Application of lactobacillus paracasei IOB413 metaplasm and composition thereof in gastric cancer

Abstract

The invention provides application of a Lactobacillus paracasei IOB413 metagen and a composition thereof in gastric cancer, and belongs to the technical field of microorganisms. The preservation number of the lactobacillus paracasei IOB413 is CGMCC (China General Microbiological Culture Collection Center) No.16022, the afterbiogen of the lactobacillus paracasei IOB413 can inhibit gastric cancer progression by inhibiting tumor cell epithelial-mesenchymal transition and regulating IL-6 / JAK2 / STAT3 signal channels, and the composition formed by the lactobacillus paracasei IOB413 and folic acid has a synergistic effect and can more effectively reduce the tumor volume and reduce the levels of proinflammatory factors and tumor markers. The medicine is high in safety and suitable for being used as an auxiliary treatment means before and after gastric cancer operation, and a new way is provided for gastric cancer treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to application of a Lactobacillus paracasei IOB413 postbiotic and a composition thereof in treating gastric cancer. Background Art

[0002] Gastric cancer (GC) is one of the most common malignancies, ranking fifth and fourth in morbidity and mortality, respectively, among malignant tumors worldwide. Currently, the chemotherapy drug 5-fluorouracil (5-fluorouracil) has anticancer effects, but it is associated with significant adverse reactions. Therefore, there is a need for effective treatments to slow the progression of GC. Studies have shown that overactivation of the interleukin-6 (IL-6) / non-receptor tyrosine kinase 2 (JAK2) / signal transducer and activator of transcription 3 (STAT3) pathway can promote epithelial-mesenchymal transition (EMT) in tumor cells and is closely associated with 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] Treatments for gastric cancer, such as chemotherapy, targeted therapy, immune checkpoint inhibitor therapy, and cell therapy, have made significant progress in the past decade. Fluorouracil, platinum, and taxanes are the main chemotherapy drugs for gastric cancer. First-line chemotherapy is based on fluoropyrimidines, combined with platinum and / or taxanes to form a two-drug or three-drug chemotherapy regimen, and second-line chemotherapy is usually taxanes. 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 stage III operable gastric cancer is surgery plus postoperative adjuvant chemotherapy.

[0004] Gastric cancer patients are generally recommended to receive adjuvant therapy before or after surgery to ensure they are in good physical condition and able to tolerate the high-intensity treatment before surgery, allowing them to better recover their body functions and achieve better treatment outcomes. Therefore, there is an urgent need for a clinical adjuvant therapy that has minimal 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 solve the above-mentioned defects and deficiencies in the prior art, the present application aims to provide an application of a Lactobacillus paracasei IOB413 postbiotic and a composition thereof in gastric cancer.

[0006] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei ) is a naturally fermented sourdough strain independently screened from the homes of residents in Tianjin. This strain has been preserved and tested for physical and chemical indicators. On March 19, 2021, a bacterial identification test was conducted at the China Food Fermentation Industry Research Institute Co., Ltd. The colonies are white, round, moist, opaque, and have neat edges. They are classified and named Lactobacillus paracasei ( Lacticaseibacillus paracasei ), was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on June 29, 2018. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No.16022.

[0007] One of the purposes of the present invention is to provide an application of a postbiotic of Lactobacillus paracasei IOB413 in preparing a medicine for treating gastric cancer. The deposit number of the Lactobacillus paracasei IOB413 is CGMCC No.16022.

[0008] Preferably, the preparation method of the Lactobacillus paracasei IOB413 postbiotic comprises: The strain in the cryopreserved tube is inoculated into a slant culture medium, cultured at 36.5-37.5°C for 18-24 hours to obtain a slant strain; the slant strain is inoculated into a liquid culture medium containing 2%-4% of, but not limited to, soybeans and yam, cultured in a sealed container at 34-38°C for 18-22 hours to obtain a seed solution; The seed liquid is inoculated into a culture medium including but not limited to soybeans and yam at a material-liquid ratio of 1:1.5, and is cultured in a closed state at 34-38°C for 24-48 hours. After fermentation, the seed liquid is inactivated at 80-95°C, dried to a moisture content of ≤10%, and then crushed to obtain the postbiotic of Lactobacillus paracasei IOB413.

