Application of no-NOHA in preparation of medicine for treating or improving social abnormality

Abstract

The invention relates to the technical field of medicines, in particular to application of nor-NOHA in preparation of a medicine for treating or improving social abnormality, the nor-NOHA can down-regulate the amplitude and frequency of a prefrontal cone neuron sEPSC, and has no influence on sIPSC. The staying time of the S-ketamine exposed mouse in the box body of a strange mouse and the time of sniffing the strange mouse are obviously prolonged. The no-NOHA also alleviates the impairment of social memory of the S-ketamine exposed mouse. In a social memory test, compared with a familiar mouse, the S-ketamine exposed mouse is more preferred for a strange mouse after the intervention of the no-NOHA, and the time of social interaction with the strange mouse in the box body is longer, but the social memory index is not obviously increased. The results jointly show that the nor-NOHA reverses neuronal morphological abnormality induced by S-ketamine by inhibiting abnormal activation and synaptic phagocytic ability of microglial cells in the prefrontal lobe, and finally, normalization of social behaviors is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to application of nor-NOHA in preparing drugs for treating or improving social abnormalities. Background Art

[0002] Approximately 1.5 million infants and young children worldwide require surgical intervention under general anesthesia each year. However, the long-term effects of early postnatal exposure to general anesthetics on neurocognitive development remain controversial. Based on evidence from large-scale multicenter clinical studies (such as the Pediatric Anesthesia Neurodevelopmental Assessment (PANDA), Mayo Anesthesia Safety for Kids (MASK), and Infant General Anesthesia or Awake Regional Anesthesia (GAS)), no differences in general intelligence quotient (IQ) were found in children and adolescents who had received short-term general anesthesia before the age of 3 years. However, parent and caregiver assessments of neurodevelopmental outcomes also mentioned that children's social skills and executive functions were altered after general anesthesia in early childhood. At the same time, a large body of evidence from preclinical studies, especially nonhuman primates (NHPs) experiments, suggests that early exposure to volatile anesthetics may lead to abnormalities in intimate social behavior and dendritic development, without significant declines in learning and memory abilities. These phenomena suggest that social cognitive functions may be more vulnerable to general anesthesia, and their effects on neurodevelopment may be masked due to the low sensitivity of existing diagnostic tools.

[0003] Synaptic development is an important biological process in the central nervous system in the early postnatal period, including synapse formation, maturation, and pruning, which contributes to the assembly of neural circuits, neuronal excitation / inhibition (E / I) balance, and long-term plasticity. Abnormal synaptic development mediates a series of psychiatric-behavioral abnormalities, such as social avoidance, repetitive stereotyped behaviors, and motor dysfunction, which are considered to be the core symptoms of autism spectrum disorder (ASD). In fact, previous studies have shown that early exposure to general anesthetics may increase the risk of autism spectrum disorder. However, whether the neurodevelopmental abnormalities associated with general anesthesia are different from autism remains unclear.

[0004] Existing technologies have made slow progress in developing molecular intervention strategies for social cognitive disorders such as ASD. Although some studies have revealed the role of microglia in neurodevelopmental disorders, how to effectively regulate their function to improve social behavior remains a challenge. Summary of the invention

[0005] The object of the present invention is to provide the use of nor-NOHA in the preparation of drugs for treating or improving social abnormalities, and nor-NOHA can downregulate the amplitude and frequency of sEPSC of prefrontal pyramidal neurons, but has no effect on sIPSC. In the three-box social experiment, nor-NOHA significantly increased the time that S-ketamine-exposed mice stayed in the box of unfamiliar mice, as well as the time they sniffed unfamiliar mice. nor-NOHA also alleviated the social memory impairment of S-ketamine-exposed mice. In the social memory test, after nor-NOHA intervention, S-ketamine-exposed mice preferred new mice more than familiar mice, and spent more time in social interaction with new mice in the box. These results collectively indicate that nor-NOHA reverses S-ketamine-induced neuronal morphological abnormalities by inhibiting abnormal activation of microglia in the prefrontal cortex and synaptic phagocytosis, ultimately contributing to the normalization of social behavior.

[0006] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0007] Application of nor-NOHA in the preparation of drugs for treating or improving social abnormalities.

[0008] Furthermore, the drug comprises an effective dose of nor-NOHA and a pharmaceutically acceptable pharmaceutical adjuvant.

[0009] Furthermore, the effective dose is 0-30 mg / kg.

[0010] Furthermore, the applications include: nor-NOHA reverses S-ketamine-induced neuronal morphological abnormalities, restores synaptic function, and prevents S-ketamine exposure-induced social disorders by inhibiting abnormal activation of microglia and synaptic phagocytosis in the prefrontal cortex;

[0011] Furthermore, nor-NOHA can restore the number of microglia branches in the prefrontal cortex of S-ketamine-exposed mice, and reverse the enhanced CD68+ lysosomal PSD95 immunoreactivity in prefrontal microglia of P28 S-ketamine-exposed mice; at the same time, it can reduce the branching complexity of the basal terminal dendrites of prefrontal pyramidal neurons caused by S-ketamine exposure.

