Bifidobacterium animalis subsp. Lactis with effect of inhibiting lung cancer growth and application

Abstract

The invention provides an animal bifidobacterium subsp. Lactis with an effect of inhibiting growth of lung cancer and application of the animal bifidobacterium subsp. Lactis. The animal bifidobacterium subsp. Lactis SDZL01 with the effect of inhibiting growth of lung cancer is obtained through separation and preserved, the animal bifidobacterium subsp. Lactis SDZL01 is named as an animal bifidobacterium subsp. Lactis SDZL01 strain, and the animal bifidobacterium subsp. Lactis SDZL01 strain is named as an animal bifidobacterium subsp. Lactis SDZL01 strain. The animal bifidobacterium subsp. Lactis is preserved in the China General Microbiological Culture Collection Center on November 18, 2024, the preservation number of the animal bifidobacterium subsp. Lactis is bifidobacterium with the preservation number of CGMCC No.32692, and the animal bifidobacterium subsp. Lactis has the effect of inhibiting the growth of lung cancer; the indole-3-acetic acid can be used for inhibiting the expression of STAT3 by down-regulating m6A modification. The strain can be used as a biological marker of lung cancer, can significantly prevent, improve or treat lung cancer, and prolongs the overall survival time of cancer patients.

Description

Technical Field

[0001] The present invention relates to the field of bifidobacteria, and in particular to an animal bifidobacterium lactis subspecies with the efficacy of inhibiting the growth of lung cancer and an application thereof. Background Art

[0002] Lung cancer is currently the most commonly diagnosed cancer and the leading cause of cancer death worldwide. In 2020, there were 820,000 new cases of lung cancer in my country, and 715,000 lung cancer-related deaths. Non-small cell lung cancer (NSCLC), as the main subtype of lung cancer, accounts for about 85% of lung cancer, which seriously threatens the life and health of people in my country and even the world. my country is a high-incidence area of ​​NSCLC, but the effects of treatment methods such as surgery, chemotherapy, and molecular targeted therapy are not satisfactory. Although immunotherapy has achieved good results in NSCLC patients in recent years, and the applicant has previously discovered that multiple biological markers can accurately screen potential patients who benefit from immunotherapy, the vast majority of advanced NSCLC patients develop resistance to immunotherapy and eventually progress, which leads to a poor prognosis for NSCLC patients. How to improve the poor prognosis of patients has always been a clinical difficulty faced by NSCLC patients in the treatment process. Therefore, in order to reduce the mortality rate of NSCLC patients, how to provide more effective and safe treatment methods for NSCLC patients is an urgent problem to be solved.

[0003] The intestinal flora has become a key player in maintaining human health, affecting not only the gastrointestinal organs, but also distal organs such as the brain, lungs, liver and heart. Changes in the composition and function of the intestinal flora, namely intestinal flora dysbiosis, can promote the occurrence and progression of various diseases, including obesity, diabetes, neurodegenerative diseases and cancer. The intestinal flora not only affects tumor progression, but also regulates the sensitivity of tumor chemotherapy, radiotherapy and immunotherapy. In lung cancer, the intestinal flora of patients is significantly changed compared with healthy people, including a decrease in the abundance of probiotics and a significant increase in the abundance of potential pathogens. The intestinal flora mainly affects the occurrence and development of lung cancer by regulating metabolic pathways, immune cell function and the production of proinflammatory factors through the gut-lung axis. In recent years, the role of intestinal flora in the treatment of NSCLC has also attracted much attention. Changes in intestinal flora are closely related to the immunotherapy and chemotherapy responses of NSCLC patients. For example, Akkermansia muciniphila in the intestine improves the efficacy of lung cancer immunotherapy by increasing the recruitment of CCR9+CXCR3+CD4+T lymphocytes; while intestinal flora imbalance promotes the transport of immunosuppressive cell subset Tr17 cells to tumor-draining lymph nodes, thereby leading to lung cancer immunotherapy resistance; in addition, the probiotic Clostridium butyricum can reduce chemotherapy-induced diarrhea and systemic inflammatory responses in lung cancer patients

[18] . The above studies all suggest that intestinal flora plays an important role in the progression of NSCLC.

