Lactobacillus gasseri BDUP for treating atopic dermatitis or eczema and application

Abstract

The invention discloses a lactobacillus gasseri BDUP (Lactobacillus gasseri) strain and application of the lactobacillus gasseri BDUP strain in preparation of drugs for treating atopic dermatitis or eczema. The preservation number of the lactobacillus gasseri BDUP strain is CGMCC (China General Microbiological Culture Collection Center) No. 18357. Different from existing probiotics which take effect mainly by resisting histamine or regulating Th1 / Th2 balance, the invention discloses a unique nerve-immune bidirectional regulation mechanism of the BDUP strain for the first time, and the BDUP strain can significantly down-regulate expression levels of a neuropeptide P substance (Substance P), a calcitonin gene-related peptide (CGRP), a nerve growth factor (NGF) and a pruritus specific factor IL-31 in skin tissues, so that the BDUP strain can be used for treating skin pruritus. Therefore, neurogenic inflammation and a pruritus conduction pathway are effectively blocked. Animal experiments show that the BDUP strain can shorten the scratching time of eczema model mice by about 80%, the itching relieving and anti-inflammatory effects of the BDUP strain are equivalent to those of potent glucocorticoid, and the BDUP strain has no side effect. The invention provides a novel efficient biological treatment scheme for solving intractable pruritus caused by eczema.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the use of Lactobacillus gasseri BDUP in the preparation of drugs for treating atopic dermatitis or eczema. Background Technology

[0002] 1. The current situation and harms of eczema

[0003] Eczema is a chronic, relapsing inflammatory skin disease prevalent worldwide. Its main clinical features include dry skin, erythema, edema, and intense, unbearable itching. It is estimated that approximately 10%-20% of children and 3%-5% of adults worldwide are affected by it. The intense itching can lead to a vicious cycle of itching-scratching-inflammation, severely impacting patients' sleep quality and mental health, resulting in a significant disease burden.

[0004] 2. Limitations of existing drug treatments

[0005] Currently, first-line drugs for the clinical treatment of eczema mainly include topical corticosteroids (TCS) and calcineurin inhibitors (TCIs). Despite their widespread use, they still have many limitations. While corticosteroids can quickly relieve inflammation, long-term use can easily lead to serious side effects such as skin atrophy, telangiectasia, pigmentation, and steroid-dependent dermatitis, and the condition is prone to recurrence after discontinuation. Calcineurin inhibitors (such as tacrolimus ointment and pimecrolimus cream) avoid some of the side effects of corticosteroids, but they still pose a potential risk of immunosuppression, and long-term use may increase the likelihood of skin infections. Furthermore, their safety in children and pregnant women remains controversial. In addition, antihistamines are commonly used clinically to treat itching, but studies have shown that the itching mechanism of eczema is complex, and many patients experience "non-histamine-dependent pruritus," making conventional antihistamines often ineffective. While biologics (such as monoclonal antibodies targeting the IL-4 / IL-13 pathway) that have been marketed in recent years are effective, their high price and the need for injection limit their widespread use.

[0006] 3. Shortcomings of current probiotic technology

[0007] With the deepening research into the relationship between microorganisms and human health, the "Gut-Skin Axis" theory has been established, and the role of probiotics in regulating the balance of the human gut microbiota and improving immune function has received increasing attention, with research on their application in the treatment of skin diseases also gradually increasing. Utilizing probiotics to regulate the gut microbiota to improve skin diseases has become a research hotspot. Existing technologies have some reports on the use of lactobacilli (such as Lactobacillus rhamnosus and Lactobacillus paracasei) and bifidobacteria in the adjunctive treatment of eczema. However, existing probiotic technologies face the following significant technical bottlenecks:

[0008] First, the mechanism of action is singular and neglects neurogenic regulation: Most existing research and patented technologies focus on regulating the systemic immune response (such as correcting the Th1 / Th2 balance and reducing IgE levels) or inhibiting mast cell degranulation (reducing histamine release).

[0009] However, the most unbearable symptom for eczema patients is the intense, non-histamine-dependent neuropathic itching, which often leads to a vicious cycle of itching and scratching. Modern neurobiological research on skin has found that the persistent itching and inflammation of eczema are largely mediated by neurogenic inflammation driven by neuropeptides (such as Substance P and calcitonin gene-related peptide CGRP) and nerve growth factor (NGF). Currently available probiotic strains have few reports of effectively downregulating these key neuropeptides or directly blocking the neuropathic itch transmission pathway. Furthermore, existing probiotics have limited effectiveness in relieving these neurogenic symptoms, failing to reach the level of clinical therapeutic drugs.

