Application of Trolox in preparation of medicine for treating neurodevelopmental disorder related diseases

Abstract

The invention discloses application of Trolox in preparation of a medicine for treating neurodevelopment disorder related diseases, and belongs to the technical field of biological medicine. The invention firstly discovers that Trolox has new application of drugs for preventing and / or treating neurodevelopment disorder related diseases; furthermore, the Trolox restores the neural connectivity by repairing the densities of the damaged neuron dendritic spines of the brain hippocampus and / or striatum; abnormal dopaminergic signal channels are corrected, so that expression of key receptors and downstream molecules thereof is recovered to be normal, and therefore, diseases related to neurodevelopment disorders are prevented and / or treated; the neuroprotective effect of Trolox depends on the activation of dopamine D1 and D2 receptor pathways; in addition, the invention discloses the potential of Trolox in the aspect of targeted repair of the core pathology link of the neurodevelopmental disorder for the first time, and a clear candidate compound and an action target are provided for developing a novel medicine for treating the diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of Trolox in the preparation of drugs for treating neurodevelopmental disorders. Background Technology

[0002] Neurodevelopmental disorders are a group of complex diseases originating in early brain development, often accompanied by persistent social, cognitive, and behavioral dysfunctions. Their pathogenesis is not fully understood, but abnormal synaptic connections and dysfunction of specific neurotransmitter systems (such as the dopamine system) are considered important pathological bases.

[0003] Valproic acid (VPA) is a known environmental risk factor. Pregnancy exposure can induce pathological phenotypes in offspring that mimic human neurodevelopmental disorders, including synaptic developmental impairment and neural pathway abnormalities. Therefore, it is widely used to study the mechanisms of related diseases and to screen drugs.

[0004] Currently, treatment options for neurodevelopmental disorders are limited, and there is a lack of disease-modifying drugs that can target core pathological processes (such as synaptic structure and neural pathway function). Therefore, there is an urgent clinical need to develop novel treatment strategies that can repair neurodevelopmental damage. However, there are currently no research reports on Trolox (6-hydroxy-2,5,7,8-tetramethyltryptane-2-carboxylic acid) in the prevention and / or treatment of neurodevelopmental disorders related to VPA. Summary of the Invention

[0005] The purpose of this invention is to provide the use of Trolox in the preparation of medicaments for treating neurodevelopmental disorders. This addresses the problem in the prior art of lacking medicaments that can effectively prevent and / or treat neurodevelopmental disorders caused by VPA.

[0006] In a first aspect, the present invention provides the use of Trolox in the preparation of medicaments for the prevention and / or treatment of neurodevelopmental disorders, which include at least one of neuronal synaptic structural damage and dopaminergic signaling pathway dysfunction; the neurodevelopmental disorders are valproic acid-induced neurodevelopmental disorders.

[0007] In this invention, the inventors have discovered for the first time that Trolox has a novel pharmaceutical use for the prevention and / or treatment of neurodevelopmental disorders. Furthermore, Trolox restores neural connectivity by repairing damaged dendritic spine density in neurons of the hippocampus and / or striatum; it corrects abnormal dopaminergic signaling pathways, restoring the normal expression of key receptors (D1 receptor (DRD1), D2 receptor (DRD2)) and their downstream molecules (DARPP32, ADCY5), thereby preventing and / or treating neurodevelopmental disorders. The neuroprotective effect of Trolox depends on the activation of the dopamine D1 and D2 receptor pathways. In addition, this invention reveals for the first time the potential of Trolox in targeting and repairing core pathological processes in neurodevelopmental disorders, providing clear candidate compounds and targets for the development of novel drugs to treat such diseases.

[0008] It is understood that neurodevelopmental disorders can be conventional neurodevelopmental disorders in the prior art, and this invention does not limit them. Furthermore, in this invention, neurodevelopmental disorders preferably include at least one of neuronal synaptic structural damage and dopaminergic signaling pathway dysfunction.

[0009] In some implementations, neuronal synaptic structure damage includes at least one of a reduction in the number of neuronal dendrites and / or a decrease in dendritic spine density in the hippocampus region of the brain, or a reduction in the number of neuronal dendrites and / or a decrease in dendritic spine density in the striatum region of the brain.

[0010] It is understood that neuronal synaptic structural damage can be any conventional neuronal synaptic structural damage in the prior art, and the present invention does not limit this. Furthermore, in the present invention, neuronal synaptic structural damage preferably includes at least one of the following: a reduction in the number of neuronal dendrites and / or a decrease in dendritic spine density in the hippocampus region of the brain, or a reduction in the number of neuronal dendrites and / or a decrease in dendritic spine density in the striatum region of the brain.

[0011] In some implementations, dysfunction of the dopaminergic signaling pathway includes elevated gene expression levels and / or protein content of dopamine D1 receptors and / or D2 receptors.

[0012] In some implementations, dopaminergic signaling pathway dysfunction also includes increased gene expression levels and / or protein content of downstream signaling molecules DARPP32 and / or ADCY5.

[0013] It is understood that the dysfunction of the dopaminergic signaling pathway can be a conventional dysfunction of the dopaminergic signaling pathway in the prior art, and the present invention does not limit this. Furthermore, in the present invention, the dysfunction of the dopaminergic signaling pathway preferably includes an increase in the gene expression level and / or protein content of the dopamine D1 receptor and / or D2 receptor; more preferably, it also includes an increase in the gene expression level and / or protein content of downstream signaling molecules DARPP32 and / or ADCY5.

