Use of scutellarein in the preparation of a drug for treating autism caused by haploinsufficiency of gene TCF20

Abstract

The application discloses application of scutellarein in preparation of a medicine for treating autism caused by haploinsufficiency of a gene TCF20, and belongs to the technical field of biological medicine. The pharmacodynamics verification is carried out by using a Tcf20 haploinsufficiency mouse model, and the experimental results show that oral administration of scutellarein can improve social deficiency, repetitive behavior, cognitive impairment and anxiety-like behavior of the model mice, and effectively alleviate the growth retardation phenotype. Single-cell transcriptome sequencing finds that Tcf20 defects cause wide down-regulation of gene expression of mitochondrial respiratory chain complex I, III, IV and V of prefrontal cortex neurons; in an in-vitro primary neuron culture system, Tcf20 defects cause a decrease of a mitochondrial membrane potential of neurons, and the membrane potential is significantly increased after scutellarein treatment. The application provides a new therapeutic medicine for autism caused by haploinsufficiency of the gene TCF20.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of scutellarin in the preparation of a drug for treating autism caused by insufficient haplo-dose of gene TCF20. Background Technology

[0002] 1. Autism spectrum disorders and their comorbidities Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder. According to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5), its core diagnostic criteria include: persistent deficits in social communication and interaction; restricted, repetitive patterns of behavior, interests, or activities. In clinical practice, ASD is often accompanied by various comorbidities, such as cognitive impairment, anxiety disorders, and growth retardation.

[0003] 2. The association between mitochondrial dysfunction and autism In recent years, numerous studies have shown that mitochondrial dysfunction plays a crucial role in the pathogenesis of ASD. Abnormal activity of the mitochondrial electron transport chain and impaired energy metabolism can lead to abnormal brain development and synaptic dysfunction. One review suggests that ASD can be considered a mitochondrial disease.

[0004] However, the manifestations of mitochondrial dysfunction under different genetic backgrounds exhibit significant brain region and cell subtype specificity, and mitochondrial dysfunction cannot be generalized. Different ASD genetic subtypes may involve completely different mitochondrial pathological mechanisms, suggesting that precise intervention strategies targeting specific genetic defects have significant research value.

[0005] 3. Haploinous TCF20 gene deficiency and autism The transcription factor 20 (TCF20) gene has been identified as a high-risk gene for acute respiratory distress syndrome (ASD) and growth retardation. Copy number deletion leading to haploinadequacy is one of its important pathogenic mechanisms.

[0006] TCF20-associated neurodevelopmental disorder is characterized by a variety of neurological abnormalities, including growth retardation, intellectual disability, and autism spectrum disorder. At the animal model level, TCF20-half-dose-insufficient mice (TCF20...) + / - They exhibit social impairments, cognitive and learning memory deficits, and generalized growth retardation. To date, there are no reports on whether TCF20 deficiency affects neuronal mitochondrial function.

[0007] 4. Current uses of scutellarin B Scutellarin (also known as wild scutellarin) is a natural flavonoid compound extracted from *Erigeron breviscapus*, a plant in the Asteraceae family. It is a key quality control component of *Erigeron breviscapus* as specified in the *Pharmacopoeia of the People's Republic of China*. Based on its pharmacological effects, such as increasing cerebral and cardiac blood flow and improving microcirculation, scutellarin preparations (such as scutellarin tablets) have been widely used clinically for the treatment of ischemic cardiovascular and cerebrovascular diseases (such as cerebral infarction and coronary heart disease).

[0008] In the field of basic research, the mitochondrial protective function of scutellarin has attracted attention. Related studies have shown that scutellarin can alleviate mitochondrial dysfunction and apoptosis by improving mitochondrial glucose oxidation and promoting mitophagy.

[0009] 5. Shortcomings of existing technology Based on the above analysis, the existing technology has at least the following shortcomings: First, although TCF20 has been identified as a high-risk gene for ASD, and Tcf20 haplo-deficient mice also exhibit ASD-like behavioral phenotypes, there is a lack of effective intervention strategies in the current technology for autism caused by TCF20 deficiency. Furthermore, the current technology also does not provide any technical insights into applying scutellarin to ASD models caused by TCF20 deficiency.

[0010] Second, current research on mitochondria with Tcf20 deficiency mainly focuses on liver and embryonic fibroblasts. Whether Tcf20 deficiency leads to neuronal mitochondrial dysfunction, and the specific molecular characteristics of this dysfunction (which respiratory chain complexes are involved and which brain cell types are affected), are all previously unknown.

[0011] In summary, existing technologies lack effective interventions for the pathological manifestations caused by insufficient TCF20 doses. Therefore, there is an urgent need in the field to develop a drug that can improve autism caused by TCF20 deficiency. This invention is proposed based on the aforementioned shortcomings of existing technologies. Summary of the Invention

[0012] In view of the shortcomings of the prior art, the purpose of this invention is to provide the application of scutellarin in the preparation of a drug for treating autism caused by insufficient haplo-dose of gene TCF20.

