Application of beta-carotenoid in preventing or treating progeny autism spectrum disorder-like behavior caused by gestational diabetes mellitus

Abstract

The invention discloses application of beta-carotenoid in preventing or treating progeny autism spectrum disorder behaviors caused by gestational diabetes mellitus. The invention develops new application of beta-carotenoid in preparation of medicines for preventing or treating progeny autism spectrum disorder behaviors caused by gestational diabetes mellitus, not only can the blank of existing treatment means be filled, but also a safe, effective and easily popularized treatment scheme can be provided, and the beta-carotenoid has important clinical value and social significance. The beta-carotenoid is applied to preparation of the medicine for preventively improving gestational diabetes mellitus progeny autism, and a new medicine is provided for treating the autism.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of a β-carotene in the prevention or treatment of autism spectrum disorder-like behaviors in offspring caused by gestational diabetes mellitus. Background Technology

[0002] Autism Spectrum Disorder (ASD) is a group of neurodevelopmental disorders characterized by impaired social communication, repetitive and stereotyped behaviors, and restricted interests. Its pathogenesis is complex, and there is currently no cure. Recent studies have confirmed that gestational diabetes mellitus (GDM) is a significant risk factor for ASD in offspring. The hyperglycemic microenvironment in pregnant women with GDM can affect fetal nervous system development through multiple pathways, including oxidative stress, inflammatory responses, and insulin resistance, leading to ASD-like behavioral abnormalities in offspring after birth. Furthermore, intervention for these offspring is more challenging, placing a heavy burden on families and society.

[0003] Currently, interventions for ASD-like behaviors in offspring of children with GDM primarily rely on behavioral therapy and rehabilitation training. Drug treatment is limited to symptomatic relief (such as improving anxiety and controlling impulsive behavior), lacking specific drugs targeting the underlying causes. Existing drugs suffer from limited efficacy, significant side effects, and a narrow applicable population, failing to meet clinical needs.

[0004] β-Carotenoids are natural, fat-soluble carotenoids widely found in vegetables and fruits. As a precursor to vitamin A, they have been proven to possess clear antioxidant, anti-inflammatory, and immunomodulatory biological activities, with high safety and no obvious toxic side effects. They have been widely used in food, beverages, and health supplements. Current research shows that β-Carotenoids may have potential protective effects against various neurodevelopmental disorders (such as cerebral palsy and cognitive impairment) through mechanisms such as scavenging reactive oxygen species, inhibiting the release of inflammatory factors, and protecting the integrity of nerve cells. However, no studies have publicly disclosed or suggested that β-Carotenoids can be used to treat autism spectrum disorder-like behaviors in offspring of children with gestational diabetes mellitus, nor have there been any reports of their application in this specific indication. Summary of the Invention

[0005] The purpose of this invention is to address the above-mentioned problems by providing an application of β-carotene in the prevention or treatment of autism spectrum disorder-like behaviors in offspring caused by gestational diabetes mellitus.

[0006] To achieve its objective, the present invention employs the following technical solution:

[0007] This invention provides the application of β-carotene in the preparation of medicaments for the prevention or treatment of autism spectrum disorder-like behaviors in offspring caused by gestational diabetes mellitus.

[0008] The autism spectrum disorder-like behaviors include social communication impairments, repetitive and stereotyped behaviors, narrow interests, or anxiety-like behaviors.

[0009] The drug uses β-carotene as its active ingredient and also contains pharmaceutically acceptable carriers or excipients.

[0010] Preferably, the pharmaceutically acceptable carrier or excipient is selected from one or more of fillers, binders, disintegrants, lubricants, solubilizers, and stabilizers.

[0011] Preferably, the dosage form of the drug is an oral preparation or an injectable preparation.

[0012] Preferably, the dosage form of the drug is an oral preparation, selected from tablets, capsules, granules, suspensions or oral liquids.

[0013] Preferably, the drug is administered to mothers with gestational diabetes and / or their offspring.

[0014] Preferably, the offspring include infants, children, or adolescents.

