阅读说明：本技术 一种采用色氨酸代谢物抑制银屑病病发的方法 (Method for inhibiting psoriasis by using tryptophan metabolite ) 是由 王刚 乔佩 党二乐 于 2021-09-09 设计创作，主要内容包括：本发明公开了一种采用色氨酸代谢物抑制银屑病病发的方法,属于银屑病治疗技术领域,所述方法采用喹啉酸治疗银屑病,所述喹啉酸通过激活芳香烃受体AhR抑制NLRP3炎症小体抑制银屑病,具体的说,皮肤表面色氨酸代谢的代谢物喹啉酸通过激活细胞核内的AhR负调控NLRP3炎性体通路的激活,从而抑制炎症细胞因子的分泌参与银屑病的发病。(The invention discloses a method for inhibiting psoriasis by using tryptophan metabolites, which belongs to the technical field of psoriasis treatment, wherein quinolinic acid is used for treating psoriasis, and the quinolinic acid inhibits NLRP3 inflammasome from inhibiting psoriasis by activating aromatic hydrocarbon receptor AhR, and specifically, the quinolinic acid, the metabolite of skin surface tryptophan metabolism, regulates the activation of NLRP3 inflammasome passage by activating AhR in nucleus cells, so as to inhibit the secretion of inflammatory cytokines from participating in the pathogenesis of psoriasis.)

1. A method of inhibiting the onset of psoriasis using a tryptophan metabolite, wherein the psoriasis is treated with quinolinic acid.

2. The method of claim 1, wherein the quinolinic acid is a metabolite of skin flora tryptophan.

3. The method of claim 1, wherein the quinolinic acid inhibits psoriasis by activating the aromatic hydrocarbon receptor AhR to inhibit NLRP3 inflammasome.

4. The method of claim 1, wherein the quinolinic acid inhibits psoriasis by inhibiting the secretion of IL-1 β protein and IL-18 protein in keratinocytes.

5. The method according to any one of claims 1 to 4, wherein the quinolinic acid is administered at a concentration of 0.1 μ g/ml to 1mg/ml in the inhibition of the psoriatic hair.

6. The method of claim 5, wherein the quinolinic acid is combined with pharmaceutically acceptable excipients to form a medicament for inhibiting psoriasis.

7. The method of claim 6, wherein the agent is an emulsion or a liniment.

Technical Field

The invention relates to the technical field of psoriasis treatment, in particular to a method for inhibiting psoriasis by using tryptophan metabolites.

Background

A large number of microbial populations inhabit the skin, share nutrients with the host, and produce metabolites that are involved in regulating skin health and immune function. Tryptophan (Trp) metabolites produced by microbial populations are of great interest because of their role in immune regulation, particularly immunosuppression. Several studies have shown that they play a role in the maturation of immune tissues and the fine-tuning of inflammatory diseases, such as inflammatory bowel disease, rheumatoid arthritis, metabolic syndrome, neuropsychiatric features, etc. Compared to the intestine, the skin is in a dry, nutritionally poor and acidic environment, and therefore the metabolic function of the skin microbiota is very different from that of the intestine. The stratum corneum of the epidermis is rich in amino acids from dead keratinized cells and broken keratin, providing an abundant substrate for tryptophan metabolism by the skin microflora. Quinolinic Acid (QA) is an important metabolite of tryptophan downstream of metabolic pathway through kynurenine, and plays an important role in various mental diseases, such as depression and stress-related diseases. Over the past decades, much of the research on QA has focused on its association with neurological diseases. In the field of treating psoriasis, it is of interest whether the metabolite QA can be utilized.

