Application of insulin-like growth factor binding protein 2 to prepare drugs for the treatment of neurodemyelinating diseases

Abstract

The present invention relates to the application of insulin-like growth factor binding protein 2 to prepare drugs for the treatment of neuronal demyelinating diseases. IGFBP2 intervention can significantly enhance the myelination level and improve the psychiatric symptoms caused by stress demyelination.

Description

[Technical field]

[0001] This application claims priority to Chinese Invention Patent Application No. 202210074291.3 filed on January 21, 2022, all of which are incorporated herein by reference.

[0002] The present invention relates to the field of medical technology, in particular to the application of insulin-like growth factor binding protein 2 for preparing drugs for the treatment of neuronal demyelinating diseases. [Background technology]

[0003] Myelin is the membrane that surrounds the outside of the nerve cell axon; in other words, the myelin sheath is composed of tens of thousands of cells and myelin cell membranes. The myelin sheath plays an insulating role, preventing the conduction of nerve electrical impulses from the axon of one neuron to the axon of another neuron. Demyelination refers to damage to the myelin sheath that occurs after the formation of the myelin sheath. Demyelinating diseases are acquired disorders with different etiologies and clinical symptoms but similar characteristics, the characteristic pathological change being that the nerve fiber is demyelinated, but the nerve cell remains relatively intact. Since normal conduction of nerve impulses depends on the insulating properties of the myelin sheath around the axon, myelin dysfunction or changes can impair learning and adaptive behavior. Research data show that even small changes in myelin throughout the life course can affect mechanisms in circuits and systems involved in cognition and behavior. The function of myelin may be broader than previously assumed: dysfunctional or maladaptive levels of myelination may be the cause of neurodegenerative and neuropsychiatric disorders. These diseases have previously been thought to be neuronal in origin.

[0004] Many human and animal studies have demonstrated that white matter demyelination in specific brain regions of the central nervous system may be an important pathophysiological feature of psychiatric disorders. White matter serves as an important neural substrate connecting distributed brain systems. Neuroimaging studies reveal abnormal microstructures in human white matter exposed to stress. White matter demyelination changes are reportedly associated with stress maladaptation and resilience. Moreover, evidence from anxiety disorder patients strongly reveals that white matter demyelination in pathways related to the prefrontal cortex is inversely correlated with trait anxiety scores. Moreover, demyelination in the mPFC has also been observed in various animal stress models accompanied by anxiety-like and depressive-like behaviors. We speculate that if myelin damage is repaired quickly and effectively, the risk of developing psychiatric disorders such as anxiety, depression, and cognitive impairment will be reduced.

[0005] Insulin growth factor binding protein 2 (IGFBP2), one of the six major members of IGF-binding proteins, is a growth factor with autocrine and paracrine activity. Studies have shown that IGFBP2 is involved not only in brain growth and development, but also in higher cognitive functions, such as regulating neuronal plasticity, spatial learning and memory, and information processing. In addition, IGFBP2 is widely present in the central system, highly expressed in the developing brain, and secreted by primary astrocytes and neurons. IGFBP2 has a low peripheral concentration, a molecular weight of 36 kD, and can cross the blood-brain barrier (BBB). However, there is no study to prove that IGFBP2 can treat psychiatric disorders after demyelination. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention aims to overcome the above-mentioned shortcomings of the prior art and provide the application of insulin-like growth factor binding protein 2 for preparing drugs for treating neurodemyelinating diseases. The drug for improving cognitive impairment of the present invention contains insulin-like growth factor binding protein 2, which is a drug for treating neuronal demyelinating diseases. Furthermore, this drug for improving cognitive impairment contains the insulin-like growth factor binding protein 2, which is a drug for enhancing the level of myelination. [Means for solving the problem]

[0007] The present invention specifically provides a technical solution of the application of insulin-like growth factor binding protein 2 to prepare drugs for treating neurodemyelinating diseases.

[0008] Preferably, the application of insulin-like growth factor binding protein 2 for the preparation of a medicament for enhancing myelination levels, wherein the enhancement of myelination levels relates to both the state of myelination and the level of myelination.

[0009] Alternatively, it relates to the application of insulin-like growth factor binding protein 2 for preparing a drug for treating neuronal demyelinating diseases.

[0010] Preferably, the neuro-demyelinating disease (demyelinating disease) comprises depression and cognitive impairment.

