CN111281965A - Application of TIR/BB ring mimetic AS-1 in preparation of drugs for treating corneal neovascular diseases - Google Patents

Abstract

The invention discloses an application of TIR/BB ring analog AS-1 in preparing a medicament for treating corneal neovascular diseases, in particular an application in preparing a medicament for treating corneal alkali burn. The invention introduces fat-soluble small molecular polypeptide into the treatment field of the corneal alkali burn for the first time, and researches the treatment effect of AS-1 on the corneal alkali burn, thereby not only providing a new targeted medicine for treating the corneal alkali burn and the corneal neogenesis lymphatic vessel, but also providing a new idea for treating the corneal alkali burn by the small molecular polypeptide.

Description

Application of TIR/BB ring mimetic AS-1 in preparation of drugs for treating corneal neovascular diseases

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a TIR/BB loop mimetic AS-1 in preparation of a medicine for treating corneal neovascular diseases.

Background

Angiogenesis and formation are fundamental biological processes, and pathological neovascularization plays an important role in a variety of eye diseases. The corneal neovascular diseases are the second approximately blindness eye diseases in China, the pathogenesis is not completely clarified, and no effective treatment method is available.

Corneal alkali burn is a clinically common eye trauma type, and has extremely high blindness rate. Corneal neolymphatics and neovascularization often form after alkali burns. These pathological vascular tissues are characterized by lack of tight junctions and high permeability, are prone to bleeding, aggravate inflammatory reactions, cause corneal clouding, and finally lead to vision loss, and can lead to blindness in severe cases. Furthermore, the formation of corneal neolymphatic and neovascular vessels breaks the "immune privileged" state of the cornea, which is also a major cause of clinical corneal graft rejection, and no effective treatment is currently available. Therefore, the research on the corneal neovascularization and corneal neovascularization is deeply carried out, and a new therapeutic drug which mainly acts on reversing the pathological vascular tissues and is safer and more effective is developed, so that the research is a key scientific problem which is dedicated to be solved by the medical community.

In the immune dysfunction caused by corneal alkali burn, the action of cell factor can not be ignored, after the corneal epithelium is stimulated, a large amount of Interleukin-1 (Interleukin-1, IL-1) can be produced, and IL-1 can also cause the migration of corneal scleral edge Langerhans cells to the center of cornea, and is involved in inflammatory cell infiltration, and a large amount of inflammatory cell factor is secreted, IL-1 β is a cell factor synthesized and released in the early stage of corneal alkali burn inflammatory reaction, not only has chemotactic effect on various inflammatory cells, but also can directly act on the cornea and cause tissue damage, and cause the generation of corneal ulcer, IL-1 β is an inflammatory factor which can recruit medullary differentiation factor (myelid differentiation factor 88, MyD88) by combining IL-1 receptor, mediates the activation of NF-kB, promotes angiogenesis factor VEGF-A, and promotes the secretion of angiogenesis factor IL-10, and the like, so that excessive angiogenesis is caused, and further causes corneal cicatrization, influences the transparency, in addition, IL-10 can reduce the accumulation of angiogenesis factor VEGF-A in a cornea-activating cell, and further reduce the corneal infiltration of corneal lymphocyte, the corneal ulcer, and the corneal ulcer after the corneal scar is treated by using rat interferon-1 receptor interferon, rat interferon- β, macrophage infiltration and mouse leukocyte infiltration of macrophage cell infiltration and macrophage cell infiltration.

The TIR/BB loop mimetic AS-1 (Hydrocinoyl-L-valylpyrrolidine, AS-1) is a fat-soluble small molecule compound which easily enters cells, and simulates a tripeptide sequence of MyD88-TIR-BB loop (F/Y) - (V/L/I) - (P/G), and can inhibit the combination of MyD88 and interleukin-1 type I receptor/auxiliary receptor (interleukin-1 receptor type I/interleukin-1 receptor access protein, IL-1RI/IL-1 RAcP). As shown by research, AS-1 has different degrees of inhibition effects on various inflammation-related diseases and fibrotic diseases.

Disclosure of Invention

The invention aims to provide application of a TIR/BB ring mimetic AS-1 in preparing a medicament for treating corneal neovascular diseases.

In order to achieve the above object, the present invention adopts the following technical means:

application of TIR/BB ring mimetic AS-1 in preparing medicines for treating corneal neovascular diseases.

Use of TIR/BB ring mimetic AS-1 for the manufacture of a medicament for the treatment of corneal alkali burn.