[0012] Beneficial effects of the present invention:

[0013] The application of nor-NOHA in the preparation of drugs for the treatment or improvement of social abnormalities is first reflected in its regulation of microglial function. Microglia maintain normal neural networks through synaptic pruning during neurodevelopment. However, S-ketamine exposure leads to excessive activation of microglia, which is manifested by enlarged cell bodies, reduced number of branches, shortened processes, and significantly enhanced phagocytosis. This excessive synaptic pruning destroys the dendritic structure of neurons and leads to dysfunction of neural networks. nor-NOHA limits the abnormal activation of microglia by inhibiting the overexpression of Arg1 (arginase 1) in microglia, thereby restoring the normal dendritic branch morphology of pyramidal neurons and significantly reducing the co-labeling level of CD68+ lysosomes and the synaptic marker PSD95. This shows that nor-NOHA effectively inhibits excessive synaptic phagocytosis, protects the dendritic structure of pyramidal neurons, and lays the foundation for the normalization of social behavior.

[0014] The application of nor-NOHA also significantly improved the dendritic structure and function of pyramidal neurons in the prefrontal cortex. S-ketamine exposure led to excessive complexity of the basal dendrites of pyramidal neurons and an increase in the proportion of immature dendritic spines (an increase in slender dendritic spines), which significantly reduced the signal transmission efficiency and synaptic stability of neurons. nor-NOHA restored the normal morphological proportion and function of dendritic spines by reducing the ability of microglia to phagocytose abnormal synapses, especially increasing the proportion of apical mushroom-type dendritic spines. This adjustment improved the accuracy of synaptic transmission and the plasticity of neurons, restored the excitation / inhibition balance (E / I balance) of the local neural network, and effectively improved the impairment of social memory caused by S-ketamine exposure.

[0015] nor-NOHA significantly reduced the excessive enhancement of excitatory synaptic transmission caused by S-ketamine exposure by downregulating the amplitude and frequency of sEPSC in prefrontal pyramidal neurons. S-ketamine exposure causes excessive enhancement of excitatory synaptic transmission in the prefrontal cortex, while inhibitory synapses (sIPSC) remain unchanged. This imbalance can lead to abnormal "signal-to-noise ratio" in the neural network and destroy the accuracy of information processing. By reducing the overactive state of sEPSC, nor-NOHA restored the E / I balance of local neural circuits and improved information processing capabilities. This regulation not only improves the accuracy of information processing in social behavior, but also significantly enhances the retrieval of social memory and the frequency of social interactions.

[0016] The application of nor-NOHA provides a new therapeutic target for diseases related to social disorders (such as autism spectrum disorder, ASD). Synaptic pruning disorders of microglia are considered to be one of the key mechanisms of the core symptoms of ASD (social disorders and stereotyped behaviors). nor-NOHA reshapes the function of the local neural network of the PFC by inhibiting the excessive pruning function of microglia and restoring dendrite development, thereby enhancing social ability. This mechanism of action is similar to the pathological mechanism of microglial synaptic pruning dysfunction in ASD, providing a new molecular intervention strategy for social cognitive disorders such as ASD. The intervention effect of nor-NOHA not only significantly improved synaptic function and dendritic plasticity, but also provided experimental evidence for the precise treatment of neurodevelopmental disorders, and has important clinical translational value.

[0017] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0019] Figure 1 Schematic diagram of behavioral changes caused by early postnatal S-ketamine exposure;

[0020] Figure 2 Schematic diagram of S-ketamine exposure disrupting dendritic structure and hyperexcitatory excitatory synaptic transmission;

[0021] Figure 3 Schematic diagram of S-ketamine exposure activating microglia and enhancing synaptic pruning;

[0022] Figure 4 Schematic diagram of the Stat1-Arg1 pathway that directly activates microglia in the PFC by S-ketamine exposure;

[0023] Figure 5 Schematic diagram of the dynamic changes in microglial morphology and synaptic pruning in CA2 after S-ketamine anesthesia;

[0024] Figure 6 Schematic diagram of nor-NOHA restoring microglial and synaptic function to prevent S-ketamine exposure-induced social deficits;

[0025] Figure 7 Schematic diagram of different types of microglia surrounding neurons;

[0026] Figure 8 Schematic diagram of the Stat1-Arg1 pathway in microglia activated by S-ketamine exposure in the PFC;

[0027] Fig. 9 Schematic diagram showing that nor-NOHA treatment can restore social memory in neonatal S-ketamine-anesthetized adolescent mice. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] The present embodiment provides the use of nor-NOHA in the preparation of a drug for treating or improving social dysfunction.

[0031] In this embodiment, the drug comprises an effective dose of nor-NOHA and a pharmaceutically acceptable pharmaceutical adjuvant.

[0032] In this embodiment, the effective dose is 0-30 mg / kg.

[0033] In this embodiment, the application includes: nor-NOHA reverses S-ketamine-induced neuronal morphological abnormalities, restores synaptic function, and prevents S-ketamine exposure-induced social disorders by inhibiting abnormal activation of microglia and synaptic phagocytosis in the prefrontal cortex;

[0034] In this example, nor-NOHA can restore the number of branches of microglia in the prefrontal cortex of S-ketamine-exposed mice, and can reverse the enhanced CD68+ lysosomal PSD95 immunoreactivity in prefrontal microglia of P28 S-ketamine-exposed mice; at the same time, it can reduce the branching complexity of the basal dendrites of prefrontal pyramidal neurons caused by S-ketamine exposure.

[0035] Example 2

[0036] Materials and methods

[0037] animal

[0038] C57BL / 6J (C57) mice were provided by Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The pups of C57 mice were obtained by breeding the mice purchased above.

[0039] Behavioral testing

[0040] Behavioral tests were performed at the same time during the day. Mice were acclimatized to the experimental room for at least 30 minutes before the test, and there was a 24-hour interval between two consecutive tests.

[0041] Standard behavioral tests were used to verify the social abnormalities of S-ketamine exposed mice and the therapeutic effects of nor-NOHA, including but not limited to the three-box social test, interactive social test, grooming test, bead burying test, open field test, elevated plus maze test, novel object recognition test, and Barnes maze test.

