Traditional Chinese medicine formula Xiaoyaosan alleviates autistic behaviors and anxiety behaviors in valproic acid-induced autistic rats

DOI: https://doi.org/10.21203/rs.3.rs-2225690/v1

Abstract

Background

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that is correlated with anxiety behavior. This study aimed to investigate the effect of Xiaoyaosan (XYS) on anxiety behavior in autism rats and its potential mechanism.

Materials and Methods

The autistic rat model was established by intraperitoneal injection of pregnant rats with sodium valproic acid (VPA) at 12.5 days of pregnancy. The newborn rats (n = 15 in each group) were given XYS solution gavage daily for 21 days. The autistic behaviors were identified by the marble-burying behavior test (MBT), ultrasonic vocalization (USV) test, three-chamber social interaction task (TCT), and novel object recognition (NOR) task. The anxiety behaviors were detected by open field test (OFT), elevated plus maze (EPM), and sucrose preference test (SPT). Heart rate variability (HRV) was used to detect the changes in the autonomic nerve. The expression of Ionized calcium-binding adaptor molecule 1 (Iba-1) in microglia of the hippocampus and amygdala was detected by immunohistochemistry.

Results

Compared with the control group, the number of buried marbles in MBT was increased, the number of vocalizations at 50 kHz in the USV test was reduced, the social ability in the TST was reduced, and the exploration time and distance of new objects in NOR task were reduced in the VPA group. In the OFT, the activity time in the central zone was reduced, the open arm activity time in EPM was reduced, and the sucrose consumption rate in SPT was reduced in autistic rats. The autonomic sympathetic balance of autistic rats was impaired. In hippocampus and amygdala regions, the number of Iba-1 positive cells was increased in VPA-induced rats. After XYS treatment, the above effects caused by VPA were reversed.

Conclusion

XYS could improve autism and anxiety behaviors. It could also maintain the balance of sympathetic and parasympathetic nerves in autistic rats. Its mechanism may be related to the inhibition of the activation of microglia in the hippocampus and amygdala.

Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disorder that begins in early childhood with core symptoms including impaired social interaction, a narrow range of interests, and repetitive stereotypic behaviors[1]. Recent reports indicated that the prevalence of ASD is 1 in 44[2]. Anxiety is one of the most common symptoms in children with ASD and severely affects the development of cognitive and social skills in children with ASD[3]. Studies have shown that the severity of anxiety is positively correlated with the severity of core symptoms in children with ASD, causing them to exhibit a variety of abnormal behaviors[4, 5]. Upwards 40% of children with autism are diagnosed with anxiety by the Diagnostic and Statistical Manual of Mental Disorders at some point in their lives, compared to 3%-24% of normal children[6].

Anxiety is a negative emotional state that humans tend to experience when faced with uncertainty, manifesting as subjective feelings of tension, apprehension, and worry, while activating the autonomic nervous system in response to potential threats[7]. Children with ASD have an impaired ability to integrate multiple sources of information and are more prone to anxiety[7]. Children with ASD have interaction and communication difficulties, are not understood and accepted by peers, and therefore have more emotional problems. children with ASD have conflict control deficits, abnormal emotion regulation, and are prone to anxiety[8]. Children with ASD and anxiety have higher baseline cortisol levels and levels of change in response to stress and exhibit more sluggish heart rate and cortisol responses[9]. In addition, hyperactivity of the hypothalamic-pituitary-adrenal axis causes frequent mobilization of organismal resources in children with ASD, triggering anxiety responses and proliferation of microglia in brain tissues such as the hippocampus and amygdala[10], which secrete a variety of inflammatory factors leading to central nervous system inflammation and impaired cellular synaptic transmission[11], further damaging the nervous system and inducing structural and functional neuronal pathological alterations, causing anxiety[12, 13].

