index, reflecting gut microbiota α-diversity; C represents β-diversity assessed using principal coordinate analysis (PCoA); D is a Venn diagram showing OTU overlap in the gut microbiota; E represents the distribution of gut microbiota at the phylum level; F–G represent the relative abundance of Firmicutes and Bacteroidetes; H represents the distribution of gut microbiota at the genus level; and I–K represent… Allobaculum The relative abundance of *Pediococcus pentosaceus*, *Pediococcus*, and *Lactobacillus*. All data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way repeated measures ANOVA followed by Tukey's post-hoc test. p< 0.05 ， ** p <0.01 ， *** p <0.001, **** p <0.0001.

[0024] Figure 5This study aimed to inhibit the recruitment of PMN-MDSCs and remodel tumor immune composition by combining Pediococcus pentosus NCU-11 with chemotherapy. In the figures, A represents single-cell analysis showing the immune cell composition in lymph nodes and tumor tissues; values ​​represent average frequencies. B–G in the figures show the cytokine expression levels in tumor tissues of each experimental group detected by qRT-PCR, detecting the relative mRNA expression levels of IL-2, IL-4, IL-10, IFN-γ, TNF-α, and TGF-β, with β-actin as an internal control. H–J in the figures show the proportion and quantitative analysis of PMN-MDSCs and M-MDSCs in tumor tissues detected by flow cytometry; the figures show representative flow cytometry pseudocolor plots and quantitative results of each subset. K–M in the figures show the protein expression levels of iNOS and Arg1 in tumor tissues detected by Western blot. N–O in the figures show the CD8+ expression levels in tumor tissues detected by flow cytometry. + IFNγ + Cell proportion and quantitative analysis. In the figure, P – Q represents the TH1 / TH2 polarization characteristics in the tumor microenvironment. In the figure, R – S represents Treg cells (CD3+) in the tumor microenvironment. + CD4 + CD25 + FOXP3 + Cell polarization characteristics. All data are expressed as mean ± standard deviation. Statistical significance was assessed using one-way repeated measures ANOVA followed by Tukey's post-hoc test. ns indicates no statistical difference (p>0.05), * p< 0.05 ， ** p <0.01 ， *** p <0.001, **** p <0.0001.

[0025] Figure 6 This study investigated the effects of Pediococcus pentosaceus NCU-11 combined with chemotherapy on serum TGF-β, IL-10, IL-4, and IFN-γ levels in melanoma-affected mice. In the figures, A represents serum IFN-γ; B represents serum IL-10; C represents serum IL-4; and D represents serum TGF-β.

[0026] Figure 7 These are representative images of colon length in each group in Example 4. Detailed Implementation

[0027] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0028] The following examples use Bifidobacterium longum (Bifidobacterium longum) Bifidobacterium longum subsp. longum ATCC 15707, Lactobacillus fermentum ( Limosilactobacillus fermentum ATCC 14931, Lactobacillus plantarum ( Lactiplantibacillus plantarum ATCC 14917, Lactobacillus salivarius ( Ligilactobacillus salivarius ATCC 11741, Leuconostoc mesenteroides ( Leuconostoc mesenteroides subsp. mesenteroide ATCC 8293 is a standard commercial strain, all purchased from Beijing Huayueyang Biotechnology Co., Ltd.

[0029] Example 1: Screening and Identification of Strains Screening process for Pediococcus pentosaceus NCU-11: a. Collect fresh feces from healthy adults and place the sample in a 15 mL sterile centrifuge tube containing 3 mL of 30% glycerol.

[0030] b. Take 1g of sample, add 3mL of sterile PBS, mix thoroughly, and centrifuge (200rpm, 5min) or allow to settle naturally. Dilute 1000g of the sample to obtain 1g of PBS. 0 ~10 7 The bacteria were spread on MRS solid medium (Lactobacillus) in a sterile operating table and incubated in an aerobic environment for 24-48 hours. Plates with 200-400 colonies were taken and, in a sterile laminar flow hood, single colonies were selected based on their morphology, size, color, edge regularity, and whether they were raised. They were then inoculated into the corresponding liquid medium for activation and culture for 24-28 hours, after which DNA was extracted and sequenced for identification.

