Lactobacillus paracasei for improving urticaria and Alzheimer's disease and product

Abstract

The invention discloses lactobacillus paracasei for improving urticaria and Alzheimer's disease and a product, and relates to the technical field of microorganisms. The lactobacillus paracasei NHNK-1701 disclosed by the invention is preserved in the China Center for Type Culture Collection on April 14, 2025, and the preservation number of the lactobacillus paracasei NHNK-1701 is CCTCC (China Center for Type Culture Collection) NO: M 2025765. The NHNK-1701 has the effects of inhibiting the growth of the klebsiella pneumoniae and / or generating alcohol, agglutinating the klebsiella pneumoniae, tolerating 10% of alcohol, reducing alcohol-induced injury of skin cutin barrier and inflammation, reducing alcohol-induced injury of brain microvascular barrier and inflammation, improving the klebsiella pneumoniae-induced injury survival rate of brain microvascular endothelial cells and improving the activity of the brain microvascular endothelial cells. Klebsiella pneumonia induced damage to brain microvascular barriers and inflammation is reduced, and Klebsiella pneumonia induced damage to intestinal barriers and inflammation is reduced.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Lactobacillus paracasei for improving urticaria and Alzheimer's disease and a product thereof. Background Art

[0002] The ocean is Earth's largest biosphere and the world's largest repository of species diversity. Marine microorganisms, including algae, bacteria, fungi, and protozoa, are among the earliest known sources of marine bioactive substances. These bioactive substances have important applications in various fields, including food, pharmaceuticals, and cosmetics.

[0003] Chronic urticaria is characterized by spontaneous wheals or angioedema with a course of more than 6 weeks, often accompanied by severe itching, which seriously affects the patient's quality of life. A study explored the intestinal microbiome of chronic urticaria patients and healthy controls through metagenomic sequencing and targeted metabolomics, and evaluated the differences in functional relevance. The results showed that the intestinal flora of chronic urticaria patients was significantly different from that of healthy controls, and chronic urticaria was associated with reduced levels of short-chain fatty acid (SCFA) producers and Klebsiella pneumoniae ( Klebsiella pneumoniae ) pathogens. Transplanting Klebsiella pneumoniae into the mouse intestine increased allergic responses in the recipient mice and increased blood lipopolysaccharide levels. LPS increases allergic and inflammatory responses in the body by promoting IgE-mediated mast cell degranulation.

[0004] Klebsiella pneumoniae is a major pathogen that causes both hospital-acquired and community-acquired infections, leading to diseases such as liver abscesses, pneumonia, and bacteremia, posing a serious threat to human health. In recent years, researchers have isolated highly ethanol-producing strains of K. pneumoniae from the feces of patients with autobrewery syndrome. This strain continuously produces alcohol in the body, causing vasodilation and increased permeability, leading to persistent damage to the liver and intestines. Endogenous alcohol produced by K. pneumoniae in the intestine reduces the expression of tight junction proteins ZO-1, claudin-1, and claudin-4, adherens junction proteins β-catenin and E-cadherin, and desmoplakin in intestinal epithelial cells, disrupting intestinal barrier integrity and increasing the severity of chronic urticaria. The core of the skin barrier is the brick wall structure formed by lipids in the stratum corneum. Due to its lipophilic nature, alcohol can penetrate and dissolve these lipids, weakening the barrier function, leading to water loss and the infiltration of external irritants. Alcohol may loosen the connections between keratinocytes, accelerating desquamation and reducing keratinocyte cohesion.

[0005] Approximately 50 million people worldwide suffer from dementia, with Alzheimer's disease (AD) being the most common form. It is characterized by impaired cognitive function, including neurodegeneration, neuronal loss, accumulation of amyloid-β (Aβ), and neurofibrillary tangles. Chronic neuroinflammation is considered a key factor in the progression of the disease. Studies have found that hospitalized patients infected with Klebsiella pneumoniae have a 1.7-fold increased incidence of dementia in a dose-dependent manner. Klebsiella pneumoniae can migrate from the intestine into the bloodstream and ultimately into the brain. Klebsiella pneumoniae infection can aggravate neuroinflammation in the brain, leading to symptoms of Alzheimer's disease. Certain Klebsiella pneumoniae proteins, such as OmpA, have a similar structure to Aβ and may trigger an autoimmune response through molecular mimicry, promoting Aβ production or inhibiting its clearance.