[0013] The solution of the present invention is: Application of scutellarin in the preparation of drugs for treating autism caused by insufficient haplo-dose of gene TCF20.

[0014] Furthermore, the autism mentioned is an autism spectrum disorder.

[0015] Furthermore, the drug improves the core and accompanying symptoms of autism.

[0016] Furthermore, the core symptoms include social impairment and / or repetitive behaviors; the accompanying symptoms include cognitive impairment, anxiety-like behaviors, and / or growth retardation.

[0017] Furthermore, the growth retardation was manifested as a decrease in body weight compared to wild-type mice in the same litter.

[0018] Furthermore, the autism is accompanied by mitochondrial dysfunction of prefrontal cortical neurons, manifested as abnormal expression of mitochondrial energy metabolism-related genes and / or abnormal mitochondrial membrane potential.

[0019] Furthermore, the drug contains a therapeutically effective amount of scutellarin and a pharmaceutically acceptable carrier.

[0020] Furthermore, the drug is administered orally.

[0021] Furthermore, the drug is an oral preparation.

[0022] Furthermore, the dosage of the oral formulation is 2 mg / kg / day; the dosage is calculated based on the clinical dosage of 120 mg / day indicated on the marketed Erigeron breviscapus tablets, using an adult standard weight of 60 kg as a benchmark, and converted through body surface area.

[0023] Since the main active ingredient in *Dendrobium nobile* tablets is scutellarin B, the clinical dosage of *Dendrobium nobile* tablets can be equivalent to the dosage of scutellarin B. Because the clinical dosage of *Dendrobium nobile* tablets can be equivalently replaced by the clinical dosage of scutellarin B, those skilled in the art can perform the conversion based on this equivalence. The clinical dosage is 120 mg / day (based on an adult standard weight of 60 kg), i.e., 2 mg / kg / day. Therefore, the dosage of the oral formulation for human use in this invention is 2 mg / kg / day.

[0024] Furthermore, the oral formulation is administered to mice at a dose of 25 mg / kg / day.

[0025] Specifically, this invention uses a Tcf20 haplo-deficient mouse model (Tcf20). + / - Efficacy verification was conducted on 5-week-old male Tcf20 pharmacokinetic animals. + / -Mice were randomly divided into a model group (HET), a low-dose group of scutellarin (SCU-L, 25 mg / kg / day), and a high-dose group of scutellarin (SCU-H, 75 mg / kg / day), with wild-type mice (WT) serving as the control group. Each group consisted of at least six mice. All treatment groups were administered orally once daily for four weeks. After treatment, behavioral tests (three-box socialization test, marble burial test, hair-stroking test, new object recognition test, Y-maze test, and black-and-white box test) were conducted. During the treatment period, mice were weighed every five days to monitor growth retardation phenotype. Single-cell transcriptome sequencing was performed on the prefrontal cortex tissues of WT and HET mice. Differentially expressed genes were screened and GO / KEGG enrichment analysis was performed focusing on excitatory neuronal clusters. Mitocarta gene sets were obtained from the MitoCarta 3.0 database to analyze changes in the expression of respiratory chain complex IV-related genes. Primary neurons were cultured from the prefrontal cortex of WT and HET fetal rats at the E18.5 stage and treated with 10 μM scutellarin for 24 h. Mitochondrial membrane potential was detected using the JC-1 probe.

[0026] Application of scutellarin in the preparation of drugs to improve mitochondrial dysfunction of prefrontal cortical neurons caused by insufficient haplo-dose of gene TCF20.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows: First, this invention is the first to observe, at a clinically equivalent dose, the ameliorative effect of scutellarin on core behavioral disorders and accompanying behavioral abnormalities in autism caused by Tcf20 gene deficiency.

[0028] Regarding core behavioral disorders, the three-box social experiment results showed that during the social phase, the social preference index of mice in the HET model group (0.35±0.03) was significantly lower than that in the WT group (0.62±0.02) (p<0.0001); the SCU-L group (0.56±0.07, p<0.05) and the SCU-H group (0.54±0.05, p<0.01) were both significantly higher than those in the HET group, and then returned to the WT level. During the social novelty phase, the novelty preference index of the SCU-H group (0.61±0.06, p<0.05) was significantly higher than that of the HET group (0.36±0.09), and then returned to near the WT level. In the marble burying experiment, the number of marbles buried in the SCU-L group (3.00±1.10, p<0.01) and the SCU-H group (2.67±1.52, p<0.05) was significantly less than that in the HET group (8.83±1.38). In the hair-stroking experiment, the cumulative hair-stroking time in the SCU-L group (20.00±4.63s, p<0.05) and the SCU-H group (16.50±3.23s, p<0.001) was significantly shorter than that in the HET group (54.83±9.05s). These results indicate that scutellarin can improve the core behavioral disorders of autism caused by Tcf20 deficiency.

