Lactobacillus paracasei for skin anti-inflammatory and acne treatment and application thereof

Abstract

The present invention relates to the field of microbial technology, and more particularly to a kind of Lactobacillus paracasei for skin anti-inflammatory and treatment of acne and its application. The present invention is isolated and identified from the feces of a 7-year-old healthy girl. Lactobacillus paracasei ProfMIC 209 can inhibit the growth of Propionibacterium acnes, reduce the flora ratio of Propionibacterium acnes and Staphylococcus epidermidis, so as to treat acne; The Lactobacillus paracasei also has the effect of promoting the expression of inflammatory factor-related factors IL 6, IL 8, IL 22, COX 2 and TRPV1 genes, and can inhibit cells from producing NO, so as to effectively alleviate or treat skin inflammation; In addition, the Lactobacillus paracasei strain can also raise the expression of barrier repair-related gene FLG. In short, the ProfMIC 209 strain provided by the present invention has considerable application prospects in the preparation of products for skin anti-inflammatory and treatment of acne.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Lactobacillus paracasei for anti-inflammatory and acne treatment of the skin and an application thereof. Background Art

[0002] The skin microbiome refers to the ecosystem composed of various microorganisms such as bacteria, fungi, viruses, mites, and arthropods, as well as the tissues, cells, secretions, and microenvironment on the skin's surface. Many skin problems are closely related to an imbalance in the skin's microbiome.

[0003] Acne is closely related to the proliferation of Propionibacterium acnes. Acne is a chronic inflammatory skin disease of the pilosebaceous follicle unit, primarily linked to factors such as excessive sebum secretion, obstruction of the pilosebaceous ducts, bacterial infection, and inflammatory reactions. After puberty, large amounts of sebum are secreted and excreted. Hair follicles harbor a variety of microorganisms, among which Propionibacterium acnes plays a crucial role. P. acnes is an anaerobic bacterium, and the obstruction of sebum excretion creates a favorable local anaerobic environment, allowing it to thrive. The lipase produced by P. acnes breaks down triglycerides in sebum, producing free fatty acids, which are the primary factor in the formation of inflammatory acne lesions. Furthermore, P. acnes produces peptides that attract neutrophils and activate complement, inducing or exacerbating inflammation.

[0004] Acne is essentially an inflammatory response, which is the earliest event in the development of acne lesions and persists throughout the course of acne. The pathogenesis of acne is associated with multiple inflammatory factors. The expression of IL-6, IL-8, IL-12, and IL-1α genes promotes hyperkeratosis of the hair follicle opening and the characteristic inflammatory lesions of acne. When the skin is exposed to various external irritants and allergens, it causes oxidative stress. Furthermore, oxidative stress caused by excessive reactive oxygen species (ROS) can also induce skin inflammation. On the one hand, inflammatory cells produce ROS, which contribute to oxidative stress. On the other hand, ROS can activate NF-κB, leading to enhanced expression of multiple inflammatory cytokines (IL-1, IL-6, IL-8), chemotaxis of inflammatory cells such as neutrophils, and an enhanced inflammatory response.

[0005] Skin inflammation is often accompanied by damage to the skin barrier. The structural foundation of the skin barrier is primarily the stratum corneum, epidermal lipids, and natural moisturizing factors. A healthy skin barrier protects the skin from external toxins, irritants, and sunlight, while also providing moisturizing and conditioning benefits. However, when the skin barrier is damaged, the skin's defenses weaken, allowing various allergens (including foreign pathogens) to easily penetrate the damaged barrier and penetrate the skin, leading to a series of skin inflammatory reactions such as dermatitis and itchy skin.

[0006] Staphylococcus epidermidis, a skin-resident bacterium, can compete with Propionibacterium acnes to inhibit its proliferation. Therefore, increasing the proportion of Staphylococcus epidermidis in the bacterial population can effectively alleviate acne inflammation, thereby maintaining skin health by regulating the balance of the skin's microecological ecosystem. Topical probiotics can balance the microbial flora on the skin's surface and promote the repair of the skin barrier. In addition, some probiotics play an important role in maintaining host homeostasis, activating the immune system, and maintaining immune balance. Studies have found that certain probiotics can control the systemic immune state by regulating the phagocytic ability of macrophages and the release of cytokines, thereby preventing and treating various inflammatory diseases. Summary of the Invention

[0007] In view of this, the problem to be solved by the present invention is to provide a Lactobacillus paracasei for skin anti-inflammatory and acne treatment and an application thereof.