[0009] Preferably, the slant culture medium comprises: peptone 4.0-7.0 g, beef extract 4.0-7.0 g, yeast powder 5.0-7.0 g, glucose 13.0-18.0 g, Tween 80 0.8-1.0 mL, dipotassium hydrogen phosphate 1.5-2.0 g, sodium acetate 3.0-4.0 g, triammonium citrate 1.0-1.2 g, magnesium sulfate 0.1-0.2 g, manganese sulfate 0.03-0.05 g, agar 12-15.0 g, pH = 6.2 ± 0.2, and the amount of water added is 1-1.2 L.

[0010] The second object of the present invention is to provide an application of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a microecological preparation for inhibiting gastric cancer tumor growth.

[0011] The third object of the present application is to provide an application of the composition of Paracaseiclovaci IOB413 probiotics in the preparation of a drug for treating gastric cancer, characterized in that the Paracaseiclovaci IOB413 has a preservation number of CGMCC No.16022; and the composition comprises Paracaseiclovaci IOB413 probiotics and folic acid.

[0012] Preferably, the mass ratio of the Paracaseiclovaci IOB413 probiotics and the folic acid in the composition is 315:1.

[0013] The beneficial effects of the present application are: The present application first found that Paracaseiclovaci IOB413 probiotics can inhibit the progression of gastric cancer through two pathways: on the one hand, it can effectively inhibit the epithelial-mesenchymal transition (EMT) process of tumor cells, up-regulate the expression of epithelial marker E-cadherin, and down-regulate the expression of mesenchymal markers N-cadherin and Vimentin, thereby reducing the invasion and metastasis 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 the pathway, and thereby inhibit tumor growth.

[0014] The composition of Paracaseiclovaci IOB413 probiotics and folic acid of the present application shows a significant synergistic effect in inhibiting the growth of gastric cancer. The experimental results show that the combination therapy group is superior to the use of probiotics or folic acid alone in reducing tumor volume, down-regulating pro-inflammatory factors (IL-6, IL-1β, TNF-α), reducing tumor markers (CEA, CA199) levels, and improving pathological tissue morphology, etc., suggesting that the combination of the two can further enhance the inhibitory effect on gastric cancer, providing a better option for clinical treatment.

[0015] Paracaseiclovaci IOB413 is derived from natural fermented sourdough, and the preparation process of its probiotics uses food-grade raw materials (such as soybeans, yam, etc.) and mild culture and inactivation processes, which is highly safe and has very low side effects. Compared with traditional chemotherapy drugs (such as 5-fluorouracil), it not only inhibits tumor progression, but also regulates the health of the gastrointestinal tract of patients, especially suitable for use by gastric cancer patients before or after surgery - it can improve the physical and physical condition of patients before surgery and improve their tolerance to high-intensity treatment; it can promote the recovery of body functions after surgery and help improve treatment effectiveness, solving the problem that existing adjuvant therapy methods may cause additional burden on patients' bodies.

[0016] In summary, the Lactobacillus paracasei IOB413 postbiotics of the present invention and their combination with folic acid have the advantages of significant effect, clear mechanism, high safety, strong synergy and easy production in inhibiting gastric cancer tumor growth, providing a new and effective means for the clinical treatment of gastric cancer, especially preoperative and postoperative adjuvant treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present invention and, together with the description, serve to explain the principles of the present invention, in which: Figure 1 Figure 2 is the tumor growth of nude mice in each group; Figure 2 The growth curve of tumor volume during oral gavage in each group of nude mice is shown; Figure 3 Figure 2 is the expression of IL-6 in the serum of nude mice in each group; Figure 4 Figure 2 is the expression of IL-1β in the serum of nude mice in each group; Figure 5 Figure 2 is the expression of TNF-α in the serum of nude mice in each group; Figure 6 Figure 2 is the expression of CEA in the serum of nude mice in each group; Figure 7 Figure 2 is the expression of CA199 in the serum of nude mice in each group; Figure 8 HE staining images of the tumor tissue pathological changes of nude mice in each group; Figure 9 HE staining images of the pathological changes of gastric antrum tissues in nude mice in each group; Figure 10 Figure 2 is the expression level of JAK2 mRNA in the tumor tissues of nude mice in each group; Figure 11 Figure 2 is the expression level of STAT3 mRNA in the tumor tissues of nude mice in each group; Figure 12 Figure 2 is the expression level of E-cadherin mRNA in tumor tissues of nude mice in each group; Figure 13 Figure 2 is the expression level of N-cadherin mRNA in the tumor tissues of nude mice in each group; Figure 14 Figure 2 is the expression level of Vimentin mRNA in the tumor tissues of nude mice in each group; Figure 15 Figure 2 is the expression level of E-cadherin protein in the tumor tissues of nude mice in each group; Figure 16 Figure 2 is the expression level of N-cadherin protein in the tumor tissues of nude mice in each group; Figure 17Figure 2 is the expression level of JAK2 protein in the tumor tissues of nude mice in each group; Figure 18 Figure 2 is the expression level of p-JAK2 protein in the tumor tissues of nude mice in each group; Figure 19 Figure 2 is the expression level of p-STAT3 protein in the tumor tissues of nude mice in each group; Figure 20 Figure 2 is the expression level of STAT3 protein in tumor tissues of nude mice in each group.

