Application of lactobacillus casei Zhang in preparation of preparation for treating skin diseases

Abstract

The invention discloses application of lactobacillus casei Zhang in preparation of a preparation for treating skin diseases, and relates to the technical field of microbial probiotic preparations. The invention provides application of lactobacillus casei Zhang in preparation of a preparation for treating or relieving psoriasis. The preservation number of the lactobacillus casei Zhang is CGMCC (China General Microbiological Culture Collection Center) No. 5469. The lactobacillus casei Zhang has a relieving effect on symptoms of psoriasis, can inhibit the weight reduction trend of psoriasis patients and reduce spleen indexes, can be used for preparing preparations for relieving psoriasis, can also be used as microecological preparations to be applied to medicines, fermented foods or health-care products, and provides a potential choice for developing a novel psoriasis treatment method.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial probiotic preparations, and specifically relates to the application of Lactobacillus casei Zhang in the preparation of a preparation for treating skin diseases. Background Art

[0002] Psoriasis is a chronic immune-mediated skin disease with a long course and easy recurrence, and the main clinical symptoms are erythema, scales, etc., and the main symptoms of the disease are itching and pain. Psoriasis is usually called psoriasis vulgaris. Clinically, psoriasis can be divided into vulgaris psoriasis, pustular psoriasis, erythrodermic psoriasis, and psoriatic arthritis.

[0003] Currently, the main clinical treatments for psoriasis are topical treatment, phototherapy, and systemic treatment. Topical medications are commonly used in the treatment of mild psoriasis patients, but the effects are not very obvious in moderate and severe psoriasis patients. Its advantage is that it can directly act on the lesion area, with a fast onset, simple application, and small side effects on other parts of the body. However, long-term use may cause local side effects. Ultraviolet light therapy is a common treatment for psoriasis, which can promote the apoptosis of T cells and delay the proliferation of lymphocytes. However, ultraviolet light therapy may cause side effects such as itching, desquamation, and redness and swelling.

[0004] Psoriasis is usually considered to be a disease that only manifests on the skin. However, studies have shown that intestinal microbiota dysbiosis is related to the occurrence and / or development of chronic low-grade inflammation. At the same time, there are a large number of dysregulated cytokines and inflammatory markers in the sera of psoriasis patients. Therefore, the systemic inflammation of psoriasis may be closely related to the dysregulation of gut and skin microbiota. Currently, exploring the relationship between health and disease from the perspective of microorganisms has gradually become a focus and hotspot. Probiotics can colonize the intestine and form a natural biological barrier with the intestinal mucosa by regulating the intestinal microbiota and increasing the expression of adhesion proteins. It has been confirmed that probiotics can inhibit the inflammatory response of atopic dermatitis and have a definite therapeutic effect, which provides inspiration for the treatment of psoriasis with probiotics.

[0005] Chinese Patent CN115708835A discloses a Bifidobacterium breve strain that can relieve psoriasis and its application. The preservation number of the Bifidobacterium breve CCFM683 described in this invention is CGMCC No. 11828. The Bifidobacterium breve CCFM683 of this invention has the effect of relieving psoriasis caused by imiquimod, specifically manifested as: (1) significantly reducing the weight loss of psoriasis mice; (2) significantly improving the pathological features such as skin scales, thickening, and redness and swelling in psoriasis-like mice; (3) regulating the levels of skin immune factors in psoriasis-like mice; (4) significantly inhibiting the proliferation of skin keratinocytes in psoriasis mice.

[0006] A Bifidobacterium adolescentis capable of alleviating psoriasis and its application are disclosed in Chinese Patent CN112812989A. The preservation number of the Bifidobacterium adolescentis in this invention is GDMCC No. 61349. The Bifidobacterium adolescentis provided by this invention ( Bifidobacterium adolescentis ) CCFM667 has the effect of alleviating psoriasis, relieving the skin lesions of psoriatic mice, inhibiting the thickening of the epidermis of the skin of psoriatic mice, reducing the spleen weight of psoriatic mice, and reducing the levels of IL-22 and IL-23 in the skin of psoriatic mice by 30.2%.

[0007] At present, there is little research on probiotics that can alleviate psoriasis, and there is an urgent need to provide a probiotic that can effectively alleviate psoriasis. Summary of the Invention

[0008] The purpose of the present invention is to provide the application of Lactobacillus casei Zhang in the preparation of a preparation for treating psoriasis.

