Drug for targeted therapy of diabetic peripheral neuropathy and neuroprotective drug for regulating Schwann cell proliferation function

Abstract

The invention discloses a drug for targeted therapy of diabetic peripheral neuropathy and a neuroprotection drug for regulating and controlling the proliferation function of Schwann cells, and relates to the technical field of biological medicines.The drug for targeted therapy of diabetic peripheral neuropathy comprises functional polypeptide CAP229, the amino acid sequence of the functional polypeptide CAP229 is shown as SEQ ID NO: 1, and the functional polypeptide CAP229 is a functional polypeptide CAP229. The functional polypeptide CAP229 is applied to a protein drug for targeted therapy of diabetic peripheral nerve demyelination, a new selection for targeted therapy of diabetic peripheral neuropathy is provided, and 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 a good transformation prospect, and can be widely applied to the field of targeted therapy of diabetic peripheral nerve demyelination. The myelin sheath loss in diabetic neuropathy can be relieved in vitro and in animal models, and the method can be used for constructing a function screening platform and a high-throughput evaluation model.

Description

Technical Field

[0001] This application relates to the field of biomedical technologies, and particularly to drugs for targeted treatment of diabetic peripheral neuropathy and neuroprotective drugs for regulating the proliferative function of Schwann cells. Background Art

[0002] Diabetic peripheral neuropathy is one of the most common and highly disabling chronic complications of diabetes. Clinical manifestations include distal sensory hypoesthesia, pain, numbness, and reduced reflexes, severely affecting the daily life of patients. Currently, the treatment of diabetic peripheral neuropathy mainly focuses on symptomatic relief, and there is no effective fundamental treatment method with significant effects. Summary of the Invention

[0003] This application provides a drug for targeted treatment of diabetic peripheral neuropathy and a neuroprotective drug for regulating the proliferative function of Schwann cells, so as to provide a drug for diabetic peripheral neuropathy.

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

[0005] SEQ ID NO: 1 is specifically: MKMADRSGKIIPGQVYIEVEYDYEYEAKDRKIVIKQGERYILVKKTNDDWWQVKPDENSKAFYVPAQYVKEVTRKALMPPVKQVAGLPNNSTKIMQSLHLQRSTENVNKLPELSSFGKPSSSVQGTGLIRDANQNFGPSYNQGQTVNLSLDLTHNNGKFNNDSHSPKVSSQNRTRSFGHFPGPEFLDVEKTSFSQEQSCDSAGEGSERIHQDSESGDELSSSSTEQIRV.

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

[0007] In a second aspect, this application provides a neuroprotective drug for regulating the proliferative function of Schwann cells, including a functional polypeptide CAP229, and the amino acid sequence of the functional polypeptide CAP229 is as 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 the drugs for 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 will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0011] Figure 1 It is a verification experimental result diagram of the encoding and translation of the CAP229 polypeptide by hsa_circ_0000231 in Example 1 of the present application. Figure 2 It is an experimental result diagram of the promotion of Schwann cell proliferation and cycle transformation by the CAP229 polypeptide in Example 2 of the present application.

[0012] Figure 3 It is an experimental result diagram of the behavioral and nerve conduction function evaluation after constructing a mouse model of diabetic peripheral neuropathy and locally delivering CAP229 in Example 3 of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0013] To make the purpose, technical solutions and advantages of the present application clearer, the following will clearly and completely describe the technical solutions of the present application in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present application.

[0014] Diabetic peripheral neuropathy is one of the most common and highly disabling chronic complications of diabetes. The clinical manifestations include distal sensory hypoesthesia, pain, numbness, and weakened reflexes, which seriously affect the daily life of patients. At present, the treatment of diabetic peripheral neuropathy mainly focuses on symptomatic relief, there is no effective fundamental intervention method, and the effect is not significant.

[0015] In view of this, the present application provides a drug for targeted treatment of diabetic peripheral neuropathy and a neuroprotective drug for regulating the proliferation function of Schwann cells to provide a drug for diabetic peripheral neuropathy.

[0016] In a first aspect, the present application provides a drug for targeted treatment of diabetic peripheral neuropathy, including the functional polypeptide CAP229, and the amino acid sequence of the functional polypeptide CAP229 is as shown in SEQ ID NO: 1.

