Use of a paracasei IOB413 probiotic and compositions thereof in gastric cancer

Abstract

This invention provides an application of *Lactobacillus paracasei* IOB413 metabiotic and its composition in gastric cancer, belonging to the field of microbial technology. *Lactobacillus paracasei* IOB413 has the preservation number CGMCC No. 16022. Its metabiotic can inhibit gastric cancer progression by suppressing epithelial-mesenchymal transition in tumor cells and regulating the IL-6 / JAK2 / STAT3 signaling pathway. The composition with folic acid has a synergistic effect, more effectively reducing tumor volume and lowering the levels of pro-inflammatory factors and tumor markers. It has high safety and is suitable as an adjuvant therapy before and after gastric cancer surgery, providing a new approach to gastric cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of a postbiotic of Lactobacillus paracasei IOB413 and its composition in gastric cancer. Background Technology

[0002] Gastric cancer (GC) is one of the most common malignant tumors, ranking 5th in incidence and 4th in mortality worldwide. Currently, 5-fluorouracil (5-FU), a chemotherapy drug for gastric cancer, has anti-cancer effects, but it has significant side effects. Therefore, there is still a need to find effective drugs to treat stomach pain and slow its progression. Studies have found that overactivation of the interleukin-6 (IL-6) / Janus kinase 2 (JAK2) / signal transducer and activator of transcription 3 (STAT3) pathway can promote epithelial-mesenchymal transition (EMT) in tumor cells, which is closely related to tumor progression. Other studies have shown that traditional Chinese medicine can inhibit cell invasion, metastasis, and EMT through the IL-6 / JAK2 / STAT3 signaling pathway.

[0003] Treatment methods for gastric cancer, such as chemotherapy, targeted therapy, immune checkpoint inhibitor therapy, and cell therapy, have made significant progress in the past decade or so. Fluorouracil, platinum, and taxanes are the main chemotherapy drugs for gastric cancer. First-line chemotherapy is based on fluorouracil drugs, combined with platinum and / or taxanes to form two- or three-drug chemotherapy regimens, while second-line chemotherapy is usually taxane-based. Studies have shown that perioperative treatment, including preoperative neoadjuvant therapy and postoperative adjuvant chemotherapy, can effectively improve the 5-year survival rate of gastric cancer patients. For example, in China and Japan, the basic treatment principle for stage II and III operable gastric cancer is surgery plus postoperative adjuvant chemotherapy.

[0004] It is generally recommended that gastric cancer patients undergo adjuvant therapy before or after surgery. This is to ensure that the patient's physical condition and strength are in good condition before surgery, enabling them to tolerate high-intensity treatment, and to better recover bodily functions after surgery, thus achieving better treatment results. Therefore, there is an urgent need for a clinical adjuvant therapy method that has extremely low side effects on the patient's body before or after surgery, and can even regulate the patient's gastrointestinal health. Summary of the Invention

[0005] In order to overcome the above-mentioned defects and deficiencies in the prior art, this application aims to provide the application of Lactobacillus paracasei IOB413 postbiotic and its composition in gastric cancer.

[0006] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei This strain was independently selected from naturally fermented sourdough from a resident's home in Tianjin. The strain has undergone strain preservation and physicochemical testing. On March 19, 2021, bacterial identification testing was conducted at the China National Research Institute of Food Fermentation Industries Co., Ltd. The colonies were white, round, moist, opaque, and with neat edges, and were classified and named *Lactobacillus paracasei*. Lacticaseibacillus paracasei It was deposited on June 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16022.

[0007] One of the objectives of this invention is to provide an application of Lactobacillus paracasei IOB413 postbiotic in the preparation of a drug for treating gastric cancer, wherein the Lactobacillus paracasei IOB413 has the accession number CGMCC No. 16022.

[0008] Preferably, the method for preparing the postbiotic of *Lactobacillus paracasei* IOB413 includes:

[0009] The bacterial strain in the cryopreservation tube was inoculated into a slant culture medium and cultured at 36.5-37.5℃ for 18-24 hours to obtain a slant culture. The slant culture was then inoculated into a liquid culture medium containing 2%-4% of, but not limited to, soybean and yam, and cultured at 34-38℃ in a sealed, static environment for 18-22 hours to obtain a seed culture.

