CN117180428A - Application of IL-4 in preparation of medicine for treating cartilage degeneration - Google Patents

Abstract

The invention belongs to the field of biological medicine, and relates to an application of IL-4 in preparing a medicine for treating cartilage degeneration. According to the research, the invention discovers that IL-4 can inhibit the nucleic acid level of various inflammatory factors in cartilage-like cells, simultaneously IL-4 can obviously relieve intervertebral disc degeneration, and IL-4 obviously reduces the expression of pain related factors in dorsal root ganglion. The invention discovers the inhibition effect of IL-4 on cartilage degeneration for the first time, and has important significance for improving the prognosis of reducing cartilage degeneration.

Description

Application of IL-4 in preparation of medicine for treating cartilage degeneration

Technical Field

The invention belongs to the field of biological medicine, and relates to an application of IL-4 in preparing a medicine for treating cartilage degeneration.

Background

Pain, including Low Back Pain (LBP), leg pain, joint pain, etc., is one of the most common symptoms in orthopedics, especially spinal surgery outpatient [1], most commonly seen in middle-aged to elderly people. Therefore, the lumbago is an urgent problem in global public health, and has great social and scientific significance for effectively relieving or radically curing the lumbago in time.

Intervertebral discs are complex fibrocartilage tissue in the human body and are also representative of cartilage tissue in the body. Which under normal anatomic conditions, a Nucleus Pulposus (NP) rich in gelatinous proteoglycans, an Annulus Fibrosus (AF) interwoven with multiple layers of collagen fibers, and the vertebral endplates together constitute healthy disc tissue. The intervertebral discs connect adjacent vertebral bodies and help the spine to realize the movement function. The cartilage tissue is even smoother on the facet facing the joint cavity, facilitating bone-to-bone movement. Cartilage itself is elastic, so that it can cushion the shock and impact of the connected bones during walking, jumping and other movements. In addition, the elasticity of cartilage and its deformability have an effect on increasing joint mobility. Normal cartilage is neither nerve nor vessel, and its nutrition is mainly supplied by the end plates, synovial fluid and arterial branches around the synovial layer of the joint capsule. Between these collagen fibers, chondrocytes and/or nucleus pulposus cells are dispersed, and gradually from the superficial layer to the deep layer, these chondrocytes maintain the normal metabolism of cartilage, and they are composed of flat-like to oval or round cells.

There are numerous causes of cartilage degeneration. The onset of cartilage degeneration progresses with age, most of the apparent altered phenotypes appear after adulthood, with more than 80% of the discs exhibiting degeneration-related changes in the population over 50 years [6]. Biomechanics loaded on cartilage, and the long-term abrasive effect of this force on cartilage, is the initiating factor for cartilage degeneration [7-9]. For example, the incidence of low back pain and disc degeneration in physical workers is higher than in the general population. All astronauts who re-enter the atmosphere after microgravity suffer from lumbago [10,11], both of which may be due to disc degeneration (intervertebral disc degeneration, IVDD) caused by over-pressurization of the nucleus pulposus. There is an inseparable link between high mechanical load bearing and cartilage degeneration [11-13].

The cartilage degeneration process implies a series of structural failure events. In early endochondral, the larger vacuolated empty cord cells (large vacuolated notochordal-like cells) in NP begin to disappear in the early stages of life (about ten years), which is thought to be the beginning of the degenerative process [14]. Endogenous progenitor cells were found in cartilage [15], indicating the potential for cartilage to self-repair, but lacking evidence of spontaneous regeneration of human cartilage. Subsequently, proteoglycan synthesis is reduced, accompanied by conversion of collagen synthesis. Wherein type II collagen is reduced, type I and type III collagen is increased [16], exemplified by degeneration of disc cartilage (FIG. 1). The content of matrix metalloproteinases (Matrix metalloproteinases, MMPs) was significantly increased [17]. Apoptotic chondrocytes were also significantly increased [18]. Accumulation of cellular metabolic waste products and activation of matrix degradation factors, which can lead to an increasing deterioration of the acidic environment within the disc, further compromising cellular function and phenotype [19]. Degradation of extracellular matrix (Extracellular matrix, ECM) and loss of proteoglycans can lead to reduced loading capacity and height of cartilage [20]. The high osmotic pressure and acidic environment in cartilage further exacerbate the stress difficulty [18]. In studying the pathogenesis of cartilage degeneration-related diseases, researchers have proposed several hypotheses including impairment of normal nutritional pathways, excessive mechanical load, genetic factors, unhealthy habits, aging and spinal infections [21-25]. Regardless of the cause, however, inflammation is always present throughout the degeneration process and has a clear relationship with pain [20,26].

Degeneration of cartilage is a significant cause of back pain [20,26]. During cartilage degeneration, cells in cartilage (NP cells or chondrocytes) exhibit a significant increase in pro-inflammatory cytokines [20,26]. Denaturation of cartilage also leads to degradation of extracellular matrix and loss of hydrophilic matrix molecules, leading to tissue structural necrosis and biomechanical changes [27]. These are all the main causes of increased endochondral inflammation, nerve ingrowth [28,29] and pain factor release [30].

In healthy cartilage, only the periphery of cartilage has a domination of nerve fibers [31]. Examination of animal models and human clinical specimens revealed that sensory innervation extends into the cartilage, and that sensory nerve ingrowth occurs up to the periphery and lining of the cartilage [32-34], is the primary cause of pain [35,36]. Cytokines including TNF- α, IL-1 and nerve growth factor (Nerve growth factor, NGF) were identified as inducers with nerve ingrowth induction function [36]. On the other hand, not all patients with cartilage degeneration have symptoms of pain [37]. Epidemiological investigation showed that the proportion of people with severe cartilage degeneration but no pain also increases and is proportional to age [38,39]. The determinant of the occurrence of symptoms in patients with cartilage degeneration may be the presence of cartilage persistent inflammation [40]. Taken together, these studies strongly suggest that there is a correlation between sensory nerve ingrowth, inflammatory mediators and disc-derived LBP.

Inflammation was originally thought to be a response to infection or tissue injury, but the scientific community is increasingly investigating its physiological role in maintaining tissue homeostasis [41,42]. In the inflammatory response caused by infection, leukocytes are recruited to the site of infection and secrete inflammatory mediators that cause activation of other cell types. A complex series of events is initiated, ultimately leading to clearance of tissue infection and resolution of inflammation and immune response. When tissue is damaged, the inflammatory response also includes vascular responses and coordinated recruitment and activation of various cell types [41,42]. However, cartilage is a vascular-free tissue, and thus in this case, the inflammatory reaction is different from that described above.

Cartilage degenerative disease is a complex process. Proinflammatory diseases may be a key factor in cartilage degeneration [20,26]. Recent studies have identified inflammatory mediators and signaling pathways as important factors in the occurrence and progression of cartilage degeneration [43]. It has been found that the accumulation of a large number of immune cells in a degenerated disc mediates inflammation, and that the degradation products and release proteins of these immune cells include: interferon-gamma (IFN-gamma), interleukins, such as IL-1, IL-6 and IL-12, and matrix metalloproteinases MMPs, etc., result in a decrease in the number of NP cells and deterioration of the disc microenvironment [44]. Long-term inflammation continues to recruit inflammatory cells, forming a long-term process of destruction of cartilage tissue, exacerbating degeneration [22]. Inflammatory molecules are elevated in the degenerated disc. Cartilage degeneration may be caused or exacerbated.

