Pharmaceutical applications of SH2B adaptor protein 1 in the treatment of osteoarthritis pain

Abstract

The pharmaceutical applications of SH2B adaptor 1 in treating osteoarthritis pain: Knockout of the SH2B1 gene significantly increased mechanohyptiform hypersensitivity in osteoarthritis model mice, while neuron-specific overexpression of SH2B1 significantly improved pain tolerance in these mice. SH2B1 knockout significantly increased articular cartilage degeneration and proteoglycan loss scores in MIA-induced mice, and also increased activation of the spinal dorsal horn, hippocampal astrocytes, and microglia. Neuron-specific overexpression of SH2B1 significantly improved cartilage degeneration and proteoglycan loss scores, and reduced activation of the spinal dorsal horn, hippocampal astrocytes, and microglia. These findings suggest that SH2B1 could be used to develop drugs for relieving or treating osteoarthritis pain.

Description

Technical Field

[0001] This invention belongs to the field of osteoarthritis treatment technology, specifically relating to the pharmaceutical use of SH2B adaptor protein 1 in the treatment of osteoarthritis pain. Background Technology

[0002] Osteoarthritis (OA) is a common joint disease, a degenerative disease characterized by joint pain, caused by fibrosis, fissures, ulceration, and loss of articular cartilage due to multiple factors. It commonly affects the knee, hip, spine, and hands. OA is prevalent in middle-aged and elderly people, and its prevalence is gradually increasing with the aging population. Reduced chondrocyte synthesis and loss of proteoglycans due to enzyme activation that degrades the cartilage matrix are considered among the earliest events in OA, followed by mechanical damage to collagen fibers. The development of OA is mediated by multiple mechanisms, mainly including extracellular matrix (ECM) degradation, chondrocyte apoptosis, inflammatory response, angiogenesis, and autophagy. Its pathological features include articular cartilage degeneration and destruction, subchondral bone sclerosis or cystic changes, osteophyte formation at joint margins, synovial inflammation, joint capsule contracture, and ligament laxity or contracture. Osteoarthritis (OA) is one of the most common causes of chronic pain. The pain caused by OA differs from pain caused by other factors, including pain after prolonged walking, pain when squatting or sitting, and pain while walking. It severely impacts patients' quality of life and places a heavy burden on patients, families, and society. Therefore, alleviating and controlling chronic pain caused by OA is particularly important. The mechanisms of chronic pain caused by OA are not fully understood. Although many medications exist for controlling OA-related pain, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and weak opioids, they still cannot achieve adequate pain control, and long-term use of analgesics is detrimental to long-term health. Therefore, identifying specific molecules that improve chronic pain caused by OA is of great significance for further elucidating the mechanisms of its development and progression, and can provide new targets and strategies for the clinical prevention and treatment of chronic pain caused by OA.

[0003] Existing research has shown that central sensitization plays a crucial role in the chronicity of osteoarthritis (OA) pain, with an incidence rate as high as 20%-40% in patients with advanced knee OA. Reducing central sensitization can effectively alleviate OA pain and improve patients' quality of life. The dorsal horn of the spinal cord is the first station for pain information transmission and modulation. In a rat model of OA induced by sodium iodoacetate (MIA), peripheral nociceptors interact with sensory neurons in the dorsal horn of the spinal cord, hypersensitizing the spinal cord pain reflex pathway. The skin of the affected side of the rat's foot showed a stronger spinal cord response to mechanical stimulation. In addition to the neuron-neuron interaction in the dorsal horn of the spinal cord, activated glial cells can release various inflammatory mediators and neurotransmitters, which in turn activate multiple signaling pathways within glial cells and neurons, participating in the generation and maintenance of pain. Other studies have found that microglia in the dorsal horn of the spinal cord are in an activated state in OA pain models; inhibiting microglia activation can reduce painful behaviors in OA rats.

