ObjectiveTo explore the role and mechanism of Hei Xiaoyaosan in regulating the protein kinase B （Akt）/glycogen synthase kinase-3β （GSK-3β）/nuclear factor E₂-related factor 2 （Nrf2）/heme oxygenase-1 （HO-1） signaling pathway in cascade modulation of oxidative stress and apoptosis for preventing and treating Alzheimer's disease （AD）. MethodsNinety male SD rats of 4 months old were randomly assigned into a control group （n=10）， a sham group （with injection of 1 μL normal saline into bilateral hippocampi， n=10）， and a modeling group （with injection of 1 μL beta-amyloid 1-42 solution into bilateral hippocampi to induce AD， n=70）. One week after modeling， 50 successfully modeled rats were selected and randomly allocated into model， donepezil hydrochloride （0.45 mg·kg^-1）， and high-， medium-， and low-dose （15.30， 7.65， 3.82 g·kg^-1， respectively） Hei Xiaoyaosan groups. The rats were administrated with corresponding drugs once daily for six consecutive weeks. The Morris water maze was used to assess the learning and memory abilities of rats. Hematoxylin-eosin （HE） staining was performed to reveal hippocampal morphological changes in AD rats. Apoptosis in the hippocampal CA3 region was detected by terminal-deoxynucleotidyl transferase-mediated Nick end labeling. Immunofluorescence was used to visualize the expression of neuronal nuclear antigen （NeuN） in the CA1 region. Additionally， enzyme-linked immunosorbent assay was performed to assess the activities of glutathione peroxidase （GSH-Px）， glutathione-S-transferase （GST）， and catalase （CAT） in the hippocampus. Real-time PCR was conducted to measure the mRNA levels of Akt， GSK-3β， Nrf2， and HO-1， while Western blot was employed to determine the protein levels of phosphorylated Akt （p-Akt）/Akt， phosphorylated GSK-3β （p-GSK-3β）/GSK-3β， Nrf2， and HO-1. ResultsCompared with the control group， the model group on day 5 showed an increase in total swimming distance （P<0.01）， a reduction in the percentage of time spent in the target quadrant （P<0.01）， reduced and disarranged neurons， nuclear condensation， varying degrees of cellular damage， increased apoptosis of hippocampal neurons （P<0.01）， decreased NeuN content （P<0.01）， weakend activities of GSH-Px， GST， and CAT （P<0.01）， and down-regulated mRNA levels of Nrf2 and HO-1 （P<0.01） and protein levels of p-Akt/Akt， p-GSK-3β/GSK-3β， Nrf2， and HO-1 （P<0.01） in the hippocampus. Compared with the model group， donepezil hydrochloride and high， medium， and low doses of Hei Xiaoyaosan shortened the total swimming distance on day 5 （P<0.05， P<0.01）， increased the percentage of time spent in the target quadrant （P<0.05， P<0.01）， improved the arrangement and morphology of neurons， reduced nuclear condensation and the apoptosis rate of hippocampal neurons （P<0.01）， increased the NeuN content （P<0.01）， enhanced the activities of GSH-Px， GST， and CAT （P<0.05， P<0.01）， and up-regulated the mRNA levels of Nrf2 and HO-1 （P<0.05， P<0.01） and the protein levels of p-Akt/Akt， p-GSK-3β/GSK-3β， Nrf2， and HO-1 （P<0.05， P<0.01） in the hippocampus. ConclusionHei Xiaoyaosan can regulate the Akt/GSK-3β/Nrf2/HO-1 pathway to enhance the antioxidant stress capacity and inhibit neuron apoptosis to exert the neuroprotective effect， thereby ameliorating the cognitive dysfunction and pathological damage in AD rats.

