ObjectiveTo observe the effect of Liuwei Dihuangwan on behavioral impairments in the rat model of autism spectrum disorder （ASD） and explore the mechanism of action. MethodsTwelve SD pregnant rats were intraperitoneally injected with valproic acid （VPA） （10 rats） or normal saline （2 rats）， and male offspring were selected to establish the model of ASD and the control rats. Rats were randomly assigned into model， low-dose （0.75 g·kg^-1） and high-dose （1.5 g·kg^-1） Liuwei Dihuangwan， vitamin D （positive drug， 3.7×10^-5 g·kg^-1）， and blank groups. Each group was administrated with the corresponding concentration of drugs or the same volume of normal saline by gavage for 2 weeks. After the intervention， the three-chamber social test was conducted to evaluate social interaction and social preference. The open field test was carried out to observe spontaneous behavior and anxiety state. Hematoxylin-eosin staining （HE） was used to observe the pathological changes of the prefrontal tissue. Transmission electron microscopy was employed to observe the ultrastructure of mitochondria in prefrontal neurons. Immunofluorescence was used to detect the expression of ionized calcium-binding adapter molecule-1 （Iba-1） in the prefrontal tissue. Enzyme-linked immunosorbent assay （ELISA） was adopted to measure the levels of tumor necrosis factor-α （TNF-α） and interleukin-6 （IL-6）. Western blot was employed to assess the expression differences of phosphorylated adenosine monophosphate-activated protein kinase （p-AMPK）， adenosine monophosphate-activated protein kinase （AMPK）， phosphorylated Unc-51-like autophagy-activating kinase 1 （p-ULK1）， Unc-51-like autophagy-activating kinase 1 （ULK1）， and FUN14 domain-containing protein 1 （FUNDC1）. ResultsCompared with the blank group， the model group spent less time sniffing stranger 1 and stranger 2 in the three-chamber social test （P<0.01） and showed reductions in the total distance traveled， average speed， distance traveled in the central area， and time spent in the central area in the open field test （P<0.01）. In addition， the model group showed extensive apoptosis of neurons， with shrunken nuclei and red-stained cytoplasm， and extensive necrosis of neurons in the prefrontal tissue， mitochondrial swelling， decreased matrix density， disrupted cristae， and autophagic lysosomes in neurons， increases in the rate of Iba-1 positive cells in the prefrontal area （P<0.01） and the levels of TNF-α and IL-6 （P<0.01）， and down-regulation in the expression of p-AMPK/AMPK， p-ULK1/ULK1， and FUNDC1 （P<0.01）. Compared with the model group， low-dose and high-dose Liuwei Dihuangwan and the vitamin D prolonged the time spent sniffing stranger 1 and stranger 2 in the three-chamber social test （P<0.05， P<0.01）， increased the total distance traveled， average speed， distance traveled in the central area， and time spent in the central area in the open field test （P<0.05， P<0.01）， restored the morphology of neurons in the prefrontal tissue， decreased the number of apoptotic cells， alleviated the swelling of mitochondria in neurons， increased the matrix density， mitigated the fragmentation and disorder of cristae， and increased the number of autophagosomes. Moreover， the drugs decreased the rate of Iba-1 positive cells in the prefrontal area （P<0.01）， lowered the levels of TNF-α and IL-6 （P<0.01）， and up-regulated the expression of p-AMPK/AMPK， p-ULK1/ULK1， and FUNDC1 （P<0.01）. ConclusionLiuwei Dihuangwan ameliorate autism-like behaviors and reduce neuronal apoptosis and neuroinflammatory damage in the rat model of ASD by promoting mitophagy mediated by the AMPK/ULK1/FUNDC1 pathway.