The ingrowth and re-innervation of nerves has been recognized by many researchers as an important process in the generation of cartilage-derived pain. Cartilage is generally thought to contain no neural structures, such as in intervertebral discs, the outer annular layer of healthy intervertebral discs has a small number of nerve fibers that extend little into the internal AF and NP [45,46]. The interior of NP and AF in chronic discogenic pain patients contains a large number of peripheral nerve fibers [32,34,35]. During degeneration of the disc, the nerve fibers gradually extend to the inner layers of AF and even NP, accompanied by the expression of the pain transmitting substance P (Neuropeptide substance P, NSP) [34] which is highly correlated with the occurrence of intervertebral disc-derived lumbago [32,34,35]. It has been proposed that increased expression of inflammatory-related cytokines is a feature of pain caused by cartilage degeneration [20,47]. In the cartilage of patients suffering from pain, the expression of IL-6, IL-8 and prostaglandin E2 was increased [48], and the levels of IL-1 beta and IL-6 were increased [49]. Vascularized granulation tissue grows inward along the fissures of the cartilage, expanding the inner layer from the outer layer, accompanied by massive macrophage and mast cell infiltration [50]. Rupture of AF behind the disc cartilage stimulates TNF- α and IL-1β expression and induces DRG inflammatory responses and mechanical hyperalgesia in rat models [51]. The above studies demonstrate that the content of pro-inflammatory mediators in the painful intervertebral disc increases. All of these factors together contribute to the occurrence of pain.

Interleukin 4 (IL-4) is produced primarily by activated T cells and plays a key role in regulating humoral and adaptive immunity [52,53]. IL-4 has entered phase II clinical trials as a tumor immunomodulator, and the U.S. FDA has approved antibody drug 611 against IL-4Rα for the treatment of atopic dermatitis [54]. IL-4 is seen to have very significant clinical transformation advantages. Recent studies have shown that IL-4 or IL-13 affects neutrophil migration by modulating chemokine receptors CXCR1 and CXCR2 to alleviate type 2 immune disorders such as asthma [55]. The IL-4/CCL22/CCR4 axis is also involved in migration of Tregs cells to osteolytic lesions, thereby inhibiting the production of inflammation and pro-inflammatory mediators to reduce the development of inflammatory bone resorption [56]. High IL-4 is highly expressed by lymphostromal cells CXCL12, which together induce cellular activation, migration and adhesion of follicular lymphoma malignant B [57].

IL-4 and IL-13 activate TH2 cells. IL-4 and IL-13 are marker cytokines in TH2 cell-mediated immune responses. Physiologically, IL-4 and IL-13 share the same receptor and are both able to activate the signal transduction and activator of transcription 6 (STAT 6) signaling pathway [58,59]. These cytokines have pro-inflammatory and pro-fibrotic effects in pulmonary diseases such as asthma. In contrast, IL-4 and IL-13 are upregulated in the synovial fluid of early stage rheumatoid arthritis, suggesting that these cytokines are part of an early-mediated response that is instead lost when patients develop well-defined arthritis [60]. This early IL-4 and IL-13 response in the synovium of the joint can trigger the following tissue damage by releasing sirens such as IL-25 and IL-33 [61]. Functionally, IL-4 and IL-13 are potent anti-arthritic cytokines [62-64], which strongly inhibit cartilage damage and osteoclastogenesis [65,66].

Previous studies have shown that worm-induced TH2 cell responses can inhibit arthritis in a mouse model, a process that is accompanied by significant down-regulation of pro-inflammatory cytokines [67,68]. However, the exact nature of how TH2 cell responses inhibit arthritis remains unclear. Studies have shown that in the intestinal worm Nippostrongylus brasiliensis-induced arthritis model, mice strongly activate STAT6 signaling in IL-4 and IL-13-induced hematopoietic cells, followed by macrophage polarization switching to the anti-inflammatory "M2-like" phenotype, responsible for inhibiting arthritis [69]. This also demonstrates the mechanism by which the IL-4/IL-4R pathway reduces osteoclastogenesis and bone destruction in mice by inhibiting the nuclear factor- κB (NF- κB) and c-JunN-terminal kinase (JNK) signaling pathways through constitutively active STAT6 fusion proteins [70].

IL-4 and IL-13 have direct anti-inflammatory effects in the synovial membrane of arthritis, which can reduce IL-1β and TNF- α produced by synovial macrophages, and their expression of CD16 and CD64 [71,72]. IL-4 administration has been tested in cancer patients, but is associated with dose limiting side effects, including gastrointestinal symptoms and vascular leak syndrome [73]. However, these limitations can be overcome by using IL-4 mimics that activate the class I IL-4 receptor (expressed on immune cells).

IL-4R is the alpha chain of the interleukin 4 receptor, a type I transmembrane protein, which binds IL-4 and IL-13 to regulate IgE antibody production in B cells and promote Th2 cell differentiation. Soluble forms of the encoded protein may be produced by proteolysis of alternative splice variants or membrane-bound proteins, which may inhibit IL-4 mediated cell proliferation and up-regulation of IL-5 by T cells. The variation of the allele of the gene encoding IL-4R is associated with atopic diseases and may be manifested as allergic rhinitis, sinusitis, asthma or eczema. IL-4 or IL-13 binding to IL-4R on the surface of macrophages results in the alternate activation of these macrophages. Activated macrophages (AAMΦ) down-regulate inflammatory mediators during the immune response [74].

In summary, cartilage degeneration and a series of diseases secondary to cartilage degeneration are important causes of clinical pain, and increase of inflammatory factors promotes cartilage degeneration and pain, inhibits or reverses inflammation progress to improve inflammatory cascade in the process of cartilage degeneration, improves microenvironment of NPCs, promotes proliferation of NPCs and resumes synthetic secretion of NPCs, delays or reverses cartilage degeneration progress, and further reduces or prevents cartilage degeneration-related pain. IL-4 acts as an anti-inflammatory factor, has a remarkable function of inhibiting inflammation, and is a cytokine with better transformation potential.

Disclosure of Invention

In one aspect, the application provides the use of an IL-4/IL-4R signaling pathway as a target in the manufacture of a medicament for treating or ameliorating cartilage degeneration and/or disc degeneration.

"IL-4/IL-4R" also refers to "IL-4-IL-4R" wherein IL-4 is a ligand in the signaling pathway and IL-4R is a receptor in the signaling pathway.

In one aspect, the application also provides the use of IL-4 or an analogue or agonist thereof in the manufacture of a medicament for the treatment of cartilage degeneration and/or disc degeneration.

In some embodiments, "analogs" include compounds or genetic tools that function the same or similar to IL-4, or which may be substituted therefor.

In some embodiments, an "agonist" includes a compound or genetic tool that can promote an increase in the amount of IL-4; either directly promoting the rise of IL-4 or acting upstream or downstream of IL-4, such that the amount of IL-4 is increased.

"IL-4 agonist" refers to any compound or substance capable of promoting/activating IL-4. In some embodiments, an "IL-4 agonist" may be a substance or means that agonizes the formation of any one of the targets in IL-4/IL-4R, or agonizes the activity of the target, or upregulates the target, including chemical or biological substances or means, and the like.

In one aspect, the application provides the use of IL-4 or an analog or agonist thereof in the manufacture of a medicament for treating a condition caused by cartilage degeneration.

In some embodiments, the disorder caused by cartilage degeneration comprises: at least one of herniated disk, spondylolisthesis, spinal stenosis, degenerative scoliosis, isthmus fracture, or fracture of vertebral body of the spine, osteochondritis, osteoarthritis, degenerative arthritis, senile arthritis, hypertrophic arthritis, and hypertrophic arthritis. Cartilage degeneration is an essential pathophysiological feature of the aforementioned diseases, which are accompanied by cartilage degeneration, and cartilage degeneration itself is the fundamental nature of these diseases.

