Lactobacillus rhamnosus for improving thrombosis and stroke and its products and applications

Abstract

The present invention discloses a Lactobacillus rhamnosus for improving thrombosis and stroke, and its products and applications, and relates to the field of microbial technology. The Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) NHNK‑604 disclosed in the present invention has been deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024417. NHNK‑604 has the functions of dissolving thrombus, regulating endothelial cell coagulation factors, reducing endothelial cell inflammation and apoptosis, reducing aging damage of brain microvascular endothelial cells, and promoting blood-brain barrier. In addition, bacterial biofilm can increase the colonization of mucin and intestinal epithelial cells, and can be used to prepare products for improving thrombosis and stroke.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus rhamnosus bacterium for improving thrombosis and cerebral stroke, and a product and application thereof. Background Art

[0002] Thrombosis refers to a clot formed by blood components in the human body in blood vessels or the heart. Thrombotic diseases include myocardial infarction, cerebral thrombosis, pulmonary embolism, limb artery embolism, limb vein thrombosis, disseminated intravascular coagulation, etc., which seriously endanger the patient's physical and mental health.

[0003] After the coagulation factors interact with fibrinogen, the resulting reticular fibrin can entangle blood cells and platelets to form a clot, thereby forming a thrombus. There is an anticoagulant system in the blood that dissolves fibrin and resists its dissolution. Tissue factor in the coagulation system ( TF ), plasminogen activator inhibitor 1 ( PAI-1 ) is an important protein that promotes thrombosis. TF and PAI-1 The expression of fibrinolytic system can reduce unnecessary thrombosis and make blood vessels smoother. Weak fibrinolysis can lead to thrombosis or excessive fibrin deposition; excessive fibrinolysis can lead to excessive consumption of blood coagulation factors. The fibrinolytic system is of great significance in limiting the extension of blood coagulation and maintaining blood flow.

[0004] When blood vessels are damaged, inflammatory cells will secrete inflammatory cytokines (such as IL-6), which will further accelerate the apoptosis of vascular stromal cells and smooth muscle cells. This also shows that inflammatory cytokines will cause secondary damage to the blood vessel wall.

[0005] The caspase family plays an important role in regulating cell apoptosis. Modern studies have shown that the CASPASE cascade system is directly involved in mediating cell apoptosis and plays a vital role in different pathways of cell apoptosis. BAX ) is the main protein in the BCL-2 family that promotes cell apoptosis. It stimulates the production of cytochrome C by improving the permeability of the mitochondrial outer membrane, producing polymeric apoptotic bodies, which are activated after being cleaved. CASPASE-3 The enzymatic properties of mitochondria are followed by a cascade of apoptotic proteases, which degrade a large number of proteins and ultimately lead to cell death.

[0006] Ischemic stroke is caused by blood clots or arterial stenosis that prevents blood from reaching the brain, resulting in brain tissue damage. Current studies have found that an important area for drug targets is the blood-brain barrier. The blood-brain barrier (BBB) ​​is a structure located around the brain that prevents unnecessary circulating cells and biomolecules from entering the brain. In the case of a stroke, the blood vessels in the brain are blocked or cut off, which compromises the integrity of the blood-brain barrier, leading to neuronal death and the accumulation of toxic byproducts, and immune cells infiltrate the brain to exacerbate inflammation.

[0007] The physiological structure of the blood-brain barrier can reduce or even prevent the brain tissue from being invaded by harmful substances in the peripheral blood, maintain the basic stability of the internal environment of the brain tissue, and has important biological significance for maintaining the normal physiological function of the central nervous system. Tight junction proteins are protein molecular complexes located at the top of brain microvascular endothelial cells, and are an important structural and functional basis for regulating the permeability of the BBB; brain microvascular endothelial cells and tight junctions are the main structures that constitute the BBB, and tight junction proteins play an important role in maintaining the stability of tight junctions and the permeability and integrity of the BBB. The BBB can selectively prevent toxins, macromolecules, inflammatory factors, immune cells and harmful substances in the blood from entering the central nervous system.