BACKGROUND:Gait analysis,as a radiation-free dynamic assessment method,may be able to assist imaging to evaluate patients with scoliosis,thereby reducing radiation exposure.OBJECTIVE:To analyze the correlation between gait parameters and imaging parameters in adolescent idiopathic scoliosis patients,and preliminarily explore the potential mechanism through OpenSim simulation modeling.METHODS:Twenty adolescent idiopathic scoliosis patients aged 10-18 years with Cobb angle of 10°-45° were selected as adolescent idiopathic scoliosisgroup. The control group was normal children of the same age and the same body mass index range,and a total of 40 subjects were included. The differences in spatiotemporal parameter,kinematic parameters,dynamic parameters,and gait deviation index of the two groups were compared to explore the correlation between pathological gait and coronal balance. The related mechanisms were preliminarily studied.RESULTS AND CONCLUSION:(1) The comparison of gait parameters between groups showed that the percentage of bilateral support phase and double support phase increased in adolescent idiopathic scoliosis patients,and the percentage of bilateral swing phase decreased (P<0.05). The maximum adduction angle of the left hip joint of patients in the adolescent idiopathic scoliosis group was greater than that of the control group,and the maximum abduction angle of the bilateral hip joints was significantly less than that of the control group (P<0.05). The ankle joints of the two groups when the bilateral heels initially touched the ground;the difference between angles was significant (P<0.05). The peak power of right hip flexor muscle group in adolescent idiopathic scoliosis patients was significantly decreased when they pushed off the ground (P<0.05). (2) The results of the correlation study showed that the maximum abductive angle of the right hip joint was significantly positively correlated with the Cobb angle (r=0.552,P=0.012),and the right peak power of right hip flexor muscle and C7 lateral offset were significantly negatively correlated (r=-0.475,P=0.034). There was a significant positive correlation between ankle angle and lateral pelvic tilt when left foot followed the ground (r=0.476,P=0.034). (3) The simulation results of muscle length showed that the change of muscle fiber length of gluteal medium muscle in adolescent idiopathic scoliosis patients was significantly reduced (P<0.05). The change of muscle fiber length of the bilateral internal oblique muscle was significantly reduced in adolescent idiopathic scoliosis patients (P<0.05). Comparing the muscle fiber length of the two groups when foot followed the ground,it was found that the muscle fiber length of the calf triceps of the adolescent idiopathic scoliosis patients was significantly shorter when foot followed the ground (P<0.05). (4) The results showed that the postural stability control of the trunk of adolescent idiopathic scoliosis patients was decreased,which was manifested as slow step frequency,increased percentage of double support phase and decreased percentage of swing phase. The common pattern of low foot striking pattern is caused by bilateral Achilles tendon tension. In adolescent idiopathic scoliosis patients,the ability of hip flexor muscle to do work is decreased,which may be related to joint angular velocity and main bend direction.

Objective:To investigate the effects of Jianpi Rougan Xifeng Decoction on the behaviors and the Ca 2+ -CaM-CaMK Ⅱ signaling pathway in the striatum of rats with tic disorder models. Methods:Seventy-two SPF-grade SD male rats were randomly divided into blank group, model group, tiapride group (15.93 mg/kg, intragastric administration), low-dose (4.32 g/kg, intragastric administration), medium-dose (8.64 g/kg, intragastric administration) and high-dose (17.28 g/kg, intragastric administration) Jianpi Rougan Xifeng Decoction groups, with 12 rats in each group. Rats in the blank group and model group were gavaged with 0.5 mg/kg of distilled water while rats in other 4 groups were gavaged with corresponding drugs, all rats were gavaged once a day for 28 days.The evaluations of motor behavior and stereotyped behavior were conducted using the Kadasah scoring method and the Diamond scoring method. Calcium content in the striatum was detected using a calcium assay kit. Immunohistochemical analysis was employed to detect the expression of dopamine transporter (DAT) and inositol 1, 4, 5-triphosphate (IP3) in the striatum of rats. Western blot was used to assess the expression of calmodulin (CaM) and calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in