Application of leuconostoc mesenteroides subsp. Mesenteroides in improvement of coronary heart disease and sclerotin

Abstract

The invention discloses application of leuconostoc mesenteroides subsp. Mesenteroides in improvement of coronary heart disease and sclerotin, and belongs to the technical field of marine microorganisms. The invention discloses a leuconostoc mesenteroides subsp. Mesenteroides NHNK-613, which is preserved in the China Center for Type Culture Collection on July 1, 2024, and the preservation number of the leuconostoc mesenteroides subsp. Mesenteroides NHNK-613 is CCTCC (China Center for Type Culture Collection) NO: M 20241431. The NHNK-613 has the capabilities of adsorbing calcium ions, up-regulating intestinal epithelial cell calcium and vitamin D absorption and transfer related gene expression, promoting intestinal absorption and transfer of calcium ions, promoting intestinal tract conversion of 1, 25-dihydroxy vitamin D3, promoting myocardial cell repair, regulating bone metabolism related genes, promoting osteoblast repair and promoting vascular endothelial cell repair. The composition can be used for preparing products for improving coronary heart disease and sclerotin.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine microorganisms, and specifically relates to the application of Leuconostoc mesenteroides subsp. mesenteroides in improving coronary heart disease and bone quality. Background Art

[0002] Calcium ion (Ca 2+ ) is a key mediator for myocardial cell contraction. Low calcium levels lead to a decrease in myocardial contraction efficiency, triggering heart failure, with clinical manifestations such as decreased exercise tolerance, dyspnea, edema, etc. Calcium ions are involved in regulating the myocardial cell membrane potential. Low calcium can prolong the action potential duration, inducing premature beats, atrial fibrillation, and ventricular tachycardia. Low calcium causes vascular smooth muscle dysfunction, inhibits the release of nitric oxide (NO), increases vascular resistance, raises blood pressure, and causes vascular relaxation disorders. At the same time, it activates calmodulin proteins, leading to vasospasm and exacerbating myocardial ischemia in coronary heart disease.

[0003] Vitamin D (VD) is a fat-soluble vitamin, and the main source of vitamin D in the human body is vitamin D3. Active vitamin D can reduce inflammatory responses and improve endothelial cell function. The conversion of vitamin D into the active form of 1,25-dihydroxyvitamin D3 requires the catalysis of two key enzymes. Vitamin D is first catalyzed by 25-hydroxylase to convert into 25-hydroxyvitamin D, and then catalyzed by 1α-hydroxylase to convert into 1,25-dihydroxyvitamin D3. Intestinal epithelial cells contain vitamin D receptors (VDR) and have both 25-hydroxylase and 1α-hydroxylase activities. Therefore, they can absorb and convert vitamin D into the physiologically active 1,25-dihydroxyvitamin D3. 1,25-dihydroxyvitamin D3 is the main storage form of vitamin D in the human body and a metabolite that can be detected in the blood. By detecting it, the total vitamin D status can be determined.

[0004] Vitamin D has a direct impact on the cardiovascular system, and vitamin D receptors (VDR) are found in endothelial cells, vascular smooth muscle cells, and myocardial cells. The vitamin D receptor is mainly activated by 1,25-dihydroxyvitamin D3. Low levels of vitamin D3 can cause enhanced inflammatory responses, which is an important factor in the occurrence of coronary heart disease. Vitamin D3 binds to the vitamin D receptor to regulate the activity of inflammatory cells and affect the process of arteriosclerosis.

[0005] There are mainly two mechanisms for the absorption and transport of calcium ions in the intestine. One is the active transcellular transport mechanism, which depends on transient receptor potential cation channel subfamily V member 6 (TRPV6), etc. The other is the passive paracellular transport mechanism. Human colon cancer cell line Caco-2 mainly expresses the TRPV6 calcium channel. Therefore, the calcium transport ability of cells can be judged by measuring the relative expression level of the TRPV6 gene. The human claudin-2 gene CLD2 can form calcium-permeable pores in intestinal epithelial cells to regulate calcium reabsorption and maintain calcium balance.