[0010] The seed culture is inoculated into a culture medium including but not limited to soybeans and yams at a material-to-liquid ratio of 1:1.5. It is then cultured in a sealed, static environment at 34-38℃ for 24-48 hours. After fermentation, the culture is inactivated at 80-95℃, dried until the moisture content is ≤10%, and then pulverized to obtain Lactobacillus paracasei IOB413 post-biotic.

[0011] Preferably, the slant culture medium comprises: 4.0-7.0 g peptone, 4.0-7.0 g beef extract, 5.0-7.0 g yeast powder, 13.0-18.0 g glucose, 0.8-1.0 mL Tween 80, 1.5-2.0 g dipotassium hydrogen phosphate, 3.0-4.0 g sodium acetate, 1.0-1.2 g triammonium citrate, 0.1-0.2 g magnesium sulfate, 0.03-0.05 g manganese sulfate, 12-15.0 g agar, pH=6.2±0.2, and 1-1.2 L of water.

[0012] The second objective of this invention is to provide an application of Lactobacillus paracasei IOB413 postbiotic in the preparation of a microecological preparation that inhibits the growth of gastric cancer tumors.

[0013] The third objective of this invention is to provide an application of a composition of *Lactobacillus paracasei* IOB413 post-biotic in the preparation of a medicament for treating gastric cancer, characterized in that the *Lactobacillus paracasei* IOB413 has the accession number CGMCC No. 16022; and the composition comprises *Lactobacillus paracasei* IOB413 post-biotic and folic acid.

[0014] Preferably, the mass ratio of the Lactobacillus paracasei IOB413 postbiotic to the folic acid in the composition is 315:1.

[0015] The beneficial effects of this invention are:

[0016] This invention is the first to discover that *Lactobacillus paracasei* IOB413 postbiotic can inhibit gastric cancer progression through a dual pathway: on the one hand, it can effectively inhibit the epithelial-mesenchymal transition (EMT) process of tumor cells, upregulate the expression of the epithelial marker E-cadherin, and downregulate the expression of the mesenchymal markers N-cadherin and Vimentin, thereby reducing the invasive and metastatic potential of tumor cells; on the other hand, it can significantly regulate the IL-6 / JAK2 / STAT3 signaling pathway, reduce the expression of key proteins such as IL-6, p-JAK2, and p-STAT3, block the overactivation of this pathway, and thus inhibit tumor growth.

[0017] The composition of *Lactobacillus paracasei* IOB413 post-biotic and folic acid of this invention exhibits a significant synergistic effect in inhibiting gastric cancer tumor growth. Experimental results show that the combined treatment group is superior to either post-biotic or folic acid alone in reducing tumor volume, downregulating pro-inflammatory factors (IL-6, IL-1β, TNF-α), reducing tumor marker levels (CEA, CA199), and improving pathological tissue morphology. This suggests that the combined use of the two can further enhance the inhibitory effect on gastric cancer, providing a better treatment option for clinical practice.

[0018] Lactobacillus paracasei IOB413 is derived from naturally fermented sourdough. Its postbiotic preparation process uses food-grade raw materials (such as soybeans and yams) and a gentle culture and inactivation process, resulting in high safety and extremely low side effects. Compared to traditional chemotherapy drugs (such as 5-fluorouracil), it not only inhibits tumor progression but also regulates the patient's gastrointestinal health, making it particularly suitable for pre- or post-operative use in gastric cancer patients. Pre-operatively, it can improve the patient's physical and mental state, enhancing tolerance to high-intensity treatments; post-operatively, it can promote physical recovery and assist in improving treatment efficacy, addressing the issue of existing adjuvant therapies potentially placing an additional burden on the patient's body.

