Antibody reagents, polypeptides, siRNA based on rim1 protein expression and uses thereof

Abstract

The application discloses an antibody reagent, a polypeptide, an siRNA based on RIM1 protein expression and application of the antibody reagent, the polypeptide and the siRNA, wherein the antibody reagent is RIM1 antibody, and the concentration is 1 mg / ml; the polypeptide is based on RIM1 protein sequence, contains aromatic amino acids, and the amino acid sequence is WNQTFVYSHVHRRDF, wherein Y is tyrosine, W is tryptophan, and F is phenylalanine; the siRNA can interfere with Rim1 gene expression, the siRNA is a double strand, the sense strand sequence is UGGAAUCAAACAUUUGUCUACtt, and the antisense strand sequence is GUAGACAAAUGUUUGAUUCCAtt.The action principles of the three drugs are to realize prolonging the hot paw withdrawal latency of a limb of a neuropathic pain mouse by reducing the glutamate release of the anterior cingulate cortex of the neuropathic pain mouse, so that a theoretical inspiration is provided for the treatment of clinical neuropathic pain.

Description

Technical Field

[0001] This invention relates to an antibody reagent based on RIM1 protein expression, a polypeptide, a double-stranded siRNA and their applications, belonging to the field of pharmaceutical technology. Background Technology

[0002] Pain is a protective response of the body to noxious external stimuli. However, even after the removal of noxious stimuli such as inflammation, local nerve compression, or trauma, pain as a symptom may not necessarily disappear; it may even worsen or become unbearable. In my country, two out of every three outpatients present with various forms of chronic pain, suggesting that there are at least 100 million chronic pain patients in the country. The development of chronic pain is a multi-step, multi-factorial process involving numerous abnormal changes at the cellular and molecular levels. In-depth exploration of the molecular mechanisms of pain occurrence and development, clarifying the causes of its long-term persistence, establishing pain treatment targets, and seeking new intervention strategies are of profound significance for improving the effectiveness of pain management.

[0003] Functional brain imaging studies have provided localization of brain regions involved in pain regulation. Research has found that in chronic pain states, in addition to the somatosensory cortex, some brain regions such as the profrontal cortex (PFC) and anterior cingulate cortex (ACC) show activity changes (Gungor and Johansen 2019; Liu et al. 2023). Studies have shown that real-time functional magnetic resonance imaging (fMRI) studies have demonstrated that activation of neurons controlling the ACC can alleviate pain (Buffington et al. 2005), and surgical resection of the ACC can also alleviate pain (Ballantine et al. 1967; LaGraize et al. 2004). Therefore, the ACC is gradually becoming a key brain region for central intervention in the clinical treatment of chronic pain. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides an antibody reagent, a polypeptide, a double-stranded siRNA based on RIM1 protein expression, and their applications.

[0005] To achieve the above objectives, the present invention employs an antibody reagent based on RIM1 protein expression. The antibody reagent is a RIM1 antibody with a concentration of 1 mg / ml. The RIM1 antibody prolongs the heat-shrinking paw latency of the affected limb in neuropathic pain mice by reducing glutamate release in the anterior cingulate cortex.

[0006] The present invention also provides a polypeptide based on the RIM1 protein sequence, the polypeptide containing aromatic amino acids, the amino acid sequence of which is: WNQTFVYSHVHRRDF, where Y is tyrosine, W is tryptophan, and F is phenylalanine. The polypeptide exerts an analgesic effect by reducing the release of glutamate from the anterior cingulate cortex of mice with neuropathic pain.

[0007] The present invention also provides an siRNA that can interfere with the expression of the Rim1 gene. The siRNA is double-stranded, with the sense strand sequence being: UGGAAUCAAACAUUUGUCUACtt and the antisense strand sequence being: GUAGACAAAUGUUUGAUUCCAtt. The siRNA exerts an analgesic effect by reducing the release of glutamate from the anterior cingulate cortex of mice with neuropathic pain.

[0008] The present invention also provides the use of the antibody reagent, the polypeptide, or the siRNA in the preparation of drugs for relieving neuropathic pain.

[0009] In some embodiments, the drug works by reducing glutamate release from the anterior cingulate cortex in neuropathic pain.

[0010] The present invention also provides a medicament for relieving neuropathic pain, comprising the antibody reagent, the polypeptide, or the siRNA.

[0011] In some embodiments, a pharmaceutically acceptable carrier may also be included.

[0012] The mechanism of this invention is as follows: Cav-1 is a major protein located in the pituitary gland, a microdomain of active cell membrane signaling. It can form complexes with various signaling molecules and play an important role in regulating bodily functions. Amino acids 82-101 of Cav-1 constitute its scaffold region, a key site for its interaction with other signaling molecules. It specifically binds to aromatic amino acid residues. Peptides containing aromatic amino acids can compete with inherently interacting signaling molecules for binding to its scaffold region, thereby inhibiting the binding of Cav-1 to signaling molecules. This invention uses immunoprecipitation experiments to demonstrate that Cav-1 can co-precipitate with RIM1 protein, and its expression is increased in the ACC region of neuropathic pain mice. These studies indicate that Cav-1 and RIM1 proteins participate in the regulation of behavior and related brain region neuroplasticity in neuropathic pain animals. This invention also uses bioinformatics methods to predict the binding region of these two proteins and detects that three drugs—a RIM1 antibody, a peptide based on the RIM1 protein sequence, and a siRNA that can interfere with Rim1 gene expression—can effectively prolong the latency period of heat-shrinking paws in the affected limbs of neuropathic pain mice.

