CN111888462B - Application of thymosin beta 4 in preparation of microecological balance regulator - Google Patents

Abstract

The invention discloses application of thymosin beta 4, active fragments or derivatives thereof in preparing a microecological balance regulator aiming at skin microbial flora imbalance and in preparing a medicament for treating diseases caused by skin microbial flora imbalance, and also discloses a medicinal preparation containing thymosin beta 4, active fragments or derivatives thereof prepared according to the application. The application and the pharmaceutical preparation disclosed by the invention can rapidly improve the clinical manifestations of patients with scalp seborrheic dermatitis, the improvement of the whole fungal community and bacterial community is obviously superior to that of the ketoconazole group treated traditionally, and the phenotype and the micro-ecological characteristics of the later period of medication and the drug withdrawal follow-up period are obviously superior to those of the ketoconazole group.

Description

Application of thymosin beta 4 in preparation of microecological balance regulator

Technical Field

The invention discloses an application of protein in preparation of medicines, cosmetics and/or health care products, and belongs to the field of preparation of medicines, cosmetics and/or health care products.

Background

Seborrheic Dermatitis (SD) is a common chronic inflammatory skin disease with recurrent episodes characteristic, with a prevalence of 1-10% in adult populations. SD is better sent to skin parts rich in sebaceous glands, such as scalp, face, chest, back, and the like, and mainly shows furfuryl-like or greasy desquamation, Erythema (Erythema), Inflammatory Papule (inflammation paste), pruritus, and the like. The scalp is the most commonly involved part of SD because of its physiological characteristics of thick hair, abundance of sweat and sebaceous glands, and high moisture content. Scalp scale (Dandruff), considered as a mild form of scalp SD, is a skin disease characterized by scalp desquamation, often accompanied by itching without significant inflammation, with a prevalence of up to 50%. The scalp SD not only causes discomfort such as itching, but also negatively affects the appearance, mind and social interaction of the patient, thereby reducing the quality of life of the patient. Research shows that factors such as high temperature, sweat, high humidity, ultraviolet rays, mental stress or insufficient sleep and the like can obviously aggravate the incidence rate of the seborrheic dermatitis. Therefore, in severe working environments such as islands, plateaus, submarines and the like, the incidence of scalp SD of young and old people is higher, and the working efficiency of workers is greatly influenced.

It is now generally accepted that scalp SD is a multifactorial disease, the major causative factors of which include the external environment, scalp microorganisms, sebum secretion and individual susceptibility. The microbial flora associated with the scalp SD is mainly Malassezia (Malassezia) among fungi, as well as Staphylococcus (Staphylococcus) and Propionibacterium (Propionibacterium) among bacteria. Malassezia has long been recognized as an important causative agent of scalp SD. The broad-spectrum antifungal ketoconazole is a first-line treatment drug for scalp SD, has good curative effect on the scalp SD, but also has the defects of relapse, drug resistance, adverse reaction and the like, and the ecological transition characteristics of scalp microorganisms in the treatment process are not clear.

Folliculitis is an inflammatory skin disease mainly involving hair follicles, and is often manifested as a red papule centered on a hair follicle with itching and pain. Folliculitis can occur in any part of the body where hair is present, the most common parts being the scalp, face, buttocks and thighs. Infectious folliculitis can be caused by bacteria, fungi, viruses, and the like. The most common daily folliculitis includes bacterial folliculitis caused primarily by staphylococcus aureus and fungal folliculitis caused by malassezia. Folliculitis and scalp SD both have skin microflora imbalance and both use malassezia and staphylococcus aureus as the main pathogens.

Thymosin beta 4(T β 4) is a small molecule polypeptide consisting of 43 amino acids with multiple biological functions, widely found in tissues, organs and cells of mammals and other vertebrates, with the amino acid sequence Ac-SDKPDMAEIEKFDKSKLKKTETQEKNPLPSKETIEQEKQAGES, and three functionally active regions of the sequence have been identified: amino acids at positions 1 to 4, 1 to 15 and 17 to 23. The amino acids at the 1 st to 4 th positions have an anti-inflammatory function, the amino acids at the 1 st to 15 th positions can inhibit apoptosis and protect cells, and the amino acids at the 17 th to 23 th positions influence cell migration, are combined with actin, promote skin wound healing, promote angiogenesis and hair growth. Currently, studies on T β 4 are mainly focused on anti-inflammation, wound healing, corneal repair, repair of myocardial injury, repair of the central nervous system, angiogenesis and hair growth. In vitro experiments prove that T beta 4 can obviously reduce cytokines and chemokines such as IL-1 beta, Macrophage Inflammatory Protein-1 alpha (Macrophage Inflammatory Protein-1 alpha, MIP-1 alpha), MIP-1 beta, MIP-2 and Monocyte chemotactic Protein (MCP-1) and the like, and can also inhibit TNF-alpha mediated Nuclear Factor-kappa B (Nuclear Factor-kappa B, NF-kappa B) activation, thereby playing a role in inhibiting Inflammatory reaction. Tang et al 2010 showed that T.beta.4 has in vitro antibacterial activity and inhibits the growth of Escherichia coli (Escherichia coli) and Staphylococcus aureus, but has no significant inhibitory effect on Candida albicans (Candida albicans) and Cryptococcus neoformans (Cryptococcus neoformans). A study in 2007 also found that T β 4 had inhibitory effects on Pseudomonas aeruginosa (Pseudomonas aeruginosa), staphylococcus aureus and staphylococcus epidermidis. Animal experiments by Ehrlich et al show that T β 4 promotes repair of damaged skin tissue by mechanisms such as promoting migration (re-epithelialization) of keratinocytes and epithelial cells, stimulating neovascularization, and upregulating laminin-5 expression. The T beta 4 can also promote hair Growth, the T beta 4 induces the expression of Vascular Endothelial Growth Factor (VEGF) and Matrix metalloproteinase-2 (MMP-2), and promotes the migration, differentiation and angiogenesis of melanocyte and hair follicle stem cells through the interaction of the VEGF and the MMP-2, thereby influencing the Growth speed of hair, the Growth mode of hair follicle and the number of hair shaft. Tbeta 4 has multiple biological functions and has good tolerance and safety for local use.