[0019] In summary, the Lactobacillus paracasei IOB413 postbiotic and its composition with folic acid of the present invention have advantages such as significant effect, clear mechanism, high safety, strong synergy and ease of production in inhibiting the growth of gastric cancer tumors, providing a new and effective means for the clinical treatment of gastric cancer, especially for preoperative and postoperative adjuvant therapy. Attached Figure Description

[0020] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention, wherein:

[0021] Figure 1 Images showing the growth of tumors in each group of nude mice;

[0022] Figure 2 The growth curve of tumor volume during gavage in each group of nude mice;

[0023] Figure 3 A graph showing the expression of IL-6 in the serum of nude mice in each group;

[0024] Figure 4 Figure showing the expression of IL-1β in the serum of nude mice in each group;

[0025] Figure 5 Figure showing the expression of TNF-α in the serum of nude mice in each group;

[0026] Figure 6 A graph showing the expression of CEA in the serum of nude mice in each group;

[0027] Figure 7 A graph showing the expression of CA199 in the serum of nude mice in each group;

[0028] Figure 8 HE staining images of pathological changes in tumor tissue of nude mice in each group;

[0029] Figure 9 HE staining images of pathological changes in the gastric antrum tissue of nude mice in each group;

[0030] Figure 10 The graph shows the expression level of JAK2 mRNA in tumor tissues of nude mice in each group;

[0031] Figure 11 The graph shows the expression levels of STAT3 mRNA in tumor tissues of nude mice in each group;

[0032] Figure 12 The graph shows the expression levels of E-cadherin mRNA in tumor tissues of nude mice in each group.

[0033] Figure 13 The graph shows the expression levels of N-cadherin mRNA in tumor tissues of nude mice in each group;

[0034] Figure 14 The graph shows the expression level of Vimentin mRNA in tumor tissues of nude mice in each group;

[0035] Figure 15 A graph showing the expression levels of E-cadherin protein in tumor tissues of nude mice in each group;

[0036] Figure 16 A graph showing the expression levels of N-cadherin protein in tumor tissues of nude mice in each group;

[0037] Figure 17 A graph showing the expression levels of JAK2 protein in tumor tissues of nude mice in each group;

[0038] Figure 18 A graph showing the expression levels of p-JAK2 protein in tumor tissues of nude mice in each group;

[0039] Figure 19 A graph showing the expression levels of p-STAT3 protein in tumor tissues of nude mice in each group;

[0040] Figure 20 This is a graph showing the expression level of STAT3 protein in tumor tissues of nude mice in each group.

[0041] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei This strain was independently selected from naturally fermented sourdough from a resident's home in Tianjin. The strain has undergone strain preservation and physicochemical testing. On March 19, 2021, bacterial identification testing was conducted at the China National Research Institute of Food Fermentation Industries Co., Ltd. The colonies were white, round, moist, opaque, and with neat edges, and were classified and named *Lactobacillus paracasei*. Lacticaseibacillus paracasei It was deposited on June 29, 2018, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 16022. Detailed Implementation

[0042] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. It should also be noted that, for ease of description, only the parts relevant to the invention are shown in the drawings. Unless otherwise specified, the embodiments and features described herein can be combined with each other. The present application will now be described in detail with reference to the accompanying drawings and embodiments.

[0043] Example 1

[0044] This embodiment describes a method for preparing IOB413 post-genetic agents, and the steps are as follows:

[0045] Strain activation: The strain in the cryopreservation tube was inoculated into the slant culture medium and placed at 37°C for 20 hours. The activated colonies were white, round, moist, opaque, and with neat edges. Microscopic examination showed that the bacteria were rod-shaped, uniform, and robust. The slant culture was harvested after activation.

[0046] The slant culture medium used was a modified MRS medium with the following formula: 5.0g peptone, 5.0g beef extract, 6.0g yeast powder, 15.0g glucose, 1.0mL Tween 80, 2.0g dipotassium hydrogen phosphate, 4.0g sodium acetate, 1.0g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15.0g agar, pH=6.2, and 1L of water.

[0047] Take a loopful of fresh slant culture and inoculate it into a liquid culture medium containing 2% soybean flour. Incubate at 34-38℃ in a sealed container for 18-22 hours to obtain the seed culture.