[0013] Compared with existing technologies, this invention provides theoretical inspiration for the treatment of clinical neuropathic pain and provides a theoretical basis and effective targets for drug development. Attached Figure Description

[0014] Figure 1 The graph shows the hydrophobicity of the RIM1 protein (analyzed using Bioedit software).

[0015] Figure 2 A is the immunoprecipitation band diagram of Cav-1 and RIM1 proteins, and B is the statistical diagram. As can be seen from the figure, Cav1 and RIM1 interact, and the expression of both Cav-1 and RIM1 proteins is increased in the anterior cingulate cortex of neuropathic pain mice (CCI group).

[0016] Figure 3 A schematic diagram showing how all three drugs can improve pain behavior in mice with neuropathic pain;

[0017] Figure 4 This is a schematic diagram illustrating how three drugs inhibit glutamate release from the anterior cingulate cortex of mice with neuropathic pain. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this application will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the embodiments of this application and the specific features in the embodiments are detailed descriptions of the technical solutions of this application, rather than limitations on the technical solutions of this application. In the absence of conflict, the embodiments of this application and the technical features in the embodiments can be combined with each other.

[0019] 1. Reagents used in this invention

[0020] RIM1 antibody: Shanghai Qifa Experimental Reagent Co., Ltd.;

[0021] Polypeptide synthesis: Shanghai Botai Biotechnology Co., Ltd.; This polypeptide (644 polypeptide) contains aromatic amino acids, and its amino acid sequence is: WNQTFVYSHVHRRDF (SEQ ID NO.1), where Y is tyrosine, W is tryptophan, and F is phenylalanine, which belongs to aromatic amino acids;

[0022] The siRNA (synthesized by Gemma Gene) of this invention (siRNA-RIM1) fragment selects the functional region of the protein corresponding to the target gene for interference; this siRNA fragment is double-stranded, the sense strand sequence is: UGGAAUCAAACAUUUGUCUACtt (SEQ ID NO.2); the antisense strand sequence is: GUAGACAAAUGUUUGAUUCCAtt (SEQ ID NO.3);

[0023] Catch and Reversible Immunoprecipitation System (17-500): Mlippore;

[0024] SuperSignal West Pico Chemiluminescent Substrate (34078): Pierce Company;

[0025] PVDF membrane: Mlippore;

[0026] Glutamic Acid Detection Kit: Shanghai Qifa Experimental Reagent Co., Ltd.

[0027] Unless otherwise specified, all other chemical reagents were purchased from Sigma.

[0028] The experimental device used in this invention is the Ugo Basil foot infrared thermal pain tester (Shenzhen Ruiwode Life Science Co., Ltd.).

[0029] 2. Experimental animals: Male C57 mice, clean grade, 20-25g, provided by the Experimental Animal Center of Xuzhou Medical University.

[0030] 3. Preparation of a mouse model of neuropathic pain: Chronic constriction injury (CCI) surgery was performed according to the method described by Bennett and Xie (Bennett and Xie 1988). Mice were anesthetized with pentobarbital (40 mg / kg, ip), and the upper-middle segment of the right sciatic nerve was exposed (in this invention, the right sciatic nerve was ligated in all cases). Three ligatures (1 mm apart) were made at the bifurcation of the sciatic nerve using 5-0 silk sutures. The control group was exposed without ligation. The incision was sutured layer by layer, and the wound was treated with antibiotics. Previous studies have shown that pain caused by CCI can last for at least several months.

[0031] Example 1

[0032] Hyperalgesia of heat:

[0033] Place an acrylic box on a 3mm thick glass plate and irradiate the area of ​​the rat's paw that is in contact with the glass plate using a thermal radiation stimulator according to the Hargreaves method. Record the time from the start of irradiation to the appearance of paw lifting, with a cutoff time of 15 seconds. Measure each animal three times and take the average value, with each measurement 5 minutes apart.

[0034] Example 2

[0035] Stereoscopic injection of three drugs:

[0036] Mice were anesthetized with pentobarbital (40 mg / kg, ip) and fixed on a mouse-specific electronic stereotaxic apparatus. The position of the ear rods was carefully monitored, and the depth of the ear rods was adjusted so that a crisp click was just heard. The height of the ear rods was aligned with the height of the two incisors. The fur was clipped, and the scalp was disinfected with iodine. An incision was made 0.5 cm posterior to the midline connecting the eyes. The incision was repeatedly wiped with a cotton swab dipped in 3% H2O2 to break the fascia and expose the skull. The anterior fontanelle was located, and the needle was positioned 1.11 mm anterior to the bregma and 0.25 mm lateral to it. 0.5 μl of the prepared antibody drug (RIM1 antibody: 1 mg / ml, dissolved in PBS), peptide (644 peptide: dissolved in physiological saline containing 10% acetic acid, peptide solution concentration 2 μg / μl), and siRNA (si RNA-RIM1: Dissolved in siRNA-Mate transfection reagent (siRNA-RIM1 concentration: 10 μg / μL) or siRNA-Scramble (control, dissolved in siRNA-Mate transfection reagent (siRNA-Scramble concentration: 10 μg / μL)). Slowly inject the siRNA into a 2.10 mm depth using a microsyringe (injection duration: 30 seconds). Leave the needle in place for 10 minutes, then slowly withdraw it. Suture the skin, disinfect with iodine, and apply antibiotic solution to the incision. Maintain room temperature at approximately 26°C throughout the process until the animal recovers.