In one aspect, the application also provides the use of IL-4 or an analogue or agonist thereof in the manufacture of a medicament for the treatment of pain.

In some embodiments, the pain is selected from pain caused by cartilage degeneration; in some embodiments, the pain is selected from the group consisting of lumbago, skelalgia, arthralgia; in some embodiments, the pain is selected from lumbago.

In some embodiments, the IL-4 or analog or agonist thereof treats pain by inhibiting matrix metalloproteinases and/or inflammatory cytokines; in some embodiments, the matrix metalloproteinase is selected from at least any one of MMP3, MMP13, ADAMTS4, and ADAMTS 5; in some embodiments, the inflammatory cytokine is selected from at least any one of TNF- α and IL-1β.

In some embodiments, the IL-4 or analog or agonist thereof treats pain by inhibiting a pain-associated factor in the dorsal root ganglion; in some embodiments, the pain-related factor is selected from CGRP.

In one aspect, the application provides a method for developing a drug for treating cartilage and/or disc degeneration, and corresponding IL-4 promoter is designed for IL-4.

In some embodiments, the IL-4 promoter is one or more of a substance, analog, upstream signaling pathway activator that promotes the production or activity of IL-4, either in whole or in part.

In some embodiments, the upstream signal pathway activator is selected from inflammatory factors and/or proteins; in some embodiments, the inflammatory factor is selected from the group consisting of: at least any one of TNF- α, IL-1β, IL-4, IL-6, IL-8, and IL-12; in some embodiments, the protein is selected from the group consisting of: at least any one of CBP and p300 proteins

In some embodiments, the agent that promotes the production or activity of IL-4, in whole or in part, is one or more of a fusion protein, polypeptide, compound.

In some embodiments, the cartilage is selected from the group consisting of articular hyaline cartilage, fibrocartilage, elastic cartilage; in some embodiments, the articular hyaline cartilage is selected from the group consisting of knee menisci, shoulder articular cartilage, hip articular cartilage; in some embodiments, the hyaline cartilage is selected from costal cartilage, nasal cartilage, laryngeal cartilage, tracheal cartilage, bronchial cartilage; the fibrocartilage is selected from the fibrous ring outside the intervertebral disc, the mandible and the collarbone; in some embodiments, the elastic cartilage is selected from the group consisting of outer ear, epiglottis.

In some embodiments, the route of administration of the drug is selected from at least any one of oral administration dosage form, topical administration dosage form, intranasal administration dosage form, systemic administration dosage form, intravenous administration dosage form, subcutaneous administration dosage form, intramuscular administration dosage form, intraventricular administration dosage form, intrathecal administration dosage form, transdermal administration dosage form.

In some embodiments, the dosage form of the medicament is selected from at least any one of a tablet, a capsule, a soft capsule, a gel, an oral preparation, a suspension, a granule, a patch, an ointment, a pill, a powder, an injection, an infusion solution, a freeze-dried injection, an intravenous emulsion, a liposome injection, a suppository, a sustained release preparation, and a controlled release preparation.

In some embodiments, "treatment" refers to a method of achieving a beneficial or desired result, including but not limited to a therapeutic benefit. Therapeutic benefits include, but are not limited to, eradication, inhibition, reduction or amelioration of the underlying disorder being treated. In addition, therapeutic benefit is achieved by eradicating one or more of the physiological symptoms associated with the underlying disorder, such that an improvement is observed in the patient, but the patient may still have the underlying disorder.

In some embodiments, "prevention" is used herein to refer to a method of achieving a beneficial or desired result, including but not limited to, a prophylactic benefit. To obtain a prophylactic benefit, the pharmaceutical composition may be administered to a patient at risk of developing a particular disease or to a patient reporting one or more physiological symptoms of a disease, even if the disease has not been diagnosed.

IL-4 is a pleiotropic cytokine produced by activated T cells and is a ligand for the IL-4 receptor. IL-4 receptors also bind IL-13. Thus, IL-13 can also be used as a targeting moiety to target IL-4 receptors.

The invention repeatedly confirms the treatment and prevention effects of IL-4/IL-4R in the cartilage degeneration and the intervertebral disc degeneration and other related diseases by adopting a cell and animal model.

The invention firstly extracts the human cartilage-like cells for culture passage, constructs a cartilage cell degeneration model by inducing and stimulating the cartilage-like cells through TNF-alpha, and then adopts IL-4 for treatment, thereby verifying that the IL-4 can inhibit the nucleic acid level of various inflammatory factors in the cells.

The invention constructs a rat posterior puncture intervertebral disc degeneration model by a lumbar 4/lumbar 5 intervertebral space posterior puncture mode, verifies the influence of IL-4 and an IL-4R receptor inhibitor Dupilumab on rat intervertebral disc degeneration by adopting an intra-intervertebral-disc injection mode, and shows that the IL-4 can obviously relieve the rat model intervertebral disc degeneration, and the IL-4R receptor inhibitor Dupilumab obviously inhibits the relieving effect.

Dupilumab (trade name Dupixent) is a monoclonal antibody drug targeting IL-4 receptor, is approved by FDA for the first time in 3 months and 28 days in 2017, can bind to IL-4 and IL-13 common receptor module IL-4Rα, simultaneously block IL-4 and IL-13 signals and downstream signal channels mediated by the IL-4 and the IL-13 signals, and has good control effect on various type 2 immune-related inflammatory diseases, such as inhibiting the occurrence and development of asthma airway inflammation, airway hyperreactivity and airway remodeling. Currently, dupilumab has acquired 3 indications, asthma, chronic rhinosinusitis with nasal polyps, and atopic dermatitis, respectively.

The invention collects the nucleus pulposus tissue of the rat model and detects inflammatory factors such as TNF-alpha, IL-1 beta and the like in the nucleus pulposus tissue, and the result shows that IL-4 obviously reduces the inflammatory factors such as TNF-alpha, IL-1 beta and the like in the nucleus pulposus tissue, the reduction of IL-1 beta is more obvious, and the reduction is obviously inhibited by the IL-4R receptor inhibitor Dupilumab.

The invention collects the Dorsal Root Ganglion (DRG) of the rat model, detects the expression of the pain related factor CGRP calcitonin gene and related peptide in the DRG, and discovers that IL-4 obviously reduces the expression of the pain related factor in the DRG after detection, and the IL-4R receptor inhibitor Dupilumab obviously inhibits the reduction.

Finally, the invention detects pain behaviours of the rat model, and detects the influence of IL-4/IL-4R on the pain behaviours of the rat by means of mechanical stimulation threshold measurement and thermal stimulation threshold measurement of the hind legs of the rat. The results show that IL-4 significantly reduces the mechanical and thermal pain thresholds in rats and reduces the pain behavior in rats, while the IL-4R receptor inhibitor Dupilumab significantly inhibits this reduction.

Drawings

Fig. 1 is a schematic representation of painful disc degeneration. Wherein during disc degeneration, the extracellular matrix primary content, type II collagen and proteoglycans are significantly reduced, while type I and III collagen are increased. The cytokines, chemokines and pain-related factors produced cooperate to promote the development of discogenic pain. IVD: a disc Lumbar intervertebral disc; NP: a nucleus pulposus; AF: and (3) fibrous ring.