[0006] Marine microorganisms have developed complex molecular adaptations to cope with harsh conditions, affecting their primary and secondary metabolic pathways. This results in unique physiological characteristics and altered metabolic processes, with marine microorganisms being more likely to synthesize structurally distinct enzymes and secondary metabolites than terrestrial microorganisms. Therefore, providing marine-related products developed using microbial technologies is of great practical significance. Summary of the Invention

[0007] In view of this, the present invention provides a Lactobacillus paracasei and a product for improving urticaria and Alzheimer's disease.

[0008] The present invention provides a method for improving urticaria and Alzheimer's disease by using Lactobacillus paracasei ( Lacticaseibacillus paracasei ), the strain is Lactobacillus paracasei NHNK-1701, which was deposited in the China Center for Type Culture Collection (CCTCC, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on April 14, 2025, and its deposit number is CCTCC NO: M 2025765.

[0009] The present invention also proposes the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ) in the preparation of products for improving urticaria and Alzheimer's disease.

[0010] Furthermore, the improvement of urticaria and Alzheimer's disease includes at least one of the following a)-h): a) Inhibit Klebsiella pneumoniae ( Klebsiella pneumoniae ) growing and / or producing alcohol; b), agglutination of Klebsiella pneumoniae; c) Tolerance to 10% alcohol growth; d) Reduce alcohol-induced damage to the skin keratin barrier and inflammation, and upregulate barrier-related genesZO-1, CLDN4 and OCLD , downregulating inflammation-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions; e) Reduce alcohol-induced damage to the brain microvascular barrier and inflammation, and upregulate barrier-related genes ZO-1, CLDN5 and OCLD , downregulating inflammation-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions; f) Improve the survival rate of brain microvascular endothelial cells induced by Klebsiella pneumoniae injury; g) Reduce Klebsiella pneumoniae-induced damage to the brain microvascular barrier and inflammation, and upregulate barrier-related genes ZO-1 、 CLDN5 and OCLD downregulate the expression of inflammatory factor-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions; h) Reduce Klebsiella pneumoniae-induced intestinal barrier damage and inflammation, and upregulate barrier-related genes ZO-1 、 CLDN4 downregulate the expression of inflammatory factor-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions.

[0011] The present invention also provides a product for improving urticaria and Alzheimer's disease, characterized in that it comprises the above-mentioned Lactobacillus paracasei ( Lacticaseibacillus paracasei ), and acceptable excipients and / or adjuvants.

[0012] The present invention discloses Lactobacillus paracasei ( Lacticaseibacillus paracasei ), with a deposit number of CCTCC NO: M 2025765. Experiments have shown that NHNK-1701 inhibits the growth and / or alcohol production of Klebsiella pneumoniae, agglutinates Klebsiella pneumoniae, tolerates growth in 10% alcohol, reduces alcohol-induced damage to the skin keratin barrier and inflammation, reduces alcohol-induced damage to the brain microvascular barrier and inflammation, increases the survival rate of brain microvascular endothelial cells induced by Klebsiella pneumoniae, reduces Klebsiella pneumoniae-induced damage to the brain microvascular barrier and inflammation, and reduces Klebsiella pneumoniae-induced damage to the intestinal barrier and inflammation.

[0013] Biological Deposit Description Lactobacillus paracasei ( Lacticaseibacillus paracasei) NHNK-1701 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 14, 2025, and its deposit number is CCTCC NO: M 2025765. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The MRS plate colony image and Gram staining image of Lactobacillus paracasei NHNK-1701 of the present invention are shown; Figure 2 This is a graph showing the agglutination test results of the inactivated Lactobacillus paracasei NHNK-1701 bacteria and Klebsiella pneumoniae in the present invention.