[0006] Osteoporosis is related to the disruption of the balance between bone formation and bone resorption. Osteoblast ROS17 / 2.8 is a common in vitro model. Runt-related transcription factor 2 gene Runx2, osteoblast-specific transcription factor gene Osterix, homeobox transcription factor Dlx5, osteocalcin gene Osteocalcin, and bone sialoprotein gene Bsp play roles in promoting osteogenesis and mineralization, while zinc finger transcription factor gene Aj18 and homeobox gene Msx2 negatively regulate osteogenesis. By quantitatively analyzing the above genes, the osteogenic state of cells can be determined.

[0007] Marine microorganisms have developed complex molecular adaptation abilities to cope with these harsh conditions, affecting their primary and secondary metabolic pathways. This has led to the evolution of unique physiological characteristics and metabolic processes. Marine microorganisms are more likely to synthesize enzymes and secondary metabolites with unique structures than terrestrial microorganisms. These bioactive substances have important application values in various fields such as pharmaceuticals, foods, and cosmetics. Therefore, it is of great practical significance to provide products related to the marine industry developed using microbial technology. Summary of the Invention

[0008] In view of this, the purpose of the present invention is to provide the application of Leuconostoc mesenteroides subsp. mesenteroides in improving coronary heart disease and bone quality.

[0009] The present invention provides the application of Leuconostoc mesenteroides subsp. mesenteroides in the preparation of products for improving coronary heart disease and bone quality. The Leuconostoc mesenteroides subsp. mesenteroides is Leuconostoc mesenteroides subsp. mesenteroides NHNK-613, which was deposited in the China Center for Type Culture Collection on July 1, 2024, and its deposit number is CCTCC NO: M 20241431.

[0010] Furthermore, the improvement of coronary heart disease and bone quality includes adsorbing calcium ions.

[0011] Furthermore, the improvement of coronary heart disease and bone quality also includes upregulating at least one of the expressions of transient receptor potential cation channel subfamily V member 6 gene TRPV6 and claudin 2 gene CLD2 related to calcium absorption and transport in human colon epithelial cells.

[0012] Furthermore, the improvement of coronary heart disease and bone quality also includes upregulating the expression of vitamin D receptor gene VDR related to promoting the physiological function of vitamin D in human colon epithelial cells.

[0013] Furthermore, the improvement of coronary heart disease and bone quality also includes promoting intestinal absorption and transport of calcium ions.

[0014] Furthermore, the improvement of coronary heart disease and bone quality also includes promoting the conversion of vitamin D3 to active vitamin D3 (1,25-dihydroxyvitamin D3) in the intestine.

[0015] Furthermore, the improvement of coronary heart disease and bone quality also includes promoting myocardial cell repair.

[0016] Furthermore, the improvement of coronary heart disease and bone quality also includes promoting vascular endothelial cell repair.

[0017] Furthermore, the improvement of coronary heart disease and bone quality also includes promoting osteoblast repair.

[0018] Furthermore, the improvement of coronary heart disease and bone quality also includes regulating at least one of the bone metabolism-related genes, upregulating the expressions of osteoblast-specific transcription factor gene Osterix and bone sialoprotein gene Bsp related to osteoblast bone formation, and downregulating the expressions of zinc finger transcription factor gene Aj18 and homeobox gene Msx2 related to bone resorption.

[0019] Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 in the present application has a preservation number of CCTCC NO: M20241431. Experiments show that NHNK-613 has the ability to adsorb calcium ions, upregulate the expressions of genes related to calcium and vitamin D absorption and transport in intestinal epithelial cells, promote intestinal absorption and transport of calcium ions, promote the conversion of 1,25-dihydroxyvitamin D3 in the intestine, promote myocardial cell repair, regulate bone metabolism-related genes, promote osteoblast repair, and promote vascular endothelial cell repair.

[0020] Biological preservation description

[0021] Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan University, Wuhan 430072) on July 1, 2024, and its deposit number is CCTCC NO: M 20241431. Description of the Drawings

[0022] Figure 1 This is a graph showing the experimental results of the colonies of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 grown on a solid plate and Gram staining microscopy in Example 1 of the present invention.

[0023] Figure 2 This is a graph showing the experimental results of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 promoting the repair of damaged cardiomyocytes H9C2 in Example 7 of the present invention.

[0024] Figure 3 This is a graph showing the experimental results of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 promoting the repair of damaged osteoblasts ROS17 / 2.8 in Example 9 of the present invention.