FIG. 2 shows that IL-4 can regulate the anabolic and inflammatory levels of NP cells. Wherein: (A-B) nucleic acid levels of Aggrecan (Aggrecan) and collagen II were analyzed by rt-PCR after 12 hours of treatment with TNF- α (50 ng/ml) and IL-4 (10 ng/ml). (C-F) MMP3, MMP13, ADAMTS4 and ADAMTS5 levels were analyzed by rt-PCR after 12 hours of treatment with TNF- α (50 ng/ml) and IL-4 (10 ng/ml). (G-H) after 12 hours of treatment with TNF- α (50 ng/ml) and IL-4 (10 ng/ml), the levels of TNF- α and IL-1β nucleic acids were analyzed by rt-PCR. Data are shown as mean.+ -. SEM (n.gtoreq.3). * p <0.05 is compared to the vector. #p <0.05 compared to previous treatments. Unless specifically mentioned, one-way anova with Bonferroni post-hoc test was used. MMP3: matrix metalloproteinase 3; MMP13: matrix metalloproteinase 13; ADAMTS4: a Disintegrin and Metalloproteinase with Thrombospondin motifs 4; ADAMTS5: a Disintegrin and Metalloproteinase with Thrombospondin motifs 5.

FIG. 3 shows the effect of IL-4 on disc degeneration IVDD (intervertebral disc degeneration) in vivo. Wherein: (A) The spinal discs of model rats were H & E stained with IL-4 and Dupilumab, sirius Red (SR) stained and Toluidine Blue (TB) stained. Panel (B) is the disc degeneration score for the disc HE staining of panel A. Scale bar: 100 μm. Each group n=8. Scale bar: 20 μm. Each group n=8. Data are shown as mean ± SEM. One-way ANOVA with the level test followed by a post-hoc test with Tukey was used. Du: dupilumab.

FIG. 4 is a graph showing the effect of IL-4 on inflammation in vivo. Wherein: immunohistochemical staining (A) and quantitative analysis of TNF- α and IL-1β in the spinal column of IVDD rats (B, C). Scale bar: 20 μm. Each group n=8. Data are shown as mean ± SEM. One-way ANOVA with the level test followed by a post-hoc test with Tukey was used. Du: dupilumab.

FIG. 5 shows the effect of IL-4 on the chemical pain factor of the IVDD rat model. Wherein: (A) CGRP (Calcitonin gene relatedpeptide calcitonin gene and related peptide) in IVDD rat DRG was immunohistochemically stained and quantitatively analyzed with IL-4 and Dupilumab. Scale bar: 100 μm. n=8 per group. (B) is a quantitative analysis of CGRP in the graph (A). P: and (3) puncturing.

FIG. 6 shows the effect of IL-4 on pain in an IVDD rat model. Wherein: (A-B) 50% withdrawal threshold and thermal stimulation withdrawal latency for sham, puncture, puncture+IL-4, and puncture+IL-4+Dupilumab group puncture models. Data are shown as mean ± SEM. In (A-B) a one-way analysis of variance with Bonferroni post-hoc test was used. Two-way repeat ANOVA with Bonferroni post-hoc testing was used to evaluate pain testing of the same animals at multiple time points.

FIG. 7 is a graph showing that in some embodiments, the IL-4/IL-4R axis promotes enhanced anabolism of cartilage-like cells by improving the microenvironment of cartilage tissue, reducing the release of inflammatory and pain factors, and ultimately, pain.

Detailed Description

The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.

Experimental equipment and materials

1. The main instrument is shown in table 1.

TABLE 1

2. The PCR experimental apparatus is shown in Table 2.

TABLE 2

3. The Western Blot assay apparatus is shown in Table 3.

TABLE 3 Table 3

4. The experimental equipment for wax sheet production is shown in table 4.

TABLE 4 Table 4

5. The rat experimental equipment is shown in table 5.

TABLE 5

6. The main reagents are shown in Table 6.

TABLE 6

7. The main reagents of fluorescent quantitative PCR are shown in Table 7.

TABLE 7

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8. Paraffin sections and staining were performed with the main reagents as shown in Table 8.

TABLE 8

9. The immunohistochemical major reagents are shown in table 9.

TABLE 9

Cell experiment

1. Human cartilage-like cell extraction, cryopreservation and culture

The following steps are completed in a sterile super clean bench: the complete nutrient broth (using antibiotics: 1% penicillin and streptomycin, DMEM broth and 5% foetal calf serum) of the cartilage-like cells prepared in advance was taken out and poured into a sterile 6cm dish. The nucleus tissue was removed from the sterile cotton gauze, removed using sterile forceps, and washed 2 times with 2 dishes containing PBS. The nucleus pulposus tissue was then immersed in the complete culture solution for 1 minute. Simultaneously, the complete medium containing 0.5% collagenase was removed and poured into a petri dish. The nucleus pulposus tissue is soaked and crushed using sterile ophthalmic scissors. Mixing with collagenase culture medium, and placing at 37deg.C, 5% CO ₂ Is kept for 2 hours. Subsequently, the soaked and digested pieces of nucleus pulposus tissue are removed. At 4 ℃. Centrifugation at 1200rpm/min for 3min, the underlying nucleus pulposus tissue was retained. Then using the prepared complete culture medium to blow and wash nucleus pulposus tissues, fully centrifuging, removing supernatant, repeating the above operation for 3 times, and finally cleaning and completely removing the nucleus pulposus tissues by 25cm ² The culture solution is added into the culture flask in advance, and then the treated nucleus pulposus tissue is added. Placed under 37 ℃ condition, 5% CO ₂ Is cultured (the cells produced in this process are P1 generation). After 24 hours, the cells were gently removed, and the cells were observed to climb out, and if the cells were small, the cells were delayed to 48 and 72 hours. The subsequent culture is preferably carried out by replacing the medium every 48 hours.

After the cells grow gradually, the cells are observed under a microscope, and the cells are passaged by cartilage-like cells in each field of view of 70-80%. Experiments were performed using 2 nd-4 th generation Nucleus pulposus (cells, NPCs).

2. Passaging of NPCs

1) The cultured cells were taken out gently every day, and the cells were subjected to microscopic examination every day, and when the cells were grown to less than 70%, only the liquid change treatment was performed, and when the cultured cells were grown to about 80% of the culture flask, the cells were considered to be passaged. The complete medium to be used is taken out of the 4-degree refrigerator in advance with PBS, the cells are taken out of the incubator to an ultra clean bench, all the medium is sucked out, and the culture flask is cleaned 2 times with 4ml PBS purchased. A total of about 1ml of 0.25% trypsin solution was added, and the mixture was removed from the incubator at 37℃for 1 min.

2) Cells were observed under a microscope to be round and floating in the medium. The flask was removed and gently placed in an ultra clean bench and digestion stopped by adding 3ml of pre-prepared complete medium thereto. Then a pipetting gun is adopted, the bottom of the culture flask is gently and repeatedly blown (can be gently tapped by hands), and finally, a 15ml centrifuge tube is gently added to all the culture medium containing cells.

3) And (3) carrying out accurate balancing, finally, putting the centrifuge tube into a centrifuge, preferably centrifuging at 1000rpm/min for 5min, taking the centrifuge tube back to an ultra-clean bench, and rapidly pouring (or sucking out) the supernatant.

4) The culture medium is put in an ultra clean bench, preferably about 2ml of the prepared complete culture medium is added, and the cells are repeatedly blown (can be gently beaten by hands) to be resuspended, and two new culture flasks prepared in advance are opened for inoculation operation. 3ml of complete medium was added to each flask in advance. 2 flasks were returned to 37℃with 5% CO ₂ Is cultured in an incubator of (a).

