Pediococcus acidilactici for improving heart rhythm, sclerotin and blood pressure as well as product and application thereof

Abstract

The invention discloses pediococcus acidilactici for improving heart rhythm, sclerotin and blood pressure as well as a product and application thereof, and relates to the technical field of microorganisms. The pediococcus acidilactici NHNK-610 disclosed by the invention is preserved in the China Center for Type Culture Collection on April 25, 2024, and the preservation number of the pediococcus acidilactici NHNK-610 is CCTCC (China Center for Type Culture Collection) NO: M 2024797. The NHNK-610 has the functions 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 blood vessels to generate nitric oxide.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly relates to a Pediococcus acidilactici for improving heart rhythm, bone mass and blood pressure, as well as products and applications thereof. Background Art

[0002] Cardiac arrhythmia can affect the heart's pumping function, leading to an increase in myocardial oxygen consumption. For example, during ventricular tachycardia, the rapid and uncoordinated contraction of ventricular muscle will cause a significant increase in myocardial oxygen consumption. At the same time, due to the cardiac pump dysfunction caused by arrhythmia, coronary perfusion will be insufficient, and in severe cases, myocardial infarction may occur.

[0003] Calcium plays a crucial role in the normal electrophysiological activities and contractile functions of the heart. The action potential of normal myocardial cells depends on the activities of multiple ion channels, among which calcium ions (Ca²⁺) play a key role. Calcium deficiency will cause imbalance among other ion channels in myocardial cells, resulting in abnormal repolarization of myocardial cells. In addition, the contractility of myocardial cells will weaken. This change in contractility will affect the heart's pumping function, leading to a decrease in cardiac output. To compensate for this decline in pumping function, arrhythmia may occur. Experimental results show that the incidence of arrhythmia in animals in the low-calcium feeding group is significantly higher than that in the normal-calcium group. The specific manifestations are various types of arrhythmias, such as ventricular premature beats, tachycardia, etc.

[0004] Gut microbiota dysbiosis is closely related to cardiovascular diseases. Dysregulation of the expression of genes related to calcium absorption in intestinal epithelial cells may lead to heart problems such as arrhythmia. Arrhythmia is one of the most common complications in patients with hypertension. The incidence of atrial fibrillation in hypertensive patients is 2-3 times that in non-hypertensive populations, and about 30%-50% of atrial fibrillation patients are accompanied by hypertension.

[0005] Endothelial cells regulate vascular smooth muscle relaxation by releasing nitric oxide (NO), thereby dilating blood vessels and reducing blood pressure. In hypertension, the ability of endothelial cells to synthesize NO decreases, resulting in enhanced vasoconstriction and further elevation of blood pressure.

[0006] The relationship between vitamin D and hypertension is a research field that has received wide attention in recent years. Animal experiments show that vitamin D deficiency causes vasoconstriction and blood pressure elevation; supplementing vitamin D can improve vascular elasticity. At the same time, it reduces the level of oxidized low-density lipoprotein (ox-LDL) and slows down vascular endothelial dysfunction. Active vitamin D activates eNOS (endothelial nitric oxide synthase) through VDR, increasing NO release and dilating blood vessels. A number of epidemiological studies have found that the prevalence of hypertension is relatively high in populations with vitamin D deficiency, and it has been found that low vitamin D levels are associated with both elevated systolic and diastolic blood pressures.

[0007] Vitamin D (VD) is a fat-soluble vitamin, consisting of two forms: vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). The two forms cannot be transported into each other, and the main source of vitamin D in the human body is vitamin D3. Active vitamin D can directly reduce inflammatory reactions, improve endothelial cell function, and delay vascular calcification. 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 (VDRs) 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 situation can be determined.

[0008] Currently, the commonly used drugs for treating osteoporosis mainly include bisphosphonates, denosumab, teriparatide, etc. However, the above drugs have side effects such as gastrointestinal reactions and a sharp drop in bone density after drug withdrawal, and even increase the risk of osteosarcoma. The commonly used antihypertensive drugs mainly include enalapril, amlodipine, metoprolol, etc. However, long-term use may cause risks such as edema, headache, and bradycardia. The antiarrhythmic drugs mainly include amiodarone, propafenone, verapamil, etc. However, most of these drugs have organ toxicity and may exacerbate heart diseases. In comparison, due to their high safety and effectiveness, probiotics have been reported in recent years for regulating osteoporosis, improving blood pressure and heart rate.