[0029] Regarding accompanying behavioral abnormalities, in the novel object recognition experiment, the recognition index of the SCU-L group (65.69±7.54%, p<0.05) was significantly higher than that of the HET group (39.78±7.92%). In the Y-maze experiment, the recognition indices of both the SCU-L group (58.00±8.21%, p<0.05) and the SCU-H group (52.36±7.02%, p<0.05) were significantly higher than those of the HET group (32.76±3.30%). In the black-and-white box experiment, the dwell time in the white box of the SCU-H group (88.90±6.67 s, p<0.05) was significantly longer than that of the HET group (62.22±6.90 s), recovering to near the WT level. Weight monitoring results showed that after 25 days of administration, the body weight of the SCU-L group (21.00±0.50 g) and the SCU-H group (21.28±0.56 g) was significantly higher than that of the HET group (18.15±0.98 g, p<0.05), but there was no significant difference compared with the WT group (21.81±0.29 g) (p>0.05). These results indicate that scutellarin has an ameliorative effect on accompanying symptoms such as cognitive impairment, anxiety-like behavior, and growth retardation caused by Tcf20 deficiency.

[0030] Second, this invention reveals for the first time the molecular pathological characteristics of mitochondrial dysfunction in prefrontal cortex neurons caused by haplo-insufficient Tcf20, and verifies in vitro primary neuron culture that scutellarin can improve this dysfunction. Single-cell transcriptome sequencing revealed that Tcf20... + / -Differentially expressed genes downregulated in excitatory neurons of the mouse prefrontal cortex were significantly enriched in mitochondrial energy metabolism-related pathways, with widespread downregulation of respiratory chain complexes I, III, IV, and V (p<0.0001). JC-1 membrane potential assays confirmed that the red / green fluorescence ratio of neurons in the HET group was significantly lower than that in the WT group (p<0.01); after treatment with scutellarin (10 μM, 24 h), the red / green fluorescence ratio significantly increased (p<0.05 vs HET), and there was no significant difference compared with the WT group (p>0.05). These findings provide experimental evidence, from molecular pathological characteristics to functional improvement, for the treatment of autism caused by Tcf20 haplo-insufficiency with scutellarin.

[0031] Third, the dosage used in this invention is calculated based on the clinically routine dosage (120 mg / day) of the marketed drug, *Dendrobium nobile* tablets, using a body surface area conversion method. *Dendrobium nobile* is a marketed drug whose safety has been clinically verified and which has good prospects for clinical translation. Attached Figure Description

[0032] Figure 1 A schematic diagram and results of the three-box social experiment; Wherein, A is a representative schematic diagram of the trajectory during the social stage; B is a statistical chart of the dwell time in the social box during the social stage; C is a statistical chart of the preference index during the social stage; D is a representative schematic diagram of the trajectory during the social novelty stage; E is a statistical chart of the dwell time in the social novelty stage; F is a statistical chart of the preference index during the social novelty stage; WT is the wild-type control; HET is Tcf20. + / - Model group; SCU-L was the low-dose group of scutellarin (25 mg / kg / day); SCU-H was the high-dose group of scutellarin (75 mg / kg / day); data are expressed as mean ± SEM, and significance markers in the figure indicate comparison with the HET group.

[0033] Figure 2 The results of the marble burial experiment and the hair-stroking experiment; In this diagram, A represents a representative image from the marble burial experiment; B shows the number of marbles buried by mice within 30 minutes; C is a schematic diagram of mouse grooming behavior; D shows the cumulative grooming time of mice during 10 minutes of free exploration; WT represents the wild-type control; and HET represents Tcf20. + / - Model group; SCU-L was the low-dose group of scutellarin (25 mg / kg / day); SCU-H was the high-dose group of scutellarin (75 mg / kg / day); data are expressed as mean ± SEM, and significance markers in the figure indicate comparison with the HET group.

[0034] Figure 3 Results of new object recognition and Y-maze experiments; In this diagram, A is a heatmap of mouse activity time in the new object recognition experiment, O represents the old object, and N represents the new object; B is a statistical analysis of the mouse head exploration time in the new object recognition experiment; C is a statistical analysis of the mouse cognitive index in the new object recognition experiment; D is a schematic diagram of the Y-maze apparatus, in which mice start from the initial arm and freely explore the old and new arms; E is a statistical analysis of the cognitive index in the Y-maze experiment; WT represents the wild-type control; and HET represents Tcf20. + / - Model group; SCU-L: low-dose group of scutellarin (25 mg / kg / day); SCU-H: high-dose group of scutellarin (75 mg / kg / day); data are expressed as mean ± SEM, and significance markers in the figure indicate comparison with the HET group.