More and more researches are searching for new methods for treating scalp SD and folliculitis, and in view of the fact that T beta 4 has the functions of resisting inflammatory reaction, inhibiting microbial growth, promoting tissue repair, promoting hair follicle development and the like, and pathophysiological processes such as inflammatory reaction, microbial excessive propagation, scalp skin barrier damage and the like are important pathogenic mechanisms of scalp SD and folliculitis, T beta 4 has potential application value in treatment of scalp SD and folliculitis. However, no study on the association of T β 4 with scalp SD and folliculitis has been found.

Therefore, this study investigated the role of microbial flora in the development of scalp SD by observational studies on the distribution of fungal and bacterial flora in scalp SD, and screened for potential biomarkers of scalp SD. Meanwhile, the interaction between microbial flora of the scalp and between the microbial flora and host factors is studied, and the pathogenesis of the scalp SD is further clarified. In addition, the research compares the curative effect of the Tbeta 4 and the broad-spectrum antifungal drug on the scalp SD through intervention research, observes the development trend and the recurrence condition of the scalp SD under different interventions and the influence of different intervention means on scalp microbial flora so as to provide basis and guidance for developing specific biological drugs of the scalp SD; in particular, specific therapeutic drugs are developed for scalp SD patients in a severe working environment for a long time, and clinical symptoms of scalp SD can be effectively treated.

Disclosure of Invention

In view of the above, the present invention provides, in a first aspect, the use of thymosin beta 4, an active fragment or derivative thereof for the preparation of a regulator of the microecological balance for the dysregulation of the skin microbial flora. Dysregulation of the skin microflora can lead clinically to a variety of diseases, such as seborrheic dermatitis of the scalp and folliculitis. The thymosin beta 4 of the invention is thymosin beta 4 with complete sequence; since proteins have the structural conserved regions and the active regions divided, the term "active fragment thereof" as used herein refers to the functional activity unit in thymosin beta 4 sequence, i.e. the active fragment of thymosin beta 4, which can be fully applied to the preparation of the microecological balance regulator for skin microflora dysregulation; on the basis of the curative effect of the medicament, in order to improve the stability of the medicament polypeptide and prolong the in vivo biological aging, the technicians in the field can prepare derivatives of the medicament polypeptide, such as acetylated polypeptide and polypeptide albumin fusion protein, and the derivatives of the medicament polypeptide are various derivatives which are prepared by modifying, adding and fusing thymosin beta 4 for the purposes of improving the stability, in vivo half-life, easy purification and the like on the basis of keeping the biological effect. The thymosin beta 4, the active fragment or the derivative thereof can be applied to the preparation of the microecological balance regulator aiming at the skin microbial flora dysregulation.

In a preferred embodiment, the microbial flora dysregulation comprises a fungal and/or a bacterial flora dysregulation.

In a more preferred embodiment, the fungal genus deregulation comprises an increased abundance of malassezia and/or a decreased abundance of aspergillus.

In another more preferred embodiment, the bacterial genus deregulation comprises an increased abundance of staphylococcus and/or a decreased abundance of pseudomonas.

More preferably, the skin is a hair-distributed skin.

Still preferably, the skin is head skin.

More preferably, thymosin beta 4, its active fragment or its derivative is prepared into medicine, cosmetics or health product.

In addition, the invention also provides application of thymosin beta 4, active fragments or derivatives thereof in preparing medicines for treating diseases caused by skin microbial flora dysregulation.

In a preferred embodiment, the disease is seborrheic dermatitis of the scalp or folliculitis.

Finally, the invention provides a pharmaceutical formulation prepared according to the above use, said formulation comprising thymosin beta 4, active fragments or derivatives thereof and a pharmaceutically acceptable pharmaceutical carrier.

In a preferred embodiment, the pharmaceutical preparation is prepared as a skin external preparation.

In a more preferred embodiment, the external preparation for skin includes gel, aqua, paste, liniment, patch, spray.

The invention adopts a metagenome sequencing method to respectively carry out cross section analysis research on scalp squamous-cell type and erythema type scalp SD and fungi and bacteria flora at skin damage parts and non-skin damage parts, and the distribution characteristics of the microorganism flora of the scalp SD are determined; analyzing and comparing the difference of scalp microflora of a scalp SD population and a scalp healthy population, and searching a potential biomarker of scalp SD; in addition, the pathogenic mechanism of scalp SD caused by microbial flora is further studied through the analysis of the correlation network between microbial flora and host factors. Researches find that the diversity of fungal communities and bacterial communities of patients with scalp SD is remarkably reduced, the limited malassezia and staphylococcus are both remarkably increased, and the pseudomonas is remarkably reduced. According to the disease characteristic bacterial spectrum, fungal and bacterial community indexes are respectively established, and the correlation coefficient between the fungal and bacterial community indexes and the disease main phenotype ASFS reaches 0.668 and 0.595 respectively. On the basis of the above research, the present invention uses thymosin beta 4 as an intervention drug, uses placebo shampoo and ketoconazole as drug controls, and carries out intervention research on scalp SD patients and healthy control groups, and the results show that: scalp flora was stable, whether in healthy controls or SD patients, and remained relatively stable during the 42 day follow-up period with placebo shampoo intervention. Ketoconazole has the obvious characteristic of inhibiting the restrictive malassezia and can quickly improve various clinical manifestations of SD, but the medicine has no obvious restoration of the whole micro-ecology, especially the bacterial community, and clinical indexes and flora show the recurrence characteristic in the later period of medication and after stopping medication. The T beta 4 intervention can rapidly improve the clinical manifestations of scalp SD patients, the decrease range of the restrictive malassezia is far smaller than that of the ketoconazole group, but the improvement of the whole fungal community and bacterial community is obviously better than that of the ketoconazole group, and the phenotype and the microecological characteristics of the later period of medication and the drug withdrawal follow-up period are better than those of the ketoconazole group. The research of the invention shows the application prospect of thymosin beta 4 in preparing microecological balance regulator aiming at the skin microbial flora imbalance and in preparing medicines for treating diseases caused by the skin microbial flora imbalance.

