Drug for targeted treatment of diabetic peripheral neuropathy and neuroprotective drug for regulating proliferation function of schwann cells

Abstract

The application discloses a drug for targeted treatment of diabetic peripheral neuropathy and a nerve protection drug for regulating Schwann cell proliferation function, relates to the technical field of biological medicine, and the drug for targeted treatment of diabetic peripheral neuropathy comprises a functional polypeptide CAP229, and the amino acid sequence of the functional polypeptide CAP229 is shown as SEQ ID NO:1. The functional polypeptide CAP229 is applied to a protein drug for targeted treatment of diabetic peripheral nerve demyelination, a new selection of targeted treatment of diabetic peripheral neuropathy is provided, the CAP229 polypeptide has the advantages of small molecule, stable structure, endogenous source and the like, is suitable for a protein drug platform and a gene delivery strategy, has good conversion prospect, can relieve myelin loss in diabetic neuropathy in vitro and in an animal model, and can be used for constructing a functional screening platform and a high-throughput evaluation model.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to drugs for the targeted treatment of diabetic peripheral neuropathy and neuroprotective drugs for regulating Schwann cell proliferation function. Background Art

[0002] Diabetic peripheral neuropathy is one of the most common and disabling chronic complications of diabetes. Clinical manifestations include distal sensory loss, pain, numbness, and diminished reflexes, severely impacting patients' daily lives. Currently, treatment for diabetic peripheral neuropathy primarily focuses on symptomatic relief, with no effective fundamental treatment available and limited success. Summary of the Invention

[0003] The present application provides a drug for the targeted treatment of diabetic peripheral neuropathy and a neuroprotective drug for regulating the proliferation function of Schwann cells, in order to provide a drug for diabetic peripheral neuropathy.

[0004] In a first aspect, the present application provides a drug for the targeted treatment of diabetic peripheral neuropathy, comprising a functional polypeptide CAP229, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0005] SEQ ID NO: 1 is specifically: MKMADRSGKIIPGQVYIEVEYDYEYEAKDRKIVIKQGERYILVKKTNDDWWQVKPDENSKAFYVPAQYVKEVTRKALMPPVKQVAGLPNNSTKIMQSLHLQRSTENVNKLP ELSSFGKPSSSVQGTGLIRDANQNFGPSYNQGQTVNLSLDLTHNNGKFNNDSHSPKVSSQNRTRSFGHFPGPEFLDVEKTSFSQEQSCDSAGEGSERIHQDSESGDELSSSSTEQIRV.

[0006] In some embodiments, the functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

[0007] In a second aspect, the present application provides a neuroprotective drug for regulating Schwann cell proliferation, comprising a functional polypeptide CAP229, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0008] In some embodiments, the functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

[0009] The present application provides a new targeted treatment option for diabetic peripheral neuropathy by applying the functional polypeptide CAP229 to a drug for the targeted treatment of diabetic peripheral nerve demyelinating lesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0011] Figure 1 This is a diagram showing the results of the verification experiment of hsa_circ_0000231 encoding and translating the CAP229 polypeptide in Example 1 of this application.

[0012] Figure 2 This is a graph showing the experimental results of CAP229 polypeptide promoting Schwann cell proliferation and cell cycle transformation in Example 2 of this application.

[0013] Figure 3 This is a graph showing the results of the behavioral and nerve conduction function evaluation experiment after constructing a diabetic peripheral neuropathy mouse model and locally delivering CAP229 in Example 3 of the present application. DETAILED DESCRIPTION

[0014] To make the objectives, technical solutions, and advantages of this application more clear, the technical solutions of this application will be clearly and completely described below in conjunction with the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of this application without making any creative efforts shall fall within the scope of protection of this application.

[0015] Diabetic peripheral neuropathy is one of the most common and disabling chronic complications of diabetes. Clinical manifestations include distal sensory loss, pain, numbness, and diminished reflexes, severely impacting patients' daily lives. Currently, treatment for diabetic peripheral neuropathy primarily focuses on symptomatic relief, with no effective fundamental intervention available and limited success.

[0016] In view of this, the present application provides a drug for the targeted treatment of diabetic peripheral neuropathy and a neuroprotective drug that regulates the proliferation function of Schwann cells, so as to provide a drug for diabetic peripheral neuropathy.