[0011] The present invention provides a method for evaluating the application of insulin-like growth factor binding protein 2 for preparing a medicament for treating a neurodemyelinating disease, the method comprising the steps of: S1: Establishing a demyelination model. S2: Select the evaluation criteria. S3: Evaluate the effects of IGFBP2 intervention. [Brief description of the drawings]

[0012] [Figure 1a] FIG. 1a shows a flow chart of drug demyelination modeling and IGFBP2 intervention. [Figure 1b] FIG. 1b shows a flow chart of stress demyelination modeling and IGFBP2 intervention. [Figure 2a] FIG. 2a shows the effect of IGFBP2 intervention on myelination levels in animals by drug demyelination. [Figure 2b] FIG. 2b shows the effect of IGFBP2 intervention on myelination levels in animals by drug demyelination. [Figure 3a]FIG. 3a shows the effect of IGFBP2 intervention on myelination levels in animals with stress demyelination. [Figure 3b] FIG. 3b shows the effect of IGFBP2 intervention on myelination levels in animals with stress demyelination. [Figure 3c] FIG. 3c shows the effect of IGFBP2 intervention on myelination levels in animals with stress demyelination. [Figure 3d] FIG. 3d shows the effect of IGFBP2 intervention on myelination levels in animals with stress demyelination. [Figure 3e] FIG. 3e shows the effect of IGFBP2 intervention on myelination levels in animals with stress demyelination. [Figure 4a] FIG. 4a shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals via drug demyelination. [Figure 4b] FIG. 4b shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals via drug demyelination. [Figure 4c] FIG. 4c shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals via drug demyelination. [Figure 4d] FIG. 4d shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals via drug demyelination. [Figure 4e] FIG. 4e shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals via drug demyelination. [Figure 4f] FIG. 4f shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals via drug demyelination. [Figure 5a] FIG. 5a shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. [Figure 5b] FIG. 5b shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. [Figure 5c]FIG. 5c shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. [Figure 5d] FIG. 5d shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. [Figure 5e] FIG. 5e shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. [Figure 5f] FIG. 5f shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. [Figure 5g] FIG. 5g shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. [Figure 5h] FIG. 5h shows the effect of IGFBP2 intervention on the post-stress demyelination behavior of animals subjected to stress demyelination. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In order to more clearly describe the technical contents of the present invention and verify the role of IGFBP2 in the treatment of neurological demyelinating diseases, further description will be given below in combination with specific examples. EXAMPLES

[0014] A demyelination model is established in SD rats.

[0015] SD rats were kept in a 12-hour light-dark environment with free access to water and food. All behavioral tests were performed in the light-dark environment of the animals.

[0016] Demyelination is performed using drugs.

[0017] First, SD rats are used to perform modeling, and anesthesia is induced with isoflurane (R510-22, RWD China, 4-5% induction, 1-2% maintenance). After deep anesthesia, the experimental group is injected through a surgical method with a 33-type syringe needle (Hamilton) according to the following stereotactic coordinates, where 1 μL of 1% hemolytic lecithin (LPC, Sigma-Aldrich) is dissolved in saline to obtain the injection solution, while the control group is also injected with the same amount of saline to obtain the injection solution. The injection solution is injected at +2.7 mm on both sides of the mPFC, 0.4 mm on the medial side of the anterior fontanelle, and 3.3 mm below the dorsal-dural surface. The volume of lysolecithin is 1 μL, and the injection time for one side is 15 min. After injection, the needle is left for 5 min to completely absorb the liquid. The Z-axis knob is slowly adjusted and the needle is pulled up. The skin is sutured to complete the brain injection.

[0018] Demyelination is performed in stress mode.

[0019] The stress mode was chronic unpredictable stress (CUMS), that is, chronic stress was performed once a day for 5 weeks in various stress modes. The specific stress modes are shown in Table 1.

[0020] [Table 1]

[0021] IGFBP2 intervention IGFBP2 intervention was performed on rats that had undergone drug-induced demyelination. Four days after LPC injection surgery, demyelination was completed and IGFBP2 intervention was performed. Eight days after surgery, behavior detection was performed. The experimental procedure is shown in Figure 1a.

[0022] IGFBP2 intervention is performed on rats that have undergone demyelination in stress mode. IGFBP2 intervention is performed on the first day after the end of stress. Habit detection is performed on the second day. The experimental procedure is shown in Figure 1b.