[0035] Figure 4 These are the results of the black-and-white box experiment; In this diagram, A represents the typical trajectory of mice freely moving between the black and white boxes; B shows the time mice spent in the white box; C shows the distance mice traveled in the white box; and D shows the number of times mice moved between the two boxes. WT represents the wild-type control; HET represents Tcf20. + / - Model group; SCU-L was the low-dose group of scutellarin (25 mg / kg / day); SCU-H was the high-dose group of scutellarin (75 mg / kg / day); data are expressed as mean ± SEM, and significance markers in the figure indicate comparison with the HET group.

[0036] Figure 5 For the effect of scutellaria baicalensis ethyls on Tcf20 + / - Effect on mouse body weight; Wherein, A is the mouse body weight change curve during the drug administration period, recorded every 5 days for 25 days; B is a bar chart of the mouse's final body weight on day 25 after drug administration; WT is the wild-type control; HET is Tcf20. + / - Model groups; SCU-L was the low-dose group of scutellarin (25 mg / kg / day); SCU-H was the high-dose group of scutellarin (75 mg / kg / day); data are expressed as mean ± SEM; #p < 0.05, ### p < 0.001 indicate comparison with the HET group; the connecting lines and ns in the figure indicate no significant difference between the two groups (compared with the WT group).

[0037] Figure 6 For single-cell transcriptome sequencing analysis; In the figures, A is the UMAP clustering diagram of prefrontal cortex cells, with excitatory neuron cell clusters marked in red; B is the statistical diagram of the number of intersections between differentially expressed genes (DEGs) of each cell type and the MitoCarta 3.0 mitochondrial gene set, with blue indicating downregulation and red indicating upregulation; C is the enrichment analysis of GO biological processes of downregulated differentially expressed genes in excitatory neurons, Top 15, with mitochondrial energy metabolism-related pathways marked in red; D is the enrichment analysis of KEGG pathway of downregulated differentially expressed genes in excitatory neurons, Top 15; E is the box plot of the overall mitochondrial function score of the Global MitoCarta Score of excitatory neurons; F is the box plot of the respiratory chain complex IV score of the OXPHOS Complex Module Scores of excitatory neurons; G is the heatmap of downregulated differentially expressed genes in respiratory chain complex (Complex) IV of excitatory neurons, with blue / red indicating down / upregulation, respectively; data are expressed as mean ± SEM; ****p < 0.0001 (compared with the WT group), ns indicates no significant difference between groups.

[0038] Figure 7 The results are for the JC-1 mitochondrial membrane potential detection. In this diagram, A represents representative JC-1 fluorescence images of primary neurons in each group, with red indicating JC-1 polymer and green indicating JC-1 monomer, and the scale bar is 10 μm; B is a bar chart of the red / green fluorescence intensity ratio, RFU 590nm / RFU 533nm; WT: wild-type fetal rat primary neurons; HET: Tcf20. + / - Fetal rat primary neurons; SCU: Tcf20 treated with scutellarin (10 μM) + / - Primary neurons from fetal rats; wild-type neurons were treated with CCCP (10 μM) as a mitochondrial uncoupling agent as a positive control; data are expressed as mean ± SEM; **p < 0.01, ***p < 0.001 (compared to the WT group); #p < 0.015 (compared to the HET group); ns indicates no significant difference compared to the WT group.

[0039] Figure 8 This is a schematic diagram illustrating the experimental process and symptom improvement of the present invention; Tcf20 + / - Male mice were administered scutellarin orally via gavage starting at 5 weeks of age for 4 consecutive weeks. Weight changes were continuously monitored during the administration period, and various behavioral tests were conducted sequentially after administration. The dashed box indicates the chemical structural formula and clinical background of scutellarin. The arrows in the right-hand box indicate the direction of improvement for each indicator; ↑ indicates increase / improvement, and ↓ indicates decrease / improvement. Social skills, cognitive memory, and weight showed an increasing trend, while stereotyped behaviors and anxiety-like behaviors showed a decreasing trend. Detailed Description of the Embodiments

[0040] In order to make the technical means, creative features, achieved objectives and effects of the present invention easy to understand, the present invention is further described below with reference to specific embodiments.

[0041] Example 1: Experimental Materials (1) Experimental Animals Tcf20 haploinsufficient mice (Tcf20 + / - , C57BL / 6J background, catalog number: NM-KO-230134) and littermate wild-type mice (WT) were purchased from Shanghai Model Organisms Center, Inc. (Animal License No.: SCXK (Shanghai) 2024-0007). All mice were housed in an SPF-grade environment at the Laboratory Animal Center of Yunnan University, with a 12-hour light / 12-hour dark cycle, temperature controlled at 22-25°C, humidity maintained at 50%-55%, and free access to drinking water and food. The animal experimental protocol was reviewed and approved by the Laboratory Animal Welfare and Ethics Committee of Yunnan University (Approval No.: YNU20230681).