[0009] To achieve the above-mentioned invention purpose, the technical solution of the present invention is as follows: On the one hand, the present invention provides the application of Lactobacillus casei Zhang in the preparation of a preparation for treating or alleviating psoriasis. The preservation number of the Lactobacillus casei Zhang is CGMCC No. 5469.

[0010] Specifically, the taxonomic nomenclature of the Lactobacillus casei Zhang is Lacticaseibacillus casei , which has been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, with the preservation number of CGMCC No. 5469 and the preservation date of November 18, 2011.

[0011] Specifically, the psoriasis includes plaque psoriasis, pustular psoriasis, erythrodermic psoriasis and psoriatic arthritis.

[0012] Specifically, the viable count of Lactobacillus casei Zhang in the preparation is 1×10 6 -1×10 11 CFU / mL or 1×10 6 -1×10 11 CFU / g.

[0013] Specifically, such as 1×10 6 CFU / mL (CFU / g), 1×10 7 CFU / mL (CFU / g), 1×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), 1×10 11CFU / mL (CFU / g), etc., and other point values within this numerical range can be selected.

[0014] Specifically, the preparation includes the supernatant of Lactobacillus casei Zhang fermentation broth, viable Lactobacillus casei Zhang, or freeze-dried powder of Lactobacillus casei Zhang.

[0015] Further, the viable bacteria are also called active flora, which can colonize and multiply in the intestine, and is beneficial to increasing the number of beneficial bacteria.

[0016] Further, the freeze-dried powder is obtained by freeze-drying the bacterial strain.

[0017] Further, the preparation includes the supernatant of Lactobacillus casei Zhang fermentation broth.

[0018] Still further, the dosage of the supernatant of the fermentation broth is 2.625 mg / kg, 5.25 mg / kg, or 10.5 mg / kg.

[0019] Specifically, the preparation method of the supernatant of Lactobacillus casei Zhang fermentation broth includes the following steps: (1) Inoculate Lactobacillus casei Zhang into the fermentation medium at an inoculum size of (1 - 10) × 10 6 CFU / g; (2) Incubate at 35 - 38 °C until the pH reaches 4 - 6; (3) Centrifuge to obtain the supernatant of the fermentation broth.

[0020] Further, the inoculum size in step (1) is 6 × 10 6 CFU / g.

[0021] Further, the fermentation medium in step (1) is selected from at least one of MRS medium, M17 medium, PDA medium, LB medium, TB medium, SOB medium, MEM medium, DMEM medium, RPMI1640 medium.

[0022] Still further, the fermentation medium in step (1) is MRS medium.

[0023] Further, the incubation condition in step (2) is: incubate at 37 °C until the pH is 4.5.

[0024] Further, the supernatant of the fermentation broth in step (3) also includes the process of filtering with a 0.22 μm filter membrane.

[0025] Further, the preparation also includes a pharmaceutically acceptable carrier.

[0026] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0027] Specifically, the dosage form of the drug includes, but is not limited to, dosage forms for gastrointestinal administration and dosage forms for non-gastrointestinal administration according to the administration route.

[0028] Furthermore, the dosage forms for gastrointestinal administration include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0029] Furthermore, the dosage forms for non-gastrointestinal administration include, but are not limited to: injection dosage forms, respiratory dosage forms, skin dosage forms, mucosal dosage forms, and cavity dosage forms.

[0030] Furthermore, the injection dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0031] Furthermore, the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder aerosols.

[0032] Furthermore, the skin dosage forms include, but are not limited to, lotions, ointments, topical solutions, plasters, pastes, and patches.

[0033] Furthermore, the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ophthalmic ointments, sublingual tablets, and film dressings.

[0034] Furthermore, the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and dripping pills.

[0035] Even further, the dosage form of the preparation is for skin administration.

[0036] Specifically, the preparation has the following effects: (1) It can inhibit the downward trend of body weight in psoriasis patients; (2) Reduce the spleen index; (3) Alleviate the erythema, epidermal desquamation, and infiltration symptoms of the back skin.

[0037] Specifically, the preparation includes a drug.