[0023] Evaluate the effects of IGFBP2 intervention First, we will observe myelin morphology through immunohistochemical analysis and electron microscopy to examine whether IGCBP2 reverses drug- or stress-induced demyelination. Immunohistochemistry: Rats were fixed on a single dissection board and perfused first with 1xPBS, then with 4% paraformaldehyde (PFA), and then fixed in 4% PFA overnight at 4°C. Next, the brain tissue was dehydrated in 30% sucrose until it was completely sunk to the bottom of a 50mL tube. After embedding in OCT, the brain was stored in a -80°C freezer. 30μm coronal sections were cut on a freezing microtome. The mPFC region sections were washed three times with PBS for 5 min each, and incubated sealed in PBS containing 4% goat serum and 0.1% Triton X-100 for 1 h at room temperature, followed by incubation with the secondary antibody MBP antibody (1:500) at 4°C overnight. The sections were washed five times in PBS and 0.1% Triton X-100 (PBST) at room temperature, incubated with fluorescent secondary antibody Alexa Fluor 488 goat anti-rabbit antibody diluted 1:1000 at room temperature for 5 min, followed by five washes in PBST in the dark for 5 min each time. Sections were counterstained with DAPI and sealed. All images were taken with a confocal microscope (Olympus) and analyzed with Fuji (NIMH, Bethesda MD, USA).

[0024] Electron microscopy experiments: The purpose of these experiments was to measure the thickness of the myelin sheath, and the thickness and length of the postsynaptic dense bodies (PSDs) in the mPFC region. 3 The mPFC brain region is trimmed to include the area. Sections are cut at 60 nm on an ultramicrotome (Leica EM UC7) and subsequently stained with uranyl acetate and lead citrate. Images are collected for each rat on a transmission electron microscope (Tecnai G2 SpiritBiotwin). The G-ratio is determined by dividing the axon diameter by the diameter of the entire myelinated nerve fiber. A FUJI (NIMH, Bethesda MD, USA) is used to measure axonal caliber, myelinated fiber diameter, and PSD thickness and length.

[0025] As shown in Figures 2a and 2b, the intervention effect of IGFBP2 is observed through immunohistochemical analysis after demyelination. Figures 2a and 2b show the myelination level in the mPFC brain region, where the myelination level is significantly decreased after drug demyelination with a significant decrease in MBP expression, and significantly increased after IGFBP2 intervention. According to two-way ANOVA, *** represents P<0.001, ** represents P<0.001, and * represents P<0.05.

[0026] As shown in Figures 3a-3e, after demyelination with stress, the results of the electron microscopy experiment show that stress causes demyelination and improves myelination after IGFBP2 intervention. The results of the immunofluorescence experiment show that stress causes a significant decrease in the fluorescence intensity of MBP and IGFBP2 intervention restores the fluorescence intensity. Here, *** represents P<0.001, ** represents P<0.001, * represents P<0.05, * represents the control group vs. the stress group, and # represents the control group vs. the IGFBP2 intervention group.

[0027] The above experimental data demonstrate that IGFBP2 intervention significantly improves myelination and enhances the level of myelination in demyelinating diseases.

[0028] Furthermore, behavioral experiments will observe whether IGFBP2 intervention improves psychiatric symptoms of demyelinating diseases.

[0029] Behavioral detection is used to verify the intervention effect of IGFBP2 on psychiatric symptoms after demyelination. All behavioral detection is performed with rats during light hours. Before behavioral detection, rats are placed in a behavioral detection chamber and adapted for 2 hours. During each experiment, rats that have completed the experiment are wiped with 70% alcohol to avoid odor influence on the next rat. Before behavioral detection, rats are handled every day for a few minutes to allow them to adapt for at least 4 days. Behavioral detection employs sugar water preference test, tail suspension test, and forced swimming test for evaluation of depression-like behavior, open space test and elevated plus maze test for evaluation of anxiety-like behavior, and novel object discrimination test and Barnes maze test for evaluation of cognitive behavior.

[0030] Sugar water preference experiment: The experimental animals were adapted to two bottles two days ago, one bottle had drinking water and the other bottle had 1% sucrose. The detection was carried out on the third day, and the animals were cut off from drinking water supply for 6 hours before the detection, where the sugar water preference value % = sugar water consumption / (sugar water consumption + water consumption) × 100.

