Use of polypeptides for the preparation of a medicament for preventing and treating central nervous system hypoxic diseases

Abstract

The application discloses application of a TAT-MK-ELE polypeptide in preparation of a central nervous system hypoxic disease prevention and treatment drug. In order to simulate the effect of MK-4, the TAT-MK-ELE polypeptide is designed and synthesized, and research proves that the TAT-MK-ELE polypeptide has a protective effect on hypoxic oligodendrocytes, can significantly improve the activity of the hypoxic oligodendrocytes, and can significantly inhibit the expression of proinflammatory factors in the hypoxic oligodendrocytes, thereby providing a reference for clinically preventing and treating central nervous system diseases caused by hypoxia by taking oligodendrocytes as a target.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of TAT-MK-ELE polypeptide in the preparation of drugs for the prevention and treatment of hypoxic diseases of the central nervous system. Background Technology

[0002] Oligodendrocytes are the myelin-forming cells of myelinated nerve fibers in the central nervous system and important components of brain white matter. In addition to the functions mentioned above, oligodendrocytes also nourish and protect axons, provide neurotrophic factors and growth factors to the central nervous system, and produce axonal growth inhibitory factors. Clinically, oligodendrocytes are closely related to central nervous system diseases such as multiple sclerosis, spinal cord injury, and brain white matter injury. Furthermore, like neurons, oligodendrocytes are extremely sensitive to hypoxia. Hypoxia-induced damage and death of central nervous system cells is the pathological basis for the occurrence and development of various brain diseases; for example, hypoxia is the leading cause of neonatal brain white matter injury.

[0003] However, current clinical drugs for treating hypoxic-ischemic injury of oligodendrocytes are not very effective. Therefore, studying the mechanism of hypoxic-ischemic injury of oligodendrocytes and finding protective agents against hypoxic oligodendrocytes will be of great significance for treating hypoxic-ischemic injuries of the central nervous system, such as white matter injury and demyelinating encephalopathy. Summary of the Invention

[0004] The purpose of this invention is to provide the application of TAT-MK-ELE polypeptide in the preparation of drugs for the prevention and treatment of hypoxic diseases of the central nervous system.

[0005] The amino acid sequence of the TAT-MK-ELE polypeptide is as follows:

[0006] GRKKRRQRRRPQGGSGHSGELETAFADAWRTPTTTVIEM(seq_1).

[0007] GRKKRRQRRRPQ is the TAT sequence that promotes the entry of the polypeptide into the cell, GGSGHSG is the linker peptide, and ELETAFADAWRTPTTTVIEM is the target sequence.

[0008] The specific artificial amino acid sequence is: Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Pro GlnGly Gly Ser Gly His Ser Gly Glu Leu Glu Thr Ala Phe Ala Asp Ala Trp Arg ThrPro Thr Thr Thr Val Ile Glu Met.

[0009] Experiments have shown that the TAT-MK-ELE peptide can significantly enhance the activity of hypoxic oligodendrocytes, while simultaneously significantly inhibiting the expression of pro-inflammatory factors within hypoxic oligodendrocytes.

[0010] The TAT-MK-ELE polypeptide provided by this invention can protect hypoxic oligodendrocytes by improving the expression of pro-inflammatory factors in them. This has promising application prospects in the design and preparation of drugs targeting oligodendrocytes for the prevention and treatment of hypoxic diseases of the central nervous system. Attached Figure Description

[0011] Figure 1 The results show the effect of TAT-MK-ELE peptide on the viability of hypoxic oligodendrocytes. Among them, # *p<0.05 vs DMSO treatment for normal oxygen content group; *p<0.05 vs DMSO treatment for hypoxia group.

[0012] Figure 2 This study investigated the effect of TAT-MK-ELE peptide on the expression of the pro-inflammatory factor IL-1β mRNA in hypoxic oligodendrocytes. The treatment groups were: N+D (DMSO treatment for normal oxygen levels); N+M (MK-4 treatment for normal oxygen levels); N+T (TAT-MK-ELE treatment for normal oxygen levels); H+D (DMSO treatment for hypoxia); H+M (MK-4 treatment for hypoxia); and H+T (TAT-MK-ELE treatment for hypoxia). # p<0.05 vs N+D group; *p<0.05 vs H+D group.