[0042] Fluorescence immunoassay

[0043] Mice were deeply anesthetized with pentobarbital (100 mg / kg, intraperitoneal injection) and then perfused transcardially with 0.01 M phosphate buffer (PBS, pH 7.4) for 5-10 minutes, followed by 4% paraformaldehyde for 15-20 minutes. The whole brain was removed and then fixed in 4% paraformaldehyde for 24 hours, and then immersed in a 30% sucrose solution containing 4% paraformaldehyde at a temperature of 4°C. Sagittal frozen sections with a thickness of 40 μm were collected and stored at -20°C using an antifreeze solution prepared from 30% ethylene glycol, 30% sucrose and 0.01 M PBS. For immunofluorescence staining, the sections were first rinsed in 0.01 M PBS to remove the antifreeze solution, and then incubated with the primary antibody prepared with 1% bovine serum albumin at 4°C overnight. After that, the 488-labeled secondary antibody was added and incubated at 37°C for 2 hours. The sections were counterstained with DAPI and then observed with a confocal laser scanning microscope. Imaris software was used to scan each group of brain sections immunostained for Iba1 to quantify the number of microglia in different brain regions. The morphology of microglia was analyzed using ImageJ software. A program written in Python was used to overlap microglia and display them as heat maps. Microglial synaptic phagocytosis was analyzed as described previously; brain sections were imaged in the z-axis using a 40x objective (total z-size of 15 μm, thickness of each slice of 0.8 μm). 3D reconstruction of CD68-positive lysosomes was performed using the surface rendering function in Imaris10.1. Postsynaptic density protein (PSD95) was labeled using the spot function. The proportion within lysosomes was calculated by dividing the PSD95 labeling within the CD68 volume by the total number of synaptic PSD95 labeling.

[0044] Organizational transparency

[0045] Neonatal mice were injected with AAV-sparse-NCSP-RFP virus at P0, with 1 μl per side. The virus was produced by packaging AAV-CMV-cre / flop plasmid and AAV-DIO / FDIO-mCherry plasmid in a single rAAV at a ratio of 1:2×105. Mouse brains were collected at P7, P14, P21, and P28, and then used Staining and clearing with Basic Tissue Clearing Kit.

[0046] Microglia isolation

[0047] Microglia were isolated using magnetic activated cell sorting (MACS) and anti-CD11b magnetic beads 6 h after exposure to S-ketamine at P7, P14, P21, and P28. Microglia were isolated from mouse brains strictly following the manufacturer's standard protocol.

[0048] RNA sequencing

[0049] Total RNA from microglia in the prefrontal cortex of P7, P14, P21, and P28 was extracted using the TRIZOL kit, and the quality of RNA samples was assessed. Agilent 2100 Bioanalyzer was used to establish a cDNA library and sequence it. RNase free agarose gel electrophoresis was used for verification, followed by RNA sequencing.

[0050] Quantitative real-time polymerase chain reaction (qRT-PCR)

[0051] Total RNA from PFC, hippocampus, and isolated microglia was extracted using the TRIZOL kit according to the manufacturer's instructions. Then, qRT-PCR was performed using the CFX96 Real-Time PCR System and SYBR Green qPCRMix.

[0052] Western blot analysis

[0053] Primary microglia were lysed in radioimmunoprecipitation analysis lysis buffer containing protease and phosphatase inhibitors. Protein concentration was determined using the BCA protein assay. Protein samples were mixed with 5× loading buffer, separated using 6% to 10% SDS-polyacrylamide gels, and finally transferred to polyvinylidene fluoride (PVDF) membranes. Blots were blocked using 5% nonfat milk, incubated with primary antibodies diluted in 5% milk in TBS containing Tween 20 overnight at 4°C, and then incubated with peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The antibodies were as follows: anti-Stat1, anti-Arg1, and anti-β-actin. Finally, proteins were visualized using ClarityWestern ECL Substrate. Proteins were normalized according to the β-actin band density and quantified using 2 -ΔΔC(t) Method determination

[0054] Cell transfection

[0055] Stat1-targeted small interfering RNA (siRNA) and corresponding control siRNA were produced by Beijing Qingke Biotechnology Co., Ltd. siRNA was transfected into cells at a concentration of 50 nM using Lipofectamine TM RNAiMAX transfection reagent combined with Opti-MEM was used for reverse transfection method according to the instructions provided by the manufacturer. RNA samples were collected 48 hours after transfection, and protein samples were collected at 72 hours. Overexpression constructs of Stat1 were created by integrating the complete cDNA of Stat1 into the GV492 expression vector to form the GV492-Stat1 overexpression vector. To improve transfection efficiency, the GV492-Stat1 overexpression vector and the GV492-NC control vector were transfected into HEK-293T cells using Lipofectamine 3000, along with the psPAX2 packaging plasmid and the pMD2.G envelope plasmid. After 48 hours, the generated lentivirus was collected and introduced into primary microglia. These cells were then selected with 6 μg / ml puromycin to further improve transfection efficiency.

[0056] Chromatin immunoprecipitation (ChIP) assay

[0057] ChIP analysis was performed using the EZ ChIP Chromatin Immunoprecipitation Kit following the manufacturer's protocol. In total, 2 × 10 7cells. Primary microglia were treated with 1% formaldehyde and then lysed. DNA fragments ranging from 0 to 2000 bp were obtained by sonication of the genome. The cell lysate was incubated with 10 micrograms of Stat1-specific antibody at 4°C overnight. Afterwards, the mixture was incubated with a 50% slurry consisting of protein A agarose and salmon sperm DNA beads. Subsequently, the DNA initially added and the DNA isolated by immunoprecipitation were purified. These samples were then identified and analyzed using quantitative PCR (qPCR).