The treatment of ASD should be based on rehabilitation education and supplemented by pharmacological treatment. Commonly used medications cannot treat the core symptoms and various complications of ASD and are prone to adverse effects with long-term use, so it is important to find medications that are effective and have few side effects. Chinese medicine has the advantages of low side effects, stable efficacy, easy acceptance by patients, and a high clinical value. In Chinese medicine, anxiety belongs to "depression" and "sleeplessness"[14], and the cause of the disease is mostly internal injury to the emotions[15], and the lesions are mostly in the heart, liver, and spleen. Xiaoyaosan (XYS) originated from the Song Dynasty official herbal remedy "Taiping Huimin Hejiao Bureau Formula", which has a history of 1,000 years and is one of the "Ten Famous Formulae of Traditional Chinese Medicine", and is the first choice for anti-anxiety in Chinese medicine[16, 17]. Clinical applications have shown that XYS relaxes the emotions and makes the mood happy, and has anxiolytic effects of detoxifying the liver and relieving depression, strengthening the spleen[17]. Modern pharmacological studies have concluded that XYS can regulate the immune function of the body, protect neurons, activate brain-derived neurotrophic factors, inhibit changes in neuroinflammatory factors, resist apoptosis of hippocampal neurons, and regulate synaptic plasticity[18–20].

In this study, the rat model of ASD was constructed using litters produced after intraperitoneal injection of valproic acid (VPA) in pregnant rats. The efficacy of XYS on the anxiety behavior of autistic rats was examined by animal behavior, the relationship between the autonomic nervous system and anxiety in autistic rats, and the role of microglia in key regions such as the hippocampal amygdala were initially investigated.

Materials And Methods

Animals

A total of 21 specific pathogen-free (SPF)-grade adult Sprague-Dawley (SD) rats were purchased from Huaxing Experimental Animal Center (Zhengzhou, Henan, China). The mean weight of rats was 200 ± 10 g. Among them, there were 14 female rats and 7 male rats. Rats were housed in 12-h day and 12-h night cages with adequate access to water.

Drugs and reagents

The components of XYS include 30 g of North Bupleurum Chinense, 30 g Scutellaria Baicalensis, 30 g of Angelica Sinensis, 30 g of fried Atractylodes Macrocephala, 30 g of Poria Umbellus, 30 g of root of Herbaceous Peony, 10 g of Mentha Haplocalyx, 15 g of roasted Radix Glycyrrhizae, and 10 g of burnt Zingiber Officinale Rosc. The herbal tablets were purchased from Zhengzhou Pharmacy of Beijing Tong Ren Tang (Zhengzhou, Henan, China) and identified by the Department of Traditional Chinese Medicine of The Fifth Affiliated Hospital of Zhengzhou University. The herbs were soaked in 2 times the volume of water for 30 min. Then, the herbs were decocted over high heat until boiling and switched to low heat for 40 min. Drug residues are filtered out. The second decoction was made with 1.5 times the volume of water of the herbs, and the method was the same as that of the first decoction. The two decoction solutions were combined, filtered, and stored in a refrigerator at 4℃.

Valproic acid-induced autistic rat model

To make valproic acid (VPA) solution, 3.0 g of VPA solid particles (Shanghai Chemical Reagent Co., Shanghai, China) were dissolved in 10 ml of saline and configured into a 0.3 g/ml solution. After 1 week of acclimatization, the rats were caged together (males vs females as 2:1), and the females were observed for the presence of vaginal pessary. When a clear, gelatinous vaginal pessary appeared, the female was successfully conceived and was recorded as 0.5 days pregnant. Nine of 14 females were pregnant and the pregnant rats were randomly divided into 2 groups. One group (n = 6) was given an intraperitoneal injection of VPA (600 mg/kg) at 12.5 days of pregnancy, and its children were randomly divided into the VPA group (n = 15) and the VPA + XYS group (n = 15). Another group (n = 3) was given the same dose of saline intraperitoneally at 12.5 days of pregnancy, and the children were in the control group (n = 15).