[0014] In some implementations, the effective dose of Trolox is 80-120 mg / kg, for example, 80 mg / kg, 90 mg / kg, 100 mg / kg, 110 mg / kg, 120 mg / kg or other values ​​within this range.

[0015] In some implementations, the effective dose for valproic acid induction is 400-600 mg / kg, for example, 400 mg / kg, 450 mg / kg, 500 mg / kg, 550 mg / kg, 600 mg / kg or other values ​​within this range.

[0016] In a second aspect, the present invention provides a pharmaceutical composition for the prevention and / or treatment of neurodevelopmental disorders, the pharmaceutical composition comprising Trolox.

[0017] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0018] In this invention, the term "pharmaceutically acceptable carrier" refers to excipients widely used in the pharmaceutical manufacturing industry. Excipients primarily serve to provide a safe, stable, and functional pharmaceutical composition, and may also provide methods for dissolving the active ingredient at a desired rate after administration to a subject, or for promoting effective absorption of the active ingredient after administration to a subject. Pharmaceutical excipients may be inert fillers or provide a function, such as stabilizing the overall pH of the composition or preventing degradation of the active ingredient. Pharmaceutical excipients may include one or more of the following: binders, suspending agents, emulsifiers, diluents, fillers, granulators, adhesives, disintegrants, lubricants, anti-adhesion agents, flow aids, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents, and sweeteners.

[0019] The pharmaceutical compositions provided by this invention can be prepared using any method known to those skilled in the art, based on the disclosure. Examples include, but are not limited to, conventional mixing, dissolving, granulation, emulsification, grinding, encapsulation, embedding, or lyophilization processes.

[0020] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of solid dosage forms, semi-solid dosage forms, and liquid dosage forms.

[0021] The pharmaceutical compositions provided by this invention can be administered in any form, including by injection (intravenous), mucosal, oral (solid and liquid formulations), inhalation, ocular, rectal, topical, or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intra-arterial, intramuscular) administration. The pharmaceutical compositions of this invention can also be controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, tablets, soft capsules, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serum preparations. Examples of parenteral formulations include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of the pharmaceutical composition include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and tablets.

[0022] The beneficial effects of this invention are as follows: Unlike existing technologies, this invention is the first to discover a novel pharmaceutical use for Trolox in preventing and / or treating neurodevelopmental disorders; furthermore, Trolox restores neural connectivity by repairing damaged dendritic spine density in neurons of the hippocampus and / or striatum; it corrects abnormal dopaminergic signaling pathways, restoring the normal expression of key receptors (D1 receptor (DRD1), D2 receptor (DRD2)) and their downstream molecules (DARPP32, ADCY5), thereby preventing and / or treating neurodevelopmental disorders; and the neuroprotective effect of Trolox depends on the activation of dopamine D1 and D2 receptor pathways; additionally, this invention reveals for the first time the potential of Trolox in targeting and repairing core pathological processes in neurodevelopmental disorders, providing clear candidate compounds and targets for developing novel drugs to treat such diseases. Attached Figure Description

[0023] Figure 1 The results of Golgi staining of rat hippocampus in different treatment groups in Example 1 of this invention; Figure 2 The results of the number of dendrites in the hippocampus of rats after Golgi staining in different treatment groups in Example 1 of the present invention; Figure 3 The above describes the density results of hippocampal dendritic spines in rats after Golgi staining in different treatment groups in Example 1 of this invention. p <0.01, p <0.001; Figure 4 The results of transcriptome analysis of rat hippocampus in different treatment groups in Example 1 of the present invention are shown. Among them, (A) is the statistical results of differentially expressed genes in the hippocampus of VPA / control group, Trolox prevention / VPA group, and Trolox treatment / VPA group; (B) is the statistical results of differentially upregulated and downregulated genes in the hippocampus of VPA / control group; (C) is the GSEA multichannel enrichment analysis results of differentially expressed genes in VPA / control group; and (D) is the statistical results of differentially upregulated and downregulated genes in the hippocampus of Trolox prevention / VPA group. Figure 5 The results of enrichment analysis of differentially expressed genes in the transcriptome analysis of rat hippocampus in different treatment groups in Example 1 of the present invention are shown. Among them, (A) and (B) are the KEGG enrichment analysis results of differentially expressed genes in VPA / control group and Trolox prevention / VPA group, respectively, and (C) is a heatmap of differentially expressed genes in control group, VPA group, Trolox prevention group and Trolox treatment group. Figure 6 The following are the expression analysis results of dopaminergic neurons and key signaling pathway genes in the hippocampus of rats in different treatment groups in Example 1 of the present invention. Among them, (A) and (D) are the mRNA expression levels of dopamine receptors DRD1 and DRD2 in the rat hippocampus, respectively; (B) and (C) are the mRNA expression levels of downstream signaling pathways DARPP32 and ADCY5 of dopamine receptors in the rat hippocampus, respectively; (E) is the WB result of dopamine receptor proteins DRD2 and DARPP32 in the rat hippocampus; and (F) and (G) are the quantitative results of dopamine receptor proteins DRD2 and DARPP32 in the rat hippocampus, respectively. p <0.05, p <0.01, p <0.001, p <0.0001, ns: no significant difference; Figure 7 The results of the dopamine receptor antagonist intervention in Example 1 of this invention on the behavior of rats in different treatment groups are shown. Among them, (A) is the flowchart of the behavioral experiment, (B) is the result of the bead embedding experiment in the puberty of rats in different treatment groups, (C) is the result of the total duration of self-grooming behavior in the puberty of rats in different treatment groups, (D) is the result of the number of sniffing in the S1 and S2 stages of the puberty adaptation test of rats in different treatment groups, (E) is the result of the number of sniffing in the S1 and S2 stages of the social preference test of rats in different treatment groups, and (F) is the result of the number of sniffing in the S1 and S2 stages of the social novelty test of rats in different treatment groups. p<0.05, p <0.01, p <0.0001, ns: no significant difference; Figure 8 The following are the Golgi staining results of the rat striatum in different treatment groups in Example 1 of the present invention: (A) is a Golgi staining result of the rat striatum in different treatment groups; (B) is the result of the number of dendrites in the rat striatum in different treatment groups; (C) is the result of the density of dendritic spines in the rat striatum in different treatment groups. p <0.001, p <0.0001; Figure 9 The following are the results of rat striatal transcriptome analysis in different treatment groups in Example 1 of this invention: (A) shows the statistical results of differentially expressed genes in the striatum of the VPA / control group, Trolox prevention / VPA group, and Trolox treatment / VPA group; (B) shows the statistical results of differentially expressed genes in the glutamate pathway between the VPA group and the control group; (C) shows the statistical results of differentially expressed genes among the VPA / control group, Trolox prevention / VPA group, and Trolox treatment / VPA group; (D) shows the KEGG pathway enrichment analysis results of differentially expressed genes in the VPA / control group; (E), (F), (G), and (H) are also included. The expression levels of DRD1, DRD2, GRIA1, GRIN2A, and DARPP32 in different treatment groups are shown below. p <0.05, p <0.01, p <0.001, p <0.0001, ns: no significant difference. Detailed Implementation