[0008] The invention provides Lactobacillus paracasei ProfMIC-209 with a preservation number of CCTCC NO: M 20211554.

[0009] Specifically, the strain is Gram-positive and rod-shaped under a microscope; when growing on MRS plates, it can form round colonies with smooth, opaque surfaces, which are white and have neat edges; it grows uniformly turbidly in MRS liquid culture medium, and the bacteria form white precipitates after long-term storage. The optimal growth temperature is 37°C.

[0010] The invention provides application of the Lactobacillus paracasei strain in preparing anti-inflammatory drugs.

[0011] The anti-inflammatory effect includes promoting the expression of genes related to inflammatory factors, and the genes include at least one of IL-6, IL-8, IL-22, COX-2 and TRPV1.

[0012] In some embodiments, the Lactobacillus paracasei ProfMIC-209 provided by the present invention downregulates the expression of inflammatory factor-related genes IL-6, IL-8, IL-22, COX-2, and TRPV-1 in HaCaT cells induced by Staphylococcus aureus, and the gene expression levels are downregulated by 19.27% to 63.08%.

[0013] The anti-inflammatory effect also includes inhibiting the production of cellular NO.

[0014] In some embodiments, the Lactobacillus paracasei ProfMIC-209 provided by the present invention reduces the nitric oxide (NO) production of mouse macrophages Raw264.7 induced by lipopolysaccharide (LPS) by 29.53% to 45.78%.

[0015] The present invention also provides application of the Lactobacillus paracasei strain in preparing medicine for treating acne.

[0016] The acne treatment comprises inhibiting the growth of Propionibacterium acnes and reducing the relative ratio of Propionibacterium acnes and Staphylococcus epidermidis.

[0017] The relative ratios among the strains described in the present invention are characterized by the relative concentration ratios among the strains in the examples of the present invention.

[0018] The present invention also provides the use of the Lactobacillus paracasei strain in preparing a medicine capable of up-regulating the barrier repair gene FLG.

[0019] In some embodiments, the Lactobacillus paracasei ProfMIC-209 provided by the present invention upregulates the expression of the barrier repair-related factor filaggrin gene FLG, and the gene expression level is increased by 1.36 times.

[0020] The present invention also provides a probiotic agent, the raw materials of which include the Lactobacillus paracasei strain.

[0021] Specifically, the raw materials of the product of the present invention include at least one of live bacteria, inactivated bacteria, lysates, extracts, culture supernatants, and derivatives of the Lactobacillus paracasei ProfMIC-209 strain; the derivatives include at least one of metabolites, metabolic biological products, prebiotics, cell walls and components thereof, exopolysaccharides, and compounds containing immunogenic components.

[0022] Preferably, the raw material of the product of the present invention is the supernatant or inactivated bacteria of the Lactobacillus paracasei ProfMIC-209 strain.

[0023] The present invention isolates and identifies Lactobacillus paracasei ProfMIC-209 from the feces of a 7-year-old healthy girl. It can inhibit the growth of Propionibacterium acnes and reduce the flora ratio of Propionibacterium acnes to Staphylococcus epidermidis, thereby treating acne. The Lactobacillus paracasei also has the effect of promoting the gene expression of inflammatory factor-related factors IL-6, IL-8, IL-22, COX-2 and TRPV1, and can inhibit cells from producing NO, thereby effectively alleviating or treating skin inflammation. In addition, the Lactobacillus paracasei strain can also upregulate the expression of barrier repair-related genes FLG. In short, the ProfMIC-209 strain provided by the present invention has considerable application prospects in the preparation of skin anti-inflammatory and acne treatment products.

[0024] Biological Deposit Certificate

[0025] Lactobacillus paracasei ProfMIC-209 was deposited in the China Center for Type Culture Collection on December 6, 2021, at Wuhan University, Wuhan, China, with the deposit number CCTCCNO: M 20211554. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results showed that ProfMIC-209 supernatant downregulated the expression of genes related to inflammatory factors. DETAILED DESCRIPTION

[0027] The invention provides a kind of Lactobacillus paracasei and application thereof that can be used for skin anti-inflammatory and treatment acne, 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 as being 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 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.

[0028] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0029] The present invention will be further described below in conjunction with the embodiments:

[0030] Example 1 Isolation of ProfMIC-209

[0031] Fecal samples were collected from a healthy 7-year-old girl. After appropriate preparation, the sample was shaken and mixed in normal saline. The supernatant was streaked onto MRS solid plates and incubated at 37°C for 24–48 hours. White colonies were selected and repeatedly inoculated and screened until a uniform single colony was obtained, designated ProfMIC-209.

