CN113274400B - Use of tetrahedral framework nucleic acids in medicaments for the treatment of multiple sclerosis - Google Patents

Abstract

The invention provides the use of tetrahedral framework nucleic acids in a medicament for the treatment of demyelinating diseases, such as multiple sclerosis. The tetrahedral framework nucleic acid is formed by base complementary pairing of 4 single-stranded DNAs; the sequences of the 4 single-stranded DNAs are respectively selected from the sequences of SEQ ID No. 1-4 one by one. The tetrahedral framework nucleic acid can restore the expression of myelin sheath related protein by inhibiting apoptosis of cells of the central nervous system, thereby accelerating remyelination and enriching the number of myelinated axons. Therefore, the tetrahedral framework nucleic acid can effectively treat demyelinating diseases, particularly multiple sclerosis, and has excellent clinical application prospect.

Description

Use of tetrahedral framework nucleic acid in a drug for the treatment of multiple sclerosis

technical field

The invention belongs to the field of biomedicine, in particular to the use of a tetrahedral framework nucleic acid in a drug for treating demyelinating diseases such as multiple sclerosis.

Background technique

Demyelinating diseases are a class of acquired diseases with different etiologies and different clinical manifestations, but with similar characteristics. The characteristic pathological changes are the demyelination of nerve fibers and the relatively intact nerve cells. The function of myelin sheath is to protect neurons and make nerve impulses transmit quickly on neurons, so the loss of myelin sheath will affect the transmission of nerve impulses.

As a disease with acute attack or subacute damage to the nerve center, if the treatment is delayed, the damaged nerve is secondary to ischemic degeneration and multiple sclerosis occurs. Multiple sclerosis (MS) is one of the most common demyelinating diseases and has affected 2-3 million people worldwide, with an incidence of about 50-3 million/100,000 in different regions. Clinically, patients present with weakness, spasticity, imbalance, fatigue, vision loss, cognitive impairment, and neuropathic pain. Most patients first experience symptoms in their 20s and 40s, and complications of MS lead to persistent neurological disability. , seriously affecting the work and life of patients, bringing huge economic and social burdens. Histopathologically, MS lesions manifest as multiple demyelinating lesions, inflammatory infiltration, axonal damage, and loss of oligodendrocytes.

So far, there is no effective cure for MS. The early treatment is mostly hormone and nutritional therapy, but the curative effect is difficult to control. When demyelination leads to severe neurological damage, secondary axonal damage will recur, and even further aggravate neurological symptoms. Three main strategies have been proposed for the treatment of MS, including anti-inflammatory immunosuppressive therapy, promotion of remyelination, and neuroprotection.

Most drugs act on the immune system, acting through immunosuppression or immunomodulation, rather than directly promoting myelin repair, such as cyclophosphamide, interferon-beta, glatiramer acetate, and mitoxantrone. These drugs can indeed achieve good clinical efficacy, but the mechanism and target of the drug action are relatively single, and no drug can ultimately cure the patient or permanently inhibit the development of the disease. And because the drug itself inhibits the body's immune system and attacks lymphocytes, the toxic and side effects of the drug are enhanced.

By promoting remyelination, the regenerated myelin re-protects neurons, transmits nerve impulses, and can better promote disease repair. Remyelination has been shown to be effective in acute animal models of MS, such as the monoclonal antibody opicinumab (which targets LINGO1), and antimuscarinic drugs such as clemastine and erythropoietin, but the results of these early stages of clinical trials have been mixed ideal.

Therefore, there is still an urgent need to develop new drugs that can achieve remyelination and repair, thereby protecting the integrity of nerve function, thereby treating MS and other demyelinating diseases.

SUMMARY OF THE INVENTION

The present invention provides the use of a tetrahedral framework nucleic acid in a drug for treating demyelinating diseases, wherein the tetrahedral framework nucleic acid is formed by base complementary pairing of four single-stranded DNAs; the sequences of the four single-stranded DNAs are Said sequences are respectively selected from SEQ ID NO. 1-4.