Drawings

FIG. 1 is an analysis chart of the scalp microflora;

FIG. 2 is a scalp microflora diversity comparison analysis chart of scalp SD group and healthy control group;

FIG. 3 is a graph of the ASFS score change trend after each group intervention;

FIG. 4 is a graph of MEA variation trend after intervention for each group;

FIG. 5 is a graph of NIP trend after intervention for each group;

FIG. 6 is a graph of analysis of the change in the abundance of fungal flora on the scalp under different interventions;

FIG. 7 is a graph of analysis of the change in the abundance of bacterial flora of the scalp under different interventions;

fig. 8 is a graph of analysis of changes in the diversity of scalp fungal and bacterial flora beta after T β 4 group intervention.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.

Example 1 scalp seborrheic dermatitis Microsporum Cross-section Studies

1.1 materials and methods

1.1.1 packet selection

Adult Chinese healthy people are selected as research objects. Inclusion criteria were as follows: (1) health (no other diseases, including chronic diseases such as hypertension, diabetes, chronic kidney disease, etc., excluding scalp SD); (2) the age is between 20 and 59 years old; (3) living in the long-lived city for more than 5 years; (4) the Han population; (6) the test protocol can be read and understood, and written consent can be signed in cooperation with the test content voluntarily.

Exclusion criteria were as follows: (1) other skin diseases simultaneously occur on the scalp; (2) obvious scars on the scalp; (3) a history of skin cancer; (4) orally administering an antibiotic, immunosuppressant, anti-inflammatory agent or antihistamine 4 weeks before administration; (5) the scalp scales, anti-seborrheic dermatitis shampoo and/or therapeutic product are administered within 2 weeks prior to administration.

After evaluation of the subjects' scalp scales, scalp erythema, inflammatory papules, etc. by a professional dermatologist, the subjects were divided into 3 groups: healthy controls (health controls), scalp scale (Dandruff) and Erythema (erythma), which together comprise scalp SD.

1.1.2 nucleic acid extraction

On the scaly scalp and erythematous scalp of the subjects, samples were taken at the skin lesion site and the non-skin lesion site, respectively, and on the scalp of the healthy control group, samples were taken twice at the same site. The sampling sites were swabbed with sterile Swabs (Catch-all Sample Collection swab, QEC091H, Epicentre, USA) for 30 seconds. Swab sections were cut with sterile scissors and placed into 2mL cryopreservation tubes for numbering. The samples were stored at-80 ℃. DNA was extracted from the scalp sample microflora using a buccal swab DNA rapid extraction kit (centrifugal column type) (Beijing BioTeke). First, the swab was transferred to a 2mL centrifuge tube and 200. mu.L of buffer was added. Add 20. mu.L proteinase K solution, vortex for 10 seconds and mix, stand at 70 ℃ for 10 minutes, vortex for several times every 2 minutes. Removing the swab to obtain a sample solution. Add 400. mu.L of binding solution to the sample solution, mix well by inversion, stand at 70 ℃ for 10 minutes, centrifuge briefly to remove the droplets on the inner wall of the tube cover. Adding 200 mu L of absolute ethyl alcohol, shaking and mixing uniformly. Adding the solution and flocculent precipitate obtained in the previous step into a micro adsorption column, and selectively adsorbing the Genomic DNA (Genomic DNA, gDNA) on the silicon substrate membrane in the centrifugal column under the high-order salt state. Then, through a series of rapid rinsing and centrifuging steps, the inhibitor removing solution and the rinsing solution are used for removing impurities such as cell metabolites, proteins and the like. And finally, transferring the micro adsorption column into a clean centrifugal tube, hanging and dropwise adding 35 mu L of elution buffer solution (the elution buffer solution is preheated at 65-70 ℃ in advance) to the middle position of the adsorption membrane, standing for 2-5 minutes at room temperature, and centrifuging at 12000rpm for 2 minutes to obtain filtrate, namely the sample DNA, obtained by eluting the pure gDNA from the silicon substrate membrane. The tube caps are marked and numbered, and the tubes are placed in a refrigerator at the temperature of minus 20 ℃ for storage.