[0004] SH2B1 is a member of the Src homology 2B (SH2B) family, a group of adapters that includes two other members, SH2B2 (formerly APS) and SH2B3 (also known as Lnk). They share the same domains: an SH2 domain, a PH domain, a dimerization domain, and numerous proline-rich regions. The SH2B1 gene encodes four isoforms (α, β, γ, and δ), differing only in the C-terminus following the SH2 domain. Because they contain conserved PH and SH2 domains, these four isoforms have similar functions. Deletion of the SH2B1 gene can lead to morbid obesity and type 2 diabetes. It can also inhibit JAK2 / IRS2 signaling, increasing appetite and / or decreasing energy expenditure in mice, leading to weight gain and potentially metabolic syndrome (polyphagia, hyperleptinemia, hyperinsulinemia, and fatty liver). In bronchogenic mice, SH2Bβ expression was significantly enhanced in inflammatory cells in the lungs, airway epithelium, and adventitia, suggesting that SH2Bβ may participate in the pathogenesis of asthma under the influence of nerve growth factor. Other studies have shown that SH2B1 inhibits inflammation, apoptosis, and ROS in cardiomyocytes during myocardial ischemia-reperfusion injury (MIRI) through the PI3K / AKT pathway. Previous research in our group found that SH2B1 deficiency may lead to impaired PLIN4 degradation, increasing the accumulation of brain lipid droplets and lipid peroxidation levels in Parkinson's disease (PD), ultimately resulting in apoptosis of dopaminergic neurons. The SH2B1 / HSC70 / PLIN4 / LD axis may be related to the pathogenesis of neurodegenerative diseases and holds promise as a novel therapeutic target. SH2B1 is involved in the pathogenesis of Parkinson's disease, diabetes, obesity and obesity-related metabolic syndrome, asthma, and MIRI. However, there are currently no reports on the role of SH2B1 in osteoarthritis. SH2B1 may be a potential drug target for the treatment of OA. Summary of the Invention

[0005] Technical problem to be solved: In order to overcome the defects and deficiencies of the prior art, the present invention has determined the relationship between SH2B1 expression and osteoarthritis, and provides a pharmaceutical use of SH2B adaptor protein 1 in the treatment of osteoarthritis pain.

[0006] Technical solution: Application of SH2B adaptor protein 1 as a drug target in screening drugs for treating osteoarthritis pain.

[0007] Application of SH2B adaptor protein 1 as a drug target in the preparation of kits for screening drugs to prevent, relieve and / or treat osteoarthritis pain.

[0008] The use of SH2B adaptor protein 1 agonists in the preparation of drugs for the prevention, relief and / or treatment of osteoarthritis pain.

[0009] A drug for the prevention, relief and / or treatment of osteoarthritis pain, comprising an agonist of SH2B adaptor protein 1.

[0010] The agonist of the aforementioned SH2B adaptor protein 1 is one of the following: oeRNA of the SH2B1 gene, RNA overexpression vector of the SH2B1 gene, and other agonists that can increase SH2B1 expression.

[0011] For example, using SH2B1 as a target gene, double-stranded oeRNAs that can increase SH2B1 expression can be designed. After being synthesized chemically, SH2B1 gene expression can be increased through RNA overexpression to treat osteoarthritis pain. Alternatively, small molecule compound agonists can be designed with SH2B1 as a target. Using in vitro cell models or animal models of SH2B1 gene overexpression, molecules that can specifically activate SH2B1 can be screened to provide new therapeutic molecular targets for the treatment of osteoarthritis pain.

[0012] Beneficial Effects: This invention discovered a novel function of the SH2B1 gene. Results showed that SH2B1 gene knockout (KO) significantly increased mechanohyperthermia in osteoarthritis model mice, while neuron-specific overexpression (Tg) of SH2B1 significantly improved pain tolerance in these mice. SH2B1 KO significantly increased articular cartilage degeneration and proteoglycan loss scores in MIA-induced mice, and also increased activation of the spinal dorsal horn, hippocampal astrocytes, and microglia. Neuron-specific overexpression of SH2B1 significantly improved cartilage degeneration and proteoglycan loss scores, and reduced activation of the spinal dorsal horn, hippocampal astrocytes, and microglia. These findings suggest that SH2B1 can be used to prepare drugs for relieving or treating osteoarthritis pain. This invention is the first to validate the therapeutic effect of SH2B1 in osteoarthritis pain, demonstrating significant market value and social benefits. Attached Figure Description

[0013] Figure 1 The expression of SH2B1 in mouse and human articular cartilage (n=3) is shown. All data are expressed as mean±SEM, *p<0.05, **p<0.01 vs normal articular cartilage OA articular cartilage. A and B are the results of immunoblotting analysis and IMAGE J image analysis of the gray values ​​of the immunoblotting protein bands.