Further, the above-mentioned tetrahedral framework nucleic acid is prepared by the following method: the four single-stranded DNAs of the tetrahedral framework nucleic acid are placed at a temperature sufficient to denature them for more than 10 minutes, and then the temperature is lowered to 2-8 °C for 20 minutes. above.

Further, the above-mentioned tetrahedral framework nucleic acid is prepared by the following method: placing the four single strands of the DNA tetrahedron at 95° C. for 10 minutes, and then lowering the temperature to 4° C. for more than 20 minutes.

Further, the above-mentioned medicine is a medicine for the treatment of demyelinating diseases, preferably for the treatment of multiple sclerosis, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis or diffuse cerebral sclerosis, more preferably for the treatment of multiple sclerosis Sexual sclerosis drugs.

Furthermore, the above-mentioned drug is a drug for promoting the regeneration of myelin sheath.

Further, the above-mentioned drug is a drug that promotes an increase in the number of myelinated fibers and the thickness of the myelin sheath.

Further, the above-mentioned drug is a drug that increases the expression of MBP and/or MOG; preferably, the drug that increases the expression of MBP is a drug that upregulates the PI3K/AKT/m-TOR signaling pathway.

Furthermore, the above-mentioned drug is a drug for alleviating the inflammatory response of the central nervous system.

Furthermore, the above-mentioned drug is a drug for inhibiting the activation of microglia and the abnormal proliferation of astrocytes.

The present invention provides new uses of tetrahedral framework nucleic acids in medicines for treating demyelinating diseases. Tetrahedral framework nucleic acids can accelerate remyelination and enrich the number of myelinated axons by inhibiting apoptosis in the central nervous system and restoring the expression of myelin-related proteins. In addition, tetrahedral framework nucleic acids can inhibit the abnormal activation and proliferation of microglia and astrocytes to alleviate the inflammatory response. These phenomena are related to the up-regulation of phosphorylation of PI3K-AKT-mTOR signaling pathway by tFNAs. Therefore, tetrahedral framework nucleic acids can effectively treat demyelinating diseases (multiple sclerosis, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic leukoencephalitis or diffuse cerebral sclerosis, etc.), especially multiple sclerosis, with Excellent clinical application prospects.

Terms of the invention: MBP: myelin basic protein; MOG: myelin oligodendrocyte glycoprotein.

Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

Description of drawings

Figure 1 is a PAGE electropherogram of a tetrahedral framework nucleic acid and its single strand.

Figure 2 is a capillary electrophoresis image of a tetrahedral framework nucleic acid and its single strand.

Figure 3 is a particle size diagram of a tetrahedral framework nucleic acid.

Figure 4 is a Zeta potential plot of a tetrahedral framework nucleic acid.

Figure 5 is a transmission electron microscope set of tetrahedral framework nucleic acids.

Fig. 6 is a graph showing the movement trajectories of mice in each group of open field experiments.

Fig. 7 is the LFB staining chart of corpus callosum myelin sheath in each group.

Figure 8 is a transmission electron microscope image of the corpus callosum of each group.

Figure 9 is the immunofluorescence image of corpus callosum MBP in each group.

Figure 10 is the corpus callosum MOG immunofluorescence image of each group.

Figure 11 is the immunofluorescence image of corpus callosum Iba1 in each group.

Figure 12 is the GFAP immunofluorescence image of the corpus callosum in each group.

Figure 13 is a TUNEL fluorescence image of the corpus callosum in each group.

Figure 14 is a WB map of the changes of MBP expression levels and related pathways in brain tissue of each group.

Detailed ways

The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

Example 1. Synthesis of DNA Tetrahedron

Dissolve the four DNA single strands (S1, S2, S3, S4) in TM Buffer (10 mM Tris-HCl, 50 mM MgCl2, pH=8.0), the final concentration of the four DNA single strands is 1 μM, mix well, and quickly heat to 95 The temperature is maintained for 10 minutes, and then the temperature is rapidly lowered to 4°C and maintained for more than 20 minutes to obtain a tetrahedral framework nucleic acid.