[0018] Lactobacillus paracasei IOB413 ( Lacticaseibacillus paracasei ) is a naturally fermented sourdough strain independently screened from the homes of residents in Tianjin. This strain has been preserved and tested for physical and chemical indicators. On March 19, 2021, a bacterial identification test was conducted at the China Food Fermentation Industry Research Institute Co., Ltd. The colonies are white, round, moist, opaque, and have neat edges. They are classified and named Lactobacillus paracasei ( Lacticaseibacillus paracasei ), was deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms (CGMCC) on June 29, 2018. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is CGMCC No.16022. DETAILED DESCRIPTION

[0019] The present application will be further described in detail below with reference to the accompanying drawings and examples. The specific embodiments described herein are intended only to explain the relevant invention and are not intended to limit the invention. It should also be noted that, for ease of description, only portions relevant to the invention are shown in the accompanying drawings. The embodiments and features in the embodiments of the present application may be combined with each other unless there is a conflict. The present application will be described in detail below with reference to the accompanying drawings and in conjunction with the examples.

[0020] Example 1 This embodiment is a method for preparing IOB413 postbiotics, and the steps are as follows: Strain activation: Inoculate the strain from the cryopreserved tube into a slant culture medium and incubate at 37°C for 20 hours. Activated colonies should be white, round, with a moist, opaque surface and neat edges. Microscopic examination reveals rod-shaped, uniform, and robust bacteria. Harvest the slant strains after activation. Among them, the slant culture medium used was a modified MRS culture medium with the following formula: 5.0 g of peptone, 5.0 g of beef extract, 6.0 g of yeast powder, 15.0 g of glucose, 1.0 mL of Tween 80, 2.0 g of dipotassium hydrogen phosphate, 4.0 g of sodium acetate, 1.0 g of triammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 15.0 g of agar, pH = 6.2, and the amount of water added was 1 L.

[0021] Take a loopful of fresh slant culture and inoculate it into liquid culture medium containing 2% soybean meal. Keep it in a sealed container at 34-38°C for 18-22 hours to obtain seed solution. The seed liquid was inoculated into soybean solid culture medium at a material-liquid ratio of 1:1.5, and cultured in a closed state at 36±2°C for 24±2h to finally obtain a solid fermentation product, which was inactivated at 85°C after fermentation and dried at 55°C to a moisture content of 8%. After crushing, the postbiotics of Lactobacillus paracasei IOB413 were obtained.

[0022] Example 2 This example is a study on the inhibitory effect of IOB413 postbiotic powder and folic acid on gastric cancer in nude mice, including: 2.1 Experimental Materials 2.1.1 Experimental animals 42 SPF-grade BALB / c male nude mice aged 6-8 weeks, weighing 17-22 g, were housed in a constant temperature and humidity environment with alternating light and dark conditions.

[0023] 2.2 Experimental methods 2.2.1 Modeling Six of 42 SPF-grade BALB / c male nude mice were randomly selected as the normal group, and the remaining 36 were used as the model group. Gastric cancer cells grown to the logarithmic phase were trypsinized and serum-free medium was added to make 1×10 7 / mL cell suspension, then 0.1mL of the cell suspension was inoculated subcutaneously in the axilla of nude mice. After model establishment, the mice were allowed free access to food and water. The general condition of the nude mice and tumor growth were observed daily. After 3 weeks, the model was successfully established when the subcutaneous tumor nodule reached a diameter of approximately 8mm.

[0024] 2.2.2 Experimental groups and intervention methods Nude mice with successfully established models 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 combination treatment group (IOB413 postbiotic + folic acid). Nude mice in each drug group were gavaged at a daily dose of 0.315 g / kg, calculated based on adult and animal equivalent doses. IOB413 postbiotics were administered 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, while the combination treatment group received 0.63 g / kg of IOB413 postbiotics and 0.002 g / kg of folic acid. The normal and model groups were given equal volumes of normal saline twice daily for 10 consecutive days.