XYS treatment

Rats in the VPA group and the control group were given distilled water gavage (10 ml/kg) daily. Rats in the VPA + XYS group were given XYS solution gavage (10 ml/kg) daily. Newborn rats were treated from 2-month-old. The gavage last 21 days. Then, the rats underwent behavioral tests.

Marble-burying behavior test

Stereotypic behavior in autism was evaluated using the marble-burying behavior test (MBT)[21]. The rats were placed in a single cage with a 5 cm thick bedding at the bottom of the cage. At the beginning of the experiment, the rats were placed in the cage without glass marbles for 5 min, and then 20 glass marbles of 1.4 cm in diameter were placed on the bedding. Glass marbles were placed 5 per row, 4 rows in total, to ensure that each marble was fully exposed. The camera was filmed for 5 min, and the rats were considered positive if more than 70% of the glass marbles were buried, and the number of buried marbles was counted at the end of the test.

Ultrasonic vocalization test

Communication disorder behavior in autism was evaluated using the ultrasonic vocalization (USV) test[22]. The rats were placed in a soundproof box (Med Associates, Fairfax, VT, USA) and acclimatized for 10 min. Then, 100 g of bedding containing heterosexual rat urine was placed in the bottom of the soundproof box. After 10 min, the rats were recorded and videotaped with USV software for 10 min. The rats were removed and the soundproof box was ventilated for 10 min before the next test.

Three-chamber social interaction task

Social impairment in autism was evaluated using the three-chamber social interaction task (TCT)[23]. The room of TCT consists of a central chamber and two side chambers (XinRun Corporation, Shanghai, China). In stage 1, the rats were placed in the central chamber with the doors on both sides closed and allowed to acclimatize for 5 min. In stage 2, the empty cage (Empty) and the unfamiliar rat (Stranger 1) were placed in two side chambers, respectively. The doors on both sides were opened, and the rat was allowed to explore the three rooms for 10 min. In stage 3, another unfamiliar rat (Stranger 2) was placed in the empty cage, the doors on both sides were opened, and the rat was allowed to explore the three rooms for 10 min, after which the three rooms were cleaned with 75% alcohol before the next test.

Novel object recognition task

Learning memory impairment in autism was evaluated using the novel object recognition (NOR) task[24]. A novel object zone and an old object zone were set up. First, two identical objects were placed, and the time and distance of the rat exploring these two objects were recorded. Then, one of the objects was replaced with a different object, and the time and distance of the rat exploring the two objects were recorded.

Evaluation of anxiety behavior

Anxiety behaviors were evaluated using the open field test (OFT)[25], the elevated plus maze (EPM)[26], and the sucrose preference test (SPT)[27]. For the OFT, the inside surface and bottom surface of the open wooden box (100 × 100 × 40 cm) were black, and a camera was placed directly above them. One hour before the experiment, the rats were moved into the test room to adapt to the environment. After the experiment started, the rats were placed in the central zone, and the heads of the rats were facing the same side for each experiment. The rats were filmed for 10 min and the total distance of movement, the time spent in the central zone, and the number of times they entered the central zone were measured, and the wooden box was cleaned with 75% alcohol after the test.

EPM was constructed of polyvinyl chloride in light grey. It consists of two open arms (45 × 15 cm) and two closed arms (45 × 15 × 30 cm), which was connected by the central zone (15 × 15 cm) in the middle. The equipment was 50 cm high from the ground, and the camera was placed directly above the central zone. Before the video recording, the light was isolated by a blackout curtain, and the surrounding environment was kept quiet. During the test, the rat was placed in the central zone, with the open arm on one side of the head, and the video was recorded for 5 min, and the number and time of the rat entering the open arm and the closed arm were recorded.

