CN112656786A - Application of oroxylin in preparation of medicine for treating psoriasis - Google Patents

Abstract

The invention discloses a new medical application of oroxylin, belonging to the technical field of medicines. Oroxylin is used for preparing the medicine for treating psoriasis, and can be used as the medicine for resisting psoriasis for subsequent development due to low toxic and side effects.

Description

Application of oroxylin in preparation of medicine for treating psoriasis

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of oroxylin in preparation of a medicine for treating psoriasis.

Background

Psoriasis is a common chronic inflammatory disease involving multiple cells, has high recurrence rate and cannot be completely cured. The pathological manifestations of the disease are hyperkeratosis and parakeratosis of epidermis, hypertrophy of cells of acanthosis, infiltration of immune cells, increase of the level of inflammatory factors and vasodilatation. At present, the worldwide prevalence rate is about 1% -2%, and the pathogenesis of the disease is not completely clarified, so that the clinical treatment is difficult, the disease can be treated symptomatically, and related treatment medicines are adopted to improve the symptoms of patients and improve the life quality of the patients.

Psoriasis can be classified into mild, moderate and severe psoriasis according to the severity of the disease, and the mild psoriasis generally adopts local medicines, including corticosteroids, methotrexate, cyclosporine and vitamin D3 analogues. For psoriasis patients with large areas and no topical application, NB-UVB (Narrowband Ultraviroet B) or PUVA (psoralen-Ultraviolet A) phototherapy is used. However, PUVA treatment cross-links psoralea fruit with DNA via UVA, is highly mutagenic and induces the development of skin cancer such as melanoma with long-term use. In more severe cases, systemic drugs, such as retinoids, MTX or cyclosporin, are added in addition to the basic therapy, and these small-molecule drugs have good therapeutic effects, are convenient to use and are inexpensive, but their high toxicity and teratogenicity to liver, kidney and bone marrow limit their application in clinical therapy.

One current explanation for the pathogenesis of psoriasis is the result of the interaction of cells of the immune system (e.g., T cells, macrophages, DC cells, etc.) with skin keratinocytes. Among them, the IL-23/TH17 signaling axis plays a key role in the development of psoriasis. Therefore, antibody-based biologics against TNF α, IL-17, etc. are currently approved for clinical treatment of psoriasis. Although the efficacy of biological agents in treating psoriasis is significant, their use in the clinic is limited due to their high cost and potential unknown risks. Therefore, the search for effective small molecular compounds with small toxic and side effects is still an important direction for treating psoriasis.

Oroxylin (OA) is one of the active ingredients of scutellaria, and has been proven to have a wide range of pharmacological actions including anticancer, anti-inflammatory, neuroprotective, anticoagulant, etc. In recent years, multiple researches show that various flavonoid traditional Chinese medicine extracts can regulate tissues and organs, immune cells and immune molecules, and meanwhile, oroxylin is reported to have a certain treatment effect on enteritis, arthritis, smoking-induced pulmonary inflammation and asthma, and the oroxylin is suggested to have a relatively broad-spectrum and safe anti-inflammatory effect.

At present, the oroxylin is not reported to be used for preparing the psoriasis treatment medicine.

Disclosure of Invention

The invention aims to provide a new medical application of oroxylin.

In order to achieve the purpose, the invention adopts the following technical scheme:

application of oroxylin in preparing medicine for treating psoriasis is provided.

The medicine is orally taken, and the effective dose of the medicine to mice is 20-80mg/kg, preferably 80 mg/kg. The positive drug is cyclosporin A (CSA).

Further, the dose of oroxylin at the cellular level is in the range of 2-50 μ M, preferably 10 μ M.

The inventor learns that the oroxylin can inhibit the proliferation of keratinocytes and inhibit the infiltration of immune cells through the research of oroxylin administration on an imiquimod-induced psoriasis mouse model, thereby realizing the treatment of the imiquimod-induced psoriasis.

The oroxylin serving as a medicament for treating psoriasis can obviously relieve local scale formation and skin thickening of psoriasis-like skin lesions induced by imiquimod, improve psoriasis pathological score, inhibit the level of a plurality of proinflammatory factors such as IL-23, TNF alpha and IL-1 beta in serum and the like, and further realize the treatment of the psoriasis.

Has the advantages that: the invention discovers the effect of oroxylin on psoriasis for the first time, and can be used as a potential medicament for resisting psoriasis for subsequent development due to low toxic and side effects.

Drawings

Figure 1 is a graph of the effect of oroxylin on weight and pathology scores in an imiquimod-induced psoriasis mouse model. (A) Making a model of a mouse picture; (B) clinical scoring of mice; (C) the body weight of the mice changed.

Figure 2 is HE of oroxylin versus imiquimod-induced psoriasis model mouse skin tissue. (A) HE staining of skin tissue; (B) and (5) counting the thickness of the epidermis.