[0042] (2) Drugs and Reagents Scutellarin (purity ≥98%, item number HY-N0751) was purchased from MCE (MedChemExpress); JC-1 Mitochondrial Membrane Potential Assay Kit (item number MA0338_A) was purchased from Dalian Meilun Biotechnology Co., Ltd.; Neurobasal medium and B-27 supplement were purchased from Gibco (Thermo Fisher Scientific); all other conventional reagents are of analytical grade.

[0043] Example 2: Animal Grouping and Administration 5-week-old male mice were selected, and Tcf20 haploinsufficient mice were randomly divided into 3 groups: Tcf20 + / - model group (HET), scutellarin low-dose group (SCU-L), scutellarin high-dose group (SCU-H); littermate wild-type mice were used as the control group (WT), with no less than 6 mice in each group.

[0044] The dosage conversion was based on the following: Referring to the standard clinical dosage of *Erigeron breviscapus* tablets (40 mg / time, 3 times daily, i.e., a total daily dose of 120 mg / day), calculated based on an adult standard weight of 60 kg, this dose is equivalent to 2 mg / kg / day. Using the body surface area conversion method (Km method, human Km=37, mouse Km=3), the equivalent dose in mice = 2 mg / kg × (37 / 3) ≈ 24.67 mg / kg, rounded to 25 mg / kg / day. The high-dose group was set at three times the low-dose group, i.e., 75 mg / kg / day. All treatment groups received oral gavage once daily for 4 consecutive weeks. The control group received an equal volume of solvent control (physiological saline solution containing 2% DMSO).

[0045] Example 3: Behavioral Testing All behavioral experiments were conducted in a quiet, temperature- and humidity-controlled environment after drug administration. The video analysis system was purchased from Shanghai Xinsoft Co., Ltd.

[0046] (1) Social behavior detection The three-box socialization test was used to assess social skills and social novelty. Socialization phase: The time mice spent with unfamiliar mouse 1 and the empty cage was recorded for 10 minutes. Social novelty phase: The time mice spent with unfamiliar mouse 2 and familiar mouse 1 was recorded for 10 minutes. Mouse trajectories and exploration time were recorded, and the preference index was calculated as either S1 exploration time / total exploration time or S2 exploration time / total exploration time.

[0047] (2) Repetitive behavior test (marble burying test and hair stroking test) Marble burying experiment: Record the number of marbles buried by mice within 30 minutes. Hair stroking experiment: Record the cumulative hair stroking time of mice during 10 minutes of free exploration in an open field device.

[0048] (3) Cognitive function testing (new object recognition experiment and Y maze experiment) New object recognition experiment: Record the time mice spend exploring new and old objects with their heads, and calculate the cognitive index (%) = new object exploration time / total exploration time × 100%. Y maze experiment: Record the time mice spend entering the new and old arms and convert it into a cognitive index, cognitive index (%) = time to enter the new arm / total time for both new and old arms × 100%, to assess the mice's preference for the new and old arms.

[0049] (4) Anxiety-like behavior detection (black and white box experiment) Record the time the mouse spent in the open box, the distance it traveled, and the number of times it shuttled between the two boxes within 5 minutes.

[0050] (5) Monitoring of growth retardation Weigh yourself every 5 days during the medication period and plot the weight change curve.

[0051] Example 4: Single-cell transcriptome sequencing (1) Collection of materials Prefrontal cortex tissues were taken from 5-week-old and 11-week-old WT and HET mice, respectively, and flash-frozen in liquid nitrogen and stored at -80°C.

[0052] (2) Sequencing Single-cell transcriptome sequencing was performed using the BGI DNBelab C4 platform, with a target of capturing >5000 cells per sample.

[0053] (3) Data Analysis Quality control was performed using Seurat v5 software (cells with <200 or >9000 genes (nFeature_RNA), <200 or >30000 UMI counts (nCount_RNA), >2% mitochondrial gene proportion, and >1% hemoglobin gene proportion were excluded), SCT normalization, Harmony batch correction, PCA and UMAP dimensionality reduction clustering, and cell type identification were performed. Focusing on excitatory neuronal clusters, the FindMarkers function (Wilcoxon rank-sum test, logfc.threshold=0.25, min.pct=0.25) was used for initial screening of differentially expressed genes between HET and WT, and further differentially expressed genes (DEGs) were identified using a corrected p-value (p_val_adj) <0.05 as a threshold. GO and KEGG enrichment analyses were performed using ClusterProfiler. Mitocarta gene sets were obtained from the MitoCarta 3.0 database, and the expression changes of respiratory chain complex IV-related genes were analyzed.

[0054] Example 5: Primary prefrontal cortex neuron culture and mitochondrial membrane potential detection (1) Primary neuron culture Fetal mice with WT and HET genotypes at the E18.5 stage were collected, and the prefrontal cortex was harvested, digested with 0.25% trypsin, and resuspended in Neurobasal+B27 medium and cultured until day 13 (DIV13).