[0058] Neuron-microglia co-culture

[0059] Neuronal cultures were prepared from PFC of embryonic day 18 (E18) or E19 mouse embryos. Neurons were cultured at 2 × 10 4 Cells / well were plated at a density of 100 μg / well on coverslips coated with poly-D-lysine and laminin in 24-well plates and maintained in NbActiv4 medium (BrainBits). Half of the medium was replaced with fresh medium every week. Primary microglia were obtained from P1-P2 pups by grinding the forebrain with a 5 ml pipette and then centrifuging at 300 × g for 5 min. The supernatant was discarded, the pellet was resuspended with a 1 ml pipette and filtered through a 75 μm filter. Primary microglia stimulated with 25 μm S-ketamine were cultured alone or with primary neurons, which were also exposed to S-ketamine at normothermia or hypothermia. Co-culture of microglia and neurons followed previously established methods. Microglia were plated at 5 × 10 per well. 4 Cells were plated in 150 μl of culture medium in a 96-well plate and incubated at 37°C in 95% air / 5% CO2. Cortical neurons were plated at 3 × 10 4 Cells / insert were plated at a density of 1:1 in 80 μl of culture medium in cell culture inserts (0.2 μm pore size) and incubated overnight at 37°C in 95% air / 5% CO2. Neurons were then co-cultured with microglia by placing the inserts into the wells containing neurons prior to S-ketamine and hypothermia treatment.

[0060] To collect neuronal conditioned medium, neurons were plated at 5 × 10 4 Cells were seeded in 150 μl of culture medium in a 96-well plate and stimulated with S-ketamine at low or room temperature. The conditioned medium was collected and centrifuged at 1,200 rpm for 2 minutes to remove debris. This conditioned medium from neurons was added to 96-well plates at 5 × 10 cells per well. 4 Cells were plated in microglia and incubated at 37°C, 95% air / 5% CO2 for 24 hours at low or normal temperatures prior to biochemical measurements.

[0061] Arginase inhibitor administration

[0062] The selective arginase inhibitor nor-NOHA was intraperitoneally injected into C57 pups at a dose of 30 mg / kg per day from P2 to P6 before receiving a single dose of S-ketamine at P7. The three-box social test, reciprocal social test, and grooming test were performed from P28 to P30, after which the brains were collected for other experiments.

[0063] Slice preparation and electrophysiology

[0064] Adolescent mice in the saline and S-ketamine groups were deeply anesthetized with pentobarbital (100 mg / kg, ip) and euthanized to collect brains, which were quickly sliced ​​for whole-cell patch clamp recordings according to established protocols. Brain slices were prepared in a slice medium consisting of 2mM CaCl2, 2mM MgCl2, 2.5mM KCl, 26mM NaHCO3, 1.25mM KH2PO4, 10mM glucose, and 220mM sucrose, each with a thickness of 300 microns, maintained at a pH of 7.4, and oxygenated with 95% O2 / 5% CO2. These slices were then immersed in synthetic cerebrospinal fluid (ACSF) containing 126mM NaCl, 26mM NaHCO3, 10mM glucose, 3mM KCl, 1.4mM NaH2PO4, 2mM CaCl2, and 1mM MgCl2, with pH maintained between 7.35-7.40 and continuous oxygenation.

[0065] In preparation for whole-cell patch clamp recordings, slices were perfused with ACSF at a rate of 4 ml / min. To record sEPSCs, a patch pipette (3-6 mω resistance) was filled with an internal solution containing 120 mM potassium methanesulfonate, 10 mM NaCl, 10 mM EGTA, 1 mM CaCl2, 10 mM HEPES, 5 mM ATP-Mg, pH was adjusted to 7.2 with KOH, and the osmotic pressure was maintained at 300 mOsm. These recordings were performed at a holding potential of -70 mV in the presence of 50 μM picrotoxin. For sIPSC recordings, pipettes contained an internal solution of 130 mM cesium methanesulfonate, 10 mM sodium methanesulfonate, 10 mM EGTA, 1 mM CaCl2, 10 mM HEPES, 5 mM lidocaine N-ethylquaternium bromide-Cl, and 2 mM ATP-Mg, pH balanced to 7.2 with CsOH, and osmolarity maintained at 300 mOsm. These recordings were performed at a holding potential of 10 mV with 20 μM CNQX and 50 μM AP5. Data from PFC and CA2 pyramidal neurons were captured using a MultiClamp 700B amplifier and recorded by pClamp9.2 software, filtered at 2 kHz, digitized at 10 kHz, and analyzed using Clampfit 10.0 software and the Mini analysis program.

[0066] Experimental Results

[0067] To investigate the effects of early postnatal S-ketamine exposure on social behavior and the differences from ASD-like symptoms, a series of behavioral tests were performed on adolescent mice ( Figure 1 A) In the three-box social experiment, control mice spent more time in direct contact with the stranger mouse than with the object during the sociability test, while mice exposed to S-ketamine showed no interest in the box containing the stranger mouse ( Figure 1 B). Sniffing time is a more sensitive parameter reflecting sociality, which is consistent with the time mice spend exploring in different boxes ( Figure 1 B) In addition, control mice preferred the box containing the unfamiliar mouse over the familiar mouse and were more likely to sniff the unfamiliar mouse in a social memory test ( Figure 1 C). In contrast, mice exposed to S-ketamine showed no preference for the unfamiliar versus familiar mouse ( Figure 1 C).