[0010] Second, the efficacy of probiotics is highly strain-specific, resulting in limited effectiveness in relieving itching: Many commercially available or reported strains, while showing some improvement in skin lesions, often fall short in alleviating the most pressing need of patients—intense itching—and rarely achieve a level of treatment comparable to medication.

[0011] 4. The technical problem solved by the present invention

[0012] Therefore, there is an urgent need in this field to screen for highly efficient, safe, and beneficial functional bacterial strains. These strains should not only possess conventional immunomodulatory capabilities but also significant neuroimmunomodulatory functions, blocking the development of eczema from both neurotransmission and immune response dimensions by downregulating substance P, CGRP, and pruritus-related cytokines (such as IL-31), particularly achieving highly effective relief of severe itching. This invention is based on the aforementioned needs. Summary of the Invention

[0013] To address the aforementioned issues, this invention screened out a strain of Lactobacillus gasseri BDUP with significant neuroimmunomodulatory function, which can block itching at the nerve conduction level and can be used as a highly effective and safe pharmaceutical preparation for the treatment of eczema.

[0014] One aspect of the present invention provides the use of cultures, metabolites, lysates, or extracts of *Lactobacillus gasseri* strain BDUP in the preparation of medicaments for treating atopic dermatitis or eczema, wherein the *Lactobacillus gasseri* strain BDUP is accessed under CGMCC No. 18357. Preferably, the medicament is used to improve neurogenic inflammation and itching symptoms caused by atopic dermatitis or eczema.

[0015] Preferably, the improvement of neurogenic inflammation and itching symptoms is achieved by downregulating the expression level of at least one factor selected from the following in skin tissue: substance P, calcitonin gene-related peptide (CGRP), nerve growth factor (NGF), or interleukin-31 (IL-31).

[0016] Preferably, the drug is used to block the itch-scratch cycle of the skin and significantly reduce the duration of scratching behavior.

[0017] According to another aspect of the invention, the drug is also used to downregulate the mRNA expression level of pruritus receptors TRPV1 or MrgprA3, or to reduce serum IgE levels.

[0018] According to another aspect of the invention, the culture of the Lactobacillus gasseri BDUP strain comprises live cells, dead cells (inactivated bacteria), fermentation supernatant, or cell lysate.

[0019] Preferably, the amount of *Lactobacillus gasseri* BDUP strain added is an effective amount sufficient to exert a therapeutic effect. More preferably, the amount of *Lactobacillus gasseri* BDUP strain added is 1 × 10⁻⁶. 6 CFU / mL to 1×10 11 CFU / mL.

[0020] According to another aspect of the invention, the medicament is an oral or topical formulation. Preferably, the oral formulation is selected from powders, granules, capsules, tablets, or liquid formulations; the topical formulation is selected from creams, ointments, gels, lotions, or skin patches.

[0021] Compared with existing technologies, the Lactobacillus gasseri BDUP strain of the present invention has the following significant advantages and unexpected technical effects: (1) A unique "neuro-immune" bidirectional regulatory mechanism. Existing technologies mainly work by inhibiting mast cell degranulation and histamine release, while the BDUP strain of the present invention reveals a completely new mechanism of action: by significantly downregulating the levels of substance P, calcitonin gene-related peptide (CGRP), and nerve growth factor (NGF) in skin tissue, it directly intervenes in neurogenic inflammation. In addition, the BDUP strain can also significantly inhibit the expression of pruritus-specific cytokine IL-31 and pruritus receptors TRPV1 and MrgprA3. This means that the BDUP strain can not only regulate the immune system, but also directly act on the "skin-nerve" crosstalk network, blocking the generation and transmission of pruritus signals from the source. (2) Excellent antipruritic effect: In the DNCB-induced eczema mouse model, the BDUP strain showed amazing antipruritic efficacy. This indicates that the BDUP strain has the potential to replace hormone drugs in relieving the core symptom of eczema (pruritus). (3) Comprehensive skin barrier repair and anti-inflammatory capabilities: BDUP strains (including both live and inactivated strains) can significantly reduce transepidermal water loss (TEWL), restore skin barrier function, and significantly downregulate the levels of pro-inflammatory factors (TNF-α, IL-4, IL-13) and increase the levels of anti-inflammatory factors (IL-10), exhibiting excellent therapeutic effects on erythema, swelling, and dry desquamation caused by eczema. (4) High safety and diverse application forms: As a probiotic preparation, BDUP avoids the side effects of hormonal drugs (such as skin thinning and dependence). In addition, studies have confirmed that the inactivated (dead) bacteria of BDUP also retain significant anti-inflammatory and neuromodulatory activities, which makes it extremely convenient for the preparation of various storage-resistant oral or topical drug preparations (such as powders and creams) that do not require cold chain.