[0004] Bifidobacterium is one of the main members of probiotics. Several recent new studies have shown that intestinal bifidobacteria can inhibit the progression of gastrointestinal tumors, but the role of intestinal bifidobacteria on tumors of distant organs has not yet been elucidated. The applicant's previous studies have shown that acetate, a metabolite of Bifidobacterium pseudolongum, can reach the liver cancer microenvironment through the intestinal-liver axis and act on the GPR43 receptor on the surface of liver cancer cells, thereby inhibiting the expression of the IL-6-JAK1-STAT3 cancer-promoting pathway, and ultimately hindering the progression of fatty liver-related liver cancer, suggesting that intestinal bifidobacteria can inhibit the occurrence and progression of tumors of distant organs by producing tumor suppressor metabolites. Although bifidobacteria play an important role in inhibiting tumor progression and regulating treatment sensitivity, the role of intestinal bifidobacteria on NSCLC has not yet been elucidated; therefore, we propose an animal bifidobacterium and its application that has the effect of inhibiting the growth of lung cancer. Summary of the invention

[0005] The purpose of the present invention is to address the problems raised by the current background technology. In order to achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: an animal bifidobacterium, including bifidobacterium, the bifidobacterium is named as Bifidobacterium animalis subsp. lactis SDZL01 strain; the animal Bifidobacterium lactis subsp. has been deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China General Microbiological Culture Collection Center on November 18, 2024, and its deposit number is CGMCC No. 32692 of the Bifidobacterium lactis subsp., and the animal Bifidobacterium lactis subsp. is used to inhibit the growth of lung cancer cells.

[0006] As a preferred technical solution of the present invention, it comprises Bifidobacterium animalis subsp. lactis SDZL01 strain and its metabolite indole-3-acetic acid.

[0007] The invention discloses an application of Bifidobacterium animalis subsp. lactis in inhibiting the growth of lung cancer. The Bifidobacterium animalis subsp. lactis SDZL01 strain has the application of inhibiting the growth of lung cancer.

[0008] As a preferred technical solution of the present invention, the indole-3-acetic acid can inhibit the expression of STAT3 by downregulating m6A modification.

[0009] As a preferred technical solution of the present invention, the Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid can activate the mechanism of AHR receptor regulating METTL3 / STAT3 to inhibit NSCLC.

[0010] As a preferred technical solution of the present invention, the Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid are used to regulate the expression of METTL3 and STAT3.

[0011] As a preferred technical solution of the present invention, the application of indole-3-acetic acid in combination with AHR receptor; the application of Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid in regulating the whole genome and STAT3 m6A modification.

[0012] As a preferred technical solution of the present invention, knocking out the AHR receptor can reverse the inhibitory effects of animal Bifidobacterium lactis subsp. lactis and indole-3-acetic acid on NSCLC.

[0013] As a preferred technical scheme of the present invention, knocking out the AHR receptor can reverse the inhibitory effect of animal Bifidobacterium lactis subspecies and indole-3-acetic acid on the expression of METTL3 and STAT3; knocking out the AHR receptor can reverse the regulatory effect of animal Bifidobacterium lactis subspecies and indole-3-acetic acid on m6A modification.

[0014] A method for preparing a product for preventing, improving or treating lung cancer based on the application of Bifidobacterium animalis subsp. lactis in inhibiting the growth of lung cancer, comprising mixing the Bifidobacterium animalis subsp. lactis SDZL01 strain with a pharmaceutically acceptable carrier.

[0015] Compared with the prior art, the present invention has the following beneficial effects: In the scheme of the present invention, the present invention isolates and preserves an animal Bifidobacterium lactis subspecies SDZL01 having the effect of inhibiting the growth of lung cancer, and names it as the animal Bifidobacterium lactis subspecies Bifidobacterium animalis subsp. lactis SDZL01 strain. The strain can be used as a biological marker for lung cancer, and can significantly prevent, improve or treat lung cancer, and prolong the overall survival time of cancer patients; therefore, the animal Bifidobacterium lactis subspecies Bifidobacterium animalis subsp. lactis SDZL01 strain can be used to prepare products for preventing, improving or treating lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The animal Bifidobacterium lactis subspecies provided by the present invention can inhibit the growth of Lewis lung cancer subcutaneous implanted tumors. (A) After Lewis lung cancer cells were subcutaneously implanted in C57BL / 6 male mice, 200uL 1x10 8 CFU of animal Bifidobacterium lactis, control bacteria and PBS were intragastrically administered every day; (BE) Animal Bifidobacterium lactis significantly inhibited the formation of NSCLC subcutaneous tumors; (F) The proportion of Ki-67 positive cells in tumor tissues of the animal Bifidobacterium lactis intragastrically administered group was significantly reduced; (G) The proportion of Tunel positive cells in tumor tissues of the animal Bifidobacterium lactis intragastrically administered group was significantly increased;