In the 1st phase of the SPT, two bottles of 1% sucrose solution were placed in each cage. After 24 h, one of the bottles was replaced with water. In the 2nd phase of the SPT, the rats fasted with food and water for 24 h. In the 3rd phase of the SPT, the rats were simultaneously fed two bottles of pre-weighed liquid: one containing 1% sucrose solution and the other one with water. Throughout the experiment, the bottles were placed at the same height on both sides of the cage. After 1 h, the two bottles of liquid were weighed and the sucrose consumption ratio was calculated. The calculation formula was: sucrose consumption rate (%) = sucrose consumption/total consumption × 100%.

Electrocardiogram examination

After the behavioral experiment, 10 rats in each group were randomly selected for a two-lead electrocardiogram (ECG) examination. The testing and recording were performed using the PowerLab data acquisition system and the LabChart 8.2.3 ECG recording software. Firstly, the electrodes and electrode plates were checked to make sure that they were firmly connected, and the interference of AC power in the environment was excluded. Young adult rats are anesthetized with an intra-peritoneal induction mixture of tiletamine-xylazine (2 mg and 1.4 g/100 g body weight, respectively).When there was no response to the thighs with hemostatic forceps and the breathing was stable, the rats were sufficiently anesthetized. The anesthesia level is constantly monitored by testing the absence of eye and hind-limb reflexes and whiskers’ spontaneous movements.The 0.1 cm acupuncture needles were inserted into the medial skin of the right forelimb, left forelimb, and left hindlimb of the rats, respectively, and the electrodes of negative, ground, and positive were clamped at the needle shanks. The ECG signals of the rats were recorded for 2 h.

Immunohistochemical staining

Six rats were randomly selected from each group, and the thoracic cavity was opened after anesthesia. After the heart was exposed, the perfusion pump needle was inserted into the ascending aorta along the left heart tip and the right heart ear was cut. The heart was rapidly injected with a frozen phosphate buffer solution at 4℃ until the fluid coming out of the right heart ear was light red and the liver was pale, the abdominal aorta was clamped with hemostatic forceps and fixed by instilling 4% paraformaldehyde solution at 4℃. After 20–30 min, when the rats were in violent convulsions and the limbs were stiff, the brain was immediately removed and quickly put into cold 4% paraformaldehyde overnight. Rat brain tissues were placed in 30% sucrose solution for dehydration, and after the tissues sank, they were sectioned at a thickness of 30 µm (Leica CM1860, Stuttgart, Germany) and stored in a -80°C refrigerator.

Frozen sections were baked at 37℃ for 10–20 min and then fixed with 4% paraformaldehyde for 20 min, incubated with antigen repair solution for 15 min, endogenous peroxidase for 25 min, goat serum for 30 min at room temperature. The primary antibody (anti-Iba-1, ab178847, Abcam, Cambridge, UK) was added and incubated at 4℃ for 24 h, then a biotinylated secondary antibody was added and incubated for 50 min at room temperature. The sections were dried, dehydrated, transparent, and sealed. The sections with similar coronal surface and hippocampal amygdala were scanned using the AperioCS2 digital pathology section scanning system, and images were acquired at 40× magnification using K-viewer software. Six sections were taken from each group, and the number of positive cells was analyzed and calculated using Image J software.

Statistical analysis

Behavioral data were imported from the Anymaze video tracking recording analysis system (Global Biotech Inc, Scarborough, ME, USA), and ECG recording results were imported from the PowerLab data acquisition system. The number of positive cells in each group was analyzed using ImageJ software, and SPSS26.0 software (IBM, Armonk, NY, USA) was used for statistical analysis. Independent samples t-test was used for comparison between two groups, and the one-way analysis of variance (ANOVA) test followed by the Tukey's post hoc test was used for comparison between multiple groups. P < 0.05 represents a statistical difference, and GraphPad Prism 9.0.0 was used for graphing.