[0022] In summary, the BDUP strain of this invention can also regulate the levels of pruritus neural pathway factors and reduce the production of pruritus-related neuropeptides, thereby weakening the activation of the pruritus response and effectively relieving erythema, swelling, dryness, and crusting caused by eczema. It can be used in the preparation of various eczema-relieving products, providing a new, safe, and effective approach for the improvement and intervention of eczema, and has broad application prospects and market value.

[0023] Preservation instructions for the strains described in this invention:

[0024] Strain name: BDUP

[0025] Latin name: Lactobacillus gasseri

[0026] Classification and nomenclature: Lactobacillus gasseri

[0027] Strain number: 18357

[0028] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0029] Collection institution abbreviation: CGMCC

[0030] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0031] Date of preservation: August 2, 2019

[0032] CGMCC Registration Number: 18357 Attached Figure Description

[0033] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 The effect of Lactobacillus gasseri BDUP on HaCaT cell viability, as described in this invention. n=6, *p<0.05, **p<0.01, ***p<0.001.

[0035] Figure 2 The levels of TNF-α (A), IFN-γ (B), IL-1β (C), IL-6 (D), and IL-10 (E) in HaCaT cells after treatment with Lactobacillus gasseri BDUP according to the present invention are shown. n=5, *p< 0.05, **p< 0.01, ***p< 0.001.

[0036] Figure 3 The serum levels of specific antibodies IgE (A) and IgG1 (B) in mice with eczema model after treatment with Lactobacillus gasseri BDUP were calculated. n=10, *p<0.05, **p<0.01, ***p<0.001.

[0037] Figure 4 The levels of inflammation-related cytokines IL-13 (A), IL-31 (B), TNF-α (C), IFN-γ (D), and IL-10 (E) in the serum of mice with eczema after treatment with Lactobacillus gasseri BDUP were determined. n=10, *p<0.05, **p<0.01, ***p<0.001.

[0038] Figure 5The serum levels of pruritus-related neuropeptides Substance P (SP) (A), calcitonin gene-related peptide CGRP (B), and nerve growth factor NGF (C) in mice with eczema model after treatment with Lactobacillus gasseri BDUP were determined. n=10, *p<0.05, **p<0.01, ***p<0.001.

[0039] Figure 6 The levels of propylene glycol (MDA) (A) and superoxide dismutase (SOD) (B) in the skin tissue of mice with eczema model treated with Lactobacillus gasseri BDUP were measured. n=10, *p< 0.05, **p< 0.01, ***p< 0.001.

[0040] Figure 7 The mRNA expression levels of pruritus factors Nppb (A), MrgprA3 (B), IL-31 (C), and TRPV1 (D) in skin tissue of mice with eczema model after treatment with Lactobacillus gasseri BDUP were determined. n=10, *p< 0.05, **p< 0.01, ***p<0.001. Detailed Implementation

[0041] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0042] Example 1. Culture and sample preparation of Lactobacillus gasseri BDUP strain

[0043] Spread BDUP bacterial culture frozen at -80℃ onto MRS solid plates and incubate upside down at 37℃ for 24 hours. Then, take a single colony and inoculate it into liquid MRS medium (10 g peptone, 10 g beef extract, 5 g yeast extract, 2 g diammonium citrate, 20 g glucose, 1 mL Tween-80, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.58 g magnesium sulfate, 0.25 g manganese sulfate, and 1000 mL distilled water). Incubate at 37℃ for 24 hours to obtain the first-generation bacterial culture. Take 10% of the first-generation bacterial culture and inoculate it into fresh MRS liquid medium. Incubate at 37℃ for 24 hours to obtain the second-generation bacterial culture. Take 10% of the second-generation bacterial culture and inoculate it into fresh MRS liquid medium. Incubate at 37℃ for 24 hours to obtain the working bacterial culture.

[0044] Centrifuge the working bacterial culture at 13000 rpm for 15 min at 4°C, collect the supernatant (fermentation supernatant), and store it at 4°C for subsequent experiments. Resuspend the bacterial pellet in physiological saline to obtain a live bacterial suspension containing viable cells. Adjust the suspension to the appropriate concentration required for animal experiments (e.g., 10⁻⁶). 9 (CFU / mL). Live bacteria can be obtained by treating them at 70℃ for 30 min to obtain inactivated Lactobacillus gasseri BDUP cells.

[0045] Example 2. Evaluation of the effect of Lactobacillus gasseri BDUP strain in improving the inflammatory response of HaCaT skin cells.