[0017] Figure 2The animal Bifidobacterium lactis subsp. conditioned medium (Ba-CM) provided by the present invention can inhibit the proliferation of human non-small cell lung cancer. (A) MTT results show that Ba-CM inhibits the viability of NSCLC cells (A549 and Calu-3); (B) Colony formation experiments confirm that Ba-CM inhibits the proliferation of NSCLC cells; (C) Ba-CM induces cell cycle G1 / S phase arrest in NSCLC cell lines; (D) Ba-CM promotes cell apoptosis in NSCLC cell lines; (E) Western blot further verifies that Ba-CM can regulate the protein levels of PCNA, Caspase3, Cleaved Caspase 3, and Cyclin D1 in NSCLC cell lines. NSCLC, Non-smallcell lung cancer; Ba-CM, B. animalis subsp. lactis conditioned medium;

[0018] Figure 3 The indole-3-acetic acid provided by the present invention is a key metabolite released by animal Bifidobacterium lactis subspecies. (A) Third-generation sequencing shows that the genes of animal Bifidobacterium lactis subspecies are mainly related to its metabolic function; (B) Principal component analysis of non-targeted metabolomics shows that the metabolites of animal Bifidobacterium lactis subspecies are significantly different from those of BHI controls; (C) KEGG enrichment analysis of non-targeted metabolomics finds that the metabolites of animal Bifidobacterium lactis subspecies are significantly related to the tryptophan biosynthesis pathway; (D) Targeted metabolomics of bacterial supernatant shows that tryptophan and indole-related metabolites in animal Bifidobacterium lactis subspecies are significantly different from those of BHI controls, and a heat map shows that indole-3-acetic acid is the most significantly increased metabolite in animal Bifidobacterium lactis subspecies; (E) Experimental design of germ-free mouse model; (F) Targeted metabolomics analysis of germ-free mice shows that indole-3-acetic acid is the only metabolite that is significantly increased in feces, serum and lung tissue of germ-free mice immunized with animal Bifidobacterium lactis subspecies. Ba-CM,B.animalis subsp.lactis conditioned medium; IAA,Indole-3-acetic acid;

[0019] Figure 4The indole-3-acetic acid provided by the present invention inhibits the progression of NSCLC both in vivo and in vitro. (AB) After Lewis lung cancer cells were implanted subcutaneously in C57BL / 6 male mice, indole-3-acetic acid and PBS were gavaged daily; indole-3-acetic acid significantly inhibited the formation of NSCLC subcutaneous tumors; (C) MTT results showed that indole-3-acetic acid could inhibit the viability of A549 cells; (D) indole-3-acetic acid inhibited the cell cycle G1 / S phase transformation of A549 cell line; (E) indole-3-acetic acid promoted cell apoptosis of A549 cell line; (F) Western blot further verified that indole-3-acetic acid could regulate the protein levels of PCNA, Caspase 3, Cleaved Caspase 3, and Cyclin D1 in NSCLC cell lines. NSCLC, Non-small cell lung cancer; IAA, Indole-3-acetic acid;

[0020] Figure 5 The animal Bifidobacterium lactis subspecies and metabolites provided by the present invention may inhibit the progression of NSCLC through the AHR / METTL3 / STAT3 axis. (A) Transcriptome analysis showed that after oral administration of animal Bifidobacterium lactis subspecies, mRNA transcription and stability-related pathways were significantly downregulated; the expression of METTL16 and METTL3 in the m6A key molecules was most significantly downregulated in the animal Bifidobacterium lactis subspecies group; (B) qRT-PCR found that the expression of METTL3 was significantly decreased in the animal Bifidobacterium lactis subspecies treatment group; (C) Western blot further verified that METTL3 was significantly decreased in A549 cells treated with Ba-CM or indole-3-acetic acid; (D) Transcriptome analysis showed that the STAT3 signaling pathway was significantly downregulated in the animal Bifidobacterium lactis subspecies gavage group; (E) Molecular docking predicted that indole-3-acetic acid and AHR can be stably combined. Ba-CM, B. animalis subsp. lactis conditioned medium; IAA, Indole-3-acetic acid; NSCLC, Non-small cell lung cancer. DETAILED DESCRIPTION

