Probiotic agent for improving ischemic stroke and application thereof

Abstract

This invention relates to a probiotic agent for improving ischemic stroke and its application. The probiotic agent comprises *Lactobacillus rhamnosus* LRa05 and *Lactobacillus acidophilus* LA85, wherein the preservation number of *Lactobacillus rhamnosus* LRa05 is CGMCC NO.24377, and the preservation number of *Lactobacillus acidophilus* LA85 is CGMCC NO.1.12735. The probiotic agent developed by this invention, through the interaction and synergy of the two strains, can effectively promote the repair of neurological function after ischemic stroke, reduce the degree of brain damage after ischemic stroke, inhibit the release of pro-inflammatory factors and promote the secretion of anti-inflammatory factors, effectively regulate the balance of inflammatory responses in the body, reduce the damage of inflammation to brain tissue, and exert a synergistic effect in improving ischemic stroke.

Description

Technical Field

[0001] This invention belongs to the field of microbial medicine technology, specifically relating to a probiotic agent for improving ischemic stroke and its application. Background Technology

[0002] Currently, treatments for ischemic stroke mainly include thrombolytic therapy, antiplatelet therapy, and neuroprotective drugs. However, thrombolytic therapy has a strict time window limitation; exceeding this window can easily lead to serious complications such as cerebral hemorrhage. Antiplatelet drugs may increase the risk of bleeding, while traditional neuroprotective drugs often have problems such as single target, limited efficacy, and a tendency to produce side effects. Therefore, finding safe, effective, and comprehensive treatment methods has become an important need in the field of ischemic stroke research.

[0003] In recent years, with the deepening research on gut microbiota, researchers have discovered a close "gut-brain axis" regulatory relationship between gut microbiota and the central nervous system. After ischemic stroke, the composition and structure of gut microbiota become significantly disordered, manifested as a decrease in the number of beneficial bacteria and an increase in the number of harmful bacteria. This imbalance further aggravates the inflammatory response and oxidative stress damage in the brain, inhibits the repair and regeneration of nerve cells, and thus affects the prognosis after stroke.

[0004] Probiotics, as live microorganisms capable of regulating gut microbiota balance, have been proven to possess various biological functions, including immunomodulation, anti-inflammation, and anti-oxidation. Existing research indicates that probiotics have good effects on improving digestive system diseases and regulating immune function. However, in-depth research on the application of probiotics in improving ischemic stroke in mice, particularly regarding their synergistic effects on post-stroke neurological function recovery, reduction of infarct volume, and correction of gut microbiota dysbiosis, is lacking. Furthermore, a systematic elucidation of the selection of probiotic strains, intervention protocols, and mechanisms of action is insufficient. Therefore, developing a probiotic agent that can effectively improve ischemic stroke has significant experimental research value and application prospects. Summary of the Invention

[0005] The purpose of this invention is to overcome the deficiencies in the prior art and provide a probiotic agent for improving ischemic stroke and its application, which can achieve synergistic improvement effects on neurological function repair, brain damage reduction and regulation of the body's inflammatory response after ischemic stroke in mice.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A probiotic agent for improving ischemic stroke includes Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85, wherein the preservation number of Lactobacillus rhamnosus LRa05 is CGMCC NO. 24377; and the preservation number of Lactobacillus acidophilus LA85 is CGMCC NO. 1.12735.

[0008] As a further technical solution, the live bacteria ratio of Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85 (0.5-1.5):1 can be 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, preferably 1:1.

[0009] As a further technical solution, the total number of live bacteria per gram or milliliter of the probiotic agent is ≥ 1×10⁻⁶. 10 CFU.

[0010] As a further technical solution, a probiotic agent for improving ischemic stroke also includes a protective agent;

[0011] As a further technical solution, the protective agent includes one or more of the following: skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol, or xylitol.

[0012] As a further technical solution, the total weight of Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85 cells to the mass ratio of the protectant is 1:5.