[0008] The main material structure of BBB is based on brain capillary endothelial cells and their intercellular tight junction proteins. The basic structure of tight junction proteins is composed of membrane-intrinsic proteins and cytoplasmic auxiliary proteins. Intrinsic membrane proteins include CLDN, OCLN and junctional adhesion molecules. Tight junction chains act as physical barriers to prevent solutes and water from freely passing through the paracellular space between epithelial or endothelial cell sheets, and also play a key role in maintaining cell polarity and signal transduction. CLDN11 ) The protein encoded by this gene belongs to the Claudin family of tight junction-related proteins. Ocln ) is an important structural component of the blood-brain barrier. Many studies have shown that Occludin protein can regulate the integrity and permeability of the blood-brain barrier. MMP2 ) mainly uses type IV collagen, laminin and fibronectin of the basement membrane of the brain capillary wall as substrates. Focal cerebral ischemia can cause changes in the permeability and structure of the basement membrane, resulting in BBB damage, which is the main cause of cerebral microvascular hemorrhage.

[0009] Marine microorganisms have developed complex molecular adaptations 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, and marine microorganisms are more likely to synthesize structurally unique enzymes and secondary metabolites than terrestrial microorganisms. Exploring the application of marine intestinal microorganisms 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 Lactobacillus rhamnosus for improving thrombosis and stroke, and a product and application thereof.

[0011] The present invention provides a Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), which is Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) NHNK-604, 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 March 7, 2024, and its preservation number is CCTCC NO: M2024417 of Lactobacillus rhamnosus.

[0012] The present invention also proposes the above-mentioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) in the preparation of products for improving thrombosis and stroke.

[0013] Furthermore, the improvement of thrombosis and stroke includes at least one of dissolving blood clots, regulating endothelial cell coagulation factors, reducing endothelial cell inflammation and apoptosis, reducing aging damage of brain microvascular endothelial cells, promoting blood-brain barrier, and increasing bacterial biofilm colonization of mucin and intestinal epithelial cells.

[0014] Furthermore, the thrombus dissolving agent has plasmin activity and dissolves the thrombus formed by fibrinogen and thrombin.

[0015] Furthermore, the regulation of endothelial cell coagulation factors includes downregulating tissue factor of human umbilical vein cell fusion cell EA.hy926 TF Plasminogen activator inhibitor 1 PAI-1 at least one of the expressions.

[0016] Furthermore, the reduction of vascular endothelial cell inflammation and apoptosis includes down-regulating the gene of interleukin-6, an inflammation-related factor in human umbilical vein cell fusion cell EA.hy926. IL-6 , apoptosis regulator gene BAX and caspase genes CASPASE-3 at least one of the expressions.

[0017] Furthermore, the reduction of aging damage of brain microvascular endothelial cells is to improve the survival ability of brain microvascular endothelial cells bEnd.3 against oxidative damage.

[0018] Furthermore, the blood-brain barrier promotion includes upregulating the bEnd.3 occluden gene of brain microvascular endothelial cells CLND11 and occludin gene OCLN ; and down-regulation of blood-brain barrier permeability-related matrix metalloproteinase genes MMP2 at least one of the expressions.

[0019] Furthermore, the bacterial biofilm increases the colonization of mucin and intestinal epithelial cells, and the adsorption capacity of the bacteria to mucin and intestinal epithelial cells Caco-2 cells is increased after the biofilm is produced.

[0020] The present invention also provides a product for improving thrombosis and stroke, comprising the above-mentioned Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), and acceptable excipients and / or adjuvants.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Experiments have shown that the Lactobacillus rhamnosus disclosed in the present invention ( Lacticaseibacillus rhamnosus NHNK-604 has the functions of dissolving blood clots, regulating endothelial cell coagulation factors, reducing endothelial cell inflammation and apoptosis, reducing aging and damage of brain microvascular endothelial cells, and promoting the blood-brain barrier. In addition, bacterial biofilms can increase the colonization of mucin and intestinal epithelial cells, and can be used to prepare products to improve thrombosis and stroke.