[0021] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0022] Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely represents some embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features and technical solutions in the embodiments can be combined with each other without conflict. It should be noted that similar numbers and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0023] Example 1: An animal Bifidobacterium lactis subspecies, including Bifidobacterium, the Bifidobacterium is named as Bifidobacterium animalis subsp. lactis SDZL01 strain; the animal Bifidobacterium has been deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China General Microbiological Culture Collection Center on November 18, 2024, and its preservation number is CGMCC No. 32692. The animal Bifidobacterium is used to inhibit the growth of lung cancer cells.

[0024] Bifidobacterium animalis subsp. lactis SDZL01 strain and its metabolite indole-3-acetic acid. Application of Bifidobacterium animalis subsp. lactis in inhibiting the growth of lung cancer. Bifidobacterium animalis subsp. lactis SDZL01 strain has the application of inhibiting the growth of lung cancer.

[0025] Indole-3-acetic acid can inhibit the expression of STAT3 by downregulating m6A modification.

[0026] The Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid can activate the AHR receptor to regulate METTL3 / STAT3 to inhibit NSCLC.

[0027] The regulation of METTL3 and STAT3 expression by Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid.

[0028] The application of indole-3-acetic acid binding to AHR receptor; Bifidobacterium animalis subsp. lactis SDZL01 strain and the regulation of indole-3-acetic acid on the whole genome and STAT3 m6A modification.

[0029] Knocking out the AHR receptor can reverse the inhibitory effects of Bifidobacterium lactis subsp. animalis and indole-3-acetic acid on NSCLC.

[0030] Knocking out the AHR receptor can reverse the inhibitory effects of animal Bifidobacterium lactis subsp. lactis and indole-3-acetic acid on the expression of METTL3 and STAT3; Knocking out the AHR receptor can reverse the regulatory effects of animal Bifidobacterium lactis subsp. lactis and indole-3-acetic acid on m6A modification.

[0031] A method for preparing a product for preventing, improving or treating lung cancer based on the application of Bifidobacterium animalissubsp. lactis in inhibiting the growth of lung cancer, comprising mixing the Bifidobacterium animalissubsp. lactis SDZL01 strain with a pharmaceutically acceptable carrier.

[0032] Experimental example:

[0033] Part I: The experiment clarified that animal Bifidobacterium lactis subsp. lactis and its metabolite indole-3-acetic acid inhibit the progression of NSCLC.

[0034] (1) Experimental verification of the reduction in the abundance of Bifidobacterium animalis subsp. lactis in the feces and the indole-3-acetic acid content in the serum of NSCLC patients: Fecal and serum samples of NSCLC patients and healthy controls were collected to detect the abundance of Bifidobacterium animalis subsp. lactis in the feces of patients and the content of indole-3-acetic acid in the serum. The correlation between Bifidobacterium animalis subsp. lactis and indole-3-acetic acid and the clinical pathological characteristics and prognosis of NSCLC patients, such as staging and grading, was verified.

[0035] (2) Experimental verification of the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on NSCLC: C57BL / 6 mice were used as animal templates, and an orthotopic tumor model was constructed using the mouse NSCLC cell line CMT167. The mice were gavaged with animal Bifidobacterium lactis, indole-3-acetic acid, and PBS, respectively, to clarify the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on NSCLC; nude mice were used as animal templates, and a subcutaneous tumor model was constructed using the human NSCLC cell line A549. The mice were gavaged with animal Bifidobacterium lactis, indole-3-acetic acid, and PBS, respectively, to further confirm the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on NSCLC.