[0015] Preferably, β-carotene inhibits the inflammatory intrauterine environment in mothers and reduces the level of inflammatory factors.

[0016] Preferably, β-carotene upregulates the expression levels of Ahi1, B9D1, and Shh proteins in the offspring brain, restores the number of 5-HT neurons in the dorsal raphe nucleus (DRN) of the midbrain, and improves ASD-like behavior.

[0017] The beneficial effects of this invention are:

[0018] 1. New application discovered for the first time: This invention is the first to demonstrate that β-Carotenoid can specifically improve autism spectrum disorder-like behavior in offspring of gestational diabetes mellitus, filling a gap in the field of therapeutic drugs and expanding the scope of medical applications of β-Carotenoid.

[0019] 2. Clear treatment mechanism: β-Carotenoid works through multiple mechanisms, including clearing excess reactive oxygen species (such as ROS) in offspring of gestational diabetes, inhibiting the expression of inflammatory factors (such as TNF-α and IL-6), protecting synaptic plasticity, and regulating gut-brain axis function, thereby improving ASD-like behavior at the etiological level and resulting in more significant treatment effects.

[0020] 3. High safety: β-Carotenoid is a naturally occurring nutrient that is well tolerated by the human body, has no obvious toxic side effects, and is highly safe for long-term use. It is especially suitable for special populations such as infants and children, solving the problem of large side effects of existing drugs.

[0021] 4. Diverse dosage forms and easy to promote: The drugs described in this invention can be prepared as oral preparations (tablets, capsules, oral liquids, etc.), which are convenient to administer, have high compliance, and have a wide range of raw material sources and low production costs, making them easy to scale up and promote in clinical practice.

[0022] Previous research by our team revealed that inflammatory conditions can induce downregulation of the Ahi1 gene in cell experiments. This invention further confirms that β-carotene can inhibit the maternal inflammatory intrauterine environment—gestational diabetes mellitus (GDM) is a chronic inflammatory state—and reduce the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in amniotic fluid. This upregulates the expression of the Ahi1 gene in the offspring's brain tissue, thereby regulating the function of the B9D1 / Shh signaling pathway, affecting the development of serotonin (5-HT) neurons, and ultimately restoring the physiological levels of 5-HT neurotransmitters in the offspring's central nervous system, achieving a therapeutic effect on the autism spectrum disorder (ASD) phenotype in offspring.

[0023] This invention provides a novel intervention strategy for offspring with ASD, namely, intrauterine treatment or prevention of fetal diseases in mothers at high risk of ASD (such as GDM). This strategy is superior to direct intervention in offspring, with the core advantage being: β-carotene metabolites are vitamin A. Direct intervention with vitamin A in offspring may lead to excessive vitamin A intake, causing increased intracranial pressure and headaches, potentially resulting in persistent headaches accompanied by dizziness, blurred vision, and other symptoms. β-carotene itself is safer, and incompletely converted β-carotene can be directly metabolized and cleared from the mother's body, avoiding the aforementioned safety risks. Furthermore, ASD is a neurodevelopmental disorder, and clinical practice has shown that the earlier the intervention, the more significant the treatment effect. The intervention strategy provided by this invention achieves prevention or treatment of fetal ASD through intrauterine means in the mother, with significantly better efficacy than intervention programs implemented after the onset of the disease in the offspring.

[0024] This invention develops a novel application of β-Carotenoid in the preparation of drugs for the prevention or treatment of autism spectrum disorder-like behaviors in offspring caused by gestational diabetes mellitus. This not only fills a gap in existing treatment methods but also provides a safe, effective, and easily scalable treatment option, possessing significant clinical value and social significance. Its application in the preparation of drugs for the preventive improvement of autism in offspring of children with gestational diabetes mellitus provides a new drug for the treatment of autism and can advance the development of autism treatment drugs. Attached Figure Description

[0025] Figure 1 The results of the three-box design experiment are as follows: (ab) The effect of β-Carotenoid on the social behavior of mice in each experimental group in the three-box experiment; *** P < 0.001, ** P < 0.01, ns P > 0.05, n ND-O = n ND+β-Carotenoid-O = n GDM-O =8 animals / group, n GDM+β-Carotenoid-O =11 animals / group.