[0009] 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 compared to terrestrial microorganisms. Exploring the applications of intestinal microorganisms in marine organisms has important practical significance in expanding the value of the marine industry. Summary of the Invention

[0010] In view of this, the present invention provides a Pediococcus acidilactici for improving heart rhythm, bone mass and blood pressure, as well as products and applications thereof.

[0011] The present invention provides a Pediococcus acidilactici for improving heart rhythm, bone mass and blood pressure ( Pediococcus acidilactici ), the strain is Pediococcus acidilactici NHNK-610, which was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on April 25, 2024, and its preservation number is CCTCC NO: M 2024797.

[0012] The present invention also proposes the above-mentioned Pediococcus acidilactici ( Pediococcus acidilactici ) in the preparation of products for improving heart rhythm, bone mass and blood pressure.

[0013] Further, the calcium and vitamin D3 absorption includes at least one of the following a)-i): a) Adsorption of calcium ions; b) Upregulation of the gene for transient receptor potential coumarin type 6 channel protein related to calcium absorption and transport in human colonic epithelial cells TRPV6 and claudin 2 gene CLD2 at least one of the expressions; c) Upregulation of vitamin D receptor genes related to the promotion of vitamin D physiological effects in human colonic epithelial cells VDR Expression; d) Promote intestinal absorption and transport of calcium ions; e) Promote the intestinal conversion of vitamin D3 into active vitamin D3; f) Promote myocardial cell repair; g) Regulate bone metabolism-related genes and upregulate osteoblast-specific transcription factor genes related to osteoblast bone formation Osterix downregulates the expression of bone resorption-related homeobox genes Msx2 , zinc finger transcription factor genes Aj18 at least one of the expression of a gene; h) Promote osteoblast repair; i) Promotes the production of nitric oxide in blood vessels.

[0014] The present invention also provides a product for improving heart rhythm, bone quality and blood pressure, characterized in that it comprises the above-mentioned Pediococcus acidilactici ( Pediococcus acidilactici ), and acceptable excipients and / or adjuvants.

[0015] The lactic acid bacteria disclosed in the present invention ( Pediococcus acidilactici ), and its deposit number is CCTCCNO: M 2024797. Experiments have shown that NHNK-610 has the functions of absorbing calcium ions, upregulating the expression of genes related to calcium and vitamin D absorption and transport in intestinal epithelial cells, promoting intestinal absorption and transport of calcium ions, promoting intestinal conversion of 1,25-dihydroxyvitamin D3, promoting myocardial cell repair, regulating bone metabolism-related genes, promoting osteoblast repair, and promoting blood vessels to produce nitric oxide.

[0016] Biological Deposit Description Pediococcus acidilactici ( Pediococcus acidilactici ) NHNK-610 was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, postal code 430072) on April 25, 2024, and its deposit number is CCTCC NO: M 2024797. Description of the Drawings

[0017] 图1 This is the MRS plate colony map and Gram staining map of Pediococcus acidilactici NHNK-610 in the present invention; 图2 This is the experimental result map of the increased survival rate of cardiomyocytes against oxidative damage by Pediococcus acidilactici NHNK-610 in the present invention; 图3 This is the experimental result map of the increased survival rate of osteoblasts against oxidative damage by Pediococcus acidilactici NHNK-610 in the present invention.

[0018] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. Specific Embodiments

[0019] The present invention provides Pediococcus acidilactici and its applications. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve. 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 alterations 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.

[0020] The Pediococcus acidilactici strain NHNK-610 of the present invention is derived from dried seaweed and is identified as Pediococcus acidilactici by 16S rDNA Pediococcus acidilactici . This strain is Gram-positive, spherical under the microscope, generally cells grow in pairs and are not arranged in chains; it grows on MRS plates and can form smooth and translucent round colonies on the surface, white, with neat edges; it grows uniformly turbid in MRS liquid medium, and the cells form white precipitates after standing for a long time. The optimum growth temperature is 37°C.

[0021] Furthermore, in the applications described in the present invention, Pediococcus acidilactici NHNK-610 provided by the present invention exists in the form of live bacteria, inactivated bacteria, or as a fermentation product (i.e., supernatant), and the derivative form is preferably selected from: metabolites, metabolic bioproducts, probiotics, cell walls and their components, exopolysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products, live bacteria, and inactivated bacteria.

