Use of lactobacillus reuteri and metabolites thereof in the preparation of a medicament for modulating ferroptosis

Abstract

The application discloses Lactobacillus reuteri and metabolites thereof in the preparation of drugs for regulating ferroptosis. The application also discloses Lactobacillus reuteri and metabolites thereof in the preparation of drugs for promoting tumor growth, establishing a tumor ferroptosis-related drug resistance model or invasion model, preparing a drug for reducing the toxicity of a ferroptosis inducer, and preparing a drug for inhibiting the lipid peroxidation level of tumor cells. The application inhibits ferroptosis through Lactobacillus reuteri and metabolites thereof, significantly reduces the lipid peroxidation level of tumor cells, protects GPX4 protein, and promotes tumor growth.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of Lactobacillus reuteri and its metabolites in regulating ferroptosis. Background Technology

[0002] Ferroprelation is an iron-dependent, non-apoptotic form of cell death characterized by lipid peroxidation and antioxidant system imbalance induced by intracellular iron excess. Its mechanisms include iron metabolism imbalance, reactive oxygen species (ROS) accumulation, and polyunsaturated fatty acid oxidation, primarily manifesting as cell membrane rupture and abnormal mitochondrial morphology (reduction or disappearance of mitochondrial cristae). Ferroprelation plays a crucial role in tumor suppression and certain pathological injuries (such as neurodegenerative diseases and ischemic injuries), and has become a hot topic in cell death research in recent years.

[0003] Meanwhile, the gut microbiota is widely recognized as an important regulator of host metabolism. Through the regulation of redox balance and iron metabolism via metabolites, the gut microbiota exerts a profound influence on the tumor microenvironment. However, novel strategies targeting gut microbiota metabolites to regulate tumor growth and treatment by intervening in ferroptosis have not yet been fully developed. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned technical problems by providing a technical solution for regulating tumor growth and treatment through intervention in ferroptosis.

[0005] To achieve the above-mentioned objectives, this invention provides the use of *Lactobacillus reuteri* and its metabolites in the preparation of a drug for regulating ferroptosis. Preferably, the metabolite is indole-3-carboxaldehyde (I3A). Preferably, the regulation includes inhibiting ferroptosis.

[0006] On the other hand, the present invention also provides the use of *Lactobacillus reuteri* and its metabolites in the preparation of medicaments for promoting tumor growth. Preferably, the metabolite is indole-3-carboxaldehyde.

[0007] On the other hand, the present invention also provides the application of *Lactobacillus reuteri* and its metabolites in establishing tumor ferroptosis-related drug resistance models or invasion models. Preferably, the metabolite is indole-3-carboxaldehyde.

[0008] On the other hand, the present invention also provides the use of *Lactobacillus reuteri* and its metabolites in the preparation of a medicament for reducing the toxicity of ferroptosis inducers. Preferably, the metabolite is indole-3-carboxaldehyde.

[0009] On the other hand, the present invention also provides the use of *Lactobacillus reuteri* and its metabolites in the preparation of a medicament for inhibiting lipid peroxidation levels in tumor cells. Preferably, the metabolite is indole-3-carboxaldehyde.

[0010] Preferably, the tumor includes colorectal cancer or melanoma.

[0011] This invention, based on the function of specific gut microbiota metabolites, proposes a novel method to inhibit ferroptosis and regulate colorectal cancer growth through the tryptophan metabolite I3A produced by *Lactobacillus reuteri*. Specifically, I3A, as a natural ligand of the aryl hydrocarbon receptor (AHR), significantly reduces lipid peroxidation levels and inhibits ferroptosis by activating the AHR-JUN-GPX4 signaling axis, ultimately promoting tumor cell survival. In in vitro experiments, I3A significantly reduced the effect of the ferroptosis inducer RSL3; in mice, its inhibitory effect on tumor ferroptosis was also verified by gavage administration of *Lactobacillus reuteri* or direct intraperitoneal injection of I3A. The unique aspect of this technology is that it achieves ferroptosis regulation through gut microbiota metabolism intervention, providing a novel tumor treatment model and expanding the application potential of gut microbiota in personalized medicine.

[0012] This invention reveals that tryptophan metabolite I3A, as a natural ligand for AHR, activates AHR transcriptional activity, downregulating c-JUN at the transcriptional level, and subsequently downregulating its protein and phosphorylation levels. This inhibits c-JUN-promoted autophagic flux and GPX4 degradation, thereby endowing I3A with ferroptosis resistance and promoting colorectal cancer growth. I3A can be administered directly, or via Lactobacillus reuteri (using Lactobacillus reuteri as a source of I3A to replace direct administration), blocking the use of Lactobacillus reuteri as a source of I3A and thus inhibiting its production in vivo. For aryl hydrocarbon receptors AHR, c-JUN, and GPX4, the specific inhibitors include RNA interference molecules or antisense oligonucleotides, small molecule inhibitors, siRNA, lentiviral infection or gene knockout substances, and specific antibodies against the receptors themselves or their upstream and downstream molecules; no specific limitations are specified here.