[0015] The following specific embodiments will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0016] The invention provides Lactobacillus paracasei and application thereof. Those skilled in the art can learn from this paper content, suitably improve process parameter and realize.It is particularly important to point out that all similar replacements and changes are obvious to those skilled in the art, and they are all considered to be included in the present invention.Method and application of the present invention have been described by preferred embodiment, and relevant personnel obviously can change or suitably change and combine the method and application of this paper without departing from content of the present invention, spirit and scope, and realize and apply the technology of the present invention.

[0017] The present invention relates to a strain of Lactobacillus paracasei, NHNK-1701, which is derived from the intestine of Qingdao sea bass and is identified as Lactobacillus paracasei by 16S rDNA. Lacticaseibacillus paracasei This strain is Gram-positive and appears as short rods under a microscope. It grows on MRS plates and forms smooth, opaque, round colonies with milky white and neat edges. It grows uniformly in MRS liquid medium and exhibits a turbid, white precipitate upon prolonged static culture. Its optimal growth temperature is 37°C.

[0018] Furthermore, in the application described in the present invention, the Lactobacillus paracasei NHNK-1701 provided by the present invention exists in the form of live bacteria or inactivated bacteria, or in the form of a fermentation product (i.e., supernatant). 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: fermentation products, live bacteria, and inactivated bacteria.

[0019] In vitro experiments show that the fermentation product of Lactobacillus paracasei NHNK-1701 of the present invention has the effect of inhibiting Klebsiella pneumoniae ( Klebsiella pneumoniae) growth, with the inhibition rate reaching 34.69%~51.06%.

[0020] In vitro experiments show that the Lactobacillus paracasei NHNK-1701 of the present invention has the effect of agglutinating Klebsiella pneumoniae, with an agglutination rate of 14.71% to 21.13%.

[0021] In vitro experiments show that the Lactobacillus paracasei NHNK-1701 of the present invention can grow in a culture medium containing 10% alcohol, with a growth rate of 200.00% to 257.14%.

[0022] In vitro experiments showed that the present invention's Lactobacillus paracasei NHNK-1701 has the effect of regulating the expression of genes related to the barrier and inflammation of HaCaT cells in skin damaged by alcohol, and upregulating the expression of zonula occludens protein gene. ZO-1 , claudin-4 gene CLDN4 and tight junction protein genes OCLD The relative expression of tumor necrosis factor-α gene was 1.64-31.85 times; TNF-α , interleukin-6 gene IL-6 and interleukin-1β gene IL-1β The relative expression levels were 0.06-0.89 times.

[0023] In vitro experiments showed that the present invention's Lactobacillus paracasei NHNK-1701 has the effect of regulating the expression of BEND3 barrier and inflammation-related genes in brain microvascular endothelial cells induced by alcohol, and upregulating the expression of zonula occludens protein gene. ZO-1 , claudin-5 gene CLDN5 and tight junction protein genes OCLD The relative expression of tumor necrosis factor-α gene was 1.31-fold to 6.63-fold; TNF-α , interleukin-6 gene IL-6 and interleukin-1β gene IL-1β The relative expression levels were 0.05-0.83 times.

[0024] In vitro experiments show that the Lactobacillus paracasei NHNK-1701 of the present invention can increase the survival of brain microvascular endothelial cells BEND3 under Klebsiella pneumoniae-induced damage, with a relative survival rate of 110.59% to 142.86%.

[0025] In vitro experiments showed that the present invention's Lactobacillus paracasei NHNK-1701 has the effect of regulating the expression of genes related to the BEND3 barrier and inflammation induced by Klebsiella pneumoniae in brain microvascular endothelial cells, upregulating the expression of zonula occludens protein gene. ZO-1 , claudin-5 gene CLDN5and tight junction protein genes OCLD The relative expression of tumor necrosis factor-α gene was 1.20-7.34 times; TNF-α , interleukin-6 gene IL-6 and interleukin-1β gene IL-1β The relative expression levels were 0.04-0.84 times.