ObjectiveTo explore the mechanism of Hei Xiaoyaosan in improving the cognitive function in Alzheimer's disease （AD） from cell apoptosis mediated by the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt）/nuclear factor kappa B （NF-κB） signaling pathway. MethodsFour-month-old SD male rats were randomly assigned into a blank group， a sham group， a model group， a donepezil hydrochloride （0.45 mg·kg^-1） group， and high-， medium-， and low-dose （15.30， 7.65， and 3.82 g·kg^-1， respectively） Hei Xiaoyaosan groups， with 10 rats in each group. The sham group received bilateral hippocampal injection of 1 μL normal saline， while the other groups received bilateral hippocampal injection of 1 μL beta-amyloid 1-42 （Aβ_1-42） solution for the modeling of AD. Rats were administrated with corresponding agents once a day for 42 consecutive days. The Morris water maze test was carried out to assess the learning and memory abilities of rats. Hematoxylin-eosin staining was employed to observe pathological changes in the hippocampus of rats. Enzyme-linked immunosorbent assay was employed to measure the levels of cysteinyl aspartate-specific proteinase-3 （Caspase-3）， B-cell lymphoma-2 （Bcl-2）， and Bcl-2-associated X protein （Bax）. Western blot was employed to determine the protein levels of PI3K， Akt， and NF-κB. A cell model of AD was established by co-culturing Aβ_1-42 and PC12 cells in vitro. Cell viability and apoptosis were detected by the cell-counting kit 8 （CCK-8） assay and flow cytometry （FC）， respectively. ResultsAnimal experiments showed that compared with the blank group， the model group had a prolonged escape latency （P<0.01）， a reduced number of crossing platforms （P<0.01）， disarrangement and a reduced number of hippocampal neurons， up-regulated expression of Bax and Caspase-3， down-regulated expression of Bcl-2 （P<0.01）， decreased p-PI3K/PI3K and p-Akt/Akt levels， and an increased p-NF-κB/NF-κB level （P<0.01）. Compared with the model group， donepezil hydrochloride and high- and medium-dose Hei Xiaoyaosan shortened the escape latency and increased the number of crossing platforms （P<0.05， P<0.01）， improved the arrangement and increased the number of hippocampal neurons， down-regulated the expression levels of Bax and Caspase-3， up-reguated the expression level of Bcl-2 （P<0.05， P<0.01）， increased the p-PI3K/PI3K and p-Akt/Akt levels （P<0.05， P<0.01）， and reduced the p-NF-κB/NF-κB level （P<0.05， P<0.01）. Cell experiments showed that compared with the blank group， the model group exhibited an increased apoptosis rate （P<0.01）. Compared with the model group， the serum containing Hei Xiaoyaosan at various doses improved the cell viability （P<0.01）， and the serum containing Hei Xiaoyaosan at the high dose decreased the cell apoptosis （P<0.01）. ConclusionHei Xiaoyaosan may improve the learning and memory abilities of AD model rats by regulating cell apoptosis， while increasing the vitality and reducing the apoptosis rate of AD model cells via the PI3K/Akt/NF-κB signaling pathway.