[0013] The animals used in the described animal tumor models are preferably non-human mammals, such as rats and mice. The product can be used as a pharmaceutical or experimental reagent in medical research, and the experimental reagent can be used in basic medical research. Attached Figure Description

[0014] Figure 1This diagram illustrates the in vitro inhibition of ferroptosis in tumor cells by I3A. The results include: (A) a schematic diagram of small molecule drug screening; (B) LDH detection of the inhibitory effects of various small molecule compounds on ferroptosis; (C) crystal violet staining detection of the inhibitory effect of I3A on RSL3-induced cell death; (D) Zombie dye detection showing the inhibitory effect of I3A on RSL3-induced cell death; (E) BODIPY-C11 581 / 591 probe detection showing the inhibitory effect of I3A on RSL3-induced lipid peroxidation; (F) a statistical analysis of the detection results in Figure E; (G) DCFH-DA probe detection showing the inhibitory effect of I3A on RSL3-induced reactive oxygen species accumulation; and (H) a statistical analysis of the detection results in Figure G.

[0015] Figure 2 This study demonstrates the inhibitory effect of I3A on tumor ferroptosis in vivo. Specifically: (A) Schematic diagram of nude mouse experiments; (B) I3A delayed the regression of RSL3-induced MC38 tumors in mice; (C) I3A delayed the reduction in size of RSL3-induced MC38 tumors; (D) I3A inhibited the reduction in tumor weight of RSL3-induced MC38 tumors; (E) I3A reduced the accumulation of MDA (dimethylaminopropyl hydroxyl) lipid peroxidation products in RSL3-induced MC38 tumors in mice; (F) I3A reduced the accumulation of 4-HNE (dimethylaminopropyl hydroxyl) lipid peroxidation products in RSL3-induced MC38 tumors in mice; (G) I3A delayed the regression of RSL3-induced B16 tumors in mice; (H) I3A delayed the regression of RSL3-induced tumors in mice. The effects of I3A on reducing the size of B16 tumors induced by RSL3; (I) I3A inhibited the reduction in B16 tumor weight induced by RSL3; (J) I3A reduced the accumulation of MDA, a lipid peroxidation product, in mouse B16 tumors induced by RSL3 treatment; (K) I3A reduced the accumulation of 4-HNE, a lipid peroxidation product, in mouse B16 tumors induced by RSL3 treatment; (L) I3A delayed the accumulation of transferrin receptor (TRFC) and the degradation of glutathione peroxidase (GPX4) in mouse B16 tumors induced by RSL3 treatment; (M) Statistical analysis of GPX4 immunohistochemical results in Figure L; (N) Statistical analysis of TFRC immunohistochemical results in Figure L.

[0016] Figure 3This study demonstrates the necessity of AHR transcriptional activity for I3A's inhibition of ferroptosis. Specifically: (A) The effect of AHR knockout in B16 cells reversing I3A's inhibition of RSL3-induced cell death and verification of the AHR knockout effect; (B) Annexin V / PI apoptosis assay showing that I3A only inhibits RSL3-induced cell death in the presence of AHR; (C) Statistical analysis of the results in Figure B; (D) Statistical analysis of DCFH-DA probe assay showing that I3A only inhibits RSL3-induced reactive oxygen species accumulation in the presence of AHR; (E) Statistical analysis of BODIPY-C11 581 / 591 probe assay showing that I3A only inhibits RSL3-induced lipid peroxidation accumulation in the presence of wild-type AHR, not the nuclear-localized AHR mutant; (F) Crystal violet staining results showing that I3A only inhibits RSL3-induced lipid peroxidation accumulation in the presence of wild-type AHR, not the nuclear-localized AHR mutant.

[0017] Figure 4 This study demonstrates how I3A inhibits ferroptosis by targeting and suppressing the autophagic degradation of GPX4. Specifically: (A) Crystal violet staining results show that knockdown of GPX4 in B16 cells reverses the effect of I3A in inhibiting RSL3-induced cell death; (B) LDH assay results show that knockdown of GPX4 in B16 cells reverses the effect of I3A in inhibiting RSL3-induced cell death, and Western blotting confirms the knockdown effect; (C) Western blotting shows that I3A inhibits RSL3-induced GPX4 protein degradation; (D) Immunofluorescence results show that I3A inhibits RSL3-induced GPX4 protein degradation; (E) Western blotting results show that I3A inhibits RSL3-induced GPX4 protein degradation via AHR. (F) Crystal violet staining results showed that the autophagy inhibitor CQ / BAF, rather than the proteasome inhibitor MG132, inhibited RSL3-induced wild-type cell death; (G) LDH assay showed that in AHR-deficient B16 cells, the autophagy inhibitor CQ / BAF, rather than the proteasome inhibitor MG132, inhibited RSL3-induced cell death and rescued I3A to inhibit RSL3-induced cell death; (H) LDH assay showed that in GPX4-deficient B16 cells, the autophagy inhibitor CQ did not inhibit RSL3-induced cell death or rescued I3A to inhibit RSL3-induced cell death.

[0018] Figure 5This study demonstrates that I3A inhibits GPX4 autophagic degradation in an AHR-dependent manner, blocking the c-JUN-BECLIN1 axis. Specifically: (A) RNA-seq results showed differential gene expression in cells treated with RSL3 alone and those treated with I3A in combination with RSL3; (B) Crystal violet staining showed that knocking out Hmox1 in B16 cells did not inhibit RSL3-induced cell death; (C) qPCR results validated the Hmox1 knockout effect; (D) qPCR results validated the effect of I3A in inhibiting RSL3-induced upregulation of c-jun mRNA levels; and (E) qPCR results validated the I3A-dependent AHR-dependent inhibition of RSL3-induced upregulation of c-jun mRNA levels. (F) Western blot analysis showed that I3A inhibited the upregulation of total protein and phosphorylation levels of c-jun induced by RSL3; (G) Western blot analysis showed that I3A-dependent AHR inhibited the upregulation of total protein and phosphorylation levels of c-jun induced by RSL3; (H) LDH analysis showed that the c-JUN inhibitor JNK-VIII inhibited the RSL3-induced cell death; (I) Western blot analysis showed that the c-JUN inhibitor JNK-VIII inhibited the degradation of GPX4 protein and the accumulation of BECLIN1 protein.