The sequence of the four single strands is as follows:

After the synthesis of the tetrahedral framework nucleic acid, PAGE gel electrophoresis (Fig. 1) and capillary electrophoresis (Fig. 2) show that the size of the tetrahedral framework nucleic acid is about 180KD, and it can be considered that the tetrahedral framework nucleic acid has been successfully synthesized. The tetrahedral particle size is about 10.15d.nm (Fig. 3), and the potential is -2.83mV (Fig. 4). Using transmission electron microscopy, the characteristic tetrahedral structure is visible under the microscope (Figure 5).

The beneficial effects of the present invention are demonstrated below through experimental examples.

Experimental Example 1. Therapeutic effect of tetrahedral framework nucleic acid on multiple sclerosis (demyelinating disease) model

1. Experimental method

Model animals: C57BL/6J mice were induced by cuprizone (CPZ) to prepare a demyelinated MS model to explore the effect and possible mechanism of tetrahedral framework nucleic acid in promoting remyelination and repair. A total of 25 C57BL/6 male mice aged 6-8 weeks were selected. They were divided into 8 rats in the normal group, 9 rats in the control group, and 8 rats in the treatment group. The normal group was fed with normal feed every day, while the control group and the treatment group were fed with mixed feed containing 0.2% CPZ every day. After 6 weeks of feeding, the treatment group and the control group were injected with tetrahedral framework nucleic acid (1000 nM) and 0.1 mL of normal saline through tail vein respectively, once every other day, for a total of 4 times.

Detection of related indicators: open field experiments were performed on the mice on the second day after the administration, and then the corpus callosum of the mice was taken, and LFB myelin staining, MBP, and MOG immunofluorescence staining were used to observe the changes of myelin in the corpus callosum area, and transmission electron microscopy was used to observe the ultramicroscopic examination of each group. The structure, IBa1 and GFAP immunofluorescence staining were used to observe the changes of microglia and astrocytes in the corpus callosum area, and TUNEL method was used to detect the apoptosis ratio in the corpus callosum. WB detected the expression level of MBP and the changes of PI3K-AKT-mTOR pathway in each group.

2. Experimental results

(1) The symptoms and signs of MS include limb weakness, paresthesia, ataxia, etc., and episodic symptoms also include tonic spasm, dysarthria, epilepsy, and pain and discomfort, which seriously affect the patient's motor function. In the present invention, it was found through the open field experiment that the total exercise distance of the treatment group increased compared with the control group, which proved that the spontaneous activity intensity and motor function of the mice were improved (Fig. 6), indicating that the tetrahedral framework nucleic acid of the present invention can effectively improve MS. Symptoms of movement disorders in patients with therapeutic effects on MS disease.

(2) Compared with the normal group, the LFB staining results of the myelin sheath in the corpus callosum area of the control group showed a phenomenon of light blue staining and loose structure. The remyelination process indicated that the tetrahedral framework nucleic acid had the function of protecting the myelin structure and promoting the remyelination (Fig. 7).

(3) Transmission electron microscopy showed that the corpus callosum in the control group had obvious myelin ultrastructural damage and axonal mitochondrial damage, with more vacuoles and dissolution, and the number of myelinated axons was significantly reduced. In the treatment group, the number of remyelinated axons was significantly increased, and the integrity of the myelin lamellar structure increased, indicating that the tetrahedral framework nucleic acid contributed to the recovery of the number of myelinated fibers and the thickness of the myelin sheath, and accelerated the process of remyelination (Figure 8). ).