[0013] As a further technical solution, the probiotic agent also includes excipients.

[0014] As a further technical solution, the excipients include one or more of the following: wetting agents, fillers, binders, disintegrants, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, colorants, pH regulators, and buffers;

[0015] As a further technical solution, the dosage form of the probiotic agent includes powder, capsule, tablet or granule.

[0016] A method for preparing a probiotic agent to improve ischemic stroke, comprising:

[0017] Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85 were cultured, centrifuged, and their cells were collected to obtain Lactobacillus rhamnosus LRa05 cells and Lactobacillus acidophilus LA85 cells, respectively. Then, Lactobacillus rhamnosus LRa05 cells and Lactobacillus acidophilus LA85 cells were mixed according to the ratio of live bacteria, a protectant was added, and then freeze-dried to obtain a probiotic agent for improving ischemic stroke.

[0018] A method for preparing a probiotic agent to improve ischemic stroke, comprising:

[0019] Lactobacillus rhamnosus LRa05 strain and Lactobacillus acidophilus LA85 strain were respectively cultured, centrifuged, and the bacterial cells were collected. Protective agents were added, and then freeze-dried to obtain Lactobacillus rhamnosus LRa05 bacterial powder and Lactobacillus acidophilus LA85 bacterial powder.

[0020] A probiotic agent for improving ischemic stroke was obtained by mixing Lactobacillus rhamnosus LRa05 bacterial powder and Lactobacillus acidophilus LA85 bacterial powder according to the ratio of live bacteria count.

[0021] The application of the above-mentioned probiotic agent for improving ischemic stroke in the preparation of drugs for improving ischemic stroke.

[0022] As a further technical solution, the improvement of ischemic stroke includes promoting the repair of neurological function after ischemic stroke, reducing the degree of brain damage after ischemic stroke, and regulating the body's inflammatory response after ischemic stroke.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] This invention develops a probiotic preparation composed of Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85. These two bacteria interact and cooperate to exert a synergistic effect in improving ischemic stroke, specifically in the following ways: 1) effectively promoting the repair of neurological function after ischemic stroke in mice; 2) reducing the degree of brain damage after ischemic stroke in mice; 3) inhibiting the release of pro-inflammatory factors and promoting the secretion of anti-inflammatory factors, effectively regulating the balance of inflammatory response in mice and reducing the damage of inflammation to brain tissue. Attached Figure Description

[0025] Figure 1 The graph shows the neurological function scores for each treatment group;

[0026] Figure 2 Figure 1 shows the infarct volume results for each treatment group;

[0027] Figure 3 The results of inflammatory factors in each treatment group are shown in the figure;

[0028] exist Figure 3 In, a: IL-6; b: TNF-α; c: IL-10. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] 1. Lactobacillus rhamnosus LRa05: Its classification name is Lactobacillus rhamnosus. Lactobacillus rhamnosus The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit date is January 24, 2022; the deposit number is CGMCC NO. 24377; the address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; it has been disclosed in several Chinese patents, including CN202210382754.2.

[0031] 2. Lactobacillus acidophilus LA85, classified as Lactobacillus acidophilus. Lactobacillus acidophilus The depositary institution is the China General Microbiological Culture Collection Center (CGMCC), the deposit date is July 20, 2020; the accession number is CGMCC No.1.12735; the address is No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0032] 3. Unless otherwise specified, all raw materials used in this invention are commercially available.

[0033] Example 1

[0034] (a) Experimental materials

[0035] 1. Experimental animals: 60 SPF-grade male C57BL / 6 mice, 8-10 weeks old, weighing 22-25g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were housed in a barrier environment with a temperature of (23±2)℃, humidity of (50±5)%, and 12h light-dark alternation, and were allowed free access to food and water.