[0019] Figure 6 This study demonstrated that *Lactobacillus reuteri* inhibits tumor ferroptosis in vivo. Among them: (A) qPCR results showed that *Lactobacillus reuteri* accumulated significantly after gavage with *Lactobacillus reuteri* but not after gavage with PBS; (B) Targeted metabolism mass spectrometry results showed that I3A accumulated significantly after gavage with *Lactobacillus reuteri* but not after gavage with PBS; (C) Gavage with *Lactobacillus reuteri* delayed the regression of RSL3-induced MC38 tumors in mice; (D) Gavage with *Lactobacillus reuteri* delayed the reduction of MC38 tumor weight induced by RSL3; (E) Gavage with *Lactobacillus reuteri* reduced the accumulation of MDA, a lipid peroxidation product, in MC38 tumors induced by RSL3; (F) TCGA dataset analysis showed that AHR levels were significantly higher in colorectal cancer specimens from patients compared with normal adjacent normal tissues; (G) TCGA dataset analysis showed that GPX4 levels were significantly higher in colorectal cancer specimens from patients compared with normal adjacent normal tissues; (H) TCGA dataset analysis showed that AHR and JUN levels were negatively correlated in tumor specimens from colorectal cancer patients. (I) Schematic hypothesis of I3A inhibiting ferroptosis and its specific mechanism. Detailed Implementation

[0020] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following embodiments.

[0021] Unless otherwise specified, all reagents mentioned below are commercially available. For simplicity, some procedures have not been detailed in terms of parameters, steps, and instruments used; it should be understood that these are well-known and reproducible to those skilled in the art.

[0022] It is worth noting that, as used herein, when referring to the term "Lactobacillus reuteri and its metabolites," it means including Lactobacillus reuteri, Lactobacillus reuteri metabolites, and / or a combination of both.

[0023] Experimental methods

[0024] 1. Cell Culture

[0025] Mouse melanoma cells B16 and mouse colon cancer cells MC38 (wild-type B16 cells were purchased from ATCC, and MC38 cells were kindly donated by Professor Yang Xuanming of Shanghai Jiao Tong University and preserved in our laboratory; both were tested and found to be free of mycoplasma contamination) were cultured in DMEM complete medium supplemented with 10% v / v fetal bovine serum, 100 U / mL penicillin and 100 μg / mL streptomycin. The cells were cultured at 37°C and 5% CO2.

[0026] 2. Culture of Lactobacillus reuteri (purchased from Beina Biotechnology, BNCC-337178)

[0027] (1) Culture conditions: 37℃, oxygen conditions: slightly aerobic.

[0028] (2) Culture medium: MRS broth medium (purchased from Qingdao Haibo Biotechnology, HB0384-1).

[0029] (4) Expanded culture: After confirming the activity by dilution and plating, expand the culture by inoculating liquid culture at 3%-5% of the total volume of the culture medium.

[0030] 3. Flow cytometry detection of cellular lipid peroxidation levels

[0031] (1) Digest the cells into a single-cell suspension and spread it evenly on a 12-well plate;

[0032] (2) When the cell density reaches 70-80%, add the tryptophan metabolite indole-3-carboxaldehyde (I3A; CAS No.: 487-89-8), add the ferroptosis inducer RSL3 after 2 hours, and incubate for 3 hours;

[0033] (3) When ferroptosis is observed in some cells, use a calcium-free solution. 2+ Cells were digested with trypsin, collected and centrifuged, and washed twice with PBS to remove serum effects.

[0034] (4) Resuspend the cells in PBS containing 10 μM of C11 BODIPY 581 / 591 / DCFH-DA fluorescent probe and incubate them in a cell culture incubator for 30 minutes in the dark.

[0035] (5) After staining, wash twice with PBS, resuspend the sample with 300 μL PBS, filter it through a 40 μM nylon mesh into a flow cytometer, and then use a flow cytometer to detect and assess the lipid peroxidation level.

[0036] 4. Cell viability detection

[0037] 4.1 Cell CCK8 Detection Experiment

[0038] (1) One day in advance, seed B16 cells at 3×103 / well in a 96-well plate with a total volume of 90μL;

[0039] (2) Dilute I3A with the corresponding culture medium, add 10 μL / well, and gently shake to mix.

[0040] (3) Then add 10 μL of CCK8 detection reagent at the specified time points. React in a 37°C cell culture incubator for 30 min-1 hr and then read the values.

[0041] 4.2 Zombie probe staining

[0042] (1) Cell collection: Cells were collected after digestion with trypsin and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells / mL.

[0043] (2) Preparation of Zombie probe working solution: From the kit (Zombie NIR) TM Remove Zombie probe powder from the Fixable Viability Kit (Biolegend, catalog number 423106) and dissolve it in DMSO (1:1000 volume dilution). Add 1 μL of Zombie dye to 99 μL of PBS. Prepare Zombie working solution;

[0044] (3) Add Zombie working solution to the cell suspension: every 10 6 Add 100 μL of Zombie working solution to each cell. Mix the cells and dye thoroughly. Incubate at room temperature in the dark for 5–10 min, then wash twice with PBS, and resuspend the cells in 300 μL of PBS for flow cytometry analysis.