The gDNA extracted from the scalp sample was amplified by Polymerase Chain Reaction (PCR). The 16S rDNA V4 region of the bacterium was amplified using 514F (GTGCCAGCMGCCCGGTAA) upstream and 805R (GGACTACHVGGGTWTCTAAT) downstream primers. The PCR cycling program was as follows: 2 minutes at 94 ℃; 30 cycles of 94 ℃ for 30 seconds, 54 ℃ for 30 seconds and 72 ℃ for 45 seconds; 5 minutes at 72 ℃. Each 25. mu.L of the reaction system contained: template DNA 0.5. mu.L, dNTP (2.5mmol/L) 2.0. mu.L, 10 XEx Taq buffer (Mg 2+) free 2.5. mu.L, Mg2+ solution (25mmol/L) 1.5. mu.L, Ex Taq DNA polymerase (2.5 units) 0.25. mu.L, 514F upstream primer and 805R downstream primer (10. mu. mol/L) each 0.5. mu.L, and double distilled water 17.25. mu.L. Similarly, the ITS1 region of the fungus was amplified using the ITS1-F (CTTGGTCATTTTAGAGGAAGTAAA) upstream primer and the ITS1-R (GCTGCGTTCTTCTTCATCGATGC) downstream primer. The PCR cycling program was as follows: 15 minutes at 94 ℃; 30 seconds at 94 ℃, 30 seconds at 50 ℃ and 1 minute at 72 ℃ for 5 cycles; 35 cycles of 95 ℃ for 30 seconds, 65 ℃ for 30 seconds, and 72 ℃ for 1 minute; 10 minutes at 72 ℃. Each 20. mu.L of the reaction system included: 10. mu.L of Maxima Hot Start PCR Master Mix (Thermo, USA), 2. mu.L of template DNA, 0.5. mu.L of ITS1-F upstream primer and ITS1-R downstream primer, respectively, and 7. mu.L of double distilled water. The primers used in this study were universal primers with barcode and synthesized by England Weiji technology services, Inc.

1.1.3 sequencing analysis

After the PCR sample was subjected to DNA concentration identification and purification, the PCR product was sequenced using Illumina Hiseq (PE 250). After the sequence was obtained, sequence analysis was performed on QIIME (v1.9.1). The original double-ended data was concatenated with SeqPrep. Low quality sequences are filtered with an acceptable minimum quality score of 20. Sequences were assigned to each sample according to the barcode, with the barcode and primers removed. And clustering bacteria and fungi to generate an operation classification Unit (OTU) to obtain a corresponding OTU table. The number of sequences per sample of bacteria and fungi were normalized to 1350 and 3230 strips, respectively.

1.1.4 parameter analysis

Statistical analysis and mapping was performed by the R programming language (v3.3.2). The Analysis of α -diversity and β -diversity of the flora was performed on QIIME and the study evaluated α -diversity and β -diversity using Shannon Index (Shannon Index) and Principal component Analysis based on unweighted UniFrac distance (PCoA), respectively. Alpha diversity is used to describe the abundance and diversity of microbial communities Within a sample (Within a community), including how many species are contained in a community (species abundance), and the number of species in each community (species uniformity); . Beta diversity was used to compare species diversity (beta-diversity) among groups, together with alpha diversity, to make up a description of overall diversity. Genera with significant differences between groups were analyzed based on Linear Discriminant Analysis Effect Size (LEfSe). The inter-group difference analysis was performed by Kruskal Walls and the statistics of differences between the two groups were performed by Wilcoxon rank sum test. The comparison between sets of continuity variables was performed using Analysis of Variance (ANOVA). From phylum to genus level, bacteria with average relative abundances above 0.01 and fungi with average relative abundances above 0.001 would appear on the graph. P <0.05 was defined as statistically significant.

1.2. As a result, the

1.2.1 physiological parameter characteristics

The scalp physiological parameter results of the subjects are shown in table 1. The differences in apical SCH (stratum corneum water content) were statistically significant in the healthy control group, the scaly scalp group, and the erythemic erythema group (P ═ 0.022). Further pairwise comparisons showed that the healthy control group had a significantly higher apical SCH (5.35 ± 4.13C.U.) than the scalp scale group (3.04 ± 2.02C.U.) and the differences were statistically significant (P ═ 0.022), whereas the healthy control group had a statistically significant difference in apical SCH from the erythema group and the scalp scale group had a statistically significant difference in apical SCH from the erythema group. There was no significant difference in scalp TEWL, sebum, pH and occipital SCH for the healthy control, scaly scalp and erythemic groups.

TABLE 1 scalp physiological parameters for different groups

^a TEWL, Trans-epidemal Water Loss through the scalp

^b SCH: stratum Corneum Hydration, Stratum Corneum Water content

^c Mean average difference of mean

P <0.05 has statistical significance

1.2.2 microbial flora distribution characteristics, comparison

As shown in fig. 1, 1 is a healthy control group, 2 is a scalp SD group including a dandruff group and an erythema group, 3 is a dandruff normal site, 4 is a dandruff skin lesion site group, 5 is an erythema normal site group, 6 is an erythema skin lesion site group, a/b: healthy control group and scalp SD group, scalp scale group, and bacterial flora in skin damage area and non-skin damage area of erythema group. c/d: healthy control group and scalp SD group, scalp scale group, and erythema group. a/c: the composition of the bacterial and fungal flora at the phylum level. b/d: bacterial and fungal flora on the genus level.

Compared with the scalp SD group (including dandruff group and erythema group), the healthy control group has similar scalp dominant flora in the skin lesion part of the dandruff group and erythema group and the skin lesion part of the erythema group and the non-skin lesion part. Among the fungi, the Basidiomycota (Basidiomycota) and the Ascomycota (Ascomycota) are the dominant phyla, accounting for more than 95% of the total fungi (fig. 1 a); at the genus level, the most predominant 8 genera are malassezia, Aspergillus (Aspergillus), exophium (exophila), Aureobasidium ((Aureobasidium)), Phaeoacremonium, Lecanicillium (Lecanicillium), Debaryomyces (Cyberlindnera) and Debaryomyces (Debaryomyces), accounting for more than 75% of the fungal genera (fig. 1 b). Of the bacteria, at the phylum level, Proteobacteria (Proteobacteria), Firmicutes (Firmicutes), Bacteroidetes (Bacteroidetes) and actinomycetes (Actinobacteria) are the most abundant 4 phyla, accounting for more than 95% of the total bacteria (fig. 1 c); at the genus level, the most important 8 genera are staphylococcus, sedimentary bacilli (Sediminibacterium), Corynebacterium (Corynebacterium), Pseudomonas (Pseudomonas), Phyllobacterium (Phyllobacterium), Bradyrhizobium (Bradyrhizobium), Sphingomonas (sphingamonas) and Bosea (Bosea), accounting for more than 50% of the bacterial genera (fig. 1 d).