[0022] Example 1 Isolation of NHNK-610 Take an appropriate amount of dried sea moss and soak it in sterile PBS for half an hour, then shake it several times. Centrifuge the sample at a low speed of 6000 r / min to remove the precipitate. Streak the supernatant onto an MRS solid plate and incubate it at 37°C for 24 h. Then pick the colonies and inoculate and screen them repeatedly until a uniform single colony is obtained, which is named NHNK-610.

[0023] Gram staining and microscopic examination: The strain NHNK-610 is a Gram-positive colony, spherical under the microscope, generally growing in pairs of cells and not arranged in chains; growing on the MRS plate, it can form a white, smooth and round semi-transparent circular colony with neat edges; growing evenly and turbidly in the MRS medium, and the bacteria will form a white precipitate after standing for a long time. As 图1 shown.

[0024] Example 2 Nucleic Acid Identification of NHNK-610 1. 16S rDNA gene sequence analysis: Pick a single colony into MRS liquid medium and incubate it at 37°C overnight. Then 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). 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, denaturation at 94°C for 15 s, annealing at 57°C for 15 s, extension at 72°C for 1 min, 35 cycles, and extension at 72°C for 10 min.

[0025] 2. Results After the PCR product is sequenced and compared with the published standard sequences in the GenBank database (BLASTN), it is found that the NHNK-610 strain is Pediococcus acidilactici Pediococcus acidilactici .

[0026] Example 3 Calcium Ion Adsorption Experiment of NHNK-610 1. Preparation of Live NHNK-610 Pick a single colony of Pediococcus acidilactici NHNK-610 and inoculate it into MRS liquid medium. Incubate statically at 37 °C for 48 h, centrifuge at 5000 rpm for 10 min, collect the precipitate, wash it twice with PBS, and then resuspend the cells with PBS and adjust the OD 600 = 1.0 to obtain viable bacteria.

[0027] 2. Experiment on the adsorption of calcium ions by NHNK-610 In the experimental group, add 1 mL of 2 mmol / L CaCl2 solution and 1 mL of viable bacteria suspension of NHNK-610 to a centrifuge tube. In the control group, add 1 mL of CaCl2 solution and 1 mL of PBS. After incubating at 37 °C for 3 h, use a calcium ion assay kit (microplate method for calcium assay kit, Nanjing Jiancheng) to measure the absorbance of the supernatant at 600 nm, and convert it to the concentration of calcium ions according to the kit instructions.

[0028] The calculation formula and results are shown in Table 1 below: Table 1 Calcium ion adsorption rate of NHNK-610

[0029] The results showed that viable bacteria of NHNK-610 could adsorb calcium ions, and the adsorption rate was 13.95% - 16.61%.

[0030] Example 4 Regulation of the expression of genes related to calcium ion and vitamin D absorption and transport in Caco-2 cells by NHNK-610 1. Preparation of NHNK-610 Pick a single colony of Pediococcus acidilactici NHNK-610 and inoculate it into fresh MRS medium. Incubate at 37 °C for 24 h. Adjust to OD 600 = 0.5 with DMEM medium (Solarbio), centrifuge at 5000 rpm to obtain the supernatant, and then filter it through a 0.22 μm filter membrane to obtain a sterile fermentation product. Collect the centrifuged precipitate of bacteria, wash it twice with sterile PBS, resuspend the bacteria with DMEM medium and adjust the OD 600 = 0.5 to obtain a viable bacteria suspension. Wash a part of the precipitated viable bacteria twice with sterile PBS and then autoclave at 121 °C for 15 min, resuspend it with DMEM medium and adjust to OD 600 = 0.5 to obtain heat-inactivated bacteria.

[0031] 2. Culture of human colon epithelial cells Caco-2 Caco-2 cells (BNCC350769, BeiNa Bio) are activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, and cultured at 37 °C and 5% CO2. After the cells reach 80 - 90% confluence, subculture or plating operations are performed.

[0032] 3. NHNK-610 Regulates the Expression of Genes Related to Calcium and Vitamin D Absorption and Transport in Caco-2 Cells Inoculate Caco-2 cells at a density of 1×10^6 cells / well in a 6-well cell culture plate and incubate for 12 h until the cells adhere. Remove the cell culture medium, wash twice with sterile PBS, add 1.9 mL of DMEM medium and 100 μL of NHNK-610 live bacteria / fermentation product / inactivated bacteria suspension to the experimental group, add an equal volume of DMEM medium to the control group, and culture at 37 °C and 5% CO2 for 24 h. After the culture is completed, discard the supernatant, wash twice with sterile PBS, then add 1 mL of cell RNA extraction reagent to each well, and extract total RNA (Trizol total RNA extraction reagent, Solarbio) according to the reagent instructions and measure the concentration and purity. After the extraction is completed, reverse transcribe it into cDNA, and use GADPH as the internal reference gene, and use qPCR to measure the expression level of transient receptor potential cation channel subfamily V member 6 gene TRPV6、 tight junction protein gene CLD2 and vitamin D receptor protein gene VDR . The relative expression fold of the control group gene F = 1, and the F value of each sample is calculated using the 2 -ΔΔCT method.