[0045] 4.3 Annexin V / Propidium Iodide (Annexin V / PI) Probe Staining (All materials used below are included in the Annexin V / PI kit, purchased from Biolegend, catalog number 556547)

[0046] (1) Cell treatment: Cells were collected after gentle digestion with EDTA-free trypsin. Cells were washed twice with PBS and centrifuged to remove culture medium and serum. Cells were resuspended in 1×Binding Buffer at a concentration of 1×10⁶ cells / mL.

[0047] (2) Preparation of staining mixture:

[0048] Prepare the staining solution (per 100 μL staining system) according to the following ratio: 100 μL 1× Binding Buffer + 5 μL Annexin V + 5 μL PI

[0049] (3) Staining: Transfer 100 μL of cell suspension (approximately 1 × 10⁵ cells) to a flow cytometry tube. Add the prepared staining solution and mix gently. Incubate at room temperature for 10-15 minutes in the dark. Add 300 μL of 1 × Binding Buffer to each sample. Gently mix the sample, avoiding the formation of air bubbles, and perform detection using a flow cytometer.

[0050] 5. Western Blot assay for proteins

[0051] 5.1 Preparation of protein samples

[0052] After collecting cells, extract proteins using protein lysis buffer (RIPA lysis buffer: PMSF = 100:1), place on ice and shake for 15 min and sonicate (if necessary), centrifuge at 4°C and 12,000 rpm for 10 min, and the supernatant is the desired protein.

[0053] 5.2 Protein Concentration Determination

[0054] Total protein concentration was determined using the BCA method or the Bradford method.

[0055] 5.3 Protein denaturation

[0056] (1) Add 5× Protein Loading Buffer (GBCBIO, catalog number G4552) to the sample EP tube with protein added and the volume made up, the loading volume is 1 / 5 of the protein sample volume, and mix well;

[0057] (2) Heat in a 98℃ metal bath for 5-10 minutes to fully denature the protein, then cool the sample on ice.

[0058] (3) After centrifugation, load the sample or store it directly at -80℃.

[0059] 5.4 SDS-PAGE electrophoresis

[0060] (1) Prepare the gel: Use stacking gel and resolving gel. Adjust the concentration of the resolving gel according to the size of the protein (generally 10%-15%).

[0061] (2) Sample loading: Add an equal amount of protein sample or a small amount of protein molecular weight standard (marker) to each well;

[0062] (3) Electrophoresis: run in constant voltage or constant current mode (80V stacking gel, 120V separating gel).

[0063] 5.5 Transfer film

[0064] (1) Preparation of the transfer device: Use tweezers to pick up the cut PVDF membrane and place it in methanol for 10 seconds. After activation, transfer it to the transfer solution.

[0065] (2) Assemble the gel and membrane in the transfer clamp (ensure there are no air bubbles);

[0066] (3) Wet transfer conditions: constant voltage 95V, transfer for 95min.

[0067] 5.6 Blocking and Antibody Incubation

[0068] (1) Sealing: After the transfer, soak the membrane in 5% skim milk powder (prepared with TBST) and seal at room temperature for 1 hour or at 4°C overnight;

[0069] (2) Primary antibody incubation: Prepare the antibody according to the verified dilution ratio and incubate overnight on a shaker at 4°C;

[0070] (3) Washing the membrane: Wash the membrane with TBST for 5-10 minutes each time, repeat 3 times;

[0071] (4) Secondary antibody incubation: Transfer the membrane to the pre-prepared corresponding secondary antibody and incubate on a shaker at room temperature for 1 hour; Wash the membrane: Same as above.

[0072] 5.7 Development

[0073] 6. Immunohistochemistry

[0074] Mouse tumor tissue was placed in 5 ml EP tubes containing 4% paraformaldehyde and sent to the Pathology Department of Sun Yat-sen University Cancer Center for sectioning. After the samples were returned, the following experiments were performed:

[0075] (1) Baking slides: Place the glass slides in a 60℃ oven and bake for 1-2 hours. The specific time depends on the complete melting of the wax on the surface of the tissue as observed by the naked eye.

[0076] (2) Dewaxing: Immerse the glass slide in xylene substitute, change the solution every 10 minutes for a total of 3 times, at room temperature.

[0077] (3) Gradient alcohol hydration: Place the glass slides into 100%, 95%, 90%, 80%, and 70% ethanol in sequence, immersing them for 5-7 minutes at each concentration.

[0078] (4) Wash the slide with PBS 3 times, 3 minutes each time.

[0079] (5) Preparation of EDTA antigen retrieval solution: Dilute the EDTA retrieval solution with pH=8.0 at a volume ratio of 1:50 and dissolve it in ultrapure water.

[0080] (6) Completely immerse the slide in the above-mentioned EDTA antigen retrieval solution, place it in a microwave oven and heat on high for 10 minutes, then on medium-low for 10 minutes, and then let it cool naturally at room temperature for 1 hour. Add EDTA antigen retrieval solution once during the process to prevent the solution from reducing and causing the slide to not be completely immersed.

[0081] (7) Wash the slides with PBS 3 times, 3 minutes each time.

[0082] (8) Remove the slide and use an immunohistochemistry pen to draw a circle around the tissue section, being careful to avoid contact with the tissue.

