CN117942398A - Application of SEMA3C-NRP1/NRP 2-GAS 6/AXL in preparation of medicine for treating onychomycosis - Google Patents

Abstract

The invention provides an application of SEMA3C-NRP1/NRP 2-GAS 6-AXL in preparation of a medicine for treating a onychomycosis. The invention discloses a novel SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal path closely related to orbital adipose tissue fibrosis or onychomycosis, and discloses a novel mechanism which is participated by the signal path and used for regulating and controlling the orbital adipose tissue fibrosis or the onychomycosis, and based on the novel mechanism, a therapeutic drug targeting the signal path is provided; the signal path is used as a drug screening platform and can be also applied to screening novel therapeutic drugs. Fibrosis is an important pathological feature of thyroiditis and is not an FDA approved drug. The novel therapeutic agent of the present invention can alleviate/treat thyroiditis by inhibiting fibrosis of cells derived from patients suffering from thyroiditis. The invention also provides a novel cell model and an animal model of the orbital adipose tissue fibrosis or the thyroidism.

Description

Application of SEMA3C-NRP1/NRP 2-GAS 6/AXL in preparation of medicine for treating onychomycosis

Technical Field

The invention belongs to the field of biological medicine, and in particular relates to SEMA3C- (NRP 1/NRP 2) to GAS6-AXL and application thereof in preparation of a medicine for treating the onychomycosis.

Background

The nystagmus disease (Thyroid EYE DISEASE, TED), a complex autoimmune disease, is accompanied by vision threat and orbital appearance effects, causing considerable physical and mental burden to the patient and its family members. Active TED patients typically exhibit inflammation, and when they progress to an inactive phase characterized by fibrosis, they experience abnormal eye movement and even vision loss, which has a great impact on their quality of life. Current treatments, including steroid treatment, radiation treatment for active TED, and orbital decompression surgery for inactive TED, do not prevent fibrosis progression and may leave behind some health-related systemic adverse effects. Thus, fibrosis remains a major challenge for TED inactive patients and clinicians.

Prior studies of the pathogenesis of TED fibrosis indicate that orbital fibroblasts (Orbital Fibroblasts, OFs) are stimulated by secretory factors, such as transforming growth factor- β (tgfβ), or direct cell-cell contact or infiltration with immune cells, resulting in excessive extracellular matrix (Extracellular Matrix, ECM), which in turn produces large amounts of collagen fibers. As an autoimmune disease, the immune response has been shown to be involved in the overall process OF fibrosis upon interaction with OF. OFs in TED is reported to cause T cell recruitment as a critical step in the onset of inflammation. T cells are thought to cause the progression of inflammation by secreting costimulatory molecules, adhesion molecules, and cytokines (including ifnγ, IL-1β, and tnfα), among others. Macrophages, a type of pro-fibrotic cell, have been found to be activated in subcutaneous and visceral adipose tissue banks, ultimately leading to fibrosis of adipose tissue. In addition, some studies have also found that macrophages and mast cells can also stimulate fibroblasts to produce excessive ECM in TED by secreting tgfβ or Platelet Derived Growth Factor (PDGF). All of these findings above suggest that complex immunomodulatory networks may exist in TED. However, the specific immune mechanisms that trigger fibrosis and activate fibroblasts are not known.

There is essentially no mature reliable treatment for the ocular of the first-stage presbyopia, one of which was approved by the FDA in 2020, but there is no evidence that it could alleviate fibrosis. Thus, although fibrosis is an important pathological feature of the onychomycosis, there is no FDA approved effective drug yet.

Whether the AXL pathway is specifically inhibited or not can be prevented or treated by inhibiting fibrosis is explored, and the method has important significance for deeply understanding the mechanism of the onychomycosis in the inactive phase, preventing or treating the onychomycosis and improving the life quality of patients.

Disclosure of Invention

The invention aims to provide a SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL pathway and application thereof in preparation of a medicine for treating the onychomycosis.

In a first aspect of the invention there is provided the use of a modulator (e.g. a down regulator or inhibitor) of the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signalling pathway for the preparation of a pharmaceutical composition for inhibiting (including preventing, alleviating or treating) orbital adipose tissue fibrosis or inhibiting (including preventing, alleviating or treating) a neurite eye disease;

Wherein the modulator is selected from the group consisting of: a modulator that inhibits or down-regulates expression, activity or secretion of SEMA3C in dpp4+ cells, a modulator that inhibits or down-regulates expression or activity of NRP1/NRP2 in M2 macrophages, a modulator that inhibits or down-regulates binding of SEMA3C to NRP1/NRP2, a modulator that inhibits or down-regulates interaction of dpp4+ cells with M2 macrophages, a modulator that inhibits or down-regulates the action of GAS6-AXL on dpp4+ cells; the DPP4+ cells are an orbital adipose tissue cell population with fibrotic differentiation potential.

In one or more embodiments, the dpp4+ cells are DAPI-/CD31-/CD45-/pdgfra+/dpp4+ cells isolated from SVF cells.

In one or more embodiments, the DPP4+ cells are present in adipose tissue within the orbit.

In one or more embodiments, the dpp4+ cells are present in adipose tissue-derived Stromal Vascular Fraction (SVF) cells within the orbit.

In one or more embodiments, the inhibiting the nystagmus disease.

In one or more embodiments, the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway, pathway genes and pathway proteins are derived from human or non-human animals (e.g., monkey, mouse, rat, rabbit, sheep, pig, etc.); preferably of human origin (e.g.recombinant expression of a gene/protein of human origin).

In one or more embodiments, the dpp4+ cells or M2 macrophages are derived from a human or non-human animal (e.g., monkey, mouse, rat, rabbit, sheep, pig, etc.); preferably of human origin (e.g.recombinant expression of a gene/protein of human origin).

In one or more embodiments, the modulator that inhibits or down-regulates SEMA3C expression, activity or secretion in DPP4+ cells comprises: agents that knock out or silence the SEMA3C gene, agents that inhibit SEMA3C protein activity; preferably, it includes: a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent aiming at a SEMA3C gene, wherein the reagent carries out the function-losing mutation on the SEMA3C, an interfering molecule which specifically interferes with the SEMA3C gene expression, and a small molecule inhibitor of SEMA3C protein; or (b)

The modulator that inhibits or down-regulates NRP1/NRP2 expression or activity in M2 macrophages comprises: agents that knock out or silence the NRP1/NRP2 gene, agents that inhibit NRP1/NRP2 protein activity; preferably, it includes: a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent aiming at NRP1/NRP2 genes, wherein the reagent carries out functional loss mutation on NRP1/NRP2, an interfering molecule which specifically interferes with NRP1/NRP2 gene expression, and a small molecule inhibitor of NRP1/NRP2 protein; or (b)

The modulator that inhibits or down-regulates SEMA3C binding to NRP1/NRP2 comprises: an agent that competitively binds to NRP1/NRP2, an agent that competitively binds to SEMA3C, thereby preventing or reducing SEMA3C binding to NRP1/NRP2; modulators (e.g., down-regulators or inhibitors) that down-regulate SEMA3C expression, activity or secretion; modulators that down-regulate NRP1/NRP2 expression or activity; or (b)

The modulators that inhibit or down-regulate the action of GAS6-AXL on dpp4+ cells include: modulators that down-regulate GAS6 or AXL expression and activity; preferably, it includes: agents that knock out or silence the GAS6 or AXL gene, agents that inhibit GAS6 or AXL protein activity; preferably, it includes: a GAS6 or AXL gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent for GAS6 or AXL genes, wherein the reagent performs a loss-of-function mutation on GAS6 or AXL, an interfering molecule which specifically interferes with the expression of GAS6 or AXL genes, a small molecule inhibitor of GAS6 or AXL proteins;

The modulators that inhibit or down-regulate the interaction of dpp4+ cells with M2 macrophages include: modulators that inhibit SEMA3C binding to NRP1/NRP2, modulators that down-regulate SEMA3C expression, activity or secretion; modulators that down-regulate NRP1/NRP2 expression or activity.