[0068] Ketamine has mild respiratory and circulatory depressive effects and is a dissociative anesthetic widely used in pediatric surgery. In recent years, the therapeutic effects of subanesthetic concentrations of ketamine or S-ketamine on mental illness have been elaborated. Although studies on rodents have shown that ketamine can impair neurocognition and dendritic development by affecting synaptic silencing, neuronal apoptosis, etc., the persistent effects of ketamine administration in infancy on subsequent cognition and synaptic development remain unclear. In this study, this example compared the effects of single exposure to S-ketamine and sevoflurane at P7 on subsequent neurocognition in adolescent mice (P28-P30). Combining brain transparency, transcriptomics and electrophysiology techniques, this example further explored the neural mechanisms of S-ketamine exposure on microglial synaptic pruning, dendritic structure development and synaptic neurotransmission. This study aims to illustrate the neural mechanisms of early postnatal S-ketamine exposure mediating social deficits in neurocognitive development.

[0069] Early postnatal exposure to S-ketamine may induce social impairment in adolescence, accompanied by the following abnormalities:

[0070] Exposure to S-Ketamine disrupts dendritic structure and overactivates excitatory synaptic transmission

[0071] Social behavior is closely related to the regulation of multiple brain regions, among which the prefrontal cortex is the central hub of the social brain, and the hippocampal CA2 subregion is considered to be a key brain region for social memory. In addition, it has been reported that structural and functional changes in pyramidal neurons can affect social behavior. Therefore, this example performed Golgi staining to detect changes in the synaptic structure of pyramidal neurons after exposure to S-Ketamine. Sholl analysis showed that the dendritic complexity of PFC pyramidal neurons in mice exposed to S-ketamine increased, especially the basal dendrites ( Figure 2 , a and b). PFC neurons exposed to S-Ketamine showed a significant increase in the number of intersections at 50-90 μm from the cell body. However, there was no difference in the complexity of the apical and basal dendrites of CA2 pyramidal neurons between the two groups. Mice exposed to S-Ketamine showed a significant increase in the number of intersections at 50-90 μm from the cell body. Figure 2 , c, and d) had lower dendritic spine densities at both the apical and basal ends than control mice. In addition, PFC pyramidal neurons from mice exposed to S-Ketamine had a higher proportion of immature dendritic spines (filamentous processes and elongated dendritic spines in both apical and basal dendrites) and a lower proportion of mature dendritic spines (mushroom dendritic spines in both apical and basal dendrites). Similarly, CA2 pyramidal neurons had a higher proportion of filamentous spines in the apical dendrites and filamentous spines in the basal dendrites, and a lower proportion of mature dendritic spines (mushroom dendritic spines in both apical and basal dendrites) ( Figure 2 , e and f).

[0072] In addition, whole-cell patch clamp recordings were performed on PFC and CA2 pyramidal neurons in this example. PFC pyramidal neurons of mice exposed to S-Ketamine showed a significant increase in the frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) compared with control mice ( Figure 2 g), while the frequency and amplitude of spontaneous inhibitory postsynaptic current (sIPSC) did not change ( Figure 2 H). Similarly, the sEPSC amplitude of CA2 pyramidal neurons increased significantly, but the frequency did not change, whereas the sIPSC frequency and amplitude did not change ( Figure 2 , g and h). These results demonstrate that mice exposed to S-Ketamine have defects in the morphology and maturation of PFC pyramidal neurons, which may lead to reduced excitability within the PFC neural network.

[0073] S-ketamine exposure activates microglia and enhances synaptic pruning

[0074] Microglia participate in synaptic remodeling through synaptic pruning in the early postnatal period, and dysregulated synaptic pruning is associated with social disorders. In order to investigate whether the destruction of the dendritic structure of pyramidal neurons is mediated by microglia, this example specifically evaluated the morphological characteristics and synaptic pruning ability of microglia in PFC and hippocampal CA2 at P7, P14, P21 and P28 ( Figure 3 A). Microglia in the PFC of mice exposed to S-chlorantheamine are highly activated ( Figure 3 B), from P7 to P14, the cell body area increased significantly ( Figure 3 C), while from P7 to P28, the number of branches decreases ( Figure 3 D), the neurite length decreases from P7 to P14 ( Figure 3 E). However, microglia in CA2 of mice exposed to S-Ketamine only showed enlarged cell bodies from P7 to P14, and reduced branching during P7 ( Figure 5 , a to d).

[0075] The cells were co-stained with Iba1 by immunostaining for CD68 (a phagolysosomal microglial marker) and PSD95 (a postsynaptic marker). Figure 3 F), it was observed that the immunoreactivity of CD68 in the PFC of the S-ketamine group was significantly enhanced from P7 to P14 ( Figure 3 G). In addition, PSD95 immunoreactivity within CD68+ lysosomes from P7 to P28 in the PFC of S-ketamine-exposed mice was significantly higher than that in control mice ( Figure 3H), which reflects the enhanced phagocytosis of excitatory synapses by microglia after early postnatal S-ketamine exposure. In the CA2 of the S-ketamine group, CD68 immunoreactivity was transiently enhanced at P7 ( Figure 5 , e and f).

