Application of porphyrin compound in preparation of medicine for treating social dysfunction

Abstract

The invention provides application of a porphyrin compound in preparation of a medicine for treating social dysfunction, and belongs to the technical field of biological medicine. It is found that the porphyrin compound TMPyP4 shows a dose effect to inhibit expression of Xlr3b, after TMPyP4 is injected into the abdominal cavity of a Dip2a gene knockout (Dip2a- / -) mouse, the phenotype of the social dysfunction of the Dip2a- / -mouse can be effectively improved, the defect of the social ability and the defect of social novelty of the Dip2a- / -mouse are improved, the abnormal social behavior of the Dip2a- / -mouse is restored to the normal level, and therefore the TMPyP4 can be used for inhibiting the expression of Xlr3b. The traditional Chinese medicine composition has an obvious effect on improving the social dysfunction and can be used for preparing the medicine for treating the social dysfunction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the application of porphyrin compounds in the preparation of drugs for treating social dysfunction. Background Art

[0002] Social behavior is the most basic behavior for human survival, which is manifested in the basic mode of interaction between individuals and others and is crucial for communication, emotional expression, social adaptation, etc. In some neuropsychiatric diseases such as autism and anxiety disorders, there are social dysfunctions, which manifest as fear, anxiety or other discomfort in social situations. At present, there is a lack of effective treatment for such diseases.

[0003] Disco-interacting protein 2homologA (DIP2A) is a member of the human DIP2 (Disco-interaction protein 2) protein family that is conserved across species. It is located in the 22.3 region (21q22.3) of the q arm of human chromosome 21. Copy number variations in this region usually cause neurodevelopmental diseases. In the applicant's previous work, Dip2a-△65kb knockout (Dip2a - / - ) mice to study the correlation between Dip2a and the onset of ASD (human autism spectrum disorder) and found that Dip2a - / - Mice can effectively simulate human autism spectrum disorders (Ma J, Zhang L, He Z, et al. Autism Candidate Gene DIP2A Regulates Spine Morphogenesis via Acetylation of Cortactin, PLOS Biology 2019), and confirmed that Dip2a - / - Mice can show two core symptoms of ASD - repetitive stereotyped behaviors and abnormal social skills. In addition, existing technologies have also proposed that Dip2a - / - Abnormally elevated Xlr3b expression in the prefrontal cortex of the mouse brain is key to the ASD-like phenotype.

[0004] TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine), tetrapyridylporphyrin is a cationic porphyrin compound with a molecular weight of 1363.60 and a molecular formula of C 72 H 66 N 8 O 12 S 4, CAS number 36951-72-1. It is mainly used as a photosensitizer. Due to its high water solubility, high cell membrane permeability and preferential accumulation in tumor cells, it is often used in photodynamic therapy to treat cancer. TMPyP4 can bind to the G-quadruplex structure of DNA and stabilize these structures, thereby inhibiting telomerase activity and leading to cancer cell death. TMPyP4 also has antibacterial applications and is also used in nanoparticle-mediated delivery, controlled drug release, biosensing and other fields. In the treatment of neurodegenerative diseases, TMPyP4 can inhibit the RAN translation of the G4C2 repeat sequence in the C9orf72 gene, which may be a potential way to treat frontotemporal lobar degeneration and amyotrophic lateral sclerosis. At the same time, TMPyP4 can also affect gene expression by stabilizing the secondary structure of nucleic acids. Specifically, TMPyP4 can stabilize G-quadruplex and i-motif structures, which may change the transcription level of genes. At present, TMPyP4 has not been found to be used in improving social dysfunction. Summary of the invention

[0005] The purpose of the present invention is to provide a means for effectively treating social dysfunction and to expand the new use of TMPyP4.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides application of porphyrin compounds in preparing medicines for treating social dysfunction.

[0008] Preferably, the porphyrin compound is TMPyP4.

[0009] Preferably, the dosage form of the drug is an injection.