[0026] Figure 2 These are the results of the open field experiment, showing the effect of β-Carotenoid on the number of times mice in each experimental group crossed the central region (a) and the time spent in the central region (b).

[0027] Figure 3 This is the result of the hair-grooming experiment.

[0028] Figure 4 This is the result of the spontaneous activity experiment of the Y maze.

[0029] Figure 5 The effects of β-Carotenoid on the abnormal intrauterine inflammatory environment in GDM mice were shown: (a) IL-1β levels in mice in each experimental group during mid-pregnancy (E10-12) were detected by ELISA; **** P < 0.0001, ns P > 0.05; (b) ELISA detection of TNF-α levels in mice during mid-pregnancy (E10-12) in each experimental group; **** P < 0.0001, ns P > 0.05; (c) ELISA detection of IL-6 levels in mice during mid-pregnancy (E10-12) in each experimental group; **** P < 0.0001, ns P > 0.05; n = 3 animals / group.

[0030] Figure 6 The results show that β-Carotenoid can upregulate the expression levels of Ahi1 and downstream B9D1 and Shh in the DRN brain region of GDM mouse progeny: (a) typical WB representation of Ahi1, B9D1 and Shh in the DRN brain region of mice in each experimental group; (b) WB statistical analysis results of Ahi1, B9D1 and Shh protein expression in each experimental group. * P < 0.05, *** P < 0.001, **** P < 0.0001, n = 4 animals / group.

[0031] Figure 7The following diagram shows the number of 5-HT neurons in the DRN brain region of β-Carotenoid-restored GDM mouse progeny: (A) Typical representative diagrams of the distribution of 5-HT neurons in the DRN brain region of mice in each experimental group; (B) Statistical analysis results of the average optical density values ​​of 5-HT neurons in each experimental group; ns, P>0.05 * P < 0.05, ** P < 0.01, n = 4 animals / group, scale bar 100 μm.

[0032] The diagram is marked as follows:

[0033] ND, normal control group; ND+β-Carotenoid, normal mice fed with β-Carotenoid; GDM group, gestational diabetes mellitus group; GDM+β-Carotenoid group, GDM mice fed with β-Carotenoid.

[0034] ND-O, male offspring of the normal control group; ND+β-Carotenoid-O, male offspring of normal mice fed with β-Carotenoid; GDM-O group, male offspring of gestational diabetes mellitus group; GDM+β-Carotenoid-O, male offspring of GDM mice fed with β-Carotenoid. Detailed Implementation

[0035] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0036] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0037] Example 1

[0038] I. Methods for establishing mouse models

[0039] 1. Experimental animals: 4-week-old SPF-grade female C57BL / 6 mice. The experimental animals were housed in an SPF-grade animal room at a temperature of 22±2℃ and a humidity of 50±5%, with a 12-hour light / dark cycle and free access to food and water.

[0040] 2. Establishment of the GDM model: After 4 weeks of high-fat diet feeding, mice were mated with normally fed male mice. At 0.5 and 0.75 days of gestation, streptozotocin (STZ, dissolved in 0.1 mol / L citrate buffer, pH=4.5) was injected intraperitoneally at a dose of 110 mg / kg body weight. Control group female mice received an equal volume of citrate buffer intraperitoneally. Fasting blood glucose (after 8 hours of fasting) was measured 3 and 7 days post-injection. A successful gestational diabetes mellitus (GDM) model was defined as a fasting blood glucose level ≥ 11.1 mmol / L for more than one week.