[0014] Figure 2The effects of sodium iodoacetate (MIA) on mechanical hyperalgesia in mice after SH2B1 conditional overexpression and knockout mice were investigated (n=10). Data are expressed as mean ± SEM. **p<0.01 vs. control group MIA group, #p<0.05 Wild-type mouse MIA group Knockout mouse MIA group ( Figure 2 :B), #p<0.05 Wild-type mouse MIA group overexpressing mouse MIA group ( Figure 2 :D). Where A and C are mouse behavioral test graphs, B and D are the areas under the curve (AUC) of ipsilateral and contralateral paw withdrawal values ​​(mechanical withdrawal) from baseline to 28 days after injection, a graph of subtracting the ipsilateral AUC from the contralateral AUC, and E is the treatment flowchart for the control group and the MIA group.

[0015] Figure 3 The effects of SH2B1 conditionally overexpressing mice and knockout mice with MIA modeling on articular cartilage degeneration in mice were investigated. Data are expressed as mean ± SEM, **p<0.01 vs control group MIA group, #p<0.05 SH2B1 KO mouse MIA group SH2B1Tg mouse MIA group. A represents microscopic results, B and C represent cartilage degeneration scores and proteoglycan loss scores. Scale bar: 100 μm.

[0016] Figure 4 The effect of MIA modeling in SH2B1 conditionally overexpressing mice and knockout mice on the activation of dorsal horn glial cells in the mouse spinal cord (n=4). Data are expressed as mean ± SEM. **p<0.01 vs control group MIA group, #p<0.05 knockout mice MIA group overexpressing mice MIA group ( Figure 4 :C), #p<0.05 knockout mice MIA group WT mice MIA group ( Figure 4 (D). A and B represent the immunofluorescence microscopic results of the L4 dorsal horn, while C and D represent the number of activated astrocytes and activated microglia in the L4 dorsal horn. Scale bar: 100 μm.

[0017] Figure 5 The effect of MIA modeling in SH2B1 conditionally overexpressing mice and knockout mice on the activation of glial cells in mouse hippocampus (n=4). Data are expressed as mean ± SEM. **p<0.01 vs control group MIA group, #p<0.05 knockout mice MIA group overexpressing mice MIA group ( Figure 5 (C, D). A and B represent the immunofluorescence microscopic results of hippocampal tissue, while C and D represent the number of hippocampal astrocytes and activated microglia. Scale bar: 100 μm. Detailed Implementation

[0018] The following embodiments are provided to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0019] Example 1

[0020] The relationship between SH2B1 expression and osteoarthritis was determined through experiments.

[0021] This invention uses articular cartilage from osteoarthritis patients and healthy controls, as well as normal mice and osteoarthritis mouse models. Total protein was extracted and subjected to SDS-PAGE-Western Blot analysis, combined with a specific SH2B1 antibody for detection, to determine the expression of SH2B1. The results showed that, compared with the control group, the expression of SH2B1 was significantly downregulated in the articular cartilage of humans and mice with osteoarthritis.

[0022] Immunoblot assay: Knee cartilage from OA patients and healthy controls was obtained from fresh tibial plateau fracture patients (ethics number 2020-SR-063, First Affiliated Hospital of Nanjing Medical University), human and mouse cartilage. After being minced in PBS at 4°C, centrifuged, and the precipitate was homogenized in RIPA solution. The homogenate was then centrifuged at 16000 rpm for 15 min at 4°C. The supernatant was transferred to 1.5 mL EP tubes, and protein concentration was determined using the BCA method. Proteins were denatured by boiling at 100°C for 5 min in sodium dodecyl sulfate (SDS) sample buffer and stored at -80°C for later use. Proteins were separated by SDS-PAGE (60 μg) and transferred to a PVDF membrane. The membrane was washed with Tris-Buffered Saline Tween (TBST), then blocked with 5% skim milk at room temperature for 1 h. Primary antibodies SH2B1 (1:600) and β-actin (1:5000) were diluted to appropriate concentrations and incubated overnight at 4°C. The next day, the membrane was washed 5-6 times with TBST, and secondary antibodies labeled with horseradish peroxidase were added. After incubation at room temperature for 1 h, the membrane was washed 5-6 times with TBST, and substrate chromogenic reagent was added. Imaging was performed using an Amersham Imager 600 system, and band intensity was quantified using IMAGEJ software.