Results

XYS could alleviate stereotypic behavior, communication disorder behavior, learning memory impairment, and social impairment in autistic rats

The MBT results showed that the number of marbles buried by rats in the VPA group was significantly more than that in the Control group (P = 0.0022), while the number of marbles buried by rats in the VPA + XYS group was reduced compared with that in the VPA group (P = 0.464, Fig. 1A & 1B). The result of the USV test indicated that at 50 kHz, the number of vocalizations was decreased in the VPA group in comparison with the Control group (P = 0.0061). After the XYS treatment, the number of vocalizations was elevated (P = 0.0134), suggesting that the communication disorder was improved (Fig. 1C & 1D). The TCT results showed that in Stage 2, rats in the VPA group spent less time sniffing the Stranger 1 and more time sniffing the empty cage than rats in the Control group (P < 0.05, Fig. 2A & 2B), while the XYS treatment reversed the effects (P < 0.05, Fig. 2C). In Stage 3, rats in the VPA group spent more time sniffing the Stranger 1 and less time sniffing the Stranger 2 than rats in the Control group (P < 0.05, Fig. 2D & 2E), while the XYS treatment reversed the effects (P < 0.05, Fig. 2F). These data indicated that social impairment in autism could be attenuated by XYS. Besides, the results of the NOR task (Fig. 3A & 3B) showed that control rats spent more time and distance exploring new objects than VPA rats (both P < 0.05), the XYS treatment also restored the time and distance exploring new objects than VPA rats (both P < 0.05, Fig. 3C & 3D).

Xys Could Alleviate Anxiety Behaviors In Autistic Rats

Anxiety behaviors were evaluated using OFT, EPM, and SPT. As shown in Fig. 4A, rats in the VPA group had less active time in the central zone than the Control group (P = 0.0112), and rats in the VPA + XYS group had more active time than the VPA group (P = 0.003). EPM results showed that compared to the Control group, rats in the VPA group had reduced access to the open arms (P = 0.0062), while rats in the VPA + XYS group had increased access to the open arms (P = 0.0356, Fig. 4B & 4C). The sucrose consumption rate of rats in the VPA group was lower than that of rats in the Control group (P < 0.0001), while the sucrose consumption rate of rats in the VPA + XYS group was higher than that of rats in the VPA group (P < 0.0001, Fig. 4D).

Xys Improved The Autonomic Sympathetic Balance Of Autistic Rats

Compared with the Control group, the root mean square of successive difference (RMSSD) of RR interval was decreased (P = 0.0002, Fig. 5A) and high frequency (HF) power was decreased (P = 0.0031, Fig. 5B) in the VPA group, indicating a decrease in parasympathetic activity in rats. Low frequency (LF) power was increased (P = 0.0057, Fig. 5C) and LF/HF power was increased (P = 0.0063, Fig. 5D) in the VPA group, suggesting that sympathetic nerves were over-aroused in rats. Compared with the VPA group, the rats in the XYS + VPA group showed elevated RMSSD (P = 0.04, Fig. 5A), elevated HF power (P = 0.0304, Fig. 5B), decreased LF power (P = 0.0376, Fig. 5C), and decreased LF/HF power (P = 0.0241, Fig. 5D), indicating that XYS improved the autonomic sympathetic balance of autistic rats.

After the immunohistochemistry of anti-Iba-1 (Fig. 5E & 5F), compared with the Control group, the number of Iba-1 positive cells in the hippocampal CA1 (P = 0.0001), CA3 (P = 0.0038) and DG (P = 0.0014) regions, and the amygdala CeA (P = 0.0002) and BLA (P = 0.0006) regions were increased in the VPA group rats (Fig. 5G), and the microglia in the VPA group rats showed amoeboid activation, and the number of Iba-1 positive cells in the hippocampal CA1 (P = 0.0219), CA3 (P = 0.0451) and DG (P = 0.0373) regions, and the amygdala CeA (P = 0.0168) and BLA (P = 0.0126) regions was reduced in the VPA + XYS group rats compared with the VPA group rats (Fig. 5G).

