Application of anethomycin in preparation of medicine for treating social novelty preference defect disorder caused by Coronin1a gene deletion

Abstract

The invention relates to the technical field of medicines. The invention provides an application of anethomycin in preparation of a medicine for treating social novelty preference defect disorder caused by Coronin1a gene deletion, and the anethomycin is used for treating the social novelty preference defect disorder by activating a JNK (Junction Natural Killer) pathway, improving the morphology of microglial cells, improving the activity of the microglial cells and enhancing the phagocytic function of the microglial cells. The invention not only provides a new drug source for preparing and treating the social novelty preference defect disorder caused by the Coronin1a gene deletion, but also explores the new medicinal value of the anethomycin.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of anisomycin in preparing a drug for treating social novelty preference deficiency disorder caused by Coronin1a gene deletion. Background Art

[0002] Preference of Social Novelty (PSN) is the behavior of an individual who tends to communicate with unfamiliar potential social objects. PSN ability is the result of human social evolution and long-term adaptation to the natural environment, enabling individuals to actively integrate into group life to maintain their own survival and development. PSN disorder, as a common phenotype of social disorder, is prevalent in the autism spectrum disorder (ASD) group. Clinically, ASD is a pervasive developmental disorder with "social communication disorder" and "stereotyped repetitive behavior" as its core characteristics, with an incidence of about 1% in China.

[0003] At present, the pathogenesis of PSN disorder is not fully understood, and the research on specific drug targets is still a blank area. These practical problems objectively hinder the development of drugs for the prevention and clinical treatment of PSN disorder and ASD. Anisomycin can be used to regulate inflammation and promote the pruning of neurons by microglia, an immune cell in the brain, which has a neuroprotective effect. At present, there are no reports on its treatment of PSN disorder. Summary of the invention

[0004] The purpose of the present invention is to provide the use of anisomycin in the preparation of a drug for treating social novelty preference deficiency disorder caused by Coronin1a gene deficiency, by activating the JNK pathway, improving the morphology of microglia, increasing the activity of microglia, and enhancing the phagocytic function of microglia to treat social novelty preference deficiency disorder.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an application of anisomycin in preparing a medicine for treating social novelty preference deficiency disorder.

[0007] The present invention provides the use of anisomycin in preparing a medicine for treating social novelty preference deficiency disorder caused by Coronin1a gene deletion.

[0008] Preferably, the anisomycin treats social novelty preference deficiency disorder by activating the JNK pathway and increasing the activity of microglia.

[0009] The present invention also provides a medicine for treating social novelty preference deficiency disorder caused by Coronin1a gene deletion.

[0010] Preferably, the composition comprises anisomycin and pharmaceutically acceptable excipients.

[0011] By adopting the above technical solution, the present invention has the following beneficial effects:

[0012] The anisomycin described in the technical solution of the present invention can activate the JNK pathway and increase the activity of microglia; it can also reverse the abnormal morphology of microglia and enhance the phagocytic function of microglia; it can also improve the abnormal expression of synapse-related proteins and inflammatory factors, thereby effectively treating the social novelty preference deficiency disorder caused by the Coronin1a gene deficiency. This not only provides a new source of drugs for the preparation and treatment of social novelty preference deficiency disorder caused by the Coronin1a gene deficiency, but also discovers the new medicinal value of anisomycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a diagram of the three-chamber social experiment model;

[0014] Figure 2 The contact time between the Coronin1a gene systemic deletion model mice and WT group mice and familiar mice or unfamiliar mice;

[0015] Figure 3 The results of fluorescence staining of the mPFC region of the brain of the Coronin1a gene systemic deletion model mice and WT group mice;

[0016] Figure 4 The number, length and connection points of microglia in the Coronin1a gene systemic deletion model mice and WT mice ( Figure 4 A represents the number of branches, B represents the length of the branch, and C represents the connection point);

[0017] Figure 5 Coronin1a gene deletion model mice, cx3cr1-cre ert The contact time between mice in each group and familiar mice or unfamiliar mice;

[0018] Figure 6 Coronin1a gene deletion model mice, cx3cr1-cre ert Fluorescence staining results of the mPFC region of the brain of mice in group A;

[0019] Figure 7 Coronin1a gene deletion model mice, cx3cr1-cre ertThe number of microglia branches, branch length and connection points of mice in the two groups ( Figure 7 A represents the number of branches, B represents the connection point, and C represents the length of the branch);

[0020] Figure 8 Coronin1a gene deletion model mice, cx3cr1-cre ert The electrophoretic band diagram of synaptic-related proteins in the mPFC region of the brain of group mice;

[0021] Fig. 9 Coronin1a gene deletion model mice, cx3cr1-cre ert The content of synaptic-related proteins in the mPFC region of the brain of the mice in the group ( Fig. 9 A in the figure is the content of PSD95, and B is the content of IL1-β). DETAILED DESCRIPTION

[0022] The present invention provides an application of anisomycin in preparing a medicine for treating social novelty deficiency disorder.