[0026] In vitro experiments showed that the present invention's Lactobacillus paracasei NHNK-1701 has the effect of regulating the expression of genes related to the Caco-2 barrier and inflammation induced by Klebsiella pneumoniae in colon epithelial cells, upregulating the expression of zonula occludens protein gene. ZO-1 and claudin-4 gene CLDN4 The relative expression of tumor necrosis factor-α gene was 1.19-2.87 times, and the ​ , interleukin-6 gene ​ and interleukin-1β gene ​ The relative expression levels were 0.13-0.89 times.

[0027] Example 1 Isolation of NHNK-1701 Lateolabrax japonicus was obtained from a marine cage aquaculture base in Qingdao. The abdominal surface of the fish was wiped with 75% alcohol. The stomach, pyloric caeca, and anterior midgut were removed with sterile instruments, and the contents were removed. The fish were rinsed twice with sterile saline. The mucus on the inner wall of each organ was scraped and added to sterile saline. After vortexing and shaking, the supernatant was streaked onto MRS solid plates (Qingdao Haibo Biotechnology Co., Ltd.). After constant temperature incubation at 37°C for 48 h, white colonies were picked and repeatedly streaked for purification until a single colony with regular and uniform shape was obtained, which was named NHNK-1701.

[0028] Gram staining microscopy: strain NHNK-1701 is Gram-positive and appears as short rods under a microscope. When grown on MRS plates, it forms round colonies with smooth, opaque surfaces, milky white, and neat edges. In MRS (Qingdao Haibo Biotechnology Co., Ltd.) liquid culture medium, it grows uniformly turbid and may produce white precipitates when left to stand for a long time. ​ shown.

[0029] Example 2 Identification of the 16S rDNA Gene Sequence of NHNK-1701 1. 16S rDNA gene sequence analysis: Single colonies were picked and incubated in MRS liquid medium at 37°C overnight. Bacteria were collected by centrifugation at 8000 rpm for 1 minute and the DNA extraction kit for Gram-positive bacteria (Beijing Solebold Technology Co., Ltd.) was used according to the instructions. Universal primers for bacterial 16S sequencing, 27F and 1492R, were used, and the PCR amplification system was 20 μL. The PCR amplification program included 35 cycles of initial denaturation at 95°C for 5 minutes, followed by 35 cycles of 94°C for 15 seconds, 57°C for 15 seconds, and 72°C for 1 minute, followed by extension at 72°C for 10 minutes.

[0030] 2. Results The PCR product was sequenced and compared with the standard sequences published in the GenBank database (BLASTN). The result showed that the NHNK-1701 strain was Lactobacillus paracasei ( ​ ).

[0031] Example 3 Experiment on the inhibition of Klebsiella pneumoniae growth by NHNK-1701 fermentation product 1. Preparation of NHNK-1701 Fermentation Products A single colony of Lactobacillus paracasei NHNK-1701 was selected and cultured in MRS liquid medium, and then cultured at 37°C for 48 h and adjusted to OD 600 =1.0, centrifuge (5000 rpm, 5 min), take the supernatant, and then filter with a 0.22 μm filter membrane to obtain a sterile fermentation product.

[0032] 2. Preparation of Klebsiella pneumoniae Suspension A single colony of Klebsiella pneumoniae CICC 10870 was inoculated into BHI (Qingdao Haibo Biotechnology Co., Ltd.) liquid medium at 1% (v / v) and cultured at 37°C with shaking for 24 hours. After the culture was completed, the cells were centrifuged (5000 rpm, 5 minutes) and the OD was adjusted with BHI. 600 =0.3 spare.

[0033] 3. Experiment on the inhibition of Klebsiella pneumoniae proliferation by NHNK-1701 fermentation products 3 mL of BHI medium and 0.3 mL of NHNK-1701 fermentation product were added to a centrifuge tube. An equal volume of MRS medium was added to the control group and inoculated with a 1% (v / v) Klebsiella pneumoniae suspension. The culture was shaken at 37°C for 24 h and the absorbance at 600 nm was measured.

[0034] The results are shown in Table 1 below: Table 1 Inhibition of Klebsiella pneumoniae growth by NHNK-1701 fermentation products

[0035] The results showed that NHNK-1701 could reduce the proliferation of Klebsiella pneumoniae, with an inhibition rate of 34.69%~51.06%.