[0032] Gram staining microscopy: Strain ProfMIC-209 is Gram-positive and rod-shaped under a microscope; it grows on MRS plates and forms white, smooth, round, opaque small colonies with neat edges; it grows uniformly turbid in MRS liquid culture medium and forms white precipitates after prolonged exposure.

[0033] Example 2 Nucleic acid identification of ProfMIC-209

[0034] 1. 16S rRNA gene sequence analysis.

[0035] Single colonies were picked and placed in MRS liquid medium. After incubation at 37°C overnight, the cells were collected by centrifugation and DNA was extracted using a DNA extraction kit. PCR amplification was then performed using the universal bacterial primers 27F and 1492R. The PCR amplification system was 50 μL and the following protocol was used: 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 40 seconds, followed by extension at 72°C for 10 minutes.

[0036] 2. Results.

[0037] The sequencing results of the PCR product were compared with the standard sequences published in GenBank by BLASTN, and the strain ProfMIC-209 was identified as Lactobacillus paracasei.

[0038] Example 3 ProfMIC-209 reduces NO production in Raw264.7 cells

[0039] 1. Preparation of ProfMIC-209 bacterial solution

[0040] ProfMIC-209 was cultured in MRS medium overnight and the OD 600 , adjust the bacterial solution concentration to OD600 = 0.2, and after centrifugation, sterilize the bacteria at 121°C for 30 min to obtain a ProfMIC-209 inactivated bacterial sample, and filter the centrifuged supernatant with a 0.22 μm filter membrane to obtain a ProfMIC-209 inactivated supernatant sample.

[0041] 2. Preparation of Raw264.7 Cells

[0042] Raw264.7 cells were digested and then cultured at 2×10 5 The cells were seeded into 24-well plates at a density of 1000 cells / well and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0043] 3. ProfMIC-209 addition and LPS stimulation

[0044] The supernatant was added to Raw264.7 cells cultured overnight at a volume ratio of 5% (V / V) and the inactivated bacteria were added to different groups at a volume ratio of 10% (V / V). After 2 hours, 0.5 ml of 0.2 μg / ml LPS solution was added to induce inflammation of the Raw264.7 cells. After 20 hours, the cell culture supernatant was collected and the NO content was detected using a NO content detection kit. Three replicates were used each time, and a total of three experiments were performed.

[0045] Results. As shown in Table 1, ProfMIC-209 has an anti-inflammatory effect and can reduce LPS-induced NO production in Raw264.7 cells. Compared with the control group, ProfMIC-209 supernatant reduced NO production by 45.78%, and ProfMIC-209 inactivated bacteria reduced NO production by 29.53%.

[0046] Table 1 ProfMIC-209 reduces NO production in Raw264.7 cells

[0047]

[0048] Example 4 ProfMIC-209 downregulates the expression of inflammatory factor-related genes in HaCaT cells

[0049] 1. ProfMIC-209 sample preparation

[0050] ProfMIC-209 was cultured with MRS overnight and the OD 600 , adjust the bacterial concentration to OD 600 =0.2, after centrifugation, the bacteria were sterilized at 121°C for 30 min under high pressure to obtain a ProfMIC-209 inactivated bacteria sample, and the centrifuged fermentation broth was filtered using a 0.22 μm filter membrane to obtain a ProfMIC-209 inactivated supernatant sample.

[0051] 2. Preparation of HaCaT cells

[0052] HaCaT cells were digested and plated at 2×10 5 The cells were seeded into 24-well plates at a density of 1000 cells / well and cultured overnight at 37°C in a 5% carbon dioxide incubator.

[0053] 3. Preparation and addition of Staphylococcus aureus

[0054] Staphylococcus aureus was inoculated into nutrient broth medium and cultured overnight at 37°C in a shaking incubator. The concentration of the bacterial solution was adjusted to OD 600 =6.0, and then 100 μl per well was added to HaCaT cells cultured overnight to stimulate the cells to produce inflammatory factors. After 3 h, the cell culture medium was discarded, and the cells were washed with PBS 5 times, and 1 ml of MEM serum-free medium was added to each well.

[0055] 4. ProfMIC-209 sample addition

[0056] The ProfMIC-209 supernatant was added to the HaCaT cells stimulated with Staphylococcus aureus at a ratio of 5% (V / V), with three replicate wells per group, and cultured overnight.