FIG. 3 shows the staining of mouse proliferation marker Ki67 with oroxylin on the skin tissue of an imiquimod-induced psoriasis model mouse and the detection of inflammatory factors (A) the immunohistochemical staining of the skin tissue Ki 67; (B) statistics of Ki67 positive area; (C) the skin tissue protein extract contains IL-1 beta and IL-17A, TNF-alpha.

Figure 4 is a fluorescent staining of oroxylin for immune cell infiltration in imiquimod-induced psoriasis model mouse skin tissue.

Detailed Description

The invention is described in further detail below with reference to the figures and the specific examples, which should not be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.

Example 1

(1) Imiquimod (IMQ) -induced psoriasis mouse model

Mice were anesthetized with 0.1mL/10g of 3.5% chloral hydrate (C57BL/6 mice, 18-22g, 6-8 weeks), the back hairs of the mice were shaved, the backs of the mice were exposed to about 2cm X3 cm of skin, the psoriasis was induced by continuously applying IMQ ointment (62.5mg) to the back skin of the mice for 6d, and the control group was administered with an equivalent dose of vaseline ointment (Sham).

Positive drug cyclosporin A (CSA, 30mg/kg/day) or individual doses of OA (20, 40, 80mg/kg/day) were gavaged during the molding cycle. During the molding process, the physiological indices (body weight, skin changes) of the mice were monitored daily. The clinical symptoms adopt a PASI scoring standard to evaluate indexes such as erythema, scale 2 and the like, scores are given in 0-4 points, and the total score is obtained by adding the 3 points. PASI scoring criteria are as follows: 0, no symptoms; 1, light; 2, moderate; 3, severe; 4, very severe.

And (3) killing the mice on the 7 th day of modeling, taking down dorsal skin, spleen and abdominal cavity macrophages of the mice, taking blood from eyeballs, centrifuging to obtain serum, and detecting inflammatory factors such as IL-1 beta, TNF alpha and the like in the serum by an ELISA method.

The results are shown in figure 1, Oroxylin (OA) significantly reduced imiquimod-induced psoriasis-like lesions local scale formation, improved pathological scores, reduced imiquimod-induced weight loss in mice, and reduced imiquimod-induced inflammatory symptoms.

(2) Hematoxylin-eosin (HE), Immunohistochemical (IHC) assay

The tissue is pretreated according to the conventional method, coated by paraffin and sliced. Paraffin section is dewaxed and then is stained in a conventional H.E. mode, or after antigen retrieval, fixation, permeation and serum sealing are carried out, primary antibody (Ki67 antibody) is incubated overnight, rinsing is carried out, biotin-labeled secondary antibody is incubated for 0.5 hour at normal temperature, secondary antibody is added for incubation for 0.5 hour, ABC compound is incubated for 0.5 hour at normal temperature, DAB substrate is incubated at normal temperature, incubation time is controlled under a mirror, reaction is stopped in water, hematoxylin counter staining is carried out, and dehydration sealing is carried out (immunohistochemistry method).

The results are shown in fig. 2 and fig. 3 (a) and (B), which show that oroxylin significantly reduces epidermal thickness and staining of keratinocytes Ki67 positive cells, and its effect is comparable to that of positive drug CSA.

(3) Enzyme linked immunosorbent assay (ELISA) method for detecting content of inflammatory factors

And collecting sample protein, and detecting the contents of the cytokines IL-1 beta, IL-17A and TNF-alpha according to the ELISA kit instruction. The general flow is as follows: add 100. mu.L of sample to the wells, 50. mu.L/well diluted Biotinylated antibody, mix well and cover the plate-sealing membrane, incubate at 37 ℃ for 90 min. The plate was washed and 100. mu.L/well was added with diluted Streptavidin-HRP. Incubation was carried out at 37 ℃ for 30min, TMB was added at 100. mu.L/well, incubation was carried out at 37 ℃ for 5-30min in the dark, and termination was judged according to the shade of blue in the wells. Stop solution was added to 100. mu.L/well to terminate the reaction and absorbance at 450nm was measured within 10 min.

The results are shown in FIG. 3 (C), which can significantly reduce the levels of IL-17A and TNF- α in serum.

(4) Immunofluorescence (IF) detection of inflammatory cell infiltration in skin tissue

Paraffin sections are dewaxed and then are subjected to conventional immunofluorescence staining, the sections are sealed for 1h after being recovered, and then primary antibodies (1:200) are incubated overnight, and secondary antibodies (1:500) are incubated for 1 h. After rinsing with PBS, the anti-fluorescence quencher is added dropwise, and the mounting is photographed and analyzed.

The results are shown in fig. 4, and the oroxylin can obviously reduce infiltration of T cells and macrophages, thereby indicating that the oroxylin can inhibit the occurrence and the development of psoriasis by inhibiting infiltration of immune cells.

Claims (2)

1. Application of oroxylin in preparing medicine for treating psoriasis is provided.

2. Use according to claim 1, characterized in that: the medicine for treating psoriasis is orally taken.

Images