Discussion

Autism is a serious neurodevelopmental disorder that has gained widespread social attention and often leads to lifelong mental disability in children. The prevalence of autism continues to climb, increasing 227-fold in the last 20 years. Anxiety disorders are common concomitant disorders of autism, often affecting the improvement of core symptoms in children with autism and increasing the difficulty of treatment. VPA-induced rat models of autism are widely used in basic and preclinical studies. A single intraperitoneal injection of 600 mg/kg of VPA to pregnant rats at day 12.5 of the rodent embryonic period, a critical period for neural tube development, induces neurogenic inflammation in neonatal rats, which exhibit ASD-like behavior and affects skeletal development, resulting in tail curvature and limb deformities in neonatal rats[28].

Studies have shown that there is a correlation between the severity of behaviors such as communication disorders and central neuroinflammation in patients with ASD[29]. There is an important link between neuroinflammation and the development of autistic anxiety[30]. Therefore, suppression of neuroinflammation has become one of the directions of neuroprotection. The basic pathogenesis of anxiety disorders is the disorder of emotion and the loss of drainage of the liver in traditional Chinese medicine. Modern pharmacological studies have shown that the main components of XYS contain chai hu saponin, angelica polysaccharide, glycyrrhetinic acid, etc., which have anti-inflammatory, immune function regulation, and antioxidant effects[18, 20]. Numerous studies have confirmed the effectiveness of XYS in the treatment of autism and anxiety disorders; therefore, the present study explores XYS from the perspective of its anti-neuroinflammatory mechanism based on the above.

The act of burying marbles has been used to study repetitive stereotypic behavior in animals[21], and our findings showed increased stereotypic behavior in rats with autism. Rats rely on the larynx to emit ultrasound for communication over short distances in underground burrows; therefore, the USV test can be used to assess the social competence of rats[22], and our findings show vocalization deficits in autistic rats with a reduced number of vocalizations at 50 kHz, indicating reduced social communication in autistic rats. TST is widely used to assess the social behavior of animals in studies[23], and the results of our experiments showed that autistic rats exhibited significant social impairment. The cognitive functions of animals are generally evaluated by learning and memory abilities, which became significantly worse in autistic rats in the NOR task[24]. All of the above findings indicate that the autistic rats were successfully modeled in this study.

The OFT used the animals' nature to explore novel environments and avoid open environments to evaluate the animals' alertness and anxiety status[25], and the reduced time spent in the central area by rats in the VPA group indicated a high level of anxiety. The EPM uses the animal's exploratory nature and fear of heights to evaluate the anxiety level of the animal[26]. The results of our study showed that rats in the VPA group spent significantly less time in the open arms and entered the closed arms more often. In addition, the sucrose consumption rate was a valid indicator of the response to the animals' lack of pleasure due to anxiety[27]. The results of our study showed that the sucrose consumption rate of rats in the VPA group was significantly lower, indicating a reduced interest and higher anxiety level. These indicators reflect the increased anxiety behavior of rats in the VPA group. In contrast, the use of XYS played a significant role in alleviating the autistic and anxious behaviors of rats.

Symptoms of ASD, such as stereotypic behaviors and difficulties with mood and emotion recognition, are closely related to autonomic nervous system (ANS) dysfunction[31]. The ANS is a pathway system connecting the heart to the central nervous system, mediated by a variety of chemicals that maintain the body's ability to adapt acutely to stress, through parasympathetic and sympathetic stability to maintain homeostasis[32]. ANS indicators can assist in assessing the severity of ASD, and patients with ASD tend to have elevated heart rate, decreased parasympathetic activity, increased sympathetic activity, and decreased cardiac vagal activity[33]. The vagus nerve is the longest in the body and represents the physiological basis of the parasympathetic nervous system. Heart rate is mainly controlled by sympathetic and parasympathetic nerves. Heart rate variability (HRV) is a commonly used measure of peripheral ANS activity[34], and HRV testing in the ASD patients has shown that patients exhibit reduced autonomic flexibility to environmental changes and reduced control of the heart due to a lack of stability of the biological system[35]. In the present study, our findings showed that rats in the VPA group had reduced HRV and autonomic dysfunction, while XYS improved autonomic function in rats with autism.