In one or more embodiments, the modulator that down-regulates NRP1/NRP2 expression or activity is a CRISPR gene editing agent that knocks out NRP1/NRP2; preferably its sgRNA sequence is: the forward sequence is shown as SEQ ID NO. 1, and the reverse sequence is shown as SEQ ID NO. 2.

In one or more embodiments, the small molecule inhibitor of GAS6 or AXL protein is a compound of the parent nucleus structure shown in formula (I) or an isomer, derivative, solvate or precursor thereof, or a pharmaceutically acceptable salt, composition or pharmaceutical composition thereof (TP 0903);

in one or more embodiments, dpp4+ cells in the intra-orbital adipose tissue express SEMA3C, SEMA3C binds to NRP1/NRP2, recruits M2 macrophages expressing NRP1/NRP2, and GAS6 in M2 macrophages acts on dpp4+ cells AXL receptor to cause fibrosis and differentiation, forming orbital adipose tissue fibrosis or a thyroidism; preferably, the modulator intervenes by means of a refined spatial modulation, or the fibrogenesis and/or the lesions occur in the active phase of the nystagmus.

In one or more embodiments, the first and second ocular conditions include: eyelid swelling, eyelid retraction (eyelid retraction), late upper eyelid fall (lagophthalmos), eyelid insufficiency, eye herniation (exophthalmos), the appearance of exposed keratitis, compression optic neuropathy, secondary glaucoma, reduced or lost vision function, or blindness.

In one or more embodiments, the compositions, pharmaceutical compositions further comprise a second therapeutic agent. In one or more embodiments, the second therapeutic agent comprises an analgesic, a non-steroidal anti-inflammatory drug, a glucocorticoid, a steroid; more preferably, the steroid comprises methylprednisolone (methylprednisolone), prednisolone (prednisolone), dexamethasone (dexamethasone).

In one or more embodiments, one or more of the following treatments are included before, simultaneously with, or after the prophylaxis or treatment: topical treatment, oral hormone, steroid treatment, surgical treatment, and radiation treatment.

In one or more embodiments, the topical treatment includes attention to eye rest, sunglasses to avoid glare and various external irritation, application of antibacterial eye cream before sleep, eye-wear, relief of double vision with single-sided eye-mask, high-sleeper position, control of salt intake, use of eye drops (ointments); the surgical treatment comprises an orbit decompression operation, an orbit bone wall decompression operation, an orbit fat decompression operation, an upper eyelid muscle extension operation and an strabismus correction operation.

In one or more embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

In a second aspect of the invention, there is provided the use of the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway for screening substances (drugs) that inhibit orbital adipose tissue fibrosis or nystagmus; preferably, in the SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal pathway, SEMA3C is expressed by DPP4+ cells in adipose tissue in the orbit, NRP1/NRP2 is expressed by M2 macrophagin, SEMA3C is combined with NRP1/NRP2 so that DPP4+ cells interact with M2 macrophages, and GAS6 in the M2 macrophages acts on the AXL receptor of the DPP4+ cells to enable the DPP4+ cells to be fibrotically differentiated, so that orbital adipose tissue fibrosis or nystagmus is formed.

In a third aspect of the present invention, there is provided a method of screening for a substance that inhibits orbital adipose tissue fibrosis or thyroiditis, the method comprising:

(1) Contacting the candidate substance with a system comprising SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathways; in the system, SEMA3C, AXL is expressed by DPP4+ cells, and NRP1/NRP2 and GAS6 are expressed by M2 macrophages; the DPP4+ cells are orbital adipose tissue cell groups with fibrosis differentiation potential;

(2) Screening for agents (including potential agents) that down-regulate (including inhibit) SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathways useful for inhibiting orbital adipose tissue fibrosis or nystagmus; preferably, the down-regulating includes: down-regulating SEMA3C expression, activity or secretion in dpp4+ cells, down-regulating NRP1/NRP2 expression or activity in M2 macrophages, inhibiting SEMA3C binding to NRP1/NRP2, down-regulating dpp4+ cells to interact with M2 macrophages, down-regulating GAS6-AXL to act on dpp4+ cells.

In one or more embodiments, step (1) comprises: in the test set, contacting the candidate substance with a system comprising SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathways; the step (2) comprises: detecting changes in each protein or gene encoding the same in the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway in the test group and comparing the changes with a control group, wherein the control group is a system containing the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway without adding the candidate substance; if a candidate substance in the test set down-regulates the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway, the candidate substance is a substance useful for inhibiting orbital adipose tissue fibrosis or a nystagmus.

In one or more embodiments, the system comprising SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway is selected from the group consisting of: a cell (culture) system, a subcellular (culture) system, a tissue (culture) system, or an animal system.

In one or more embodiments, the cell (culture) system comprises: dpp4+ cells (culture) and M2 macrophages (culture) systems.

In one or more embodiments, the tissue (culture) system comprises: orbital tissue (culture) system, intra-orbital adipose tissue (culture) system.

In one or more embodiments, the candidate substance includes (but is not limited to): regulatory molecules (such as, but not limited to, interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands)), CRISPR constructs, small molecule compounds, compounds from a library of compounds, etc., designed for SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway, or its pathway proteins, or its upstream or downstream proteins, genes, or signaling pathway.

In a fourth aspect of the present invention, there is provided a disease model system for orbital adipose tissue fibrosis or thyroidism, characterized in that the disease model system is a cell model system comprising dpp4+ cells and M2 macrophages, which are mixed and interacted;

Preferably, in the cell model system, the SEMA3C- (NRP 1/NRP 2) -GAS6/AXL signal pathway is used for mediating the occurrence of diseases, SEMA3C, AXL is expressed by DPP4+ cells in adipose tissues in the eye sockets, NRP1/NRP2 and GAS6 are expressed by M2 giant and fine, the SEMA3C is combined with the NRP1/NRP2 to enable the DPP4+ cells to interact with M2 macrophages, and GAS6 in the M2 macrophages acts on the DPP4+ cells AXL receptor to enable the DPP4+ cells to be fibrotically differentiated, so that the adipose tissue fibrosis or the thyropathy is formed in the eye sockets.

In one or more embodiments, when the DPP4+ cells and the M2 macrophages are mixed, the DPP4+ cells and the M2 macrophages are 1:100-100:1 in terms of cell number; such as 1:50 to 50:1; 1:30-30:1; 1:20 to 20:1; 1:10-10:1; 1:5 to 5:1, etc.

In a fifth aspect of the present invention, there is provided a method of preparing a disease model system for orbital adipose tissue fibrosis or nystagmus, characterized in that the disease model system is a cell model system, the method comprising: mixing DPP4+ cells and M2 macrophages so that the DPP4+ cells and the M2 macrophages interact with each other to form a cell model system; or (b)

The disease model system is an animal model system, the method comprising: the DPP4+ cells and M2 macrophage mixed cells are transplanted into the orbital adipose tissue of a non-human animal, and the two cells interact to form an animal model system of orbital adipose tissue fibrosis or onychomycosis.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, DPP4+ cells have strong interactions with macrophages and co-localize;

The upper panel, a partial representative result of a cellular communication analysis;

middle panel, HE & Masson staining of orbital adipose tissue in the nystagmus;

the following graph shows the immunofluorescence results of DPP4+ cells, macrophages (CD 68) and co-localization of both, wherein red fluorescence is CD68, green fluorescence is DPP4, blue fluorescence is DAPI, and Merge represents co-localization.