(4) The results of immunofluorescence staining of the corpus callosum on the expression levels of MBP and MOG, respectively, showed that the fluorescence signals of MBP and MOG in the corpus callosum of mice induced by CPZ were significantly weakened, while administration of tetrahedral framework nucleic acid could improve the expression levels of MBP and MOG in the corpus callosum induced by CPZ. The expression levels of MBP and MOG indicated that the administration of tetrahedral framework nucleic acid had a significant promoting effect on the regeneration and repair of myelin sheath (Fig. 9 and Fig. 10).

(5) The proliferation of astrocytes and microglia in MS is currently considered to be one of the obstacles to remyelination, because they not only participate in inflammatory and immune responses, but also form glial scars that hinder regeneration. myelination process. In the experiment, the immunofluorescence staining of Iba1 (Figure 11) and GFAP (Figure 12) showed that the fluorescence intensity of Iba1 and GFAP in the treatment group were weakened, indicating that the tetrahedral framework nucleic acid can inhibit the abnormal proliferation of astrocytes and microglia. activation to alleviate the inflammatory response.

(6) TUNEL assay showed that lower levels of apoptosis were detected in the treatment group ( FIG. 13 ), indicating that the tetrahedral framework nucleic acid could inhibit CPZ-induced apoptosis in the corpus callosum.

(7) Brain tissue WB showed that the expression of MBP in the treatment group was significantly increased, and the PI3K-AKT-mTOR phosphorylation pathway was activated (Fig. 14), indicating that the tetrahedral framework nucleic acid can up-regulate the phosphorylation of the PI3K-AKT-mTOR signaling pathway. Promote MBP expression, thereby promoting remyelination repair.

In conclusion, the present invention provides new uses of tetrahedral framework nucleic acids in medicines for the treatment of demyelinating diseases. Tetrahedral framework nucleic acids can accelerate remyelination and enrich the number of myelinated axons by inhibiting apoptosis in the central nervous system and restoring the expression of myelin-related proteins. In addition, tetrahedral framework nucleic acids can inhibit the abnormal activation and proliferation of microglia and astrocytes to alleviate the inflammatory response. These phenomena may be related to the up-regulation of phosphorylation of PI3K-AKT-mTOR signaling pathway by tFNAs. Therefore, tetrahedral framework nucleic acids can effectively treat demyelinating diseases, especially multiple sclerosis, and have excellent clinical application prospects.

SEQUENCE LISTING

<110> Sichuan University

<120> Use of tetrahedral framework nucleic acid in a drug for the treatment of multiple sclerosis

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Claims (7)

1. Use of a tetrahedral framework nucleic acid in the manufacture of a medicament for the treatment of multiple sclerosis, wherein the tetrahedral framework nucleic acid is formed from 4 single stranded DNAs through base complementary pairing; the sequences of the 4 single-stranded DNAs are respectively selected from the sequences of SEQ ID NO. 1-4.

2. The use according to claim 1, wherein said tetrahedral framework nucleic acid is prepared by a method comprising: 4 single-stranded DNAs of tetrahedral framework nucleic acid are placed at a temperature sufficient for denaturation and maintained for more than 10min, and then the temperature is reduced to 2-8 ℃ and maintained for more than 20 min.

3. The use of claim 2, wherein said tetrahedral framework nucleic acid is prepared by a method comprising: the 4 single strands of the DNA tetrahedron are placed at 95 ℃ for 10min, and then the temperature is reduced to 4 ℃ for more than 20 min.

4. The use of claim 1 wherein the medicament is a medicament for promoting remyelination in the treatment of multiple sclerosis.

5. The use of claim 4, wherein the medicament is a medicament for treating multiple sclerosis by promoting an increase in the number of myelin fibers and myelin sheath thickness.

6. The use according to claim 4 or 5, wherein the medicament is a medicament for treating multiple sclerosis by increasing MBP and/or MOG expression.

7. The use of claim 6, wherein the agent that increases MBP expression for the treatment of multiple sclerosis is an agent that upregulates the PI3K/AKT/m-TOR signaling pathway for the treatment of multiple sclerosis.

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