To explore the flora differences of scalp bacteria and fungi between scalp SD patients and healthy controls, we performed a diversity analysis on their scalp microbial flora, the results of which are shown in fig. 2, wherein a: scalp SD group was compared with healthy control group for scalp fungal flora diversity. b: scalp SD group and healthy control group have diversity of scalp bacterial flora. The Shannon index (the number of all species contained in the colony, and the proportion of each species in the population, both affected by the abundance and the average of the species) of the fungal flora of scalp SD group was significantly lower than that of the healthy control group (P ═ 0.011), indicating that the scalp fungal flora of healthy control group had higher alpha diversity than that of scalp SD patients. The Shannon index indicates that there was no significant difference in alpha diversity of scalp bacterial flora between the scalp SD group and the healthy control group (P ═ 0.28). However, a significant difference in the beta diversity of the scalp bacterial flora was observed between PCoA (PC1 and PC2 represent the principal component analysis) (PERMANOVA, P ═ 0.001). A Random Forest Model (Random Forest Model) is respectively constructed based on the specificity and the sensitivity of the fungal flora and the bacterial flora, and the results show that the Area Under the Curve (Area Under Curve, AUC) of a Receiver Operating Characteristic (ROC) Curve of the fungal flora Model is 73.82%, and the AUC of the bacterial flora Model is 66.71%, which shows that the fungal flora Model has good distinguishing capability, and the bacterial flora Model has weaker distinguishing capability than the fungal flora Model.

1.2.3 biomarker analysis

The scalp SD groups were further analyzed for a significant difference in fungal and bacterial populations to search for a potential biomarker for microbial populations in the scalp SD based on LEfSe (Linear Discriminant Analysis Effect Size, a calculation used to discover and reveal group characteristics, which identifies species abundance features and correlations between different groups, with a powerful recognition function). The results showed that malassezia and Mycosphaerella (mycosphaeroella) among the fungi and staphylococcus and Brevibacterium (Brevibacterium) among the bacteria were enriched on the scalp of scalp SD patients, while 5 mycosis of aspergillus, Ganoderma (Ganoderma), black ear (Exidia), pilatopterus and lateral tooth (enterodotium) and 5 mycosis of pseudomonas, myceliophthora (Hyphomicrobium), Proteus (Proteus), tevorax (Devosia) and Bacteroides (Bacteroides) were enriched on the scalp of healthy controls.

To summarize: through the above analysis, it was found that the genus malassezia, the genus aspergillus in fungi and the genus staphylococcus and the genus pseudomonas in bacteria are potential biomarkers for scalp SD. The distribution characteristics of fungal and bacterial communities in the scalp of seborrhoeic dermatitis patients suggest potential interactions between the fungi, bacteria and host. SD may be a disease associated with the ecological balance of skin microorganisms, rather than an infectious disease. Scalp microecological balance is expected to be a new target for risk assessment, prevention and treatment of SD diseases.

Example 2 intervention study of thymosin beta 4 and traditional drugs for scalp SD

2.1 materials, methods

2.1.1 grouping and intervention scheme

Each group was expected to have 25 people enrolled for a total of 4 groups and 100 people enrolled, with at least 20 people/group for the final validation data. The grouping intervention is performed according to table 2. The duration of the intervention experiment was 6 weeks. Wherein, the blank control group A, B (A is the blank control group of healthy people, B is the blank control group of seborrheic dermatitis) always uses the blank control product; the scalp SD intervention group C, D (C group is seborrheic dermatitis positive intervention group, D group is seborrheic dermatitis T beta 4 intervention group) is stopped after 4 weeks by using corresponding intervention products (C group uses ketoconazole lotion, D group uses T beta 4 gel), and relapse is observed after 2 weeks.

TABLE 2 subject grouping intervention Condition Table

a: lishi soft and bright shampoo (Union Rihua (China) Co., Ltd., test subjects used according to the instructions of the product)

b: keloconazole lotion (2%) (Nanjing Baijing pharmaceuticals Ltd. subjects used a placebo shampoo and then used the ketoconazole lotion.)

c: tss 4 gel (0.5mg/mL) (homemade, prepared with reference to CN 201010521842.3. Subjects applied topically Tss 4 gel to scalp skin lesions after placebo shampoo.)

During the process of the intervention test, scalp clinical evaluation, physiological parameter determination and scalp microorganism sampling and preservation are carried out on each subject at five time nodes of D0, D7, D14, D28 and D42 (corresponding to T0, T1, T2, T3 and T4 respectively).

2.1.2 assay protocol

2.1.2.1 scalp clinical assessment

The clinical evaluation of the indicators of the scalp adhesion scale score (ASFS), the Number of Inflammatory Papules (NIP) in the lesion area, the Maximum Erythema Area (MEA) in the lesion area, etc. was carried out for each subject at five time nodes (T0-T4).

2.1.2.2 scalp physiological parameter measurement

At each of five time nodes (T0-T4), physiological parameters TEWL, SCH, sebum, and pH were measured for each subject.