FIG. 2, M2 macrophages are the predominant macrophage subtype and can progress DPP4+ cells toward fibrosis;

Upper panel, macrophages are divided into M1 and M2 subtypes and their duty cycle;

In the middle panel, the change in biomarkers when dpp4+ cells and M2 macrophages were cultured (Col 1a1, col3a1, eln, fn1, acta2, timp1, ppar γ, cebp α, cd 36);

The lower panel shows the change in fibrosis biomarkers when SVF cells and M2 macrophages are cultured (Col 1a1, col3a1, eln, fn1, acta2, timp1, ppar γ, cebp α, cd 36); in the figure, CM is a conditioned medium, and M2 CM is a culture supernatant of M2 type macrophages.

FIG. 3, partial results of single cell signaling analysis of the signal pathway in which DPP4+ cells are involved;

the upper panel, single cell signaling analysis of M2 macrophages and DPP4+ cells in part of the statistics of the signal pathway;

the following figure shows the interaction relationship between single cell signaling analysis M2 macrophage and DPP4+ cell.

FIG. 4, cell (patient fresh tissue derived cells) experiments suggest that M2 macrophages and DPP4+ cells progress to fibrosis through the GAS6-AXL mode; AXL-specific inhibitors can very significantly inhibit fibrosis;

A, GAS6 expression in macrophages of different subtypes (left); GAS6 expression in different subtype macrophage secretions (medium) (right);

b, schematic representation of GAS 6-mediated conversion of M2 macrophages into dpp4+ cells to promote fibrosis;

C, DPP4+ cells and DPP4+ cells treated with GAS6 (50 ng/mL) were treated with TP0903 (125 nM), respectively, changes in fibrosis biomarkers;

d, DPP4+ cells and THP-1 derived M2 macrophages, treated with TP0903 (125 nM), change in fibrosis biomarker, respectively.

Detailed Description

The inventor has conducted intensive studies to reveal for the first time a novel signal pathway closely related to orbital adipose tissue fibrosis or thyroiditis: SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signaling pathway. The invention discloses a novel mechanism which is participated in by the signal path and used for regulating and controlling orbital adipose tissue fibrosis or onychomycosis, and a therapeutic drug targeting the signal path is proposed based on the novel mechanism; the signal path is used as a drug screening platform and can be also applied to screening novel therapeutic drugs. The invention also provides a novel cell model and an animal model of the orbital adipose tissue fibrosis or the thyroiditis.

Terminology

In the present invention, the term "treatment" in relation to a given disease or condition means: including, but not limited to, inhibiting a disease or disorder, e.g., arresting the development of a disease or disorder; alleviating a disease or condition, e.g., causing regression of a disease or condition; or to alleviate a condition caused by or resulting from a disease or disorder, e.g., to alleviate, prevent or treat symptoms of a disease or disorder.

In the present invention, "orbital adipose tissue fibrosis" is an indication that indicates an abnormal state of orbital adipose tissue, in which visible fibrotic lesions occur. Fibrosis is a characteristic disorder of thyroiditis, commonly found in the stationary phase of thyroiditis, and is currently clinically lacking in effective drugs.

As used herein, "thyroiditis" or "TED" is an autoimmune disease characterized by eyelid edema, herniation, palpebral recession, and enlargement of the eyelid fissure, also known as "thyroid-related eye disease". The course of thyroid-related eye disease generally falls into two phases, namely active and inactive (stationary). Since the initial stage of onset is characterized by alternating deterioration and remission, it is called active phase. Active patients typically exhibit an inflammatory state. The release of pro-inflammatory cytokines is associated with inflammation of the ocular surface and lacrimal glands, and periorbital skin erythema and edema. The resultant tissue remodeling, fibrosis and fat deposition can impart permanent physical changes to the ocular accessories. For the judgment of the active period, a method known in the art may be used, including but not limited to a CAS scoring method, a magnetic resonance method, a tear inflammation judgment method. After the active periods with different lengths, the inactive period occurs when the eye performance is stable. When the patient progresses to the inactive period, the patient may experience abnormal eye movement and even vision loss, which has a great influence on the quality of life.

In the present invention, the term "prevention" in relation to a given disease or condition means: if no disease occurs, preventing the onset of the disease; preventing a disease or disorder from occurring in a subject who may be susceptible to the disease or disorder but has not yet been diagnosed as having the disorder or disorder, and/or preventing further disease/disorder progression if already present.

In the present invention, the terms "comprising," having, "" including, "or" comprising "include" consisting essentially of … …, "" consisting essentially of … …, "and" consisting of … …; "consisting essentially of … …", "consisting essentially of … …" and "consisting of … …" are the underlying concepts of "containing", "having", "containing" or "including".

As used herein, the term "combination" refers to administration sequentially or simultaneously by the same route or by different routes of administration. When administered sequentially, the time between administrations is selected to benefit, inter alia, from the therapeutic efficacy and/or safety of the combination therapy. May be administered for the same duration or, alternatively, for a longer or shorter duration than the latter. In some embodiments, the administration of a compound of the invention, or a salt, composition, or pharmaceutical composition thereof, is a first treatment method, and the second treatment method is administered before, simultaneously with, or after the first treatment method. The second treatment method and the first treatment method may be the same or different. In some embodiments, the compounds of the invention are prepared as a first pharmaceutical composition and the second therapeutic agent is prepared as a second pharmaceutical composition, wherein the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously, sequentially or separately.

As used herein, the term "pharmaceutically acceptable" ingredient is a substance suitable for use in humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response), commensurate with a reasonable benefit/risk ratio.

As used herein, the term "pharmaceutically acceptable carrier" is a pharmaceutically or food acceptable solvent, suspending agent or excipient for delivering the compounds of the present invention to an animal or human. The carrier may be a liquid or a solid.

As used herein, the term "animal model (system)" may also be referred to as "model animal (system)", including non-human mammals, preferably including (but not limited to): rodents (including mice, rats, hamsters, etc.), non-human primates (e.g., monkeys, chimpanzees, etc.), domestic animals (e.g., cows, sheep, dogs, pigs, rabbits, etc.).

SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL and application thereof

As used herein, the term "signaling pathway" refers to a signaling system formed by the interaction or interaction between a series of genes or proteins or their metabolites (synthetic or processed products), as well as the interaction of pathway proteins with other elements or organelles within a cell, and sometimes also the co-participation of genes or proteins upstream and downstream thereof, which generally results in the occurrence of some cellular event. The SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal path mainly comprises the following elements: SEMA3C gene (and/or protein encoded thereby), NRP1 and/or NRP2 (and/or protein encoded thereby), GAS6/AXL gene (and/or protein encoded thereby).

As used herein, the "(signal) pathway" is used interchangeably with "(signal) pathway".

The amino acid sequence of the SEMA3C protein is shown, for example, in GenBank accession No. np_001337049.1 (human).

The amino acid sequence of the NRP1 protein is shown, for example, in GenBank accession NP-001019799.2 (human).

The amino acid sequence of the NRP2 protein is shown, for example, in GenBank accession NP-003863.2 (human).

The amino acid sequence of the GAS6 protein is shown, for example, in GenBank accession No. np_000811.1 (human).

The amino acid sequence of the AXL protein is shown, for example, in GenBank accession No. np_001265528.1 (human).

In the present invention, unless otherwise indicated, information about other pathway proteins/genes associated with the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway or proteins/genes upstream and downstream thereof is also known to those skilled in the art.

The above proteins (polypeptides) also include variants thereof, including (but not limited to): deletion, insertion and/or substitution of several (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10, still more preferably 1 to 8, 1 to 5) amino acids, and addition or deletion of one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and/or the N-terminus. Any protein having high homology to the protein (such as 70% or more homology to the polypeptide sequence; preferably 80% or more homology; more preferably 90% or more homology, such as 95%,98% or 99%) and having the same function as the protein is also included in the present invention. The invention also includes a mutant form of the protein or protein truncations, as long as the mutant protein or truncations substantially retain the function of the full-length protein.