[0046] (1) Experimental methods

[0047] This embodiment uses HaCaT immortalized human keratinocytes for the following experiments. Cells were cultured in DMEM medium (supplemented with 10% fetal bovine serum) at 5% CO2 and 37°C. HaCaT cells in logarithmic growth phase were seeded into 96-well plates (5 × 10⁶ cells / well). 3 The cells were cultured in wells for 22-24 hours, and then grouped according to experimental requirements: blank group (NC) (normal culture conditions), model group (MC) (TNF-α added to a final concentration of 25 μg / L), strain treatment group (BDUP) (TNF-α to a final concentration of 25 μg / L + BDUP strain culture supernatant with a volume fraction of 10%), BDUP inactivated bacteria group (p-BDUP), and dexamethasone (Dex) group (TNF-α to a final concentration of 25 μg / L + Dex to a final concentration of 1 μM). The cells were cultured for another 24 hours, with dexamethasone Dex serving as a positive control.

[0048] After treating the cells with the bacterial strain, the culture supernatant from each group was aspirated and collected. The cells were washed twice with sterile PBS, and then 10% CCK-8 solution was added to each well. After incubation in a constant temperature incubator for 2 h, the absorbance at 450 nm was measured. The cell supernatant from each group was collected, and the levels of TNF-α, IFN-γ, IL-1β, IL-6, and IL-10 were detected using the corresponding inflammatory factor ELISA kits. Specific procedures were followed according to the instructions of each kit.

[0049] (2) Experimental results

[0050] Compared with untreated cells (NC group), the viability of HaCaT cells treated with TNF-α was significantly reduced in the model group (p<0.001). Figure 1The cell survival rate was 82.28%, which may be due to cell damage caused by TNF-α-induced inflammatory response. After applying Dex and BDUP, the cell survival rate increased to 92.45% and 96.49%, respectively, indicating that Dex and BDUP can significantly alleviate TNF-α-induced inflammatory damage and restore cell viability to normal.

[0051] Regarding the levels of inflammatory factors, compared with the NC group ( Figure 2 In the Model group, the levels of pro-inflammatory factors TNF-α, IFN-γ, IL-1β, and IL-6 in the HaCaT cell culture supernatant were significantly increased (p < 0.001), while the level of the anti-inflammatory factor IL-10 was decreased, indicating the presence of skin inflammation. Compared with the MC group, in the Dex-treated group, the levels of TNF-α decreased to 86.39 pg / mL (P < 0.001, a decrease of 24.91%), IFN-γ decreased to 13.43 pg / mL (P < 0.001, a decrease of 33.91%), IL-6 decreased to 49.00 pg / mL (P < 0.001, a decrease of 51.58%), and IL-1β decreased to 18.55 pg / mL (P < 0.001, a decrease of 41.53%); while the level of IL-10 increased to 14.23 pg / mL (P < 0.001, an increase of 65.59%).

[0052] In the experimental group treated with *Lactobacillus gasseri* BDUP, compared with the model group, the TNF-α content decreased to 89.08 pg / mL (P<0.001, a decrease of 22.58%), the IFN-γ content decreased to 15.45 pg / mL (P<0.1, a decrease of 23.97%), the IL-1β content decreased to 23.80 pg / mL (P<0.001, a decrease of 25.01%), and the IL-6 content decreased to 58.72 pg / mL (P<0.001, a decrease of 41.98%); the IL-10 content also increased to 10.91 pg / mL (P<0.1, an increase of 26.97%). These results indicate that the *Lactobacillus gasseri* BDUP strain described in this invention can reduce the content of pro-inflammatory factors and increase the level of anti-inflammatory factors at the cellular level, thereby improving skin inflammation.

[0053] Example 3. Evaluation of the effect of Lactobacillus gasseri BDUP strain on improving the condition of eczema model mice.

[0054] (1) Experimental methods

[0055] ① Establishment of a mouse model of eczema

[0056] Fifty 6-week-old SPF-grade male BALB / c mice (n=10 per group, Vitaliva) were randomly divided into four groups based on body weight after 7 days of laboratory acclimatization: control group (NC), model group (MC), BDUP live bacterial culture group (BDUP), BDUP inactivated bacterial culture group (p-BDUP), and positive control group (0.1% dexamethasone group (Dex). The BDUP bacterial culture group was fed a diet containing *Lactobacillus gasseri* BDUP (15 mL of *Lactobacillus gasseri* BDUP fermentation broth mixed with 40 g of feed) twice daily for one week to allow the bacteria to colonize the mouse intestines. The control and model groups were fed an equal volume of physiological saline. Subsequently, the back hair of mice in the model and probiotic groups was removed using a razor and depilatory agent, and skin sensitization was induced for 3 consecutive days with 50 μL of 2,4-dinitrochlorobenzene (DNCB, 2%). After a 2-day interval, the treatment was repeated for 3 consecutive days with 50 μL of DNCB (1%) in the same area. Then, the mice in the BDUP bacterial culture group continued to be fed diets containing Lactobacillus gasseri BDUP for 21 days.