[0010] Beneficial Effects

[0011] The present invention found that the porphyrin compound TMPyP4 showed a dose-dependent inhibition of Xlr3b expression. - / - ) mice after intraperitoneal administration, which can effectively improve Dip2a - / - Dip2a improves social dysfunction in mice - / - Defects in sociability and social novelty in mice restored by Dip2a - / - The abnormal social behavior of mice is restored to normal levels, which has a significant effect on improving social dysfunction and can be used to prepare drugs for treating social dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The regulatory effect of TMPyP4 on the expression of Xlr3b in neurons cultured in vitro in Example 1;

[0013] Figure 2 This is a flow chart of drug treatment and behavioral testing in Example 2;

[0014] Figure 3 The effect of TMPyP4 on Dip2a in Example 2 - / - The impact on the sociability of mice;

[0015] Figure 4 The effect of TMPyP4 on Dip2a in Example 2 - / - The effects of social novelty in mice;

[0016] Figure 5 The quantitative PCR detection of the regulatory effect of TMPyP4 on the expression of Xlr3b mRNA in different groups of mice in Example 3;

[0017] Figure 6 The protein immunoblot analysis method in Example 3 was used to detect the effect of TMPyP4 on the expression of Xlr3b in different groups of mice. DETAILED DESCRIPTION

[0018] 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.

[0019] Example 1 TMPyP4 can significantly inhibit the expression of Xlr3b in vitro

[0020] Gonadotropin-releasing hormone cell line NLT cells were used for in vitro culture to detect the regulatory effect of TMPyP4 on Xlr3b expression. The cells were derived from brain tumors of transgenic mice, expressed neuronal cell-specific markers and secreted gonadotropin-releasing hormone (Zhen et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY 1997; 272(19): 12620–12625). High-glucose DMEM + 10% fetal bovine serum complete medium was used, and the cell culture conditions were 37°C with 5% CO 2 The cells showed adherent growth. When the cell density reached 80%-90%, they were digested with 2.5% trypsin and subcultured. After incubating the cells with different concentrations of TMPyP4 (the concentration of TMPyP4 was set to: 0, 12.5, 25, 50, 100 μM) for 48 hours, the expression level of Xlr3b mRNA in the cells of each treatment group was detected by quantitative PCR and protein immunoblotting analysis. The results are shown in Figure 1As shown: After three independent experiments, one way ANOVA statistical analysis showed that 12.5 μM TMPyP4 had a statistically significant inhibitory effect on Xlr3b (p=0.0377); changes in TMPyP4 concentration showed a certain dose-dependent effect on Xlr3b inhibition (25 μM, p=0.0499; 50 μM, p=0.0153; 100 μM, p=0.0097). Figure 1 These results indicate that TMPyP4 inhibits the expression of Xlr3b and that the inhibitory effect of TMPyP4 on Xlr3b is dose-dependent.

[0021] Example 2 Effect of TMPyP4 on the improvement of social behavior of Dip2a knockout mice

[0022] To illustrate the improvement effect of TMPyP4 on social behavior, we used Dip2a knockout mice with social behavior disorders identified in previous work and randomly + / + Dip2a - / - Mice were divided into Control group (Dip2a + / + + saline), negative control group (Dip2a - / - + saline), drug control group (Dip2a + / + +TMPyP4) and drug treatment group (Dip2a - / - +TMPyP4), 10 mice per group. The drug was prepared in physiological saline at a concentration of 1 mg / ml and injected intraperitoneally at 10 mg / kg body weight. For example, if the mouse weighed 20 grams, the injected drug was 0.2 mg (0.2 ml). From the third to the eighth week after birth (P21–P56), the drug was administered three times a week for a total of 18 times over 6 weeks ( Figure 2 A), the control group and the negative control group were given the same volume of saline. After the treatment, the classic three-box social behavior test was used to test the social ability of mice. The behavioral test was conducted in three rounds (each round lasted 10 minutes): Adaptation phase: Figure 2 As shown in B, there is an inverted small cage (object, O) in the left and right corners of the box. The test mice are placed in the box from the middle and freely explore the three boxes. Mice with no obvious position preference will participate in subsequent experiments.

[0023] In the social ability experiment, a strange mouse (S1) was placed in the upside-down cage in the right corner. The test mice were more willing to spend more time exploring the S1 mouse rather than the empty cage (Object), which was defined as social ability.