Application of TIR/BB ring mimetic AS-1 in preparing medicines for treating corneal alkali burn neovascularization.

Application of TIR/BB ring mimetic AS-1 in preparing medicines for treating corneal alkali burn neolymphangiogenesis.

The TIR/BB loop mimic AS-1 inhibits the activation of a transcription factor NF-B by blocking the combination of IL-1R and MyD88, reduces the secretion of inflammatory factors and the infiltration of inflammatory cells, reduces the level of VEGF-A, VEGF-C, and finally relieves the neovascularization and lymphangiogenesis of the alkali burn cornea, thereby achieving the purpose of treatment.

The invention introduces fat-soluble small molecular polypeptide into the treatment field of the corneal alkali burn for the first time, and researches the treatment effect of AS-1 on the corneal alkali burn, thereby not only providing a new targeted medicine for treating the corneal alkali burn and the corneal neogenesis lymphatic vessel, but also providing a new idea for treating the corneal alkali burn by the small molecular polypeptide.

Has the advantages that:

1. AS-1 has good application prospect in treating corneal neovascular diseases, and the possibility of being converted into an ophthalmic preparation is provided for increasing research strength.

2. The research on the treatment effect of AS-1 on the corneal alkali burn not only provides a new treatment medicine for treating the corneal neovascular and corneal neolymphatic vessels after the alkali burn, but also provides a new idea for relieving or even blocking clinical corneal transplantation rejection.

Drawings

FIG. 1 is a design route of an experiment according to an embodiment of the present invention.

Fig. 2 is a slit-lamp white-light photograph and a cobalt blue-light photograph of AS-1 reversing the neovascularization of alkali-burned cornea in example 1, with n being 6.

FIG. 3 shows the growth of corneal neovascularization and neovascularization in mice after alkali burn in example 1. Wherein: a is a slit lamp photo, B is a mouse cornea paraffin embedded tissue H & E staining photo, C, D, E is Westernblot and a quantitative graph thereof, and n is 6.

FIG. 4 is a graph showing the expression of the vascular markers IB4, the lymphatic vascularization markers LYVE1, VEGF-C in the corneal tissues of each group after an alkali burn in example 2. Wherein: a is expression of LYVE1/IB4, B is expression of VEGF-C/IB4, and n is 6.

FIG. 5 is a graph showing cytokine levels in rat corneal tissue after alkali burn in example 2. Wherein A, B, C, D, E, F is used for detecting the level of 35 cytokines in the corneal tissue by an MSD electrochemiluminescence method, G is used for detecting the level of mRNA of the cytokines in the corneal tissue by Real-time PCR, and n is 3.

FIG. 6 is a graph showing that AS-1 inhibits the level of VEGF-A, VEGF-C, LYVE1 by blocking the binding of IL-1R to MyD88 in example 3. Wherein: A. b is a graph for detecting the combination and the quantification of MyD88 and IL-1R in the corneal endothelial cells treated by NaOH through immunoprecipitation, C is the co-localization of MyD88 and IL-1R in alkali burn tissues through immunofluorescence detection, D is the expression of angiogenesis and lymphangiogenesis promoting related proteins in the corneal endothelial cells treated by NaOH through Western blot detection, and n is 3.

FIG. 7 is a graph of inhibition of NF-. kappa.B activation in NaOH-treated corneal endothelial cells by blocking nuclear translocation of p65 in example 3. Wherein: a is Western blot for detecting the level of p65 in cytoplasmic nucleus, B is immunofluorescence for detecting nuclear translocation of p65, C is EMSA for detecting the activation of NF-kB, and n is 3.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples.

As shown in figure 1, the invention respectively takes an alkali burn animal model and human corneal cells AS research objects, and further researches (1) the treatment effect of AS-1 on alkali burn cornea and (2) the molecular mechanism of AS-1 for regulating the neovascularization of alkali burn cornea and the corneal neovascularization from the tissue function to the cell level.

Example 1

1. Experimental animals: the experimental animal used in the invention is Sprague-Dawley rat (SPF grade), and is raised in the experimental animal center of Min Hospital in Sn-free city.