[0017] In a first aspect, the present application provides a drug for the targeted treatment of diabetic peripheral neuropathy, comprising a functional polypeptide CAP229, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0018] SEQ ID NO: 1 is specifically: MKMADRSGKIIPGQVYIEVEYDYEYEAKDR KIVIKQGERYILVKKTNDDWWQVKPDENSKAFYVPAQYVKEVTRKALMPPVKQVAGLPNNSTKIMQSLHLQRSTENVNKLPELSSFGKPSSSVQGTGLIR DANQNFGPSYNQGQTVNLSLDLTHNNGKFNNDSHSPKVSSQNRTRSFGHFPGPEFLDVEKTSFSQEQSCDSAGEGSERIHQDSESGDELSSSSTEQIRV.

[0019] In combination with the first aspect, in some embodiments provided herein, the functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

[0020] In a second aspect, the present application provides a neuroprotective drug for regulating Schwann cell proliferation, comprising a functional polypeptide CAP229, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0021] In combination with the second aspect, in some embodiments provided herein, the functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

[0022] The present application provides a new targeted treatment option for diabetic peripheral neuropathy by applying the functional polypeptide CAP229 to a drug for the targeted treatment of diabetic peripheral nerve demyelination.

[0023] The CAP229 polypeptide has the advantages of small size, stable structure, and endogenous origin. It is suitable for protein drug platforms and gene delivery strategies, has good translational prospects, can alleviate myelin loss in diabetic neuropathy, and can be used to construct functional screening platforms and high-throughput evaluation models, which will facilitate the development of neuropathy drugs and mechanism research.

[0024] The technical solutions provided by this application are described in detail below with reference to the examples. In the accompanying drawings of experimental results, "*, **, and ***" are typically used to indicate the significance level of a statistical test, corresponding to different p-value ranges, where "*" represents P < 0.05, "**" represents P < 0.01, and "***" represents P < 0.001.

[0025] Example 1 Verification of hsa_circ_0000231 Encoding Translation CAP229

[0026] Verify whether hsa_circ_0000231 can encode and translate into a 229-amino acid polypeptide (CAP229), and confirm the molecular weight, amino acid sequence, and expression characteristics of the polypeptide.

[0027] 1) Construction of hsa_circ_0000231 overexpression vector with FLAG tag

[0028] Based on the sequence information of hsa_circ_0000231 (derived from CircBase, ID: hsa_circ_0000231), the hsa_circ_0000231 sequence containing an open reading frame (ORF) was designed and synthesized, and a 3×FLAG tag was introduced at the end of the ORF to facilitate subsequent detection.

[0029] The sequence was cloned into the GV689 circular RNA expression vector (GeneChem, China) to construct an hsa_circ_0000231+3×FLAG overexpression circular RNA expression vector and packaged into a virus.

[0030] 2) Cell culture and transfection

[0031] 293T cells were selected and cultured in high glucose medium at 37°C in a 5% CO2 environment.

[0032] The hsa_circ_0000231-3×FLAG expression vector was transfected into 293T cells, and the empty vector was used as a control (no FLAG expression).

[0033] 48 hours after transfection, the stably transfected cell population was selected using 2 μg / mL puromycin.

[0034] 3) Western blot detection of FLAG expression

[0035] FLAG is not expressed in normal cells. If FLAG is detected, it indicates that the circRNA has the function of translating protein.

[0036] Cells were harvested, and total protein was extracted using RIPA lysis buffer (containing protease inhibitors) and quantified using the BCA assay. A 30 μg protein sample was separated by SDS-PAGE and transferred to a membrane. The membrane was blocked with 5% skim milk powder for 1 hour and incubated with a primary antibody (anti-FLAG, Proteintech, 1:1000) and an HRP-conjugated secondary antibody (1:5000) for ECL development.

[0037] 4) Immunoprecipitation (IP) and LC-MS / MS analysis

[0038] The hsa_circ_0000231-3×FLAG-overexpressing cell lysate was immunoprecipitated using anti-FLAG magnetic beads (MedChemExpress, USA).

[0039] The eluted protein was separated by SDS-PAGE and stained with Coomassie Brilliant Blue, and a band of approximately 28.9 kDa was observed (the theoretical molecular weight of CAP229 is approximately 25.7 kDa, and it is approximately 28.9 kDa after binding to the 3×FLAG tag).

[0040] After gel excision, the protein was digested with trypsin (Trypsin Gold, Promega), and its amino acid sequence was analyzed by LC-MS / MS (Thermo Scientific Q Exactive HF-X mass spectrometer).