[0083] (9) Perform subsequent immunohistochemical staining using the Kangwei Century DAB kit. First, add an appropriate amount of Solution A white solution (blocking solution), incubate at room temperature for 10 min, and then rinse thoroughly with PBS.

[0084] (10) Add an appropriate amount of white Solution B solution, incubate at room temperature for 10 min, and then spin dry.

[0085] (11) Primary antibody incubation: Dilute the primary antibody working solution with antibody diluent at a ratio of 1:100, then add an appropriate amount of diluted primary antibody working solution to cover the tissue section, place it in a humidified box to prevent the section from drying out, and incubate overnight at 4°C in a refrigerator.

[0086] (12) The next day, take the humidified box out of the refrigerator and let it warm up to room temperature for 10 minutes. Rinse the slides with PBS.

[0087] (13) Add an appropriate amount of Solution C yellow solution, incubate at room temperature for 10 min, and rinse with PBS.

[0088] (14) Add an appropriate amount of Solution D solution, incubate at room temperature for 10 min, and rinse with PBS.

[0089] (15) Prepare DAB colorimetric solution: DAB-A solution: DAB-B solution are mixed at a volume ratio of 1:19. Note that it should be prepared and used immediately.

[0090] (16) Add 50-100ul of DAB chromogenic solution according to the size of the tissue section, let it stand at room temperature for 5-7 minutes, and then wash it with distilled water (observe the color under a microscope to determine the duration of DAB action).

[0091] (17) Hematoxylin counterstaining: Add 50 μL of hematoxylin for counterstaining for 30 seconds to 1 minute. Rinse with PBS or tap water to restore blue color.

[0092] (18) Dehydration and clearing: Immerse the tissue sections in 70%, 80%, 90%, 95% and 100% ethanol gradients for 5 minutes each. After gradient dehydration, allow them to air dry naturally.

[0093] (19) Mounting: After drying, mount the slide with neutral resin. Read the slide after the neutral resin dries.

[0094] 8. Animal experiments

[0095] 8.1 Subcutaneous tumor formation and intraperitoneal administration in nude mice

[0096] (1) Animal preparation: 5-6 week old BALB / c nu / nu mice were purchased from Beijing Vital River and placed in the SPF-grade animal room of Sun Yat-sen University. They were stabilized and quarantined for one week in preparation for subsequent experiments.

[0097] (2) Cell preparation: Prepare cells according to the required amount to be injected, ensuring that the injected cells are in good condition and in the logarithmic growth phase. Digest the cells into a single-cell suspension and dilute with PBS to the appropriate number.

[0098] (3) Subcutaneous tumor formation: 1×10 6 B16 / MC38 cells were injected subcutaneously into nude mice to prevent cell suspension from leaking out.

[0099] (4) Preparation for drug injection: After the tumor has formed, intraperitoneal injection shall be started when the tumor grows to be visible to the naked eye. I3A / RSL3 shall be prepared in the ratio of DMSO:PEG300:Tween80:H2O = 3:30:10:57 (final concentration of RSL3 is 100mg / kg, and final concentration of I3A is 50mg / kg).

[0100] (5) Drug injection: The mice were fixed in a supine position with their heads facing down, and the drugs were injected into the abdominal cavity.

[0101] (6) Wait until the largest tumor tissue grows to 2000 mm 3 When necessary, mice are euthanized, tumors are removed, weighed, photographed, and fixed or cryopreserved for subsequent experiments.

[0102] 8.2 Gavage administration to nude mice

[0103] (1) The concentration of Lactobacillus reuteri cultured was detected based on the OD value.

[0104] (2) Centrifuge Lactobacillus reuteri at 4000 rpm for 5 min.

[0105] (3) Resuspend in PBS and centrifuge again at 4000 rpm for 5 min.

[0106] (4) Adjust the lactobacillus concentration to 1×10⁻⁶ using PBS. 10 CFU / ml.

[0107] (5) Mice were divided into PBS+PBS group, Lactobacillus reuteri+PBS group, PBS+RSL3 group, and Lactobacillus reuteri+RSL3 group.

[0108] (6) Each mouse was given 100ul PBS / Lactobacillus reuteri culture by gavage every day, and 100ul PBS or 100mg / kg RSL3 was injected intraperitoneally every three days until the end of the entire experimental period.

[0109] Experimental results

[0110] 1. I3A significantly inhibits ferroptosis

[0111] 1.1 In vitro experiments

[0112] (1) Through screening of 26 endogenous metabolites of tryptophan in vitro, it was found that indole formaldehyde I3A, an indole branch metabolite of tryptophan, has significant resistance to B16 ferroptosis of tumor cells.

[0113] 1) Schematic diagram of small molecule drug screening ( Figure 1 A).

[0114] 2) Tumor cells B16 were pretreated with various small molecule compounds for 2 hours, followed by treatment with classic ferroptosis inducers RSL3 (GPX4 inhibitor) or Erastin (xCT inhibitor) for 24 hours. The supernatant was collected, and the lactate dehydrogenase (LDH) content was measured and a heatmap was plotted. The results showed that I3A had the most significant inhibitory effect on ferroptosis. Figure 1 B).

[0115] 3) After pretreatment with I3A (500 μM) and the ferroptosis inhibitor Fer-1 (Ferrostatin-1) (10 μM) for 2 hours, followed by treatment with the ferroptosis inducer RSL3 (1 μM) for 24 hours, cells were stained with crystal violet. The results showed that I3A significantly inhibited RSL3-induced cell death. Figure 1 C).