[0025] 2.3 Index test results 2.3.1 General observation of nude mice The body weight of each group of nude mice was weighed at the beginning of administration, and then the body weight was recorded once every 7 days. The general condition of the nude mice and the growth of the tumor were observed every day after administration, such as Figure 1 The long diameter and short diameter of the tumor were measured with a vernier caliper, the data were recorded, and the tumor volume growth curve was drawn, as shown in Figure 2 The tumor volume of each intervention group of nude mice was significantly reduced compared with the model group during the gavage period, and the effect of the high-dose treatment group, the positive control group and the combined treatment group was more obvious, among which the combined treatment group was the best. This shows that IOB413 probiotics has the effect of delaying tumor growth, and has a synergistic effect when combined with drugs, increasing the therapeutic effect of drugs on tumors.

[0026] 2.3.2 ELISA determination of inflammatory factors in nude mice The expression of IL-6, IL-1β and TNF-α in the serum of nude mice was detected, as shown in Figure 3 、 Figure 4 and Figure 5 Compared with the normal group, the pro-inflammatory cytokines IL-6, IL-1β and TNF-α in the serum of the model group of nude mice were extremely significantly increased, promoting the gastric cancer pathological reaction; after gavage intervention, compared with the model group, the IL-6, IL-1β and TNF-α levels in each intervention group showed a decreasing trend to varying degrees, and the IL-6, IL-1β and TNF-α levels in the high-dose treatment group, the positive control group and the combined treatment group decreased significantly, among which the combined treatment group had the most significant decrease in pro-inflammatory factor levels. This shows that IOB413 probiotics has the effect of inhibiting the progression of gastric cancer, and greatly improves the inhibitory effect when combined with drugs.

[0027] 2.3.3 ELISA determination of tumor markers in nude mice The expression of CEA and CA199 in the serum of nude mice was detected, and the overexpression of tumor marker CEA (carcinoembryonic antigen) often indicates digestive tract cancer, which is commonly seen in colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, lung cancer, and medullary thyroid cancer. The overexpression of CA199 is mainly related to the occurrence of pancreatic cancer, bile duct cancer and colorectal cancer.

[0028] Figure 6 and Figure 7 It is shown that compared with the normal group, the CEA and CA199 in the serum of the model group of nude mice were extremely significantly increased, indicating the occurrence of gastric cancer pathological reaction; after gavage intervention, compared with the model group, the CEA and CA199 levels in each intervention group showed a decreasing trend to varying degrees, and the CEA and CA199 levels in the high-dose treatment group, the positive control group and the combined treatment group decreased significantly, among which the combined treatment group had the most significant decrease in tumor marker levels. This shows that IOB413 probiotics has the effect of inhibiting the progression of gastric cancer, and greatly improves the inhibitory effect when combined with drugs.

[0029] 2.3.4 HE staining to observe pathological changes in nude mice The tumor and antral tissue of each group of nude mice were fixed in 4% paraformaldehyde, dehydrated with ethanol, transparent, immersed in wax, embedded, sectioned, deparaffinized, stained, and sealed. After sealing, the pathological changes of tumor tissue in each group of nude mice were observed under a microscope.

[0030] As shown in Figure 8 , there were obvious differences in histological features between the model group and each treatment group. The cancer cells in each group showed typical malignant features, including large and irregularly stained nuclei and rare lightly stained cytoplasm. The cancer tissue destroyed the normal gastric mucosa structure to form irregular masses, accompanied by inflammatory cell infiltration. The sections of each treatment group showed that probiotic intervention may have an inhibitory effect on cancer cell growth and mucosal destruction, with the combination therapy group showing a more significant trend of tissue structure improvement.

[0031] As shown in Figure 9 , the normal group had a complete and regular gastric mucosa structure, while the model group had disordered gastric mucosa cell arrangement, significant nuclear atypia, and increased mitotic figures, among other malignant manifestations. Each dose of probiotic treatment group showed a dose-dependent trend of tissue improvement, with reduced inflammatory cell infiltration and fibrosis, suggesting that probiotics may have an inhibitory effect on the progression of gastric cancer. The combination therapy group further showed a synergistic effect of glandular structure repair and reduced inflammatory infiltration, with the most significant effect.