[0036] (3) Experimental verification of the abundance of Bifidobacterium lactis subsp. animalis in mice inoculated with Bifidobacterium lactis subsp. animalis: feces of mice inoculated with Bifidobacterium lactis subsp. animalis at different time points were collected, and the qRT-PCR technology was used to detect the changes in the abundance of Bifidobacterium lactis subsp. animalis in the feces of mice inoculated with Bifidobacterium lactis subsp. animalis; intestinal tissues of mice inoculated with Bifidobacterium lactis subsp. animalis were collected, and the colonization of Bifidobacterium lactis subsp. animalis in the intestinal tissues of mice was detected by PCR technology.

[0037] (4) Experimental verification of the content of indole-3-acetic acid in mice inoculated with animal Bifidobacterium lactis subsp. lactis: Feces, serum, lung tissue and tumor tissue were collected from the mouse models of orthotopic tumors and subcutaneous tumors inoculated with animal Bifidobacterium lactis subsp. lactis, and targeted metabolomics was used to verify whether indole-3-acetic acid was increased in the feces, serum, lung tissue and tumor tissue of mice inoculated with animal Bifidobacterium lactis subsp. lactis.

[0038] Part 2: The experiment illustrates that animal Bifidobacterium lactis subsp. lactis and indole-3-acetic acid downregulate m6A modification through METTL3 and thus inhibit the expression of STAT3.

[0039] (1) The experiment clarified the regulation of METTL3 and STAT3 expression by Bifidobacterium animalis subsp. lactis and indole-3-acetic acid: Human NSCLC cells were treated with culture supernatant of Bifidobacterium animalis subsp. lactis (Ba-CM) and indole-3-acetic acid, and then the cell pellets were collected and the expression changes of methyltransferase METTL3, STAT3 and phosphorylated STAT3 were detected. In addition, the expression differences of METTL3, STAT3 and phosphorylated STAT3 in tumor tissues of mice gavaged with Bifidobacterium animalis subsp. lactis and indole-3-acetic acid and control mice were detected.

[0040] (2) Experiments to clarify the regulation of Bifidobacterium animalis subsp. lactis and indole-3-acetic acid on whole-genome and STAT3 m6A modification: Human NSCLC cells were treated with Ba-CM and indole-3-acetic acid, and then the cell pellets were collected to extract RNA. MeRIP-Seq / m6A-Seq technology was used to verify the regulatory effects of Bifidobacterium animalis subsp. lactis and its metabolite indole-3-acetic acid on whole-genome m6A methylation modification and STAT3 m6A methylation modification in NSCLC cells.

[0041] Part III: The mechanism by which Bifidobacterium animalis subsp. lactis and indole-3-acetic acid activate AHR receptor to regulate METTL3 / STAT3 and inhibit NSCLC.

[0042] (1) The specific mechanism of IAA binding to AHR receptor was investigated by using surface plasmon resonance (SPR) technology to analyze the binding affinity of IAA to AHR receptor. The IAA pull-down experiment was used to explore whether IAA could directly bind to the recombinant AHR receptor protein.

[0043] (2) Whether experimental knockout of the AHR receptor can reverse the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on NSCLC: The AHR receptor of the human NSCLC cell line was knocked out, and the human NSCLC cells with AHR receptor knockout were treated with Ba-CM and indole-3-acetic acid. In vitro experiments were conducted to explore whether the inhibitory effects of Ba-CM and indole-3-acetic acid on NSCLC were eliminated after the knockout of the AHR receptor. A subcutaneous tumor model was constructed using the human NSCLC cell line with AHR receptor knockout, and mice were gavaged with animal Bifidobacterium lactis, indole-3-acetic acid and PBS to determine whether the inhibitory effects of animal Bifidobacterium lactis and indole-3-acetic acid on the growth of subcutaneous tumors in mice were eliminated after the knockout of the AHR receptor.

[0044] (3) Whether experimental knockout of the AHR receptor can reverse the inhibitory effects of Bifidobacterium animalis subsp. lactis and indole-3-acetic acid on the expression of METTL3 and STAT3: The AHR receptor of the human NSCLC cell line was knocked out, and the human NSCLC cells with AHR receptor knockout were treated with Ba-CM and indole-3-acetic acid, and the expression changes of METTL3, STAT3 and phosphorylated STAT3 were detected by qRT-PCR and Western blot techniques; a subcutaneous tumor model was constructed using the human NSCLC cell line with AHR receptor knockout, and mice were gavaged with Bifidobacterium animalis subsp. lactis, indole-3-acetic acid and PBS. The tumor tissues were collected to extract RNA and protein, and the expression changes of METTL3, STAT3 and phosphorylated STAT3 were detected by qRT-PCR and Western blot techniques.