[0116] 4) After pretreatment with I3A (500 μM) and the ferroptosis inhibitor Fer-1 (10 μM) for 2 hours, followed by treatment with the ferroptosis inducer RSL3 (1 μM) for 6 hours, the dead cells were stained with Zombie dye; flow cytometry results showed that I3A significantly reduced RSL3-induced cell death. Figure 1 D).

[0117] 5) Flow cytometry

[0118] After pretreatment with I3A (500 μM) and the ferroptosis inhibitor Fer-1 (10 μM) for 2 hours, followed by treatment with the ferroptosis inducer RSL3 (1 μM) for 3 hours, staining was performed using BODIPY-C11581 / 591, DFCH-DA, or mito-SOX fluorescent probes. Flow cytometry results showed that I3A significantly reduced the accumulation of RSL3-induced lipid peroxidation in tumor cells. Figure 1 (EH).

[0119] 1.2 Animal Experiments

[0120] 1×10 in the body 6 Wild-type B16 cells (WT-B16) or wild-type MC38 cells (WT-MC38) were subcutaneously injected into BALB / c nu / nu mice. When tumors were clearly visible, I3A (50 mg / kg, every other day) or the ferroptosis inducer RSL3 (100 mg / kg, twice a week) was injected intraperitoneally. Tumors were allowed to grow to 2000 mm. 3 Mice were euthanized, tumors were removed, photographed, and weighed. The tumor tissue was divided into three parts for: (1) detecting MDA content using a kit; (2) detecting 4-HNE content using a kit; and (3) detecting GPX4 and ferroptosis marker TFRC levels using immunohistochemistry. Three fields of view were randomly selected from the tumor tissue of each mouse for photographing. The experimental procedure is shown in the figure below. Figure 2 As shown in Figure A.

[0121] In vivo experimental results showed that I3A significantly inhibited the therapeutic effect of RSL3 on tumor cells B16 or MC38, reduced the levels of MDA and 4-HNE in mouse tumor tissues, rescued GPX4 levels, and inhibited the accumulation of the ferroptosis marker TFRC. Figure 2 (BN).

[0122] In summary, I3A, an indole metabolite of tryptophan, significantly inhibited tumor ferroptosis both in vivo and in vitro.

[0123] 2. I3A-dependent AHR transcriptional activity inhibits ferroptosis in tumor cells.

[0124] Numerous studies have reported that most metabolites of the tryptophan-indole branch (including I3A) are natural ligands of AHR (aromatic hydrocarbon receptor), inducing AHR entry into the cell nucleus and exerting transcriptional activity through binding to AHR. To investigate whether I3A-conferred ferroptosis resistance in tumor cells depends on its AHR activation activity, Ahr was knocked down or eliminated in B16 cells using siRNA or CRISPR-Cas9 technology, and Western blotting was used to verify the knockdown efficiency of Ahr in tumor cells. The following experiments were conducted to explore whether I3A's resistance to tumor cells depends on AHR and its transcriptional activity.

[0125] 1) Stable tumor cells B16 with knocked-down / non-knocked-down Ahr were treated with I3A (500 μM) and RSL3 (1 μM). After incubation with I3A / RSL3 for 24 hours, the cells were observed under a microscope and photographed. The results showed that when Ahr was knocked down, the inhibitory effect of I3A on ferroptosis completely disappeared. Figure 3 A).

[0126] 2) After pretreating tumor cells with I3A (500 μM) and the ferroptosis inhibitor Fer-1 (10 μM) for 2 hours, followed by treatment with the ferroptosis inducer RSL3 (1 μM) for 8 hours, the cells were stained with Annexin V / PI probe and analyzed by flow cytometry. The results showed that when Ahr was knocked down or eliminated, I3A could not protect cell survival; however, when WT-Ahr was reintroduced, the effect of I3A in protecting cells from ferroptosis was restored. Figure 3 ,BC).

[0127] 4) After pretreating tumor cells with I3A (500 μM) and the ferroptosis inhibitor Fer-1 (10 μM) for 2 hours, and then treating them with the ferroptosis inducer RSL3 (1 μM) for 3 hours, the cells were stained with BODIPY-C11581 / 591 or DCFH-DA fluorescent probes and analyzed by flow cytometry. The flow cytometry results showed that when Ahr was knocked down or eliminated, I3A no longer reduced the accumulation of RSL3-induced lipid peroxidation in tumor cells; however, when WT-Ahr was reintroduced, the protective effect of I3A against ferroptosis was restored; when Ahr with nuclear localization loss was reintroduced (the mutant dNLS-Ahr loses its transcriptional activity because Ahr cannot enter the nucleus), I3A still could not reduce the accumulation of RSL3-induced lipid peroxidation in tumor cells, nor could it protect cells from ferroptosis. Figure 3 ,DF).

[0128] In summary, the resistance of I3A to tumor cell ferroptosis depends entirely on the transcriptional activity of AHR.

[0129] 3. AHR confers resistance to ferroptosis to I3A by inhibiting the autophagic degradation of GPX4.

[0130] Glutathione peroxidase (GPX4) is a classic gene that inhibits ferroptosis. It uses glutathione (GSH) as a reducing agent to reduce lipid peroxides (LOOH) to harmless lipid alcohols (LOH), thereby preventing oxidative damage to membrane lipids. GPX4 effectively inhibits ferroptosis by scavenging lipid peroxides, protecting the cell membrane, and preventing the accumulation of reactive oxygen species (ROS). To investigate the role of GPX4 in regulating I3A's resistance to ferroptosis, I3A was administered to B16 cells (si1-Gpx4, si2-Gpx4) with GPX4 knockdown. It was found that when the GPX4 gene was absent in the cells, the resistance of I3A to ferroptosis was no longer present. Figure 4 , AB).