3. Cell resuscitation

1) Injecting clean water into the water bath kettle in advance, setting the temperature at 37 ℃ in advance, waiting for 30min, rapidly taking out the freezing tube from the liquid nitrogen when the water temperature of the water bath kettle is stable at 37 ℃, rapidly bathing in 37 ℃ water according to the principle of slow freezing and instant dissolving, and taking the freezing tube out of the water bath kettle after no solid exists in the freezing tube.

2) Repeatedly sterilizing with 75% ethanol spray, and placing on ice bath.

3) Cells were aspirated into 4ml of complete medium.

4) 1000rpm/min, centrifuging for 5min, and reserving bottom cells.

5) 4ml of medium was added, gently swirled, the cells were thoroughly resuspended, and added to a new flask. And (5) placing the mixture back into an incubator for culture.

6) In the experimental record, the storage time, place, name of the resuscitated cells, and number are described in detail.

4. Treatment of chondrocytes with drugs

1) The cultured 2 nd-4 th generation human cartilage-like cells were inoculated in purchased 6, 12, 24-well plates.

2) The addition of cytokines such as TNF-alpha (sum) or IL-4 requires waiting for the cell to adhere to the wall (typically waiting for 2-6 h) and for the cell to grow to 60-70% of the under-the-mirror field.

3) The treated NPCs are subjected to related experiments such as PCR, WB, immunofluorescence and the like respectively.

Real-time PCR experiments

mRNA measurements of Aggrecan and Collagen II and MMP3, MMP13, ADAMTS4 and ADAMTS5 were performed on the treated cells with internal references to beta-actin and GAPDH.

1 extraction of RNA from cells

The TaKaRa RNA extraction kit was used. The method comprises the following specific steps:

1) Taking out transfected and medicated cells in incubator, washing out all the culture medium, then gently and repeatedly washing the cells with PBS at 4deg.C for three times, sucking out all PBS, and taking care that 10 μl pipette is used to completely suck out PBS as much as possible;

2) Adding cell lysate according to the content of 30 mu l of each hole of a 6-hole plate, covering all cells by the lysate, fully mixing the cells, and sucking the cells into a 1.5ml enzyme-free EP tube;

3) A volume of 200 μl of chloroform was drawn in advance, added to each EP tube before the next operation, and the two liquids were allowed to come into full contact (most of them were operated on an oscillator) by repeated shaking for about 40s, followed by standing for 3min;

4) The centrifuge was pre-chilled, followed by centrifugation of the resting EP tube at 12000rpm/min for 10min at 4 ℃. Transferring the uppermost layer of liquid of the lysate after centrifugation, and adding a new tube;

5) 500 μl of isopropanol was added to the EP tube, and the mixture was left to stand for 12min after shaking;

6) Centrifuging again at 12000rpm/min at 4deg.C for 10min to obtain RNA precipitate in the EP tube, thoroughly absorbing supernatant, soaking in 75% ethanol for 2 times;

7) DEPC water was added in an amount of 20. Mu.l per well to a 6-well plate, and left for 10min.

2 detection of the concentration of Total RNA in cells

1) Washing the Nanodrop ultraviolet spectrophotometer by using TE buffer solution, and then correcting;

2) 2. Mu.l of RNA was taken from each EP tube, diluted to 400. Mu.l with TE solution, and measured for A260 using a Nanodrop ultraviolet spectrophotometer;

3) A260 multiplied by the dilution factor and multiplied by 40 equals the total RNA concentration.

3 construction of the Total cDNA within the cell

This operation was carried out with reference to the reverse transcription kit of TAKARA corporation of japan, and the reaction system is shown in table 10:

table 10

Using a PCR apparatus, reverse transcription reaction: 30℃for 10min,42℃for 60min,99℃for 5min, and 55℃for 5min (1 cycle).

4 PCR amplification

Reference SYBR Premix Ex Taq (Takara) operations. The reaction system is shown in Table 11:

TABLE 11

The two-step PCR amplification was performed by adding fluorescent dye (Takara), sequence and RT products according to the instructions, and the amplification procedure used in the Bio-Rad Real-time PCR apparatus was mainly shown in Table 12:

table 12

Animal model building

Animal experiments are essential for studying painful disc degeneration and inflammation. The establishment of the intervertebral disc cartilage degeneration model can provide an experimental method for researching painful intervertebral disc degeneration. In an ideal situation, primates are the ideal choice for studying LBP among all species, as they are the species closest to humans. However, using primates as animal models is expensive and involves ethical problems in experimental animals. As are many other large animal models. Therefore, rodent models are the most widely used animals to study LBP. Because of the generally long time for establishing the IVDD model, according to the analysis of the existing intervertebral disc degeneration and pain model, a rat intervertebral disc posterior puncture model is selected in rodents, and the model is currently a world-recognized model with definite phenotype, high stability, relatively easy realization and relatively low price [75].

1 laboratory animal

In this subject, sprague-Dawley (SD) rats, about 230g, were used for animal experiments, and about 8-12 weeks of age. Purchased from a first hospital animal experiment center affiliated to the university of Zhongshan. Raising was performed in a professional SPF-class animal house. Compliance with regulations of the ethics committee of animals affiliated with the first hospital of university of chinese and receiving examinations and spot checks by the animal laboratory and ethics committee.

2 modeling

1) Surgical instruments ready for purchase in advance are left for use after autoclaving at a public laboratory platform of the university of Zhongshan, traditional Chinese medical college. A 21 gauge needle, two ophthalmic scissors, two ophthalmic forceps, two small gauge scissors, two small gauge forceps, iodophor, 75% ethanol, sodium pentobarbital, etc. were prepared in advance in the laboratory.

2) The following operations were completed at the SPF-grade animal laboratory affiliated with the first hospital at the university of Zhongshan. The pentobarbital solution is prepared by using sterile physiological saline in advance, and is anesthetized according to the measurement of 40mg/kg by adopting a professional intraperitoneal injection mode. The skin preparation operation is gentle, the prone position is adopted, the special shaver is adopted to shave the hairs on the chest, back, waist and back of the rat, and the hairs are sterilized once in advance by using iodophor and alcohol.

3) The rat is moved to an aseptic operating table prepared in advance, an aseptic operation towel is laid, and the prone position posture is adopted, so that the limbs are fixed. The bilateral iliac spines were identified by hand stroking, using the principle that the location of the line connecting the highest points of the iliac spines was relative to the L5/L6 intervertebral space. A line drawing pen is adopted to draw a line, and in an experiment, the puncture point is L4/L5 intervertebral space. According to the professional surgical procedure, the iodophor is sterilized, and then alcohol is used for deiodination according to the procedure.

4) The intervertebral space of L4/L5 above the drawn line is used as an operation center, the back is cut in a middle way, the back skin is cut in order for about 3cm at a time, the small animal hemostatic forceps blunt separate subcutaneous tissues, stop bleeding in time, cling to the left spinous process, cut and separate the muscles beside the vertebrae, stop bleeding in time, the muscles beside the vertebrae are abundant and more in blood supply, the operation can be carefully performed, the damage to excessive waist and back muscles is avoided, the vertebral lamina of the spine is seen after all muscles are separated, and the articular process is found up and down. Gradually exposing left articular processes of L4/L5 and cleaning surrounding tissues and muscles.