[0041] Mass spectrometry data were analyzed using Peaks Studio software, and the matched amino acid sequences were aligned with the predicted CAP229 sequence.

[0042] 5) Prepare anti-CAP229 antibodies and detect their expression in Schwann cells and peripheral nerves.

[0043] Anti-CAP22 antibodies were generated (Abclonal, China); a control vector with an ATG start codon mutation (ATG→ACG) was constructed (this vector cannot produce translated CAP229) to verify translation dependency.

[0044] Human Schwann cells (SCs, ATCC, CRL-3392™) were cultured in SC medium (ATCC, 30-2002™, containing 5% FBS) and transfected with the hsa_circ_0000231 overexpression vector. Empty vector and mutant vector served as controls.

[0045] CAP22 expression was detected in Schwann cells and peripheral nerve specimens. Based on SDS-PAGE mobility shift and LC-MS / MS analysis, the molecular weight of the identified peptide was confirmed to be approximately 25.7 kDa, significantly different from the 108.5 kDa of the parent protein ARHGAP12.

[0046] The experimental results are as follows Figure 1 As shown, Figure 1 (A) Schematic diagram of the circular RNA hsa_circ_0000231-3×FLAG encoding the translation of CAP229-3×FLAG; Figure 1(B) Western blot analysis showed that FLAG protein was not expressed in 293T cells, but FLAG protein was produced after overexpression of hsa_circ_0000231-3×FLAG, confirming that hsa_circ_0000231 has coding and translation function; Figure 1 (C) The hsa_circ_0000231-3×FLAG overexpressing cell lysate was immunoprecipitated with anti-FLAG antibody, and its amino acid sequence was analyzed by SDS-PAGE separation and LC-MS / MS. Figure 1 (D) Western blot verification using anti-CAP229 antibody showed that CAP229 was expressed in peripheral nerve tissue. Figure 1 (E) Overexpression of hsa_circ_0000231 in Schwann cells increased the expression level of CAP229, while mutation of the ATG start codon significantly decreased the expression level of CAP229.

[0047] (1) Western blot results showed that an obvious FLAG-positive band with a molecular weight of 25-30 kDa was detected in 293T cells transfected with has_circ_0000231-3×FLAG, while no FLAG signal was detected in the control, confirming that has_circ_0000231 can encode a translated polypeptide.

[0048] (2) After Coomassie brilliant blue staining, a protein band of approximately 28.9 kDa was specifically detected in the has_circ_0000231-3×FLAG overexpression group. LC-MS / MS analysis of this band clearly identified it to have a total of 229 amino acids, and the sequence was completely consistent with the amino acid sequence of the has_circ_0000231 open reading frame predicted by the circBank database.

[0049] (3) Western blot analysis using anti-CAP229 antibody further confirmed that CAP229 protein is expressed in Schwann cells and peripheral nerve tissue. Overexpression of has_circ_0000231 increased the expression level of CAP229 protein, while the expression level of CAP229 protein decreased significantly after the ATG start codon mutation, further confirming that CAP229 protein is encoded by has_circ_0000231.

[0050] (4) Combining mass spectrometry results and Western blot detection, it was confirmed that hsa_circ_0000231 can be translated to produce a polypeptide consisting of 229 amino acids with a molecular weight of approximately 25.7 kDa.

[0051] Example 2 CAP229 promotes Schwann cell proliferation and cell cycle transformation

[0052] 1) Construction of CAP229 overexpression vector and viral packaging

[0053] Based on the sequence information of the has_circ_0000231 coding region, the full-length CAP229 sequence was synthesized and cloned into the GV492 expression vector (GeneChem, China) to construct a CAP229 overexpression plasmid and package it into virus.

[0054] 2) Schwann cell culture and CAP229 overexpression

[0055] Human Schwann cells (SCs, ATCC CRL-3392) were cultured in a dedicated Schwann cell medium (ATCC, 30-2002) supplemented with 5% fetal bovine serum at 37°C and 5% CO2. When the cells reached 30–50% confluency, they were transfected with a CAP229 overexpressing virus. Forty-eight hours after transfection, 2 μg / mL puromycin was used for selection for five days to establish a stable overexpressing cell line. A control group consisted of Schwann cells infected with an empty vector (vector).