[0045] (4) Whether experimental knockout of the AHR receptor can reverse the regulatory effects of animal Bifidobacterium lactis and indole-3-acetic acid on m6A modification: The AHR receptor of human NSCLC cells was knocked out, and the human NSCLC cells with AHR receptor knockout were treated with Ba-CM and indole-3-acetic acid. The cell pellets were collected to extract RNA. The MeRIP-Seq / m6A-Seq technology was used to explore whether after knocking out the AHR receptor, animal Bifidobacterium lactis and its metabolite indole-3-acetic acid had a regulatory effect on m6A methylation modification and STAT3 m6A methylation modification in the whole NSCLC genome.

[0046] Specific experimental process:

[0047] (1) Bifidobacterium animalis subsp. lactis inhibits the progression of NSCLC both in vivo and in vitro.

[0048] In order to verify the effect of animal Bifidobacterium lactis subsp. lactis on NSCLC, we orally administered the isolated animal Bifidobacterium lactis subsp. lactis strains into Lewis lung cancer subcutaneous tumor-implanted mouse models, and simultaneously orally administered PBS and other bifidobacteria isolated from healthy human feces as control groups ( Figure 1 A), the results showed that animal Bifidobacterium lactis subsp. can significantly inhibit tumor growth ( Figure 1 BD) and significantly reduced the tumor burden in mice ( Figure 1 E). In addition, animal Bifidobacterium lactis subsp. lactis can significantly reduce the number of Ki-67 positive cells in tumor tissues ( Figure 1 F), and increased the number of Tunel-positive cells ( Figure 1 G). These results suggest that Bifidobacterium animalis subsp. lactis can inhibit the progression of NSCLC in vivo.

[0049] To further clarify the effect of animal Bifidobacterium lactis subsp. lactis on NSCLC, we used the culture supernatant of animal Bifidobacterium lactis subsp. lactis (Ba-CM) to treat human NSCLC cell lines (A549 and Calu-3) and normal lung epithelial cell line (BEAS-2B). Both cell proliferation and colony formation experiments showed that Ba-CM could significantly inhibit the viability of A549 and Calu-3 cell lines, but had no effect on the normal lung epithelial BEAS-2B cell line ( Figure 2 In addition, Ba-CM could change the cell cycle of A549 and Calu-3 cell lines, including increasing the proportion of cells in G1 phase and decreasing the proportion of cells in S phase ( Figure 2 C), and can induce apoptosis in A549 and Calu-3 cells ( Figure 2 D). At the same time, we used Western blot to further verify that Ba-CM treatment of cells can reduce the expression level of cell proliferation index PCNA and cell cycle regulatory molecule CyclinD1, and increase the expression level of apoptosis-related protein CleavedCaspase 3 ( Figure 2 E). These results suggest that Bifidobacterium animalis subsp. lactis can inhibit the proliferation of NSCLC cells in vitro, induce cell cycle arrest and promote apoptosis.

[0050] (2) Indole-3-acetic acid is a key metabolite released by Bifidobacterium animalis subsp. lactis.