the striatum. RT-PCR was utilized to detect the expression of CaM and CaMK Ⅱ mRNA in the striatum. All data were processed using SPSS 26.0 software, and comparisons among multiple groups were performed using one-way ANOVA and Kruskal-Wallis test.Results:(1) After four weeks of pharmacological intervention, statistically significant differences were observed in the locomotor activity scores and stereotyped scores among the six groups of rats ( H=41.20, 44.24, both P<0.01). Specifically, the locomotor activity scores(3.00(3.00, 3.25), 1.00(0.75, 1.25), 1.00(0.75, 2.00), 1.00(0, 1.00)) and stereotyped scores(3.00(3.00, 4.00), 1.00(0.75, 2.00), 2.00(0.75, 2.00), 1.00(0, 1.25)) in the tiapride group and the medium- and high-dose Jianpi Rougan Xifeng Decoction groups were significantly lower than those in the model group (all P<0.01). (2) Results from the calcium assay kit revealed statistically significant differences in striatal calcium content among the six groups of rats ( F=146.67, P<0.01). The calcium content in the tiapride group and the medium- and high-dose Jianpi Rougan Xifeng Decoction groups was significantly lower than that in the model group (all P<0.01). Additionally, the calcium content in the medium-dose ((0.40±0.02)mmol/g) and high-dose ((0.30±0.03)mmol/g) Jianpi Rougan Xifeng Decoction groups was lower than that in the low-dose group ((0.48±0.02)mmol/g) (both P<0.01). (3) Immunohistochemical results showed that there were statistically significant differences in the mean optical density values of DAT and IP3 in the striatum among the six groups of rats ( F=25.57, 154.98, both P<0.01). The IP3 mean optical density in the tiapride group and the Jianpi Rougan Xifeng Decoction groups with medium and high doses exhibited lower values compared to the model group (all P<0.05), whereas the DAT mean optical density displayed higher values in these groups compared to the model group (all P<0.05). The low-dose Jianpi Rougan Xifeng Decoction group also exhibited a lower optical density value of IP3 compared to the model group( P<0.05). The optical density values of IP3 (2.68±0.21, 2.40±0.22) in the medium-dose and high-dose Jianpi Rougan Xifeng Decoction groups were lower than that in the low-dose group (4.27±0.23) (both P<0.01). (4) Western blot results indicated that there were statistically significant differences in the protein expression levels of CaM and CaMK Ⅱ in the striatum among the six groups of rats ( F=233.03, 118.60, both P<0.01). The protein expression levels of CaM and CaMK Ⅱ in the tiapride group and the low-dose, medium-dose, and high-dose Jianpi Rougan Xifeng Decoction groups were lower than those in the model group (all P<0.01). The protein expression levels of CaM (1.02±0.06, 0.84±0.02) and CaMK Ⅱ (0.48±0.03, 0.40±0.02) in the medium-dose and high-dose Jianpi Rougan Xifeng Decoction groups were lower than those in the low-dose group (1.21±0.03, 0.57±0.02)) (all P<0.05). Additionally, the protein expression level of CaM in the high-dose Jianpi Rougan Xifeng Decoction group was lower than that in the medium-dose group( P<0.05).(5) The RT-PCR results indicated significant variations in the mRNA expression levels of CaM and CaMK Ⅱ within the striatum across the six groups rats ( F=30.54, 20.78, both P<0.01). The mRNA expression levels of CaM and CaMK Ⅱ in the tiapride group and the high-dose Jianpi Rougan Xifeng Decoction group were lower than those in the model group (both P<0.05). The mRNA expression levels of CaMK Ⅱ (1.38±0.17) in the high-dose Jianpi Rougan Xifeng Decoction group were lower than those in the low-dose group (1.99±0.27) ( P<0.01). Conclusion:Jianpi Rougan Xifeng Decoction can improve locomotor activity and stereotyped behavior in rats with tic disorder model. The mechanism may be through inhibiting the Ca 2+ -CaM-CaMK Ⅱ signaling pathway and regulating the expression of DAT and IP3, thereby modulating the release and recovery of dopamine and reducing the occurrence of tic symptoms.