[0017] SEQ ID NO: 1 is specifically as follows: MKMADRSGKIIPGQVYIEVEYDYEYEAKDRKIVIKQGERYILVKKTNDDWWQVKPDENSKAFYVPAQYVKEVTRKALMPPVKQVAGLPNNSTKIMQSLHLQRSTENVNKLPELSSFGKPSSSVQGTGLIRDANQNFGPSYNQGQTVNLSLDLTHNNGKFNNDSHSPKVSSQNRTRSFGHFPGPEFLDVEKTSFSQEQSCDSAGEGSERIHQDSESGDELSSSSTEQIRV.

[0018] In combination with the first aspect, in some embodiments provided in the present application, the functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

[0019] In a second aspect, the present application provides a neuroprotective drug for regulating the proliferative function of Schwann cells, including the functional polypeptide CAP229, and the amino acid sequence of the functional polypeptide CAP229 is as shown in SEQ ID NO: 1.

[0020] In combination with the second aspect, in some embodiments provided in the present application, the functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

[0021] The present application provides a new targeted treatment option for diabetic peripheral neuropathy by applying the functional polypeptide CAP229 to drugs for targeted treatment of diabetic peripheral nerve demyelination.

[0022] The CAP229 polypeptide has advantages such as small molecule size, stable structure, and endogenous source, is suitable for protein drug platforms and gene delivery strategies, has good translational prospects, can alleviate myelin loss in diabetic neuropathy, can be used to construct a functional screening platform and a high-throughput evaluation model, and is helpful for the research and development of neuropathy drugs and mechanism research.

[0023] The technical solutions provided in the present application are described in detail below in combination with embodiments. Among the accompanying drawings of the experimental results, "*", "**", "***" are usually used to represent the significance levels of statistical tests, corresponding to different p-value ranges respectively, where "*" represents P < 0.05, "**" represents P < 0.01, and "***" represents P < 0.001.

[0024] Example 1 Verification of the translation of CAP229 encoded by hsa_circ_0000231 Verify whether hsa_circ_0000231 can encode and translate a polypeptide (CAP229) of 229 amino acids, and confirm the molecular weight, amino acid sequence and expression characteristics of this polypeptide.

[0025] 1) Construct an overexpression vector of hsa_circ_0000231 with a FLAG tag According to the sequence information of hsa_circ_0000231 (from CircBase, ID: hsa_circ_0000231), design and synthesize the hsa_circ_0000231 sequence containing an open reading frame (ORF), and introduce a 3×FLAG tag at the end of the ORF for subsequent detection.

[0026] Clone this sequence into the GV689 circular RNA expression vector (GeneChem, China) to construct an overexpression circular RNA expression vector of hsa_circ_0000231 + 3×FLAG, and package it into a virus.

[0027] 2) Cell culture and transfection Select 293T cells and culture them in high-glucose medium at 37°C in a 5% CO2 environment.

[0028] Transfect the hsa_circ_0000231-3×FLAG expression vector into 293T cells, and use the empty vector as a control (not expressing FLAG).

[0029] 48 hours after transfection, screen the stably transfected cell population with 2 μg / mL puromycin.

[0030] 3) Western blot detection of FLAG expression FLAG is not expressed in normal cells. Detection of FLAG indicates that this circRNA has the function of translating proteins.

[0031] Collect cells, extract total proteins using RIPA lysis buffer (containing protease inhibitors), and quantify by BCA method. Take 30 μg of protein samples, separate them by SDS-PAGE and transfer to a membrane, block with 5% skim milk for 1 hour, incubate with the primary antibody (anti-FLAG, Proteintech, 1:1000) and the HRP-labeled secondary antibody (1:5000) respectively, and develop with ECL.

[0032] 4) Immunoprecipitation (IP) and LC-MS / MS analysis Use anti-FLAG magnetic beads (MedChemExpress, USA) to immunoprecipitate the lysate of hsa_circ_0000231-3×FLAG overexpressing cells.

[0033] The eluted protein was separated by SDS-PAGE, stained with Coomassie Brilliant Blue, and the band at 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 the 3×FLAG tag).

[0034] After cutting the gel, it 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).

[0035] The mass spectrometry data was analyzed using Peaks Studio software, and the amino acid sequence was matched and compared with the predicted CAP229 sequence.

[0036] 5) Prepare anti-CAP229 antibody and detect its expression in Schwann cells and peripheral nerves.