[0057] ②Detection indicators

[0058] Each mouse was videotaped for 60 minutes every week to record scratching behavior and to determine the duration of scratching. Transepidermal water loss (TEWL) was measured using a Tewameter™ 300 instrument (TM300-MDD, Germany) to reflect the status of skin barrier function. Four indicators were scored on the skin on the back of the mice: erythema / bleeding, edema / swelling, dryness / desquamation, and crusting / scratching. Each indicator was scored from 0 to 3 points, with a maximum total score of 12 points.

[0059] After the experiment, the thickness of the right ear of each group of mice was measured using calipers. Immediately after the mice were euthanized, ear tissue samples were extracted using a 6 mm perforator and weighed. The formula for calculating ear swelling is: Swelling = Right ear thickness - Left ear thickness.

[0060] The body weight of mice in each group was measured, and skin, thymus and spleen tissues were collected. The thymus and spleen were weighed, and the thymus index and spleen index were calculated respectively.

[0061] Thymus index = thymus weight / mouse body weight

[0062] Spleen index = spleen weight / mouse body weight.

[0063] Blood was collected from the orbital sinus of mice in each group. After standing at room temperature for 30 min, the blood was centrifuged at 3000 r / min for 10 min to obtain serum. The serum was collected, transferred to centrifuge tubes, and stored at -20℃ for later use. Then, the levels of immunoglobulins IgE and IgG1, inflammatory factors IL-13, IL-31, TNF-α, IFN-γ, and IL-10, as well as pruritus-related neuropeptides SP, CGRP, and NGF in mouse serum were detected using the corresponding ELISA kits.

[0064] Appropriate amounts of dorsal skin tissue from each group of mice were collected and homogenized thoroughly in an ice bath using a tissue homogenizer. The levels of oxidative stress markers MDA and SOD in the skin tissue were then detected using an ELISA kit. In addition, total RNA was extracted using Trizol reagent. The qualified total RNA was reverse transcribed into cDNA using a reverse transcription kit. RT-qPCR was performed using the SYBR Green dye method on a real-time quantitative PCR instrument. Each sample was tested in triplicate, and the relative expression levels of pruritus factors Nppb, MrgprA3, IL-31, and TRPV1 in the skin tissue were calculated using a relative quantification method.

[0065] (2) Experimental results

[0066] ① BDUP strain alleviates skin condition and itching behavior in eczema model mice.

[0067] After the experiment, observation of the mice revealed that the model group mice exhibited obvious allergic reactions such as ear swelling, erythema, edema, and papules, indicating successful modeling. The skin lesion scoring method was used to assess the degree of skin inflammation in each group of mice, with a scoring range of 0-12 points. Total scores: 0–3 for mild eczema, 4–7 for moderate eczema, and 8–12 for severe eczema. According to Table 1, the average score of the blank group was 0.7±0.2, the average score of the model group was 8.6±0.9, and the average score of the Dex-positive control group was 3.0±0.6. The average score of the *Lactobacillus gasseri* BDUP live bacteria intervention group was 3.8±0.7, significantly lower than the model group (P<0.001). The average score of the BDUP inactivated bacteria group (p-BDUP) was 5.1±0.4, significantly lower than the model group (P<0.01), indicating that the BDUP strain can effectively alleviate the redness, swelling, dryness, and desquamation of eczema.

[0068] Regarding scratching behavior, the average scratching time of mice in the model group surged from 14 seconds in the control group to 110 seconds (experimental results are shown in Table 1), indicating severe itching symptoms. After intervention with BDUP live bacteria, the scratching time of mice dramatically decreased to 21 seconds, a reduction of approximately 81%. Notably, this effect significantly outperforms the approximately 50% relief achieved by conventional probiotics in existing technologies, and there was no statistically significant difference between this effect and the effect observed in the group treated with the potent steroid drug dexamethasone (Dex) (19 seconds). This suggests that the BDUP strain has the potential to replace or assist steroid drugs in alleviating the core symptom of eczema—severe itching—and can effectively break the vicious cycle of 'itch-scratching'.