[0031] Open-area experiment: The experimental animals were placed in a 50 cm x 50 cm box, and the video was recorded for 10 minutes. The time the animals spent in the central area was calculated.

[0032] Elevated plus maze experiment: Rats are placed in a cross maze from the central compartment toward the open arms, and the number of times and the time spent by the rat in the open arms, and the number of times and the time spent by the rat in the closed arms are recorded for 5 minutes.

[0033] Water maze experiment: Rats are trained for 4 days, the time it takes for the rats to find the platform is recorded, and on the final day the time the animals spend in the destination quadrant is recorded.

[0034] For the experimental animals that were demyelinated with drugs, Figures 4a-4d show the results of the open-space experiment, and Figures 4e and 4f show the results of the elevated plus maze experiment, where the ratio of time that the experimental animals spent in the open arms was the time spent in the open arms / (time spent in the open arms+time spent in the closed arms). According to two-way ANOVA, *** represents P<0.001, ** represents P<0.001, and * represents P<0.05.

[0035] The results of the wide-space experiment show that the time spent in the central zone by animals in the demyelination group (LPC group) was significantly lower than that of the control group, but after IGFBP2 intervention, the time spent in the central zone by animals in the demyelination group (IGFBP2 intervention group) was significantly higher.

[0036] The results of the elevated plus maze experiment show that the time spent in the open arms of animals in the demyelination group (LPC group) is significantly lower than that of the control group, but IGFBP2 intervention significantly increases the time spent in the open arms of animals in the demyelination group (IGFBP2 intervention group).

[0037] For the experimental animals that were demyelinated by stress, stress caused demyelination of the mPFC brain region. Figures 5a-5c show the results of the open space experiment, Figures 5d and 5e show the results of the elevated plus maze experiment, where the ratio of time that the experimental animals stayed in the open arms was the time spent in the open arms / (time spent in the open arms+time spent in the closed arms), Figure 5f shows the results of the sugar-water preference experiment, where the ratio of sugar-water preference of the animals = sugar-water consumption / (sugar-water consumption+water consumption), and Figures 5g and 5h show the results of the water maze experiment, where the time that the animals found the platform during training. According to two-way ANOVA, *** represents P<0.001, ** represents P<0.001, * represents P<0.05.

[0038] The results of the open-space experiment show that IGFBP2 intervention does not affect the time spent in the central zone of the control group animals. The time spent in the central zone of the stress group animals is significantly lower than that of the control group, but after IGFBP2 intervention, the time spent in the central zone of the stress group animals is significantly higher (P<0.001).

[0039] The results of the elevated plus maze experiment show that the time spent in the open arms of the animals in the stress group is significantly lower than that of the control group. IGFBP2 intervention does not affect the time spent in the open arms of the animals in the control group. However, IGFBP2 intervention significantly increases the time spent in the open arms of the animals in the stress group.

[0040] The results of the sugar water preference experiment showed that the sugar water preference value of the stressed animals was significantly lower than that of the control group, indicating significant anhedonic behavior. IGFBP2 intervention did not affect the sugar water preference value of the control animals, but significantly increased the sugar water preference value of the stressed animals.

[0041] The results of the water maze experiment show that the animals in the stress group spend more time finding the platform during training, but after IGFBP2 intervention, the animals in the stress group significantly reduce the time to find the platform.

[0042] The results of the open space experiment and elevated plus maze experiment show that IGFBP2 can effectively improve anxiety-like behavior caused by demyelination. The results of the sugar-water preference experiment show that IGFBP2 intervention can effectively improve depression-like behavior caused by demyelination. The results of the water maze experiment show that IGFBP2 intervention can effectively improve cognitive dysfunction caused by demyelination.

[0043] In summary, it is demonstrated that IGFBP2 intervention can significantly enhance myelination levels and improve psychiatric symptoms caused by stress demyelination. [Industrial Applicability]

[0044] In this specification, the present invention is described with reference to specific embodiments thereof. However, it will be apparent that various modifications and alterations can still be made without departing from the spirit and scope of the present invention. Accordingly, the specification is to be regarded as illustrative rather than restrictive.

Claims

1. A drug for improving cognitive impairment, comprising insulin-like growth factor binding protein 2, which is a drug for treating neuronal demyelinating diseases.

2. The drug for improving cognitive impairment according to claim 1, characterized in that it contains the insulin-like growth factor binding protein 2, which is a drug for enhancing the level of myelination.

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