[0025] Figure 4 This is a graph showing the experimental results of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 promoting the repair of damaged vascular endothelial cells EA.hy926 in Example 10 of the present invention.

[0026] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0027] The present invention provides the application of Leuconostoc mesenteroides subsp. mesenteroides in improving coronary heart disease and bone quality. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0028] Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 described in the present invention is derived from dried seaweed velvet and is identified as Leuconostoc mesenteroides subsp. mesenteroides by 16S rDNA. This strain is Gram-positive and spherical under the microscope, with some in pairs or arranged in short chains; it grows on MRS plates and can form opaque round colonies that are milky white, smooth and round on the surface, with neat edges; it can grow evenly and turbidly in MRS medium, and the bacteria form a white precipitate after long-term placement. The optimum growth temperature is 30 °C, it is microaerophilic and grows well under anaerobic culture.

[0029] Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 is deposited with the China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University. The deposit date is July 1, 2024, and the deposit number is CCTCC NO: M20241431.

[0030] Furthermore, in the applications described in the present invention, Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 exists in the form of viable bacteria without sterilization, inactivated bacteria after sterilization, or in the form of fermentation products (i.e., supernatant). The preferred forms of the derivatives are selected from: metabolites, metabiolites, probiotics, cell walls and their components, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products, viable bacteria, inactivated bacteria.

[0031] It should be noted that the reagents and consumables used in the present invention are all ordinary commercially available products. Now, in combination with the examples, the present invention will be further elaborated:

[0032] Example 1 Isolation of NHNK-613

[0033] Please refer to Figure 1 , take an appropriate amount of dried seaweed velvet, soak it in sterile PBS (1×PBS buffer, Solarbio, Beijing) for half an hour, shake it several times, centrifuge the sample at a low speed of 6000 r / min to remove the precipitate, take the supernatant and streak it onto MRS (HaiBo Bio, Qingdao) solid medium, and incubate it at 30 °C for 48 h. Then, pick the white colonies and streak them repeatedly for purification until regular and uniform single colonies are obtained, named NHNK-613.

[0034] Gram staining microscopy: The strain NHNK-613 is a Gram-positive colony, spherical under the microscope, and some are paired or arranged in short chains; it grows on MRS plates and can form opaque round colonies that are milky white, smooth and round on the surface, with neat edges; it can grow uniformly and turbidly in MRS medium, and the bacteria form white precipitates after standing for a long time.

[0035] Example 2 Nucleic acid identification of NHNK-613

[0036] 1. 16S rDNA gene sequence analysis

[0037] Pick a single colony and inoculate it into MRS liquid medium. After culturing overnight at 30 °C, centrifuge at 8000 rpm for 1 min to collect the bacteria, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit (bacterial DNA extraction kit, Solarbio, Beijing). The primers used are the universal primers 27F and 1492R for bacterial 16S sequencing, and the PCR amplification system is 20 μL. The PCR amplification program is pre-denaturation at 95 °C for 5 min, 94 °C for 15 s, 57 °C for 15 s, 72 °C for 1 min, 35 cycles, and extension at 72 °C for 10 min.

[0038] 2. Results

[0039] The sequencing result of the PCR product is shown in SEQ ID NO.1. After homology comparison (BLASTN) with the published standard sequence in GenBank, it is concluded that the strain NHNK-613 is Leuconostoc mesenteroides subsp. mesenteroides.

[0040] Example 3 Calcium ion adsorption experiment of NHNK-613

[0041] 1. Preparation of viable cells of NHNK-613

[0042] Pick a single colony of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 and inoculate it into MRS liquid medium. Incubate statically at 30 °C for 48 h, centrifuge at 5000 rpm for 10 min to obtain the precipitate, wash it twice with PBS, and then resuspend the bacteria with PBS and adjust OD 600 = 1.0 to obtain viable cells.