[0048] The seed culture was inoculated into soybean solid culture medium at a material-to-liquid ratio of 1:1.5 and cultured in a sealed environment at 36±2℃ for 24±2h to obtain solid fermentation product. After fermentation, the product was inactivated at 85℃ and then dried at 55℃ until the moisture content was 8%. After pulverization, Lactobacillus paracasei IOB413 post-biotic was obtained.

[0049] Example 2

[0050] This example illustrates the study of the inhibitory effects of IOB413 post-biotic powder and folic acid on gastric cancer in nude mice, including:

[0051] 2.1 Experimental Materials

[0052] 2.1.1 Laboratory Animals

[0053] 42 male nude mice aged 6-8 weeks, weighing 17-22g, SPF grade BALB / c, were housed in a constant temperature and humidity environment with alternating day and night light and dark conditions.

[0054] 2.2 Experimental Methods

[0055] 2.2.1 Modeling

[0056] Forty-two SPF-grade male BALB / c nude mice were randomly selected, with six mice forming the normal control group and the remaining 36 forming the model group. Gastric cancer cells that had grown to the logarithmic growth phase were digested with trypsin and added to serum-free culture medium to prepare a 1×10⁻⁶ m² / g medium. 7 0.1 mL of cell suspension was then inoculated subcutaneously into the axilla of nude mice. After modeling, the mice were allowed free access to food and water, and their general condition and tumor growth were observed daily. After 3 weeks, when the diameter of the subcutaneous tumor nodule reached approximately 8 mm, the model was considered successfully established.

[0057] 2.2.2 Experimental Grouping and Intervention Methods

[0058] Nude mice that successfully developed the model were randomly divided into a model group, a high-dose treatment group, a medium-dose treatment group, a low-dose treatment group, a positive control group (folic acid), and a combined treatment group (IOB413 post-biotic + folic acid). All drug groups were administered the medication via gavage. Based on the equivalent dose for adults and animals, the daily dose for nude mice was 0.315 g / kg. IOB413 post-biotic was administered via gavage at high, medium, and low doses of 1.26, 0.63, and 0.315 g / kg, respectively. The positive control group received 0.002 g / kg of folic acid via gavage, while the combined treatment group received 0.63 g / kg of IOB413 post-biotic and 0.002 g / kg of folic acid via gavage. The normal group and the model group received an equal volume of physiological saline twice daily for 10 consecutive days.

[0059] 2.3 Indicator Test Results

[0060] 2.3.1 General observation of nude mice

[0061] The weight of nude mice in each group was recorded at the start of drug administration, and then every 7 days thereafter. The general condition and tumor growth of the nude mice were observed daily after drug administration. Figure 1 As shown, the major and minor axes of the tumor were measured using vernier calipers, the data were recorded, and a tumor volume growth curve was plotted. The results are as follows. Figure 2 As shown, during gavage, the tumor volume in nude mice in all intervention groups was significantly reduced compared to the model group. The high-dose treatment group, positive control group, and combined treatment group showed the most significant effects, with the combined treatment group showing the best results. This indicates that IOB413 post-biotic has the effect of delaying tumor growth and has a synergistic effect when used in combination with drugs, increasing the therapeutic effect of drugs on tumors.

[0062] 2.3.2 ELISA determination of inflammatory factors in nude mice

[0063] The expression levels of IL-6, IL-1β, and TNF-α in the serum of nude mice were detected, such as... Figure 3 , Figure 4 and Figure 5 As shown, compared with the normal group, the serum levels of pro-inflammatory cytokines IL-6, IL-1β, and TNF-α in the model group nude mice were significantly increased, promoting the gastric cancer lesion response. After gavage intervention, compared with the model group, the levels of IL-6, IL-1β, and TNF-α in each intervention group showed varying degrees of decrease. The high-dose treatment group, positive control group, and combination treatment group showed the most significant decreases in IL-6, IL-1β, and TNF-α levels, with the combination treatment group showing the most significant decrease in pro-inflammatory cytokine levels. This indicates that IOB413 postbiotic has an inhibitory effect on the progression of gastric cancer, and the inhibitory effect is greatly enhanced when used in combination with drugs.