[0031] Sequencing revealed the 16S rDNA sequence of the screened strain as shown in SEQ ID No. 1. The strain was identified as *Pediococcus pentosaceus*. Pediococcus pentosaceus It was named Pediococcus pentosaceus NCU-11.

[0032]

[0033] Example 2: In vitro characterization of candidate probiotic strains The in vitro characteristics of the screened Pediococcus pentosaceus NCU-11 were evaluated, and Bifidobacterium longum (ATCC 15707), Lactobacillus fermentum (ATCC 14931), Lactobacillus plantarum (ATCC 14917), Lactobacillus salivarius (ATCC 11741), and Leuconostoc mesenteroides (ATCC 8293) were selected as reference strains. According to existing literature, all five strains have potential antitumor activity.

[0034] In vitro characterization methods include: (1) Growth curve: The probiotic strain was activated overnight and then inoculated into MRS medium. The optical density (OD) at 600 nm was measured every 2 hours. 600 The growth curve is plotted with time on the x-axis and OD value on the y-axis until it stops increasing (indicating a stable period).

[0035] (2) Bile salt tolerance: To assess bile salt tolerance, 0%, 0.1%, 0.3% and 0.5% (w / v) bovine bile salts were added to MRS medium, respectively. After the culture reached the stationary phase, 10 μL of bacterial solution was spread on the corresponding solid agar plates for viable count.

[0036] (3) Acid tolerance: Take 100 μL of activated bacterial culture and perform 10-fold serial dilutions with PBS as the diluent to prepare 10¹, 10³, and 10⁻⁶ solutions sequentially. 5 The bacterial culture was diluted 10 times (10 parallel EP tubes per gradient), centrifuged at 2500 rpm for 4 min, the supernatant was discarded, and the culture was resuspended in PBS buffer at pH 1.0, 3.0, 5.0 and 7.0 respectively. The culture was then placed under anaerobic or aerobic conditions for 4 h. 10 μL of the bacterial culture was then evenly spread on the corresponding solid plates for viable cell counting.

[0037] (4) Antioxidant capacity: After activation, the supernatant of the strain was collected by centrifugation, and its antioxidant activity was measured. ① DPPH free radical scavenging capacity: DPPH methanol solution and deionized water were added to the sample supernatant. After reacting at room temperature in the dark, the absorbance was measured at 517 nm. Deionized water was used as a control instead of DPPH solution and the sample was used as a control. ② Hydroxyl free radical scavenging capacity: FeSO4 solution, H2O2 and sample supernatant were added in sequence. After standing, salicylic acid was added. After reacting at room temperature, the absorbance was measured at 510 nm. Deionized water was used as a control instead of the sample. ③ Superoxide free radical scavenging capacity: Tris-HCl buffer and pyrogallol solution were added to the sample. After rapid mixing, the absorbance was measured at 330 nm every 30 s for 150 s. The pyrogallol self-oxidation rate was measured as a control. ④ Fe² + Chelating ability: Take the sample and add FeSO4 solution, mix well, then add VC solution and NaOH solution sequentially, react at room temperature, add trichloroacetic acid, centrifuge to remove protein, take the supernatant and add o-phenanthroline, react at room temperature and measure the absorbance at 536 nm. At the same time, deionized water and FeSO4 are used as controls to replace the sample. ⑤ Total reducing power: Take the sample and add phosphate buffer and K3[Fe(CN)6] solution, mix well and incubate, add trichloroacetic acid and shake to mix well, take the mixture and add deionized water and FeCl3 solution, let stand and measure the absorbance at 700 nm. Deionized water is used as a control to replace the sample.