The sequences of the above genes also include sequences degenerate thereto. The polynucleotides (genes) encoding the proteins may be natural genes or degenerate sequences thereof.

As used herein, the terms "inhibit (agent)" and "down-regulate (agent)" are used interchangeably and also include: a blocking agent, an antagonistic agent, etc., a weakening agent, etc.

The inventor finds that in the signal path, SEMA3C expression, activity or secretion in DPP4+ cells is down-regulated, NRP1/NRP2 expression or activity in M2 macrophages is down-regulated, SEMA3C and NRP1/NRP2 binding is inhibited, DPP4+ cells and M2 macrophages are down-regulated to interact, GAS6 is down-regulated to act on the DPP4+ cells, and orbital adipose tissue fibrosis or thyroidism can be inhibited. Thus, drugs suitable for targeted modulation can be screened or designed by this mode of action.

It will be appreciated that after the function of the SEMA3C- (NRP 1/NRP 2), SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway (and preferably also the upstream and downstream proteins or genes thereof) is known, the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway can be modulated using a variety of methods well known to those skilled in the art. For example, expression of the pathway protein may be modulated or deleted using a variety of methods well known to those skilled in the art. Or by methods well known to those skilled in the art to attenuate the occurrence of interactions between proteins or between cells.

As a preferred mode of the invention, there is provided a modulator that down-regulates expression, activity or secretion of SEMA3C in DPP4+ cells, a modulator that down-regulates expression or activity of NRP1/NRP2 in M2 macrophages, a modulator that inhibits binding of SEMA3C to NRP1/NRP2, a modulator that down-regulates interaction of DPP4+ cells with M2 macrophages, and a modulator that down-regulates action of GAS6-AXL on DPP4+ cells. The modulator means any substance which can reduce the activity of SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway protein, reduce the stability of SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway protein, down regulate the expression of SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway protein, reduce the effective duration of SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway protein, inhibit the transcription and translation of SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway gene, and can be used in the present invention as a substance useful for inhibiting orbital adipose tissue fibrosis or a presbyopia. They may be chemical compounds, chemical small molecules, biological molecules. The biomolecules may be nucleic acid-level (including DNA, RNA) or protein-level. For example, the modulators are: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of GGPS1 or RAB7A or genes upstream thereof; or specific editing GGPS1 or RAB7A or upstream gene editing reagent thereof, etc.

The invention also provides a method for down-regulating SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signal pathway, which comprises the steps of carrying out targeted mutation, gene editing or gene recombination on SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway genes, so as to realize down-regulation. As a more specific example, by any of the methods described above, the SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL pathway protein may be converted into a loss-of-function truncate or mutant. As a more specific embodiment, gene editing is performed using the CRISPR/Cas system, thereby knocking out or down-regulating the target gene. Suitable sgRNA target sites will lead to higher gene editing efficiency, so suitable target sites can be designed and found before proceeding with gene editing. After designing specific target sites, in vitro cell activity screening is also required to obtain effective target sites for subsequent experiments.

As another embodiment of the present invention, there is provided a method for down-regulating expression of SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway gene, comprising: the interfering molecules that interfere with the expression of the SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL pathway gene are transferred into cells, or the cells are treated by a suitable route to be introduced into cells, for example, by designing a transmembrane domain to have the ability to penetrate a membrane.

When used as a target for artificial regulation or when a screening system is artificially established, the above protein or coding gene may be naturally occurring, for example, it may be purified and isolated from a mammal; it may also be recombinantly produced, e.g., recombinant proteins may be produced according to conventional genetic recombination techniques. In addition, any variant that does not affect the biological activity of these proteins, such as derivatives or variants whose function is not altered, may be used.

In a specific embodiment of the present invention, there is provided a small molecule inhibitor for inhibiting orbital adipose tissue fibrosis or inhibiting onychomycosis by inhibiting SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway protein, which is a compound having a parent nucleus structure represented by structural formula (I):

the compound with the parent nucleus structure shown in the structural formula (I) is called as a compound (I) in short, and is called as TP0903 or 2- ((5-chloro-2- ((4- ((4-methylpiperazine-1-yl) methyl) phenyl) amino) pyrimidine-4-yl) amino) -N, N-dimethylbenzenesulfonamide in short, wherein the CAS number is 1341200-45-0.

The present invention also includes isomers, solvates, precursors, or pharmaceutically acceptable salts thereof of the above-described compound having the parent structure of formula (I), as long as they also have the same or substantially the same function as the compound having the parent structure of formula (I). The term "pharmaceutically acceptable salts" refers to salts formed by the reaction of a compound with an inorganic acid, an organic acid, an alkali metal or alkaline earth metal, and the like. These salts include (but are not limited to): (1) a salt with a mineral acid: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) Salts with organic acids such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts with alkali or alkaline earth metals (such as sodium, potassium, calcium or magnesium) in the form of esters, carbamates, or other conventional "prodrugs". The compounds have one or more asymmetric centers. These compounds can therefore exist as racemic mixtures, individual enantiomers, individual diastereomers, diastereomeric mixtures, cis-or trans-isomers.

The term "precursor of a compound" refers to a compound which, when administered by a suitable method, undergoes a metabolic or chemical reaction in a patient to convert to a parent structure of formula (I), or a salt or solution of a parent structure of formula (I).

It will be appreciated by those skilled in the art that, after knowing the structure of the compounds of the present invention, the compounds of the present invention may be obtained by a variety of methods well known in the art, using well known starting materials, such as chemical synthesis or extraction from organisms (e.g., animals or plants) or modification upon extraction, all of which are encompassed by the present invention.

The compounds of the present invention can be synthesized by known methods; the synthesized compound can be further purified by column chromatography, high performance liquid chromatography, etc. In addition, the compounds of the present invention may also be obtained by commercial means.

The invention also provides a composition (e.g., a pharmaceutical composition) comprising an effective amount of a compound of the parent nucleus structure of formula (I), or an isomer, solvate, or precursor thereof.

As a preferred mode, the composition is a pharmaceutical composition, and the composition further comprises pharmaceutically acceptable salts and/or pharmaceutically acceptable carriers or excipients.

In the invention, the pharmaceutical composition contains 0.001-50% of a compound shown in a parent nucleus structure shown in a formula (I) or pharmaceutically acceptable salt thereof according to a weight ratio. Preferably, the pharmaceutical composition contains 0.05-30% of a compound shown in a mother nucleus structure shown in a formula (I) or pharmaceutically acceptable salt thereof according to a weight ratio; more preferably, the pharmaceutical composition contains 0.01-20% of a compound shown in a mother nucleus structure shown in a formula (I) or pharmaceutically acceptable salt thereof according to a weight ratio. It will be appreciated by those skilled in the art that other weight ratios are possible depending on the actual clinical needs or the drug design in the pharmaceutical industry.

The dosage form of the pharmaceutical composition of the present invention may be various, as long as it is a dosage form capable of allowing the active ingredient to reach the mammalian body effectively. For example, it may be selected from: a gel, aerosol, tablet, capsule, powder, granule, syrup, solution, or suspension. The type of disease to be treated with the compounds of the invention can be selected by those skilled in the art as a convenient dosage form for use. Preferred pharmaceutical compositions are solid compositions, especially tablets and solid filled or liquid filled capsules, from the standpoint of ease of preparation and administration. The compounds of the present invention or pharmaceutical compositions thereof may also be stored in a disinfecting device suitable for injection or instillation. The effective dosage of the core structure compound of formula (I) as an active ingredient may vary depending on the mode of administration and the severity of the disease to be treated.