[0076] In order to clarify the sensitivity of early postnatal microglia to S-ketamine exposure and the potential connection between microglia and neuronal dendrites, this example detected the contact strength between microglia and dendrites. In this example, the AAV-sparse-NCSP-RFP virus was injected into the lateral ventricle at P0, and the tissue clearing method combined with immunostaining was used to detect microglia at P7, 14, 21, and 28. Figure 3 I). Through three-dimensional reconstruction, this example divides the microglia surrounding neurons into four types according to the contact strength, including no contact type (type 1), protrusion contact type (type 2), cell body contact type (type 3) and cell body wrapping type (type 4) ( Figure 3 , J and K)( Figure 2 , a and b). Compared with the control group, the number of non-contacting microglia around neurons in the PFC of the S-ketamine group was significantly reduced at P7, while the number of process-contacting, cell body-contacting, and cell body-wrapping microglia was greatly increased ( Figure 3 L). Similar results were obtained for P14 and P21 ( Figure 3 , M, and N). However, on P28, the differences between the saline group and the S-Ketamine group were only found in the untouched and cell body-encapsulated microglia ( Figure 3 O).

[0077] S-ketamine exposure directly activates the Stat1-arginase 1 (Arg1) pathway in prefrontal microglia

[0078] Considering that S-ketamine exposure affects microglial activity and function in different brain regions, brains were collected at P7, P14, P21, and P28, and their microglia were screened for further analysis by RNA sequencing ( Figure 4 A). The results showed that there were hundreds of differentially expressed genes (DEGs) after S-ketamine exposure ( Figure 4 B); however, there were only 28 co-varied DEGs at the four time points. Considering that the expression of key genes may not be significantly changed immediately after S-ketamine exposure, 148 co-varied DEGs at P14, P21, and P28 were also included in the analysis ( Figure 4 C). Gene ontology (GO) analysis of these 176 DEGs showed that biological processes related to defense responses to external stimuli were significantly enriched ( Figure 4D). It is noteworthy that DEGs within arginine biosynthesis (Arg 2, Nos2, Ass1, Arg1) accounted for the highest proportion in the KEGG pathway ( Figure 4 E).

[0079] In addition, the protein-protein interaction (PPI) network of these 176 DEGs showed that Stat1, which is closely related to Arg1 expression, was differentially expressed between control and S-ketamine-exposed mice at P14, P21, and P28 ( Figure 5 a). In addition, Stat1 was included in the NOD-like receptor signaling pathway, which contained the most DEGs among the 176 DEGs ( Figure 4 E). The Arg1 mRNA level detected by qRT-PCR was consistent with the sequencing results ( Figure 4 , F and G). The mRNA levels of Stat1 in microglia of both groups were also confirmed ( Figure 8 b) To verify the functional relationship between Stat1 and Arg1, this example performed a ChIP assay and found that Stat1 is a transcription factor of Arg1 because they have obvious effective binding sites ( Figure 8 c). In addition, Stat1 knockout (si-Stat1-1 had the highest knockout efficiency) significantly downregulated the mRNA and protein levels of Arg1 ( Figure 8 d), while Stat1 overexpression upregulated the mRNA and protein levels of Arg1 ( Figure 8 , e and f).

[0080] To explore whether S-ketamine-induced neuronal abnormalities are mediated by microglia, a co-culture model of primary microglia and neurons was performed in vitro, which is a transwell co-culture model ( Figure 4 H). The results showed that when microglia were cultured alone or co-cultured with neurons, the mRNA levels of Arg1, CD11b, and CD18 were significantly increased after S-ketamine exposure ( Figure 4 , I, K, and L). However, there was no difference between the mRNA levels of CD68 in isolated microglia from the control and S-ketamine groups ( Figure 4 J). Compared with microglia cultured alone, microglia co-cultured with neurons showed higher mRNA levels of Arg1, CD68, and CR3, indicating that microglia exerted a stronger dendritic phagocytic function ( Figure 4 , I to L). Together, these findings suggest that the Arg1 signaling pathway is involved in S-ketamine-induced enhancement of microglial activation and synaptic pruning.

[0081] nor-NOHA restores microglial and synaptic function and prevents social impairment induced by S-ketamine exposure

[0082] The study also explored the role of the selective arginase inhibitor nor-NOHA. Neonatal C57 mice were injected with nor-NOHA (30 mg / kg) daily from P2 to P6 before S-ketamine injection at P7. Figure 6 A). Overlay heatmap showing that nor-NOHA restored the number of microglial branches in the PFC of mice exposed to S-Ketamine ( Figure 6 B). Similarly, at P28, nor-NOHA reversed the enhanced PSD95 immunoreactivity in CD68+ lysosomes of PFC microglia in S-Ketamine-exposed mice ( Figure 6 C). In addition, nor-NOHA reduced the complexity of basal but not apical dendrites of PFC pyramidal neurons induced by S-Ketamine exposure ( Figure 6 , D and E), accompanied by an increase in the density of apical and basal dendritic spines ( Figure 6 , F and G), a decrease in the proportion of slender dendritic spines, and an increase in the proportion of mushroom-shaped dendritic spines in apical but not basal dendrites ( Figure 6 , H and I). In addition, nor-NOHA also downregulated the amplitude and frequency of sEPSCs in PFC pyramidal neurons ( Figure 6 , J and K), but had no effect on sIPSC ( Figure 6 , L and M).

[0083] In addition, in the sociability test of the three-box social experiment, nor-NOHA significantly increased the time the experimental mice stayed in the box with the unfamiliar mice and the time they sniffed the unfamiliar mice ( Figure 6 N). nor-NOHA also alleviated the impaired social memory of mice exposed to S-ketamine, as they showed more preference for unfamiliar mice over familiar mice in a social memory test ( Fig. 9 , a and b), and spent more time socially interacting with stranger mice in standard cages ( Figure 6 O), but the social memory index did not increase significantly ( Figure 6 P). Together, these results suggest that nor-NOHA reverses S-Ketamine-induced abnormal neuronal morphology by inhibiting prefrontal microglial activation and elevated synaptic phagocytosis, ultimately promoting normalization of social and grooming behaviors.