[0056] 3) Protein expression verification

[0057] Western blot analysis using CAP229 antibody (Abclonal, China) was performed to verify the expression level of CAP229 protein after transfection. The results showed that CAP229 protein expression was significantly upregulated in the overexpression group.

[0058] 4) Cell proliferation assay

[0059] CAP229-overexpressing and control SCs were seeded in 96-well plates at 5000 cells per well and cultured for 24, 48, and 72 h.

[0060] At each time point, 10 μL of CCK-8 reagent (Dojindo, Japan) was added, and the absorbance (OD) was measured at 450 nm after 1 h of incubation.

[0061] The DNA synthesis capacity of Schwann cells was assessed using the BeyoClick™ EdU-594 cell proliferation assay kit (Beyotime, China). Schwann cells were seeded in 96-well plates at 2 × 10 cells per well. 4 The cells were fixed, permeabilized, and stained after 2 h of incubation. Five fields of view were randomly selected using a fluorescence microscope (Olympus, Japan) to calculate the proportion of EdU-positive cells.

[0062] 5) Flow cytometry analysis of cell cycle

[0063] Schwann cells from the CAP229 overexpression and control groups were collected, rinsed twice with PBS, and fixed overnight with ice-cold 70% ethanol. The next day, the cells were washed with PBS and stained with 100 μg / mL RNase A and 50 μg / mL propidium iodide (PI) in a staining solution. The cells were incubated in the dark for 40 minutes. Flow cytometry (BD FACS Canto II) was used to analyze cell cycle distribution and the proportions of G1, S, and G2 / M phases. Data were processed using ModFit software.

[0064] The experimental results are as follows Figure 2 As shown, Figure 2 (A) Western blot analysis showed that the protein level of CAP229 was significantly increased after overexpression in Schwann cells; Figure 2 (B) and Figure 2 (C) EdU and CCK8 assay results showed that CAP229 promoted Schwann cell proliferation; Figure 2 (D) Cell cycle analysis showed that the G2 / M phase ratio increased and the G0 / G1 phase ratio decreased in Schwann cells after CAP229 overexpression.

[0065] (1) CAP229 significantly promotes SCs proliferation

[0066] The CCK-8 assay showed that the OD values ​​of SCs in the CAP229 overexpression group were significantly higher at 48 and 72 hours compared to the control group, indicating that CAP229 enhances SC proliferation. Consistent with the results of the EdU assay, the proportion of EdU-positive cells in the CAP229 group was significantly increased.

[0067] (2) CAP229 promotes G1 / S phase transition

[0068] Flow cytometry analysis of the cell cycle showed that the proportion of cells in the G0 / G1 phase decreased significantly in the CAP229 overexpression group, while the proportion of cells in the G2 / M phase increased significantly. This result suggests that CAP229 promotes the transition of SCs from the G1 phase to the G2 phase, accelerating cell cycle progression.

[0069] Example 3 Construction of a diabetic peripheral neuropathy mouse model and evaluation of behavior and nerve conduction function after local delivery of CAP229

[0070] 1) Establishment of a diabetic peripheral neuropathy (DPN) mouse model

[0071] Eight-week-old male C57BL / 6J mice were selected and divided into a DPN group and a DPN+CAP229 treatment group.

[0072] After six weeks of high-fat diet, the DPN model was treated with intraperitoneal injections of streptozotocin (50 mg / kg, Sigma-Aldrich) for three consecutive days, while continuing the high-fat diet. Starting on the seventh day after injection, a diabetic model was considered successfully established if fasting blood glucose levels were ≥16.7 mmol / L (300 mg / dL) for three consecutive times. Phenotypic confirmation of DPN included decreased mechanical and thermal pain thresholds and slowed electrophysiological nerve conduction velocity.

[0073] 2) CAP229 overexpression virus injection

[0074] After the DPN model was established, CAP229 overexpression virus was injected into the sciatic nerve of mice. A Hamilton microsyringe (10 μL, equipped with a 33G needle) was used to slowly inject 2.5 μL of a total of 5×10 6 The TU-derived CAP229-overexpressing lentivirus was injected approximately 5 mm distal to the sciatic nerve at an injection rate of approximately 0.2 μL / min. The needle was held in place for 5 minutes and then slowly withdrawn to prevent backflow. A control group of DPN mice was injected with an equal volume of empty virus.

[0075] 3) Pain threshold assessment

[0076] Eight weeks after virus injection, the pain threshold of mice in each group was evaluated.