[0055] (2) Drug treatment DIV13 neurons included a WT control group, a HET model group, and an SCU treatment group. A stock solution (10 mM) of scutellarin was prepared by dissolving it in DMSO. This stock solution was then diluted to a working concentration (10 µM) in complete neuronal culture medium, and the neurons were incubated for 24 h. The WT and HET groups were incubated in culture media containing their respective carrier solvents.

[0056] (3) Membrane potential detection Mitochondrial membrane potential (ΔΨm) is a direct functional reading of electron transport activity in the mitochondrial respiratory chain, and its level reflects the energy metabolism status of mitochondria. The JC-1 probe (2 µM) was incubated at 37°C in the dark for 30 minutes and imaged using a Zeiss LSM800 confocal microscope. Red fluorescence indicates that the dye has entered the mitochondria and formed polymers, while green fluorescence indicates that the dye exists in monomeric form. A decrease in the red / green fluorescence ratio indicates a decrease in mitochondrial membrane potential. The red / green fluorescence ratio was analyzed using ImageJ software to represent the mitochondrial membrane potential level. Each biological study was repeated three times.

[0057] Example 6: Statistical Analysis Statistical data were analyzed using GraphPad Prism 9.5 software. Unpaired t-tests were used for comparisons between two groups, and one-way ANOVA and Tukey's post-hoc test were used for comparisons among multiple groups. JC-1 ratios were logarithmically transformed to stabilize variance and satisfy the normality assumptions for parametric tests before statistical analysis. A p-value < 0.05 was considered statistically significant.

[0058] Example 7: Ethyl benzoate from *Erigeron breviscapus* improves Tcf20 + / - Core behavioral deficits and growth retardation in mice (1) Ethyl styrax var. chinensis improves Tcf20 + / - Social behavioral deficits in mice Figure 1 ) Social behavior was assessed using a three-box social experiment.

[0059] In the social stage ( Figure 1 Compared with the WT group (preference index: 0.62±0.02, n=10), the social preference index of mice in the HET model group was significantly reduced (0.35±0.03, n=6, p<0.0001), indicating that mice with haplo-insufficient Tcf20 doses had social deficits. The social preference indices of mice in the SCU-L group (0.56±0.07, n=6, p<0.05) and the SCU-H group (0.54±0.05, n=6, p<0.01) were significantly higher than those in the HET group, and there was no significant difference between them and the WT group (p>0.05). Figure 1 C). The box access time results are consistent with the trend of the preference index. Figure 1 B).

[0060] During the social novelty stage ( Figure 1The novelty preference index of mice in the HET model group (0.36±0.09) was significantly lower than that in the WT group (0.62±0.03, n=10, p<0.01), indicating a weakened preference for unfamiliar individuals. The novelty preference index of mice in the SCU-H group (0.61±0.06, n=6, p<0.05) was significantly higher than that in the HET group, and returned to levels close to those of the WT group (p>0.05 vsWT). Although the novelty preference index of mice in the SCU-L group (0.54±0.05, n=6, p=0.116) showed an increasing trend, it did not reach statistical significance compared to the HET group. Figure 1 F). The box visit time results are consistent with the trend of the preference index ( Figure 1 E).

[0061] The above results indicate that continuous administration of scutellarin for 4 weeks can improve Tcf20. + / - Social deficits in mice.

[0062] (2) Ethyl scutellaria baicalensis improves Tcf20 + / - Repetitive behaviors in mice Figure 2 ) The marble burial test and the hair-stroking test were used to evaluate the repetitive behaviors of mice.

[0063] In the marble burial experiment ( Figure 2 The number of embedded marbles in the HET model group (8.83±1.38, n=6) was significantly increased compared to the WT group (1.90±0.46, n=10) (p<0.0001), indicating that mice with a single-dose under-dose Tcf20 exhibited repetitive stereotyped behaviors. Compared to the HET group, the number of embedded marbles in the SCU-L group (3.00±1.10, n=6, p<0.01) and the SCU-H group (2.67±1.52, n=6, p<0.05) was significantly decreased. Figure 2 AB).

[0064] In the hair-stroking experiment ( Figure 2 The cumulative grooming time in the HET model group (54.83±9.05 s, n=6) was significantly longer than that in the WT group (28.30±3.64 s, n=10) (p<0.01), indicating that mice with a single-dose under-dose Tcf20 exhibit repetitive self-grooming behavior. The grooming time in both the SCU-L group (20.00±4.63 s, n=6, p=0.01 vs HET) and the SCU-H group (16.50±3.23 s, n=6, p<0.001 vs HET) was significantly shorter than that in the HET group; however, there was no significant difference between the SCU-L group and the WT group (p>0.05), while the SCU-H group had a shorter duration than the WT group (p<0.05). Figure 2 CD).

[0065] The above results indicate that continuous administration of scutellarin for 4 weeks can significantly improve Tcf20. + / - Repetitive behaviors in mice.