5) Nerve was dissected under the scope of a small animal surgical microscope, and L4 left hemilamina (laminectomy) and L4/L5 facet joint resections (facetectomy) were performed using tools such as mini rongeurs and small animal surgical retractors. The bone that is snapped off each time is very small and carefully handled. Gradually exposing dorsal root ganglion on left side of L4 and nerve root on left side of L5, cleaning blood clot and tissue, stopping bleeding in time, and displaying spinal cord. The nerve stripper was used to gently pull the L5 nerve root and spinal cord to the right, and the puncture window and L4/5 disc were visible to the naked eye. The 21G injector is adopted, and the direction parallel to the end plate avoids other tissues, so that the intervertebral disc is directly penetrated, and the obvious penetration feeling can be felt after the intervertebral disc is penetrated. Nerve stripping is properly loosened during the puncturing process, and nerve roots and spinal cords are not continuously pulled. The puncture needle enters the annulus fibrosus for about 3mm, maintains the puncture state and keeps still, so that the puncture needle is slowly pulled out after about 1 minute in the intervertebral disc. After puncture, nerve stripping is removed, nerve roots and spinal cords are checked to be harmless, and after no obvious bleeding points are found, suturing is started.

6) The rat spinal disc to be treated was punctured by the same method using a microinjector (Shanghai Gao Ge), and 2. Mu.l each of the formulated Dupilumab and IL-4 was slowly injected using a microinjector.

7) Muscles were sutured layer by layer using 3-0 filaments, and the skin was carefully sutured with 4-0 filaments. Removing blood clot, and stopping bleeding by gelatin sponge. After awakening was observed, the animals were returned to the rearing room and the rearing conditions were observed daily.

8) Control rats were sutured as described above until after exposure of the L4/L5 disc.

Pain behavioural detection

Pain behavioural detection is a common method for detecting pain perception in animals by detecting changes in their behaviour.

The rats in the experimental group and sham group were tested using pain behavioural.

Rats were placed in laboratory cages 1h in advance, so that the rats were used in an experimental environment in advance, and the state was stable and stress behavior was not being generated. The time points for detection of pain behavior were performed every 3 days, on preoperative days 5 and 2, and on postoperative days 1 to 37, for a total of 13 post-operative years.

The pain behavioural test contents include: detection of mechanical stimulation threshold of hind limb and thermal stimulation threshold of hind limb.

1. Mechanical stimulation threshold determination

1) The mechanical stimulation threshold was measured on the rat hind paw using von Frey fiber pen [75,76] to determine the change in mechanical pain in the rat. Depending on its particular fiber structure, the test pen has a total of 10 mechanical strengths including 3.61, 3.84, 4.08, 4.17, 4.31, 4.56, 4.74, 4.93, 5.07 and 5.18g. Before the fiber pen is used, the bending degree and the cleaning degree of the fiber pen hair are checked, so that the measurement accuracy is ensured.

2) The rats to be tested are placed in a specific metal net cage in advance, so that the rats are quietly adapted to the environment for 1h. Rats were calm without stress and after no stress behavior, the experiment was started.

3) The von Frey pen penetrates through the bottom of the metal net cage and penetrates deep into the inner cage to puncture the sole and sole part of the rat, and each time, the strength starts from 4.31 g. If the rat does not have obvious response to puncture, replacing the fiber pen with primary strength for puncture again; if the rat has obvious foot withdrawing behavior, the fiber pen with the primary strength is replaced for puncture again. In the pain behavioural experiment, a positive response was determined if the following was present: when the von frey microscopic hairs are vertical, the rat is stimulated under the foot (the sole palm center part), and when the rat is in a slightly bent state due to stress, the rat is kept for 5-10s, if rapid foot withdrawal occurs, a positive reaction can be judged. At the last moment, the moment of stimulation out of the rat sole was also counted as a positive response. If the rats have walking activities and other activities for various reasons (abnormal sounds, light, article movement or vibration, etc.) during the measurement, the rats need to wait for about 10 minutes and then test again after they have newly entered a non-emergency resting state. The hind feet of the tested rats should have enough rest time of more than 10 s. The stimulation and response of the left and right hind legs were recorded. The 50% withdrawal threshold (50%withdrawal threshold) was measured using the report of Chaplan et al [77 ]:

50％withdrawal threshold＝(10[Xf+κδ])/10000

Wherein Xf = the log of the last test intensity; the kappa number is obtained according to a foot withdrawal reaction mode table (table 13); delta = mean of log differences between 10 von frey fiber pens.

TABLE 13

2. Thermal stimulation threshold determination

1) And (3) measuring the samples by using a thermal plate tester [75,76], and judging the condition of the rat that the thermal stimulus pain sense is sensitive by using a threshold measurement mode. Before using the hot plate tester, checking whether the instrument switch and the timing are good or not, and ensuring the measurement accuracy.

2) As in the above experiment, rats to be tested were placed in a specific glass cage in advance to make them quietly fit for 1h. Rats were calm without stress and after no stress behavior, the experiment was started.

3) The incubation period of heat stimulation for removing feet was measured by using a thermal plate tester for focusing a heat radiation light source, penetrating through the glass of the cage bottom, and directing the rat plantar heel directly (paw withdraw thermal latency).

4) In order to protect the rats from scalding tissues such as skin due to the reduction of the thermal stimulation sensitivity, the thermal stimulation time is set to be not longer than 24 seconds at maximum. In most cases, the withdrawal response occurs in rats at about 15-20 seconds of exposure to the heat radiation source. The hot light source will be automatically turned off. In general, when the foot is removed, the tester presses the switch, the light source is turned off, and the thermal flat plate tester automatically records the whole time length of the thermal stimulation, and the time recorded by the tester is the latency period value of the foot removal of the thermal stimulation. Both feet should be tested and alternately, and the hind feet of the tested rats should have enough rest time of more than 10 s. Referring to the methods reported by scholars such as George, the average value was calculated for statistical analysis on the double hind legs of rats 3 times each [78].

Sample collection, preservation, decalcification and tissue paraffin embedding section

The intervertebral discs and DRGs of rats subjected to the Sham group, the puncture +C646 group and the puncture +CTPB group are obtained, morphological staining is carried out on the intervertebral discs, immunohistochemical detection is carried out on the expression of TNF-alpha and IL-1 beta, and immunofluorescence detection is carried out on the expression of CGRP of the DRG.

1 sample collection and preservation

Rats in the Sham group, the puncture group, the puncture+inhibitor C646 group and the puncture+activator CTPB group were anesthetized in the same manner as described above. A thoracotomy procedure was performed, cannulated through the aorta, rapidly perfused with heparin saline, followed by perfusion with 4% paraformaldehyde. After the tissue is fixed, the animal is dissected, the dorsal root ganglion tissue at the left side of the waist 5 is taken out, the whole of the L4 and L5 vertebral bodies is taken out, 4% paraformaldehyde is quickly put in, and the total amount of the formaldehyde in the fixing solution is 25 times the volume of the fixed tissue.

2 Paraffin embedding section of tissue

1) In the fume hood, tissue is removed from the fixative solution and the spinal tissue is trimmed again using a surgical knife.

2) For spinal bone tissue, decalcification. A common method is to use a chelating agent which is an organic compound, and combine with metal ions in the specimen, and decalcification with sodium Ethylene Diamine Tetraacetate (EDTA) does not substantially affect staining. Stepwise soaking (3%, 8%, 11%, 14% and 18%) in gradient concentration EDTA solution. Decalcification at room temperature is carried out by placing decalcified bottle on shaking table and shaking continuously for 30-60 days.