The present inventors have found that the parent nucleus structural compound represented by formula (I) can prevent or treat orbital adipose tissue fibrosis or a paronychia disease, which acts by inhibiting the occurrence and development of fibrosis in orbital adipose tissue fibrosis or a paronychia disease, and/or improving fibrotic lesions.

As an application mode of the present invention, there is provided use of SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway modulator (down regulator/inhibitor such as TP 0903) for preparing a medicament (composition) for preventing or treating orbital adipose tissue fibrosis or a thyroiditis, and/or inhibiting fibrosis occurrence and development in orbital adipose tissue fibrosis or a thyroiditis, and/or improving fibrotic lesions.

It can also be said that SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL pathway modulators (e.g., down-regulators or inhibitors) of the present invention, such as TP0903 or an analog thereof, can reduce (i.e., down-regulate) the expression of one or more markers associated with orbital adipose tissue fibrosis or thyroiditis. In some embodiments, the marker is one that is expressed at a high level or a low level in a fibrotic disease (e.g., a fibrotic disease in a thyroiditis). Examples of related markers include Col1a1, col3a1, eln, fn1, acta2, timp1, and the like.

As one mode of the present invention, the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway modulator (e.g., a down regulator or inhibitor) is administered during the active phase of the thyroiditis, thereby inhibiting the fibrogenesis and/or the lesion. In a preferred mode of the invention, the fibrosis is adipose tissue fibrosis.

Macrophages in adipose tissue, as described herein, are referred to as tissue-resident macrophages, which are small circular cells dispersed in adipose tissue, and are also the most prominent inflammatory cells in adipose tissue, and are involved in maintaining the homeostasis of adipose tissue. Macrophages exhibit greater plasticity in different microenvironments, and are generally classified as classical activated (also known as M1) and alternative activated (also known as M2) types. Orbital adipose tissue fibrosis progression, as described herein, is dominated by M2 type macrophages in tissue.

Dipeptidyl peptidase 4 (DPP 4/CD 26), a serine protease according to the present invention, genBank accession number NP-001366533.1, cleaves the penultimate amino acid of a variety of proteins and is also a characteristic cell surface marker of adipose stem cells. As used herein, "dpp4+ cells" are a group of adipose tissue progenitor cells having fibrotic differentiation potential, wherein DPP4 expression is positive.

Symptoms of a onychomycosis, as described herein, include, but are not limited to: eyelid swelling, eyelid retraction (eyelid retraction), late upper eyelid fall (lagophthalmos), eyelid insufficiency, herniation (exophthalmos), appearance of exposed keratitis, compression optic neuropathy, secondary glaucoma, hypopsia or loss of vision, blindness.

As an application mode of the invention, the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL channel modulator (such as a lower regulator or inhibitor) can be used for preventing or treating the onychomycosis.

In some embodiments, the treatment comprises use in combination with other methods of treatment. That is, one or more additional therapeutic methods are administered to a subject prior to, concurrently with, or subsequent to the prophylaxis or treatment described herein. Other methods of treatment include, but are not limited to: topical treatment (e.g., eye rest, sunglasses to avoid glare and various external stimuli, antibacterial eye cream before sleep, eye mask wearing, relief of double vision with unilateral eye mask, high occipital position, control of salt intake, use of eye drops (cream), oral hormone, steroid treatment, surgical treatment (e.g., orbital decompression, orbital bone wall decompression, orbital fat decompression, extension of upper eyelid muscle, strabismus correction), radiation treatment. In some embodiments, the subject is, has been, or is about to undergo treatment or prevention by one or more methods of treatment.

In some embodiments, SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway modulators (e.g., down-regulation or inhibitors) described herein can be used in combination with a second therapeutic agent. In some embodiments, the second therapeutic agent includes, but is not limited to: analgesics, non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, steroids (such as, but not limited to, methylprednisolone (methylprednisolone), prednisolone (prednisolone), dexamethasone (dexamethasone)).

According to the present invention, as one mode of application of the present invention, there is provided a kit/kit for preventing or treating a first sudden eye disease, comprising: SEMA3C- (NRP 1/NRP 2) to GAS6-AXL pathway modulator (e.g., down regulator or inhibitor) or pharmaceutical compositions containing the same.

Drug screening

Based on the new findings of the present inventors, there are various uses for the study of the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway, including: screening for substances that modulate the signal pathway for inhibiting orbital adipose tissue fibrosis or nystagmus. Wherein said adjusting comprises: downregulating SEMA3C expression, activity or secretion agent in DPP4+ cells, downregulating NRP1/NRP2 expression or activity in M2 macrophages, inhibiting SEMA3C binding with NRP1/NRP2, downregulating interaction between DPP4+ cells and M2 macrophages, downregulating GAS6-AXL acting on DPP4+ cells, and the like.

The invention provides a method for screening modulators that down-regulate the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway by adding a candidate to be screened to a system containing the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway and observing the change or interaction of each protein or gene in the SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathway. If the candidate substance causes some downregulation as described in the preceding paragraph, the candidate substance is a substance useful for inhibiting orbital adipose tissue fibrosis or a thyroiditis.

As used herein, the terms "down-regulate," "inhibit," "prevent," "reduce," or "attenuate" are used interchangeably in some instances to mean "down-regulate," "inhibit," "prevent," "reduce," or "attenuate" that are statistically significant. For example, the level of "down-regulation", "inhibition", "prevention", "reduction" or "attenuation" is 5% or more, 10% or more, 20% or more, 30% or more, 50% or more, 80% or more, 100% or more, 200% or more, 500% or more, etc., as compared to a control or substrate.

The system containing SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal pathway is selected from the following: a cell system (or cell culture system), a subcellular system (or subcellular culture system), a solution system, an animal system or tissue system (or tissue culture system), and the like.

As a preferred mode of the present invention, the method further comprises: further cellular and/or animal experiments are performed on the potential material obtained to further select and determine, from among candidate materials, materials useful for inhibiting orbital adipose tissue fibrosis or nystagmus.

When screening is performed, various techniques well known in the art can be used to determine the change in the protein or its encoding gene and the interaction.

A variety of conventional techniques can be used to identify transcription or expression of genes in a system. These techniques include, but are not limited to: oligonucleotide hybridization techniques (e.g., probes), polymerase Chain Reaction (PCR), polyacrylamide gel electrophoresis, and the like. The detection of the protein-protein interaction and the strength of the interaction may be carried out by a variety of techniques known to those skilled in the art, such as co-immunoprecipitation techniques, GST sedimentation techniques, phage display techniques, or yeast two hybrid systems. The nuclear localization of proteins is also a well known technique in the art.

The substances preliminarily screened by the method can form a screening library so that people can screen substances which can be really useful for inhibiting the orbital adipose tissue fibrosis or the thyroidism from the screening library.

The invention also provides a potential substance which can be used for inhibiting orbital adipose tissue fibrosis or the onychomycosis and is obtained by adopting the screening method.

The present invention also provides a method of preparing a medicament for inhibiting orbital adipose tissue fibrosis or a nystagmus (particularly, inhibiting orbital adipose tissue fibrosis or a nystagmus), the method comprising: the substances useful for inhibiting orbital adipose tissue fibrosis or nystagmus obtained by the aforementioned screening are synthesized and/or purified as a drug for inhibiting orbital adipose tissue fibrosis or nystagmus.

The substances obtained which are useful for inhibiting orbital adipose tissue fibrosis or nystagmus can be used for the preparation of pharmaceutical compositions, as described hereinabove for the present invention.

Methods for screening for substances that act on a target site, either on a protein or on a gene or on a specific region thereof, are well known to those skilled in the art and can be used in the present invention. The candidate substance may be selected from: peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. Depending on the kind of substance to be screened, it is clear to the person skilled in the art how to select a suitable screening method.