[0023] Biological Deposit Description

[0024] Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) NHNK-604, 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 March 7, 2024, and its deposit number is CCTCC NO: M 2024417. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a plate image of artificial thrombus of Lactobacillus rhamnosus NHNK-604 in the present invention;

[0026] Figure 2 This is a graph showing the experimental results of the present invention showing that Lactobacillus rhamnosus NHNK-604 improves the viability of oxidatively damaged bEnd.3 cells;

[0027] Figure 3The adsorption diagrams of the bacterial solution and biofilm of Lactobacillus rhamnosus NHNK-604 on mucin in the present invention are shown in FIG. 1 , wherein (a) is the adsorption diagram of the bacterial solution of Lactobacillus rhamnosus NHNK-604 on mucin, and (b) is the adsorption diagram of the biofilm of Lactobacillus rhamnosus NHNK-604 on mucin;

[0028] Figure 4 These are the adsorption diagrams of the Lactobacillus rhamnosus NHNK-604 bacterial solution and biofilm on intestinal epithelial cells in the present invention, wherein (a) is a diagram of intestinal epithelial cells, (b) is a diagram of the adsorption of the Lactobacillus rhamnosus NHNK-604 bacterial solution on intestinal epithelial cells, and (c) is a diagram of the adsorption of the Lactobacillus rhamnosus NHNK-604 biofilm on intestinal epithelial cells.

[0029] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0030] The present invention provides Lactobacillus rhamnosus and its application. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0031] The present invention is lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus NHNK-604, from the intestine of Abalone variegatedis, was identified as Lactobacillus rhamnosus by 16S rDNA ( Lacticaseibacillus rhamnosus ). This strain is Gram-positive and short rod-shaped under a microscope; it grows on MRS plates to form white, raised, moist, smooth and rounded opaque round colonies with neat edges; it grows evenly in MRS liquid culture medium and can show white precipitation when left to stand for a long time. The optimal growth temperature is 37°C. Lacticaseibacillus rhamnosus )NHNK-604, depository unit: China Center for Type Culture Collection, address: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, deposit date: March 7, 2024, deposit number: CCTCC NO: M 2024417.

[0032] Further, the present invention provides Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus) In the application described in the present invention, NHNK-604 exists in the form of sterilization or non-sterilization, or in the form of fermentation product (i.e., supernatant), and the derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides and compounds containing immunogenic components, preferably selected from: live bacteria, fermentation broth, fermentation products.

[0033] In vitro experiments show that the Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus ) NHNK-604 has the function of dissolving blood clots. NHNK-604 has plasminogen activitv and can form a transparent ring on the artificial blood clot plate with a diameter of 6.55 mm to 6.84 mm.

[0034] In vitro experiments show that the Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus NHNK-604 has the function of down-regulating tissue factor in human umbilical vein cell EA.hy926 fusion cells TF , plasminogen activator inhibitor 1 PAI-1 The relative expression multiple was 0.17 to 0.80.

[0035] In vitro experiments show that the Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus NHNK-604 has the effect of down-regulating the gene of interleukin-6, an inflammatory factor, in human umbilical vein cell fusion cells EA.hy926 IL-6、 Apoptosis regulator gene Bax and caspase genes CASPASE-3 The relative expression multiples were 0.06-0.69.

[0036] In vitro experiments show that the Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus ) NHNK-604 has the ability to enhance the survival of oxidatively damaged brain microvascular endothelial cells bEnd.3, with a relative growth rate of 25.54%~32.87%.

[0037] In vitro experiments show that the Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus NHNK-604 upregulates the beed.3 occludin gene in brain microvascular endothelial cells CLND11 and occludin gene OCLN The relative expression multiple was 1.20 to 1.44 times. Down-regulation of matrix metalloproteinase genes MMP2 The relative expression multiples were 0.15 to 0.42 times.