[0064] 2.3.3 ELISA determination of tumor markers in nude mice

[0065] The expression levels of CEA and CA199 in the serum of nude mice were detected. Elevated levels of the tumor marker CEA (carcinoembryonic antigen) often indicate gastrointestinal cancers, commonly seen in colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, and medullary thyroid carcinoma. Elevated levels of CA199 are primarily associated with the development of pancreatic cancer, bile duct cancer, and colorectal cancer.

[0066] Figure 6 and Figure 7 The results showed that, compared with the normal group, serum CEA and CA199 levels in the model group of nude mice were significantly elevated, indicating the occurrence of gastric cancer lesions. After gavage intervention, compared with the model group, the levels of CEA and CA199 in each intervention group showed varying degrees of decrease. The high-dose treatment group, positive control group, and combination treatment group showed the most significant decreases in CEA and CA199 levels, with the combination treatment group showing the most significant decrease in tumor marker levels. This indicates that IOB413 postbiotic has an inhibitory effect on the progression of gastric cancer, and the inhibitory effect is greatly enhanced when used in combination with drugs.

[0067] 2.3.4 HE staining to observe pathological changes in nude mice

[0068] Tumors and gastric antrum tissues from nude mice in each group were fixed in 4% paraformaldehyde. After dehydration with ethanol, clearing, paraffin impregnation, embedding, sectioning, dewaxing, staining, and mounting, the pathological changes of tumor tissues from each group of nude mice were observed under a microscope.

[0069] like Figure 8 As shown, there were significant differences in histological characteristics between the model group and each treatment group. Cancer cells in each group exhibited typical malignant features, including large, irregular, deeply stained nuclei and sparse, lightly stained cytoplasm. The cancerous tissue destroyed the normal gastric mucosal structure, forming irregular masses, accompanied by inflammatory cell infiltration. Sections from each treatment group showed that metagenetic intervention may have an inhibitory effect on cancer cell growth and mucosal destruction, with the combined treatment group showing a more significant trend of improved tissue structure.

[0070] like Figure 9 As shown, the normal group exhibited intact and regular gastric mucosal structure, while the model group showed malignant manifestations such as disordered gastric mucosal cell arrangement, significant nuclear atypia, and increased mitotic figures. All doses of postbiotic treatment groups showed a dose-dependent trend of tissue improvement, manifested as reduced inflammatory cell infiltration and fibrosis, suggesting that postbiotics may have an inhibitory effect on gastric cancer progression. The combined treatment group further demonstrated a synergistic effect of glandular structure repair and reduced inflammatory infiltration, with the most significant effect.

[0071] 2.3.5 RT-PCR detection of the expression of EMT and IL-6 / JAK2 / STAT3 pathway-related mRNAs

[0072] Tumor tissue was selected, total RNA was extracted, and cDNA was synthesized according to the kit instructions. Polymerase chain reaction was then performed, with β-actin used as an internal control. -AACt Target gene analysis was performed using this method. ACt = target gene Ct - internal reference gene Ct, AACt = control group ACt - ACt of each sample; 2 -AACt The ratio reflecting the expression level of the target gene in each sample relative to the control group sample.

[0073] from Figure 10 , Figure 11 , Figure 12 , Figure 13 and Figure 14 The results showed that each treatment group had a significant impact on the expression of tumor-related genes: E-cadherin expression was highest in the combination therapy group, indicating that the treatment effectively enhanced epithelial cell adhesion properties; while the expression levels of mesenchymal markers N-cadherin and Vimentin were lowest in the combination therapy group, suggesting that the treatment significantly inhibited the epithelial-mesenchymal transition (EMT) process. qPCR analysis using β-actin as an internal control revealed that the combination therapy exhibited a synergistic effect in regulating key EMT molecules. By bidirectionally regulating E-cadherin upregulation and N-cadherin / Vimentin downregulation, it may effectively inhibit tumor metastatic potential, providing important molecular-level experimental evidence for subsequent mechanistic studies.