[0057] 5. Detection of relative expression of inflammatory factor mRNA by qPCR

[0058] After discarding the culture medium from the above cells, RNA was extracted using an RNA extraction kit, and the RNA concentration and purity were tested. All samples were adjusted to 1 μg, and RT-PCR and qPCR were performed using a reverse transcription kit and a SYBR Green qPCR kit. The relative expression fold F of the inflammatory factor genes IL-8 and TRPV1 was then calculated.

[0059] Formula: F = 2 -ΔΔCT

[0060] 6. Results. As shown in Table 2, ProfMIC-209 can downregulate the expression of inflammatory factor-related genes in HaCaT cells induced by Staphylococcus aureus, with the expression levels decreasing by 19.27% to 63.08%. Therefore, ProfMIC-209 has an anti-inflammatory effect. The statistical results of ProfMIC-209 downregulating the expression of inflammatory factor-related genes are shown in Figure 1 .

[0061] Table 2 ProfMIC-209 down-regulates the expression of inflammatory factor genes

[0062]

[0063] Example 5 Effect of ProfMIC-209 on treating acne by changing the proportion of bacterial flora

[0064] 1. Preparation of Lactobacillus paracasei ProfMIC-209 bacterial solution:

[0065] The activated Lactobacillus paracasei ProfMIC-209 bacterial solution was cultured in MRS liquid medium in a 37°C incubator for 16-18 hours, and the OD was adjusted to 600 =2.0, then inactivated at 121°C for 30 min, centrifuged to obtain the supernatant, and filtered through a 0.22 μm filter membrane to obtain the ProfMIC-209 inactivated supernatant sample.

[0066] 2. Preparation of skin flora solution:

[0067] Propionibacterium acnes CGMCC 1.5003 and Staphylococcus epidermidis CGMCC 1.4260 were cultured in BHI medium at 37°C for 18 h, and the OD values were adjusted to 600 =0.2.

[0068] 3. Experiment on the effect of adding supernatant on skin flora growth

[0069] The inactivated supernatant was added to the two skin flora solutions at a ratio of 10% (V / V), cultured at 37°C for 16 h, and the relative concentrations of the two bacterial solutions (OD600 ) ratio was used as an indicator to evaluate the effect of ProfMIC-209 on the growth of skin flora.

[0070] The calculation formula of the relative concentration ratio is: relative concentration ratio of bacteria A and bacteria B = (concentration of bacteria A in the experimental group / concentration of bacteria A in the control group) / (concentration of bacteria B in the experimental group / concentration of bacteria B in the control group).

[0071] 4. Results. As shown in Table 3, ProfMIC-209 exhibited significant inhibitory effects against Propionibacterium acnes, while its inhibitory effect on Staphylococcus epidermidis was much lower. ProfMIC-209 significantly reduced the relative concentration ratio of Propionibacterium acnes to Staphylococcus epidermidis, thereby reducing the relative proportion of Propionibacterium acnes to Staphylococcus epidermidis, thereby achieving the goal of treating acne.

[0072] Table 3 Effects of ProfMIC-209 on the growth of acne-related flora

[0073]

[0074] Example 6 Experiment on ProfMIC-209 Promoting HaCaT Barrier Repair-Related Gene Expression

[0075] Immortalized human keratinocytes HaCaT (5×10 5 Cells were plated (cells / well) in 6-well plates and cultured overnight until adherent. 5% (v / v) inactivated supernatant of the ProfMIC-209 strain was added and cultured for 24 hours. Lysis buffer was then added to extract total cellular RNA, and RNA concentration and purity were determined. All samples were adjusted to 1 μg and reverse-transcribed into cDNA for qPCR analysis of FLG gene expression. Fold change in expression was calculated using the formula.

[0076] Formula: F = 2 -ΔΔCT The results are shown in Table 4. The addition of ProfMIC-209 has the effect of promoting skin barrier repair.

[0077] Table 4 ProfMIC-209 supernatant upregulates the expression of barrier repair-related genes

[0078]

[0079] 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. Lactobacillus paracasei ProfMIC-209 (CCTCC NO: M20211554) Lactobacillus paracasei ProfMIC-209).

2. Use of Lactobacillus paracasei ProfMIC-209 according to claim 1 in preparing a medicine for treating acne, wherein the treatment of acne comprises inhibiting the growth of Propionibacterium acnes and reducing the relative ratio of Propionibacterium acnes and Staphylococcus epidermidis.

3. application of Lactobacillus paracasei ProfMIC-209 according to claim 1 in preparing a medicine for promoting skin barrier repair.

4. A probiotic agent, characterized in that The invention comprises the Lactobacillus paracasei ProfMIC-209 according to claim 1.

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