Orbital adipose tissue fibrosis or thyroiditis model

Orbital adipose tissue fibrosis or thyroiditis is a disease that occurs in a specific location and has a unique pathogenesis, differing from fibrosis of other tissues of the body. In view of the specificity of their mechanisms and the complexity of the disease, there is also a need in the art for more cellular or animal models of such diseases.

In the invention, the inventor firstly separates stroma vascular component (SVF) cells from adipose tissues in eyeorbits and further separates DPP4+ cells from the stroma vascular component (SVF) cells; preferably, the cell is a DAPI-/CD31-/CD45-/PDGFRA+/DPP4+ cell. On the basis of obtaining the cells, the inventors found that it is an orbital adipose tissue cell population with fibrotic differentiation potential.

More importantly, in further research analysis, the inventors found that dpp4+ cells were able to recruit to M2 macrophages, which interacted with each other, which promoted the progress of fibrosis of dpp4+ cells.

Based on these new findings, the present inventors have established a novel disease model system for orbital adipose tissue fibrosis or nystagmus, including a cell model system or an animal model system.

As one embodiment, the disease model system is a cell model system comprising dpp4+ cells and M2 macrophages, which are mixed and interact. In the cell model system, SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal pathway mediates the occurrence of diseases, SEMA3C is expressed by DPP4+ cells in adipose tissue in the eye sockets, NRP1/NRP2 is expressed by M2 megaly, the combination of SEMA3C and NRP1/NRP2 enables the DPP4+ cells to interact with M2 macrophages, and GAS6 in the M2 macrophages acts on the DPP4+ cells to enable the DPP4+ cells to be fibrotically differentiated, so that the adipose tissue fibrosis or the thyroidism is formed.

As another embodiment, the disease model system is an animal model system. By transplanting the cells mixed by DPP4+ cells and M2 macrophages into the orbital adipose tissue of a non-human animal, the two cells interact to form an animal model system of orbital adipose tissue fibrosis or onychomycosis.

In the present invention, the method of preparing the disease model system for orbital adipose tissue fibrosis or nystagmus is not directly aimed at disease diagnosis or disease treatment.

A wide variety of biological agents or small molecule compounds can be used as candidate substances in the above model system to observe whether they have an ameliorating effect on disease states, and thus screen potential drugs useful for alleviating or treating orbital adipose tissue fibrosis or thyroidism.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which are not specifically noted in the examples below, are generally carried out according to conventional conditions such as those described in J.Sam Brookfield et al, molecular cloning guidelines, third edition, scientific Press, or according to the manufacturer's recommendations.

Materials and methods

1. Adipose tissue Stromal Vascular Fraction (SVF) cell acquisition

And (3) cutting intra-orbital adipose tissue obtained through orbital decompression operation, then using collagenase type I to digest for 1-2h at a water bath kettle at 37 ℃, removing erythrocytes by using erythrocyte lysate after digestion, simultaneously removing mature adipocytes floating on the upper layer, and removing excessive impurities through a 40 mu m cell sieve to obtain single cell suspension which is a single cell suspension containing SVF components.

2. Single cell sequencing belief analysis

By single cell sequencing library establishment, the gene expression in each cell in the SVF single cell suspension is subjected to sequencing analysis, then cell clustering analysis is carried out by using Seurat packages, subgroup marker gene analysis is carried out by using FIND ALL MARKERS, and the potential interaction mode of DPP4+ cell population, GPC3+ cell population and other cell subgroups in the disease composition fiber cell subgroup are predicted by using CellChat packages for communication analysis.

3. DPP4+ cell acquisition

After SVF was obtained, cells were screened layer by layer using white blood cells (CD 31/CD 45), fibroblast markers (PDGFRA) and DAPI, and then the PDGFRA+ viable cell population was finally screened using DPP4 to select DAPI-/CD31-/CD45-/PDGFRA+/DPP4+ cells.

4. Immunofluorescence analysis of dpp4+ cells and co-localization with macrophages (CD 68)

For immunofluorescent labeling, intraorbital adipose tissue from TED and control samples was first fixed with 4% paraformaldehyde overnight, then dehydrated by a series of ethanol washes, and then embedded in paraffin for serial sectioning. These sections were stained with hematoxylin, eosin (H & E) and Masson using standard procedures and finally examined by light microscopy.

In addition, serial sections were used for immunofluorescence analysis of dpp4+ cells and their co-localization with macrophages (CD 68) by the following procedure: first, paraffin-embedded sections were dewaxed, rehydrated and subjected to antigen retrieval treatment, and then blocked in 5% bsa+5% normal goat serum in PBS for 1h at room temperature. After an inherent overnight consistency with primary antibody at 4 ℃, sections were washed 3 times with PBS and incubated with secondary antibody at room temperature for 1 hour. The primary antibodies used were as follows: DPP4 (goat; 1:250;R&D Systems), CD68 (mouse); 1:100; invitrogen). The mounting was performed using ProLong gold anti-quencher and DAPI (Invitrogen P36931). Fluorescence images were obtained using a zeiss LSM 880 confocal microscope.

5. M2 macrophage induction

Macrophages grown in 1640 medium with 10% fetal bovine serum (v/v), 100IU/mL penicillin and 50 μg/mL streptomycin sulfate, induced from two types of monocytes: THP-1 cells from a cell bank and monocytes extracted from peripheral blood samples of four TED patients. The extraction of patient monocytes is according to conventional methods.

Phosphomyristate (PMA) (Sigma, 16561-29-8) was used at 100ng/mL for 48 hours to induce differentiation of THP-1 cells from monocytes to M0 macrophages, whereas M-CSF (obtained from STEMCELL ^TM, 78057) was added to the medium at 50ng/mL for 8 days to induce differentiation of M0 macrophages.

Lipopolysaccharide (LPS) (Sigma, SMB00610, 250 ng/ml) was used for M1 macrophage differentiation, and IL-4 (obtained from STEMCELL ^TM, 8045, 20 ng/ml) and IL-13 (obtained from STEMCELL ^TM, 78029, 20 ng/ml) were used for M2 macrophage polarization for 48h.

M1/M2 induction was confirmed by following the expression of specific marker genes (M1 markers: TNFα, IL1B, IL6, CCR7; M2 markers: IL10, CCL18, CCL22, MRC1, TGFB 1).

The expression of the GAS6 gene was quantified by qRT-PCR in M0, M1 and M2 macrophages, respectively.

6. DPP4+ cell-macrophage interaction cell assay

DPP4+ cells are incubated with macrophage medium DPP4+ or SVF cells, and an intercellular interaction experiment is performed between DPP4+ cells and macrophages. To eliminate the interference of the above-mentioned inducers on cell-cell interaction experiments, M1/M2 macrophage medium was replaced with fresh medium for 48h and then mixed with DPP4+ or SVF cell medium in the ratio (1:1). After 48h interaction with M1/M2 macrophage supernatant, DPP4+ and SVF cells were harvested for RNA extraction for qRT-PCR. In addition, supernatants of M0, M1 and M2 macrophages without inducer were analyzed using ELISA kit (Abclonal, china, RK 01443). In terms of inhibitor assay, AXL-specific inhibitor TP0903 was first added to dpp4+ or SVF cell culture medium, dpp4+ or SVF cells were pre-treated for 24h at 125nM, then the medium to be provided with inhibitor was removed and conditioned with M2 macrophage secretion medium (DMEM/F12 supplement) at 37 ℃ to 1:1, mixing and post-treating the cells for 48 hours, extracting RNA of the cells, and carrying out expression analysis on fibrosis and fat differentiation related indexes by qRT-PCR.

7. Fibrosis analysis of dpp4+ cells

Dpp4+ cells were seeded in 24-well plates, cultured in high-sugar DMEM/F12 medium containing 20% FBS and antibiotics to a cell growth of about 80%, and cultured with medium containing M2 macrophage secretion and dpp4+ medium 1 as described above: 1 mixing 37 ℃ and incubating dpp4+ cells for 48h. After 48h, the cells were collected and RNA was extracted and the expression of the fibrosis index was detected by qRT-PCR.