[0041] II. Experimental Grouping and Animal Handling

[0042] (1) Maternal experimental animals: Fifty 4-week-old SPF-grade female C57 BL / 6 mice were selected. The GDM model was constructed using STZ as described above. The experimental mice were divided into four groups:

[0043] ① Normal control group (ND): Eight 4-week-old SPF-grade female C57 BL / 6 mice. Experimental animals were housed in an SPF-grade animal room at 22±2℃ and 50±5% humidity, with a 12-hour light / dark cycle, and free access to food and water. They were fed a breeding diet (nutritional composition according to standard GB 14924.3-2010). After one week of acclimatization, formal experiments began. Mice in the ND group were mated at 9 weeks of age. On days 0.5 and 7.5 of gestation, they were intraperitoneally injected with the same volume of citrate buffer (0.1M, pH 4.4-4.5) as the STZ group. After birth, the litter size was adjusted to 6-8 mice, with male offspring used for subsequent experiments.

[0044] ② Normal control + 0.02‰ β-Carotenoid (ND+β-Carotenoid): Eight 4-week-old SPF-grade female C57BL / 6 mice. From the start of the formal experiment at 6 weeks of maternal age until 21 days after birth, 0.02‰ β-Carotenoid (i.e., 0.02g β-Carotenoid per 1000g of animal feed, Sigma-Aldrich) was added to the maternal feed. Mice had free access to food and water, and all other feeding conditions were exactly the same as the ND group. Mice in the ND+β-Carotenoid group began mating at 9 weeks of age. On days 0.5 and 7.5 of gestation, they were intraperitoneally injected with the same volume of citrate buffer (0.1M, pH 4.4-4.5) as the STZ group. After all mice in the experimental groups were born, the litter size was adjusted to 6-8 mice, and the male offspring were used for subsequent experiments.

[0045] ③GDM Group (GDM): Four-week-old SPF-grade female C57BL / 6 mice were acclimatized to a normal breeding diet for one week, then fed a high-fat diet (60% of calories from fat, Research Diets) until their offspring were 21 days old. Mice had free access to food and water, and all other feeding conditions were identical to the ND group. At 6:00 PM on the first day of 9 weeks of age, GDM mice were mated with male C57BL / 6J mice on a normal diet at a female-to-male ratio of 2:1. Vaginal plugs were checked between 8:00 and 9:00 AM the following day; the presence of a plug was marked as day E0. GDM mice were intraperitoneally injected with STZ (110 mg / kg) on ​​days 0.5 and 7.5 of gestation. 36-72 hours after the second STZ injection, eight mice with fasting blood glucose levels between 10-20 mmol / L were selected for the GDM model. After birth, the litter size was adjusted to 6-8 mice per litter, with male offspring used for subsequent experiments.

[0046] ④GDM+0.02‰ β-Carotenoid group (GDM+β-Carotenoid): 4-week-old SPF-grade female C57BL / 6 mice were acclimatized to a normal breeding diet for 1 week, then fed a high-fat diet supplemented with 0.02‰ β-Carotenoid (i.e., 0.02g β-Carotenoid per 1000g animal feed, Sigma-Aldrich) until 21 days after birth. Mice had free access to food and water, and all other feeding conditions were identical to the GDM group. At 6:00 PM on the first day of 9 weeks of age, they were mated with male C57BL / 6J mice on a normal diet at a female-to-male ratio of 2:1. Vaginal plugs were checked between 8:00 and 9:00 AM the following morning; the presence of a plug was marked as day E0. GDM group mice received intraperitoneal injections (STZ, 110 mg / kg) on ​​days 0.5 and 7.5 of gestation. 36-72 hours after the second STZ injection, eight mice with fasting blood glucose levels between 10-20 mmol / L were selected to enroll in the GDM model. After birth, the number of newborn mice in the experimental group was adjusted to 6-8 mice per litter, and the male offspring were used for subsequent experiments.

[0047] In the four experimental groups above, the offspring mice were weaned 21 days after birth. After weaning, the offspring mice were fed with ordinary breeding feed until they were euthanized, and had free access to food and water.

[0048] (2) Parental experimental animals: 16 male C57 BL / 6 mice (female:male = 2:1, for mating) were randomly divided into four groups of 4 mice each. The male mice were fed normal breeding feed and water until 8 weeks of age, and then switched to maintenance feed. Mating between males and females began at 9 weeks of age.

