Use of a synthetic peptide targeting inhibition of GRP in the preparation of a medicament for the treatment of diabetic neuropathic pain

Abstract

This invention relates to the field of pharmaceutical technology, disclosing the application of a synthetic peptide that targets and inhibits GRP in the preparation of a drug for treating diabetic neuropathy. The synthetic peptide sequence is SWYLILRIENGKLVKEVVHVPD. This sequence targets and blocks the GRP / vimentin signaling axis. Vimentin is specifically expressed by astrocytes and is a characteristic protein of astrocyte activation. Astrocyte activation can cause central sensitization, leading to chronic pain and other chronic diseases. This synthetic peptide sequence is used in the preparation of a drug for treating diabetic neuropathy. This invention provides a GRP-targeting inhibitory peptide that has a good therapeutic effect on chronic pain, laying the foundation for clinical treatment of chronic pain and possessing significant application and promotion value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically the application of a synthetic peptide that targets and inhibits GRP in the preparation of a drug for treating diabetic neuropathy. Background Technology

[0002] Diabetic neuropathy, one of the most common complications of diabetes, clinically manifests as hyperalgesia, spontaneous pain, and touch-induced pain. Diabetic peripheral neuropathy falls under the category of neuropathic pain, resulting from damage to peripheral nerves caused by a pathological process triggered by hyperglycemia. Its pathogenesis is extremely complex and remains not fully understood.

[0003] This painful neuropathy severely impacts patients' physical and mental health and quality of life, and also presents a challenging problem in clinical treatment. Currently, medications used to relieve pain generally suffer from significant side effects, short duration of action, and limited therapeutic efficacy, failing to meet clinical needs and causing considerable distress to both doctors and patients. Therefore, there is an urgent need to develop new analgesic drugs.

[0004] The applicant's previous research demonstrated for the first time that galactolectin-associated protein (GRP) can activate astrocytes by binding to vimentin expressed by astrocytes, thereby triggering diabetic neuropathy. Furthermore, chemogenetic methods to inhibit astrocyte activity effectively alleviated symptoms. Based on this, developing an inhibitory peptide that can block the GRP / vimentin signaling axis holds promise as a novel and effective treatment for diabetic neuropathy. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the above-mentioned technical defects and provide an application of a synthetic peptide that targets and inhibits GRP in the preparation of a drug for treating diabetic neuropathic pain.

[0006] To address the aforementioned problems, the technical solution of this invention is: the application of a synthetic peptide that targets and inhibits GRP in the preparation of a drug for treating diabetic neuropathic pain, wherein the sequence of the synthetic peptide is SWYLILRIENGKLVKEVVHVPD.

[0007] Furthermore, the synthetic peptide sequence targets and blocks the GRP / vimentin signaling axis.

[0008] Furthermore, vimentin is specifically expressed by astrocytes and is a characteristic protein of astrocyte activation. Astrocyte activation can cause central sensitization, which in turn leads to chronic diseases such as chronic pain.

[0009] Furthermore, the synthetic peptide sequence is used in the preparation of a drug for treating diabetic neuropathic pain.

[0010] The advantages of this invention compared to existing technologies are as follows: This invention provides a GRP protein inhibitory peptide that has a good therapeutic effect on chronic pain, laying the foundation for the clinical treatment of chronic pain and having significant application and promotion value. Attached Figure Description

[0011] Figure 1 This is a schematic diagram illustrating the construction of a diabetic neuropathic pain model according to the present invention;

[0012] Figure 2 This is a schematic diagram illustrating the increased GRP secretion in diabetic neuralgia rats according to the present invention;

[0013] Figure 3 This is a schematic diagram illustrating the pain-induced behavior in rats by intracranial administration of GRP according to the present invention;

[0014] Figure 4 This is a schematic diagram of the pain-relieving behavior in rats using the method of knocking down GRP expression in the anterior cingulate cortex according to the present invention.

[0015] Figure 5 This is a schematic diagram illustrating the direct binding of GRP to vimentin, a protein specifically expressed in astrocytes, according to the present invention.

[0016] Figure 6 This is a schematic diagram of the blocking peptide designed based on the binding site of GRP and vimentin in this invention;

[0017] Figure 7 This is a schematic diagram illustrating how intracranial administration of synthetic peptides to rats can alleviate GRP-induced pain behavior. Detailed Implementation

[0018] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0019] The application of a synthetic peptide that targets and inhibits GRP in the preparation of a drug for treating diabetic neuropathic pain, wherein the sequence of the synthetic peptide is SWYLILRIENGKLVKEVVHVPD.

[0020] Verification Experiment 1: Detection of GRP secretion protein expression in cerebrospinal fluid and ACC dialysate of diabetic neuropathy model rats

[0021] 1. Experimental Method:

[0022] (1) Establishment of a rat model of diabetic neuralgia

[0023] SPF-grade male Sprague-Dawley rats (180–200 g) were fasted overnight and then injected intraperitoneally with 70 mg / kg fresh streptozotocin (STZ) dissolved in 0.1 M citrate buffer (pH 4.5). Control animals received an equal volume of citrate buffer. Blood glucose levels were assessed via the tail vein using a glucometer 72 hours after STZ injection. Rats with blood glucose levels ≥16.7 mmol / L were considered diabetic and included in the following study. Rats with neuropathic pain were defined as having a mechanical withdrawal threshold (MWT) induced at 3 weeks of von Frey fiber stimulation of the left hind paw, with the ratio of the induced MWT to the baseline MWT <0.8.