Example 1 identification of DPP4+ cells and their interactions with macrophages

And (3) performing enzyme digestion on the intra-orbital adipose tissue obtained through the orbital decompression operation, and removing erythrocytes and redundant impurities to obtain adipose tissue matrix vascular component (SVF) cells.

First, the inventors performed a cell communication analysis. Based on extensive analysis and screening, the inventors identified dpp4+ cells as a group of adipose tissue progenitor cells with fibrotic differentiation potential (fig. 1, top panel).

Further, the inventors performed HE & Masson staining analysis of orbital adipose tissue in the mastoid disease (fig. 1, middle panel), dpp4+ cells, macrophages (CD 68), and immunofluorescence analysis of co-localization of both (fig. 1, bottom panel). The results show that DPP4+ cells have strong interactions and co-localization with macrophages.

EXAMPLE 2 macrophage progression of DPP4+ cells towards fibrosis

Macrophages are classified into M1 and M2 subtypes, with M2 macrophages being the predominant subset of macrophages, with a ratio (natural ratio in patient samples) of about 82.5% (fig. 2, top panel).

The present invention analyzes the cells in which DPP4+ cells interact with M2 macrophages, and uses DPP4+ cells without M2 macrophages as a control. As a result, it was found that the fibrotic biomarkers Col1a1, col3a1, eln, fn1, acta2, timp1 were significantly increased in the DPP4+ cell M2 macrophage co-cultured group compared to the DPP4+ cell control group; whereas the adiposity indicators Ppar γ, cebp α were significantly reduced, as was Cd36 (fig. 2, middle panel).

The invention analyzes the interaction between SVF cells and M2 macrophages, and takes SVF cells without M2 macrophages as a control. Similar changes in fibrotic biomarkers (Col 1a1, eln, acta 2) were also observed compared to the SVF control group (fig. 2, lower panel).

The results in FIG. 2 demonstrate that macrophages can progress DPP4+ cells toward fibrosis.

Example 3, signal pathway involving DPP4+ cells

The intracellular signal pathway is complicated, and in order to further define the functions of DPP4+ cells and the intracellular signal pathway involved in the DPP4+ cells, the inventor establishes a single-cell sequencing library and performs single-cell signaling analysis on SVF.

FIG. 3 shows partial results of single cell signaling analysis of the signal pathway involved in DPP4+ cells. From among the many intracellular signaling pathways, the inventors found that the SEMA3C- (NRP 1/NRP 2) pathway promotes the conversion of dpp4+ cells to M2 macrophages, whereas the GAS6-AXL pathway inhibits the conversion of dpp4+ cells to M2 macrophages. Under the condition of thyroid eye symptoms, DPP4+ fibroblasts recruit M2 macrophages expressing NRP1/NRP2 by secreting SEMA3C, and after spatial localization change, the M2 macrophages secrete GAS6 to act on the DPP4+ fibroblasts to promote fibrosis.

After cell culture, the identification of the expression/secretion products showed that dpp4+ cells expressed and secreted SEMA3C (SEMA 3C). Further experiments on gene expression regulation showed that M2 macrophages expressed NRP1/NRP2 and were recruited to M2 macrophages by the binding of SEMA3C to NRP1/NRP 2. In the interaction process of DPP4+ cells and M2 macrophages, the M2 macrophage phase acts on the DPP4+ cells through GAS6-AXL to promote fibrosis. The inventors prepared sgrnas for efficient NRP1 knockout based on CRISPR/CAS9 system:

Forward direction: 5'-GAGGGTTTATGGGGGACACT-3' (SEQ ID NO: 1);

Reversing: 5'-CAGGACATCTGGGGCTACAT-3' (SEQ ID NO: 2);

with the above sgrnas, knockouts were performed based on CRISPR-Cas9 technology. The results show that when NRP1/NRP is knocked out, the situation where fibrosis is promoted no longer occurs.

The results demonstrate that M2 macrophages and DPP4+ cells interact through the pathway of SEMA3C- (NRP 1/NRP 2) interaction with GAS6-AXL (SEMA 3C- (NRP 1/NRP 2) -GAS 6-AXL pathway).

Example 4 TP0903 exerts its inhibitory effect by inhibiting GAS6-AXL pathway in DPP4+ cells

To further verify the single cell prediction mechanism and to find potential therapeutic targets, the inventors examined the expression of GAS6 in M2 macrophages and in the analyte, and found that it was highly expressed in both M2 macrophages and in the secretion medium (as shown in fig. 4A). Dpp4+ cells, after reacting with M2 macrophage secretion, activate themselves through GAS6-AXL pathway and develop into myofibroblasts capable of secreting collagen fibers and the like, thereby secreting fibrous molecules and proteins in large amounts (as shown in fig. 4B).

Through screening and research analysis, the present inventors treated dpp4+ cells with TP 0903. The results show that TP0903 can inhibit the GAS6-AXL pathway such that the fibrosis markers (Fn 1, col1a1, col3a1, fn1, acta2, timp 1) are significantly reduced, thereby inhibiting fibrosis (as shown in fig. 4C).

After incubation of dpp4+ cells with the secretion-bearing medium for 48h at 37 ℃, cellular RNAs were extracted and expression of the fibrosis index was detected using qRT-PCR. The expression of fibrosis indices (Acta 2, timp1, col1a1, fn1, col3a 1) in DPP4+ cells was significantly reduced in the TP0903 group compared to the control group with the AXL-specific inhibitor TP0903 treatment (as shown in fig. 4D).

These results indicate that TP0903 exerts its inhibitory effect by inhibiting the GAS6-AXL pathway in dpp4+ cells. M2 macrophages and DPP4+ cells progress to fibrosis through the GAS6-AXL mode, and fibrosis can be improved better by using an AXL specific inhibitor.

Fibrosis is an important pathological feature that leads to the disease of the nystagmus, so TP0903 can alleviate/treat the nystagmus. Meanwhile, in the experiment, the cells are cells derived from fresh tissues of patients, so the result shows that TP0903 can also have the effect of inhibiting fibrosis on in vivo (in vivo) cells, thereby relieving/treating the onychomycosis.

EXAMPLE 5 establishment of cell model and drug screening Using Signal pathway

As described in example 1, a cell culture system was obtained in which DPP4+ cells interacted with macrophages as a cell model. Screening for drug candidates was performed by the following method:

test group: a cell culture system in which dpp4+ cells interact with macrophages, and administering a candidate substance;

control group: cell culture systems in which dpp4+ cells interact with macrophages, do not administer candidate substances.

And detecting the change condition of SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal channel genes or proteins in the test group and the control group respectively, and observing the interaction condition of DPP4+ cells and macrophages. If SEMA3C expression, activity or secretion in DPP4+ cells of the test group is inhibited by the candidate substance, NRP1/NRP2 expression or activity in M2 macrophages is inhibited by the candidate substance, the mutual combination of SEMA3C and NRP1/NRP2 is blocked or weakened by the candidate substance, the interaction of DPP4+ cells and M2 macrophages is blocked or weakened by the candidate substance, and GAS6-AXL acts on DPP4+ cells and is blocked or weakened by the candidate substance; then the candidate substance is a substance or potential substance that inhibits orbital adipose tissue fibrosis or inhibits the condition of the nystagmus. The inhibition, weakening, etc. are all calculated according to a statistical mode, so as to achieve significance.

The foregoing examples illustrate only a few embodiments of the application and are described in detail herein without thereby limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims. All documents referred to in this disclosure are incorporated by reference herein as if each was individually incorporated by reference.