ObjectiveTo investigate the effects of Hei Xiaoyaosan on cognitive impairment and the histone deacetylase sirtuin-1 （SIRT1）/tumor suppressor p53/solute carrier family 7 member 11 （SLC7A11） signaling pathway in the rat model of Alzheimer's disease （AD）. MethodsA total of 90 16-week-old SPF-grade SD male rats were randomly assigned in a blank group （n=10）， a sham group （n=10， with injection of 1 μL normal saline into the bilateral hippocampi）， and an AD modeling group （n=70， with injection of 1 μL β amyloid 1-42 solution into the bilateral hippocampi）. According to the random number table method， fifty successfully modeled rats were assigned into model， donepezil hydrochloride （0.45 mg·kg^-1）， and high-， medium-， and low-dose （15.30， 7.65， and 3.82 g·kg^-1， respectively） Hei Xiaoyaosan groups， and they were administrated with corresponding agents via gavage once a day for 42 consecutive days. Morris water maze test was carried out to examine the cognitive function of rats. Nissl staining was employed to observe the morphology of hippocampal neurons in each group， and Prussian blue staining was used to detect iron deposition in the hippocampal tissue. Biochemical kits were used to measure the levels of superoxide dismutase （SOD）， malondialdehyde （MDA）， and iron ion （Fe²⁺） in the hippocampal tissue. Western blot and real-time quantitative polymerase chain reaction （Real-time PCR） were employed to determine the protein and mRNA levels， respectively， of SIRT1， p53， SLC7A11， glutathione peroxidase 4 （GPX4）， and acyl-CoA synthetase long-chain family member 4 （ACSL4） in the hippocampus. ResultsCompared with the blank group， the model group showed reductions in target quadrant movement distance （P<0.01） and target quadrant residence time （P<0.05）， disarrangement of hippocampal neurons， increased ferroptosis deposition in the hippocampus， a lowered level of SOD， risen levels of MDA and Fe²⁺ （P<0.05， P<0.01）， down-regulated protein and mRNA levels of SIRT1， SLC7A11， and GPX4 （P<0.05， P<0.01）， up-regulated protein and mRNA levels of p53 and ACSL4 （P<0.01）， and aggravated pathological process of AD. Compared with the model group， donepezil hydrochloride extended the target quadrant residence time and the target quadrant movement distance （P<0.05， P<0.01）. High- and medium- doses of Hei Xiaoyaosan extended the target quadrant residence time and the target quadrant movement distance （P<0.05， P<0.01）， improved the neuron arrangement and reduced the ferroptosis deposition in the hippocampus， elevated the SOD level， lowered the MDA and Fe²⁺ levels （P<0.05， P<0.01）， up-regulated the protein and mRNA levels of SIRT1， SLC7A11， and GPX4 （P<0.01， P<0.05）， and down-regulated the protein and mRNA levels of p53 and ACSL4 （P<0.05， P<0.01）. ConclusionHei Xiaoyaosan can regulate the SIRT1/p53/SLC7A11 signaling pathway to mitigate oxidative stress and inhibit ferroptosis， thereby ameliorating the cognitive dysfunction in AD rats.

Temporomandibular joint (TMJ) diseases are common clinical conditions. The number of patients with TMJ diseases is large, and the etiology, epidemiology, disease spectrum, and treatment of the disease remain controversial and unknown. To understand and master the current situation of the occurrence, development and prevention of TMJ diseases, as well as to identify the patterns in etiology, incidence, drug sensitivity, and prognosis is crucial for alleviating patients′suffering.This will facilitate in-depth medical research, effective disease prevention measures, and the formulation of corresponding health policies. Cohort construction and research has an irreplaceable role in precise disease prevention and significant improvement in diagnosis and treatment levels. Large-scale cohort studies are needed to explore the relationship between potential risk factors and outcomes of TMJ diseases, and to observe disease prognoses through long-term follw-ups. The consensus aims to establish a standard conceptual frame work for a cohort study on patients with TMJ disease while providing ideas for cohort data standards to this condition. TMJ disease cohort data consists of both common data standards applicable to all specific disease cohorts as well as disease-specific data standards. Common data were available for each specific disease cohort. By integrating different cohort research resources, standard problems or study variables can be unified. Long-term follow-up can be performed using consistent definitions and criteria across different projects for better core data collection. It is hoped that this consensus will be facilitate the development cohort studies of TMJ diseases.

Objective To analyze the epidemiological characteristics and epidemic situation of children with Kashin-Beck disease (KBD) in China,and provide the basis for formulating prevention and control measures. Methods Fixed-point monitoring,moving-point monitoring,and full coverage of monitoring were promoted successively from 1990 to 2023. Some children (7-12 years old) underwent clinical and right-hand X-ray examinations every year. According to the KBD diagnosis criteria,clinical and X-ray assessments were used to confirm the diagnosis. Results In 1990,the national KBD detectable rate was 21.01％. X-ray detection decreased to below 10％ in 2003 and below 5％ in 2007. Between 2010 and 2018,the prevalence of KBD in children was less than 0.4％,which fluctuated at a low level,and has decreased to 0％ since 2019. Spatial epidemiological analysis indicated a spatial clustering of adult patients prevalence rate in the KBD areas. Conclusion The evaluation results of the elimination of KBD in China over the last 5 years showed that all villages in the monitored areas have reached the elimination standard. While the adult KBD patients still need for policy consideration and care.