2. Establishment of alkali burn animal model

Healthy SPF grade 8 week male C57BL/6 mice or 200-220g male SD rats were examined by ophthalmic slit lamp microscopy and indirect ophthalmoscopy to exclude ocular diseases. After anesthesia, the oxybuprocaine eye drops are dropped into eyes for three times of surface anesthesia, after an eyelid opener opens the eyes, the redundant liquid of the conjunctival sac is removed, the round Whatman No. 3 filter paper is soaked in 1N Na0H solution for 30s and is placed in the center of the cornea for 40s, then the cornea and the conjunctival sac are washed by 40mL of physiological saline for 60s, and the conjunctival sac is coated with erythromycin eye ointment to prevent infection. The progression of corneal lesions in rats was recorded by slit-lamp microscopy and photography on days 3, 7 and 14 after the alkali burn.

3. AS-1 mode of administration

The corneal injury and the number of new blood vessels are observed by AS-1 subconjunctival injection and 3 times per day eye drop dripping of levofloxacin eye drops to C57BL/6 mice or SD rats modeled after 1NNaOH alkali burn through AS-1, and the corneal injury and the number of new blood vessels are observed by sodium fluorescein contrast or HE staining and the number of corneal new lymphatic vessels is observed by VEGF-C immunohistochemistry or immunofluorescence staining after alkali burn. ELISA was used to detect the levels of VEGF-A and VEGF-C. And (3) detecting AS-1 on the cornea neovascularization and cornea neovascularization caused by alkali burn.

AS shown in fig. 2 and 3, after AS-1 administration, the numbers of corneal neovascularization and corneal neovascularization lymphatics were significantly lower than those of the solvent control group; AS-1 reverses the area of corneal ulceration injury caused by alkali burn, AS well AS inflammatory cell infiltration in the stroma and fibroplasia and stromal disorganization.

Example 2

Culturing human corneal cells, pretreating AS-1, treating with 0.01N NaOH, detecting TUNEL apoptosis, flow cytometry, EdU staining and scratch experiment to determine regulation and control of AS-1 on cell proliferation, apoptosis and migration, detecting the level of lymphatic growth factor VEGF-C, and revealing the influence of AS-1 on neovascularization of alkali-burned cornea and corneal neolymphangiogenesis from cell level.

As shown in FIG. 4, the expression of the vascular marker IB4, the lymphatic vascularization marker LYVE1, VEGF-C in the corneal tissues of each group was significantly reduced in the AS-1 group after alkali burn.

As shown in FIG. 5, 35 kinds of cytokines were detected in the corneal tissue, which were significantly increased in the alkali burn group, particularly at 3d and 7d, AS-1 inhibited the levels of inflammatory factors in the cornea, particularly IL-1 β, IL-6, MCP1, IL-17A, MIP-1 α, etc., by blocking the binding of IL-1R to MyD88, and the increase of angiogenesis promoting factors such AS IL-10, VEGF, EPO, MIP-2, etc., caused by alkali burn was also significantly reversed in the AS-1 group, and further confirmed in the RT-PCR experiment.

Example 3

Discloses a molecular mechanism of AS-1 for regulating neovascularization and corneal neovascularization of alkali-burned cornea.

1) Adopting a signal channel to find a chip and screening channel specific genes, and further verifying the chip by Western-blot; further blocking or activating corresponding signal transduction pathways, detecting the influence of AS-1 on cell proliferation, and revealing a signal transduction system participating in regulation and control of neovascularization of alkali-burned cornea and cornea neovascularization of AS-1.

2) The change condition of AS-1 on the neovascularization of the alkali burn cornea and the corneal neolymphangiogenesis of the cornea is detected by respectively activating and blocking IL-1R signal paths through overexpression and siRNA, and a mechanism participating in reversing the neovascularization of the alkali burn cornea and the corneal neolymphangiogenesis of the AS-1 is disclosed.

As shown in FIGS. 6 and 7, in corneal endothelial cells, AS-1 inhibited NaOH-induced binding of IL-1R to MyD88, inhibited Erk (1/2) phosphorylation and NF- κ B transcriptional activity, and thereby inhibited elevated levels of VEGF-A, VEGF-C, and LYVE 1.

Claims (4)

1. The application of TIR/BB ring mimetic AS-1 in preparing a medicament for treating corneal neovascular diseases.
2. Use of TIR/BB ring mimetic AS-1 for the manufacture of a medicament for the treatment of corneal alkali burn.
3. The application of the TIR/BB ring analog AS-1 in preparing the medicines for treating the neovascularization caused by corneal alkali burn.
4. The application of the TIR/BB ring mimetic AS-1 in preparing the medicine for treating the neolymphangiogenesis caused by the corneal alkali burn.

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