[0038] The results are as follows Figure 1 As shown, growth curve analysis revealed that all strains entered the logarithmic growth phase after 6–8 hours of culture, but the peak bacterial counts varied. Specifically, *Lactobacillus plantarum* and *Lactobacillus salivarius* reached their peak counts earliest at 14–16 hours, followed by *Lactobacillus fermentum* and *Pediococcus pentosus* NCU-11, while *Bifidobacterium longum* and *Leuconostoc mesenteroides* entered the stationary phase at 18–22 hours. Figure 1 (A) In terms of antioxidant capacity, *Pediococcus pentosaceus* NCU-11 exhibited excellent free radical scavenging activity, with DPPH and hydroxyl radical scavenging rates reaching 88.24% and 96.94%, respectively, significantly superior to other strains. However, there were no statistically significant differences among the strains in ferrous ion chelation capacity (*Lactobacillus plantarum* 21.2%, *Lactobacillus fermentum* 20.19%, *Pediococcus pentosaceus* NCU-11 24.52%, *Lactobacillus salivarius* 21.55%, *Bifidobacterium longum* 21.62%, *Leuconostoc mesenteroides* 20.72%), superoxide radical scavenging (*Lactobacillus plantarum* 84.49%, *Lactobacillus fermentum* 89.18%, *Pediococcus pentosaceus* NCU-11 94.89%, *Lactobacillus salivarius* 91.18%, *Bifidobacterium longum* 86.63%, *Leuconostoc mesenteroides* 89.4%), and total reducing power. Figure 1 B in Figure 1F in the text). Gastrointestinal tolerance tests confirmed that all strains could survive at a 0.5% (w / v) bile salt concentration, with *Pediococcus pentosaceus* NCU-11, *Leuconostoc mesenteroides*, and *Bifidobacterium longum* showing strong tolerance to ox bile salts; and all strains exhibited good acid resistance, surviving at pH 1.0 (*Lactobacillus plantarum* 4.91 μg CFU / mL, *Lactobacillus fermentum* 6.51 μg CFU / mL, *Pediococcus pentosaceus* NCU-11 6.91 μg CFU / mL, *Lactobacillus salivarius* 3.20 μg CFU / mL, *Bifidobacterium longum* 6.83 μg CFU / mL, *Leuconostoc mesenteroides* 6.10 μg CFU / mL). Figure 1 G in Figure 1 (H in the text). In summary, Pediococcus pentosaceus NCU-11 has good probiotic properties and can be considered as a candidate probiotic strain.

[0039] Example 3: Pediococcus pentosaceus NCU-11 synergistically inhibits melanoma growth with chemotherapy by regulating apoptosis and proliferation pathways. The continued progression of melanoma depends primarily on apoptosis arrest, abnormal proliferation, and enhanced invasiveness, processes tightly regulated by key proteins such as Bax, Bcl2, and MMP9. This study systematically evaluated the effects of Pediococcus pentosaceus NCU-11 combined with chemotherapy on melanoma cell apoptosis, proliferation, and invasion.

[0040] By constructing a B16-F10 subcutaneous melanoma-bearing mouse model, the adjuvant antitumor effect of Pediococcus pentosaceus NCU-11 combined with chemotherapy regimens of 2 mg / kg cisplatin and 75 mg / kg dacarbazine was systematically evaluated. The specific methods are as follows: 1. Animal experiments Six-week-old C57BL / 6J mice, weighing 19 g–21 g, were housed at the Animal Center of Nanchang University under controlled conditions of 24 ℃ and a 14-hour light / 8-hour dark cycle, with free access to food and water. The experimental procedures were approved by the Animal Welfare and Ethics Committee of Nanchang University, approval number NCULAE-20250609001.

[0041] 2. Experimental Grouping like Figure 2 As shown in Figure A, after a one-week acclimatization period, a mouse subcutaneous melanoma model was established by inoculating B16-F10 cells (purchased from Hefei Wanwu Biotechnology Co., Ltd.). After tumor formation, mice were randomly divided into groups of six to begin treatment. Cisplatin and dacarbazine were administered intraperitoneally at doses of 2 mg / kg and 75 mg / kg body weight, respectively, using 100 µL of PBS or physiological saline as a solvent. Pediococcus pentosaceus NCU-11 was administered orally by gavage, with each mouse receiving 100 µL of 1 × 10⁻⁶ cells.9 Colony-forming units (CFU) of viable bacteria were prepared using a gelatin-saline solution (gelatin to saline solution volume ratio 0.1:100). Control group mice received an equal volume of saline solution. Experimental groups included: (1) MP group: as the model control group, they did not receive treatment; (2) HP group: i.e., chemotherapy group, receiving cisplatin and dacarbazine treatment; (3) MWP group: received combination therapy with cisplatin, dacarbazine and Pediococcus pentosus NCU-11.