[0024] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] Experimental methods not specified in the examples are generally performed under conventional conditions and as described in the manual, or as recommended by the manufacturer. Unless otherwise specified, the general equipment, materials, reagents, etc. used are commercially available.

[0026] Example 1 In this embodiment, valproic acid (VPA) was first used to induce the rat model of neurodevelopmental disorders to establish a rat model. Then, Trolox was administered to the rat model of neurodevelopmental disorders for prevention and treatment to study the preventive and therapeutic effects of Trolox on neurodevelopmental disorders.

[0027] Specifically, it includes the following steps: 1. Establishment of a rat model of neurodevelopmental disorders induced by valproic acid (VPA) and the Trolox dosing regimen. 1) Grouping and Treatment: Wistar pregnant mice were randomly divided into a control group (saline), a VPA model group, a Trolox prevention group, and a Trolox treatment group. On day 12.5 of pregnancy, the pregnant mice in the latter three groups were intraperitoneally injected with VPA (500 mg / kg). The Trolox prevention group received Trolox via gavage at a dose of 100 mg / kg half an hour before the intraperitoneal injection of VPA solution. The Trolox treatment group received Trolox via gavage at a dose of 100 mg / kg for the offspring starting on day 25 (P25) after birth, once daily for two weeks.

[0028] 2) Sample collection: After the offspring rats reached adolescence (P35-P42), brain tissues such as the hippocampus and striatum were collected for subsequent analysis.

[0029] 2. Rats' heart perfusion and tissue sampling After the behavioral tests were completed, the rats were anesthetized and dissected for tissue collection. The steps are as follows: 1) Preparation stage: Connect the perfusion kit and perfusion needle and drain them; pre-cool physiological saline and paraformaldehyde; prepare surgical instruments.

[0030] 2) Rat anesthesia: Rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital. The anesthesia was considered successful if the rats did not exhibit a pinching pain reflex.

[0031] 3) Rat surgery: After anesthetization, the rats were fixed on the perfusion platform. The abdomen of the rat was cut open with scissors to expose the ribs. The diaphragm was removed, and the thoracic cavity was cut open from the left rib of the rat until the heart was exposed. The rat ribs were then turned over.

[0032] 4) Perfusion: Gently grasp the rat's heart with forceps and insert a needle into the left ventricle. Locate the right atrial appendage and cut it open. Once blood flows out, turn on the peristaltic pump to begin perfusion. Perfuse with approximately 100 mL of physiological saline. Successful perfusion is indicated by observing the liver change from dark red to white.

[0033] 5) Tissue Collection: After perfusion, the rat skull was cut open and removed, and the entire rat brain tissue was soaked in pre-cooled physiological saline for about 2 minutes. According to the rat brain atlas, the rat brain regions (hippocampus and striatum) were removed and placed in RNase-free EP tubes, frozen with liquid nitrogen and stored at -80°C; for tissue Golgi staining, the removed brain was immersed in Golgi fixative.