[0024] In the social novelty behavior experiment, another strange mouse (S2) was placed on the left side, and the S1 mouse from the previous test was still on the right side. Under physiological conditions, the test mice were more willing to spend more time exploring the S2 mouse than exploring the S1 mouse, which was defined as social novelty ( Figure 2 B) The results are as follows Figure 3 As shown in the sociability experiment: Dip2a + / + The mice in the +TMPyP4 group (drug control) explored the unfamiliar mouse S1 for about 3 minutes, and the preference index was similar to Dip2a. + / + + There was no difference in the saline group (Control). Dip2a - / - The exploration time of mice in the + saline group (negative control) for the unfamiliar mouse S1 was less than 2 minutes, and the preference index was significantly lower than that of Dip2a + / + +Saline group (Control) mice and Dip2a + / + +TMPyP4 group (drug control) mice confirmed that intraperitoneal injection of normal saline did not improve Dip2a - / - Abnormal social behavior in mice. However, after TMPyP4 treatment, Dip2a - / - The +TMPyP4 group (drug treatment group) increased the exploration time of S1 mice, the mice's social ability was significantly improved, and the social preference index was increased, indicating that TMPyP4 effectively improved the defects in social ability. For wild-type mice, no regulatory effect on social behavior was found after administration of TMPyP4. Figure 3 A is a representative heat map of social ability. The exploration path of mice captured by the infrared camera changes from blue to red, which means that the time the mice spend exploring increases from less to more. Figure 3 B is the duration of exploring unfamiliar mice and empty cages in the sociability behavior test; Figure 3 C is the preference index of sociability.

[0025] Similarly, Figure 4 As shown in the social novelty behavior experiment, similar results can be observed: TMPyP4 treatment does not significantly improve the social novelty ability of wild mice, but can make Dip2a - / - The mice spent more time exploring the unfamiliar mouse S2 and their preference index increased to that of wild-type mice, confirming that TMPyP4 treatment can improve the social novelty deficit of Dip2a knockout mice. Figure 4 A is a representative heat map of the social novelty behavior test. Figure 4 B is the social novelty exploration time; Figure 4 C is the social novelty index.

[0026] In summary: TMPyP4 treatment can significantly restore Dip2a - / - Abnormal social behavior in mice to normal levels.

[0027] Example 3 Regulatory Effects of TMPyP4 Treatment on Xlr3b Expression in Dip2a Knockout Mice

[0028] After the social behavior test, the experimental mice in each group in Example 2 were killed by cervical sciatic nerve, and the frontal lobe brain area was taken for qPCR and WB detection of Xlr3b expression. The experimental results are as follows: Figure 5 and Figure 6 (After t-test statistical analysis, * indicates significant difference; **** indicates extremely significant difference) as shown:

[0029] Figure 5 and Figure 6 The results showed that TMPyP4 could reduce the abnormally high expression of Xlr3b in the brain of Dip2a knockout mice. Figure 5 ) and protein ( Figure 6 ) levels were significantly improved, and it is speculated that it affects gene expression by stabilizing the secondary structure of nucleic acids.

[0030] Existing literature shows that the Xlr3b promoter region contains a G-quadruple structure. Some ligand compounds with special molecular structures, namely G-quadruple binding ligands, can specifically bind to the G-quadruple, exert transcriptional repression, and inhibit the expression of Xlr3b. Therefore, it is speculated that the mechanism of action of TMPyP4 in regulating the expression of the Xlr3b gene involves the stability of the G-quadruple structure. As an RNA binding protein, Xlr3b may regulate RNA stability, the recognition of ribosomes and mRNA, and play a role through protein translation.

[0031] In summary, TMPyP4 may regulate the expression of Xlr3b gene by interacting with G-quadruplex, effectively improve the phenotype of social dysfunction in Dip2a gene-deficient mice, and can be used to treat social dysfunction.

[0032] 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. Application of porphyrin compounds in the preparation of drugs for treating social dysfunction.

2. The use according to claim 1, characterized in that: The porphyrin compound is TMPyP4.

3. The use according to claim 2, characterized in that: The dosage form of the drug is injection.

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