[0069] Furthermore, the transepidermal water loss (TEWL) values ​​of the back skin showed that, compared with the control group, the TEWL of the model group was significantly increased (38.8±5.1 g / m² / h vs. 6.3±2.1 g / m² / h), while that of the BDUP live bacteria group was significantly decreased to 15.2±3.7 g / m² / h, close to the level of the Dex positive control group (12.6±4.5 g / m² / h). Simultaneously, TEWL was also reduced after BDUP dead bacteria intervention, with a value of 20.1±4.3 g / m² / h. Elevated TEWL indicates impaired skin barrier function; the higher the value, the more severe the damage. These results suggest that the BDUP strain of this invention, including both live and dead bacteria, can significantly improve skin barrier function.

[0070] Table 1. Skin condition, scratching behavior, and TEWL evaluation in eczema mice

[0071] Group Skin lesion score Scratching time (s) Number of scratches TEWL score (g / m² / h) Blank group 0.7±0.2 14±1.19 8±0.60 6.3±2.1 Model group 8.6±0.9 110±3.56 89±1.46 38.8±5.1 Dex Group 3.0±0.6（***） 19±2.24（***） 16±0.71（***） 12.6±4.5（***） BDUP group 3.8±0.7（***） 21±0.88（***） 20±1.07（***） 15.2±3.7（***） p-BDUP group 5.1±0.4（***） 41±2.26（***） 32±1.24（***） 20.1±4.3（***）

[0072] Note: Significant differences are compared with the model group.

[0073] ② BDUP strains improved the thymus index and spleen index in eczema model mice.

[0074] Ear swelling is the average thickness of the right ear. The degree of ear swelling reflects the severity of inflammation. During the experiment, DNCB induced eczema in mice, and the skin condition gradually worsened. The model group mice showed significant swelling in the right ear, with a swelling degree of 0.29 mm (see Table 2). In contrast, the BDUP live bacteria strain treatment group showed significant improvement in ear swelling, with a swelling degree of 0.06 mm, and the allergic reaction effect was similar to that of the positive control Dex group (swelling degree of 0.05 mm). The BDUP inactivated bacteria group also alleviated the swelling and inflammation of the mouse ears, but the effect was slightly worse than that of the live bacteria group (swelling degree of 0.10 mm). This indicates that both live and inactivated Lactobacillus gasseri BDUP can alleviate the inflammatory phenotype in mice with eczema.

[0075] Table 2. Thickness and swelling of the right ear in mice with eczema

[0076] Group Average right ear thickness (mm) Average swelling (mm) Blank group 0.25±0.01 0.02±0.00 Model group 0.80±0.01 0.29±0.02 Dex Group 0.23±0.01（***） 0.05±0.00（***） BDUP group 0.27±0.01（***） 0.06±0.01（***） p-BDUP group 0.37±0.01（***） 0.10±0.00（***）

[0077] Note: Significant differences are compared with the model group.

[0078] The results are shown in Table 3. After the experiment, there was no significant difference in the body weight of mice in each group, ranging from 29.73 to 30.62 g, indicating that DNCB-induced eczema did not affect the growth of mice. The effects of eczema on the thymus and spleen indices of each group were observed. Compared with the control group, the thymus index (2.06 vs 3.2) and spleen index (4.0 vs 5.4) of the model group mice were significantly increased. However, after treatment with *Lactobacillus gasseri* BDUP (live / inactivated bacteria), the thymus and spleen indices of the eczema mice were significantly reduced, essentially returning to normal levels. The effect of the inactivated bacteria group on both indices was slightly weaker than that of the live bacteria group. These results indicate that DNCB-induced eczema affects the immune organs of mice, while *Lactobacillus gasseri* BDUP can protect the thymus and spleen of eczema mice.

[0079] Table 3. Mouse body weight, thymus and spleen index

[0080] Group Initial body weight (g) Final body weight (g) Thymus index Spleen index Blank group 21.75±0.55 30.09±0.62 2.06±0.07 4.0±0.07 Model group 22.36±0.61 30.62±0.87 3.20±0.08 5.4±0.05 Dex Group 21.95±0.69 (ns) 29.73±0.56 (ns) 2.40±0.05（***） 4.3±0.06（***） BDUP group 22.05±0.45 (ns) 29.84±0.58 (ns) 2.68±0.07（***） 4.2±0.11（***） p-BDUP group 22.28±0.57 (ns) 30.17±0.53 (ns) 2.89±0.06（*） 4.8±0.07（***）

[0081] Note: Significant differences are compared with the model group.

[0082] ③ BDUP strains reduce specific antibodies and immune cytokines in the serum of eczema model mice.