[0049] (3) Offspring experimental animals: After weaning at 21 days, 10 male mice were randomly selected from each of the four groups (8 litters) of offspring: normal control group (ND), normal control + 0.02‰ β-Carotenoid (ND+β-Carotenoid), GDM group (GDM), and GDM+0.02‰ β-Carotenoid group (GDM+β-Carotenoid). The mice were fed with ordinary breeding feed and had free access to food and water. All other feeding conditions were exactly the same as those mentioned above. Behavioral experiments were completed at 42-52 days of age. After 63 days of age, the mice were sacrificed and the DRN brain region of the midbrain was harvested for subsequent experiments.

[0050] III. The effect of β-Carotenoid on the amelioration of ASD-like behavior in male offspring mice of GDM

[0051] After the intervention, 8-11 male offspring from each experimental group, totaling 32 mice, were collected. At 42, 45, 48, and 52 days of age, behavioral tests including grooming, open field, Y-maze, and three-box socialization tests were conducted to assess the improvement in ASD-like behaviors.

[0052] 1) Three-chamber social test: This test is a quantitative and qualitative assessment of the social behavior and social ability of mice based on the principles of their social nature and the formation of social memory. The experimental procedure was implemented with slight modifications to the previously reported method. The specific plan is as follows: The experiment was conducted from 9:00 to 18:00 every day. The experimental mice were placed in the test room 2 hours in advance to familiarize themselves with the environment. The entire experiment was conducted in a low-light (40 lux), quiet (below 30 dB), and constant-temperature (24-26℃) environment. The experiment was divided into three stages: (1) Adaptation stage: Two restraint cages (15 cm × 8 cm) were placed in the left and right chambers of the three-chamber social test chamber (60 cm × 20 cm × 20 cm, consisting of three chambers: left, middle, and right). The test mice were placed in the central chamber of the behavior box with their backs to the experimenters. The small doors on the left and right partitions (5 cm × 5 cm, allowing the mice to move freely) were opened, and the mice were allowed to move freely for 10 minutes to fully familiarize themselves with the environment. (2) Social ability test stage: After 10 minutes, the test mice were put back into the breeding cages, the small doors on the left and right partitions were closed, and a strange mouse of the same species, same sex, and similar age (Stranger 1) was randomly placed into one of the restraint cages in the left and right chambers. The other restraint cage was left empty. The test mice were placed in the central chamber of the three-box test box, the left and right partitions were opened, and the mice were allowed to move freely for 10 minutes. The VisuTrack software automatically recorded the interaction time between the test mice and Stranger 1 and Empty respectively. (3) Social novelty test stage: After 10 minutes, the mice were put back into the breeding room, and the small doors on the left and right partitions were closed. Stranger 1 was removed and randomly placed back into any restraint cage. Another unfamiliar mouse of the same species, similar age, and same sex (Stranger 2) was placed in another restraint cage. The test mouse was then placed in the central chamber of the three-box experimental setup. The left and right side doors were opened, allowing the mouse to move freely for 10 minutes. The interaction time between the test mouse and Stranger 1 and Stranger 2 was recorded. After the experiment, the mouse was returned to its cage. Animal feces and foreign objects were cleaned from the three-box social experiment chamber, and the chamber was wiped with 75% alcohol to remove odor.

[0053] In the three-box social experiment, the interaction time of the tested mice with the empty restraint cage, unfamiliar mice 1, and unfamiliar mice 2 was recorded during the social ability test and the social novelty test, respectively. The results showed (see...) Figure 1In the social competence test, there was no statistically significant difference in the interaction time between the GDM group offspring mice and Stranger 1 and Empty. In the social novelty test, there was also no statistically significant difference in the interaction time between the GDM group offspring mice and Stranger 2 and Stranger 1, indicating that the GMD offspring mice exhibited social dysfunction. Meanwhile, in the social competence test, when the GDM+β-Carotenoid group offspring mice interacted with Stranger 1 and Empty, the tested mice showed a statistically significant preference for interacting with Stranger 1; in the social novelty test, the tested mice showed a statistically significant preference for interacting with Stranger 2, with a statistically significant difference in interaction time, suggesting that β-Carotenoid intervention significantly improved the social dysfunction in the GMD offspring mice.