Objective:To investigate the effects of Jianpi Rougan Xifeng Decoction on the behaviors and the Ca 2+ -CaM-CaMK Ⅱ signaling pathway in the striatum of rats with tic disorder models. Methods:Seventy-two SPF-grade SD male rats were randomly divided into blank group, model group, tiapride group (15.93 mg/kg, intragastric administration), low-dose (4.32 g/kg, intragastric administration), medium-dose (8.64 g/kg, intragastric administration) and high-dose (17.28 g/kg, intragastric administration) Jianpi Rougan Xifeng Decoction groups, with 12 rats in each group. Rats in the blank group and model group were gavaged with 0.5 mg/kg of distilled water while rats in other 4 groups were gavaged with corresponding drugs, all rats were gavaged once a day for 28 days.The evaluations of motor behavior and stereotyped behavior were conducted using the Kadasah scoring method and the Diamond scoring method. Calcium content in the striatum was detected using a calcium assay kit. Immunohistochemical analysis was employed to detect the expression of dopamine transporter (DAT) and inositol 1, 4, 5-triphosphate (IP3) in the striatum of rats. Western blot was used to assess the expression of calmodulin (CaM) and calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) in the striatum. RT-PCR was utilized to detect the expression of CaM and CaMK Ⅱ mRNA in the striatum. All data were processed using SPSS 26.0 software, and comparisons among multiple groups were performed using one-way ANOVA and Kruskal-Wallis test.Results:(1) After four weeks of pharmacological intervention, statistically significant differences were observed in the locomotor activity scores and stereotyped scores among the six groups of rats ( H=41.20, 44.24, both P<0.01). Specifically, the locomotor activity scores(3.00(3.00, 3.25), 1.00(0.75, 1.25), 1.00(0.75, 2.00), 1.00(0, 1.00)) and stereotyped scores(3.00(3.00, 4.00), 1.00(0.75, 2.00), 2.00(0.75, 2.00), 1.00(0, 1.25)) in the tiapride group and the medium- and high-dose Jianpi Rougan Xifeng Decoction groups were significantly lower than those in the model group (all P<0.01). (2) Results from the calcium assay kit revealed statistically significant differences in striatal calcium content among the six groups of rats ( F=146.67, P<0.01). The calcium content in the tiapride group and the medium- and high-dose Jianpi Rougan Xifeng Decoction groups was significantly lower than that in the model group (all P<0.01). Additionally, the calcium content in the medium-dose ((0.40±0.02)mmol/g) and high-dose ((0.30±0.03)mmol/g) Jianpi Rougan Xifeng Decoction groups was lower than that in the low-dose group ((0.48±0.02)mmol/g) (both P<0.01). (3) Immunohistochemical results showed that there were statistically significant differences in the mean optical density values of DAT and IP3 in the striatum among the six groups of rats ( F=25.57, 154.98, both P<0.01). The IP3 mean optical density in the tiapride group and the Jianpi Rougan Xifeng Decoction groups with medium and high doses exhibited lower values compared to the model group (all P<0.05), whereas the DAT mean optical density displayed higher values in these groups compared to the model group (all P<0.05). The low-dose Jianpi Rougan Xifeng Decoction group also exhibited a lower optical density value of IP3 compared to the model group( P<0.05). The optical density values of IP3 (2.68±0.21, 2.40±0.22) in the medium-dose and high-dose Jianpi Rougan Xifeng Decoction groups were lower than that in the low-dose group (4.27±0.23) (both P<0.01). (4) Western blot results indicated that there were statistically significant differences in the protein expression levels of CaM and CaMK Ⅱ in the striatum among the six groups of rats ( F=233.03, 118.60, both P<0.01). The protein expression levels of CaM and CaMK Ⅱ in the tiapride group and the low-dose, medium-dose, and high-dose Jianpi Rougan Xifeng Decoction groups were lower than those in the model group (all P<0.01). The protein expression levels of CaM (1.02±0.06, 0.84±0.02) and CaMK Ⅱ (0.48±0.03, 0.40±0.02) in the medium-dose and high-dose Jianpi Rougan Xifeng Decoction groups were lower than those in the low-dose group (1.21±0.03, 0.57±0.02)) (all P<0.05). Additionally, the protein expression level of CaM in the high-dose Jianpi Rougan Xifeng Decoction group was lower than that in the medium-dose group( P<0.05).(5) The RT-PCR results indicated significant variations in the mRNA expression levels of CaM and CaMK Ⅱ within the striatum across the six groups rats ( F=30.54, 20.78, both P<0.01). The mRNA expression levels of CaM and CaMK Ⅱ in the tiapride group and the high-dose Jianpi Rougan Xifeng Decoction group were lower than those in the model group (both P<0.05). The mRNA expression levels of CaMK Ⅱ (1.38±0.17) in the high-dose Jianpi Rougan Xifeng Decoction group were lower than those in the low-dose group (1.99±0.27) ( P<0.01). Conclusion:Jianpi Rougan Xifeng Decoction can improve locomotor activity and stereotyped behavior in rats with tic disorder model. The mechanism may be through inhibiting the Ca 2+ -CaM-CaMK Ⅱ signaling pathway and regulating the expression of DAT and IP3, thereby modulating the release and recovery of dopamine and reducing the occurrence of tic symptoms.