[0037] The results are as follows Figure 3 , Figure 4 As shown, Figure 3 The results showed that the heat-shrinkage latency of mice with neuropathic pain was significantly shortened. After stereotactic injection of the three drugs of the present invention into the ACC brain region, the heat-shrinkage latency of mice with neuropathic pain was effectively prolonged. Figure 4 The results showed that glutamate release in the anterior cingulate cortex of mice with neuropathic pain was significantly increased. After stereotactic injection of the three drugs of the present invention into the ACC brain region, all three drugs could reduce glutamate release in the anterior cingulate cortex of mice with neuropathic pain.

[0038] Analysis shows that the antibody reagent based on protein RIM1, a polypeptide, and a siRNA of the present invention can all alleviate pain behavior in mice by inhibiting the release of glutamate from the anterior cingulate cortex of addicted mice.

[0039] Example 3

[0040] Immunoprecipitation technique for detecting whether Cav1 coprecipitates with RIM1:

[0041] Mice that successfully modeled CCI were decapitated, and brain tissue was dissected on ice. A section of the cerebral cortex measuring 2.34 mm anterior to 0.22 mm posterior to bregma and 0.6 mm lateral to bregma was harvested; this section constitutes the ACC region. 80 μl of whole-cell lysis buffer was added to one side of the ACC region from each mouse, followed by sonication and incubation on ice for 30 minutes. The tissue was then centrifuged at 10,000 rpm for 5 minutes, and the supernatant was used as the whole-cell lysis buffer. (Specific instructions follow Millipore 17-500A|Catch and...) The v2.0 Reversible ImmunoprecipitationSystem operation is performed.

[0042] The results are as follows Figure 1 , Figure 2 As shown in Table 1, which is a hydrophobicity analysis diagram of the RIM1 protein, it can be seen that amino acids 644-658 (WNQTFVYSHVHRRDF) are hydrophilic, which is the polypeptide involved in this invention. According to information from https: / / www.uniprot.org / uniprotkb / Q99NE5 / entry#sequences, this peptide is located in the C2 domain of the RIM1 protein and is easy to interact with other molecules. Figure 2 Immunoprecipitation of Cav-1 and RIM1 proteins was observed, and analysis revealed that Cav-1 and RIM1 interact. Furthermore, the expression of both Cav-1 and RIM1 proteins was increased in the anterior cingulate cortex of neuropathic pain mice (CCI group).

[0043] Example 4

[0044] Mice that successfully modeled CCI were decapitated, and brain tissue was dissected on ice. A section of the cerebral cortex, 2.34 mm anterior to 0.22 mm posterior to bregma and 0.6 mm lateral to bregma, was taken, which is the ACC area of ​​the brain tissue. The tissue was rinsed three times with physiological saline and then hardened in ice-cold physiological saline for 3 minutes. It was then sliced ​​into 300 μm sections using a microtome and placed in pre-prepared artificial cerebrospinal fluid containing 100 mM KCl at 37°C. After 30 minutes, the supernatant was collected and centrifuged (12,000 rpm for 2 minutes) for analysis. The tissue was then dried and used as an internal control.

[0045] The glutamate detection procedure was strictly performed according to the Biovision kit K629-100 instruction manual.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions or improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide, characterized in that, The polypeptide contains aromatic amino acids with the following amino acid sequence: WNQTFVYSHVHRRDF, where Y is tyrosine, W is tryptophan, and F is phenylalanine. The polypeptide exerts an analgesic effect by reducing glutamate release from the anterior cingulate cortex of mice with neuropathic pain.

2. A siRNA, characterized in that, The siRNA is double-stranded, with the sense strand sequence being: UGGAAUCAAACAUUUGUCUACtt; and the antisense strand sequence being: GUAGACAAAUGUUUGAUUCCAtt. The siRNA exerts its analgesic effect by reducing glutamate release from the anterior cingulate cortex in mice with neuropathic pain.

3. The use of the polypeptide of claim 1 or the siRNA of claim 2 in the preparation of a drug for relieving neuropathic pain.

4. The application according to claim 3, characterized in that, The drug works by reducing glutamate release from the anterior cingulate cortex in neuropathic pain.

5. A drug for relieving neuropathic pain, characterized in that, Includes the polypeptide of claim 1 or the siRNA of claim 2.

6. The drug for relieving neuropathic pain according to claim 5, characterized in that, It also includes pharmaceutically acceptable carriers.

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