[0054] 2) Open Field Test: This test is a widely used method for assessing anxiety behavior in mice and rats. In the open field test, mice are naturally afraid of open spaces and tend to avoid them. However, they also exhibit curiosity about the center of an open space and want to explore it. This psychological conflict leads to anxiety. Clinical data reports that anxiety behavior is a common accompanying symptom in patients with ASD. The specific protocol for the open field test is as follows: The test is conducted daily from 9:00 AM to 6:00 PM. Two hours before the test, the mice are placed in the test room to familiarize themselves with the environment. The entire test is conducted in a low-light (40 lux), quiet (below 30 dB), and constant-temperature (24-26℃) environment. The experiment uses an open field behavior box to detect spontaneous activity behavior in mice to assess their anxiety level. The box used in the experiment (50 cm × 50 cm × 50 cm, white background) is an open cube. During the experiment, the mice are placed in the center of the behavior box with their backs to the experimenter. VisuTrack software automatically records and analyzes the mice's free movement data within the behavior box for 5 minutes. After each experiment, the test chamber was treated with 75% alcohol and wiped with paper to remove odors and foreign matter.

[0055] In the open field experiment, the time mice spent in the central region of the open field and the distance they traveled were recorded; the results showed (see...). Figure 2 The GDM mouse offspring showed significantly shorter dwell time in the central region and reduced movement distance (P<0.02), and β-Carotenoid intervention significantly improved this abnormality (P<0.05), suggesting a reduction in anxiety-like behavior.

[0056] 3) Grooming Experiment: Stereotyped behaviors are one of the core symptoms of ASD. Grooming behavior is widely used in ASD mouse models as an indicator of repetitive and stereotyped behaviors. Common grooming actions include forelimb grooming, facial rubbing, and cleaning of the abdomen, tail, and genitals. The specific experimental procedure is implemented with slight modifications according to previously reported methods. The specific protocol for the grooming (stroking) experiment is as follows: The experiment is conducted daily from 9:00 to 18:00. Two hours before the experiment, the mice are placed in the test room to familiarize themselves with the environment. The entire experiment is conducted in a low-light (40 lux), quiet (below 30 dB), and constant-temperature (24-26℃) environment. The test mice are placed in a 20 cm × 20 cm × 20 cm cage, allowed to move freely, and a high-definition digital camera is used to record the cumulative grooming time within 10 minutes. The cumulative grooming time within 10 minutes is recorded. Figure 3 The results showed that GDM offspring mice exhibited significantly more repetitive stereotyped behaviors than the normal control group (P<0.01), and the GDM+β-Carotenoid group significantly reduced this behavior (P<0.01), suggesting a reduction in repetitive stereotyped behaviors.

[0057] 4) Y-maze spontaneous selection experiment: If mice identify the corresponding environment and objects and it is difficult to change their selection behavior, they are considered to have repetitive and stereotyped biased behaviors. The behavioral experiment protocol was slightly modified from previous literature reports. The specific experimental protocol is as follows: The experiment was conducted daily from 9:00 to 18:00. Two hours before the experiment, the mice were placed in the test room to familiarize themselves with the environment. The entire experiment was conducted in a low-light (40 lux), quiet (below 30 dB), and constant-temperature (24-26℃) environment. The experiment used a Y-maze device, which consists of three arms of equal length (35 cm × 5 cm × 15 cm) and a connecting area. The angle between the arms is 120°, and the connecting area where the three arms intersect has an openable door for easy entry and exit of the mice. During the experiment: (1) First, select one arm as the starting arm, place the test mouse with its back to the experimenter and facing the center of the Y maze, open the small door, and when the test mouse selects to enter an arm, close the door and allow the mouse to move freely in the selected arm for 2 minutes. Record the arm the mouse enters as the target arm and the other arm as the non-target arm. (2) Put the mouse back into the starting arm, open the door, and record the number of times the test mouse enters the target arm or the non-target arm. (3) Repeat the operation in (2) 10 times, record and count the cumulative number of times the test mouse enters the target arm and the non-target arm. The percentage of entering the target arm is the number of times the target arm is entered / 10 × 100%. After each mouse experiment, clean the animal feces and foreign objects in the Y maze spontaneous selection experimental box, and wipe the box with 75% alcohol to remove the odor.