[0036] Example 4: NHNK-1701 Agglutination of Klebsiella pneumoniae 1. Preparation of NHNK-1701 inactivated bacteria A single colony of Lactobacillus paracasei NHNK-1701 was picked and placed in MRS liquid medium, incubated at 37°C for 48 h, centrifuged at 5000 rpm for 10 min, and the precipitate was washed twice with PBS (Beijing Solebold Technology Co., Ltd.). The cells were then resuspended in PBS and the OD was adjusted. 600 =0.3. Autoclave at 121℃ for 15 min to obtain inactivated bacteria.

[0037] 2. Preparation of Klebsiella pneumoniae suspension A single colony of Klebsiella pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured at 37°C with shaking for 24 hours. After the culture was completed, the cells were centrifuged (5000 rpm, 5 minutes) and the OD was adjusted with PBS. 600 =0.3 spare.

[0038] 3. Coagulation experiment The bacterial suspension of Klebsiella pneumoniae and NHNK-1701 inactivated bacteria was mixed in a volume ratio of 1:1. After 30 minutes of reaction, samples of the reaction liquid of NHNK-1701 alone, Klebsiella pneumoniae, and the mixed reaction liquid of NHNK-1701 and Klebsiella pneumoniae were taken. The sampling range was the top 50 μL of the liquid surface. After aspiration, the sample was transferred to a 96-well plate and its absorbance at OD = 600 nm was measured. At the same time, the agglutination precipitate was stained with Gram stain and its bacterial agglutination state was observed. ​ shown.

[0039] Calculation formula: Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%; Note: Ax: OD measured by NHNK-1701 alone at the reaction time 600 Value; Ay: OD of Klebsiella pneumoniae alone measured at the reaction time 600 Value; Amix: OD measured at the reaction time after NHNK-1701 and Klebsiella pneumoniae are mixed 600 Numeric value.

[0040] The results are shown in Table 2 below: Table 2 Agglutination of Klebsiella pneumoniae by NHNK-1701

[0041] The results showed that after 30 minutes of reaction, NHNK-1701 inactivated bacteria could agglutinate Klebsiella pneumoniae.

[0042] Example 5 Alcohol tolerance test of NHNK-1701 Pick a single colony of NHNK-1701 and place it in fresh MRS medium and culture it at 37°C for 24 hours. After the culture is complete, inoculate it into MRS medium containing 10% alcohol at a ratio of 2% (v / v). Measure the absorbance at OD = 600nm and record the OD before and after 48 hours of culture at 37°C. 600 Calculation formula: Growth rate (%) = (A1-A0) / A0*100% (A1: absorbance value after culture; A0: absorbance value before culture). The results are shown in Table 3 below: Table 3 Alcohol tolerance test of NHNK-1701

[0043] The results showed that NHNK-1701 has the ability to tolerate alcohol and can grow in a culture medium containing 10% alcohol with a growth rate of 200.00%~257.14%.

[0044] Example 6 NHNK-1701 regulates alcohol-induced keratinocyte-related gene expression 1. Preparation of NHNK-1701 Pick a single colony of NHNK-1701 in fresh MRS liquid medium and culture at 37°C for 24 h. Use DMEM medium (Beijing Solebow Technology Co., Ltd.) to adjust the OD 600 =1.0, centrifuge (5000rpm, 5min), take the supernatant, and then filter with a 0.22μm filter membrane to obtain a sterile fermentation product. Collect the centrifugal precipitate, wash twice with sterile PBS, resuspend the bacteria in DMEM medium and adjust the OD 600 =0.5, and a live bacterial suspension was obtained. Part of the precipitated live bacteria was washed twice with sterile PBS and then sterilized by autoclave at 121°C for 15 min. The suspension was then resuspended in DMEM medium and adjusted to OD 600 =0.5, and inactivated bacteria were obtained.

[0045] 2. Culture of human immortalized keratinocytes HaCaT HaCaT cells (BNCC339817, Beijing Beina Biotechnology) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin and cultured at 37°C in 5% CO2. Cells were passaged or plated after reaching 80-90% confluence.