[0042] All mice were housed under identical environmental conditions and were treated and monitored strictly according to the established protocol throughout the experiment.

[0043] 3. After 7 days of feeding, the mice were euthanized, representative tumors were collected, and the following tests were performed: (1) TUNEL detection Tissue sections were first permeabilized with proteinase K and then incubated in a labeling solution containing biotin-labeled dUTP and terminal deoxynucleotidyl transferase. Subsequently, the sections were reacted with FITC-labeled streptavidin at 37°C for 1 hour, followed by counterstaining with DAPI in the dark. Samples were observed under a fluorescence microscope. Cells exhibiting green fluorescence were considered TUNEL-positive cells, i.e., apoptotic cells. The apoptosis index (%) was calculated as the proportion of TUNEL-positive cells to the total number of cells.

[0044] (2) Hematoxylin-eosin staining and immunohistochemistry Tumor specimens were fixed in 4% paraformaldehyde, dehydrated in a gradient manner, and then embedded in paraffin. Longitudinal sections (5 μm thick) were obtained using a Leica CM1850 microtome for histological analysis. Sections were stained with H&E and mounted, and morphologically evaluated under an optical microscope according to standard histological procedures.

[0045] Immunohistochemistry: Paraffin-embedded tumor sections were first dewaxed and antigen-retrieval performed, then incubated with Ki67 primary antibody. After washing with PBS, they were incubated with HRP-labeled secondary antibody at room temperature for 50 minutes. Signal visualization was performed using 3,3'-diaminobenzidine as the chromogenic substrate. After counterstaining, dehydration, and mounting, images were acquired using an Olympus IX71 microscope. Staining intensity and distribution were assessed using a German semi-quantitative scoring system.

[0046] (3) Real-time quantitative PCR Total RNA was extracted from tumor tissue using TRIzol reagent, and RNA purity and concentration were determined using a NanoDrop 2000 spectrophotometer. To eliminate genomic DNA contamination, cDNA synthesis was performed using a reverse transcription kit (PrimeScript RT Reagent Kit, containing a gDNA eraser). RT-qPCR was performed on a ViiA 7 real-time PCR system using PCR amplification premix (Hieff qPCR SYBR Green Master Mix). Reaction conditions: 95°C pre-denaturation for 5 minutes; 40 cycles: 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 20 seconds. Two... -ΔΔCt The relative gene expression levels were calculated using the method described in Supplementary Table 1. Primer sequences are shown in Supplementary Table 1.

[0047] Table 1 (4) Western blot Tumor tissue was lysed in RIPA lysis buffer containing protease and phosphatase inhibitors, centrifuged at 12,000 × g for 15 min to remove debris, and the protein concentration of the supernatant was quantified using the BCA method. An equal volume of total protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane. After membrane blocking, the membrane was incubated overnight at 4°C with primary antibody, followed by incubation at room temperature for 1 hour with HRP-labeled secondary antibody. Protein signals were detected using the Bio-Rad ChemiDoc XRS+ system, and band quantification was performed using ImageJ software. Detailed antibody information is provided in Supplementary Table 2.

[0048] Table 2 The results are as follows Figure 2 As shown, tumor phenotypic analysis revealed that tumor growth was significantly inhibited in the MWP group, with tumor volume and mass being superior to those in the HP group. Figure 2 B in Figure 2 (C in the original text). The continued progression of melanoma mainly depends on apoptosis arrest, abnormal proliferation, and enhanced invasiveness, processes tightly regulated by key proteins such as Bax, Bcl2, and MMP9. This invention systematically evaluated the effects of Pediococcus pentosaceus NCU-11 combined with chemotherapy on melanoma cell apoptosis, proliferation, and invasion. Histological examination using H&E staining and TUNEL assays showed accumulation of immune inflammatory cells, extensive tumor tissue necrosis, and significantly increased apoptosis levels in the MWP group. Figure 2 D in Figure 2 In the F). Ki67 immunohistochemical staining showed that the tumor cell proliferation activity in the MWP group was significantly lower than that in the HP group (F). Figure 2 G in Figure 2To clarify whether Pediococcus pentosaceus NCU-11 regulates apoptosis and invasion-related indicators at the molecular level, this invention used qRT-PCR and Western blotting to detect changes in the expression of key genes and proteins. The results showed that, compared with the MP and HP groups, the MWP group exhibited significantly upregulated mRNA and protein expression levels of the pro-apoptotic factor Bax, and increased expression of cleaved-caspase-3 protein; while the mRNA and protein expression levels of the anti-apoptotic factor Bcl2, the proliferation marker survivin, and the invasion-related protein MMP9 were significantly downregulated. Figure 2 I in Figure 2 (Q in the text). In summary, Pediococcus pentosaceus NCU-11 significantly enhanced the therapeutic effect of cisplatin combined with dacarbazine on melanoma by regulating apoptosis and proliferation-related signaling pathways.