[0036] 2. Probiotic strains: Lactobacillus rhamnosus LRa05 was isolated from the feces of healthy infants in Longcai Village, Dari County, Golog Tibetan Autonomous Prefecture, Qinghai Province; Lactobacillus acidophilus LA85 was isolated from pickled vegetable samples from Group 2 in Shimiao Village, Jintang County, Chengdu, Sichuan Province.

[0037] 3. Main reagents and instruments: 2,3,5-triphenyltetrazolium chloride (TTC, Sigma); TNF-α, IL-6, IL-10 ELISA kits (Shanghai Enzyme-Link Biotechnology Co., Ltd.); bacterial genomic DNA extraction kit (Tiangen Biotech Co., Ltd.); Illumina MiSeq high-throughput sequencing platform (Illumina); stereotaxic instrument (Stoelting); image analysis software ImageJ (National Institutes of Health).

[0038] (II) Experimental Methods

[0039] 1. Preparation of probiotic freeze-dried powder formulation:

[0040] (1) Inoculate L. rhamnosus LRa05 and L. acidophilus LA85 into MRS liquid medium and culture them at 37℃ under anaerobic conditions for 18-24h until the OD600 value of the bacterial solution reaches 1.0-1.2.

[0041] (2) Centrifuge the cultured bacterial solution at 4℃ and 8000r / min for 10min, collect the bacterial precipitate, wash it twice with sterile physiological saline, and then mix the bacterial precipitate with the protective agent at a mass ratio of 1:5 and mix thoroughly.

[0042] (3) Place the mixture in a freeze dryer, pre-freeze at -50℃ for 2 hours, and then freeze-dry under vacuum for 24 hours to obtain a probiotic freeze-dried powder preparation. Determine its viable count to ensure that the total number of viable bacteria per gram of freeze-dried powder is not less than 1×10⁻⁶. 10 CFU was dispensed to obtain Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85, which were stored at -20℃ for later use.

[0043] (4) Before use, prepare LRa05 bacterial suspensions of Lactobacillus rhamnosus and Lactobacillus acidophilus LA85 (total concentration of 2×10⁻⁶). 9 CFU / mL), LA85 bacterial suspension (concentration of 2×10⁻⁶ CFU / mL), 9 CFU / mL) and LA85+ LRa05 combined bacterial suspension (total concentration 2×10) 9 (CFU / mL).

[0044] 2. Establishment of a mouse model of ischemic stroke: A mouse model of middle cerebral artery occlusion (MCAO) was established using the suture occlusion method.

[0045] Mice were anesthetized by intraperitoneal injection of 10% chloral hydrate (350 mg / kg) and fixed supine on the operating table. A midline incision was made in the neck, and the right common carotid artery, external carotid artery, and internal carotid artery were separated. The external carotid artery and its branches were ligated, and the proximal end of the common carotid artery was clamped with an arterial clamp. A small incision was made at the distal end, and a 0.26 mm diameter nylon suture was inserted. The suture was slowly advanced to the origin of the middle cerebral artery (approximately 9-10 mm). The internal carotid artery was ligated near the suture, and the incision was sutured. The suture was removed 2 hours postoperatively to achieve reperfusion and establish an ischemic stroke model.

[0046] 3. Experimental grouping and intervention: Sixty mice were randomly divided into four groups of 15 mice each: model control group, LRa05 single bacteria group, LA85 single bacteria group, and compound probiotic (LRa05+LA85) group.

[0047] Model control group: An ischemic stroke model was constructed. From 7 days before modeling to 21 days after modeling, each animal was given 0.5 mL of physiological saline by gavage daily.

[0048] LRa05 single-strain group: An ischemic stroke model was established. From 7 days before modeling to 21 days after modeling, 0.5 mL of LRa05 bacterial suspension per animal was administered by gavage daily (concentration of 2×10⁻⁶). 9 CFU / mL);

[0049] LA85 single-strain group: An ischemic stroke model was established. From 7 days before modeling to 21 days after modeling, 0.5 mL of LA85 bacterial suspension per animal was administered by gavage daily (concentration of 2×10⁻⁶). 9 CFU / mL);

[0050] Compound probiotic group: An ischemic stroke model was established. From 7 days before modeling to 21 days after modeling, each animal was administered 0.5 mL of LA85 + LRa05 combined bacterial suspension by gavage daily (total concentration 2 × 10⁻⁶). 9 CFU / mL, viable bacteria ratio of 1:1).