2.1.2.3 scalp microorganism sampling and preservation

Scalp microbes were sampled from each subject at five time nodes (T0-T4). The scalp seborrheic dermatitis group was sampled in the area of skin lesions, the area with the highest ASFS score among 8 areas of scalp. The healthy scalp control group was sampled from any area of the scalp. The number of samples was two, one for DNA extraction and sequencing and the other was kept as a backup. Scalp Microbiologics sampling A sterile swab (Catch-all Sample Collection swab, QEC091H, Epicentre, USA) was used to swab the sampling site for 30 seconds. Swab sections were cut with sterile scissors and placed into 2mL cryopreservation tubes for numbering. The samples were stored at-80 ℃.

2.1.2.4 DNA extraction

Combining a binding solution/proteinase K (Thermo, USA) to rapidly crack cells and inactivate nuclease in the cells, selectively adsorbing genomic DNA to a silicon substrate membrane in a centrifugal column under a high-order salt state, performing a series of rapid rinsing-centrifuging steps, removing impurities such as cell metabolites and proteins by using an inhibitor removing solution and a rinsing solution, and finally eluting pure genomic DNA from the silicon substrate membrane by using a low-salt elution buffer solution to obtain sample DNA.

Amplification of 2.1.2.5 target fragment

The 16S rDNA V4 fragment and ITS1 were amplified using the DNA extracted as a template.

(1)16Sv4 fragment amplification: bacterial 16S rDNA V4 fragment was selected as a universal primer for PCR amplification. The upstream primer V4F (5 'GTGTGCCAGCMGCCGCGGTAA 3') and the downstream primer V4R (5 'CCGGACTACHVGGGTWTCTAAT 3') were used, and the primers were universal primers with barcode and synthesized by Ensifier Techikuchiki technology services, Ltd. PCR amplification of each sample corresponds to the information of the corresponding barcode upstream and downstream primers, and a SYBR GREEN Master Mix system is adopted, wherein the amplification system is 20 mu l, and the total DNA template is 4 mu l. The PCR cycling program was as follows: 5 minutes at 95 ℃; 35 cycles of 95 ℃ for 30 seconds, 52 ℃ for 30 seconds, and 72 ℃ for 45 seconds; 30 seconds at 72 ℃ and 5 minutes at 72 ℃. Each 20. mu.L of the reaction system included: SYBR Green Mix (Vazyme, Nanjing) 10. mu.L; mu.L of template DNA, 0.4. mu.L of each of the V4F upstream primer and V4R downstream primer, and 4.8. mu.L of deionized water.

(2) Amplification of ITS1 fragment: the universal primers for fungal ITS1 fragments were selected for PCR amplification. The upstream primer ITS1F (5'-CTTGGTCATTTAGAGGAAGTAA-3') and the downstream primer ITS1R (5'-GCTGCGTTCTTCATCGATGC-3') were selected. The primers are universal primers with barcode, are synthesized by Weiji technology services GmbH, and PCR amplifies the information of the corresponding barcode upstream and downstream primers of each sample. The PCR cycling program was as follows: 15 minutes at 95 ℃; 5 cycles of 95 ℃ for 30 seconds, 50 ℃ for 30 seconds, and 72 ℃ for 1 minute; 35 cycles of 95 ℃ for 30 seconds, 65 ℃ for 30 seconds and 72 ℃ for 45 seconds; 10 minutes at 72 ℃. The amplification system is a 20 μ l system, and the specific amplification conditions are as follows: SYBR Green Mix (Vazyme, Nanjing) 10. mu.L; mu.L of template DNA, 0.4. mu.L of ITS1F upstream primer and ITS1R downstream primer, and 6.8. mu.L of deionized water.

2.1.2.6 scalp microbiological sequencing analysis

(1) And mixing the samples. And (3) carrying out relative quantification on the amplified samples, and mixing all the samples according to the proportion of uniform concentration so as to balance the final detection concentration of each sample and reduce sequencing depth difference among different samples.

(2) And (5) purifying. And (3) placing the AMPure XP magnetic beads on an oscillator, and oscillating for 5 minutes at a high vibration speed to ensure that the magnetic beads are fully and uniformly mixed. Another new 1.5mL EP tube was added with 50 μ l of the mixed total PCR product solution and 50 μ l of the well-mixed AMPure XP magnetic beads, shaken well for 2 minutes, left to stand at room temperature for 5 minutes, placed on a magnetic rack for more than 2 minutes until the liquid was clear, removed, and carefully pipetted onto the magnetic beads. Adding 200 μ l of freshly prepared 80% ethanol, shaking, mixing, placing on magnetic frame for more than 2 min until the liquid is clear, and removing the liquid. Adding 200 μ l of freshly prepared 80% ethanol, shaking, mixing, placing on a magnetic frame for more than 2 min until the liquid is clear, removing the liquid, removing the excessive ethanol at the bottom of the tube with a small gun head, and carefully sucking the magnetic beads. Drying at room temperature for 12-15 minutes to ensure no ethanol remains. 27.5. mu.l of 10mM Tris HCl (pH 8.5) was added, and the mixture was stirred well for 2 minutes (small amplitude shaking) and allowed to stand at room temperature for 2 minutes. Place on magnetic rack for more than 2 minutes until the liquid is clear, pipette the liquid into a new EP tube, carefully separate the magnetic beads. The nucleic acid concentration of the purified sample was measured using a qubit3.0 nucleic acid concentration detector, and the average concentration of the total PCR product after purification (converting the concentration unit ng/ml to ug/ml) was determined, along with the name of the purification tube, the date, and the concentration of the purified DNA. The nucleic acid concentration was determined and diluted to the concentration required for sequencing.

(3) And (5) sequencing. Double-ended (PE250, cycle 300) sequencing was performed by the Illumina iSeq100 platform.