[0131] Western blot analysis of GPX4 protein levels in WT-B16 cells after I3A supplementation revealed that I3A significantly inhibited RSL3-induced GPX4 degradation, a finding also confirmed by immunofluorescence assays. Figure 4 When I3A was added to Ahr-knockout B16 cells (sgAhr-B16), Western blot analysis revealed that I3A was unable to inhibit RSL3-induced GPX4 degradation. This suggests that I3A may exert its anti-ferroptosis effect by targeting GPX4. To further verify this hypothesis, the Ahr gene was reintroduced into sgAhr-B16 cells (WT-Ahr). Western blot analysis showed that the protective effect of GPX4 protein levels caused by Ahr deficiency disappeared, but was restored upon reintroduction of wild-type Ahr (Ahr-OE). Figure 4 The above results suggest that I3A inhibits RSL3-induced GPX4 degradation through AHR activity, thus resisting ferroptosis.

[0132] In eukaryotic cells, damaged proteins or organelles are primarily cleared through two independent but interconnected pathways: the proteasome pathway and the lysosome pathway. Generally, the proteasome degrades short-lived proteins and soluble misfolded proteins via the ubiquitin-proteasome system, while the lysosome degrades long-lived proteins, insoluble protein aggregates, and other macromolecules, bacteria, and organelles via endocytosis, phagocytosis, or autophagy. Previous literature has reported that GPX4 can undergo autophagic degradation mediated by autophagy receptor molecules, or via ubiquitinated proteasome degradation mediated by molecular chaperones. To investigate the I3A-mediated inhibition of RSL3-induced GPX4 degradation, WT-B16 cells were pretreated with the late autophagy inhibitor CQ / BAF or the proteasome inhibitor MG132 for 2 hours before being incubated with RSL3 (1 μM). After 24 hours of incubation, cells were stained with crystal violet. The results showed that the autophagy inhibitor CQ / BAF significantly inhibited RSL3-induced cell death, suggesting that RSL3 induces autophagic degradation of GPX4. Figure 4 F)

[0133] To further investigate whether I3A is involved in regulating this process, sgAhr-B16 cells were treated with CQ / BAF / MG132 and supplemented with I3A. After a 2-hour pretreatment, RSL3 (1 μM) was added, and after 24 hours of incubation, the cells were stained with crystal violet. The results showed that the autophagy inhibitor CQ / BAF not only significantly inhibited RSL3-induced cell death but also further restored the anti-ferroptosis effect of I3A. The results of the LDH experiment further confirmed that, even in sgAhr cells, the addition of the autophagy inhibitor CQ / BAF still demonstrated that I3A retains its anti-ferroptosis effect. Figure 4 Conversely, in tumor cells B16 with knocked-down Gpx4, supplementation with the autophagy inhibitor CQ failed to rescue RSL3-induced ferroptosis. Figure 4 H).

[0134] In summary, the results show that AHR confers resistance to ferroptosis to I3A by inhibiting the autophagic degradation of GPX4.

[0135] 4. The AHR-JUN axis endows I3A with ferroptosis resistance by inhibiting GPX4 autophagic degradation.

[0136] The previous results confirmed that the I3A-AHR axis targets GPX4 to regulate ferroptosis. The next step is to explore how GPX4 participates in regulating I3A-mediated ferroptosis resistance. RNA-seq analysis revealed that co-incubation with I3A and RSL3 significantly inhibited the upregulation of hmox1 and c-JUN by RSL3. To investigate whether these two significantly altered genes are involved in regulating I3A-mediated ferroptosis resistance, homx1 (sghomx1) was knocked out in B16 cells using CRISPR-Cas9. Crystal violet staining showed no significant change in the sensitivity of sghomx1-B16 cells to RSL3, suggesting that changes in hmox1 may not be related to I3A-mediated ferroptosis. Figure 5 AC).

[0137] Next, the changes in total protein and phosphorylation levels of c-JUN after I3A and RSL3 supplementation were examined in WT or sgAhr-B16 cells and B16 cells replenished with Ahr (sgAhr-OE). Western blot analysis showed that RSL3 supplementation in WT-B16 cells induced a significant upregulation of c-JUN and pc-JUN protein levels, which was inhibited by I3A treatment. However, when Ahr was knocked out, the inhibitory effect of I3A on c-JUN and pc-JUN disappeared. This suggests that Ahr may negatively regulate the expression and activity of c-JUN. Figure 5 (DG).

[0138] Subsequently, tumor cells were treated with the c-JUN inhibitor JNK-VIII in combination with RSL3. It was found that JNK-VIII significantly inhibited RSL3-induced tumor cell death, and Western blotting revealed that JNK-VIII could rescue RSL3-induced GPX4 degradation. These results suggest that c-JUN may act as a downstream agent of AHR in regulating GPX4 degradation. Figure 5 ,HI).