[0046] 3. NHNK-1701 regulates alcohol-induced keratinocyte barrier damage and inflammation-related gene expression HaCaT cells were seeded in 6-well cell culture plates at a rate of 1×10^6 cells / well and cultured for 12 hours until the cells adhered. The cell culture medium was removed, and the cells were washed twice with sterile PBS. Then, 1.9 mL of DMEM culture medium and 100 μL of NHNK-1701 fermentation product / inactivated bacteria were added. An equal volume of DMEM culture medium was added to the control group. After 3 hours of treatment, 6% (v / v) alcohol was added to induce damage, and the cells were cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the supernatant was discarded, and the cells were washed twice with sterile PBS. Then, 1 mL of cell RNA extraction reagent (Beijing Solebold Technology Co., Ltd.) was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA and determined by qPCR. ​ and ​ The relative expression of the gene in the control group is F=1, and the expression of the gene in the control group is F=1. -ΔΔCT The F value of each sample was calculated by this method.

[0047] The results are shown in Tables 4 and 5 below: Table 4 NHNK-1701 fermentation products regulate the expression of genes related to alcohol-induced keratinocyte barrier damage and inflammation

[0048] Table 5 NHNK-1701-inactivated bacteria regulate the expression of genes related to alcohol-induced keratinocyte barrier damage and inflammation

[0049] The results showed that NHNK-1701 upregulated barrier-related genes ​ 、 ​ and ​ downregulates the expression of pro-inflammatory factor-related genes ​ 、 ​ and ​ NHNK-1701 can alleviate alcohol-induced keratinocyte barrier damage and reduce inflammation.

[0050] Example 7 NHNK-1701 regulates alcohol-induced brain microvascular endothelial cell barrier and inflammation-related gene expression 1. Preparation of NHNK-1701 fermentation products The preparation method of NHNK-1701 fermentation product is shown in Example 3.

[0051] 2. Culture of brain microvascular endothelial cells Brain microvascular endothelial cells BEND3 (BNCC339490, Beina Chuanglian Biotechnology Co., Ltd.) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin and cultured at 37°C in 5% CO2. Cells were passaged or plated after reaching 80-90% confluence.

[0052] 3. NHNK-1701 regulates alcohol-induced damage to brain microvascular endothelial cell barrier and inflammation-related gene expression BEND3 cells were seeded in 6-well cell culture plates at 1×10^6 cells / well and cultured for 12 hours until the cells adhered. The cell culture medium was removed, and the cells were washed twice with sterile PBS. 1.9 mL of DMEM culture medium and 100 μL of NHNK-1701 fermentation product were added respectively, and an equal volume of DMEM culture medium was added to the control group. After treatment for 3 hours, 6% alcohol was added to induce damage, and the cells were cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the supernatant was discarded, and the cells were washed twice with sterile PBS. Subsequently, 1 mL of cell RNA extraction reagent was added to each well, and the total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA and determined by qPCR. ​ ​ and ​ The relative expression of the gene in the control group is F=1, and the expression of the gene in the control group is F=1. -ΔΔCT The F value of each sample was calculated by this method.

[0053] The results are shown in Table 6 below: Table 6 NHNK-1701 fermentation products regulate the expression of genes related to alcohol-induced damage to brain microvascular endothelial cells barrier and inflammation

[0054] The results showed that NHNK-1701 fermentation products can upregulate vascular barrier-related genes ​ and ​ downregulates the expression of pro-inflammatory factor-related genes ​ and ​ NHNK-1701 can alleviate alcohol-induced brain microvascular endothelial cell barrier damage and reduce inflammation.

[0055] Example 8 NHNK-1701 improves the survival rate of brain microvascular endothelial cells damaged by Klebsiella pneumoniae 1. Preparation of NHNK-1701 fermentation products The preparation method of NHNK-1701 fermentation product is shown in Example 3.

[0056] 2. Preparation of Klebsiella pneumoniae suspension A single colony of Klebsiella pneumoniae CICC 10870 was inoculated into BHI liquid medium at 1% (v / v) and cultured at 37°C with shaking for 24 hours. After the culture was completed, the cells were centrifuged at 5000 rpm, washed three times with PBS, and resuspended in DMEM to adjust the OD value. 600 =0.1 spare.