[0066] (3) Ethyl styrax var. chinensis improves Tcf20 + / - Cognitive function in mice ( Figure 3 ) In the new object recognition experiment ( Figure 3 The recognition index of mice in the AC, WT group (59.89±2.32%, n=9) and SCU-L group (65.69±7.54%, n=6, p=0.039 vs HET) was significantly higher than that in the HET group (39.78±7.92%, n=6). The SCU-H group (60.11±4.57%, n=6, p=0.051 vs HET) showed an increasing trend, and both groups recovered to the WT level (p>0.05 vsWT). Figure 3 C). The exploration time results are consistent with the trend of the identified index ( Figure 3 B).

[0067] In the Y-maze experiment ( Figure 3 The recognition index of mice in the DE group (32.76±3.30%, n=6) was significantly lower than that in the WT group (67.65±3.19%, n=10) (p<0.0001); the recognition index of mice in the SCU-L group (58.00±8.21%, n=6, p<0.05 vs HET) and the SCU-H group (52.36±7.02%, n=6, p<0.05 vs HET) was significantly higher, with the SCU-L group recovering to the WT level (p>0.05 vs WT), while the SCU-H group remained lower than the WT group (p<0.05 vs WT). Figure 3 E).

[0068] The above results indicate that continuous administration of scutellarin for 4 weeks can improve Tcf20. + / - Cognitive impairment in mice.

[0069] (4) Ethyl styrax var. chinensis improves Tcf20 + / - Anxiety-like behavior in mice Figure 4 ) The black-and-white box experiment was used to assess anxiety-like behaviors in mice, such as... Figure 4 As shown in the representative schematic diagram of the trajectory of A, the activity trajectories of the HET group mice in the white box area are significantly sparse compared to those of the other groups.

[0070] Regarding the time spent in the white box ( Figure 4B), the HET model group mice (62.22±6.90 s, n=6) had a significantly shorter time than the WT group (110.70±8.35 s, n=10) (p=0.001), indicating that Tcf20 + / - The mice exhibited anxiety-like behaviors. The SCU-L group (84.06±7.55 s, n=6, p=0.059 vs HET; p<0.05 vs WT) showed an improving trend, while the SCU-H group (88.90±6.67 s, n=6, p=0.020 vs HET; p>0.05 vs WT) showed a significantly prolonged and recovered time to near WT levels. Figure 4 B). White box travel distance ( Figure 4 C) and the number of times the black and white boxes were traversed ( Figure 4 The trend of change in D) is consistent with that described above.

[0071] The above results indicate that high-dose administration of scutellarin B for 4 weeks can improve Tcf20. + / - Anxiety-like behavior in mice tends to improve with low doses.

[0072] (5) Ethyl styrax var. chinensis improves Tcf20 + / - Mouse growth retardation phenotype ( Figure 5 ) Before drug administration (day 0), the body weight of mice in the HET group was significantly lower than that in the WT group (WT: 18.98±0.28 g, n=10 vs. HET: 13.5±1.12 g, n=6, p<0.001), indicating that the growth retardation phenotype was established. Figure 5 A).

[0073] Ten days after administration, the body weight of mice in both the SCU-L and SCU-H groups showed an increasing trend (SCU-L: 16.96±0.94 g, n=6; SCU-H: 17.67±0.79 g, n=6; HET: 14.78±0.90 g, n=6), and the SCU-H group was significantly higher than the HET group (p<0.05). Figure 5 A). After 25 days of drug administration, the body weight of mice in the SCU-L group (21.00±0.50 g, n=6) and the SCU-H group (21.28±0.56 g, n=6) was significantly higher than that in the HET group (18.15±0.98 g, n=6, p<0.05), but there was no significant difference compared with the WT group (21.81±0.29 g, n=10) (p>0.05). However, the body weight of mice in the HET group was still significantly lower than that in the WT group (p<0.001). Figure 5 B).

[0074] The above results indicate that continuous administration of scutellarin for 25 days can improve Tcf20. + / - Growth retardation phenotype in mice.

[0075] Example 8: Single-cell transcriptome sequencing reveals Tcf20 + / - Mitochondrial dysfunction in mouse neurons ( Figure 6 ) Single-cell transcriptome sequencing was performed on the prefrontal cortex of WT and HET mice (collected at approximately 11 weeks of age) after behavioral testing. UMAP cluster analysis identified major cell types including excitatory neurons, inhibitory neurons, astrocytes, oligodendrocytes, and microglia, with excitatory neurons being the dominant neuronal group in this brain region. Figure 6 A).

[0076] Functional enrichment analysis was performed on differentially expressed genes downregulated in excitatory neuronal clusters. GO biological process enrichment analysis showed that mitochondrial energy metabolism-related pathways dominated the top 15 enriched pathways, including oxidative phosphorylation, aerobic respiration, and cellular respiration (p. adjust < 10). -21 KEGG pathway enrichment analysis further showed that the "oxidative phosphorylation" pathway ranked highly (p. adjust < 10). -8 () Figure 6 C, 6D). Intersection of differentially expressed genes in each cell type with the MitoCarta 3.0 mitochondrial gene set revealed that excitatory neurons had the highest number of downregulated genes in the intersection. Figure 6 B).