[0077] Mechanical pain threshold: von Frey fibers (Stoelting, 0.04-2 g) were used for testing, and the 50% pain threshold was determined using the Dixon up-down method.

[0078] 4) Nerve conduction function test

[0079] The sciatic nerve conduction function was evaluated using electrophysiological testing: nerve conduction velocity and action potential amplitude.

[0080] 5) Electron microscopy to detect myelin lesions in the sciatic nerve

[0081] Eight weeks after virus injection, sciatic nerves were harvested and electron microscopy was used to examine the effect of CAP229 on diabetic peripheral nerve demyelination.

[0082] The experimental results are as follows Figure 3 As shown, Figure 3 (A) Improvement of mechanical pain threshold after transplantation of CAP229 overexpression virus into the sciatic nerve of DPN mice; Figure 3 (B) and Figure 3 (C) Improvement of nerve conduction velocity and action potential amplitude after CAP229 overexpression virus transplantation into the sciatic nerve of DPN mice; Figure 3(D) Electron microscopy shows improvement of demyelination lesions after CAP229 overexpression virus was transplanted into the sciatic nerve of mice with diabetic peripheral neuropathy.

[0083] (1) Improvement of pain threshold

[0084] After local delivery of CAP229, the von Frey mechanical pain threshold of mice with diabetic peripheral neuropathy was significantly increased, indicating a significant improvement in mechanical pain sensitivity.

[0085] (2) Recovery of nerve conduction velocity and action potential

[0086] CAP229 treatment significantly increased nerve conduction velocity (NCV) and compound muscle action potential (CMAP) amplitude in mice with diabetic peripheral neuropathy, indicating that CAP229 helps restore nerve signal conduction ability.

[0087] (3) Pathological results

[0088] Combined with the results of electron microscopy observation, the structural integrity of the sciatic nerve myelin sheath of mice in the CAP229 treatment group was significantly improved, supporting its neuroprotective effect.

[0089] This application systematically evaluated the therapeutic potential of CAP229 in a mouse model of diabetic peripheral neuropathy. After local delivery of CAP229, the mechanical pain threshold of mice was significantly increased, suggesting that it has potential application value in alleviating DPN-related hyperalgesia. The recovery of nerve conduction velocity and CMAP amplitude further demonstrated that CAP229 can effectively improve the neuroelectrophysiological function of DPN mice, suggesting that it helps maintain the integrity and functional state of nerve fibers. Combined with the pathological results, CAP229 significantly alleviated the myelin pathological damage of the sciatic nerve of DPN mice, suggesting that it exerts a neuroprotective effect by promoting Schwann cell proliferation and maintaining the stability of myelin structure. These results provide a strong experimental basis for the development of CAP229 as a potential therapeutic drug for DPN.

[0090] The terms "including" and "having" and any variations thereof in the specification and claims of this application and the above-mentioned drawings are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or equipment comprising a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units that are not listed, or may optionally include other steps or units inherent to these processes, methods, products or equipment. The terms "first", "second" and "third" are used to distinguish different objects, etc., and do not represent a sequence, nor do they limit the "first", "second" and "third" to different types.

[0091] In the description of the embodiments of this application, the words "exemplary," "for example," or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary," "for example," or "for example" in the embodiments of this application should not be construed as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary," "for example," or "for example" is intended to present the relevant concepts in a concrete manner.

[0092] In the description of the embodiments of the present application, unless otherwise specified, “ / ” means or, for example, A / B can mean A or B; “and / or” in the text is merely a description of the association relationship of associated objects, indicating that three relationships may exist, for example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. In addition, in the description of the embodiments of the present application, “multiple” refers to two or more than two.

[0093] In some processes described in the embodiments of the present application, multiple operations or steps are included that appear in a specific order. However, it should be understood that these operations or steps may not be performed in the order in which they appear in the embodiments of the present application or may be performed in parallel. The sequence numbers of the operations are only used to distinguish between different operations, and the sequence numbers themselves do not represent any order of execution. In addition, these processes may include more or fewer operations, and these operations or steps may be performed in sequence or in parallel, and these operations or steps may be combined.

[0094] The above are only preferred embodiments of the present application and do not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the description and drawings of this application, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. Use of a functional polypeptide CAP229 in the preparation of a targeted therapeutic drug for diabetic peripheral neuropathy, characterized in that: The amino acid sequence of the functional polypeptide CAP229 is shown in SEQ ID NO: 1, and the functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

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