[0034] 3. Golgi staining experiment Fixation of rat brain samples: Brain tissue was obtained via perfusion and cut into 0.5 cm thick pieces using a scalpel. The tissue pieces were then picked up with sterile forceps and transferred to a mixture containing 10 mL of GENMED fixative A and 10 mL of GENMED fixative B. The mixture was incubated at room temperature for 2 weeks, avoiding light. Then, it was transferred to a 30% sucrose solution and incubated at 4°C for 48 hours, protected from light. The tissue pieces were blotted dry with tissue paper and frozen at -20°C. The tissue pieces were then slowly frozen and sectioned at -20°C, mounted on anti-detachment slides, and stored at -20°C. Sample staining: A 20 mL plastic bottle was prepared, containing 10 mL of deionized water. Then, 5 mL of GENMED chromogenic solution A and GENMED chromogenic solution B were added separately, mixed well, and labeled as GENMED working solution. The solution was then protected from light.

[0035] First, take out a 20-100 μm thick frozen tissue section to be tested; at room temperature, carefully place the section into 50 mL of GENMED cleaning solution (Rcagent C) and incubate for 1 min; carefully remove the GENMED cleaning solution (Rcagent C) from the section; carefully add 500 μL of GENMED staining solution (Rcagent D), covering the entire sample surface; incubate at room temperature for 30 min; next, carefully remove the GENMED staining solution (Rcagent D) from the section; at room temperature, carefully place the section into 50 mL of GENMED cleaning solution (Rcagent C) and incubate for 1 min; then, carefully remove the GENMED cleaning solution (Rcagent C) from the section; carefully add 500 μL of GENMED developing solution, covering the entire sample surface; incubate at room temperature for 10-30 min, until it turns black, then immediately stop and avoid light exposure; carefully remove the GENMED developing solution from the section; finally, at room temperature, carefully place the section into 50 mL of GENMED staining solution (Rcagent D) and incubate for 10-30 min, until it turns black, then immediately stop and avoid light exposure; carefully remove the GENMED developing solution from the section; finally, at room temperature, carefully place the section into 50 mL of GENMED cleaning solution (Rcagent C) and incubate for 10-30 min, until it turns black. Incubate in GENMED cleaning solution (Reagent C) for 1 min; carefully remove the GENMED cleaning solution (Reagent C) from the slides; dehydrate with anhydrous ethanol and clear with xylene; air-dry the slides and mount with neutral resin; observe under a general optical microscope: neurons, pyramidal cells, glial cells, oligosynaptic glial cells (oval like beads) and other processes appear black.

[0036] 4. Transcriptome sequencing Transcriptome sequencing was commissioned to Shanghai Paisennong Biotechnology Co., Ltd. The submitted samples were hippocampal tissues from the control group, VPA model group, Trolox prevention group, and Trolox treatment group. Six hippocampal samples from each group were combined into one sample, for a total of four samples, which were then sequenced. First, the raw data was filtered, and the resulting high-quality sequences (Clean Data) were aligned to the reference genome for that species. Based on the alignment results, the expression level of each gene was calculated. Further differential expression analysis, enrichment analysis, and cluster analysis were then performed on the samples. The aligned reads were then assembled to reconstruct the transcriptome sequences. The data results were analyzed on the Paisennong Gene Cloud Platform.

[0037] 5. Real-time quantitative PCR 1) Tissue RNA extraction a) Cut approximately 30 mg of hippocampal tissue, add 1 mL of Trizol reagent, and sonicate in an ultrasonic cell disruptor until no tissue is visible to the naked eye. The entire process should be performed on ice and allowed to stand for 5 minutes.

[0038] b) Centrifuge the tissue homogenate at 4℃ and 12000g for 5 min.

[0039] c) Transfer 800 μL of supernatant to a new RNase-free centrifuge tube, add 200 μL of chloroform, and slowly invert for 15 seconds until the solution turns milky white. Incubate on ice for 5 minutes, then centrifuge at 4°C and 12000g for 15 minutes.

[0040] d) Take 400 µL of the colorless upper aqueous phase into a new RNase-free centrifuge tube, add an equal volume of pre-chilled isopropanol, mix by inverting, and let stand on ice for 10 min. Centrifuge again at 4 °C and 12000 g for 15 min.

[0041] e) Discard the liquid in the centrifuge tube, add 1 mL of 75% ethanol (prepared with DEPC water) to wash the precipitate. Centrifuge at 4℃ and 12000g for 5 min.

[0042] f) Remove the liquid from the tube and place it on ice to dry. Add 30 µL of RNase-free water to dissolve the precipitate, thus obtaining an aqueous RNA solution.

[0043] 2) RNA concentration measurement RNA concentration and purity were determined using an ultra-micro spectrophotometer, and the final concentration was diluted to 500 ng / µL based on the results.

[0044] 3) RNA reverse transcription to cDNA Add 5 µL of 4×All in-one-qRT SuperMix, 1 µL of Enzyme Mix, 12 µL of RNase-free ddH2O, and 2 µL of template RNA sequentially to a PCR tube. Mix well, heat at 37°C for 2 min, then at 55°C for 15 min, and immediately heat at 85°C for 5 min to obtain the reverse transcription product cDNA.

[0045] 4) PCR amplification Add 10µL of 2×Taq Pro Universal SYBR qPCR Master Mix, 0.4µL each of the front primer (10µM) and back primer (10µM), 5.2µL of ddH2O, and 4µL of reverse transcription product according to the formula. Mix well, centrifuge, and then load onto the PCR instrument. Amplification conditions: 95℃ pre-denaturation for 30s, followed by 95℃ denaturation for 10s, and 60℃ denaturation for 30s, for 40 cycles. The PCR primer sequences used are shown in Table 1 below.