[0023] The present invention provides the use of anisomycin in preparing a drug for treating social novelty deficiency disorder caused by Coronin1a gene deletion.

[0024] In the present invention, anisomycin is a protein synthesis inhibitor and a JNK activator. The anisomycin can activate microglia in the brain by activating the JNK pathway. Glial cells are macrophages in brain tissues and play a core role in the brain's inherent immunity. The function of microglia helps maintain brain homeostasis. The anisomycin described in the present invention can also reverse the abnormal morphology of microglia and enhance the phagocytic function of microglia; it can also improve the abnormal expression of synapse-related proteins and inflammatory factors, thereby treating social novelty deficiency disorder.

[0025] The present invention also provides a drug for treating social novelty deficiency disorder caused by Coronin1a gene deletion.

[0026] In the present invention, the drug comprises anisomycin and pharmaceutically acceptable excipients. The drug in the present invention belongs to tetraene substances, and its water-soluble component is a basic nucleoside substance.

[0027] In the present invention, the administration of the drug preferably includes intraperitoneal injection.

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1 Obtaining conditional coronin1a gene knockout model mice

[0030] Coronin1a flox / flox Mice (purchased from Saiye (Guangzhou) Biotechnology Co., Ltd.) and cx3cr1-cre ert Mice (purchased from Jax Lab) were mated and bred, and 8-week-old male double-positive mice with Coronin1a were selected. flox / flox Mice were used as a model of whole-body deletion of the Coronin1a gene. Eight-week-old male cx3cr1-cre mice were generated in the same cage. ert *Coronin1a flox / flox The mice were used as microglial Coronin1a gene deficiency model mice (i.e. mice with microglia-specific knockout of the Coronin1a gene).

[0031] Example 2

[0032] Healthy male 8-week-old Coronin1a gene whole body deletion model mice (CORO1 - / - ) and control mice (WT), among which the Coronin1a gene whole body deletion model mice were divided into CORO1 - / - Nacl group and CORO1 - / - AN group, 10 mice in each group, CORO1 - / - The Nacl group was treated with intraperitoneal administration of Nacl (drug solvent). - / - The AN group was treated with anisomycin intraperitoneally. - / - Nacl group and CORO1 - / - The AN group received two weeks of intraperitoneal injection intervention, 100ug / kg / d, and was fed normally until the 8th week of the experiment.

[0033] (I) Three-room social experiment

[0034] The three-chamber social experiment was used to detect whether the model mice had social novelty impairment.

[0035] The three-chamber social apparatus is a rectangular box (60×40×20 cm, length×width×height) divided into three interconnected chambers. First, a mouse to be tested is habituated to a three-chamber apparatus separated by transparent plexiglass for 10 minutes; then a mouse that matches the genotype, gender, and age of the mouse to be tested is placed in the apparatus, and the mouse to be tested is allowed to get familiar with the environment for 10 minutes; during the third period of time, a new mouse (strange mouse) that matches the genotype, gender, and age of the mouse to be tested is placed in the apparatus, and the mouse to be tested can interact with the familiar mouse or the strange mouse for 10 minutes. The experiment is as follows Figure 1 As shown. WT group, CORO1 - / -Nacl group and CORO1 - / - The mice in the AN group were used as test mice for the above experimental operations, and then the Etho VisionXT 10 software package was used to record and measure the contact time between the test mice and the familiar mice or the strange mice.

[0036] according to Figure 2 It can be seen that in the comparison of the contact time between familiar mice and unfamiliar mice, the contact time of WT mice with unfamiliar mice was significantly longer than that with familiar mice, but CORO1 - / - The preference of mice in the Nacl group for unfamiliar mice was not very significant, indicating that the Coronin1a gene systemic deletion model mice had social novelty defects; CORO1 - / - The contact time of mice in the AN group with unfamiliar mice was significantly increased, indicating that anisomycin can effectively improve the social novelty deficiency disorder caused by the Coronin1a gene systemic deficiency model mice. (two)

[0038] Fluorescent staining was performed on the mPFC region of the brain of the above-mentioned groups of mice to observe the morphological changes of microglia.

[0039] After apical perfusion, the mouse brain was removed and immersed in 4% PFA for 12 hours, and dehydrated with 20% and 30% sucrose gradient. After dehydration, it was embedded in OCT and frozen at -80 degrees. After sectioning, it was bleached and stained with 1:500 IBA1 antibody at 4 degrees for 12 hours, washed three times with PBS, 10 minutes each time, and the corresponding 1:1000 fluorescent secondary antibody was used. After staining for 60 minutes, PBS was washed three times, and DAPI was used to stain the cell nucleus. After PBS washing, the patch was mounted and photographed. Observation was performed.