[0023] Example 2

[0024] Construction of SH2B1 KO mice and neuron-specific transgenic (Tg) mice.

[0025] The construction method of SH2B1 KO mice (129Sv / C57BL / 6 gene background) was based on the literature (Duan, C., et al. 2004. Disruption of the SH2-B gene causes age-dependent insulin resistance and glucose intolerance. Mol. Cell. Biol. 24:7435–7443.). The full-length rat SH2B1β sequence (neuron-specific enolase promoter / rat growth hormone enhancer) marked with Myc was constructed. The constructed sequence was used to construct fertilized embryos (C57BL / 6J x SJL) by microinjection to obtain specific SH2B1 transgenic mice (Ren D1, et al. 2007 Neuronal SH2B1 is essential for controlling energy and glucose homeostasis. 117(2):397-406).

[0026] Example 3

[0027] Effects of SH2B1 conditional knockout and overexpression on sodium iodoacetate (MIA) OA model mice

[0028] Male wild-type mice (WT) with a genetic background of C57BL / 6J, SH2B1 KO mice, and SH2B1 Tg mice were housed in groups of five, fed standard diets, with free access to water. The room temperature was maintained at (24±2)℃, humidity at 50%-60%, and with good ventilation. The daily light and dark periods were 12 hours each. Mice underwent a 3-day acclimatization period in this system before the experiment. All animal experiments complied with guidelines. The animal experiment protocol was approved by the Animal Protection and Ethics Committee of Nanjing Medical University.

[0029] Mice (WT, KO, and Tg) of each species were randomly divided into two groups: a control group and a MIA group. All mice were treated with 0.1 mL / 10 g of physiological saline for 28 consecutive days. However, on day 7, mice in the control group received an intra-articular injection of 10 μL of physiological saline, while mice in the MIA group received an intra-articular injection of 0.3 mg (10 μL) of MIA in their right knee joint. Behavioral tests were performed on days 0, 3, 7 (6 hours after treatment), 10, 14, 17, 21, and 28. All mice were euthanized on day 28, and tissue samples were collected for histological examination.

[0030] Mechanical pain sensitivity testing: von Frey fibers were used for testing. Starting 3 days prior to the test, mice were placed in a 30×30×30 cm glass box with a metal mesh bottom in the laboratory. 3Internal acclimatization for 2 hours. The laboratory should be kept quiet and free of noise, with temperature and humidity controlled like an animal housing to ensure experimental stability. On the day of testing, allow the mice to acclimatize in the chamber for at least 0.5 hours. Once the mice are calm, pain sensitivity testing can be performed. Apply von Frey fibers perpendicularly to the surface of the hind paw of the mouse, stimulating the center area as much as possible, applying force to slightly bend the paw, and hold for 5 seconds. The stiffness of the hair increases logarithmically. To prevent interference from previous stimuli, there should be a 5-minute interval between stimuli. Each mouse is tested 3 times, and the average threshold is measured. When an animal responds to 0.4g or less, it is considered to have a rhythmic abnormality. A normal response is in the range of 1-2g.

[0031] Immunohistochemistry: After euthanizing mice, femoral condyle and tibial plateau cartilage tissues were dissected and collected. The specimens were decalcified in 10% EDTA solution at 4°C for 2 weeks, then placed in 30% high glucose solution for 48 hours. Afterward, the tissues were embedded in cryo-section embedding medium (OCT) and prepared into 4µm thick frozen sections using a cryostat. These sections were stored at -20°C. Toluidine blue staining was then performed. Cartilage degeneration was scored in sections using a cartilage degeneration score (0 = normal cartilage; 1 = mild surface area; 2 = slight extension to the upper central region; 3 = moderate in the central region; 4 = extension to the deep region; 5 = severe degeneration) and a proteoglycan loss score (0 = normal cartilage; 1 = reduced but incomplete loss of calcification with toluidine blue staining; 2 = focal loss of toluidine blue staining in non-calcified cartilage (<30% surface area); 3 = diffuse loss of toluidine blue staining in non-calcified cartilage (>30% surface area)). (Di Paola, R., et al., Adelmidrol, in combination with hyaluronic acid, displays increased anti-inflammatory and analgesic effects against monosodium iodoacetate-induced osteoarthritis in rats. Arthritis Res Ther, 2016. 18(1): p. 291. Janusz, MJ, et al., Detection of aggrecanase-and MMP-generated) catabolicneoepitopes in the rat iodoacetate model of cartilagedegeneration. Osteoarthritis Cartilage, 2004.12(9):p.720-8).