Methods: Patients with dry eye who were treated in the Ophthalmology Ward and Outpatient Department of the First Hospital of Hunan University of Chinese Medicine from October 1, 2020, to October 30, 2021 were enrolled as the research participants in the study. They were assigned to two groups based on traditional Chinese medicine (TCM) syndrome types: heat stagnation in liver meridian pattern group and Qi-Yin deficiency pattern group. Healthy volunteers who underwent health check-ups in the Health Management Department were included as healthy group following the random number table method. The tears of the patients and the healthy volunteer participants were tested by high-performance liquid chromatography-mass spectrometry (LC-MS). The differential metabolites were screened out by multivariate statistical analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment was performed on the differential metabolites. Finally, the association analysis of differential proteins and metabolites was conducted to verify and supplement the metabolites. Results: A total of 32 dry eye patients were enrolled, including 16 cases with heat stagnation in liver meridian pattern and 16 cases with Qi-Yin deficiency pattern. Fourteen healthy volunteers were included as healthy group. There were no significant differences in baseline characteristics among the three groups (P > 0.05). A total of 412 biomarkers were determined in Qi-Yin deficiency pattern group, mainly including lipids, lipid-like molecules, organic acids and their derivatives, organic heterocyclic compounds, and nucleosides and their analogues. For heat stagnation in liver meridian pattern group, 112 metabolites were determined, mainly including organic acids and their derivatives, lipids, and lipid-like molecules. The KEGG enrichment results of pathways and the relative content analysis of differential markers demonstrate that purine metabolism and caffeine metabolism pathways are common metabolic characteristics of all dry eyes. Among them, deoxyinosine monophosphate (dIMP) and 2-(formamido)-N1-(5-phospha-D-ribosyl) acetamidine can serve as their biomarkers. The main characteristics of Qi-Yin deficiency syndrome pattern were the significant enhancement of metabolic pathways such as lysine degradation, ovarian steroidogenesis, cholesterol metabolism, pyrimidine metabolism, and bile secretion (P < 0.05). Dry eye associated with the heat stagnation in liver meridian pattern is mainly characterized by inhibition of the valine, leucine, and isoleucine biosynthesis pathways (P < 0.05). Conclusion Metabolomics can be used as an effective basis for TCM syndrome classification. Different patterns of dry eye syndrome exhibit typical characteristics in the types and concentrations of metabolites, which correspond to the syndrome classification in TCM. This study initially confirms the rationality of TCM syndrome classification and provides significant reference for the mechanism of dry eye and drug development.

Objective To investigate the incidence rate,clinical characteristics,and prognosis of neonatal stroke in Shenzhen,China.Methods Led by Shenzhen Children's Hospital,the Shenzhen Neonatal Data Collaboration Network organized 21 institutions to collect 36 cases of neonatal stroke from January 2020 to December 2022.The incidence,clinical characteristics,treatment,and prognosis of neonatal stroke in Shenzhen were analyzed.Results The incidence rate of neonatal stroke in 21 hospitals from 2020 to 2022 was 1/15 137,1/6 060,and 1/7 704,respectively.Ischemic stroke accounted for 75％(27/36);boys accounted for 64％(23/36).Among the 36 neonates,31(86％)had disease onset within 3 days after birth,and 19(53％)had convulsion as the initial presentation.Cerebral MRI showed that 22 neonates(61％)had left cerebral infarction and 13(36％)had basal ganglia infarction.Magnetic resonance angiography was performed for 12 neonates,among whom 9(75％)had involvement of the middle cerebral artery.Electroencephalography was performed for 29 neonates,with sharp waves in 21 neonates(72％)and seizures in 10 neonates(34％).Symptomatic/supportive treatment varied across different hospitals.Neonatal Behavioral Neurological Assessment was performed for 12 neonates(33％,12/36),with a mean score of(32±4)points.The prognosis of 27 neonates was followed up to around 12 months of age,with 44％(12/27)of the neonates having a good prognosis.Conclusions Ischemic stroke is the main type of neonatal stroke,often with convulsions as the initial presentation,involvement of the middle cerebral artery,sharp waves on electroencephalography,and a relatively low neurodevelopment score.Symptomatic/supportive treatment is the main treatment method,and some neonates tend to have a poor prognosis.