[0032] 2.3.5 RT-PCR to detect the expression of EMT and IL-6 / JAK2 / STAT3 pathway-related mRNA Tumor tissue was selected, total RNA was extracted, and cDNA was synthesized according to the kit instructions. Polymerase chain reaction was performed, and B-actin was used as an internal reference to analyze target genes using 2 -AACt ACt= target gene Ct-internal reference gene Ct, AACt= control group ACt-each sample ACt; 2 -AACt reflects the ratio of the expression level of each sample relative to the control sample.

[0033] From Figure 10 , Figure 11 , Figure 12 , Figure 13 and Figure 14Each treatment group significantly impacted tumor-related gene expression: E-cadherin expression was highest in the combined treatment group, indicating that the treatment effectively enhanced epithelial cell adhesion. Mesenchymal markers N-cadherin and Vimentin expression were lowest in the combined treatment group, suggesting that the treatment significantly inhibited epithelial-mesenchymal transition (EMT). qPCR analysis using β-actin as an internal control revealed that the combined treatment regimen exhibited a synergistic effect in regulating key EMT molecules. By bidirectionally upregulating E-cadherin and downregulating N-cadherin / Vimentin, the combined treatment regimen may effectively inhibit tumor metastasis potential, providing important molecular-level experimental evidence for subsequent mechanistic studies.

[0034] 2.3.6 Western Blot analysis of EMT and IL-6 / JAK2 / STAT3 pathway-related protein expression levels Tumor tissue from each group was homogenized, added to 1 mL of lysis buffer, and lysed on ice for 30 minutes. The cells were precooled to 4°C and centrifuged at 12,000 rpm for 10 minutes. The supernatant was collected and the protein concentration was determined using the BCA assay. An appropriate amount of total protein was transferred to a PVDF membrane for electrophoresis and electrophoresis was terminated 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, and the membrane was incubated with the primary antibodies. The next day, secondary antibodies were added. ECL luminescent solution was evenly applied to the PVDF membrane and the membrane was scanned. The results were analyzed using ImageLab software, and the expression levels of each protein were calculated using β-actin as an internal reference.

[0035] Figure 15 、 Figure 16 、 Figure 17 、 Figure 18 、 Figure 19 and Figure 20 Western Blot analysis revealed 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 mesenchymal marker N-cadherin, indicating that the treatment effectively inhibited EMT progression. Key proteins in 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 decrease in their expression, confirming that the treatment inhibited tumor progression by blocking this pathway. These data collectively indicate that this treatment regimen exerts its anti-metastatic effect on gastric cancer by dually regulating EMT markers and the IL-6 / JAK2 / STAT3 signaling pathway, with the combination treatment group showing the most significant effect.

Claims

1. An application of a Lactobacillus paracasei IOB413 postbiotic in the preparation of a medicine for treating gastric cancer, characterized in that: The deposit number of the Lactobacillus paracasei IOB413 is CGMCC No.16022; The medicines for treating gastric cancer include medicines for inhibiting the growth and metastasis of cancer cells.

2. The use according to claim 1, characterized in that The preparation method of the Lactobacillus paracasei IOB413 postbiotic comprises: The strain in the cryopreserved tube is inoculated into a slant culture medium, cultured at 36.5-37.5°C for 18-24 hours to obtain a slant strain; the slant strain is inoculated into a liquid culture medium containing 2%-4% of, but not limited to, soybeans and yam, cultured in a sealed container at 34-38°C for 18-22 hours to obtain a seed solution; The seed liquid is inoculated into a culture medium including but not limited to soybeans and yam at a material-liquid ratio of 1:1.5, and is cultured in a closed state at 34-38°C for 24-48 hours. After fermentation, the seed liquid is inactivated at 80-95°C, dried to a moisture content of ≤10%, and then crushed to obtain the postbiotic of Lactobacillus paracasei IOB413.

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

4. Application of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a microecological preparation for inhibiting gastric cancer tumor growth and cancer cell metastasis.

5. An application of a composition of a postbiotic of Lactobacillus paracasei IOB413 in the preparation of a medicine for treating gastric cancer, characterized in that: The preservation number of the Lactobacillus paracasei IOB413 is CGMCC No.16022; the composition includes Lactobacillus paracasei IOB413 postbiotics and folic acid; the drug for treating gastric cancer includes a drug for inhibiting cancer cell growth and metastasis; the mass ratio of the Lactobacillus paracasei IOB413 postbiotics to the folic acid in the composition is 315:1.

Images