[0013] Figure 3 This study investigated the effect of TAT-MK-ELE peptide on the expression of the pro-inflammatory factor IL-6 mRNA in hypoxic oligodendrocytes. The treatment groups were categorized as follows: N+D: DMSO treatment for normal oxygen levels; N+M: MK-4 treatment for normal oxygen levels; N+T: TAT-MK-ELE treatment for normal oxygen levels; H+D: DMSO treatment for hypoxia; H+M: MK-4 treatment for hypoxia; and H+T: TAT-MK-ELE treatment for hypoxia. # p<0.05 vs N+D group; *p<0.05 vs H+D group.

[0014] Figure 4This study investigated the effect of TAT-MK-ELE peptide on the expression of the pro-inflammatory factor Tnf-α mRNA in hypoxic oligodendrocytes. The treatment groups were: N+D (DMSO treatment for normal oxygen levels); N+M (MK-4 treatment for normal oxygen levels); N+T (TAT-MK-ELE treatment for normal oxygen levels); H+D (DMSO treatment for hypoxia); H+M (MK-4 treatment for hypoxia); and H+T (TAT-MK-ELE treatment for hypoxia). #p<0.05 vs N+D group; *p<0.05 vs H+D group. Detailed Implementation

[0015] Menaquinone-4 (MK-4), also known as vitamin K2 (VK2), is converted into MK-4 in all organs except the liver. MK-4 is the main active form of vitamin K in the brain. Studies have shown that MK-4 can promote the survival of neurons in the late embryonic cortex, hippocampus, and striatum; MK-4 can inhibit lipopolysaccharide-induced microglial inflammation; and MK-4 can reduce oxidative stress damage to developing embryonic cortical neurons and oligodendrocyte precursor cells. These studies indicate that MK-4 plays a role in anti-oxidative stress and anti-inflammation. Research has also confirmed that MK-4 has a protective effect against hypoxic damage to oligodendrocytes. In order to mimic the effects of MK-4 and find a drug that can be synthesized in large quantities and is easy to use in clinical practice, the inventors designed and synthesized the TAT-MK-ELE peptide. At the same time, the study proved that the TAT-MK-ELE peptide has a protective effect on hypoxic oligodendrocytes, providing a reference for the clinical prevention and treatment of hypoxic central nervous system diseases by targeting oligodendrocytes.

[0016] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0017] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0018] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0019] Example 1

[0020] I. Experimental Materials

[0021] TAT-MK-ELE (Qiangyao Biotechnology), Rat Olendenocytes Oln-93 (Beijing Zhongke Quality Inspection Biotechnology Co., Ltd.), DMEM-F12 (Thermo), Fetal Bovine Serum (Gibco), Cell Counting Kit-8 (CCK-8, Dojindo Laboratories), TRI Reagnet (Sigma), SYBR Green PCR Kit (Thermo).

[0022] II. Experimental Methods

[0023] 1. Preparation of TAT-MK-ELE polypeptide solution

[0024] The lyophilized peptide powder was dissolved in sterile water and stored at a concentration of 1 mg / mL. It was dispensed in 100 μL containers and stored at -80°C.

[0025] 2. Establishment of cell culture and hypoxia model

[0026] Oln-93 cells were cultured in DMEM-F12 medium containing 10% fetal bovine serum and passaged after reaching confluence. Oln-93 cells were seeded into culture plates and divided into a normal oxygen content group and a hypoxia group. The normal oxygen content group was cultured in DMEM-F12 medium containing 10% FBS in an incubator at 5% CO2 / 95% air and 37°C. The hypoxia group was cultured in a DWS H35 hypoxia workstation (5% CO2 / 95% N2, 37°C) with glucose-free and serum-free conditions to establish a hypoxic oligodendrocyte model, subjected to hypoxia for 12 h.