[0033] Formula: F = 2 -ΔΔCT , where: △CT 实验 = CT 实验 - CT 内参（实验）; △CT 对照 = CT 对照 - CT 内参（对照）; △△CT = △CT 实验 - △CT 对照。

[0034] The results are shown in Tables 2 to 4 below: Table 2 NHNK-610 Live Bacteria Regulate the Expression of Genes Related to Calcium and Vitamin D Absorption and Transport

[0035] Table 3 NHNK-610 Inactivated Bacteria Regulate the Expression of Genes Related to Calcium and Vitamin D Absorption and Transport

[0036] Table 4 NHNK-610 Fermentation Product Regulates the Expression of Genes Related to Calcium and Vitamin D Absorption and Transport

[0037] The results show that NHNK-610 upregulates the genes related to calcium and VD absorption TRPV6 , CLD2 and the vitamin D receptor gene VDR , thus being able to promote the calcium absorption of intestinal epithelial cells and enhance the physiological function of vitamin D.

[0038] Example 5 Experiment on NHNK-610 Promoting Calcium Ion Transport in Caco-2 Cells 1. Preparation of viable and inactivated cells of NHNK-610 The preparation methods of viable and inactivated cells of NHNK-610 refer to Example 4.

[0039] 2. Culture of human colon cells Caco-2 After Caco-2 cells are activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, they are cultured at 37°C and 5% CO2. After the cells are fused to 80-90%, subculture or plating operations are carried out.

[0040] 3. Experiment on NHNK-610 Promoting Calcium Ion Transport in Caco-2 Cells When Caco-2 cells are fused to 80%, the cells are collected and the concentration is adjusted to 1×10^5 cells / mL with DMEM medium. Add 0.5 mL of cell suspension to the upper chamber of the transwell plate, and add 1.5 mL of DMEM medium to the lower chamber. Culture at 37°C and 5% CO2, and change the medium every other day. Measure the transmembrane resistance value (TEER) of Caco-2 cells every day. When the TEER value is greater than 300 Ω / cm 2 , it can be used for the calcium ion transport experiment. Prepare a 2 mmol / L CaCl2 solution with D-Hanks solution, and add 200 μL to each well in the upper chamber of the transwell plate; add 200 μL of viable or inactivated cell suspension of NHNK-610 to the upper chamber of the transwell plate in the experimental group, and add an equal volume of DMEM medium to the control group. Culture at 37°C and 5% CO2 for 3 h, measure the absorbance value of the lower chamber liquid at 600 nm with a calcium ion assay kit (microplate method for calcium assay kit, Nanjing Jiancheng), and convert the calcium ion concentration according to the kit instructions.

[0041] The calculation formula and results are shown in Table 5 below: Table 5 NHNK-610 Promoting Calcium Ion Transport in Caco-2 Cells

[0042] The results show that both viable and inactivated cells of NHNK-610 can increase the calcium ion transport of intestinal epithelial cells, and the relative growth rate is between 109.43% and 115.44%.

[0043] Example 6 Experiment on NHNK-610 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells 1. Preparation of viable and inactivated cells of NHNK-610 The preparation methods of viable and inactivated cells of NHNK-610 refer to Example 4.

[0044] 2. Culture of human colon cells Caco-2 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%, subculture or plating operations were carried out.

[0045] 3. Experiment on NHNK-610 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells 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. Add 0.5 mL of cell suspension to the upper chamber of the transwell plate and 1.5 mL of DMEM medium to the lower chamber, and culture at 37 °C and 5% CO2, changing the medium every other day. Measure the transmembrane resistance value (TEER) of Caco-2 cells daily. When the TEER value is greater than 300 Ω / cm 2 it can be used for the experiment. Prepare a 100 μg / mL vitamin D3 solution with D-Hanks solution and add 200 μL to each well in the upper chamber of the transwell plate; add 200 μL of viable or inactivated cell suspension of NHNK-610 to the upper chamber of the transwell plate in the experimental group, and add an equal volume of DMEM medium to the control group. Culture at 37 °C and 5% CO2 for 3 h, and measure the concentration of DHVD3 in the lower chamber liquid with a 1,25-dihydroxyvitamin D3 (DHVD3) enzyme-linked immunosorbent assay kit (Nanjing Jiancheng).