[0051] In order to clarify what substances animal Bifidobacterium lactis subsp. lactis produces to regulate the progression of NSCLC, we first performed functional annotation analysis on the third-generation sequencing results of animal Bifidobacterium lactis subsp. lactis and found that the genes of animal Bifidobacterium lactis subsp. lactis were mainly related to its metabolic function ( Figure 3 A). Therefore, animal Bifidobacterium lactis subsp. lactis exerts its effects mainly by releasing metabolites. We further performed non-targeted metabolomics analysis on Ba-CM and BHI controls, and principal component analysis showed that the metabolites of animal Bifidobacterium lactis subsp. lactis were significantly different from those of BHI controls ( Figure 3 B), KEGG enrichment analysis found that the metabolites of Bifidobacterium animalis subsp. lactis were significantly associated with the tryptophan biosynthesis pathway ( Figure 3 C). Therefore, we speculate that animal B. lactis subsp. lactis exerts its effects mainly by producing tryptophan-related metabolites. We then performed metabolomics analysis targeting the tryptophan pathway on Ba-CM and BHI controls. Principal component analysis revealed that tryptophan / indole-related metabolites in animal B. lactis subsp. lactis were significantly different from those in BHI controls ( Figure 3 D). To further clarify what tryptophan / indole-related metabolites produced by animal Bifidobacterium lactis subsp. lactis reach the lungs through the "gut-lung axis" to exert their effects, we constructed a germ-free mouse model and gavaged the germ-free mice with animal Bifidobacterium lactis subsp. lactis and PBS, respectively ( Figure 3 E). At the end of the experiment, we collected feces, serum, and lung tissues from germ-free mice for metabolomics analysis targeting the tryptophan / indole pathway. The results showed that indole-3-acetic acid was the only one that was both increased in Ba-CM ( Figure 3 D), and metabolites significantly increased in feces, serum and lung tissues of germ-free mice inoculated with animal Bifidobacterium lactis subsp. Figure 3 F). In summary, we preliminarily confirmed that indole-3-acetic acid is a key metabolite produced by animal Bifidobacterium lactis subsp.

[0052] (3) Indole-3-acetic acid inhibits NSCLC progression in vitro and in vivo.

[0053] In order to clarify whether Bifidobacterium animalis subsp. lactis exerts its inhibitory effect on NSCLC through indole-3-acetic acid, we used indole-3-acetic acid and PBS to gavage Lewis lung cancer subcutaneous tumor mouse model ( Figure 4 A), the results showed that indole-3-acetic acid can significantly inhibit the growth of NSCLC tumors ( Figure 4 AB). To further clarify the effect of indole-3-acetic acid on NSCLC, we treated A549 cell line with indole-3-acetic acid and PBS respectively. Cell proliferation experiments showed that indole-3-acetic acid could significantly inhibit the proliferation of A549 cells ( Figure 4 C). In addition, IAA can significantly change the A549 cell cycle, including increasing the proportion of cells in the G1 phase and decreasing the proportion of cells in the S phase ( Figure 4 D) and can induce apoptosis of A549 cells ( Figure 4E). At the same time, we further used Western blot technology to verify that indole-3-acetic acid can significantly reduce the expression level of proliferation index PCNA and cell cycle regulatory protein CyclinD1, and increase the expression level of apoptosis-related protein Cleaved Caspase 3 ( Figure 4 F). These results suggest that indole-3-acetic acid, a metabolite of Bifidobacterium animalis subsp. lactis, inhibits the progression of NSCLC both in vitro and in vivo.

[0054] (4) Bifidobacterium animalis subsp. lactis and its metabolite indole-3-acetic acid may inhibit the progression of NSCLC through the AHR / METTL3 / STAT3 axis.

[0055] To explore the specific mechanism of Bifidobacterium lactis and its metabolite indole-3-acetic acid in inhibiting NSCLC, we performed transcriptome sequencing analysis on Lewis lung cancer subcutaneous tumors administrated with Bifidobacterium lactis or PBS. The results showed that Bifidobacterium lactis was closely related to the pathway regulating mRNA transcription and stability ( Figure 5 A). Previous studies have shown that m6A, as the most abundant internal mRNA modification, plays multiple roles in various steps of mRNA processing and metabolism. Therefore, we speculated that animal Bifidobacterium lactis subsp. lactis can regulate m6A modification. We then screened known m6A key molecules, and the heat map analysis of transcriptome data showed that the expression of methyltransferases METTL16 and METTL3 was most significantly downregulated in the animal Bifidobacterium lactis subsp. lactis group ( Figure 5 A). Further qRT-PCR and Western blot analysis revealed that METTL3 was significantly decreased in the Ba-CM or I3A treatment groups, so we speculated that animal Bifidobacterium lactis subsp. lactis and I3A mainly regulated the expression of METTL3 ( Figure 5 BC). Previous studies have shown that METTL3 can induce m6A modification of STAT3 mRNA to promote tumor progression. We further analyzed the transcriptome data and found that the STAT3 signaling pathway was significantly downregulated in the animal Bifidobacterium lactis subspecies ( Figure 5 D). Based on the above, we speculate that animal Bifidobacterium lactis subsp. lactis and its metabolite indole-3-acetic acid affect the m6A modification of STAT3 by downregulating the expression of METTL3, thereby inhibiting the progression of NSCLC. AHR is a key receptor for indole metabolites to exert their effects in the body, so we used molecular docking to predict the binding activity of indole-3-acetic acid with AHR. The results showed that indole-3-acetic acid can stably bind to AHR ( Figure 5E). Studies have shown that AHR activation can directly inhibit the transcription of METTL3 and thus regulate the changes in RNA methylation in the whole genome. The above results suggest that indole-3-acetic acid is likely to inhibit the m6A methylation modification of STAT3 by activating the AHR receptor and downregulating METTL3, thereby playing a role in preventing the progression of NSCLC.