[0038] In vitro experiments show that the Lactobacillus rhamnosus of the present invention ( Lacticaseibacillus rhamnosus ) NHNK-604 has the functional ability of bacterial biofilm to increase colonization of mucin and intestinal epithelial cells.

[0039] The reagents and consumables used in the present invention are all common commercially available products. Now, in conjunction with the embodiments, the present invention is further described:

[0040] Example 1 Separation of NHNK-604

[0041] The sample was obtained from an adult individual of Abalone variegata obtained from a farm, with a shell length of 5 cm and fasting for 3 days. The digestive tract was obtained by dissection with sterile tools, rinsed with sterile saline for 3 times, and the intestinal tissue was broken by homogenization and grinding. The homogenate was collected in 1 ml of sterile saline, and the supernatant was streaked on an MRS solid plate after gradient dilution. After constant temperature culture at 37°C for 24 to 48 hours, white colonies were picked and repeatedly inoculated and screened until a uniform single colony was obtained, named NHNK-604.

[0042] Gram staining microscopy: strain NHNK-604 is a Gram-positive colony, which appears as short rods under a microscope. When grown on MRS plates, it forms white, raised, moist, smooth and rounded opaque circular colonies with neat edges. It can grow evenly and turbidly in MRS culture medium, and the bacteria will show white precipitation after being placed for a long time.

[0043] Example 2 Nucleic acid identification of NHNK-604

[0044] 1. 16S rDNA gene sequence analysis:

[0045] Pick a single colony in MRS liquid medium, culture at 37℃ overnight, collect the bacteria by centrifugation at 8000 rpm for 1min, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 57℃ for 15s, 72℃ for 1min, 35 cycles; 72℃ extension for 10min.

[0046] 2. Results

[0047] The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN). It was concluded that the NHNK-604 strain was Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ).

[0048] Example 3 Fibrinolytic activity of NHNK-604

[0049] 1. Preparation of NHNK-604 fermentation solution

[0050] A single colony of NHNK-604 was selected and placed in MRS liquid medium. After culturing at 37°C for 48 h, the OD was adjusted to 0. 600=1.0, that is, fermentation liquid is obtained.

[0051] 2. Artificial thrombus plate culture medium

[0052] Sample A: Dissolve 22 mg of fibrinogen in 10 mL of normal saline and incubate in a 37°C water bath for 5 to 10 min.

[0053] Sample B: Take 5 mg of thrombin (concentration of 40 U / mg) and add it to 2 mL of normal saline and incubate in a 37°C water bath for 5-10 min;

[0054] Sample C: Weigh 0.1 g agarose and dissolve it in 8 mL saline. Heat to fully dissolve the agarose.

[0055] Quickly add sample B into sample C, and finally add sample A. Pour the mixture into a plate after mixing evenly. Wait for the artificial thrombus plate to solidify before use.

[0056] 3. NHNK-604 fibrinolytic activity assay

[0057] Take 2 μL of NHNK-604 fermented bacteria liquid and apply it on the artificial thrombus plate. Set up three parallels. The control group is MRS culture medium. Incubate in a 37℃ incubator for 24 hours. Fibrinogen will form fibrin artificial thrombus through the action of thrombin. After the incubation, observe whether there is a transparent circle formed by dissolving the artificial thrombus and measure the diameter of the transparent circle. Figure 1 shown.

[0058] The results are shown in Table 1:

[0059] Table 1: NHNK-604 fibrinolytic activity transparent zone diameter measurement table

[0060]

[0061] The results showed that NHNK-604 has plasminogen activiy, which makes the artificial thrombus plate present a transparent circle with a diameter of 6.55 mm to 6.84 mm, and can dissolve the fibrin artificial thrombus formed by thrombin.

[0062] Example 4 NHNK-604 regulates the expression of coagulation factor-related genes in endothelial cells EA.hy926

[0063] 1. Cell culture

[0064] Human umbilical vein endothelial cell fusion cells (EA.hy926) were cultured in DMEM medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO. 2 When the cell fusion reaches 80-90%, subculture is performed.