[0040] like Figure 3 and Figure 4 As shown in the figure, the brain microglia of the mice with whole body deletion of Coronin1a gene were compared with those of the control mice. The results showed that the number of branches, branch length, and connection points of the microglia of the mice with whole body deletion of Coronin1a gene were abnormally increased. This indicates that the morphology of microglia in the mice with whole body deletion of Coronin1a gene changed and cell activation was inhibited. - / - The microglial morphology of mice in the AN group can be effectively improved, which also indicates that anisomycin can improve the microglial morphological changes caused by Coronin1a gene deficiency.

[0041] Example 3

[0042] Healthy male 8-week-old microglial Coronin1a gene deletion model mice (cx3cr1-creert *Coro1a f / f ) and control mice (cx3cr1-cre ert ), among which the microglial Coronin1a gene deletion model mice are divided into cx3cr1-cre ert *Coro1a f / f NaCl group and CX3CR1-Cre ert *Coro1a f / f AN group, 9-10 mice per group, cx3cr1-cre ert *Coro1a f / f The NaCl group was treated with NaCl (drug solvent) intraperitoneally, and the cx3cr1-cre ert *Coro1a f / f The AN group was treated with anisomycin intraperitoneally. ert *Coro1a f / f NaCl group and CX3CR1-Cre ert *Coro1a f / f The AN group received two weeks of intraperitoneal injection intervention, 100ug / kg / d, and was fed normally until the 8th week of the experiment.

[0043] (I) Three-room social experiment

[0044] The three-chamber social experiment was used to detect whether the model mice had social novelty disorder. The specific operation was the same as in Example 2. The Etho VisionXT 10 software package was used to record and measure the contact time between the tested mice and the familiar mice or the unfamiliar mice.

[0045] according to Figure 5 It can be found that in the comparison of contact time between familiar mice and unfamiliar mice, cx3cr1-cre ert The mice in the cx3cr1-cre ert *Coro1a f / f The preference of mice in the NaCl group for unfamiliar mice was not very significant, indicating that the microglial Coronin1a gene deletion model mice had social novelty defects; cx3cr1-cre ert *Coro1a f / f The contact time of mice in the AN group with unfamiliar mice was significantly increased, indicating that anisomycin can effectively improve the social novelty deficit disorder caused by microglial Coronin1a gene deficiency model mice. (two)

[0047] Fluorescence staining was performed on the mPFC region of the brain of the mice in each group to observe the morphological changes of microglia. The specific operation was the same as that in Example 2.

[0048] like Figure 6 and Figure 7 As shown, brain microglia of microglial Coronin1a gene-deficient mice were co-expressed with CX3CR1-Cre ert Compared with the brain microglia of the mice in the control group, the results showed that the number of branches, branch length, and connection points of microglia in the mice with Coronin1a gene deficiency model increased abnormally. This indicates that the morphology of microglia in the mice with Coronin1a gene deficiency model changed and cell activation was inhibited. ert *Coro1a f / f The microglial morphology of mice in the AN group can be effectively improved, which also indicates that anisomycin can improve the microglial morphological changes caused by Coronin1a gene deficiency. (three)

[0050] The levels of synaptic-related proteins (PSD95, IL1-β) in the mPFC region of the brain of the mice in the above groups were detected by immunoprotein electrophoresis to analyze the phagocytic function of microglia. GAPDH was used as an internal reference protein.

[0051] Brain tissues of mice in each group were taken, lysed and quantified, and then protein buffer was added and boiled at 95°C for 5 minutes. The protein amount was detected using 10% SDS-PAGE gel.

[0052] Microglia can phagocytize and prune synapses. When the activity of microglia decreases, their phagocytic ability weakens, resulting in an abnormal increase in synapse-related proteins. Protein bands were obtained by immunoprotein electrophoresis, such as Figure 8 The results showed that the content of synaptic-related proteins in mPFC of microglial Coronin1a gene-deficient mice was significantly increased, but the cx3cr1-cre ert *Coro1a f / f The content of synaptic-related proteins in the mPFC of mice in the AN group was significantly reduced. This suggests that anisomycin may enhance the phagocytic function of microglia and promote the ability of microglia to prune synapses (such as Fig. 9 ).

[0053] In summary, it can be seen that the anisomycin described in the technical solution of the present invention can increase the activity of microglia, reverse the abnormal morphology of microglia, and enhance the phagocytic function of microglia; it can also improve the abnormal expression of synapse-related proteins and inflammatory factors, thereby effectively treating the social novelty preference deficiency disorder caused by Coronin1a gene deletion.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The application of anisomycin in the preparation of drugs for the treatment of social novelty preference deficiency disorder.

2. The use of anisomycin in the preparation of a drug for treating social novelty preference deficiency disorder caused by Coronin1a gene deletion, characterized in that: The anisomycin treats social novelty preference deficiency disorder by activating the JNK pathway and increasing the activity of microglia.

3. A drug for treating social novelty preference deficiency disorder caused by Coronin1a gene deficiency, characterized in that: The invention comprises anisomycin and pharmaceutically acceptable excipients.

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