[0027] 3. CCK-8 cell viability assay

[0028] Oln-93 cells were used at 10 per well 4 Cells were seeded in 96-well plates with 100 μL of cell suspension per well and placed in an incubator. The next day, cells were divided into normal oxygen and hypoxia groups, and the corresponding culture media were used. Cells were pretreated for 20 min with 0.01% DMSO, 8 μmol / L MK-4, or 20 μg / mL TAT-MK-ELE peptide. Cells were then incubated for 12 h in a normal oxygen incubator or a hypoxia workstation. After incubation, 10 μL of CCK-8 solution was added to each well, and the cells were incubated for 2 h. The absorbance at 450 nm was measured using a Tecan M200 microplate reader. Each concentration was tested in triplicate, and the experiment was repeated three times.

[0029] 4. Detection of changes in the expression of inflammatory factor mRNA

[0030] Cells were pretreated with 0.01% DMSO, 8 μmol / L MK-4, or 30 μg / mL TAT-MK-ELE peptide for 20 min, followed by incubation under normal oxygen or hypoxia for 12 h, respectively. Total RNA was extracted from cells in each experimental group using TRI Reagnet, and cDNA was synthesized using a reverse transcription kit. Primers for interleukin-1 beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and β-actin were designed using Primer 3 software version 1.0 (Table 1).

[0031] Table 1. Primer sequences for real-time quantitative RT-PCR

[0032]

[0033]

[0034] III. Experimental Results

[0035] 1. TAT-MK-ELE peptide enhances the activity of hypoxic oligodendrocytes.

[0036] MK-4 can protect oligodendrocytes and significantly enhance the activity of hypoxic oligodendrocytes. Oln-93 cells were pretreated for 20 min with 0.01% DMSO, 8 μmol / L MK-4, or 20 μg / mL TAT-MK-ELE peptide, respectively, followed by 12 h of hypoxia. The results showed that after 12 h of hypoxia, the activity of Oln-93 cells significantly decreased, but MK-4 and TAT-MK-ELE peptide significantly improved the activity of hypoxic oligodendrocytes. Figure 1 More importantly, TAT-MK-ELE peptide is more effective than MK-4 in enhancing the activity of hypoxic oligodendrocytes. Figure 1 ).

[0037] 2. TAT-MK-ELE peptide inhibits the expression of pro-inflammatory factors in hypoxic oligodendrocytes.

[0038] To further understand the mechanism of the protective effect of the small molecule peptide TAT-MK-ELE on hypoxic oligodendrocytes, the effect of TAT-MK-ELE on intracellular pro-inflammatory factors in hypoxic oligodendrocytes was detected by real-time quantitative RT-PCR. The results showed that after 12 h of hypoxia in Oln-93 cells, the mRNA levels of intracellular pro-inflammatory cytokines IL-1β, IL-6, and Tnf-α were significantly increased; however, when hypoxic cells were treated with 8 μmol / L MK-4 or 30 μg / ml TAT-MK-ELE, the mRNA levels of intracellular IL-1β, IL-6, and Tnf-α were significantly decreased compared to the DMSO-treated hypoxic group. Figures 2-4 More importantly, the TAT-MK-ELE peptide is more effective than MK-4 in inhibiting the expression of pro-inflammatory factors in hypoxic cells. Figures 2-4 This result indicates that hypoxia can lead to increased expression of pro-inflammatory factors in oligodendrocytes, causing cellular inflammatory responses. However, the TAT-MK-ELE peptide can effectively reduce hypoxia-induced inflammatory responses in oligodendrocytes, thereby protecting the cells.

Claims

1. The use of a TAT-MK-ELE polypeptide in the preparation of a drug for preventing and treating a central nervous system hypoxic disease, characterized in that, the amino acid sequence of the TAT-MK-ELE polypeptide is: GRKKRRQRRRPQGGSGHSGELETAFADAWRTPTTTVIEM.

2. Use according to claim 1, characterized in that, the TAT-MK-ELE polypeptide improves the activity of hypoxic oligodendrocytes.

3. Use according to claim 1, characterized in that, the TAT-MK-ELE polypeptide inhibits the expression of pro-inflammatory factors in hypoxic oligodendrocytes.

4. A prophylactic or therapeutic drug for central nervous system anoxic diseases, characterized by comprising the compound of claim 1 or 2 as an active ingredient. the TAT-MK-ELE polypeptide is used as an effective active ingredient.

5. The central nervous system hypoxic disease-preventing and treating drug according to claim 4, characterized in that, a pharmaceutically acceptable carrier is further included.

Images