[0046] Table 6 NHNK-610 Promoting the Conversion of 1,25-Dihydroxyvitamin D3 by Caco-2 Cells

[0047] 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-625 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 28.04% and 33.87%. Therefore, NHNK-610 can promote the intestinal absorption, transport and utilization of vitamin D3.

[0048] Example 7 NHNK-610 Promoting the Repair of Myocardial Cell Injury 1. Preparation of NHNK-610 viable bacteria suspension The preparation method of NHNK-610 viable bacteria suspension refers to Example 4.

[0049] 2. Culture of rat cardiomyocytes H9C2 After H9C2 cells (BNCC337726, BeiNa Bio) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, they were cultured at 37 °C and 5% CO2. After the cells were fused to 80-90%, passage or plating operations were performed.

[0050] 3. Experiment on NHNK-610 promoting the repair of cardiomyocyte injury H9C2 cells were seeded into 96-well cell culture plates 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-610 viable 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. As 图2 shown. 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. The calculation formula and results are shown in Table 7 below: Table 7 NHNK-610 viable bacteria increase the survival rate of cardiomyocytes with oxidative injury

[0051] The results showed that NHNK-610 viable bacteria could increase the survival rate of H9C2 under oxidative injury, and the growth rate was 21.50% - 34.64%.

[0052] Example 8 NHNK-610 regulates the expression of osteoblast ROS17 / 2.8 bone metabolism-related genes 1. Preparation of NHNK-610 fermentation product and viable bacteria suspension The preparation methods of the fermentation product and viable bacteria suspension refer to Example 4.

[0053] 2. Culture of osteoblast ROS17 / 2.8 After ROS17 / 2.8 cells (BNCC359280, BeiNa Bio) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin, they were cultured at 37 °C and 5% CO2. After the cells were fused to 80-90%, passage or plating operations were performed.

[0054] 3. NHNK-610 Regulates the Expression of Genes Related to ROS17 / 2.8 Bone Metabolism Inoculate ROS17 / 2.8 cells at a density of 1×10^6 cells / well in a 6-well cell culture plate and incubate for 12 h until the cells adhere. Remove the cell culture medium, wash twice with sterile PBS, add 1.9 mL of DMEM medium, 100 μL of the fermentation product or live bacteria suspension of NHNK-610 to the experimental group, add an equal volume of DMEM medium to the control group, and culture at 37 °C and 5% CO2 for 24 h. After the culture, discard the supernatant, wash twice with sterile PBS, then add 1 mL of cell RNA extraction reagent to each well, and extract total RNA and measure its concentration and purity according to the reagent instructions (Trizol total RNA extraction reagent, Solarbio). After extraction, reverse transcribe it into cDNA, and use GADPH as the internal reference gene, and use qPCR to measure the osteoblast-specific transcription factor gene related to bone formation Osterix and the transcription factor gene related to bone resorption Aj18 , homeobox gene Msx2 expression levels. The relative expression fold of the control group gene F = 1, and the 2 -ΔΔCT method is used to calculate the F value of each sample.

[0055] The results are shown in Tables 8 to 9 below: Table 8 Fermentation Product of NHNK-610 Regulates Genes Related to ROS17 / 2.8 Cell Bone Metabolism

[0056] Table 9 Live Bacteria of NHNK-610 Regulates Genes Related to ROS17 / 2.8 Cell Bone Metabolism

[0057] The results showed that the fermentation product and live bacteria of NHNK-610 could regulate the osteoblast-specific transcription factor gene related to bone formation Osterix and the homeobox gene related to bone resorption Msx2、 zinc finger transcription factor gene Aj18 expression, thereby promoting bone formation and reducing bone resorption.

[0058] Example 9 NHNK-610 Promotes the Repair of Osteoblast Injury 1. Preparation of Live Bacteria Suspension of NHNK-610 The preparation method of the live bacteria suspension of NHNK-610 refers to Example 4.

[0059] 2. Culture of Rat Osteoblasts ROS17 / 2.8 After activation 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 was performed.