[0049] Example 4: Pediococcus pentosaceus NCU-11 supplementation reduces chemotherapy-induced systemic toxicity and protects intestinal barrier function Cisplatin combined with dacarbazine chemotherapy is often accompanied by systemic toxicity and intestinal barrier dysfunction, which limits its clinical safety. Therefore, the following measurements were performed on mice in the MP group, HP group, and MWP group after the experiment in Example 3: 1. Complete blood cell count Fresh blood was collected from mice and transferred to anticoagulant tubes containing ethylenediaminetetraacetic acid (EDTA). The tubes were gently inverted to mix. The blood samples were analyzed using an automated blood analyzer. Each sample was tested three times, and the results were recorded and averaged.

[0050] 2. Gastrointestinal motility measurement Mice were fasted overnight (with free access to water) to empty their gastrointestinal contents. The following day, each mouse was administered 0.3 mL of a 1.5% sodium carboxymethyl cellulose solution containing 2.5% Evans blue by gavage, and sacrificed 30 minutes later. The furthest migration distance from the pylorus to the Evans blue was measured as the intestinal transit distance. The length of the colon from the distal cecum to the anus was also measured.

[0051] 3. The ALT, AST and Cr levels of mice were detected using the corresponding biochemical kits (purchased from Nanjing Jiancheng Bioengineering Institute). The kits were operated according to the instructions and detected using a fully automated biochemical analyzer or a UV-Vis spectrophotometer. A standard curve was plotted using the standards provided with the kits for quantification. Blank controls and quality control samples were also provided to ensure accurate and reliable results.

[0052] 4. Immunofluorescence Detection: Colon tissue sections were used for immunofluorescence staining. The tissue was first fixed with 4% paraformaldehyde and then permeabilized with 0.5% Triton X-100. To reduce background staining, samples were incubated with 2% BSA for 1 hour at room temperature to block non-specific binding sites. Primary antibody was added, and the samples were incubated at room temperature for 1 hour or overnight at 4°C. After washing, secondary antibody labeled with Alexa Fluor dye (1:500 dilution) was added. Cell nuclei were stained with DAPI (1 µg / mL). Occludin and ZO-1 antibodies were pre-evaluated before incubation, tested on fixed cells for 30 minutes to ensure optimal reactivity.

[0053] The results are as follows Figure 3 As shown, hematological analysis of mice revealed that the white blood cell, granulocyte, platelet, hemoglobin, and red blood cell counts in the HP group were significantly lower than those in the MP group, while the above indicators were significantly restored in the MWP group, indicating that Pediococcus pentosaceus NCU-11 effectively alleviated cisplatin- and dacarbazine-induced myelosuppression. Figure 3 A in Figure 3 The E in the text is missing. Further serum biochemical tests showed that serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), and serum creatinine (Cr) levels were significantly higher in the HP group than in the MP group, while these indicators were significantly lower in the MWP group, suggesting that Pediococcus pentosaceus NCU-11 has a clear protective effect against chemotherapy-induced liver and kidney damage. Figure 3 F- Figure 3 (H in the text). Regarding intestinal barrier integrity, the colon length in the HP group was significantly shorter than that in the MP group, and intestinal peristalsis was significantly slower; the colon length in the MWP group was significantly restored, and intestinal peristalsis was basically normal. Figure 3 I in Figure 3 K in; Figure 7 H&E staining showed that the colonic mucosa in each group was relatively intact, with no obvious ulcers or extensive submucosal edema. However, scattered inflammatory cell infiltration was observed in the crypt stroma of the HP group (with an increased number of deeply stained small round nuclei compared to the MP group). Notably, the inflammatory cell infiltration was significantly reduced in the MWP group, and the stroma structure returned to density, indicating that Pediococcus pentosaceus NCU-11 effectively alleviated chemotherapy-induced mild intestinal inflammation. Figure 3 L in the text). Tight junction proteins play a crucial role in maintaining the intestinal physical barrier. Immunofluorescence staining showed that the fluorescence signals of ZO-1 and occludin were significantly weakened and discontinuous in the HP group, while the fluorescence intensity was significantly enhanced in the MWP group, showing a continuous and dense ring distribution. Figure 3 M in Figure 3 (P in the text). In summary, Pediococcus pentosaceus NCU-11 effectively protects against chemotherapy-induced intestinal barrier damage by restoring intestinal morphology, restoring intestinal peristalsis, and enhancing tight junction protein expression.