[0046] Table 1 qRT-PCR primer sequences

[0047] 5) Calculation of mRNA expression results: PCR data were calculated using a semi-quantitative analysis method, and the results were expressed in 2... -ΔΔCt express.

[0048] 6. Western blot for protein immunoblotting 6.1 Extraction and Sample Preparation of Hippocampal Proteins 1) Protein Extraction and Sample Preparation: Based on the total number of samples to be tested, a protein extraction solution was prepared using RIPA lysis buffer: PMSF (1×): protease inhibitor (100×): phosphatase inhibitor (50×) = 96:1:1:2. After mixing, the solution was placed on ice. The hippocampus was weighed, and the protein extraction solution was added at a ratio of hippocampus sample weight: protein extraction solution = 1 mg: 10 µL. The solution was then sonicated on ice. The sonication conditions used in this experiment were: 2 seconds on, 3 seconds off, 2 minutes on, and 16% power. The lysed tissue protein extract was left to stand on ice for 10 minutes, then centrifuged at 12000g for 15 minutes at 4°C. After centrifugation, the supernatant was collected in a 1.5 mL centrifuge tube. This is the extracted hippocampal tissue protein sample.

[0049] 2) Protein concentration detection using the BCA method: Add 1.2 mL of protein standard preparation solution to 30 mg of BSA, dissolve thoroughly to prepare a 25 mg / mL protein standard solution. Before use, further dilute to a 2 mg / mL protein standard solution. Prepare the BCA working solution according to a reagent A: reagent B ratio of 50:1. Add the protein standard solution to 96-well plates at volumes of 0, 2, 4, 6, 8, 10, 16, and 20 μL, and add PBS solution to bring the total volume to 20 μL. Add 200 μL of BCA working solution to each well and incubate at 37°C for 30 min. Then, measure the absorbance of each well at 545 nm using a microplate reader. Finally, calculate the protein concentration of each sample based on the standard curve.

[0050] 6.2 Western blot experiment 1) Sample preparation: Add 5×SDS loading buffer to the extracted protein supernatant at a ratio of 4:1, mix well, heat in a 95℃ metal bath for 10 min, and then cool to room temperature on ice to obtain the protein sample required for WB experiment. Store at -20℃.

[0051] 2) Gel Preparation: Prepare two sets of 1mm glass plates (large and small), clean and dried, and clamp them together in the gel preparation clamp to prevent leakage, while ensuring the bottom of the glass plates is flat. This experiment uses a one-step PAGE gel rapid preparation kit (10% and 12.5%) to prepare SDS-PAGE gels. First, pipette 2.7 mL of the lower gel buffer and lower gel solution, then add 60 µL of modified coagulant to the mixture. Mix well and immediately add the mixture to the glass plate, ensuring the liquid level is at least 0.5 cm longer than the distance to the comb teeth. Next, pipette 0.75 mL of the upper gel buffer and upper gel solution, then add 15 µL of modified coagulant to the mixture. Mix well and immediately add the mixture to the glass plate until it covers the upper edge of the short glass plate. Insert the sample comb slowly, taking care to avoid air bubbles. After the stacking gel and separating gel have solidified, remove the sample comb, add the sample, and perform electrophoresis.

[0052] 3) Sample loading and electrophoresis: Place the -20℃ frozen sample in a 100℃ constant temperature metal bath for 5 minutes, then vortex for 30 seconds and centrifuge for 10 seconds in a handheld centrifuge to ensure thorough mixing. Adjust the measured protein concentration of the sample using 1×SDS loading buffer. After loading, proceed with electrophoresis. First, electrophoresis at a constant voltage of 80V until the marker layer separates, then adjust the voltage to 120V and electrophoresis until the bromophenol blue is about to detach from the gel, then stop electrophoresis.

[0053] 4) Transfer: Activate the PVDF membrane with methanol beforehand. Wet the sponge and filter paper required for transfer in the tray with transfer buffer. Then, from bottom to top, stack the activated PVDF membrane and the gel taken after electrophoresis in the following order: "sandwich clip (black) - sponge - filter paper - gel - membrane - filter paper - sponge - sandwich clip (transparent)". Gently remove the air bubbles between the membrane and gel, fix them in place, and put them into the transfer tank together. Use a constant current of 300mA for transfer. The transfer time is adjusted according to the size of the target protein.

[0054] 5) Blocking: Pour the blocking solution into the incubation chamber beforehand. After the transfer is complete, remove the PVDF membrane, cut it according to the size of the target protein and mark it. Then quickly immerse it in the blocking solution to prevent the membrane from drying out. The blocking experiment is performed at room temperature. Place the incubation chamber on a shaker and shake it slowly for 30 minutes. After the experiment, wash three times with TBST buffer for 5 seconds each time to remove any residual skim milk powder.

[0055] The sealing solution is prepared as follows: the sealing solution is prepared according to the ratio of skim milk powder: TBST = 1g: 20mL, and after preparation, it needs to be thoroughly vortexed and mixed.

[0056] 6) Incubation of primary antibody: After blocking and cleaning the PVDF membrane, add the corresponding primary antibody dilution solution and incubate overnight on a shaker at 4°C with slow shaking.