[0043] 2. Calcium ion adsorption experiment of NHNK-613

[0044] In the experimental group, 1 mL of 2 mmol CaCl2 solution and 1 mL of NHNK-613 live bacterial suspension were added to the centrifuge tube, and in the control group, 1 mL of CaCl2 solution and 1 mL of PBS were added. After incubation at room temperature for 3 h, the absorbance of the supernatant at 600 nm was measured using a calcium ion determination kit (calcium test kit microplate method, Nanjing Jiancheng Biology), and converted to calcium ion concentration according to the kit instructions. The calculation formula and results are shown in Table 1:

[0045] Table 1

[0046]

[0047] The results showed that NHNK-613 could adsorb calcium ions with an adsorption rate of 13.62% to 15.11%.

[0048] Example 4 NHNK-613 regulates the expression of genes related to calcium ion and vitamin D3 absorption and transport in Caco-2 cells

[0049] 1. Preparation of NHNK-613

[0050] A single colony of Leuconostoc mesenteroides subsp. mesenteroides NHNK-613 was picked and cultured in fresh MRS liquid medium at 30°C for 24 h. DMEM medium (Beijing Solebow) was used to adjust the OD to 600 =0.5, 5000rpm to take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product. Collect the centrifugal precipitate, wash twice with sterile PBS, resuspend the bacteria in DMEM medium and adjust the OD 600 =0.5, and a live bacterial suspension was obtained. Some of the precipitated live bacteria were washed twice with sterile PBS, sterilized by high pressure at 121°C for 15 min, and resuspended in DMEM medium to adjust the OD 600 =0.5, and inactivated bacteria were obtained.

[0051] 2. Cultivation of human colonic epithelial cells Caco-2

[0052] Caco-2 cells (BNCC350769, Beina Biotechnology) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, cultured at 37°C and 5% CO2, and subcultured or plated after the cells were fused to 80% to 90%.

[0053] 3. NHNK-613 regulates the expression of genes related to calcium ion and vitamin D3 absorption and transport in Caco-2 cells

[0054] Caco-2 cells were seeded at a density of 1×10^6 cells per well in a 6-well cell culture plate and cultured for 12 h until the cells adhered. The cell culture medium was removed and the cells were washed twice with sterile PBS. In the experimental group, 1.9 mL of DMEM medium and 100 μL of NHNK-613 live bacteria / fermentation product / inactivated bacteria suspension were added, and in the control group, an equal volume of DMEM medium was added. The cells were cultured at 37 °C under 5% CO2 for 24 h. After the culture, the supernatant was discarded and the cells were washed twice with sterile PBS. Subsequently, 1 mL of cell RNA extraction reagent was added to each well, and total RNA was extracted according to the instructions of the reagent (Trizol total RNA extraction reagent, Beijing Solarbio) and the concentration and purity were measured. After extraction, the RNA was reverse transcribed into cDNA. Using GADPH as the internal reference gene, the expression levels of transient receptor potential cation channel subfamily V member 6 gene TRPV6, vitamin D receptor protein gene VDR, and human claudin-2 gene CLD2 were measured by qPCR. The relative expression fold of the control group gene F = 1, and the F value of each sample was calculated using the 2 -ΔΔCT -method.

[0055] Formula: F = 2 -ΔΔCT , where:

[0056] △CT 实验 = CT 实验 - CT 内参(实验) ;

[0057] △CT 对照 = CT 对照 - CT 内参(对照) ;

[0058] △△CT = △CT 实验 - △CT 对照 .

[0059] The results are shown in Table 2 (NHNK-613 fermentation product upregulates calcium and vitamin D in Caco-2 cells), Table 3 (NHNK-613 live bacteria upregulate calcium and vitamin D in Caco-2 cells), and Table 4 (NHNK-613 inactivated bacteria upregulate calcium and vitamin D in Caco-2 cells):

[0060] Table 2

[0061]

[0062] Table 3

[0063]

[0064] Table 4

[0065]

[0066] The results showed that NHNK-613 up-regulated the genes related to calcium ion and VD absorption, namely transient receptor potential cation channel subfamily V member 6 (TRPV6), claudin-2 (CLD2), and vitamin D receptor (VDR), thus promoting calcium absorption in intestinal epithelial cells and the physiological effects of vitamin VD.