[0057] 3. Culture of mouse brain microvascular endothelial cells The culture method of brain microvascular endothelial cells BEND3 is referred to Example 7.

[0058] 4. Experiment on NHNK-1701 improving the survival rate of brain microvascular endothelial cells damaged by Klebsiella pneumoniae BEND3 cells were seeded at 1×10^4 cells / well in a 96-well cell culture plate and cultured for 24 hours until the cells adhered. The culture medium was removed, and 100 μL of DMEM containing 1% (v / v) live NHNK-1701 bacteria or fermentation products was added to the experimental group, while an equal volume of DMEM was added to the control group. After a 3-hour incubation, 1 μL of Klebsiella pneumoniae suspension was added to each well, and the cells were incubated at 37°C and 5% CO2 for 24 hours. 10 μL of CCK-8 reagent (GLPbio, USA) was added to each well, and the absorbance at 450 nm was measured after incubation at 37°C for 3 hours. The calculation formula and results are shown in Table 7 below: Table 7 NHNK-1701 improves the survival rate of brain microvascular endothelial cells damaged by Klebsiella pneumoniae

[0059] The results showed that NHNK-1701 fermentation products could increase the relative survival rate of brain microvascular endothelial cells BEND3 cells under Klebsiella pneumoniae-induced injury.

[0060] Example 9 NHNK-1701 regulates the expression of genes related to Klebsiella pneumoniae-induced damage to brain microvascular endothelial cells 1. Preparation of NHNK-1701 live bacteria and fermentation products The preparation method of NHNK-1701 is as described in Example 3.

[0061] 2. Preparation of Klebsiella pneumoniae suspension The preparation method of Klebsiella pneumoniae suspension is as described in Example 8.

[0062] 3. Culture of brain microvascular endothelial cells The culture method of brain microvascular endothelial cells BEND3 is referred to Example 7.

[0063] 4. NHNK-1701 regulates the expression of genes related to Klebsiella pneumoniae-induced damage to brain microvascular endothelial cells BEND3 cells were seeded in a 6-well cell culture plate at 1×10^6 cells / well and cultured for 12 hours until the cells adhered. The cell culture medium was removed, washed twice with sterile PBS, and then 1.9 mL of DMEM culture medium and 100 μL of NHNK-609 live bacteria / fermentation product were added. An equal volume of DMEM culture medium was added to the control group. After 3 hours of treatment, 20 μL of Klebsiella pneumoniae suspension was added to induce damage, and cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA and determined by qPCR. ​ 、 ​ and ​ The relative expression of the gene in the control group is F=1, and the expression of the gene in the control group is F=1. -ΔΔCT The F value of each sample was calculated by this method.

[0064] The results are shown in Tables 8 and 9 below: Table 8 NHNK-1701 live bacteria regulates Klebsiella pneumoniae-induced damage to BEND3 cell barrier and inflammation-related gene expression

[0065] Table 9 NHNK-1701 fermentation products regulate the expression of genes related to BEND3 cell barrier damage and inflammation induced by Klebsiella pneumoniae

[0066] The results showed that NHNK-1701 upregulated vascular barrier genes ​ 、 ​ and ​ downregulates the expression of inflammation-related genes ​ 、 ​ and ​ The expression of WT11 can reduce Klebsiella pneumoniae-induced brain microvascular endothelial cell barrier damage and reduce inflammation.

[0067] Example 10 NHNK-1701 regulates Klebsiella pneumoniae-induced damage to intestinal epithelial cell barrier and expression of inflammation-related genes 1. Preparation of live and inactivated NHNK-1701 bacteria The preparation method is as in Example 6.

[0068] 2. Preparation of Klebsiella pneumoniae suspension The preparation of Klebsiella pneumoniae suspension was carried out as described in Example 9.

[0069] 3. Culture of human intestinal epithelial cells Caco-2 Caco-2 cells (BNCC350769, Beina Chuanglian Biotechnology Co., Ltd.) were activated with DMEM medium containing 10% FBS and 1% penicillin-streptomycin and cultured at 37°C in 5% CO2. Cells were passaged or plated after reaching 80-90% confluence.