[0037] Prepare anti-CAP22 antibody (Abclonal, China); construct a control vector with a mutation in the ATG start codon (ATG→ACG) (this vector cannot generate the translation of CAP229) to verify translation dependence.

[0038] Human Schwann cells (SCs, ATCC, CRL-3392™) were selected and cultured in SC medium (ATCC, 30-2002™, containing 5% FBS). The overexpression vector of hsa_circ_0000231 was transfected into SCs, and the empty vector and the mutant vector were used as controls.

[0039] Detect the expression of CAP22 in Schwann cells and peripheral nerve specimens. According to the SDS-PAGE migration rate and LC-MS / MS analysis results, it was confirmed that the molecular weight of the identified peptide segment was approximately 25.7 kDa, which was significantly different from the 108.5 kDa of the parental protein ARHGAP12.

[0040] The experimental results are as Figure 1 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 detection showed that FLAG protein was not expressed in 293T cells, but FLAG protein was produced after overexpressing hsa_circ_0000231-3×FLAG, confirming that hsa_circ_0000231 has the function of encoding and translation; Figure 1(C) Immunoprecipitation of the hsa_circ_0000231-3×FLAG overexpressing cell lysate was performed with anti-FLAG antibody, and its amino acid sequence was separated by SDS-PAGE and analyzed by LC-MS / MS. Figure 1 (D) Western blot verification was performed using anti-CAP229 antibody, and CAP229 was expressed in peripheral nerve tissue. Figure 1 (E) The expression level of CAP229 increased after overexpression of hsa_circ_0000231 in Schwann cells, while the expression level of CAP229 decreased significantly after mutation of the ATG start codon.

[0041] (1) Western blot results showed that obvious FLAG positive bands were detected in 293T cells transfected with has_circ_0000231-3×FLAG, with a molecular weight of 25-30 kDa, while no FLAG signal was detected in the control, confirming that has_circ_0000231 can encode and translate polypeptides.

[0042] (2) After Coomassie Brilliant Blue staining, a protein band of about 28.9 kDa was specifically detected in the has_circ_0000231-3×FLAG overexpression group. This band was analyzed by LC-MS / MS, and it was clearly identified that it had a total of 229 amino acids, and the sequence was completely consistent with the amino acid sequence predicted by the circBank database for the open reading frame of has_circ_0000231.

[0043] (3) Western blot detection using anti-CAP229 antibody further confirmed the expression of CAP229 protein in Schwann cells and peripheral nerve tissue. The expression level of CAP229 protein increased after overexpression of has_circ_0000231, while the expression level of CAP229 protein decreased significantly after mutation of the ATG start codon, further confirming that CAP229 protein is encoded and translated by has_circ_0000231.

[0044] (4) Combining the mass spectrometry results and Western blot detection, it was confirmed that hsa_circ_0000231 can translate to produce a polypeptide composed of 229 amino acids, with a molecular weight of about 25.7 kDa.

[0045] Example 2 CAP229 Promotes Proliferation and Cell Cycle Transition of Schwann Cells 1) Construction of CAP229 Overexpression Vector and Viral Packaging According to the sequence information of the coding region of has_circ_0000231, the full-length CAP229 sequence was synthesized and cloned into the GV492 expression vector (GeneChem, China) to construct a CAP229 overexpression plasmid, which was then packaged into a virus.

[0046] 2) Schwann cell culture and CAP229 overexpression Human Schwann cells (SCs, ATCC CRL-3392) were cultured in a dedicated Schwann cell medium containing 5% fetal bovine serum (ATCC, 30-2002) at 37°C and 5% CO2. When the Schwann cells grew to 30-50% confluence, they were transfected with the CAP229 overexpression virus. After 48 hours of transfection, they were screened with 2 μg / mL puromycin for 5 days to establish a stable overexpression cell line. The control group was Schwann cells infected with the empty vector (Vector).

[0047] 3) Protein expression verification Western blot detection was performed using a CAP229 antibody (Abclonal, China) to verify the expression level of the CAP229 protein after transfection. The results showed that the expression of the CAP229 protein was significantly upregulated in the overexpression group.

[0048] 4) Cell proliferation detection The CAP229 overexpressing and control SCs were respectively seeded into 96-well plates at 5000 cells per well and cultured for 24, 48, and 72 hours.

[0049] 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 hour of incubation.