[0065] 2. Preparation of NHNK-604 fermentation products

[0066] A single colony of NHNK-604 was selected and placed in MRS liquid medium. After culturing at 37°C for 48 h, the OD was adjusted to 0. 600 =1.0, 5000 rpm to take the supernatant, and then filter with a 0.22 μm filter membrane to obtain the fermentation product. The prepared fermentation product was added to the DMEM culture medium at a volume ratio of 0.2%.

[0067] 3. NHNK-604 regulates the expression of coagulation factor genes in EA.hy926

[0068] When the degree of confluence of human umbilical vein endothelial cell EA.hy926 reached about 90%, the cells were collected with trypsin and the cell suspension was added to a 6-well plate and incubated at 37°C and 5% CO 2 After culturing for 24 h, the culture medium was removed and the cells were washed once with sterile PBS. 2 ml of DMEM medium containing 0.2% (v / v) NHNK-604 fermentation product was added to the experimental group, and an equal volume of DMEM medium containing MRS was added to the control group. After overnight culture, the cells were collected.

[0069] 4. Fluorescence quantitative PCR and data processing

[0070] RNA was extracted from cells using the Trizol method and reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect coagulation factor-related genes TF and PAI-1 expression.

[0071] The relative expression fold of the gene in the control group was F=1, and 2 -ΔΔCT The F value of each sample is calculated by this method. Formula: F=2 -ΔΔCT ,in:

[0072] △CT 实验 =CT 实验 -CT 内参（实验） ;

[0073] △CT 对照 =CT 对照 -CT 内参（对照） ;

[0074] △△CT=△CT 实验 -△CT 对照 .

[0075] The results are shown in Table 2:

[0076] Table 2: Results of NHNK-604 regulating the expression of coagulation factor genes in EA.hy926

[0077]

[0078] The results showed that NHNK-604 fermentation products could down-regulate coagulation factor-related genes in EA.hy926 cells TF and PAI-1 expression, reducing coagulation factors to reduce thrombosis.

[0079] Example 5 NHNK-604 reduces inflammation and apoptosis in endothelial cells EA.hy926

[0080] 1. Cell culture

[0081] The cell culture method is as in Example 4.

[0082] 2. Preparation of NHNK-604 fermentation products

[0083] The preparation method of NHNK-604 fermentation product is as described in Example 4. The prepared fermentation product is added to DMEM medium at a volume ratio of 0.2%.

[0084] 3. NHNK-604 regulates the expression of inflammation- and apoptosis-related genes in EA.hy926 cells

[0085] When the degree of fusion of human umbilical vein endothelial cell EA.hy926 reached about 90%, the adherent cells were digested with 0.25% trypsin to form a cell suspension and added to a 6-well plate at 37°C and 5% CO 2 After the culture was completed, 2 ml of 1.5 mM H 2 O 2 DMEM medium, 37°C, 5% CO 2 The cells were incubated for 3 h under the same conditions, and the culture medium was discarded after 3 h. The experimental group was added with DMEM medium containing 0.2% NHNK-604 fermentation product, and the control group was added with DMEM medium containing an equal volume of MRS, and the culture was continued for 24 h.

[0086] After culture, cells were collected, cell RNA was extracted using the Trzol method, and RNA was reverse transcribed into cDNA using a kit. The internal reference gene was glyceraldehyde-3-phosphate dehydrogenase GADPH , using qPCR fluorescence quantitative technology to detect IL-6 , BAX and CASPASE- 3 The relative expression of the gene in the control group was F=1, and the expression of -ΔΔCT The F value of each sample was calculated by this method.

[0087] The results are shown in Table 3:

[0088] Table 3: NHNK-604 regulates the expression of inflammation and apoptosis-related genes in EA.hy926

[0089]

[0090] The results showed that NHNK-604 could inhibit H 2 O 2 Induced inflammatory response and cell apoptosis, down-regulation of inflammatory factor genes IL-6 , apoptosis-related genes BAX and CASPASE-3 expression.