[0067] Example 5: Experiment on NHNK-613 promoting calcium ion transport in Caco-2 cells

[0068] 1. Preparation of viable and inactivated cells of NHNK-613

[0069] Refer to Example 4;

[0070] 2. Culture of human colon cells Caco-2

[0071] After activation with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, Caco-2 cells were cultured at 37°C and 5% CO2. After the cells were fused to 80% - 90%, passage or plating operations were performed;

[0072] 3. Experiment on NHNK-613 promoting calcium ion transport in Caco-2 cells

[0073] When Caco-2 cells were fused to 80%, the cells were collected and adjusted to a concentration of 1×10^5 cells / mL with DMEM medium. 0.5 mL of the cell suspension was added to the upper chamber of the transwell plate, and 1.5 mL of DMEM medium was added to the lower chamber. The cells were cultured at 37°C and 5% CO2, and the medium was changed every other day. The transmembrane resistance value (TEER) of Caco-2 cells was measured daily. When the TEER value was greater than 300 Ω / cm 2 it could be used for the calcium ion transport experiment. A 2 mmol / L CaCl2 solution was prepared with D-Hanks solution (Solarbio, Beijing), and 200 μL was added to each well in the upper chamber of the transwell plate; in the experimental group, 200 μL of the viable or inactivated cell suspension of NHNK-613 was added to the upper chamber of the transwell plate, and an equal volume of DMEM medium was added to the control group. The cells were cultured at 37°C and 5% CO2 for 3 h, and the calcium ion concentration in the lower chamber liquid was detected using a calcium ion assay kit.

[0074] The calculation formula and results are shown in Table 5:

[0075] Table 5

[0076]

[0077] The results showed that both the viable and inactivated cells of NHNK-613 could increase the transport of calcium ions by intestinal epithelial cells, with relative growth rates of 109.40% - 118.63%.

[0078] Example 6: Experiment on NHNK-613 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells

[0079] 1. Preparation of viable and inactivated cells of NHNK-613

[0080] Refer to Example 4.

[0081] 2. Culture of human colon cells Caco-2

[0082] After activation with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, Caco-2 cells were cultured at 37°C and 5% CO2. After the cells reached 80%-90% confluence, subculture or plating was performed.

[0083] 3. Experiment on NHNK-613 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells

[0084] When Caco-2 cells reached 80% confluence, the cells were collected and adjusted to a concentration of 1×10^5 cells / mL with DMEM medium. 0.5 mL of the cell suspension was added to the upper chamber of the transwell plate, and 1.5 mL of DMEM medium was added to the lower chamber. The cells were cultured at 37°C and 5% CO2, and the medium was changed every other day. The trans-epithelial electrical resistance (TEER) of Caco-2 cells was measured daily. When the TEER value was greater than 300 Ω / cm 2 it could be used for the vitamin D3 transport experiment. A vitamin D3 solution of 100 μg / mL was prepared with D-Hanks solution, and 200 μL was added to each well in the upper chamber of the transwell plate; in the experimental group, 200 μL of the viable or inactivated cell suspension of NHNK-613 was added to the upper chamber of the transwell plate, and an equal volume of DMEM medium was added to the control group. The cells were cultured at 37°C and 5% CO2 for 3 h, and the concentration of DHVD3 in the lower chamber fluid was measured using a 1,25-dihydroxyvitamin D3 (DHVD3) enzyme-linked immunosorbent assay kit (Nanjing Jiancheng Bioengineering Institute). The results are shown in Table 6:

[0085] Table 6

[0086]

[0087] Intestinal epithelial cells can absorb and convert vitamin D3 into 1,25-dihydroxyvitamin D3 by themselves. The results showed that viable and inactivated cells of NHNK-613 could further promote the absorption and conversion of vitamin D3 by intestinal epithelial cells Caco-2, increase the content of 1,25-dihydroxyvitamin D3 in the lower chamber, and the relative growth rate was between 151.38% and 194.87%. Therefore, NHNK-613 can promote the activation and utilization of vitamin D3.

[0088] Example 7: NHNK-613 increases the survival rate of H9C2 cardiomyocytes under oxidative damage

[0089] 1. Preparation of NHNK-613 live bacteria suspension

[0090] Refer to Example 4.

[0091] 2. Culture of osteoblast ROS17 / 2.8

[0092] The H9C2 cells (BNCC337726, BeiNa Bio) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2. After the cells were fused to 80% - 90%, subculture or plating operations were performed.