[0050] The BeyoClick™ EdU-594 Cell Proliferation Detection Kit (Beyotime, China) was used to evaluate the DNA synthesis ability of Schwann cells. Schwann cells were seeded into 96-well plates at 2×10 4 cells per well, 10 μM EdU was added, and after 2 hours of incubation, they were fixed, permeabilized, and stained. Five random fields of view were selected through a fluorescence microscope (Olympus, Japan) to calculate the proportion of EdU-positive cells.

[0051] 5) Flow cytometry cell cycle analysis Schwann cells in the CAP229 overexpression group and the control group were collected, rinsed twice with PBS, and fixed overnight with pre-cooled 70% ethanol. The next day, after washing with PBS, a staining solution containing 100 μg / mL RNase A and 50 μg / mL propidium iodide (PI) was added, and the cells were incubated in the dark for 40 minutes. The cell cycle distribution was detected using a flow cytometer (BD FACS Canto II), the proportions of G1, S, and G2 / M phases were analyzed, and the data were processed using ModFit software.

[0052] The experimental results are as Figure 2 shown, Figure 2 (A) Western blot detection showed that the protein level of CAP229 was significantly increased after overexpression in Schwann cells; Figure 2 (B) and Figure 2 (C) The results of EdU and CCK8 assays showed that CAP229 promoted the proliferation of Schwann cells; Figure 2 (D) Cell cycle analysis showed that the proportion of G2 / M phase cells increased while the proportion of G0 / G1 phase cells decreased in Schwann cells after CAP229 overexpression.

[0053] (1) CAP229 significantly promoted the proliferation of SCs The results of the CCK-8 assay showed that compared with the control group, the OD values of SCs in the CAP229 overexpression group were significantly higher than those in the control group at 48 and 72 hours, indicating that CAP229 enhanced the proliferation ability of SCs. The results of the EdU assay were consistent, and the proportion of EdU-positive cells in the CAP229 group was significantly increased.

[0054] (2) CAP229 promoted the G1 / S phase transition Flow cytometric cell cycle analysis showed that the proportion of G0 / G1 phase cells in the CAP229 overexpression group was significantly reduced, while the proportion of G2 / M phase cells was significantly increased. This result suggests that CAP229 promotes SCs to enter the G2 phase from the G1 phase, accelerating the cell cycle process.

[0055] Example 3 Evaluation of behavioral and nerve conduction functions after establishing a mouse model of diabetic peripheral neuropathy and locally delivering CAP229 1) Establishment of a mouse model of diabetic peripheral neuropathy (DPN) Eight-week-old male C57BL / 6J mice were selected and divided into the DPN group and the DPN+CAP229 treatment group.

[0056] After the DPN model was established by feeding a high-fat diet for 6 weeks, streptozotocin (STZ, 50 mg / kg, Sigma-Aldrich) was intraperitoneally injected for 3 consecutive days, and the high-fat diet was continuously fed. From the 7th day after injection, if the fasting blood glucose value was ≥16.7 mmol / L (300 mg / dL) for 3 consecutive times, it was determined that the diabetes model was successfully established. The confirmation of the DPN phenotype included a decrease in mechanical pain threshold, a decrease in thermal pain threshold, and a slowdown in electrophysiological nerve conduction velocity.

[0057] 2) Injection of CAP229 overexpression virus After the DPN model was established, an injection of CAP229 overexpression virus was performed in the sciatic nerve of the mice. Using a Hamilton microsyringe (10 μL, equipped with a 33G needle), 2.5 μL of a CAP229 overexpression lentivirus with a total amount of 5×10 6 TU was slowly injected at a position about 5 mm distal to the sciatic nerve, and the injection rate was about 0.2 μL / min. The needle was kept in place for 5 minutes and then slowly withdrawn to prevent backflow. Control DPN mice were injected with an equal volume of empty virus.

[0058] 3) Evaluation of pain threshold Eight weeks after the virus injection, the pain thresholds of mice in each group were evaluated respectively.

[0059] Mechanical pain threshold: Test was performed using von Frey filaments (Stoelting, 0.04 - 2 g), and the 50% pain threshold was determined using the Dixon up-down method.

[0060] 4) Detection of nerve conduction function The electrophysiological instrument was used to evaluate the sciatic nerve conduction function: nerve conduction velocity and action potential amplitude.