[0054] Example 5: Pediococcus pentosaceus NCU-11 effectively remodels gut microbiota dysbiosis in chemotherapy-bearing tumor mice. Subsequently, the changes in the gut microbiota of tumor-bearing mice in each group were systematically evaluated. The changes in the gut microbiota of mice in the MP group, HP group, and MWP group after the experiment in Example 3 were measured: 16S rRNA sequencing: Mouse fecal samples were collected and stored at -80°C. Total genomic DNA was extracted from the feces using a commercial DNA extraction kit. The V3-V4 region of the 16S rRNA gene was amplified and high-throughput sequencing was performed by Shanghai Paiseno Biotechnology. The sequences were clustered into operational taxonomic units using the UPARSE workflow with a 97% similarity threshold. The α diversity index was calculated after library normalization. Representative sequences of each OTU were aligned to the RDP database for species classification annotation. The 16S rRNA high-throughput sequencing data of this study have been submitted to the National Center for Biotechnology Information (NCBI) database, BioProject submission number SUB16397560.

[0055] The results are as follows Figure 4 As shown, 16S rRNA sequencing revealed that cisplatin combined with dacarbazine significantly reduced the Chao1 and Shannon indices in tumor-bearing mice. Figure 4 A in Figure 4 The B in the figure indicates that the chemotherapy regimen reduced the species richness and evenness of the gut microbiota. Pediococcus pentosaceus NCU-11 supplementation significantly restored these indices, suggesting its ability to reverse the chemotherapy-induced decline in gut microbiota diversity. β-diversity PCoA analysis showed that the gut microbiota structure in the HP group significantly deviated from that in the MP group, while the MWP group showed a trend towards approaching that of the MP group. Figure 4 (C in the text). Venn diagram analysis shows that the MP, HP, and MWP groups have a total of 345 OTUs, with 2,663, 1,734, and 3,316 unique OTUs for each group, respectively. Figure 4 (D in the text). Notably, the MWP group shared 1,098 OTUs with the MP group, far more than the 429 OTUs shared with the HP group, indicating that Pediococcus pentosus NCU-11 supplementation significantly increased intestinal species diversity in chemotherapeutic mice and effectively alleviated the intestinal flora dysbiosis induced by cisplatin combined with dacarbazine.

[0056] At the phylum level, the gut microbiota of the MP, HP, and MWP groups were dominated by Firmicutes and Bacteroidetes. Figure 4 (E in the text). Compared with the MP group, the relative abundance of Firmicutes in the HP group showed a decreasing trend, while the relative abundance of Bacteroidetes increased (E in the text). Figure 4 F- Figure 4(G in the text). After Pediococcus pentosaceus supplementation, the Firmicutes / Bacteroidetes ratio partially recovered. At the genus level, there were also significant differences in the composition of the gut microbiota among the groups (G in the text). Figure 4 The H in the group. Compared with the HP group, the MWP group had more Pediococcus spp. ( Pediococcus ) and Heterobacter spp. ( Allobaculum The abundance of *Lactobacillus* significantly increased, while there were no significant changes in the abundance of these two genera between the HP and MP groups. Furthermore, the abundance of *Lactobacillus* remained relatively stable across the three groups. Figure 4 I in Figure 4 In summary, Pediococcus pentosaceus NCU-11 may help restore chemotherapy-induced decline in gut microbiota diversity and reverse the structural dysregulation of gut microbiota in tumor-bearing mice.