[0093] 3. Experiment on NHNK-613 increasing the survival rate of H9C2 cardiomyocytes under oxidative damage

[0094] Please refer to Figure 2 , the H9C2 cells were seeded into a 96-well cell culture plate at a density of 1×10^4 cells / well and cultured for 24 h until the cells adhered. The medium was removed, and 100 μL of DMEM medium containing 1 mmol / L H2O2 was added to each well. Incubate at 37°C and 5% CO2 for 2 h. After incubation, the medium was discarded. In the experimental group, 100 μL of DMEM medium containing 1% (v / v) NHNK-613 live bacteria was added, and in the control group, an equal volume of DMEM medium was added. Incubate at 37°C and 5% CO2 for 18 h. 10 μL of CCK-8 reagent was added to each well, and the absorbance at 450 nm was measured after culturing at 37°C for 4 h to quantify the number of live cells. The calculation formula and results are shown in Table 7:

[0095] Table 7

[0096]

[0097] The results showed that NHNK-613 live bacteria could increase the survival rate of H9C2 cardiomyocytes under oxidative damage, and the growth rate was 29.79% - 33.52%. That is, it can promote the repair of cardiomyocytes.

[0098] Example 8: NHNK-613 regulates the expression of genes related to bone metabolism of osteoblast ROS17 / 2.8

[0099] 1. Preparation of NHNK-613 fermentation product and live bacteria suspension

[0100] Refer to Example 4.

[0101] 2. Culture of osteoblast ROS17 / 2.8

[0102] The ROS17 / 2.8 cells (BNCC359280, BeiNa Bio) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin and cultured at 37 °C under 5% CO2. After the cells reached 80% - 90% confluence, subculture or plating was performed.

[0103] 3. NHNK-613 regulates the expression of genes related to bone metabolism in ROS17 / 2.8 cells

[0104] The ROS17 / 2.8 cells were seeded into a 6-well cell culture plate at a density of 1×10^6 cells / well and cultured for 12 h until the cells adhered. The cell culture medium was removed and the cells were washed twice with sterile PBS. In the experimental group, 1.9 mL of DMEM medium, 100 μL of NHNK-613 fermentation product or live bacteria suspension were added, and in the control group, an equal volume of DMEM medium was added. The cells were cultured at 37 °C under 5% CO2 for 24 h. After the culture, the supernatant was discarded and the cells were washed twice with sterile PBS. Subsequently, 1 mL of cell RNA extraction reagent was added to each well, and total RNA was extracted according to the reagent instructions and the concentration and purity were measured. After extraction, it was reverse transcribed into cDNA. Using GADPH as the internal reference gene, the expression levels of the osteoblast-specific transcription factor gene Osterix related to bone formation, the bone sialoprotein gene Bsp, the zinc finger transcription factor gene Aj18 related to bone resorption, and the homeobox gene Msx2 were measured by qPCR. The relative expression fold of the genes in the control group was F = 1, and the F value of each sample was calculated using the 2 -ΔΔCT method. The results are shown in Table 8 (NHNK-613 fermentation product regulates genes related to bone metabolism in ROS17 / 2.8 cells) and Table 9 (NHNK-613 live bacteria regulate genes related to bone metabolism in ROS17 / 2.8 cells):

[0105] Table 8

[0106]

[0107] Table 9

[0108]

[0109] The results showed that NHNK-613 could up-regulate the expression of the bone formation-related genes Osterix and Bsp and down-regulate the expression of the bone resorption-related genes Aj18 and Msx2, thereby promoting bone formation and reducing bone resorption.

[0110] Example 9 NHNK-613 increases the survival rate of osteoblasts under oxidative damage

[0111] 1. Preparation of NHNK-613 live bacteria suspension

[0112] Refer to Example 4.

[0113] 2. Cultivation of osteoblast ROS17 / 2.8

[0114] After being activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, ROS17 / 2.8 cells were cultured at 37°C and 5% CO2. After the cells reached 80% - 90% confluence, subculture or plating operations were performed.