Psoriasis is an immunological disorder disease under the polygenic genetic background, and is emphasized in the clinical and scientific research fields of dermatology because of the characteristics of high morbidity, repeated attack and the like. The NLRP3 inflammasome is a member of the receptor family recognized by innate immune systems and is highly expressed in psoriatic lesions, in addition to which NLRP3 pathway-associated genes (such as NLRP3 itself, caspase-1 (cysteine-containing aspartate hydrolase) and IL-1 β) may carry gain-of-function mutations that lead to a constitutive pro-inflammatory pattern. Numerous studies in the treatment of psoriasis have shown that the community diversity of skin microbiota of psoriasis patients varies significantly compared to healthy subjects, such as streptococci and staphylococcus aureus. However, most of these studies have focused on the variation of bacterial abundance between psoriatic lesions and healthy skin, and the metabolism of the skin microbiota remains largely unknown, which hinders a comprehensive understanding of the functional importance of the interaction of the skin microbiota with the host, particularly the regulatory relationship between skin microbiota metabolism and NLRP3 inflammasome, and also hinders the progress of developing new psoriasis treatment regimens through conventional techniques.

Disclosure of Invention

Accordingly, it is an object of the present invention to provide a method for inhibiting psoriatic hair using tryptophan metabolites by studying the effect of the tryptophan metabolite QA produced by the skin microbiota on psoriasis. QA plays a regulating function in an AhR dependent mode in the onset of psoriasis to inhibit NLRP3 inflammatory corpuscles, can obviously reduce psoriasis inflammation, inhibit the expression of cytokines in skin inflammation and further inhibit the psoriasis.

The invention solves the technical problems by the following technical means:

a method for inhibiting the development of psoriasis using a tryptophan metabolite comprises treating psoriasis with quinolinic acid.

Further, the quinolinic acid is a metabolite of tryptophan of the skin flora.

Furthermore, the quinolinic acid inhibits the psoriasis by activating an aromatic hydrocarbon receptor AhR to inhibit NLRP3 inflammatory corpuscle, and particularly, the quinolinic acid which is a metabolite of skin surface tryptophan metabolism negatively regulates the activation of NLRP3 inflammatory body channels by activating AhR in cell nucleus, so that the secretion of inflammatory cytokines is inhibited to participate in the pathogenesis of the psoriasis.

Further, the quinolinic acid inhibits psoriasis by inhibiting secretion of IL-1 β protein and IL-18 protein in keratinocytes.

Furthermore, the concentration of the quinolinic acid is 0.1 mu g/ml to 1mg/ml when the psoriasis disease is inhibited.

Further, the quinolinic acid is mixed with pharmaceutically acceptable auxiliary materials to prepare a medicament for inhibiting psoriasis. If the composition is administered to human body, it is preferably formulated into cream with reference to pharmaceutical base, such as common emulsion/cream/liniment base such as vaseline, lanolin, and alcohols.

Has the advantages that:

the skin flora tryptophan metabolite quinolinic acid QA can obviously inhibit the psoriasis phenotype, resist cell proliferation and inflammation, inhibit the expression of cell factors in skin inflammation and further inhibit the psoriasis. Meanwhile, the QA has good medicinal prospect for treating psoriasis, has stable chemical property, and can be prepared into cream and liniment for use.

Drawings

FIG. 1: a liquid chromatography detection result chart;

FIG. 2: QA inhibits the psoriasis and sends the result picture;

FIG. 3: a western blot detection result graph;

FIG. 4: an immunofluorescence data plot;

FIG. 5: graph of therapeutic effect of AhR specific siRNA;

FIG. 6: staining of mouse ear tissue sections.

Detailed Description

The present invention will be described in detail with reference to specific examples below:

example 1: decreased QA levels in skin lesions and non-skin lesions of patients with psoriasis

To verify that QA participates in the pathogenesis of psoriasis, QA level analysis was performed on the skin of psoriatic patients where skin lesions and non-skin lesions were removed.