[0084] Figure 1(A) Schematic diagram of the experimental design of S-ketamine exposure and behavioral tests. (B and C) Graphs (top) and the time spent in each box (lower left) and sniffing time to O and S1 (lower right) in the three-box social test in the sociability test (B), and the time spent in each box (lower left) and sniffing time to S1 and S2 (lower right) in the social memory test (C). (D) Interaction social test, including three trials in direct interaction between two groups of mice (top), social exploration time (lower left), and social memory index (lower right). (E) Representative images (left) and quantitative results (right) of bead burying in the bead burying test of two groups of mice. (F) Grooming pattern of mice (left), the proportion of grooming duration in each body part (middle), and the percentage of interruptions and incorrect transitions in different parts (right). Saline group, 10 mice, S-ketamine group, 11 mice; data are expressed as mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.

[0085] Figure 2(a) Sample images of Golgi staining of pyramidal neurons in PFC. Scale bar: 50 μm; (b) Sholl analysis of branching complexity of apical dendrites (left) and basal dendrites (right) at P28. Saline group, n = 12 neurons; S-Ketamine, n = 12 neurons. (c, d) Sample images of dendritic branches (c) and quantification of apical (left) and basal (right) dendritic spine density (d). Saline group, n = 41 dendritic segments; S-Ketamine group, n = 43 dendritic segments. Scale bar: 2 μm. (e) Different dendritic spine morphologies from immature to mature states. (f) The proportion of different types of dendritic spines in apical dendrites (left) and basal dendrites (right). The data in (b, d, f) are from 5 mice in each group. (g, h) Sample traces (top), amplitude (bottom left), and frequency (bottom right) of sEPSC (g) and sIPSC (h) in the PFC of control and S-Ketamine-exposed mice. (g) Saline group, n = 12 neurons; S-Ketamine group, n = 13 neurons. (h) Saline group, n = 11 neurons; S-Ketamine group, n = 10 neurons. Data in (g, h) are from 3 mice in each group. (i) Sample Golgi staining images of pyramidal neurons in CA2. Scale bar: 50 μm. (j) Sholl analysis of branching complexity of apical dendrites (left) and basal dendrites (right) at P28. (k, l) Sample images of dendritic branches (k) and quantification of dendritic spine density (l); apical (left), basal (right). Saline group, n = 26 dendritic segments; S-Ketamine group, n = 21 dendritic segments. Scale bar: 2 μm. (m) Different dendritic spine morphologies from immature to mature states. (n) Proportions of different types of dendritic spines in apical dendrites (left) and basal dendrites (right).

[0086] Figure 3(A) Schematic illustration of the experimental scheme for microglia assessment. (B) Heatmaps of overlaid microglia to assess their morphological changes at different developmental stages in the PFC of saline and S-ketamine groups. Scale bar: 10 μm. (C to E) Quantification of microglial morphological parameters, including cell body area (C), number of branches (D), and average neurite length (E) in the PFC. n = 3 mice per group, average 5-8 cells per mouse. (F) Representative confocal images of Iba1 (green), CD68 (red), and PSD95 (blue) in the PFC of saline and S-ketamine-exposed mice. Scale bar: 10 μm. (G and H) Ratio of CD68+ area in microglia (G) and quantification of PSD95+ area in CD68+ punctate cells in the PFC of both groups (H). n = 3 mice per group, average 4-7 cells per mouse. (I) Schematic diagram of the experimental design for virus injection, brain clearing, and 3D reconstruction in neonatal mice. (J, K) Representative orthogonal views of microglia and neurons in PFC (J) and schematic diagrams showing different contact strengths (K). Microglia can be divided into 4 types, including non-contact type (type 1), process contact type (type 2), cell body contact type (type 3), and cell body wrapping type (type 4). Scale bar: 10 μm. (L to O) Comparison of the proportions of different types of microglia around pyramidal neurons in the saline group and S-ketamine group at P7 (L), P14 (M), P21 (N), and P28 (O). n = 3 mice per group, with an average of 4-6 neurons per mouse. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

[0087] Figure 4(A) Microglia were isolated from mice at P7, P14, P21, and P28. Five to ten brains were dissected for each biological replicate. Brains were ground on ice before Percoll gradient centrifugation. Microglia were sorted by magnetic separation, flow cytometry, and subsequently RNA sequencing. (B) Heatmap showing gene profile expression in both groups. (C) Venn diagram of co-regulated DEGs at different developmental stages following S-ketamine exposure. (D and E) Biological enrichment processes (D) and KEGG pathways (E) of the co-regulated 176 DEGs. (F) Volcano plot of Arg1 at P14, P21, and P28. Red represents relatively increased expression, and blue represents relatively decreased expression. (G) qRT-PCR validation of Arg1 at different developmental stages. n = 4 independent experiments. (H) Schematic diagram of neuron-microglia co-culture. (I to L) Arg1 (I), CD68 (J), CD11b (K), and CD18 (L) mRNA levels in primary microglia cultured alone or co-cultured with neurons after S-ketamine exposure. n = 5 independent experiments. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

[0088] Figure 5 (a) Heatmap of overlapping microglia in CA2 of control and S-ketamine groups to assess morphological changes at different developmental stages. Scale bar: 10 μm. (bd) Quantification of morphological parameters of microglia in CA2, including cell body area (b), number of branches (c), and average process length (d), n = 3 mice per group, average 5-8 cells per mouse. (e) Representative confocal images of Iba1 (green), CD68 (red), and PSD95 (blue) in CA2 of saline- and S-ketamine-exposed mice. Scale bar: 10 μm. (f, g) Ratio of CD68+ area in microglia in the two groups (f) and quantification of PSD95+ area in CD68+ puncta (g). (hm) Levels of CD68 (h), CD11b (i), CD18 (j), Cx3cr1 (k), Trem2 (l), and P2ry12 (m) mRNA in CA2. n = 5 independent experiments. Data are presented as mean ± SEM. *p < 0.05, ***p < 0.001.