[0057] 7) Washing: The primary antibody dilution solution was recovered the next day, and the PVDF membrane was rapidly washed three times with TBST buffer on a shaker at room temperature for 10 minutes each time.

[0058] 8) Incubation of secondary antibody: Add secondary antibody dilution solution, and incubate slowly on a shaker at room temperature for 1 hour.

[0059] 9) Washing: After incubation, recover the secondary antibody dilution solution and wash the PVDF membrane three times with TBST buffer on a shaker at room temperature for 10 minutes each time.

[0060] 10) Development and grayscale analysis: After washing, the PVDF film was immersed in ECL chemiluminescent solution for a few seconds, then removed and placed in the developing instrument for imaging and photographing. The resulting strips were then analyzed for grayscale values ​​using ImageJ.

[0061] 7. Dopamine receptor antagonist intervention experiment 7.1 Receptor Antagonist Intervention Experimental Protocol From day 28 (P28) to day 35 (P35) after the offspring were born, rats in the corresponding treatment groups were treated with D1 receptor antagonist (SKF83566) and D2 receptor antagonist (Raclopride) at a dose of 0.5 mg / kg every other day.

[0062] 7.2 Behavioral testing to assess the effectiveness of antagonist intervention A series of behavioral tests were performed on offspring rats during their adolescence (P35-P42) to assess the effectiveness of the antagonist intervention.

[0063] 7.2.1 Bead embedding experiment This experiment was used to assess stereotyped, repetitive exploratory behaviors. A clean cage (with a 5cm thick corncob bedding layer) was placed in a quiet environment. Twenty clean glass beads (1.5cm in diameter) were evenly distributed on the bedding surface. A single rat was placed in the cage and allowed to explore freely for 30 minutes. After the experiment, the rat was removed, and the number of glass beads buried in the bedding for more than 2 / 3 of its volume was counted. Each group should contain at least 8 animals. The cages were thoroughly cleaned after the experiment.

[0064] 7.2.2 Self-combing test This experiment was used to assess stereotyped and anxiety-like behaviors. Rats were placed alone in a clean, open testing cage. After acclimatization for 5 minutes, the total duration of spontaneous grooming behavior over the following 10 minutes was recorded using a top-mounted camera. Grooming behavior included continuous actions such as licking, scratching the body, and cleaning the face. The test was conducted in a quiet, low-light environment.

[0065] 7.2.3 Three-box social test This experiment was used to assess social orientation and social cognition. The apparatus consisted of a rectangular box divided into three equal-sized compartments by transparent partitions, with doors allowing the animal to move freely between them. The test was conducted in three phases, each lasting 10 minutes. 1) Adaptation phase: The test rats were placed in the middle box and were allowed to freely explore the three empty boxes.

[0066] 2) Social Preference Test: A wire cage containing a strange, same-species rat (Stranger 1, S1) was placed in one box, and an identical empty cage was placed in the other box. The time the test rat spent in the two boxes and the time it actively sniffed the cage were recorded.

[0067] 3) Social novelty test: The empty cage was replaced with a cage containing a new, unfamiliar rat (Stranger 2, S2), while the cage containing the old rat S1 remained unchanged. The exploration time of the test rats in cages S1 and S2 was recorded.

[0068] 8. Results and Analysis 8.1 Trolox restored valproic acid-induced neuronal connectivity in the rat hippocampus. Dendritic spines are tiny projections extending from the dendritic trunk. Excitatory synapses reside in these specialized structures and facilitate the detection and transmission of chemical and electrical signals between neurons in the brain. Notably, spine density is often used as an indicator of neural connectivity.

[0069] To investigate the effects of Trolox on neural connectivity, Golgi staining was performed on the hippocampus of rats in different treatment groups. The results showed that VPA-induced hippocampal basal dendrites were significantly reduced, accompanied by a corresponding decrease in the number of apical dendrites, and the overall spine density of both basal and apical dendrites in the hippocampus decreased by approximately 40%; while in the Trolox prevention and treatment groups, the number of basal and apical dendrites, and the overall spine density of both basal and apical dendrites in the hippocampus were correspondingly increased. Figures 1-3 In summary, VPA significantly impairs dendritic branching and spine density in the rat hippocampus. These changes are thought to affect neuronal connectivity and synaptic transmission. Prevention or treatment with Trolox can restore dendritic morphology and promote neuronal connectivity, thereby preventing and / or treating neurodevelopmental disorders.

[0070] 8.2 Trolox for the prevention and treatment of valproic acid exposure-induced damage to dopaminergic neuronal pathways in the hippocampus of rats and restoration of pathway activity First, the present invention performed transcriptome sequencing analysis on the hippocampus tissue of a rat model, covering the control group, VPA model group, Trolox prevention group, and Trolox treatment group.

[0071] Relevant sequencing data were obtained from NCBI SRA Bioproject ID PRJNA1363884 (rat). Differential gene expression analysis was then performed on different treatment groups. Statistical analysis was conducted on the significantly differentially expressed gene sets obtained from the differential expression analysis results. Figure 4 A). A bar chart was then generated to show the number of upregulated and downregulated differentially expressed genes in each comparison group. Compared to the VPA group, a total of 183 differentially expressed genes were identified in the hippocampus of the control group rats, including 120 upregulated genes and 63 downregulated genes. Figure 4 B). Compared with the Trolox prevention group, a total of 395 differentially expressed genes were identified in the hippocampus of rats in the VPA group, including 66 upregulated genes and 329 downregulated genes. Figure 4D). This invention performed bidirectional clustering analysis on the joint set of all differentially expressed genes in each comparison group and sample. The clustering was based on the expression level of the same gene in different samples and the expression patterns of different genes within the same sample. Samples with similar expression patterns were grouped into one class. Box plots show the expression patterns of genes in each class in each sample. Figure 4 C).