[0057] Example 6: Pediococcus pentosaceus NCU-11 combined with chemotherapy inhibits PMN-MDSCs recruitment and regulates tumor immune cell infiltration. The immune status of the tumor microenvironment determines the efficacy of chemotherapy. Therefore, this embodiment further analyzes the immunomodulatory role of Pediococcus pentosaceus NCU-11 after cisplatin-dacarbazine chemotherapy. First, the relevant NCBI single-cell transcriptome datasets (GSM8563006, GSE268778) were referenced and re-analyzed. These datasets systematically describe the dynamic evolution of intratumoral immune cell lineages in melanoma-bearing mice after apoptosis-driven therapy.

[0058] Flow cytometry: Single-cell suspensions were prepared from mouse tumor tissue. Tumors were cut into 1 mm³–2 mm³ fragments and digested in RPMI 1640 containing collagenase IV (1 mg / mL) and DNase I (100 μg / mL) at 37°C for 30 minutes, with gentle pipetting every 10 minutes. The digest was filtered through a 70 μm cell sieve, neutralized with PBS containing 2% FBS, washed twice, and adjusted to a volume of 1 × 10⁻⁶ cells / mL. 7 Cells / mL.

[0059] Surface stain: 1 × 10 6 Cells were incubated with CD3-FITC, CD4-APC-Cy7, CD25-PerCP / Cy5.5, CD8-APC-Cy7, CD45-PE-Cy7, CD11b-APC, Ly6C-FITC and Ly6G-PE at 4°C in the dark for 30 minutes.

[0060] Intracellular cytokine staining: Cells were stimulated with a cell activation cocktail for 4–6 hours, then stained with CD3-FITC and CD4-APC-Cy7. After fixation and permeabilization, cells were stained with IFN-γ-PE and IL-4-APC to identify TH1 cells (CD3-FITC and CD4-APC-Cy7). +CD4 + IFN-γ + ) and TH2 cells (CD3) + CD4 + IL-4 + ).

[0061] FoxP3 staining: After surface staining, the cells were fixed and permeabilized. Treg cells (CD3+) were identified by FoxP3-Alexa Fluor 700 staining. + CD4 + CD25 + FOXP3 + ).

[0062] Stained cells were analyzed by flow cytometry (≥10,000 events per lymphocyte gate). Compensation was performed using a single-stain control. Data were analyzed using FlowJo (v10.8). Gating strategies: lymphocytes (FSC-A / SSC-A), single cells (FSC-H / FSC-A), and live cells. CD8 + IFNγ + The cells are CD3 + CD8 + IFN-γ + T cells; CD4 + T cells are CD3 + CD4 + T cells. TH1, TH2, and Treg subsets are IFN-γ... + IL-4 + and CD25 + FoxP3 + MDSCs at CD45 + The cells are phylogenetic, and PMN-MDSCs cells are CD11b + Ly6G + Ly6C lo M-MDSCs cells are CD11b + Ly6G lo Ly6C hi The experiment was repeated three times, and the data are expressed as mean ± standard deviation.

[0063] The results are as follows Figure 5 As shown, single-cell analysis revealed that treatment induced significant changes in the composition of intratumoral immune cells: CD8 + T cells, proliferating CD8 + T cells and CD4 + T cells increased significantly, while MDSCs and Treg cells decreased, see details below. Figure 5 A in the middle.

[0064] Based on the above single-cell findings, this invention validated the effects of the Pediococcus pentosaceus NCU-11 combination regimen on these key subsets in a B16-F10 melanoma model. At the molecular level, the MWP group exhibited a shift from immunosuppression to an anti-tumor immune state within the tumor, characterized by significant upregulation of pro-inflammatory cytokines IL-2, IFN-γ, and TNF-α, while downregulation of immunosuppressive factors IL-4, IL-10, and TGF-β. Figure 5 B in Figure 5 (G in the text). This immune regulation is not limited to the tumor site but is also reflected at the systemic level—serum TGF-β, serum IL-10, and serum IL-4 levels are decreased, while serum IFN-γ secretion is significantly increased (G in the text). Figure 6 ).