[0060] 3. Experiment on NHNK-610 promoting the repair of osteoblast injury ROS17 / 2.8 cells were seeded at 1×10^4 cells / well in a 96-well cell culture plate 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. The cells were incubated 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) live NHNK-610 was added, and in the control group, an equal volume of DMEM medium was added. The cells were incubated at 37°C and 5% CO2 for 18 h. As 图2 shown. 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. The calculation formula and results are shown in Table 10 below: Table 10 Live NHNK-610 increases the survival rate of osteoblasts with oxidative damage

[0061] The results showed that live NHNK-610 could increase the survival rate of ROS17 / 2.8 under oxidative damage, and the growth rate was 22.61% - 26.38%.

[0062] Example 10 NHNK-610 promotes the production of nitric oxide by vascular endothelial cells 1. Fermentation products and live bacteria of NHNK-610 The preparation methods of the fermentation products and live bacteria refer to Example 4.

[0063] 2. Culture of vascular endothelial cells EA.hy926 cells (BNCC342387, Beina Bio) were grown in DMEM high-glucose medium containing 10% FBS and 1% penicillin-streptomycin, and cultured in a saturated humidity incubator at 37°C and 5% CO2. Expansion culture was performed in a 1:2 ratio.

[0064] 3. NHNK-610 promotes the production of nitric oxide by injury-induced vascular endothelial cells EA.hy926 cells in the logarithmic growth phase were seeded in 6-well plates and cultured in an incubator with saturated humidity at 37°C and 5% CO2. When the cell confluence reached 70%, the cells were treated with DMEM medium containing 1.5 mM H2O2 for 6 h. In the experimental group, DMEM medium containing 1% (v / v) fermentation product of NHNK-610 or live NHNK-610 bacteria was added, and in the control group, an equal volume of DMEM medium was added. The experimental group and the control group were incubated in the incubator for 24 h. After the culture, the cells were collected and washed twice with PBS. The cell pellet was collected, 0.3 mL of normal saline was added, and the cells were ultrasonically disrupted under ice-water bath conditions. The nitric oxide content in the experimental group and the control group was measured using a nitric oxide (vascular endothelial relaxation factor, i.e., NO) detection kit.

[0065] Calculation formula: Growth rate (%) = (NO content in the experimental group - NO content in the control group) / NO content in the control group × 100% Table 11 NHNK-610 promotes NO production in EA.hy926 cells induced by injury

[0066] Nitric oxide (NO), a relaxation factor synthesized and released by vascular endothelial cells, plays a key role in regulating blood pressure. The results showed that both live NHNK-610 bacteria and its fermentation product could promote NO production in EA.hy926 cells induced by injury.

[0067] The above are only the preferred embodiments 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. A Pediococcus acidilactici strain for improving heart rhythm, bone mass and blood pressure ( Pediococcus acidilactici ), which is Pediococcus acidilactici NHNK-610 and was deposited at the China Center for Type Culture Collection on April 25, 2024, with the deposit number CCTCC NO: M 2024797.

2. The application of Pediococcus acidilactici ( Pediococcus acidilactici ) in the preparation of products for improving heart rhythm, bone mass and blood pressure.

3. The application according to claim 2, wherein The improvement of heart rhythm, bone mass and blood pressure includes at least one of the following a)-i): a) Adsorbing calcium ions; b) Upregulating at least one of the genes related to calcium absorption and transport, namely transient receptor potential cation channel subfamily V member 6 (TRPV6) and tight junction protein 2 (TJP2), in human colon epithelial cells TRPV6 and tight junction protein 2 gene CLD2 ; c) Upregulating the vitamin D receptor gene related to the physiological action of vitamin D in human colon epithelial cells VDR of expression; d) Promoting intestinal absorption and transport of calcium ions; e) Promoting the conversion of vitamin D3 to active vitamin D3 in the intestine; f) Promoting cardiomyocyte repair; g) Regulate bone metabolism-related genes, up-regulate the expression of osteoblast-specific transcription factor genes related to osteoblast bone formation Osterix and down-regulate at least one of the expression of homeobox genes related to bone resorption Msx2 , zinc finger transcription factor genes Aj18 gene expression; h) Promoting osteoblast repair; i) Promoting the production of nitric oxide by blood vessels.

4. A product for improving heart rhythm, bone mass, and blood pressure, characterized in that, Comprising Pediococcus acidilactici as described in claim 1 ( Pediococcus acidilactici ), and acceptable excipients and / or adjuvants.

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