[0077] Based on the above enrichment analysis results, the focus was further placed on mitochondrial function in excitatory neurons. The Neuronal Global MitoCarta Score showed that the overall score of the mitochondrial gene set in the HET group was significantly lower than that in the WT group (p<0.0001). Figure 6 E). Neuronal OXPHOS Complex Module Scores analysis showed that complexes I, III, IV, and V were significantly downregulated in the HET group (p<0.0001 vs WT), while complex II showed no significant difference ( Figure 6 F), the heatmap of differentially regulated genes in the respiratory chain complex showed the same trend (F). Figure 6 G).

[0078] A similar trend was observed in 5-week-old mice. These results indicate that haplo-insufficiency of Tcf20 leads to overall downregulation of the mitochondrial gene set in prefrontal cortex neurons and widespread inhibition of respiratory chain complexes I, III, IV, and V.

[0079] Example 9: Ethyl benzoate from *Erigeron breviscapus* improves Tcf20 + / - mitochondrial membrane potential of neurons Figure 7 ) The widespread downregulation of respiratory chain complexes I, III, IV, and V in Example 8 suggests impaired mitochondrial electron transport chain function. To assess the ameliorative effect of scutellarin on this functional change, the mitochondrial membrane potential of primary prefrontal cortex neurons was measured—a direct functional readout of electron transport chain activity.

[0080] The results show that ( Figure 7 ),like Figure 7 As shown in the representative JC-1 fluorescence image of A, the red fluorescence representing mitochondrial membrane potential in the neurons of the HET group was significantly reduced. Figure 7 Quantitative statistical results of B showed that, compared with the WT group (red / green ratio: 2.78±0.51, n=3), the red / green fluorescence ratio of neurons in the HET model group was significantly reduced (0.67±0.08, n=3, p<0.01), indicating that the Tcf20 gene defect led to a decrease in neuronal mitochondrial membrane potential. Compared with the HET group, the red / green fluorescence ratio of the SCU treatment group (1.85±0.19, n=3, p<0.05) significantly increased, and there was no significant difference compared with the WT group (p>0.05). The red / green ratio of the positive control group CCCP (mitochondrial uncoupling agent) treatment group (0.32±0.09, n=3) was further reduced to below that of the WT group (p<0.001 vs WT), verifying the sensitivity of the JC-1 detection system to membrane potential changes.

[0081] The above results indicate that scutellarin can improve the decrease in neuronal mitochondrial membrane potential caused by gene Tcf20 deficiency.

[0082] Example 10: Schematic diagram of the experimental procedure and symptom improvement of the present invention ( Figure 8 ) Tcf20 + / - Male mice were given oral administration of gentian root extract for four consecutive weeks starting at 5 weeks of age. They were then subjected to behavioral tests (three-box social test, marble burial test, fur grooming test, new object recognition test, Y maze test, and black and white box test), and their weight changes were continuously recorded. Figure 8The diagram illustrates the experimental procedure and symptom improvement of this invention. The diagram shows various test indicators and improvement trends (↑: increase / improvement; ↓: decrease / improvement) such as social skills, stereotyped behaviors, cognitive memory, anxiety-like behaviors, and weight changes. Specific data can be found in Examples 7 to 9.

[0083] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. Application of scutellarin in the preparation of drugs for treating autism caused by insufficient haplo-dose of gene TCF20.

2. The application according to claim 1, characterized in that: The autism mentioned refers to autism spectrum disorder.

3. The application according to claim 1, characterized in that: The medication improves the core and accompanying symptoms of autism.

4. The application according to claim 3, characterized in that: The core symptoms include social impairment and / or repetitive behaviors; the accompanying symptoms include cognitive impairment, anxiety-like behaviors, and / or growth retardation.

5. The application according to claim 2, characterized in that, The autism is accompanied by mitochondrial dysfunction of prefrontal cortical neurons, manifested as abnormal expression of genes related to mitochondrial energy metabolism and / or abnormal mitochondrial membrane potential.

6. The application according to claim 1, characterized in that: The drug contains a therapeutically effective amount of scutellarin and a pharmaceutically acceptable carrier.

7. The application according to claim 1, characterized in that: The drug is administered orally.

8. The application according to claim 7, characterized in that: The drug is an oral preparation.

9. The application according to claim 8, characterized in that: The dosage of the oral formulation is 2 mg / kg / day; the dosage is calculated based on the clinical dosage of 120 mg / day indicated on the marketed Erigeron breviscapus tablets, using an adult standard weight of 60 kg as a benchmark, and converted through body surface area.

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