[0038] On the other hand, the present invention provides a preparation for treating or alleviating psoriasis, which includes Lactobacillus casei Zhang, and the preservation number of Lactobacillus casei Zhang is CGMCC No. 5469.

[0039] Specifically, the preparation includes the supernatant of Lactobacillus casei Zhang fermentation broth, viable Lactobacillus casei Zhang, and freeze-dried powder of Lactobacillus casei Zhang.

[0040] Further, the preparation includes the supernatant of Lactobacillus casei Zhang fermentation broth.

[0041] Still further, the dosage of the fermentation broth supernatant is 2.625 mg / kg, 5.25 mg / kg, or 10.5 mg / kg.

[0042] Specifically, the method for preparing the supernatant of Lactobacillus casei Zhang fermentation broth includes the following steps: (1) Inoculate Lactobacillus casei Zhang into the fermentation medium at an inoculum amount of (1 - 10)×10 6 CFU / g; (2) Incubate at 35 - 38 °C until the pH reaches 4 - 6; (3) Centrifuge to obtain the fermentation broth supernatant.

[0043] Further, the inoculum amount in step (1) is 6×10 6 CFU / g.

[0044] Further, the fermentation medium in step (1) is selected from at least one of MRS medium, M17 medium, PDA medium, LB medium, TB medium, SOB medium, MEM medium, DMEM medium, and RPMI1640 medium.

[0045] Still further, the fermentation medium in step (1) is MRS medium.

[0046] Further, the culture condition in step (2) is: incubate at 37 °C until the pH is 4.5.

[0047] Further, the fermentation broth supernatant in step (3) also includes the process of filtering with a 0.22 μm filter membrane.

[0048] Further, when the preparation is a drug, the drug also includes a pharmaceutically acceptable carrier.

[0049] Still further, the pharmaceutically acceptable carrier is selected from one or more of excipients, stabilizers, diluents, binders, preservatives, and lubricants.

[0050] Specifically, the dosage form of the drug includes, but is not limited to, enteral dosage forms and parenteral dosage forms according to the administration route.

[0051] Furthermore, the dosage forms for gastrointestinal administration include, but are not limited to, tablets, powders, granules, solutions, capsules, emulsions, suspensions, and oils.

[0052] Furthermore, the parenteral dosage forms include, but are not limited to: injection dosage forms, respiratory dosage forms, dermal dosage forms, mucosal dosage forms, and cavity dosage forms.

[0053] Still further, the injection dosage forms include, but are not limited to, intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, and intracavitary injections.

[0054] Still further, the respiratory dosage forms include, but are not limited to, sprays, aerosols, and powder aerosols.

[0055] Still further, the dermal dosage forms include, but are not limited to, lotions, ointments, topical solutions, plasters, pastes, and patches.

[0056] Still further, the mucosal dosage forms include, but are not limited to, eye drops, nasal drops, ophthalmic ointments, sublingual tablets, and film dressings.

[0057] Still further, the cavity dosage forms include, but are not limited to, suppositories, aerosols, effervescent tablets, drops, and dripping pills.

[0058] Even further, the dosage form of the drug is for dermal administration.

[0059] The beneficial effects of the present invention are as follows: The present invention has demonstrated through experiments that the intervention of Lactobacillus casei Zhang can inhibit the decreasing trend of body weight and reduce the spleen index of psoriasis mice. At the same time, it can effectively alleviate the severity of psoriasis in mice and relieve psoriasis symptoms. Description of the Drawings

[0060] Figure 1 It is the experimental design for the remission of psoriasis by the active substances of Lactobacillus casei Zhang.

[0061] Figure 2 It is the effect of the active substances of Lactobacillus casei Zhang on the spleen volume of mice. From left to right in the figure are the normal group, the model group, the low-dose group, the medium-dose group, and the high-dose group.

[0062] Figure 3 It is the effect of the active substances of Lactobacillus casei Zhang on the body weight of mice.

[0063] Figure 4 It is the effect of the active substances of Lactobacillus casei Zhang on the spleen index of mice.

[0064] Figure 5Effects of the active substance of Lactobacillus casei Zhang on the damaged skin of psoriasis mice. From top to bottom in the figure are the normal group, the model group, the low-dose group, the medium-dose group, and the high-dose group.

[0065] Figure 6 Effects of the active substance of Lactobacillus casei Zhang on the PASI score of mice.