[0032] Immunofluorescence staining: After euthanasia of mice, the dorsal horn of the spinal cord (L3-L5 segment) and hippocampus were rapidly removed, fixed overnight with 4% paraformaldehyde, dehydrated, decalcified, and cut into 20 μm thick sections. The sections were washed three times with PBS (5 min each time) at room temperature. The prepared sections were blocked with 5% BSA (prepared with PBS / 0.1% Triton X-100) at room temperature for 2 h. GFAP and Iba-1 antibodies were added; after incubation at room temperature for 2 h, the sections were incubated overnight at 4°C. The sections were washed three times with PBS (5 min each time), and then incubated with the corresponding secondary antibodies at room temperature in the dark for 2 h. The sections were washed three times with PBS (5 min each time), and then mounted with DAPI-containing mounting medium. The sections were then observed under a fluorescence microscope, photographed, and the fluorescence intensity was measured using morphological image analysis software.

[0033] Experimental results:

[0034] Figure 1 In an OA mouse model, the expression of SH2B1 protein in the articular cartilage was significantly lower than that in the control group (sham-operated group) (p < 0.01). Similarly, the expression of SH2B1 protein in the knee articular cartilage of OA patients was significantly lower than that in healthy controls (p < 0.05). Reduced expression of SH2B1 in articular cartilage is associated with the pathogenesis of OA.

[0035] Figure 2 Behavioral tests in MIA-induced osteoarthritis (OA) model mice showed that WT group mice exhibited significant mechanohyperthermia, and SH2B1 knockout exacerbated this pain compared to WT group mice (p < 0.05), while SH2B1 conditional overexpression significantly improved pain tolerance (p < 0.05). SH2B1 knockout aggravated chronic pain in MIA-induced osteoarthritis model mice, while SH2B1 Tg alleviated MIA-induced chronic pain.

[0036] Figure 3 Intra-articular injection of 0.1 mg / 10 μL MIA can induce knee cartilage degeneration, as shown by toluidine blue staining. Figure 3 A) The MIA group showed a lack of chondrocyte layer, reduced cell structure, and delamination and destruction of the articular surface. In SH2B1 KO mice, the cartilage degeneration score and proteoglycan loss score were significantly increased after MIA modeling. Figure 3 (p < 0.01) In contrast, the cartilage degeneration score and proteoglycan loss score of SH2B1 Tg mice after MIA modeling were significantly improved compared with the WT model group (p < 0.05).

[0037] Figure 4In the dorsal horn tissue of the spinal cord, there was no significant difference in the number of activated glial cells among the SH2B1 KO, SH2B1 Tg, and WT mice in the control group. In MIA-induced OA model mice, the number of activated astrocytes and microglia in the SH2B1 KO, SH2B1 Tg, and WT groups was significantly increased compared with the control group (p < 0.01). The activation of astrocytes in the SH2B1 Tg group was significantly decreased compared with the SH2B1 KO group (p < 0.05), and the activation of microglia in the SH2B1 KO group was significantly increased compared with the WT group (p < 0.05).

[0038] Figure 5 In the hippocampus, there was no significant difference in the number of activated glial cells among the SH2B1 KO, SH2B1 Tg, and WT mice in the control group. In MIA-induced OA model mice, the number of activated astrocytes and microglia in the SH2B1 KO, SH2B1 Tg, and WT groups was significantly increased compared with the control group (p < 0.01), while the activation of astrocytes and microglia in the SH2B1 Tg group was significantly reduced compared with the SH2B1 KO group (p < 0.05).

Claims

1. Application of SH2B adaptor protein 1 as a drug target in screening drugs for treating osteoarthritis pain.

2. Application of SH2B adaptor protein 1 as a drug target in the preparation of kits for screening drugs to prevent, relieve and / or treat osteoarthritis pain.

3. Application of SH2B adaptor protein 1 agonists in the preparation of drugs for the prevention, relief and / or treatment of osteoarthritis pain.

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