[0091] Example 6 NHNK-604 increases the ability of brain microvascular endothelial cells bEnd.3 to resist oxidative damage

[0092] 1. Cell culture

[0093] Brain microvascular endothelial cells bEnd.3 were seeded in DMEM complete medium containing 10% FBS and incubated at 37°C with 5% CO 2 Cultivated under conditions.

[0094] 2. Preparation of NHNK-604 fermentation products

[0095] A single colony of NHNK-604 was selected and placed in MRS liquid medium. After culturing at 37°C for 48 h, the OD was adjusted to 0. 600 =1.0, 5000rpm to take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product. The prepared fermentation product was added to the DMEM culture medium at a volume ratio of 0.2%.

[0096] 3. NHNK-604 fermentation products improve the survival of oxidatively damaged bEnd.3 cells

[0097] When the confluency of bEnd.3 cells in the T25 culture flask reached about 90%, the cell suspension was digested with 0.25% trypsin and the cell concentration was adjusted to 50,000 cells / mL with complete culture medium. 100 μL of the cell suspension was added to a 96-well plate and placed at 37°C and 5% CO. 2 After the culture was completed, the old culture medium was discarded and washed three times with sterile PBS. 100 μL of 1.5 mM H 2 O 2 The normal group was added with the same volume of DMEM medium, and the 96-well plate was incubated at 37°C and 5% CO 2After 3 hours, the old culture medium was discarded and the cells were washed with sterile PBS for 3 times. The experimental group was added with 100 μL of DMEM containing 1% NHNK-604 fermentation product, the control group was added with 100 μL of DMEM containing 1% MRS, the normal group was added with the same volume of DMEM, and the blank group was added with 100 μL of DMEM without cells. Three parallels were set for each group. The 96-well plate was placed at 37°C and 5% CO 2 The samples were cultured for 24 h under the microscope. Figure 2 Then, 10 μL of CCK-8 detection reagent was added to each well and incubated at 37°C and 5% CO 2 The cells were placed in an incubator for 1.5 h, and the absorbance at 450 nm was measured using an enzyme-labeled instrument.

[0098] Calculation formula: Relative growth rate (%) = (experimental group - control group) / (normal group - blank group) * 100%.

[0099] The results are shown in Table 4:

[0100] Table 4: Results of NHNK-604 fermentation products improving the viability of oxidatively damaged bEnd.3 cells

[0101]

[0102] The results showed that the fermentation products of NHNK-604 could reduce cell death caused by oxidative damage in bEnd.3 cells and improve their survival ability, with a relative growth rate of 25.54%~32.87%.

[0103] Example 7 NHNK-604 regulates the expression of blood-brain barrier-related genes in bEnd.3 cells

[0104] 1. Cell Culture

[0105] Brain microvascular endothelial cells bEnd.3 were inoculated in DMEM medium containing 10% FBS serum and incubated at 37°C and 5% CO 2 When the cell fusion reached 80%, the cells were collected and inoculated in a 6-well plate. After overnight culture, the supernatant was discarded. 100 μL and 1 mL of serum-free DMEM medium were added to each well of the experimental group, and 100 μL of MR and 1 mL of serum-free DMEM medium were added to each well of the control group. The cells were incubated at 37°C and 5% CO 2 The cells were cultured for 16 h under the same conditions.

[0106] 2. Preparation of NHNK-604 fermentation products

[0107] The preparation method of NHNK-604 fermentation product is referred to Example 4.

[0108] 3. NHNK-604 regulates the expression of blood-brain barrier-related genes in bEnd.3 cells

[0109] When the confluence of brain microvascular endothelial cells bEnd.3 reached about 90%, the adherent cells were digested with 0.25% trypsin to form a cell suspension and added to a 6-well plate at 37°C and 5% CO 2 After the culture was completed, 200 μL of NHNK-604 fermentation product and 2 mL of DMEM medium were added, and an equal volume of MRS and DMEM medium were added to the control group. The cells were incubated at 37°C and 5% CO 2 Incubate for 18 h.