[0051] 4. Indicator Testing:

[0052] 1) Neurological function scoring: At 1, 3, 7, 14, and 21 days after modeling, two researchers, unaware of the group assignments, assessed the neurological function of the mice using the Longa scale. Results are shown below. Figure 1 ;

[0053] Specific criteria: 0 points, no neurological deficit symptoms; 1 point, unable to fully extend the contralateral forelimb; 2 points, turning in circles to the contralateral side; 3 points, falling to the contralateral side; 4 points, unable to walk independently, loss of consciousness.

[0054] Infarct volume determination: 21 days after modeling, 6 mice were randomly selected from each group, euthanized after anesthesia, and the whole brain was quickly removed. The olfactory bulb, cerebellum, and brainstem were removed. The brain tissue was frozen at -20℃ for 15 min, and then cut into coronal sections with a thickness of 2 mm using a brain tissue slicer. The sections were placed in 2% TTC solution and incubated at 37℃ in the dark for 30 min. Normal brain tissue was stained red, while infarcted brain tissue appeared white. After rinsing the sections with physiological saline, they were fixed in 4% paraformaldehyde solution for 24 h. The infarct area and total brain area of ​​each brain tissue section were measured using ImageJ image analysis software, and the percentage of infarct volume to total brain volume was calculated. The results are shown in [Figure number missing]. Figure 2 ;

[0055] Cerebral infarction volume % = (sum of infarct areas in each slice × slice thickness / total brain volume) × 100%.

[0056] 3) Detection of inflammatory factors: 21 days after modeling, 6 mice were randomly selected from each group. Blood was collected from the orbital venous plexus, incubated at room temperature for 30 min, and then centrifuged at 3000 r / min for 15 min. Serum was collected, and the levels of TNF-α, IL-6, and IL-10 in the serum were detected according to the ELISA kit instructions. Results are shown in […]. Figure 3 .

[0057] 4. Statistical analysis: Data were analyzed using SPSS 22.0 statistical software. The results are expressed as mean ± standard deviation (x ± s). One-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons. P < 0.05 was considered statistically significant.

[0058] (III) Experimental Results

[0059] 1) Improve neurological function: By scoring the neurological function of mice ( Figure 1 The study found that, compared with the model control group (mice with ischemic stroke who did not receive probiotic intervention), mice treated with the compound probiotic preparation of this invention showed a significant decrease in neurological function scores starting 7 days after model establishment (P<0.05), and the scores continued to decrease with the extension of intervention time. At 21 days, the neurological function scores were more than 40% lower than those of the model control group. This result indicates that compound probiotics can more effectively promote the repair of neurological function after ischemic stroke in mice.

[0060] 2) Reduce the volume of the cerebral infarction: From Figure 2It was found that after 21 days of intervention, the percentage of cerebral infarction volume in the mice in the compound probiotic intervention group was (12.3±2.1)%, significantly lower than that in the model control group (28.5±3.2)%. P The result was <0.01, indicating that the compound probiotics could significantly reduce the degree of brain damage after ischemic stroke in mice.

[0061] 3) Regulating the inflammatory response: From Figure 3 The results showed that the levels of pro-inflammatory factors TNF-α and IL-6 in the serum of mice in the compound probiotic intervention group were (25.6±3.5) pg / mL and (32.8±4.1) pg / mL, respectively, which were significantly lower than those in the model control group (58.7±6.2) pg / mL and (76.3±8.5) pg / mL (P<0.01); while the level of anti-inflammatory factor IL-10 was (45.2±5.3) pg / mL, which was significantly higher than that in the model control group (18.9±2.6) pg / mL (P<0.01). This indicates that the compound probiotics can effectively regulate the inflammatory response balance in mice by inhibiting the release of pro-inflammatory factors and promoting the secretion of anti-inflammatory factors, thereby reducing the damage of inflammation to brain tissue, and the effect is stronger than that of the single probiotic group.