[0066] Figure 7 Effects of Lactobacillus casei Zhang, the active substance of Lactobacillus casei LC09, on the body weight of mice in the model group. In the figure: a, b, c represent the differences between groups ( P <0.05). Detailed implementation manners

[0067] In order to make the technical means, creative features, achieved purposes and effects of the present invention easy to understand, the following combines specific embodiments to further clarify the present invention. However, the following embodiments are only the preferred embodiments of the present invention, not all. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operation methods used are all conventional operation methods, the equipment used is all conventional equipment, and the equipment materials used in each embodiment are the same.

[0068] Lactobacillus casei Zhang in the present invention has been disclosed in Patent CN105802876A.

[0069] Example 1 Preparation of the active substance of Lactobacillus casei Zhang Lactobacillus casei Zhang was inoculated into MRS liquid medium at an inoculation amount of 6×10 6 CFU / g, and then cultured at 37°C until the pH reached 4.5 to obtain a fermentation broth. After centrifuging the fermentation broth, the supernatant was taken, and then filtered through a 0.22 μm filter membrane to obtain the active substance of Lactobacillus casei Zhang.

[0070] Example 2 Alleviating effect of the active substance of Lactobacillus casei Zhang on psoriasis 1 Experimental materials Experimental animals: 40 SPF-grade male C57BL / 6 mice, 6 - 8 weeks old, with a body weight of 20 - 25 g. The experimental animals, maintenance feed, and rat bedding were all purchased from Beijing Spebef Biotechnology Co., Ltd.

SCXK (Jing) 2019 - 0014

[0071] Imiquimod cream was purchased from Mingxin Lidi, and petrolatum cream was purchased from Zhaonuo.

[0072] 2 Experimental Design Forty 6-week-old male BALB / c mice were selected. After one week of adaptation, the hair on the central area of the mice's backs was shaved off, about 3×3 cm 2 in area. They were randomly divided into a normal group, a model group, a low-dose group (2.625 mg / kg of the active substance solution of Lactobacillus casei Zhang), a medium-dose group (5.25 mg / kg of the active substance solution of Lactobacillus casei Zhang), and a high-dose group (10.5 mg / kg of the active substance solution of Lactobacillus casei Zhang), a total of 5 groups (n = 8); the normal group was topically applied with vaseline on the backs of the mice once a day (90 mg), and the other 4 groups were applied with imiquimod cream (5% IMQ, 90 mg) in the same way to establish a psoriasis model. The experimental design is as Figure 1 shown.

[0073] 2.1 Calculation of the spleen index of mice The body weight of the mice was weighed, the spleens were isolated, the surrounding redundant tissues were removed, washed in physiological saline to remove blood stains, blotted dry with filter paper, and the spleen weight was weighed on an electronic balance to calculate the spleen index. Spleen index = spleen weight (mg) / body weight of mice (g).

[0074] 2.2 Scoring of the severity index of skin lesions (PASI) The changes in skin lesions were recorded by daily observation. According to the PASI scoring standard, the dorsal skin of the mice was scored for PASI from three aspects: erythema, epidermal desquamation, and infiltration degree at the skin lesion sites of the mice. The scoring standard was: 0 points (asymptomatic); 1 point (mild); 2 points (significant); 3 points (severe); 4 points (very severe). The sum of the scores in the three aspects was the total PASI score. The PASI scoring standard is shown in Table 1 below.

[0075] Table 1 PASI scoring

[0076] 3 Experimental Results The spleen volume of the mice was as Figure 2 shown. Compared with the normal group, the spleen color of the mice in the model group turned black and the volume was significantly swollen; compared with the model group, the spleen volumes of the mice in the low-, medium-, and high-dose groups of the active substance were significantly reduced. The changes in the body weight of the mice were as Figure 3 shown. On the 9th day, the body weight of the mice in the high-dose group of the active substance was significantly higher than that in the model group (P < 0.01). The low- and medium-dose groups had a certain improvement effect, but there was no statistical difference. The results showed that the low-, medium-, and high-doses of the active substance could all inhibit the trend of body weight loss in psoriasis mice.