[0110] After culture, cells were collected, cell RNA was extracted using Trizol method, and RNA was reverse transcribed into cDNA using a kit. qPCR fluorescence quantitative technology was used to detect CLND11 , OCLN and MMP2 The relative expression level of mRNA, with β-actin as internal reference ACTB The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0111] The results are shown in Table 5:

[0112] Table 5: Results of NHNK-604 regulating the expression of blood-brain barrier-related genes in bEnd.3 cells

[0113]

[0114] The results showed that NHNK-604 fermentation products could upregulate genes related to blood-brain barrier tightness CLND11 and OCLN down-regulates the expression of blood-brain barrier permeability-related factor genes MMP2 expression.

[0115] Example 8 NHNK-604 forms biofilm

[0116] 1. Formation of NHNK-604 biofilm

[0117] Pick a single colony of NHNK-604 and place it in MRS liquid medium, incubate it at 37°C for 24 h, and adjust the OD to 0. 600 =0.2, 100 μl of bacterial solution was added to each well of a 96-well plate, with 3 parallels per group, and then cultured at 37 degrees for 24 hours.

[0118] 2. Crystal violet staining

[0119] After the incubation, discard the supernatant, add 100 μL sterile PBS to each well and wash twice, then add 100 μL 4% paraformaldehyde fixative to each well and fix at room temperature for 30 minutes. Discard the fixative, add 100 μL crystal violet to each well and stain at room temperature for 30 minutes. After staining, wash twice with sterile PBS and dry, add 100 μL anhydrous ethanol to each well, let stand for 1 minute, and measure the absorbance at 600 nm.

[0120] The results are shown in Table 6:

[0121] Table 6: NHNK-604 biofilm formation

[0122]

[0123] The results showed that NHNK-604 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0124] Example 9 NHNK-604 adsorbs mucin

[0125] 1. Preparation of NHNK-604 live bacterial suspension

[0126] Pick a single colony of NHNK-604 in fresh MRS medium and culture at 37℃ with shaking for 24h. Collect the cells by centrifugation at 5000rpm for 10min, wash twice with sterile PBS, resuspend the cells in DMEM medium and adjust the OD 600 =0.5, and a live bacterial suspension was obtained.

[0127] 2. Preparation of NHNK-604 biofilm

[0128] Pick a single colony of NHNK-604 and place it in MRS liquid medium, incubate it at 37°C for 24 h, and adjust the OD to 0. 600 =0.2 was added to the bacterial culture dish, and then cultured at 37 degrees for 24 hours. After the culture was completed, the upper culture medium was discarded, the bottom biofilm was resuspended after washing twice with PBS, and the bacteria were collected by centrifugation at 5000rpm for 10min, and the bacteria were resuspended with DMEM culture medium and the OD was adjusted. 600 =0.5, and a live biofilm bacterial suspension was obtained.

[0129] 3. Determination of mucin adsorption capacity

[0130] (1) Reagent preparation

[0131] Mucin solution: Weigh 10 mg of mucin and dissolve it in 10 mL of 50 mM Tris-HCL solution at 4°C overnight.

[0132] Blocking solution: Dissolve bovine serum albumin in PBS solution at a ratio of 2% (m / v).

[0133] Washing solution: Dissolve bovine serum albumin in PBS solution at a ratio of 0.1% (m / v).

[0134] (2) Adsorption experiment of NHNK-604 bacterial liquid and biofilm on mucin

[0135] Immerse the coverslip in the mucin solution and coat it overnight at 4°C. After coating, discard the supernatant. Wash twice with cleaning solution, soak in blocking solution, and incubate for 2 hours at room temperature. After incubation, rinse twice with cleaning solution. Take 100μL of NHNK-604 live bacterial suspension or biofilm bacterial suspension, add it dropwise to the mucin-coated coverslip, and incubate at 37°C for 2 hours. After incubation, rinse twice with cleaning solution to remove non-adherent bacteria. After drying, fix with 4% paraformaldehyde solution for 30 minutes, then perform Gram staining and observe under a microscope. Figure 3 shown.