[0062] In summary, the probiotic preparation of this invention, composed of Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85, interacts and cooperates with each other to effectively promote the repair of neurological function after ischemic stroke in mice, reduce the degree of brain damage after ischemic stroke in mice, inhibit the release of pro-inflammatory factors and promote the secretion of anti-inflammatory factors, effectively regulate the balance of inflammatory response in mice, reduce the damage of inflammation to brain tissue, and has the effect of improving ischemic stroke. It can be used to prepare drugs for improving ischemic stroke.

[0063] The embodiments described above are merely preferred embodiments of the present invention, and not an exhaustive list of all possible implementations of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. The application of a probiotic agent for improving ischemic stroke in the preparation of drugs for improving ischemic stroke, characterized in that, The probiotic agent for improving ischemic stroke is composed of Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85, wherein the preservation number of Lactobacillus rhamnosus LRa05 is CGMCC NO.24377; and the preservation number of Lactobacillus acidophilus LA85 is CGMCC NO.1.12735.

2. The application according to claim 1, characterized in that, The ratio of viable Lactobacillus rhamnosus LRa05 to Lactobacillus acidophilus LA85 was (0.5-1.5):

1.

3. The application according to claim 1, characterized in that, The total live bacteria count of the probiotic agent described for improving ischemic stroke is ≥ 1×10⁻⁶ per gram or milliliter. 10 CFU.

4. The application according to claim 1, characterized in that, The probiotic agent for improving ischemic stroke also includes a protectant; the protectant includes one or more of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinylpyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

5. The application according to claim 4, characterized in that, The total weight ratio of Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85 to the protectant is 1:

5.

6. The application according to claim 1, characterized in that, The probiotic agent for improving ischemic stroke also includes excipients, which include one or more of the following: wetting agents, fillers, binders, disintegrants, emulsifiers, solubilizers, osmotic pressure regulators, colorants, and pH adjusters.

7. The application according to claim 1, characterized in that, The dosage forms of the probiotic agents that improve ischemic stroke include powder, capsules, tablets, or granules.

8. The application according to claim 1, characterized in that, The improvement of ischemic stroke includes promoting the repair of neurological function after ischemic stroke, reducing the degree of brain damage after ischemic stroke, and regulating the body's inflammatory response after ischemic stroke.

9. The application as described in any one of claims 1-8, characterized in that, The method for preparing the probiotic agent for improving ischemic stroke includes: Lactobacillus rhamnosus LRa05 and Lactobacillus acidophilus LA85 were cultured, centrifuged, and their cells were collected to obtain Lactobacillus rhamnosus LRa05 cells and Lactobacillus acidophilus LA85 cells, respectively. Then, Lactobacillus rhamnosus LRa05 cells and Lactobacillus acidophilus LA85 cells were mixed according to the ratio of live bacteria, a protectant was added, and then freeze-dried to obtain a probiotic agent for improving ischemic stroke.

10. The application as described in any one of claims 1-8, characterized in that, The method for preparing the probiotic agent for improving ischemic stroke includes: Lactobacillus rhamnosus LRa05 strain and Lactobacillus acidophilus LA85 strain were respectively cultured, centrifuged, and the bacterial cells were collected. Protective agents were added, and then freeze-dried to obtain Lactobacillus rhamnosus LRa05 bacterial powder and Lactobacillus acidophilus LA85 bacterial powder. A probiotic agent for improving ischemic stroke was obtained by mixing Lactobacillus rhamnosus LRa05 bacterial powder and Lactobacillus acidophilus LA85 bacterial powder according to the ratio of live bacteria count.

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