[0139] Beclin1 plays a crucial role in the initiation of autophagy; its absence prevents autophagy activation. Furthermore, Bcl-2 and its family member Bcl-xl can bind to Beclin1 via hydrophobic grooves, thereby inhibiting the autophagy-inducing function of Beclin1 and its complex. Activation of c-JUN N-terminal kinase 1 (JNK1) leads to Bcl-2 phosphorylation, resulting in the dissociation of Bcl-2 from Beclin1 and activation of autophagy. In cardioblasts, JNK1 activation disrupts the Bcl-2 / Beclin1 interaction, promoting autophagy and resisting cardiomyocyte apoptosis. In cells supplemented with the c-JUN inhibitors JNK-VIII and RSL3, Beclin1 protein levels were significantly inhibited by JNK-VIII supplementation, suggesting that c-JUN may promote GPX4 autophagic degradation by activating Beclin1. Figure 5 AHR inhibits this process, protecting GPX4 protein levels and thus conferring I3A resistance to ferroptosis.

[0140] In summary, I3A inhibits GPX4 autophagy and degradation through the AHR-JUN axis, thereby suppressing ferroptosis.

[0141] 5. Lactobacillus reuteri is a source of I3A, which helps resist ferroptosis in colon cancer.

[0142] The gut microbiota *Lactobacillus reuteri* has the ability to produce the tryptophan metabolite I3A. The results mentioned above indicate that I3A has a significant inhibitory effect on ferroptosis in vivo and in vitro.

[0143] MC38 tumors were implanted in BALB / c nude mice and the mice were administered *Lactobacillus reuteri* via gavage. 16S rRNA analysis confirmed a significant increase in the relative abundance of *Lactobacillus reuteri* in the gavage group. LC-MS analysis demonstrated that gavage with *Lactobacillus reuteri* significantly increased the abundance of I3A in mouse serum. Figure 6 Furthermore, compared with PBS by gavage, the Lactobacillus reuteri group showed decreased sensitivity to RSL3 treatment, accompanied by a significant decrease in the lipid peroxidation end product MDA. Figure 6 These results validate that *Lactobacillus reuteri* can directly act on tumor cells by secreting I3A, thereby promoting subcutaneous tumor growth by inhibiting ferroptosis. Furthermore, analysis of the mRNA levels of AHR and GPX4 in colorectal cancer patients using the TCGA dataset revealed high expression of AHR and GPX4 in colorectal tumors compared to normal adjacent tissues, suggesting that ferroptosis resistance may play a role in tumor growth. Figure 6In colorectal tumor samples, the expression levels of AHR and JUN were negatively correlated, consistent with the experimental results mentioned above. Figure 6 H). Figure 6 The invention also illustrates the principle by which the gut microbiota metabolite I3A regulates tumor development by inhibiting ferroptosis. Figure 6 I).

[0144] application

[0145] This invention not only has significant value in basic research, but also provides innovative solutions in various practical application scenarios, specifically including the following aspects:

[0146] 1. Rapid construction of tumor models

[0147] Based on the technology of this invention, the ferroptosis process can be precisely controlled, and tumor ferroptosis-related drug resistance or invasion models can be established. These models can be used to study the tumor microenvironment, cell death mechanisms, and related molecular targets, providing a reliable platform for anti-tumor drug development.

[0148] 2. Drug screening and development

[0149] By intervening in gut microbiota metabolites, novel drug screening methods can be developed, such as small molecule compound screening models centered around I3A. This platform can be used to discover anti-ferroptosis drugs, optimize tumor treatment regimens, reduce the side effects of existing drugs, and improve treatment efficacy.

[0150] 3. Precision medicine and personalized treatment

[0151] Based on the characteristics of a patient's gut microbiota, this invention proposes a personalized treatment approach. By regulating the patient's gut microbiota, it precisely intervenes in the sensitivity of tumor cells to ferroptosis, providing a more efficient and targeted solution for cancer treatment.

[0152] 4. Novel anti-tumor combination therapy

[0153] This invention can be combined with existing treatments such as immunotherapy and chemotherapy, utilizing the anti-ferroptosis properties of I3A to improve efficacy or reduce treatment side effects. For example, by administering Lactobacillus reuteri via gavage, the toxicity of ferroptosis inducers can be reduced, leading to safer tumor treatment.

[0154] 5. Development of Biomaterials and Reagents

[0155] The key metabolites (such as I3A) and related biomaterials in this invention can be developed into experimental reagents that can be widely used in basic tumor research, cell function research and biomedical development.

[0156] Through these extended applications, the innovation and practicality of this invention are further enhanced, serving both basic scientific research and providing new ideas and technical support for tumor treatment and drug development.

Claims

1. Application of Lactobacillus reuteri and its metabolites in the preparation of drugs for regulating ferroptosis.

2. The application according to claim 1, characterized in that, The metabolite is indole-3-carboxaldehyde.

3. The application according to claim 1 or 2, characterized in that, The regulation includes inhibiting ferroptosis.

4. Application of Lactobacillus reuteri and its metabolites in the preparation of drugs for promoting tumor growth.

5. The application according to claim 4, characterized in that, The metabolite is indole-3-carboxaldehyde.

6. Application of Lactobacillus reuteri and its metabolites in establishing tumor ferroptosis-related drug resistance or invasion models.

7. The application according to claim 6, characterized in that, The metabolite is indole-3-carboxaldehyde.

8. Application of Lactobacillus reuteri and its metabolites in the preparation of drugs for reducing the toxicity of ferroptosis inducers.

9. The application according to claim 1, characterized in that, The metabolite is indole-3-carboxaldehyde.

10. Application of Lactobacillus reuteri and its metabolites in the preparation of drugs for inhibiting lipid peroxidation levels in tumor cells.

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