[0136] The results showed that NHNK-604 could adsorb mucin, the main component of the mucosal layer, and that NHNK-604 could increase its adsorption capacity for mucin after coating the biofilm formed by itself.

[0137] Example 10 NHNK-604 adsorption to intestinal epithelial cells

[0138] 1. Preparation of NHNK-604 live bacterial suspension

[0139] The method for preparing the bacterial suspension is as in Example 9.

[0140] 2. Preparation of NHNK-604 biofilm

[0141] The biofilm preparation method is described in Example 9.

[0142] 3. Cultivation of intestinal epithelial cells Caco-2

[0143] Human colorectal adenocarcinoma cells Caco-2 were inoculated in DMEM complete medium containing 10% FBS and 1% penicillin-streptomycin at 37°C and 5% CO. 2Culture under the same conditions for 1-2 days, and collect the cells when the cell fusion reaches 80%~90%. First, place a cell slide at the bottom of the 6-well plate, and then inoculate the cells into the 6-well plate at 1×10^6 / well and culture for 24 hours. After the cells are attached, remove the culture medium, wash twice with PBS, then add 1.5mL DMEM culture medium and 0.5mL NHNK-604 bacterial suspension to each well, and incubate at 37°C for 2 hours. After the incubation, remove the slide, fix it with 4% paraformaldehyde solution for 30 minutes, then perform Gram staining and observe under a microscope. Figure 4 shown.

[0144] The results showed that NHNK-604 could adsorb Caco-2 cells, and the adsorption capacity of NHNK-604 on Caco-2 cells could be increased after the biofilm formed by NHNK-604 itself was coated.

[0145] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Lactobacillus rhamnosus strain that improves thrombosis and stroke ( Lacticaseibacillus rhamnosus ), which is Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ) NHNK-604 was deposited in the China Center for Type Culture Collection on March 7, 2024, and its deposit number is CCTCC NO: M 2024417.

2. The Lactobacillus rhamnosus as claimed in claim 1 ( Lacticaseibacillus rhamnosus ) in the preparation of products for improving thrombosis and stroke.

3. The use according to claim 2, characterized in that: The improvement of thrombosis and stroke includes at least one of dissolving thrombus, regulating endothelial cell coagulation factors, reducing endothelial cell inflammation and apoptosis, reducing aging damage of brain microvascular endothelial cells, and promoting blood-brain barrier.

4. The use according to claim 3, characterized in that: The thrombolytic agent has plasmin activity and dissolves the thrombus formed by fibrinogen and thrombin.

5. The use according to claim 3, characterized in that: The method for regulating the coagulation factor of vascular endothelial cells includes down-regulating the tissue factor of human umbilical vein cell fusion cell EA.hy926 TF Plasminogen activator inhibitor 1 PAI-1 at least one of the expressions.

6. The use according to claim 3, characterized in that: The method for reducing vascular endothelial cell inflammation and apoptosis comprises down-regulating the gene of interleukin-6, an inflammation-related factor of human umbilical vein cell fusion cell EA.hy926 IL-6 , apoptosis regulator gene BAX and caspase genes CASPASE-3 at least one of the expressions.

7. The use according to claim 3, characterized in that: The method of reducing aging damage of brain microvascular endothelial cells is to improve the survival ability of brain microvascular endothelial cells bEnd.3 against oxidative damage.

8. The use according to claim 3, characterized in that: The method for promoting the blood-brain barrier comprises upregulating the bEnd.3 occluden gene of brain microvascular endothelial cells CLND11 and occludin gene OCLN ; and down-regulation of blood-brain barrier permeability-related matrix metalloproteinase genes MMP2 at least one of the expressions.

9. A product for improving thrombosis and stroke, characterized in that: comprising the Lactobacillus rhamnosus as claimed in claim 1 ( Lacticaseibacillus rhamnosus ), and acceptable excipients and / or adjuvants.

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