[0072] Furthermore, GO enrichment analysis was performed on the differentially expressed genes in the above rat hippocampal transcriptome analysis.

[0073] The results showed that, compared with the control group and the Trolox prevention group, VPA-regulated genes were mainly enriched in biological processes such as behavior, nervous system development, chemical synaptic transmission, cognition, and evolution. Enrichment was also observed in cellular components, including cellular processes and the cell membrane. KEGG pathway enrichment analysis was performed using Metascape, and the top 20 enriched pathways were visualized. Compared with the control group and the Trolox prevention group, VPA-regulated genes were mainly enriched in neurotransmitter transmission-related pathways, including neuroactive ligand-receptor interactions, calcium signaling, cAMP signaling, and dopaminergic synaptic transmission. Figure 5 A- Figure 5 C).

[0074] Then, qRT-PCR and Western blot analysis were performed on the hippocampus of rats in the control group, VPA model group, Trolox prevention group and Trolox treatment group to study the effects of different treatment groups on the expression levels of key mRNAs and proteins in the dopaminergic synaptic signaling pathway in the rat hippocampus.

[0075] Among them, DRD1 (dopamine D1 receptor) and DRD2 (dopamine D2 receptor) are genes that encode dopamine receptors, while ADCY5 and DARPP32 are key downstream genes of dopamine receptors.

[0076] The results showed that, compared with the control group, the expression levels of DRD1 and DRD2 in the VPA group were abnormally elevated; however, after receiving Trolox prevention and treatment, the expression levels of DRD1 and DRD2 mRNA in both groups decreased, approaching the normal values ​​observed in the control group. Figure 6 A, Figure 6 D). Compared with the control group, the expression levels of DARPP32 and ADCY5 mRNA were significantly increased in the VPA group; however, after Trolox prophylaxis and treatment, the mRNA expression levels of these genes were significantly decreased, approaching the normal values ​​observed in the CT group. Figure 6 B. Figure 6C). Exposure to VPA leads to an abnormal increase in the expression of DRD2 and DARPP32 receptor proteins in rat hippocampal dopamine (DA) neurons, resulting in aberrant activation of key downstream signaling pathways; however, after administration of Trolox for prevention or treatment, the expression levels of DRD2 and DARPP32 proteins in the rat hippocampus returned to normal. Figure 6 E- Figure 6 G). The results showed that Trolox prevention or treatment restored the expression of key receptors (DRD1, DRD2) and their downstream molecules (DARPP32, ADCY5) to normal, thereby preventing and / or treating neurodevelopmental disorders.

[0077] Finally, the effects of dopamine receptor antagonist intervention on the behavior of rats in different treatment groups were investigated.

[0078] Among them, behavioral tests were conducted on day P35. Figure 7 A), that is, 7 days after intraperitoneal injection of Raclopride (D2 receptor antagonist), this treatment was applied to the offspring of VPA-treated rats protected by Trolox.

[0079] In the bead implantation experiment, the number of beads implanted in rats treated with Raclopride was similar to that in the VPA group. In addition, the number of beads implanted in the Raclopride group was significantly higher than that in the Trolox prevention group. Figure 7 B); The results showed that blocking D2 receptor activation with Raclopride prevented Trolox from reversing stereotyped and anxiety-like behaviors exhibited in the offspring of VPA-treated rats. In the self-grooming experiment, rats treated with Raclopride showed a significant increase in anxiety-like behaviors (B). Figure 7 (C) This suggests that Raclopride can counteract the beneficial effects of Trolox on anxiety-like behaviors in VPA-treated rat offspring. However, compared to the Trolox prevention group, no significant differences were observed in the number of grooming sessions and the duration of grooming in rats treated with SKF83566 (a DRD1 receptor antagonist). Blocking D1 dopamine receptor activation with SKF83566 did not prevent Trolox from reversing stereotyped and anxiety-like behaviors in VPA-treated rat offspring.

[0080] In a three-box social interaction experiment, after administering either Racloprid or SKF83566 to the offspring of VPA-exposed rats treated with Trolox for 7 days, rats treated with SKF83566 (a DRD1 receptor antagonist) showed significant deficiencies in social preference and social novelty. In contrast, rats treated with Racloprid (a D2 receptor antagonist) did not show significant deficiencies in social ability. Notably, inhibiting D1 receptor activation with SKF83566 eliminated the protective effect of Trolox on social preference and social novelty deficiencies in the offspring of VPA-exposed rats. Figure 7 D- Figure 7 In summary, blocking D2 receptor activation significantly reduced the beneficial effects of Trolox on stereotyped and anxiety-like behaviors in VPA progeny; while inhibiting D1 receptor activation significantly reduced Trolox's ability to combat social preference and novelty deficits. These results indicate that the neuroprotective effects of Trolox depend on the activation of dopamine D1 and D2 receptor pathways.