2.1.2.7 scalp microbiome data processing and analysis

(1) And (4) controlling the quality of sequencing data. Amplifying specific regions (a 16s rDNAV4 region and an ITS1 region) by using barcode primers respectively, carrying out Illumina double-end sequencing on PCR products of all samples, reading original fa sequence files obtained by the double-end sequencing respectively by using a Perl script, carrying out sequence sorting and splicing, carrying out sequence quality control on the merged FASTQ files by using QIIME software (QIIMEv1.9.1), removing low-quality data, and comparing the merged FASTQ files with a database to obtain a representative sequence of each sample.

(2) Species annotation and evaluation. In sequencing analysis of amplicons, OTU clustering was first performed on the sequenced sequences with QIIME.

(3) And analyzing the flora composition of the sample. According to the species annotation, each sample is annotated to various classification levels (kingdom, phylum, class, order, family, genus).

(4) Sample richness analysis (α diversity).

(5) Comparative analysis between groups (. beta.diversity analysis).

(6) Species differences between groups were analyzed. LDA size effect analysis (LEfSe) was used to find species that were significantly different between different groups.

(7) And (5) Index modeling. And (3) finding out the genera with significant difference in the abundance among groups according to the flora analysis of different groups, and establishing an index (index) by using the content and the influence (LDA value) of the key genera as the flora score of the sample.

2.1.2.8 statistical method

Statistical analysis and mapping was performed by Python (v3.7) and R (v3.6.3) programming languages. Descriptive statistics are expressed as a sum of percentage (classification data) and mean ± standard deviation (continuity data). Comparisons between groups of clinical assessment data (including epidemiological data, scalp subjective scores, and scalp physiological parameters) were analyzed by ANOVA and covariance analysis (ANCOVA). Microbiological analysis, comparison between the two groups according to different conditions, using a two-sample Wilcoxon test, and correlation analysis using a Spearman correlation analysis. The effective range of r is between-1 and 1. P <0.05 was defined as statistically significant.

2.2 results

2.2.1 trends in the change in ASFS scores after group intervention

The ASFS score trend is shown in fig. 3, where a shows the trend of the total ASFS score of each group over time, and b shows the trend of the average change percentage of the ASFS of each group. As can be seen from fig. 3-a, the ASFS score for the A, B group did not change much at each revisit time point. The total ASFS score of group C showed a marked decline at T0-T2 and increased back after cessation of drying (during T3-T4). The total ASFS score of the group D is in a obviously continuous descending trend from T0 to T4. As can be seen from fig. 3-b, the average percent change in ASFS score of A, B, C, D groups (% vs. baseline at T0) was significantly reduced in groups D from T0 to T4 at each revisit time point. The results indicate that the improvement effect of the Tbeta 4 on the ASFS is better than that of ketoconazole, and the ASFS is not easy to relapse after the intervention is finished.

2.2.2 trends in the Change in Dry prognosis MEA for each group

The trend of the Maximum Erythema Area (MEA) of the scalp for each group is shown in fig. 4, where a shows the trend of MEA for each group over time and b shows the trend of Δ MEA for each group. As can be seen in FIG. 4-a, the MEA's in group B tended to increase from T0 to T4; the MEA of the group C is in a descending trend at T0-T2, and is raised again after the drying is stopped (T3-T4); the MEA of group D showed a significant and continuous downward trend from T0 to T4. As can be seen from FIG. 4-b, the mean change for the MEA of A, B, C, D groups (. DELTA.MEA, compared with the baseline of T0) at each revisit time point showed a significant downward trend for group D from T0 to T4. The results suggest that ketoconazole does not significantly improve the scalp erythema, T β 4 does improve the scalp erythema, and the efficacy is maintained 2 weeks after the intervention.

2.2.3 trends in the prognosis of Dry NIP for each group

The Number of Inflammatory Papules was represented by NIP (Number of Inflammatory Papules), and the amount of change in the Number of Inflammatory Papules was represented by Δ NIP (comparison with T0 as a baseline). The variation trend of NIP is shown in fig. 5, wherein a shows the variation trend of NIP of each group with time, and b shows the variation trend of Δ NIP of each group. As can be seen from FIGS. 5-a and 5-B, the inflammatory papular numbers NIP and Δ NIP of group B did not change much at different time points; the inflammatory papule numbers NIP and delta NIP of group C showed a downward trend at T0-T3, and rose back after the cessation of dry prognosis (T3-T4); the number NIP and the delta NIP of inflammatory papules in group D both decreased from T0 to T4. The results suggest that ketoconazole and T β 4 have an improving effect on inflammatory papules, and that after the intervention was completed, the improving effect of T β 4 on inflammatory papules was maintained better than ketoconazole.

2.2.4 comparison of outcomes of different intervention groups scalp physiological parameters

Analysis compared trends in change by transepidermal water loss (TEWL), changes in scalp stratum corneum water content (SCH), changes in scalp sebum and pH. The results suggest that ketoconazole and T β 4 also have an improving effect on the scalp skin barrier (TEWL) and sebum secretion, and that T β 4 has a more pronounced improving effect on TEWL than ketoconazole. Tss 4 promotes re-epithelialization, stimulates neovascularization, upregulates laminin-5 expression, and thus repairs damaged skin barriers. Compared with ketoconazole, T beta 4 also has the effect of regulating the pH of the scalp. After 2 weeks of discontinuation of intervention, the pH of the T β 4 intervention group decreased significantly more than the ketoconazole intervention group. Tss 4 lowers the pH of the scalp and is more conducive to combating microbial attack, thereby helping to maintain the balance of the scalp microflora.