[0070] 4. NHNK-1701 regulates Klebsiella pneumoniae-induced damage to intestinal epithelial cell barrier and inflammation-related gene expression Caco-2 cells were seeded in 6-well cell culture plates at 1×10^6 cells / well and cultured for 12 hours until the cells adhered. The cell culture medium was removed and washed twice with sterile PBS, followed by addition of 1.9 mL of DMEM culture medium and 100 μL of NHNK-1701 live / inactivated bacteria. An equal volume of DMEM culture medium was added to the control group. After 3 hours of treatment, 20 μL of Klebsiella pneumoniae suspension was added to induce damage and cultured for 24 hours at 37°C and 5% CO2. After the culture was completed, the supernatant was discarded, the cells were washed twice with sterile PBS, and then 1 mL of cell RNA extraction reagent was added to each well. Total RNA was extracted according to the reagent instructions and the concentration and purity were determined. After the extraction was completed, it was reverse transcribed into cDNA and determined by qPCR. ​ 、 ​ and ​ The relative expression of the gene in the control group is F=1, and the expression of the gene in the control group is F=1. -ΔΔCT The F value of each sample was calculated by this method.

[0071] The results are shown in Tables 10 and 11 below: Table 10 NHNK-1701 live bacteria regulates Klebsiella pneumoniae-induced damage to Caco-2 cell barrier and inflammation-related gene expression

[0072] Table 11 NHNK-1701 inactivated bacteria regulates Klebsiella pneumoniae-induced Caco-2 cell barrier damage and inflammation-related gene expression

[0073] The results showed that NHNK-1701 could upregulate intestinal barrier-related genes ​ 、 ​ downregulate the expression of inflammatory factor-related genes ​ 、 ​ and ​ NHNK-1701 can reduce intestinal permeability and reduce inflammation caused by Klebsiella pneumoniae, thereby reducing the possibility of Klebsiella pneumoniae passing through the intestine and entering the circulatory system.

[0074] 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 paracasei strain for improving urticaria and Alzheimer's disease ( Lacticaseibacillus paracasei ), the strain is Lactobacillus paracasei NHNK-1701, which was deposited in the China Center for Type Culture Collection on April 14, 2025, and its deposit number is CCTCC NO: M 2025765.

2. Lactobacillus paracasei as claimed in claim 1 ( Lacticaseibacillus paracasei ) in the preparation of products for improving urticaria and Alzheimer's disease.

3. The use according to claim 2, characterized in that The improvement of urticaria and Alzheimer's disease includes at least one of the following a)-h): a) Inhibit Klebsiella pneumoniae ( Klebsiella pneumoniae ) growing and / or producing alcohol; b), agglutination of Klebsiella pneumoniae; c) Tolerance to 10% alcohol growth; d) Reduce alcohol-induced damage to the skin keratin barrier and inflammation, and upregulate barrier-related genes ZO-1、CLDN4 and OCLD , downregulating inflammation-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions; e) Reduce alcohol-induced damage to the brain microvascular barrier and inflammation, and upregulate barrier-related genes ZO-1、CLDN5 and OCLD , downregulating inflammation-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions; f) Improve the survival rate of brain microvascular endothelial cells induced by Klebsiella pneumoniae injury; g) Reduce Klebsiella pneumoniae-induced damage to the brain microvascular barrier and inflammation, and upregulate barrier-related genes ZO-1 、 CLDN5 and OCLD downregulate the expression of inflammatory factor-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions; h) Reduce Klebsiella pneumoniae-induced intestinal barrier damage and inflammation, and upregulate barrier-related genes ZO-1 、 CLDN4 downregulate the expression of inflammatory factor-related genes TNF-α 、 IL-6 and IL-1β at least one of the expressions.

4. A product for improving urticaria and Alzheimer's disease, characterized in that: comprising the Lactobacillus paracasei as claimed in claim 1 ( Lacticaseibacillus paracasei ), and acceptable excipients and / or adjuvants.

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