Microglia are the resident immune cells of the central nervous system, and the morphology is divided into branched and amoeboid forms. In the resting state, microglia appear as branching cells with small cytosomes and long protrusions, and in response to neuroinflammatory stimuli, microglia are activated and appear as amoeboid cells with larger cytosomes and shorter protrusions[36]. Activated microglia can promote the release of cytotoxic factors, leading to neuronal damage. Studies have shown increased neuroinflammation, microglial activation, and increased density in hippocampal and amygdala tissues of rats with autism[37]. Ionized calcium-binding adaptor molecule 1 (Iba-1) is a protein marker specifically expressed by microglia[38]. In the current study, Iba-1 positive cells were increased and showed activation in the brain tissues of rats in the VPA group. In the VPA + XYS group, the brain Iba-1 levels of rats tended to normalize, suggesting that after 21 days of XYS treatment, the autistic and anxious behaviors of the rats were improved, suggesting that XYS inhibits the excessive activation of microglia, alleviates inflammation in brain tissues, and reduces the accumulation of neurotoxic products.

The hippocampus is located in the medial temporal lobe of the brain and is responsible for functions such as memory, learning, and emotion management. The four hippocampal subregions are CA1, CA2, CA3, and CA4. The CA3 region and CA2 region are adjacent to the dentate gyrus on both sides. The CA3 region is the largest in the hippocampus, which receives nerve fibers from the dentate granule cells on its proximal dendrites[38]. Previous studies have shown that impaired axonal activation of pyramidal cells in the CA1 region of the rodent hippocampus and abnormalities in presynaptic neurotransmitter release in the CA3 region may contribute to anxiety behavior[3]. In addition, there is a complex relationship between the amygdala and core symptoms of ASD and anxiety. The basolateral amygdala (BLA) region and the central amygdala (CeA) region are the most critical regions for generating anxiety and fear. Increased excitability in the BLA region leads to increased anxiety and further exacerbates social deficits in children with autism[39]. The dendritic density of neurons in the CeA region is reduced in anxious rats[40]. This study investigated the positive number and morphology of microglia in hippocampal and amygdala brain regions as well as the effect of XYS on microglia number and activation. Our findings indicated that XYS could alleviate autistic behaviors and anxiety behaviors in valproic acid-induced autistic rats, which could be mediated by the inhibition of microglial activation.

In conclusion, XYS can significantly improve anxiety behavior, improve autonomic function, inhibit microglial activation in the hippocampus and amygdala in rats with autism, and thus reduce central nervous system inflammation, which has potential for clinical application. However, XYS has a complex composition, and its molecular mechanism for neuroinflammation deserves further exploration.

Declarations

Ethical statement

This study was approved by The Fifth Affiliated Hospital of Zhengzhou University. All animal experiments complied with the ARRIVE guidelines（NO.KY2022004）and were carried out following the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Funding

This study was supported by the key project jointly built by the Henan Provincial Health Commission and the Ministry of Health (Grant No. SBGJ202002123) ,the Henan Provincial Department of Education Project (Grant No. 21A320058),General project of Henan Natural Science Foundation(Grant No. 212300410399) and Zhengzhou collaborative innovation major project(Grant No. 18XTZX12003).

Availability of data and material

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Acknowledgments

None.

Author contributions

Enyao Li designed the study and obtained grant funding. . Yanyan Li and Junjie Yang searched and analyzed the literature, and performed the data analysis. Xianwen Dong and Yanyan Li wrote the manuscript. Honglei Li and Chengming Xu assisted in the implementation of the study.All authors read and approved the final manuscript.

Competing interests 

All authors declare that they have no competing interests.

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