[0074] 2.3.6 Western Blot analysis of the expression levels of EMT and IL-6 / JAK2 / STAT3 pathway-related proteins

[0075] Tumor tissues from each group were selected, homogenized, and lysed on ice for 30 min with 1 mL of lysis buffer. The tissues were pre-cooled to 4°C and centrifuged at 12000 rpm for 10 min. The supernatant was collected, and protein concentration was determined using the BCA method. An appropriate amount of total protein was transferred to a PVDF membrane for electrophoresis. Electrophoresis was stopped when bromophenol blue reached the bottom of the gel. After transfer, blocking, and washing, primary antibodies against JAK2, STAT3, E-cadherin, N-cadherin, and Vimentin were added. The membrane was incubated with the primary antibodies, and the next day, secondary antibodies were added. ECL luminescent buffer was evenly dropped onto the PVDF membrane, and the membrane was scanned. The results were analyzed using ImageLab software, with β-actin as an internal control, to calculate the expression levels of each protein.

[0076] Figure 15 , Figure 16 , Figure 17 , Figure 18 , Figure 19 and Figure 20Western blot analysis showed that in a nude mouse gastric cancer model, the high-dose treatment group significantly upregulated the expression of the epithelial marker E-cadherin and downregulated the stromal marker N-cadherin, indicating that the treatment effectively inhibited the EMT process. Key proteins of the IL-6 / JAK2 / STAT3 pathway (IL-6, p-JAK2, and p-STAT3) were highly expressed in the model group, while the high-dose treatment group showed a significant decreasing trend in their expression levels, confirming that the treatment can inhibit tumor progression by blocking this pathway. These data collectively indicate that this treatment regimen exerts its anti-gastric cancer metastasis effect by dually regulating EMT markers and the IL-6 / JAK2 / STAT3 signaling pathway, with the combined treatment group showing the most significant effect.

Claims

1. The use of a composition of *Lactobacillus paracasei* IOB413 as a postbiotic in the preparation of a medicament for treating gastric cancer, characterized in that, The accession number of the Lactobacillus paracasei IOB413 is CGMCC No. 16022; The drugs mentioned for treating gastric cancer include those that inhibit the growth and metastasis of cancer cells; The composition comprises Lactobacillus paracasei IOB413 post-biotic and folic acid; the mass ratio of Lactobacillus paracasei IOB413 post-biotic and folic acid in the composition is 315:

1.

2. The application as described in claim 1, characterized in that, The method for preparing the Lactobacillus paracasei IOB413 postbiotic includes: The bacterial strain in the cryopreservation tube is inoculated into an agar slant culture medium and cultured at 36.5-37.5℃ for 18-24 hours to obtain an agar slant culture. The agar slant culture is then inoculated into a liquid culture medium containing 2%-4% of, but not limited to, soybeans and yams, and cultured at 34-38℃ in a sealed, static environment for 18-22 hours to obtain a seed culture. The seed culture is then inoculated into a culture medium containing, but not limited to, soybeans and yams, at a material-to-liquid ratio of 1:1.5 and cultured at 34-38℃ in a sealed, static environment for 24-48 hours. After fermentation, the bacteria are inactivated at 80-95℃, dried until the moisture content is ≤10%, and then pulverized to obtain the post-biotic of Lactobacillus paracasei IOB413.

3. The application as described in claim 2, characterized in that, The slant culture medium comprises: 4.0-7.0 g peptone, 4.0-7.0 g beef extract, 5.0-7.0 g yeast powder, 13.0-18.0 g glucose, 0.8-1.0 mL Tween 80, 1.5-2.0 g dipotassium hydrogen phosphate, 3.0-4.0 g sodium acetate, 1.0-1.2 g triammonium citrate, 0.1-0.2 g magnesium sulfate, 0.03-0.05 g manganese sulfate, 12-15.0 g agar, pH=6.2±0.2, and 1-1.2 L of water.

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