[0056] The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Although the present invention has been described in detail with reference to the above embodiments, the present invention is not limited to the above specific implementation methods. Therefore, any modification or equivalent replacement of the present invention; and all technical solutions and improvements that do not depart from the spirit and scope of the invention are included in the scope of the claims of the present invention.

Claims

1. An animal Bifidobacterium lactis subsp., characterized in that Including bifidobacterium, the bifidobacterium is named as Bifidobacterium animalis subsp. lactis SDZL01 strain; the Bifidobacterium animalis subsp. lactis has been deposited in the China General Microbiological Culture Collection Center (CGMCC) of the China General Microbiological Culture Collection Center on November 18, 2024, and its preservation number is CGMCC No. 32692. The Bifidobacterium animalis subsp. lactis is used to inhibit the growth of lung cancer cells.

2. An animal Bifidobacterium according to claim 1, characterized in that It contains Bifidobacterium animalis subsp. lactis SDZL01 strain and its metabolite indole-3-acetic acid.

3. The use of animal Bifidobacterium lactis subspecies in inhibiting the growth of lung cancer according to any one of claims 1 to 2, characterized in that: The Bifidobacterium animalis subsp. lactis SDZL01 strain has the application of inhibiting the growth of lung cancer.

4. The use of animal Bifidobacterium lactis subspecies in inhibiting the growth of lung cancer according to claim 3, characterized in that: Indole-3-acetic acid can inhibit the expression of STAT3 by downregulating m6A modification.

5. The use of animal Bifidobacterium lactis subspecies in inhibiting the growth of lung cancer according to claim 4, characterized in that: The Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid can activate the AHR receptor to regulate the mechanism of METTL3 / STAT3 to inhibit NSCLC.

6. The use of animal Bifidobacterium lactis subspecies in inhibiting the growth of lung cancer according to claim 5, characterized in that: The Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid are used to regulate the expression of METTL3 and STAT3.

7. The use of animal Bifidobacterium lactis subspecies in inhibiting the growth of lung cancer according to claim 6, characterized in that: The application of indole-3-acetic acid in combination with AHR receptor; the Bifidobacterium animalis subsp. lactis SDZL01 strain and indole-3-acetic acid in regulating the whole genome and STAT3 m6A modification.

8. The use of animal Bifidobacterium lactis subspecies in inhibiting the growth of lung cancer according to claim 7, characterized in that: Knocking out the AHR receptor can reverse the inhibitory effects of Bifidobacterium lactis subsp. animalis and indole-3-acetic acid on NSCLC.

9. The use of animal Bifidobacterium lactis subspecies in inhibiting the growth of lung cancer according to claim 8, characterized in that: Knocking out the AHR receptor can reverse the inhibitory effects of animal Bifidobacterium lactis subsp. lactis and indole-3-acetic acid on the expression of METTL3 and STAT3; Knocking out the AHR receptor can reverse the regulatory effects of animal Bifidobacterium lactis subsp. lactis and indole-3-acetic acid on m6A modification.

10. A method for preparing a product for preventing, improving or treating lung cancer based on the efficacy of Bifidobacterium animalis subsp. lactis in inhibiting the growth of lung cancer, comprising mixing the Bifidobacterium animalis subsp. lactis SDZL01 strain according to any one of claims 1 to 2 with a pharmaceutically acceptable carrier.

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