1. Collecting metabolites on the surface of the skin (skin lesion and non-skin lesion) of healthy subjects (healthy skin) and patients with psoriasis by using a gel patch method to obtain a skin surface microbiota sample;

2. comparing the levels of Trp metabolites on the skin surface of the psoriatic patient and healthy subjects using liquid chromatography-tandem mass spectrometry analysis of the collected samples;

the results obtained are shown in FIG. 1, with the following conclusions:

among metabolites at the level of Trp, QA is present at significantly lower levels in skin lesions (lesions) and non-lesions (non-lesions) in psoriatic patients than in healthy subjects. Furthermore, there is a significant negative linear correlation between QA levels and the area of psoriasis and the patient severity index (PASI), indicating that the more severe the psoriatic patient, the lower the expression of QA on the skin surface. These data above indicate that there are abundant metabolites of Trp of microorganisms present on the skin surface of psoriatic patients and that QA levels on the skin surface of psoriatic patients are reduced, indicating that QA may be involved in the pathogenesis of psoriasis.

Example 2: QA attenuated IMQ-induced psoriatic dermatitis and inflammatory cell infiltration in mice

Based on the experiments of example 1, QA was used to inhibit the incidence of psoriasis and to attenuate inflammatory cell infiltration. Experiments were performed using a conventional IMQ-induced psoriasis-like mouse model.

Experimental groups: smearing IMQ on mouse ear at concentration of 5%, 25 mg/ear/day, smearing QA at concentration of 10 μ g/ml (dimethyl sulfoxide dilution) on mouse skin at IMQ smearing position 60 min, continuously using for 7 days, and observing the skin at drug application position on 5 days and 7 days;

control group: the mice were treated with IMQ alone at a concentration of 5% in each ear for 7 consecutive days, and the skin of the drug application site was observed on the 5 th and 7 th days.

The results obtained are shown in FIG. 2, and the conclusions are as follows:

(1) the thickness of the ears of mice treated with a combination of IMQ and QA was significantly lower than the thickness of the ears of mice treated with IMQ alone (control), and the degree of visible scaling and redness of the ears treated with IMQ plus QA was significantly reduced.

(2) Hematoxylin and eosin staining showed reduced epidermal thickness and reduced inflammatory cell infiltration of the ears in the IMQ plus QA group of mice.

Based on the experimental results, the flow cytometry detection method is adopted for analysis, and the results show that: IMQ + QA group mouse skin CD4+ T cells or Ly6G⁺Cells were significantly less than in the IMQ group, with no difference in CD8+ T cell numbers;

real-time PCR quantification to detect the expression of cytokine transcripts (IL-1. beta., IL-6, IL-17, IL-18, IL-23, TNF-. alpha., S100A8, CXCL1 and CCL20) showed: the IMQ plus QA group had significantly reduced IL-1 β, IL-6, IL-18, S100A8, and CXCL1 compared to the IMQ group.

Taken together, these data indicate that topical application of QA reduces inflammatory cell infiltration and inhibits the expression of cytokines in IMQ-induced skin inflammation, with a significant effect on inhibiting the onset of psoriasis.

Based on the above experiments, it was demonstrated that QA inhibits the onset of psoriasis. At the same time, western blot analysis was performed on the mouse samples of the experimental groups, and the results are shown in fig. 3, which show: expression of NLRP3 was significantly down-regulated in keratinocytes after stimulation with mixed cytokines (20ng/ml IL-1. alpha., 20ng/ml IL-17,20ng/ml IL-22,50ng/ml TNF-. alpha., 20ng/ml Oncostatin M) plus QA; however, the expression of NLRC4 and AIM2 was not altered. Furthermore, the effect of QA on NLRP3 inflammasome in keratinocytes was determined by Western blotting and it was found that the protein levels of ASC, caspase-1, mature IL-1 β and IL-18 were reduced in keratinocytes after stimulation with mixed cytokines plus QA, consistent with immunofluorescence data. Thus, our results again indicate that QA inhibits activation of NLRP3 inflammatory bodies and secretion of IL-1 β and IL-18 in keratinocytes and that inhibition of psoriatic disease using QA is effective.

Example 3: inhibition of NLRP3 inflammasome by activation of the aromatic hydrocarbon receptor AhR inhibits psoriasis

Based on the above examples, it was verified that it is effective to suppress psoriasis using QA, and further investigation is still needed as to how QA suppresses NLRP3 inflammasome.