[0024] (2) Cerebrospinal fluid collection

[0025] After anesthetizing rats with sodium pentobarbital, the hair on the back of their heads (from above the eyes, between the ears, to the base of the neck) was clipped. The rats were then secured to a stereotaxic apparatus using a palatal clamp and ear rods. The skin of the rats was incised to expose the dura mater, which was then dried with a dry cotton swab (the area is triangular, typically with 1-2 large blood vessels passing through it; the optimal location for capillary insertion to collect cerebrospinal fluid is on either side of or between the blood vessels). Using a micromanipulator, the tip of a syringe was inserted through the dura mater to aspirate cerebrospinal fluid, which was then transferred to a collection tube (containing a protease inhibitor). The collected cerebrospinal fluid sample can be stored at -80°C for further analysis.

[0026] (3) Microdialysis

[0027] The microdialysis probe was implanted unilaterally into the anterior cingulate cortex (ACC) using a guiding cannula, and artificial cerebrospinal fluid was infused via a syringe pump at a rate of 1.5 μL / min. After equilibration for 30 minutes, the dialysate (90 μL / sample) was collected, rapidly frozen, and stored at -80°C.

[0028] 2. Experimental Results:

[0029] (1) STZ-induced diabetic neuropathy in rats

[0030] As attached Figure 1 As shown, within one week after injection, the blood glucose levels in the STZ model group rats were significantly higher than those in the control group, and remained elevated throughout the experiment. Despite a significant increase in food and water intake, the body weight of the STZ-treated rats decreased significantly during the 5-week observation period. Furthermore, the von Frey test showed that these STZ rats exhibited enhanced hypersensitivity, with a significant decrease in mechanical nociception in the hind paws at week 2 after STZ administration. The lowest threshold was reached at week 3 and persisted until week 5. Based on these results, a follow-up diabetic neuropathy experiment was conducted three weeks after STZ injection.

[0031] (2) Diabetic neuralgia induces increased GRP secretion in cerebrospinal fluid and ACC dialysate of rats.

[0032] As attached Figure 2 As shown, GRP levels were quantified using an ELISA kit, and the results showed that GRP levels were increased in the cerebrospinal fluid and ACC dialysis fluid of the model rats.

[0033] Verification Experiment 2: GRP Involved in Regulating Diabetic Neuropathic Pain Behavior

[0034] 1. Experimental Method:

[0035] (1) Nucleus virus injection reduces neuronal GRP expression:

[0036] According to experimental requirements, rats were injected with viruses such as AAV-U6-shLGALSL-CMV-EGFP / AAV-EF1α-rLGALSL-3×FLAG into the ACC. First, after an intraperitoneal injection of sodium pentobarbital (50 mg / kg), the rats were fixed in a stereotaxic apparatus. After disinfection with iodine, the scalp was cut open, and the anterior fontanelle Bregma point and posterior fontanelle Lambda were located. The apparatus was leveled, and the location of the nuclei requiring localization was determined using brain mapping. A micro-perforated dental drill was used to create a hole, and then the virus was vertically injected (total injection volume not exceeding 300 nl per side) using a 1 μl Hamilton microsyringe connected to a self-made glass microelectrode. After localization injection, the scalp was sutured, thus completing the stereotaxic injection of the brain. Different behavioral tests could be performed 21 days after viral expression.

[0037] (2) Validation of virus knockout efficiency by Western blot detection

[0038] Mice were perfused by first deeply anesthetizing them with isoflurane, then opening the thoracic cavity to expose the heart. A perfusion needle was inserted into the left ventricle, and the right atrial appendage was cut open. Approximately 50 ml of physiological saline was rapidly perfused. Once the mouse limbs and liver turned white, the mouse brain was removed, and ACC tissue was excised from a 500 μm thick vibratory microtome. Total protein was extracted using ice-cold RIPA buffer containing Tris-HCl (50 mM, pH 7.6), Triton X-100 (1%), NaCl (150 mM), SDS (0.1%), sodium deoxycholate (0.5%), and a mixture of protease inhibitors.

[0039] For Western blot analysis, protein samples were mixed with RIPA buffer, boiled for 5–10 minutes, and then electrophoresed on a 10% SDS-PAGE gel. Proteins were transferred to nitrocellulose membranes and blocked with 5% skim milk. The membranes were incubated overnight at 4°C with primary antibodies, including GRP and β-actin, and then incubated at room temperature for 1.5 hours with peroxidase-labeled secondary antibodies. Protein bands were visualized using a high-sensitivity ECL reagent and analyzed using ImageJ software.