3) Dehydrating: paraffin waxes are difficult to blend with biological tissues because they contain a large amount of moisture. It is necessary to remove the water using a dehydrating agent. In capped bottles, 40%, 50%, 75%, 80%, 75%, 100% to full dehydration were performed starting from 20% ethanol. And (3) carrying out dehydration treatment on the DRG specimen and the decalcified spine specimen.

4) Embedding: treating the above tissue, embedding liquid: commercial paraffin wax. Embedding is to completely wrap the above-mentioned tissue block impregnated with wax in prepared paraffin. Slowly pouring the constant-temperature pure molten wax into a box, taking out the tissue blocks by using preheated tweezers, horizontally placing the tissue blocks at the bottom of the box, enabling the tangential direction to be downward, gently taking the box, putting cold water into the box for cooling, taking out the box for 30min, and finally putting the box into an embedding frame, preferably all marks.

5) Slicing: observing the slicing knife under a magnifying glass to determine that the slicing knife is sharp and has no obvious defect, otherwise, the slicing knife is incomplete, and the slicing knife is off. The wax block is placed so as to be parallel to the tangential plane. Allowing it to leak out a small portion. After the blade is arranged, the tangent plane and the blade form an included angle of 15 degrees, and the upper edge and the lower edge of the wax block are parallel to the knife edge. The machine runner is rotated fast by the right hand, the writing brush is held by the left hand and is continuously arranged below the knife edge, all the cut wax strips are supported, the wax strips to be cut are formed to a certain length, the rotation is stopped as soon as possible, the other writing brush is used for slowly and gently picking up and horizontally placing the writing brush in the paper box, the slicing speed cannot be too high, and the cutting speed can be evenly used for forcefully rotating. And after slicing, wiping the slicing machine clean.

6) And (3) sticking: the thickness of the sheet was 4. Mu.m. The tissue was flattened at 37℃with warm water, and the tissue was removed using a slide and placed in a 60℃oven. And (5) baking the water. And then taking out and storing for standby.

Morphological staining and immunohistochemistry of spinal specimens

1HE staining

1) Dewaxing and dewatering the slice, putting the slice into the prepared xylene I for 20min, adding the slice into the xylene II for 20min, adding the slice into the absolute ethyl alcohol I for 10min, adding the slice into the absolute ethyl alcohol II for 10min, adding the slice into the absolute ethyl alcohol I for 5min, adding the slice into the absolute ethyl alcohol II for 5min, adding the slice into the absolute ethyl alcohol for 90% for 5min, adding the slice into the absolute ethyl alcohol for 5min, adding the slice into the absolute ethyl alcohol for 80% for 5min, adding the slice into the absolute ethyl alcohol for 70% for 5min, and washing the slice by distilled water.

2) Staining nuclei (staining solution: hematoxylin): the sections were slowly soaked with Harris hematoxylin for 8min, washed with clean tap water, differentiated with 1% hydrochloric acid for about 10s, rinsed again with clean tap water, then returned to blue with 0.6% ammonia water, and finally rinsed again with clean running water.

3) Using eosin chromatin: after the sections were gently placed in eosin dye solution, they were stained for 2-5min.

4) And (3) removing the water sealing piece: adding the processed slice into 95% alcohol I for 5min, then adding into 95% alcohol II for 5min, adding into absolute ethanol for 5min, then adding into absolute ethanol II for 5min, then adding into xylene I solution for 5min, then adding into xylene II solution for 5min, taking out the slice, completely airing, and sealing with the finished neutral resin.

5) Microscopic examination, image acquisition and analysis.

2 immunohistochemistry

1) And (3) conventionally dewaxing and dehydrating the paraffin slices after the preparation, putting the slices into a prepared xylene I solution for 20min, then adding the slices into a xylene II solution for 20min, then adding the slices into an absolute ethanol I solution for 10min, then continuously adding the slices into the absolute ethanol II solution for 10min, then adding the slices into a 95% ethanol solution for 5min, then adding a 90% ethanol solution for 5min, then adding the slices into a 80% ethanol solution for 5min, finally adding the slices into a 70% ethanol solution for 5min, and finally washing the slices by using distilled water.

2) Antigen retrieval: the whole slice prepared above is gradually added with antigen retrieval buffer solution, and the antigen retrieval operation is carried out in a microwave oven. EDTA buffer is filled up, and the microwave oven is turned to medium and low. Lasting for 8min,8min and 7min, respectively. And subsequently cooled. The sections are washed with PBS, preferably 3 times in total, for approximately 5min.

3) Endogenous peroxidases: the above treated sections were gradually dripped with 4% hydrogen peroxide and incubated in the dark for 30min, after which the sections were washed with PBS for a total of 3 washes, preferably for approximately 5min.

4) The blocking process was completed with formulated BSA: the pencil was assembled, and a complete circle was drawn at the far point of the tissue border, and blocked with 5% BSA for 30min.

5) Adding an antibody: gently discard the blocking solution, drop first antibody, refrigerator overnight.

6) Adding a secondary antibody: the PBS was washed 3 times, 5 min/time. The whole surface was covered with secondary antibody (HRP-labeled), incubated for 1h.

7) DAB color development: on a shaker, the slices were washed 3 times with PBS prepared in advance, each time lasting approximately 5min. Fresh DAB color development liquid prepared in advance is added dropwise. Positive brown yellow color appears, the time is controlled, and finally distilled water is used for washing.

8) The Harris hematoxylin stained sections were slowly soaked for 8min, washed with clean tap water, and differentiated with 1% hydrochloric acid for about 10 s. The mixture was rinsed again with clean tap water, then blued with 0.6% ammonia water, and finally washed with distilled water.

9) And (3) removing the water sealing piece: and adding the processed slice into 95% alcohol I solution for 5min, then adding into 95% alcohol II solution for 5min, adding into absolute alcohol solution for 5min, then adding into absolute alcohol II for 5min, then adding into xylene I solution for 5min, then adding into xylene II solution for 5min, taking out the slice, completely airing, and sealing the slice by adopting a finished neutral resin.

10 Microscopy, image acquisition and analysis.

11 Immunohistochemical interpretation: hematoxylin-stained nuclei were blue and DAB showed positive expression as brown yellow.

Statistical method

The subject uses SPSS statistical analysis applications to analyze data. Statistical analysis SPSS Statistics MAC statistical analysis software was used for each statistical analysis. Results are expressed as mean ± standard deviation, number or percentage. Unpaired Student's t-test and paired sample t-test were used to compare between two sets of data for a normalization test using the shape-Wilk test. One-way analysis of variance and level variance homogeneity test, and data from more than three groups were compared using Tukey post test or Bonferroni post test. Two-way repeated measures analysis of variance (ANOVA) of Bonferroni post hoc tests were used to evaluate pain testing of the same animals at multiple time points. P <0.05 indicates that the difference is statistically significant. The p-value is recorded in a statistical chart. Sample size is incorporated into text and graphic legends, with each replicated individual data point depicted as a small circle in all figures. The number of independent replicates for each experiment is included in the graphical illustration.

Example 1

Dissolving 50ng/ml TNF-alpha in PBS for 24 hr is the method of inducing cartilage-like cell to construct cartilage cell degeneration model. Human cartilage-like cells were first extracted for culture passage, and when the cells were cultured to the third generation, serum-free low-sugar culture medium was used for changing the liquid and the cell culture box was left for 12 hours, then the medium was aspirated, and the cell culture medium containing 50ng/ml TNF-alpha and/or IL-410ng/ml cytokines was re-added for 24 hours, finally it was verified that IL-4 could inhibit the nucleic acid levels of various inflammatory factors in the cells (FIG. 2).