[0061] 5) Detection of myelin sheath lesions in the sciatic nerve by electron microscopy Eight weeks after the virus injection, the sciatic nerve was sampled, and electron microscopy was used to detect the effect of CAP229 on diabetic peripheral nerve demyelination lesions.

[0062] The experimental results are as Figure 3 shown, Figure 3 (A) shows the 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) show the improvement of nerve conduction velocity and action potential amplitude after transplantation of CAP229 overexpression virus into the sciatic nerve of DPN mice; Figure 3 (D) shows the improvement of demyelination lesions shown by electron microscopy after transplantation of CAP229 overexpression virus into the sciatic nerve of mice with diabetic peripheral neuropathy.

[0063] (1)Improvement of pain threshold 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.

[0064] (2)Recovery of nerve conduction velocity and action potential Treatment with CAP229 significantly increased the nerve conduction velocity (NCV) and compound muscle action potential (CMAP) amplitude in mice with diabetic peripheral neuropathy, indicating that CAP229 helps to restore the nerve signal conduction ability.

[0065] (3)Pathological results Combined with the results of electron microscopy observation, the integrity of the myelin sheath structure of the sciatic nerve in the CAP229-treated group of mice was significantly improved, supporting its neuroprotective effect.

[0066] 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 the mice was significantly increased, suggesting its potential application value in relieving DPN-related hyperalgesia. The recovery of nerve conduction velocity and CMAP amplitude further demonstrated that CAP229 could effectively improve the neuroelectrophysiological function of DPN mice, suggesting that it helps to maintain the integrity and functional state of nerve fibers. Combined with the pathological results, CAP229 significantly alleviated the myelin sheath pathological damage of the sciatic nerve in DPN mice, suggesting that it plays a neuroprotective role by promoting Schwann cell proliferation and maintaining the stability of the myelin sheath structure. These results provide strong experimental evidence for the development of CAP229 as a potential therapeutic drug for DPN.

[0067] The terms "comprising" and "having" and any variations thereof in the specification, claims and above-mentioned drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps or units is not limited to the listed steps or units, but optionally further includes steps or units not listed, or optionally further includes other steps or units inherent to these processes, methods, products or equipment. The descriptions of terms such as "first", "second" and "third" are used to distinguish different objects, etc., and do not represent a sequence, nor do they limit that "first", "second" and "third" are of different types.

[0068] In the description of the embodiments of this application, words such as "exemplary", "for example", or "for illustration purposes" are used to indicate examples, illustrations, or explanations. Any embodiment or design solution described as "exemplary", "for example", or "for illustration purposes" in the embodiments of this application should not be construed as being more preferred or having more advantages than other embodiments or design solutions. Rather, the use of words such as "exemplary", "for example", or "for illustration purposes" is intended to present the relevant concepts in a specific manner.

[0069] In the description of the embodiments of this application, unless otherwise specified, " / " means "or". For example, A / B can mean A or B. The "and / or" in the text is merely a description of the association relationship between associated objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. Additionally, in the description of the embodiments of this application, "a plurality of" means two or more than two.

[0070] In some of the processes described in the embodiments of this application, there are multiple operations or steps that appear in a specific order. However, it should be understood that these operations or steps may not be executed in the order in which they appear in the embodiments of this application or may be executed in parallel. The serial numbers of the operations are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. Additionally, these processes may include more or fewer operations, and these operations or steps may be executed in order or in parallel, and these operations or steps may be combined.

[0071] The above are only the preferred embodiments of this application and do not limit the patent scope of this application. Any equivalent structural or equivalent process transformation made using the content of the specification and drawings of this application, or directly or indirectly applied in other related technical fields, shall similarly be included within the patent protection scope of this application.

Claims

1. A drug for targeted treatment of diabetic peripheral neuropathy, characterized in that, It includes the functional polypeptide CAP229, and the amino acid sequence of the functional polypeptide CAP229 is shown as SEQ ID NO:

1.

2. The drug for targeted treatment of diabetic peripheral neuropathy according to claim 1, characterized in that, The functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

3. A neuroprotective drug for regulating the proliferation function of Schwann cells, characterized in that, It includes the functional polypeptide CAP229, and the amino acid sequence of the functional polypeptide CAP229 is shown as SEQ ID NO:

1.

4. The neuroprotective drug for regulating the proliferation function of Schwann cells according to claim 3, wherein The functional polypeptide CAP229 is encoded by hsa_circ_0000231 in Schwann cells.

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