[0115] 3. Experiment on NHNK-613 increasing the survival rate of osteoblasts under oxidative damage

[0116] Please refer to Figure 3 , inoculate ROS17 / 2.8 cells at 1×10^4 cells / well into a 96-well cell culture plate and culture for 24 h until the cells adhere. Remove the medium, add 100 μL of DMEM medium containing 1 mmol / L H2O2 to each well, and incubate at 37°C and 5% CO2 for 2 h. After incubation, discard the medium. Add 100 μL of DMEM medium containing 1% (v / v) viable NHNK-613 to the experimental group, and add an equal volume of DMEM medium to the control group. Incubate at 37°C and 5% CO2 for 18 h. Add 10 μL of CCK-8 reagent to each well, and measure the absorbance at 450 nm after culturing at 37°C for 4 h to quantify the number of viable cells. The calculation formula and results are shown in Table 10:

[0117] Table 10

[0118]

[0119] The results showed that viable NHNK-613 could increase the survival rate of ROS17 / 2.8 under oxidative damage, and the growth rate was 13.51% - 21.92%. That is, it can promote the repair of osteoblasts.

[0120] Example 10 NHNK-613 increasing the survival rate of vascular endothelial cells EA.hy926 under oxidative damage

[0121] 1. Preparation of NHNK-613 fermentation products and viable bacteria

[0122] Refer to Example 4.

[0123] 2. Cultivation of vascular endothelial cells

[0124] Human umbilical vein cell fusion cells EA.hy926 (BNCC342387, BeiNa Bio) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37°C and 5% CO2. After the cells reached 80% - 90% confluence, subculture or plating operations were performed.

[0125] 3. NHNK-613 improving the survival rate of vascular endothelial cells under oxidative damage

[0126] Please refer to Figure 4 Prepare the EA.hy926 cells in the logarithmic growth phase into a cell suspension of 10,000 cells / mL, inoculate 100 μL per well in a 96-well plate, and culture in a saturated humidity incubator at 37 °C and 5% CO2 for 16 h. Treat with DMEM medium containing 1.5 mM H2O2 for 6 h. After the treatment, discard the old medium. Add DMEM medium containing 1% (v / v) NHNK-613 fermentation product or live NHNK-613 bacteria to the experimental group, and add an equal volume of DMEM medium to the control group. Incubate the experimental group and the control group in the incubator for 24 h. Discard the old medium and wash twice with PBS. Add 100 μL of DMEM medium and 10 μL of CCK-8 reagent and incubate in the incubator for 2 h. After 2 h, use a microplate reader to detect the absorbance value at 450 nm to quantify the number of live cells.

[0127] Calculation formula: Relative survival rate (%) = 100% × (OD of the experimental group 450 / OD of the control group 450 ).

[0128] The results are shown in Table 11 as follows:

[0129] Table 11

[0130]

[0131] The results show that NHNK-613 can increase the survival rate of EA.hy926 under oxidative damage, and the relative survival rate is 100.91% - 111.60%. That is, it can promote the repair of vascular endothelial cells.

[0132] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of Leuconostoc mesenteroides subsp. mesenteroides in preparing products for improving coronary heart disease and bone quality, characterized in that: The Leuconostoc mesenteroides subspecies mesenteroides is Leuconostoc mesenteroides subspecies mesenteroides NHNK-613, which was deposited in the China Center for Type Culture Collection on July 1, 2024, and its preservation number is CCTCC NO: M 20241431.

2. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality includes the absorption of calcium ions.

3. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes upregulating at least one of the expressions of human colon epithelial cell calcium absorption and transport-related receptor potential cation channel subfamily V member 6 gene TRPV6 and human claudin 2 gene CLD2.

4. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes up-regulating the expression of the vitamin D receptor gene VDR related to promoting the physiological effects of vitamin D in human colon epithelial cells.

5. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes promoting intestinal absorption and transport of calcium ions.

6. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes promoting the intestinal conversion of vitamin D3 into active vitamin D3.

7. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes promoting the repair of myocardial cells.

8. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes promoting the repair of vascular endothelial cells.

9. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes promoting osteoblast repair.

10. The use according to claim 1, characterized in that: The improvement of coronary heart disease and bone quality also includes at least one of regulating bone metabolism-related genes, upregulating the expression of osteoblast-specific transcription factor gene Osterix and bone sialoglycoprotein gene Bsp related to osteoblast bone formation, and downregulating the expression of zinc finger transcription factor gene Aj18 and homeobox gene Msx2 related to bone absorption.

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