[0081] 8.3 Trolox for the prevention and treatment of valproic acid exposure-induced damage to glutamatergic neurons in the striatum of rat brains To assess the effects of Trolox on neural connectivity in brain regions related to motor regulation and reward, Golgi staining analysis was performed on the striatum of rats under different treatments.

[0082] The results showed that, consistent with the observations in the hippocampus, VPA-induced rat striatum also exhibited a significant reduction in basal dendrites, a decrease in the number of apical dendrites, and a significant reduction in the overall dendritic spine density of both basal and apical dendrites of striatal neurons, approximately 40%. This indicates that VPA exposure caused widespread damage to the dendritic structure and synaptic connectivity of the striatum, similar to that in the hippocampus, and that Trolox prevention and treatment effectively reversed these structural damages in the striatum. Figure 8 A- Figure 8 (C) The results further support the mechanism by which Trolox exerts its neuroprotective effect by restoring the morphology and connectivity of neurons in multiple brain regions.

[0083] Furthermore, in addition to dopamine receptors, glutamatergic neurons were also investigated. Striatal transcriptome sequencing was performed on rat groups (including control group, VPA group, Trolox prevention group, and Trolox treatment group).

[0084] Subsequently, differential gene expression analysis was performed on different treatment groups. Statistical analysis was conducted on the sets of genes with significant differential expression in the differential expression analysis results. Figure 9 A). Then, a bar chart is generated to show the number of differentially expressed genes that are upregulated and downregulated in each comparison group. Figure 9B shows the differentially expressed genes in the glutamate pathway between the control group and the VPA group. Further bar charts show the number of differentially expressed genes upregulated and downregulated in each comparison group. Figure 7 C). KEGG enrichment analysis of differentially expressed genes in VPA / control group ( Figure 9 D). The VPA group showed abnormally decreased expression levels of DRD1 and DRD2. After VPA prevention and treatment, the expression levels of D1 and D2 receptors in the striatum were significantly increased ( Figure 9 E- Figure 9 F).

[0085] qRT-PCR analysis showed significant differences in the expression levels of GRIA1, a key gene in the glutamatergic neuron signaling pathway, in the striatum of the control group, VPA group, Trolox prevention group, and Trolox treatment group, with the VPA group showing an abnormally high expression level compared to the control group. However, after Trolox prevention and treatment, the expression of GRIA1 mRNA in both groups decreased to near-normal levels. Figure 9 The results showed that VPA exposure led to an abnormal increase in GRIA1 levels in glutamatergic neurons of the rat hippocampus, thereby damaging these neurons; after prevention or treatment with Trolox, the signal transduction pathways of these neurons returned to normal levels in the striatum. Furthermore, the expression levels of GRIN2A and DARPP32 mRNA were low in the control and VPA groups, while Trolox treatment significantly increased mRNA expression levels. Figure 9 The above results indicate that Trolox can activate the expression of D1 and D2 receptors in the striatum.

[0086] In summary, this invention is the first to discover a novel pharmaceutical use for Trolox in preventing and / or treating neurodevelopmental disorders. Furthermore, Trolox restores neural connectivity by repairing damaged dendritic spine density in neurons of the hippocampus and / or striatum; it corrects abnormal dopaminergic signaling pathways, restoring normal expression of key receptors (DRD1, DRD2) and their downstream molecules (DARPP32, ADCY5), thereby preventing and / or treating neurodevelopmental disorders. Moreover, the neuroprotective effect of Trolox depends on the activation of dopamine D1 and D2 receptor pathways.

[0087] It should be noted that all the above embodiments belong to the same inventive concept, and the descriptions of each embodiment have different focuses. Where the description in a particular embodiment is not detailed, please refer to the description in other embodiments.

[0088] The embodiments described above are merely illustrative of implementation methods of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The use of Trolox in the preparation of medicaments for the prevention and / or treatment of neurodevelopmental disorders, characterized in that, The neurodevelopmental disorders include at least one of neuronal synaptic structural damage and dopaminergic signaling pathway dysfunction. The neurodevelopmental disorders mentioned are neurodevelopmental disorders induced by valproic acid.

2. The application according to claim 1, characterized in that, The neuronal synaptic structure damage includes at least one of the following: a decrease in the number of neuronal dendrites and / or a decrease in dendritic spine density in the hippocampus region of the brain, or a decrease in the number of neuronal dendrites and / or a decrease in dendritic spine density in the striatum region of the brain.

3. The application according to claim 1, characterized in that, The dysfunction of the dopaminergic signaling pathway includes increased gene expression levels and / or protein content of dopamine D1 receptors and / or D2 receptors.

4. The application according to claim 1, characterized in that, The abnormality of the dopaminergic signaling pathway also includes an increase in the gene expression level and / or protein content of downstream signaling molecules DARPP32 and / or ADCY5.

5. The application according to any one of claims 1-4, characterized in that, The effective dose of Trolox is 80-120 mg / kg.

6. The application according to claims 1-4, characterized in that, The effective dose for valproic acid induction is 400-600 mg / kg.

7. A pharmaceutical composition for the prevention and / or treatment of neurodevelopmental disorders, characterized in that, The pharmaceutical composition includes Trolox.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the pharmaceutical composition includes at least one of solid dosage form, semi-solid dosage form, and liquid dosage form.

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