The results of culturing HaCaT cells according to the prior art and treating keratinocytes with a concentration of 100. mu.g/ml QA are shown in FIG. 4: the results indicate that QA-treated keratinocytes show significantly increased levels of AhR and CYP1A1 (the major downstream molecule of AhR activation), both mRNA and protein, indicating that the AhR signal is activated by QA in keratinocytes, and further confirmed this result by immunofluorescence data.

Small interfering RNA was also used to interfere with AhR expression in keratinocytes, followed by the addition of mixed inflammatory cytokines to mimic the in vitro psoriasis model. The addition of QA significantly suppressed the expression of NLRP3 inflammasome and its related molecules. Nevertheless, inhibition of NLRP3 inflammasome and IL 1-beta, IL-18 and caspase-1 secretion by QA was restored to varying degrees after interference with AhR. These results above indicate that AhR is essential for QA inhibition of NLRP3 in the pathogenesis of psoriasis, further inflammation using a mouse model.

2.5nmol of AhR-siRNA and 5mg of cream matrix are mixed to obtain AhR specific siRNA (the mixture is used in the amount of 1 ear), the AhR specific siRNA is locally applied to the ear skin of an IMQ treated mouse, and the AhR specific siRNA is applied to the ear of the mouse every other day for 4 times.

The results obtained are shown in FIG. 5 and show that: after the use of the above drugs, the psoriasis-like lesions were effectively exacerbated, showing increased ear thickness and increased erythema. Hematoxylin and eosin staining of mouse ear tissue sections after topical treatment with AhR specific siRNA showed significant epidermal changes, increased thickness and proliferation. Suggesting that IMQ-induced psoriasis phenotype is exacerbated by AhR expression intervention.

The above experiment was repeated after knockdown of AhR. Analysis by western blotting and RT-PCR showed that, as shown in figure 5: the knockdown of AhR leads to the expression reversion of NLRP3, ASC, mature caspase-1 and IL-1 beta on mRNA and protein levels, and indicates that the knockdown of AhR influences the expression level of NLRP3 inflammasome and related molecules.

Taken together, these results indicate that QA, a metabolite of skin surface tryptophan metabolism, negatively regulates the activation of NLRP3 inflammatory body pathways by activating AhR in the nucleus, thereby inhibiting the secretion of inflammatory cytokines to participate in the pathogenesis of psoriasis.

According to the experimental results of example 1-example 3, QA can inhibit the psoriasis by activating the aromatic hydrocarbon receptor AhR to inhibit NLRP3 inflammasome, i.e. the tryptophan metabolite QA can significantly reduce the psoriasis inflammation, inhibit the expression of cytokines in the skin inflammation, and further inhibit the psoriasis.

Example 4: QA dose concentration

QA can be directly used after being diluted with DMSO (dimethyl sulfoxide), and can be made into cream with reference to pharmaceutical base, such as common emulsion/cream base such as vaseline, lanolin, and alcohols.

The QA administration concentration is tested by using a conventional IMQ-induced psoriasis-like mouse model, the test is carried out according to the test group of the embodiment 2, the QA concentration is respectively 0.01ug/ml, 0.1ug/ml, 1ug/ml and 10ug/ml, the obtained results are shown in figure 6, according to the results, when the effective concentration is less than or equal to 0.1ug/ml and less than or equal to 10ug/ml, hematoxylin and eosin staining shows that the epidermis thickness is reduced and inflammatory cell infiltration is reduced, meanwhile, the visible scale and redness degree of the mouse ear are obviously reduced, and when the effective concentration is in the range of 0.1-10 ug/ml, the psoriasis can be inhibited and the psoriasis condition can be alleviated.

Meanwhile, the cell administration concentration is tested, and the optimal cell administration concentration is 0.1mg/ml-1 mg/ml.

Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention as defined in the appended claims. The techniques, shapes, and configurations not described in detail in the present invention are all known techniques.