[0065] This invention then focuses on myeloid-derived suppressor cells (MDSCs)—a core barrier to anti-tumor immunity. Pediococcus pentosus NCU-11 combined with cisplatin and dacarbazine significantly reduced MDSC infiltration in tumor tissues, and this inhibitory effect exhibited subtype specificity: primarily blocking the recruitment of PMN-MDSCs, while the proportion of M-MDSCs remained unaffected. Figure 5 H- Figure 5 (J in the text). Simultaneously, the protein expression levels of key immunosuppressive molecules iNOS and Arg1 in tumor tissue were significantly downregulated, indicating that MDSCs-mediated immunosuppressive function was inhibited at the protein level. Figure 5 K in Figure 5 (M in the text). Furthermore, combined intervention with Pediococcus pentosaceus NCU-11 significantly enhanced the infiltration and cytotoxic function of effector T cells within the tumor, manifested in CD8+ in tumor tissue. + IFN-γ + The proportion of T cells increased ( Figure 5 N- Figure 5 O in CD4. + T cell polarization analysis showed that this combined regimen induced TH1 cell polarization and inhibited chemotherapy-induced Treg cell proliferation. However, compared with the HP group, this intervention had no significant effect on the proportion of TH2 cells. Figure 5 P in Figure 5 In summary, Pediococcus pentosaceus NCU-11 inhibits PMN-MDSCs recruitment and Treg amplification while enhancing CD8+. + T cells and TH1 responses improve the immune microenvironment of melanoma.

[0066] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A strain of Pediococcus pentosaceus ( Pediococcus pentosaceus NCU-11, characterized in that, The Pediococcus pentosaceus NCU-11 was deposited on June 5, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, China, with accession number CCTCC NO: M2023898.

2. The Pediococcus pentosaceus NCU-11 according to claim 1, characterized in that, The Pediococcus pentosaceus NCU-11 exhibits DPPH and hydroxyl radical scavenging rates of 88.24% and 96.94%, respectively; and the Pediococcus pentosaceus NCU-11 can survive at a bile salt concentration of 0.5% w / v and at pH=1.

0.

3. The use of Pediococcus pentosaceus NCU-11 as described in claim 1 in the preparation of a medicament for adjuvant treatment of melanoma.

4. The use of Pediococcus pentosaceus NCU-11 as described in claim 1 in the preparation of a medicament for alleviating or reducing the side effects caused by chemotherapy for melanoma, characterized in that, The melanoma chemotherapy was performed using cisplatin and dacarbazine.

5. The use of Pediococcus pentosaceus NCU-11 as described in claim 1 in the preparation of a melanoma chemotherapy sensitizer.

6. The use of Pediococcus pentosaceus NCU-11 in combination with cisplatin and dacarbazine as described in claim 1 in the preparation of a medicament for treating melanoma.

7. The application according to claim 6, characterized in that, The Pediococcus pentosaceus NCU-11 can enhance the efficacy of cisplatin and dacarbazine in the treatment of melanoma and reduce the chemotoxicity of cisplatin and dacarbazine.

8. The application according to claim 6, characterized in that, The drug has at least one of the following effects: a. Upregulates the expression levels of the pro-apoptotic factor Bax mRNA and protein; b. Downregulate the expression levels of the anti-apoptotic factor Bcl2 and / or the proliferation marker Survivin and / or the invasion-related protein MMP9 mRNA and protein; c. Reduce chemotherapy-induced intestinal inflammation; d. Restore intestinal morphology and / or restore intestinal peristalsis; e. Enhance the expression of tight junction proteins; f. Increase the abundance of Pediococcus and Heterobacterium in the gut; g. Restore the Firmicutes / Bacteroidetes ratio; h. Upregulates the levels of pro-inflammatory cytokines IL-2 and / or IFN-γ and / or TNF-α; i. Downregulate the levels of immunosuppressive factors IL-10 and / or TGF-β; j. Downregulate the protein expression levels of key immunosuppressive molecules iNOS and / or Arg1 in tumor tissues; k. Increase CD8 in tumor tissue + IFN-γ + The proportion of T cells.

9. A melanoma chemotherapy sensitizer, characterized in that, The active ingredient of the melanoma chemotherapy sensitizer includes Pediococcus pentosaceus NCU-11 as described in any one of claims 1 to 2.

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