[0058] In the Y-maze spontaneous selection experiment, the number of times the mouse entered the target arm 10 times was recorded; such as Figure 4 As shown, the percentage of offspring mice in the GDM model group entering the target arm was significantly higher than that in the control group, and the GDM+β-Carotenoid intervention group significantly reduced this behavior (P<0.01), suggesting that repetitive stereotyped behavior was improved.

[0059] IV. Inhibitory effect of β-Carotenoid on inflammatory factors in amniotic fluid of pregnant mice (maternal offspring)

[0060] ELISA was used to detect the levels of inflammatory factors TNF-α, IL-1β, and IL-6 in the amniotic fluid of pregnant mice (maternal offspring). Pregnant mice were anesthetized by inhalation of isoflurane (induction concentration 5%, maintenance concentration 2%, 3 mice / group in total, 4 groups). 50 μL of amniotic fluid was collected from the mid-pregnancy (E12) groups of the ND group, GDM group, ND+β-Carotenoid group, and GDM+β-Carotenoid group (abdominal puncture method; pregnant mice were retained for subsequent experiments: in a sterile environment in a laminar flow hood, under the guidance of a small animal ultrasound instrument, the amniotic sac was punctured transabdominally with a 33G needle, collecting 10 μL / fetus. After collection, the mice were placed back in the feeding room after waking up on the warming rack). The amniotic fluid was centrifuged at 3000 rpm at 4℃ for 10 min, and the supernatant was carefully aspirated and stored at -20℃ for later use. Specific detection methods were performed according to the kit instructions. The ELISA results (…) Figure 5 We can see that, compared with GDM, the levels of TNF-α, IL-1β and IL-6 inflammatory factors in the amniotic fluid of the GDM+β-Carotenoid group in the second trimester (E10-12 days) were significantly reduced, indicating that the level of inflammation in the maternal amniotic fluid of the GDM+β-Carotenoid group was significantly suppressed and the intrauterine inflammatory environment was improved.

[0061] V. β-Carotenoid upregulates proteins related to the development of 5-HT neurons in the midbrain of GDM offspring.

[0062] Sixty-three days after the completion of the behavioral experiments in each experimental group, mice were anesthetized by intraperitoneal injection of 2.5% sodium pentobarbital (0.1 mL / 10 g). The dorsal raphe nucleus (DRN) region of the midbrain was harvested according to the stereotaxic atlas of the mouse brain (Keith BJ & George Paxinos). The blood membrane was removed to obtain the target brain tissue. Blood stains were washed off with ice-cold PBS, and water stains were blotted dry with absorbent paper. The brain tissue was accurately weighed, placed in EP tubes, and the weight was recorded. The tissue was then stored in liquid nitrogen for later use. Based on brain tissue weight, PIPA lysis buffer (RIPA lysis buffer: PMSF: phosphatase inhibitor = 100:1:1) was added at a ratio of tissue weight / 20 mg × 150 μL. Tissue proteins were extracted by grinding with magnetic beads, and protein concentration was determined using the BCA method. Western blot (WB) assays were performed to detect relevant protein content (primary antibody concentration: Anti-Ahi1 polyclonal antibody 1:1000; Anti-B9D1 polyclonal antibody 1:1000; Anti-Shh polyclonal antibody 1:1000; Anti-β-Tublin polyclonal antibody 1:7000, incubated overnight at 4℃; secondary antibody: goat anti-rabbit IgG 1:10000; goat anti-mouse IgG 1:9000, incubated at 37℃ for 1 hour). ECL chemiluminescence imaging and photographing were performed, and grayscale analysis was conducted using image analysis tools. WB results showed ( Figure 6 In GDM progeny mice, the expression levels of key regulatory proteins Ahi1, B9D1, and Shh proteins affecting the differentiation and development of 5-HT neurons in the DRN brain region were lower than those in the normal group. However, β-Carotenoid effectively reversed the expression levels of Ahi1, B9D1, and Shh proteins in the DRN brain region of GDM+β-Carotenoid-O mice, and the differences were statistically significant, suggesting that the function of proteins related to the development of 5-HT neurons was improved.