[0083] like Figure 3 As shown, compared with the control group, the serum levels of total IgE and IgG1 in the eczema model group were significantly increased, indicating that the Th2-type inflammatory response in eczema mice modeled by DNCB was enhanced. Compared with the model group, the IgE level was significantly reduced after treatment with live BDUP to 6.76 μg / mL (P<0.001, a decrease of 39.2%), and the IgG1 level was 780.92 μg / mL (a decrease of 41.08%); the IgE level in the inactivated strain (p-BDUP) group was 7.93 μg / mL (a decrease of 28.6%), and the IgG1 level was 1991.84 μg / mL (a decrease of 25.16%). This indicates that Lactobacillus gasseri BDUP can reduce the abnormal secretion of IgE and IgG1 induced by inflammation and inhibit the increase of specific antibodies in mouse serum.

[0084] Figure 4As shown, compared with the control group, the serum levels of Th2 cytokines IL-13 and IL-31 in DNCB-induced eczema mice in the model group were significantly increased, the secretion of pro-inflammatory TNF-α was increased, while the secretion of Th1 cytokine IFN-γ and anti-inflammatory IL-10 was decreased, demonstrating the successful establishment of the mouse eczema model. In the positive control Dex group, analysis of the secretion of related inflammatory factors showed that Dex intervention could alleviate inflammation by downregulating the Th2 inflammatory pathway.

[0085] In the BDUP strain group, the levels of Th2 cytokines IL-13 and IL-31 in mouse serum were significantly reduced by 22.88% and 53.35%, respectively (P<0.001), and the secretion of the potent pro-inflammatory cytokine TNF-α was also reduced by 44.80%. Conversely, the levels of Th1 cytokine IFN-γ and anti-inflammatory cytokine IL-10 in serum increased by 29.35% and 53.94%, respectively. These results demonstrate that the live bacterial solution of this strain has a significant ameliorative effect on inflammation.

[0086] In the p-BDUP strain group, serum levels of Th2 cytokines IL-13 and IL-31 in mice were significantly reduced by 16.77% and 32.12%, respectively (P<0.001), and the secretion of the potent pro-inflammatory cytokine TNF-α was also reduced by 34.49%. However, serum levels of Th1 cytokine IFN-γ and anti-inflammatory cytokine IL-10 increased by 21.88% and 42.12%, respectively. This indicates that inactivated BDUP treatment can also reduce inflammatory cytokine levels, but the effect is slightly lower than that of live BDUP treatment.

[0087] ④ BDUP strains reduce pruritus-related factors in the serum of eczema model mice.

[0088] Figure 5The table shows the serum levels of neurogenic inflammatory mediators. Substance P (SP) and calcitonin gene-related peptide (CGRP) are key neuropeptides driving neurogenic inflammation and pain / itch transmission. The results showed that the BDUP live bacteria treatment group significantly downregulated the expression of SP (significantly lower than the model group) and CGRP, and also significantly inhibited nerve growth factor (NGF). Specifically, the SP levels in the BDUP live bacteria group and the p-BDUP inactivated bacteria group were 161.68 pg / mL and 208.64 pg / mL, respectively. In CGRP detection, both the BDUP live bacteria group (201.86 pg / mL) and the p-BDUP inactivated bacteria group (255.74 pg / mL) were significantly lower than the model group. The effect of the BDUP live bacteria group was comparable to that of the Dex group, while the p-BDUP inactivated bacteria group was slightly higher but still lower than the MC group. Regarding NGF, the BDUP live bacteria group also showed a significant decrease (85.75 pg / mL), which was not different from the Dex group; the p-BDUP inactivated bacteria group also showed a decrease (120.87 pg / mL).

[0089] This result confirms that the BDUP strain of this invention has a unique mechanism of action: it is not limited to traditional immune regulation, but directly intervenes in the release of neuropeptides through the gut-brain-cortex axis, thereby blocking the occurrence of neurogenic inflammation. This is a characteristic rarely reported in conventional probiotics in the prior art.

[0090] In summary, BDUP treatment can modulate pruritus-related factors. Live BDUP strains showed better efficacy than inactivated strains and were close to the positive reference Dex, indicating that the BDUP strains of this invention can alleviate pruritus by reducing the levels of related neuropeptides.

[0091] ⑤ BDUP strain alleviates oxidative damage associated with skin inflammation in mice

[0092] like Figure 6 As shown, compared with the normal group, the MDA content and SOD activity in the skin tissue of mice in the model group were significantly increased and significantly decreased. After intervention, the MDA content in both the Dex group and the BDUP live bacteria group was significantly lower than that in the model group, while the SOD activity was significantly higher than that in the model group. The effect of the p-BDUP inactivated bacteria group was worse than that of the BDUP live bacteria group, but still showed a significant difference from the model group. Specifically, the MDA content in the BDUP live bacteria group was 2.29 nmol / mg protein, and the SOD content was 40.69 U / mg protein; the MDA content in the p-BDUP inactivated bacteria group was 3.12 nmol / mg protein, and the SOD content was 37.14 U / mg protein. The results indicate that the BDUP live bacteria group has a better antioxidant stress response than the p-BDUP inactivated bacteria group, but slightly weaker than the Dex group.