BACKGROUND:Gait analysis,as a radiation-free dynamic assessment method,may be able to assist imaging to evaluate patients with scoliosis,thereby reducing radiation exposure.OBJECTIVE:To analyze the correlation between gait parameters and imaging parameters in adolescent idiopathic scoliosis patients,and preliminarily explore the potential mechanism through OpenSim simulation modeling.METHODS:Twenty adolescent idiopathic scoliosis patients aged 10-18 years with Cobb angle of 10°-45° were selected as adolescent idiopathic scoliosisgroup. The control group was normal children of the same age and the same body mass index range,and a total of 40 subjects were included. The differences in spatiotemporal parameter,kinematic parameters,dynamic parameters,and gait deviation index of the two groups were compared to explore the correlation between pathological gait and coronal balance. The related mechanisms were preliminarily studied.RESULTS AND CONCLUSION:(1) The comparison of gait parameters between groups showed that the percentage of bilateral support phase and double support phase increased in adolescent idiopathic scoliosis patients,and the percentage of bilateral swing phase decreased (P<0.05). The maximum adduction angle of the left hip joint of patients in the adolescent idiopathic scoliosis group was greater than that of the control group,and the maximum abduction angle of the bilateral hip joints was significantly less than that of the control group (P<0.05). The ankle joints of the two groups when the bilateral heels initially touched the ground;the difference between angles was significant (P<0.05). The peak power of right hip flexor muscle group in adolescent idiopathic scoliosis patients was significantly decreased when they pushed off the ground (P<0.05). (2) The results of the correlation study showed that the maximum abductive angle of the right hip joint was significantly positively correlated with the Cobb angle (r=0.552,P=0.012),and the right peak power of right hip flexor muscle and C7 lateral offset were significantly negatively correlated (r=-0.475,P=0.034). There was a significant positive correlation between ankle angle and lateral pelvic tilt when left foot followed the ground (r=0.476,P=0.034). (3) The simulation results of muscle length showed that the change of muscle fiber length of gluteal medium muscle in adolescent idiopathic scoliosis patients was significantly reduced (P<0.05). The change of muscle fiber length of the bilateral internal oblique muscle was significantly reduced in adolescent idiopathic scoliosis patients (P<0.05). Comparing the muscle fiber length of the two groups when foot followed the ground,it was found that the muscle fiber length of the calf triceps of the adolescent idiopathic scoliosis patients was significantly shorter when foot followed the ground (P<0.05). (4) The results showed that the postural stability control of the trunk of adolescent idiopathic scoliosis patients was decreased,which was manifested as slow step frequency,increased percentage of double support phase and decreased percentage of swing phase. The common pattern of low foot striking pattern is caused by bilateral Achilles tendon tension. In adolescent idiopathic scoliosis patients,the ability of hip flexor muscle to do work is decreased,which may be related to joint angular velocity and main bend direction.

Objective:To investigate the protective effect and mechanism of silymarin on bronchiolitis caused by respiratory syncytial virus (RSV) in mice.Methods:A mouse model of bronchiolitis was established by intranasal instillation of RSV. After successful modeling, the mice were randomly divided into a model group, a positive control group (ribavirin, 10 mg/kg), a low-dose silymarin group (25 mg/kg), a medium-dose silymarin group (50 mg/kg), and a high-dose silymarin group (100 mg/kg). In addition, a control group was established, with 12 mice in each group. The pulmonary index and RSV virus load were determined in each group of mice. Hematoxylin-eosin (HE) staining was used to observe the pathological changes in the lungs. The levels of interleukin-4 (IL-4), interferon-γ (IFN-γ), transforming growth factor-β (TGF-β), IL-17, and IL-10 in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). Flow cytometry was used to detect the proportion of helper T cells 17 (Th17) and regulatory T cells (Treg) in peripheral blood. Western blot was used to detect the expression levels of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), nuclear factor κB subunit p65 (NF-κB p65), and phosphorylated nuclear protein p-NF-κB p65 in lung tissue.Results:Compared with the model group, the pulmonary injury and inflammatory response were significantly improved in the medium-and high-dose silymarin groups. The pulmonary indexes were (1.27±0.17)% and (0.94±0.10)%, respectively, and the RSV virus loads were (2.65±0.19) and (2.13±0.14), respectively, which were significantly lower than those in the model group (all P<0.05). The proportion of Th17 cells in peripheral blood was (4.47±0.19)% and (3.52±0.13)%, respectively, which was significantly lower than that in the model group (all P<0.05), while the proportion of Treg cells in peripheral blood was (0.88±0.08)% and (1.33±0.12)%, respectively, which was significantly higher than that in the model group (all P<0.05). The expression levels of IL-4 and IL-17 in BALF and the protein expression levels of TLR4/NF-κB signaling pathway related proteins in lung tissue were significantly lower than those in the model group (all P<0.05), while the expression levels of IFN-γ, TGF-β, and IL-10 in BALF were significantly higher than those in the model group (all P<0.05). Conclusions:Silymarin can regulate immune function and inhibit inflammatory response, thereby improving airway inflammation in bronchiolitis mice. The mechanism may be related to inhibit activation of TLR4/NF-κB signaling pathway.