[0077] The spleen can produce a variety of immunologically active cytokines. An increase in the spleen index indicates that the body's inflammatory immune response activates the proliferation of spleen cells, which reflects abnormal immune function of the body to a certain extent. The spleen index was obtained by synthesizing the spleen mass and body mass of mice. The results are as Figure 4 shown. Compared with the model group, the spleen index of mice in the high-dose active substance group was significantly decreased, indicating that the intervention of the active substance effectively alleviated the skin lesions and systemic inflammatory response related to psoriasis.

[0078] As Figure 5 shown, compared with the model group, after the intervention of the active substance, the thickness of the skin lesions, erythema and scaling symptoms on the backs of mice were significantly reduced. The PASI (Psoriasis Area and Severity Index) score was used to evaluate the severity of psoriasis. The results are as Figure 6 shown. From the 5th day, the daily PASI scores of mice in the low-, medium- and high-dose groups were significantly lower than those in the model group (P < 0.0001), indicating that the low, medium and high doses of the active substance can effectively reduce the severity of psoriasis in mice and relieve the symptoms of psoriasis.

[0079] In summary, the intervention of the active substance of Lactobacillus casei Zhang can inhibit the weight loss trend of psoriasis mice, reduce the spleen index, and at the same time, can effectively reduce the severity of psoriasis in mice and relieve the symptoms of psoriasis.

[0080] Comparative Example 1 The method of applying to the backs of mice in this comparative example was the same as that in Example 2, except that the active substance of Lactobacillus casei Zhang was replaced with the active substance of Lactobacillus casei LC09. The preparation method of the active substance of Lactobacillus casei LC09 was the same as that in Example 1. The weight changes of mice in each group were recorded according to the time nodes of the original experiment. The average weight of mice in each group at different time points was calculated and compared with the model group and the high-dose Lactobacillus casei Zhang group.

[0081] The experimental results are as Figure 7 shown. Compared with the model group, after the intervention of the active substance of Lactobacillus casei LC09, the weight loss of mice was significantly reduced, but the effect was worse than that of the active substance of Lactobacillus casei Zhang. By the 9th day, the weights of the three groups of mice were the active substance group of Lactobacillus casei Zhang (Zhang group), the active substance of Lactobacillus casei LC09 (LC09 group), and the model group in turn, and there were significant differences ( P < 0.05), indicating that the active substance of Lactobacillus casei Zhang has a better effect on improving psoriasis than the active substance of Lactobacillus casei LC09.

[0082] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of Lactobacillus casei Zhang in preparing a preparation for treating or alleviating psoriasis, characterized in that: The deposit number of the Lactobacillus casei Zhang is CGMCC No. 5469.

2. The use according to claim 1, characterized in that: The psoriasis includes psoriasis vulgaris, pustular psoriasis, erythrodermic psoriasis and arthritic psoriasis.

3. The use according to claim 1, characterized in that: The viable count of Lactobacillus casei Zhang in the preparation is 1×10 6 -1×10 11 CFU / mL or 1×10 6 -1×10 11 CFU / g.

4. The use according to claim 1, characterized in that: The preparation comprises lactobacillus casei Zhang fermentation liquid supernatant, lactobacillus casei Zhang live bacteria or lactobacillus casei Zhang freeze-dried powder.

5. The use according to claim 4, characterized in that: The preparation comprises the supernatant of Lactobacillus casei Zhang fermentation liquid.

6. The use according to claim 5, characterized in that: The preparation method of the described Lactobacillus casei Zhang fermentation liquid supernatant comprises the following steps: (1) Lactobacillus casei Zhang was added at a rate of 1×10 6 -1×10 7 The inoculum of CFU / g was inoculated into the fermentation medium; (2) Cultivate at 35-38°C until the pH reaches 4-6; (3) Centrifuge to obtain the fermentation liquid supernatant.

7. The use according to any one of claims 1 to 6, characterized in that: The preparation also includes a pharmaceutically acceptable carrier.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable carrier is selected from one or more of an excipient, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

9. The use according to claim 1, characterized in that: The preparation has the following effects: (1) It can inhibit the weight loss trend of psoriasis patients; (2) Reduce spleen index; (3) Alleviate erythema, epidermal desquamation and infiltration symptoms on the back skin.

10. A preparation for treating or alleviating psoriasis, characterized in that: The preparation comprises Lactobacillus casei Zhang, and the preservation number of the Lactobacillus casei Zhang is CGMCC No. 5469.

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