[0063] VI. Immunofluorescence results of β-Carotenoid upregulation of the number of 5-HT neurons in the midbrain DRN region of GDM progeny

[0064] Sixty-three days after the completion of behavioral experiments in each experimental group, mice were anesthetized by intraperitoneal injection of 2.5% sodium pentobarbital (0.1 mL / 10 g). The heart was perfused with room-temperature PBS until the liquid clarified, then perfused with 4% paraformaldehyde solution at 4°C. The entire brain tissue was completely removed and fixed by immersion in 4% neutral paraformaldehyde at 4°C for 12 h. The tissue blocks were fixed to the sample holder of a vibratory microtome with 502 glue and vibrated sections were prepared in the coronal or sagittal planes, with a section thickness of 45 μm. Midbrain DRN slices were collected. Brain tissue sections were placed sequentially in 24-well plates for subsequent immunofluorescence staining experiments (primary antibody Anti-5-HT polyclonal antibody 1:5000, overnight at 4°C; secondary antibody Cy5 fluorescence 1:400, incubated at 37°C in the dark for 1 hour; nucleus stained with DAPI stock solution at 37°C for 20 min). After washing with PBS, the slides were mounted, and images were acquired under a confocal microscope. The optical density values ​​were analyzed using image software. Immunofluorescence results ( Figure 7 The results showed that the number of 5-HT positive neurons in the DRN brain region of the offspring of GDM model mice was significantly reduced compared with the control group, while the β-Carotenoid intervention group could effectively reverse the number of 5-HT neurons and restore the number of 5-HT neurons in the DRN brain region of the offspring of GDM mice, thereby improving ASD-like behavior.

Claims

1. Application of β-carotene in the preparation of drugs for the prevention or treatment of autism spectrum disorder-like behaviors in offspring caused by gestational diabetes mellitus.

2. The application according to claim 1, characterized in that, The autism spectrum disorder-like behaviors include social communication impairments, repetitive and stereotyped behaviors, narrow interests, or anxiety-like behaviors.

3. The application according to claim 1, characterized in that, The drug uses β-carotene as its active ingredient and also contains pharmaceutically acceptable carriers or excipients.

4. The application according to claim 3, characterized in that, The pharmaceutically acceptable carrier or excipient is selected from one or more of fillers, binders, disintegrants, lubricants, solubilizers, and stabilizers.

5. The application according to claim 3, characterized in that, The drug is available in oral or injectable form.

6. The application according to claim 5, characterized in that, The dosage form of the drug is an oral preparation, selected from tablets, capsules, granules, suspensions, or oral liquids.

7. The application according to claim 1, characterized in that, The drug is intended for mothers with gestational diabetes and / or their offspring.

8. The application according to claim 7, characterized in that, The offspring mentioned include infants, children, or adolescents.

9. The application according to claim 1, characterized in that, β-carotene inhibits the inflammatory intrauterine environment in mothers and reduces the level of inflammatory factors.

10. The application according to claim 1, characterized in that, β-carotene upregulates the expression levels of Ahi1, B9D1, and Shh proteins in offspring brain regions, restores the number of 5-HT neurons in the dorsal raphe nucleus (DRN) of the midbrain, and improves ASD-like behavior.

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