[0040] 2. Experimental Results:

[0041] As attached Figure 4 As shown, AAV-shGRP virus significantly reduced GRP expression. Examination of the mechanical pain threshold in STZ rats injected with AAV-shGRP virus and the control virus AAV-EGFP revealed that after GRP knockout, the mechanical pain threshold almost recovered to the original control level, but not in the STZ control animals expressing AAV-EGFP. This indicates that GRP knockdown alleviates STZ-induced abnormal pain in rats.

[0042] Verification Experiment 3: Demonstrating that GRP can specifically bind to vimentin

[0043] 1. Experimental Methods:

[0044] (1) A vimentin-His and GRP-HA fusion plasmid was constructed for the purification of the recombinant protein. The plasmid and protein were constructed and purified by Anhui Qiancheng Biotechnology Co., Ltd.

[0045] (2) Pull-down experiment

[0046] The experimental protocol for Anti-HA immunomagnetic beads from Selleck was slightly modified as follows:

[0047] ① Gently pipette the resuspended Anti-HA immunomagnetic beads and transfer 20 μL of the bead suspension into a new centrifuge tube.

[0048] ② Add 500 μL TBS (50 mM Tris HCl, 150 mM NaCl, pH 7.4), gently pipette to resuspend the magnetic beads, let stand on a magnetic rack for 10 seconds, remove the supernatant, and repeat the above steps twice.

[0049] ③ Add 1 μg each of vimentin-His and GRP-HA recombinant protein to the above precipitate, gently pipette to resuspend the magnetic beads, and then incubate at room temperature for 2 h.

[0050] ④ After standing on the magnetic rack for 10 seconds, transfer the supernatant to a new centrifuge tube for later use (the supernatant can be used to detect whether there is any residue of HA-tag protein).

[0051] ⑤ Add 500 μL of PBST (NaCl 136.89 mM; KCl 2.67 mM; Na2HPO4 8.1 mM; KH2PO4 1.76 mM; 0.5% Tween 20) to the above precipitate, gently tap or blow to redisperse the magnetic beads, and then invert the sample for 5 min. Remove the supernatant after magnetic separation.

[0052] ⑥ Repeat the above steps three times.

[0053] ⑦ Add 40 μL of 2× protein loading buffer to the precipitate obtained above, boil for 5 min, cool to room temperature and let stand on a magnetic rack for 10 sec.

[0054] ⑧ Take the supernatant for SDS-PAGE analysis.

[0055] 2. Test Results:

[0056] The results are attached. Figure 5 GRP can specifically bind to vimentin.

[0057] Verification Experiment 4: Design and Synthesis of an Inhibitory Peptide that Blocks GRP / vimentin

[0058] Bioinformatics has revealed that amino acids in the GRP sequence contain two potential sites for binding to vimentin (see appendix). Figure 6 Therefore, based on the active site and hotspot residues (A37, A49, and A52), we designed 20-25 peptides that bind to GRP proteins. Hdock is a highly efficient protein-protein interaction docking tool. By calculating the binding energy scores of the designed peptides and the A protein, it can more effectively identify protein interactions with better stability and stronger affinity. The top 20 data with the highest binding energies were selected and transferred to AlphaFold3 to calculate the pTM+ipTM values. The higher the ipTM and pTM scores, the more reliable AlphaFold3's prediction of the protein complex structure and binding site. Based on these three indicators, we selected one of the peptide sequences: SWYLILRIENGKLVKEVVHVPD.

[0059] Experiment 5: Experiment on the effect of synthetic peptides on GRP-induced pain behavior

[0060] 1. Experimental Methods:

[0061] Intracranial injection: For targeted drug delivery, a cannula was stereotactically implanted into the ACC and secured with dental acrylic and cranial screws. GRP protein (1 μg / side / day) was infused at a rate of 200 nL / min using a 10 μL Hamilton syringe and a micro-infusion pump (micro 4, WPI). Artificial cerebrospinal fluid (250 nL) was used as a control. Von Frey assays were initiated 30 minutes after infusion.

[0062] 2. Experimental Results:

[0063] As attached Figure 7 As shown, intracranial administration of GRP (0.1 μg) induced mechanopathic pain in rats. We administered a synthetic peptide (1 μg) concurrently with intracranial GRP injection and measured the mechanopathic pain threshold 30 minutes post-injection. The results indicate that the synthetic peptide effectively alleviated the mechanopathic pain induced by intracranial GRP injection.

[0064] In summary, this application demonstrates that GRP specifically binds to vimentin, and based on this effect, technologies, tools, and drugs targeting GRP can be developed. This invention also demonstrates that synthetic peptides that block the binding of GRP / vimentin can effectively relieve diabetic neuropathy and can be used in the development of analgesic drugs.

[0065] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0066] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. The application of a synthetic peptide that targets and inhibits GRP in the preparation of a drug for treating diabetic neuropathy, characterized in that, The synthetic peptide sequence is SWYLILRIENGKLVKEVVHVPD.

2. The application of the synthetic peptide targeting and inhibiting GRP according to claim 1 in the preparation of a drug for treating diabetic neuropathy, characterized in that: The synthetic peptide sequence is used to prepare a drug for treating diabetic neuropathic pain.

Images