[0089] Figure 6(A) Schematic diagram of nor-NOHA treatment and behavioral testing. (B) Heat map of overlapping microglia in PFC at P28 (left) and quantification of the number of microglial branches (right). Scale bar: 10 μm. n = 3 mice per group, average 4-8 cells per mouse. (C) Representative confocal images (upper left) and 3D reconstructed images (lower left) of Iba1 (green), CD68 (red), PSD95 (blue) and quantification of PSD95+ area in CD68+ punctate structures in PFC of the four groups at P28 (right). Scale bar: 10 μm. n = 3 mice per group, average 4-7 cells per mouse. (D and E) Sholl analysis of apical (D) and basal dendrite (E) branch complexity at P28. Veh+Sal group, n = 10 neurons, Veh+S-ket group, n = 10 neurons, nor-NOHA+Sal group, n = 9 neurons, nor-NOHA+S-ket group, n = 12 neurons. (F and G) Sample images (left) and quantification results (right) of apical dendritic spine density (F) and basal dendritic spine density (G). Veh+Sal group, n = 31 segments, Veh+S-ket group, n = 28 segments, nor-NOHA+Sal group, n = 26 segments, nor-NOHA+S-ket group, n = 32 segments. Scale bar: 2 μm. (H and I) The proportion of different types of dendritic spines in apical dendrites (H) and basal dendrites (I). Data in (D to I) are from 5 mice in each group. (J and K) from PFC Sample traces of representative sEPSC (J) and sEPSC (K) from mid pyramidal neurons; amplitude (left), frequency (right). Veh+Sal group, n=14 neurons, Veh+S-ket group, n=11 neurons, nor-NOHA+Sal group, n=10 neurons, nor-NOHA+S-ket group, n=15 neurons. (L and M) Sample traces of representative sIPSC (L) and sIPSC (M) from mid pyramidal neurons in PFC, amplitude (left), frequency (right). Veh+Sal group, n=14 neurons, Veh+S-ket group, n=11 neurons Figure 2. Neurons of the nor-NOHA+Sal group, n=9 neurons, and nor-NOHA+S-ket group, n=15 neurons. Data are from 3 mice in each group. (N) Time spent in each box (left) and sniffing time of O and S1 (right) in the sociability test of the three-box social experiment. (O and P) Social exploration time (O) and social memory index (P) in direct interaction of the four groups of mice. Veh+Sal group, n=9, Veh+S-ket group, n=9, nor-NOHA+Sal group, n=10, and nor-NOHA+S-ket group, n=9. Data are expressed as mean ± SEM.*P<0.05, **P<0.01, ***P<0.001.

[0090] Figure 7 (a) Representative orthogonal views of neurons and peripheral microglia. Scale bar: 30 μm. (b) Magnified orthogonal views and 3D reconstructed images of four types of microglia surrounding neurons. Scale bar: 10 μm.

[0091] Figure 8 (a) PPI analysis of 176 DEGs co-regulated by S-ketamine exposure and different developmental stages (b) qRT-PCR validation of Stat1 at different developmental stages. (c) Workflow of ChIP assay (left) and enrichment of Stat1 in the Arg1 promoter (right). (d) Stat1 mRNA levels in microglia were analyzed by qRT-PCR after transfection of three siRNAs or control siRNA. (e, f) Effects of overexpression and knockdown of Stat1 in microglia on Arg1 levels were detected by qRT-PCR (e) and western blotting (f). (e) n = 4 independent experiments, (f) n = 3 independent experiments. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

[0092] Fig. 9 (a) Representative heatmap of the three-box social test test of mice pretreated with saline / S-Ketamine. The time mice spent in each box (left) and the time they spent closely interacting with S1 and S2 (right) during the social memory stage. Veh+Sal, n=9, Veh+S-ket, n=9, nor-NOHA+Sal, n=10, nor-NOHA+S-ket, n=9. Data are presented as mean ± SEM. *P<0.05, **P<0.01.

[0093] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of nor-NOHA in the preparation of drugs for treating or improving social abnormalities.

2. The use according to claim 1, characterized in that: The medicine comprises an effective dose of nor-NOHA and a pharmaceutically acceptable adjuvant.

3. The use according to claim 2, characterized in that: The effective dose is 0-30 mg / kg.

4. The use according to claim 1, characterized in that: The applications include: nor-NOHA reverses S-ketamine-induced neuronal morphological abnormalities, restores synaptic function, and prevents S-ketamine exposure-induced social disorders by inhibiting abnormal activation of microglia and synaptic phagocytosis in the prefrontal cortex.

5. The use according to claim 1, characterized in that: nor-NOHA can restore the number of microglia branches in the prefrontal cortex of S-ketamine-exposed mice, and reverse the enhanced CD68+ lysosomal PSD95 immunoreactivity in prefrontal microglia of P28 S-ketamine-exposed mice; at the same time, it can reduce the branching complexity of the basal terminal dendrites of prefrontal pyramidal neurons caused by S-ketamine exposure.

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