Claims (10)

1. Use of a modulator of sema3c- (NRP 1/NRP 2) to GAS6-AXL signaling pathway for the preparation of a pharmaceutical composition for inhibiting orbital adipose tissue fibrosis or inhibiting a process eye disease;

   Wherein the modulator is selected from the group consisting of: a modulator that inhibits or down-regulates expression, activity or secretion of SEMA3C in dpp4+ cells, a modulator that inhibits or down-regulates expression or activity of NRP1/NRP2 in M2 macrophages, a modulator that inhibits or down-regulates binding of SEMA3C to NRP1/NRP2, a modulator that inhibits or down-regulates interaction of dpp4+ cells with M2 macrophages, a modulator that inhibits or down-regulates the action of GAS6-AXL on dpp4+ cells; the DPP4+ cells are an orbital adipose tissue cell population with fibrotic differentiation potential.
2. 2. The use according to claim 1, wherein the modulator that inhibits or down-regulates SEMA3C expression, activity or secretion in dpp4+ cells comprises: agents that knock out or silence the SEMA3C gene, agents that inhibit SEMA3C protein activity; preferably, it includes: a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent aiming at a SEMA3C gene, wherein the reagent carries out the function-losing mutation on the SEMA3C, an interfering molecule which specifically interferes with the SEMA3C gene expression, and a small molecule inhibitor of SEMA3C protein; or (b)

   The modulator that inhibits or down-regulates NRP1/NRP2 expression or activity in M2 macrophages comprises: agents that knock out or silence the NRP1/NRP2 gene, agents that inhibit NRP1/NRP2 protein activity; preferably, it includes: a CRISPR gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent aiming at NRP1/NRP2 genes, wherein the reagent carries out functional loss mutation on NRP1/NRP2, an interfering molecule which specifically interferes with NRP1/NRP2 gene expression, and a small molecule inhibitor of NRP1/NRP2 protein; or (b)

   The modulator that inhibits or down-regulates SEMA3C binding to NRP1/NRP2 comprises: an agent that competitively binds to NRP1/NRP2, an agent that competitively binds to SEMA3C, thereby preventing or reducing SEMA3C binding to NRP1/NRP2; modulators that down-regulate SEMA3C expression, activity or secretion; modulators that down-regulate NRP1/NRP2 expression or activity; or (b)

   The modulators that inhibit or down-regulate the action of GAS6-AXL on dpp4+ cells include: modulators that down-regulate GAS6 or AXL expression and activity; preferably, it includes: agents that knock out or silence the GAS6 or AXL gene, agents that inhibit GAS6 or AXL protein activity; preferably, it includes: a GAS6 or AXL gene editing reagent, a homologous recombination reagent or a site-directed mutagenesis reagent for GAS6 or AXL genes, wherein the reagent performs a loss-of-function mutation on GAS6 or AXL, an interfering molecule which specifically interferes with the expression of GAS6 or AXL genes, a small molecule inhibitor of GAS6 or AXL proteins;

   The modulators that inhibit or down-regulate the interaction of dpp4+ cells with M2 macrophages include: modulators that inhibit SEMA3C binding to NRP1/NRP2, modulators that down-regulate SEMA3C expression, activity or secretion; modulators that down-regulate NRP1/NRP2 expression or activity.
3. 3. The use according to claim 2, wherein the modulator that down-regulates NRP1/NRP2 expression or activity is a CRISPR gene editing agent that knocks out NRP1/NRP2; preferably its sgRNA sequence is: the forward sequence is shown as SEQ ID NO. 1, and the reverse sequence is shown as SEQ ID NO. 2.
4. 4. The use of claim 2, wherein the small molecule inhibitor of GAS6 or AXL protein is a compound of the parent nucleus structure of formula (I) or an isomer, derivative, solvate or precursor thereof, or a pharmaceutically acceptable salt, composition or pharmaceutical composition thereof;
5. 5. The use of claim 1, wherein dpp4+ cells in the intra-orbital adipose tissue express SEMA3C, SEMA3C binds to NRP1/NRP2, recruiting NRP1/NRP2 expressing M2 macrophages, and GAS6 in M2 macrophages acts on dpp4+ cells AXL receptor to fibrose and differentiate, forming orbital adipose tissue fibrosis or a thyroidism; preferably, the modulator intervenes by means of a refined spatial modulation, or

   The fibrosis development and/or the lesions occur during the active phase of the nystagmus disease.
6. 6. The use according to claim 1, wherein said onychomycosis comprises: eyelid swelling, eyelid retraction, late upper eyelid closure, eye protrusion, appearance of exposed keratitis, compression optic neuropathy, secondary glaucoma, visual hypofunction or loss, blindness.
7. Use of sema3c- (NRP 1/NRP 2) to GAS6-AXL signaling pathway for screening substances inhibiting orbital adipose tissue fibrosis or nystagmus; preferably, in the SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal pathway, SEMA3C is expressed by DPP4+ cells in adipose tissue in the orbit, NRP1/NRP2 is expressed by M2 macrophagin, SEMA3C is combined with NRP1/NRP2 so that DPP4+ cells interact with M2 macrophages, and GAS6 in the M2 macrophages acts on the AXL receptor of the DPP4+ cells to enable the DPP4+ cells to be fibrotically differentiated, so that orbital adipose tissue fibrosis or nystagmus is formed.
8. 8. A method of screening for a substance that inhibits orbital adipose tissue fibrosis or a nystagmus, the method comprising:

   (1) Contacting the candidate substance with a system comprising SEMA3C- (NRP 1/NRP 2) to GAS6-AXL signaling pathways; in the system, SEMA3C, AXL is expressed by DPP4+ cells, and NRP1/NRP2 and GAS6 are expressed by M2 macrophages; the DPP4+ cells are orbital adipose tissue cell groups with fibrosis differentiation potential;

   (2) Screening out substances which down regulate SEMA3C- (NRP 1/NRP 2) -GAS 6-AXL signal paths, wherein the substances are useful substances for inhibiting orbital adipose tissue fibrosis or onychomycosis; preferably, the down-regulating includes: down-regulating SEMA3C expression, activity or secretion in dpp4+ cells, down-regulating NRP1/NRP2 expression or activity in M2 macrophages, inhibiting SEMA3C binding to NRP1/NRP2, down-regulating dpp4+ cells to interact with M2 macrophages, down-regulating GAS6-AXL to act on dpp4+ cells.
9. 9. A disease model system of orbital adipose tissue fibrosis or thyroidism, which is characterized in that the disease model system is a cell model system comprising DPP4+ cells and M2 macrophages, which are mixed and interacted;

   Preferably, in the cell model system, the SEMA3C- (NRP 1/NRP 2) -GAS6/AXL signal pathway is used for mediating the occurrence of diseases, SEMA3C, AXL is expressed by DPP4+ cells in adipose tissues in the eye sockets, NRP1/NRP2 and GAS6 are expressed by M2 giant and fine, the SEMA3C is combined with the NRP1/NRP2 to enable the DPP4+ cells to interact with M2 macrophages, and GAS6 in the M2 macrophages acts on the DPP4+ cells AXL receptor to enable the DPP4+ cells to be fibrotically differentiated, so that the adipose tissue fibrosis or the thyropathy is formed in the eye sockets.
10. 10. A method of preparing a disease model system for orbital adipose tissue fibrosis or nystagmus, wherein the disease model system is a cell model system, the method comprising: mixing DPP4+ cells and M2 macrophages so that the DPP4+ cells and the M2 macrophages interact with each other to form a cell model system; or (b)

    The disease model system is an animal model system, the method comprising: the DPP4+ cells and M2 macrophage mixed cells are transplanted into the orbital adipose tissue of a non-human animal, and the two cells interact to form an animal model system of orbital adipose tissue fibrosis or onychomycosis.