[0093] ⑥ The BDUP strain alleviates eczema-like discomfort in mice by downregulating the pruritus neural pathway.

[0094] like Figure 7 As shown, RT-qPCR was used to detect the expression of genes closely related to pruritus transmission. The results are as follows:

[0095] IL-31 (Interleukin-31): As a core 'pruritus cytokine' in atopic dermatitis, IL-31 directly stimulates sensory nerve endings. BDUP treatment significantly inhibited IL-31 production, reducing the accumulation of pruritus factors at their source.

[0096] TRPV1 and MrgprA3: These are key itch receptors on the skin's sensory nerves. BDUP treatment significantly downregulated the expression of these receptors, implying an increased itch threshold and reduced skin sensitivity to external stimuli.

[0097] Nppb (B-type natriuretic peptide): Nppb is considered a key neurotransmitter that transmits itch signals from peripheral nerves to the dorsal horn of the spinal cord. Experimental results showed that the live BDUP bacteria group significantly reduced Nppb mRNA expression (to about 1 / 3 of the model group, superior to the inactivated bacteria group), indicating that the BDUP strain can directly interrupt the transmission pathway of itch signals to the central nervous system, which is one of the key mechanisms of its potent antipruritic effect.

[0098] In summary, the BDUP strain establishes the mechanistic basis for its excellent antipruritic effect at the molecular level through a triple mechanism of "reducing pruritus factor (IL-31)," "downregulating pruritus receptor (TRPV1 / MrgprA3)," and "blocking signal transduction messenger (Nppb)," which is fundamentally different from the existing technology that relies solely on immune regulation.

[0099] The above embodiments strongly demonstrate that the *Lactobacillus gasseri* BDUP described in this invention not only exerts its anti-inflammatory effect by regulating the balance of Th1 / Th2 cytokines, but its more significant innovation lies in its ability to effectively block neurogenic inflammatory pathways and pruritus signal transduction by downregulating key factors such as substance P, CGRP, NGF, and IL-31. This 'neuro-immune' dual regulatory mechanism explains why this strain can achieve an antipruritic rate of up to 81% in animal models. Therefore, the BDUP strain is particularly suitable for preparing drugs to treat atopic dermatitis or eczema accompanied by severe itching, providing a new treatment strategy for neuropruritus that is difficult to relieve clinically with conventional antihistamines.

Claims

1. The use of cultures, metabolites, lysates, or extracts of *Lactobacillus gasseri* strain BDUP in the preparation of medicaments for treating atopic dermatitis or eczema, characterized in that, The Lactobacillus gasseri BDUP strain has the accession number CGMCC No. 18357.

2. The use according to claim 1, characterized in that, The medication is used to improve neurogenic inflammation and itching symptoms caused by atopic dermatitis or eczema.

3. The use according to claim 2, characterized in that, The improvement in neurogenic inflammation and itching symptoms is achieved by downregulating the expression levels of at least one of the following factors in skin tissue: substance P, calcitonin gene-related peptide (CGRP), nerve growth factor (NGF), or interleukin-31 (IL-31).

4. The use according to claim 2 or 3, characterized in that, The drug is used to block the itch-scratch cycle of the skin and significantly reduce the duration of scratching behavior.

5. The use according to claim 1, characterized in that, The drug is also used to downregulate the mRNA expression levels of pruritus receptors TRPV1 or MrgprA3, or to reduce serum IgE levels.

6. The use according to claim 1, characterized in that, The culture of the Lactobacillus gasseri BDUP strain includes live cells, dead cells, fermentation supernatant, or cell lysate.

7. In the use according to claim 1, the amount of *Lactobacillus gasseri* BDUP strain added is an effective amount sufficient to exert a therapeutic effect.

8. In accordance with the use described in claim 7, the amount of *Lactobacillus gasseri* BDUP strain added is 1 × 10⁻⁶. 6 CFU / mL to 1×10 11 CFU / mL.

9. The use according to claim 1, characterized in that, The drug is an oral or topical preparation.

10. The use according to claim 9, characterized in that, The oral preparations are selected from powders, granules, capsules, tablets, or liquid preparations; the topical preparations are selected from creams, ointments, gels, lotions, or skin patches.

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