2.2.5 variation of different intervention groups of scalp microflora

2.2.5.1 changes in the abundance of scalp microflora following intervention

Changes in the abundance of the scalp fungal and bacterial flora after different interventions were compared using the statistical analysis indices PD white Tree (influenced primarily by the abundance of the sample species) and Shannon index (influenced by both the abundance and the average of the species), respectively, in fig. 6 and 7, a shows the change in the PD white Tree index, b shows the change in the Shannon index, the solid line is the mean result of the set of indices, and the dotted line is the median result of the set of indices. The results of the fungal flora analysis are shown in FIG. 6. Before intervention, the abundance of scalp fungal flora of B, C, D groups (scalp SD groups) is lower than that of healthy groups, and after the intervention, the abundance of scalp fungal flora of C groups and D groups is obviously increased to reach the level similar to that of the healthy groups, and the scalp fungal flora is maintained for 2 weeks after the intervention. The analysis results of the bacterial flora are shown in fig. 7, before intervention, the abundance of the scalp bacterial flora of B, C, D groups (scalp SD groups) is lower than that of the healthy groups, after the intervention, the abundance of the scalp bacterial flora of group D is obviously increased to reach the level similar to that of the healthy groups, and the scalp bacterial flora is maintained for 14 days after the intervention. Whereas the abundance of the scalp bacterial flora of group B, C was still at a low level during the intervention and within 2 weeks after the end of the intervention.

2.2.5.2 changes in the beta-diversity of the scalp microflora following intervention

And (3) performing principal component analysis (PCoA) on the UniFrac distance by using QIIME to respectively obtain standardized (Weighted) and non-standardized (Unweighted) clustering results, and comparing the similarity of the flora structures among the groups according to the clustering distances among the groups. Fig. 8 a and b show the trend of the diversity of the T β 4 group in the scalp fungal and bacterial flora β at different intervention stages, respectively, grey points: and T beta 4 group. Black dots: healthy scalp group. The result of the change of the fungal flora shows that the ketoconazole intervention group has obvious change after the intervention for 7 days (T1) compared with the step before the intervention (T0), but the change is not close to the state of healthy people, and simultaneously, as the intervention time is prolonged, the sample starts to gradually return to the position before the intervention, which indicates that the intervention for 7 days can obviously affect the fungal flora, but as the time is changed, the influence of the intervention on the fungal flora is reduced, the flora gradually returns to the original state, and the ketoconazole intervention does not enable the scalp fungal flora to return to the state of healthy flora. Compared with the T0 group before intervention, the T beta 4 intervention group has obvious change after 7 days (T1) of intervention, and T1-T4 have the tendency of approaching the fungal flora of healthy people. The scalp fungal flora is changed after Tbeta 4 intervention for 7 days, the healthy flora state is recovered, and a certain effect can be maintained even if the dry prognosis is stopped.

The bacterial flora change result shows that the intervention of ketoconazole has no obvious influence on the scalp bacterial flora of the scalp SD population; the Tss 4 intervention group tended to be close to the healthy population flora 4 weeks after intervention (T28) and 2 weeks after intervention (T42). It is suggested that T beta 4 exerts an improvement effect on the bacteria flora of the scalp after a certain period of time, and has a certain maintenance effect even if the dry prognosis is stopped.

2.2.5.3 changes in Critical genera of scalp following intervention

Ketoconazole significantly reduced the relative abundance of the limiting Malassezia during the early intervention period, but did not decrease or increase inversely after 2 weeks of intervention and after the end of the intervention. Whereas T β 4 intervention decreased the relative abundance of the restricted malassezia bacteria more slowly, but its effect was maintained after 2 weeks of intervention. Ketoconazole intervention had no apparent effect on staphylococcus, whereas T β 4 intervention significantly reduced the relative abundance of staphylococcus.

In conclusion, T beta 4 has a significant improvement effect on both the dermatophytes and the bacterial flora, and the effect is maintained for a long time, and the effect can still be maintained for a period of time (2 weeks) after the intervention is stopped. After T beta 4 stem prognosis, the abundance of scalp fungi and bacterial flora is obviously improved, the relative abundance of restrictive malassezia is reduced, the abundance of spherical malassezia is improved, the relative abundance of staphylococcus is obviously reduced, the scores of fungi and bacterial flora are obviously reduced, and the effect of stem prognosis stopping can still be maintained. Combining the intervention results of ketoconazole and Tbeta 4 subjective scores on the scalp, compared with ketoconazole, Tbeta 4 has better treatment effect on scalp SD and has less relapse after the intervention is stopped, wherein T beta 4 group scalp scale adhesion score (ASFS) is reduced by about 4.4 times compared with ketoconazole group, Maximum Erythema Area (MEA) of scalp is reduced by about 8.3 times compared with ketoconazole group, inflammatory papule Number (NIP) is reduced by about 1.3 times compared with ketoconazole group, and transepidermal water loss (TEWL) is reduced by about 3.9 times compared with ketoconazole group after 14 days of drug withdrawal. This is probably due to T β 4 correcting the microflora imbalance in the scalp SD population, which leads to a scalp microflora profile towards healthy populations.

Claims (7)

1. Use of thymosin beta 4 for the preparation of a regulator of the microecological balance directed at the dysregulation of the microbial flora of the skin.

2. Use according to claim 1, wherein the dysbiosis of a microbial flora comprises a fungal and/or a bacterial flora dysbiosis.

3. Use according to claim 2, wherein the fungal flora imbalance comprises an increased abundance of malassezia and/or a decreased abundance of aspergillus.

4. Use according to claim 3, wherein the bacterial genus deregulation comprises an increased abundance of Staphylococcus and/or a decreased abundance of Pseudomonas.

5. The use according to any one of claims 1 to 4, wherein the skin is a hair-laden skin.

6. Use according to claim 5, wherein the skin is the skin of the head.

7. The use according to any one of claims 1 to 4, wherein thymosin β 4 is prepared as a pharmaceutical, cosmetic or nutraceutical product.

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