Aggrecan (Aggrecan) is a large proteoglycan that contains large numbers of chondroitin sulfate and keratan sulfate chains, giving articular cartilage the ability to withstand compressive loads. It exists in the extracellular matrix in the form of proteoglycan aggregates, in which many aggrecan molecules interact with hyaluronic acid and function as a junction protein and stabilize the interactions between each protein. The structure of aggrecan is not constant throughout life, but varies due to synthetic and degradation events. Any substance that causes cartilage aggrecan degradation can be considered detrimental to cartilage function. This process results in depletion of aggrecan and susceptibility to cartilage erosion. Collagen II is the basis for articular cartilage and hyaline cartilage, and the fibrous network that forms allows cartilage to capture proteoglycan aggregates and provide tensile strength to tissue. Both Aggrecan and Collagen II decrease marked the occurrence of cartilage degeneration.

Matrix metalloproteinases (including MMP3, MMP13, ADAMTS4, and ADAMTS 5) play a major role in aggrecan degradation, and their production is intimately associated with cartilage degeneration and up-regulation of inflammatory mediators (including TNF- α and IL-1β). Thus, aggrecan and its degradation related enzymes are involved in cartilage health and survival.

FIG. 2 shows that Aggrecan and Collagen II are significantly reduced (A-B) in cartilage-like cells after TNF- α addition by constructing a model of cartilage-like cell degeneration, while MMP3, MMP13, ADAMTS4 and ADAMTS5, etc. are significantly elevated (C-F), indicating that cartilage-like cells are significantly functionally degenerated. At the same time, TNF- α itself can induce the production of a number of inflammatory mediators (G-H), including TNF- α and IL-1β; subsequently, IL-4 alone was found not to promote the levels of Aggrecan and Collagen II in cartilage-like cells upon addition of IL-4, but it significantly inhibited matrix degradation-related enzymes MMP3, MMP13, ADAMTS4, ADAMTS5, and the like, and inflammatory cytokines (including TNF- α and IL-1β), indicating their role in protecting cartilage-like cells.

Example 2

A rat posterior puncture intervertebral disc degeneration model is constructed by a lumbar 4/lumbar 5 intervertebral space posterior puncture mode, the influence of IL-4 and an IL-4R receptor inhibitor Dupilumab on rat intervertebral disc degeneration is verified by adopting an intra-discal injection mode, and the result shows that the IL-4 can obviously relieve the rat model intervertebral disc degeneration, and the IL-4R receptor inhibitor Dupilumab obviously inhibits the relieving effect (figure 3).

Fig. 3B is a histological score of disc degeneration, each point representing the score of an independent sample.

Histological grading of disc degeneration (Thompson score)

The degree of spinal degeneration in the motion segment was assessed using Thompson scoring. Rat disc HE sections were scored by senior researchers and doctor without knowledge of study design. The Thompson scoring protocol is a five-class protocol for assessing the overall morphology of the lumbar disc. The scale is from 1 to 5, where 1 represents health and 5 represents end-stage disc degeneration, with the evaluation control table as follows.

TABLE 14 Thompson intervertebral disc histological grading Standard

Example 3

The results of collecting the nucleus pulposus tissue of the rat model of example 2 and detecting inflammatory factors such as TNF-alpha and IL-1β in the nucleus pulposus tissue showed that IL-4 significantly reduced inflammatory factors such as TNF-alpha and IL-1β in the nucleus pulposus tissue and that IL-1β was reduced more significantly, whereas the IL-4R receptor inhibitor Dupilumab significantly inhibited this reduction (FIG. 4).

Example 4

The Dorsal Root Ganglion (DRG) of the rat model of example 2 was collected and examined for the expression of the pain-associated factor CGRP in the DRG, and IL-4 was found to significantly reduce the expression of the pain-associated factor in the DRG, while the IL-4R receptor inhibitor Dupilumab significantly inhibited this reduction (FIG. 5).

Example 5

The rats of the rat model of example 2 were tested for pain behavior by means of mechanical stimulation threshold measurement and thermal stimulation threshold measurement of the rat hind paw, and the effect of IL-4/IL-4R on the pain behavior of the rats was tested. The results show that IL-4 significantly reduced the mechanical and thermal pain thresholds in rats and reduced the pain behavior in rats, whereas the IL-4R receptor inhibitor Dupilumab significantly inhibited this reduction (FIG. 6).

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Claims (10)

1. The use of IL-4/IL-4R signaling pathway as a target in the manufacture of a medicament for the treatment or alleviation of cartilage degeneration and/or disc degeneration.
2. Use of il-4 or an analogue or agonist thereof for the manufacture of a medicament for the treatment of cartilage degeneration and/or disc degeneration.
3. Use of il-4 or an analogue or agonist thereof in the manufacture of a medicament for a condition caused by cartilage degeneration;

   preferably, the disease caused by cartilage degeneration comprises: at least one of herniated disk, spondylolisthesis, spinal stenosis, degenerative scoliosis, isthmus fracture, or fracture of vertebral body of the spine, osteochondritis, osteoarthritis, degenerative arthritis, senile arthritis, hypertrophic arthritis, and hypertrophic arthritis.
4. Use of il-4 or an analogue or agonist thereof in the manufacture of a medicament for the treatment of pain.
5. 5. The use according to claim 4, wherein the pain is selected from pain caused by cartilage degeneration;

   preferably, the pain is selected from lumbago, skelalgia, and arthralgia;

   preferably, the pain is selected from lumbago.
6. 6. The use according to claim 4, wherein the IL-4 or an analog or agonist thereof treats pain by inhibiting matrix metalloproteinases and/or inflammatory cytokines;

   preferably, the matrix metalloproteinase is selected from at least any one of MMP3, MMP13, ADAMTS4, and ADAMTS 5;

   preferably, the inflammatory cytokine is selected from at least any one of TNF- α and IL-1β.
7. 7. The use of claim 4, wherein the IL-4 or analog or agonist thereof treats pain by inhibiting a pain-associated factor in the dorsal root ganglion;

   preferably, the pain-related factor is selected from CGRP.
8. 8. A drug development method for treating cartilage degeneration and/or intervertebral disc degeneration is characterized in that a corresponding IL-4 promoter is designed for IL-4.
9. 9. The method of claim 8, wherein the IL-4 promoter is one or more of an agent, an analog, and an activator of an upstream signaling pathway that promotes the production or activity of IL-4 in whole or in part;

   Preferably, the upstream signal pathway activator is selected from inflammatory factors and/or proteins;

   preferably, the inflammatory factor is selected from: at least any one of TNF- α, IL-1β, IL-4, IL-6, IL-8, and IL-12;

   preferably, the protein is selected from: at least any one of CBP and p300 proteins;

   preferably, the substance promoting the whole or partial generation or activity of IL-4 is one or more of fusion protein, polypeptide and compound.
10. 10. The use according to any one of claims 1, 2, 4-7, or the development method according to claims 8-9, wherein said cartilage is selected from the group consisting of articular hyaline cartilage, fibrocartilage, elastic cartilage;

    preferably, the articular hyaline cartilage is selected from the group consisting of knee menisci, shoulder cartilage, and hip cartilage;

    preferably, the hyaline cartilage is selected from costal cartilage, nasal cartilage, laryngeal cartilage, tracheal cartilage, and bronchial cartilage;

    preferably, the fibrocartilage is selected from the group consisting of the lateral annulus fibrosus of the intervertebral disc, the mandible, the collarbone;

    preferably, the elastic cartilage is selected from the group consisting of outer ear, epiglottis.