Objective To compare strain and gender differences between ICR and C57BL/6J mouse chronic fatigue syndrome models and provide experimental evidence for the selection of model animals for chronic fatigue syndrome.Methods ICR and C57BL/6J mice(half male and half female)were injected intraperitoneally with polyinosinic-polycytidylic acid(Poly I∶C)every three days and forced to swim daily.The modeling was performed for 15 consecutive days,during which the weight and food intake of the mice were measured,and fatigue scores were assessed.After the modeling was completed,behavioral tests were carried out,including exhaustive swimming,tail suspension,mechanical pain threshold,and elevated plus maze.Results Compared with the control group,both strains of model mice had significantly decreased exhaustion times in the exhaustive swimming test(P＜0.05,P＜0.01),significantly increased immobility time in the tail suspension test(P＜0.05,P＜0.01),and a significantly decreased mechanical pain threshold(P＜0.05,P＜0.01),and male model mice had significantly decreased open arm entry time and frequency in the elevated plus maze(P＜0.05,P＜0.01).The weight of model male C57BL/6J mice significantly decreased(P＜0.05,P＜0.01).The weight of model female ICR mice increased after a significant reduction(P＜0.05).The exhaustion time of control C57BL/6J mice was significantly lower than that of control ICR mice(P＜0.01).The immobility time of model C57BL/6J mice was significantly greater than that of model ICR mice(P＜0.01).Conclusions There were differences between the two strains of mice in terms of weight change,fatigue level,and depression.Within the same strain,there were differences between males and females,and the anxiety level of males was higher than that of females.

To establish a simple,convenient,sensitive,and specific method for rapid detection of Zi-ka virus(ZIKV),the whole genome sequences of ZIKV isolated from different times and regions were analyzed.The specific primers and probes were designed based on the screened target se-quences located in the conserved region of the ZIKV NS5 gene.By combining RT-LAMP isother-mal amplification technology and immunochromatography technology,a reverse transcription loop mediated isothermal amplification nucleic acid and flow visualization strip(RT-LAMP-VF)detec-tion method for ZIKV was established.The results showed that the method had good specificity and sensitivity.When the ratio of inner,outer,and ring primers(FIP∶LF∶F3)was 4∶2∶1,the detection method can specifically detect 102 copies/pL RNA transcripts or 2.15 pfu ZIKV at 61 ℃for 45 minutes,with no cross reaction with other flaviviruses such as Japanese encephalitis virus and classical swine fever virus.Other RNAs in blood tissue samples did not affect the sensitivity and specificity of RT-LAMP-VF,indicating that the method can be applied to clinical practice.The ZIKV RT-LAMP-VF detection method established in this study is easy to perform and does not require special instruments and equipment.It is particularly suitable for the rapid detection of ZIKV in grassroots units,providing technical support and material support for the establishment of on-site rapid detection and early warning and prediction systems for ZIKV disease.

The PCR-amplified severe fever with thrombocytopenia syndrome virus(SFTSV)Gn-DⅢ-Ⅲ gene was inserted into the pET-30a(+)prokaryotic expression vector to generate the re-combinant plasmid pET-SFTSV-Gn-D Ⅲ-Ⅲ.The plasmid was transformed into E.coli BL21(DE3)for Gn-DⅢ-m protein expression and the expression conditions were optimized.The Gn-DⅢ-Ⅲ protein purified with Ni-NTA column affinity chromatography was applied as the captured antigen to establish an indirect ELISA method for the detection of SFTSV antibody.The results demonstrated that the recombinant plasmid pET-SFTSV-Gn-D Ⅲ-Ⅲ was successfully constructed as identified by PCR and sequencing.The recombinant protein SFTSV Gn-D m-Ⅲ was soluble ex-pression in E.coli under the optimal induction conditions of 0.4 mmol/L IPTG at 25 ℃ for 4 h,and the protein purity was 91.77％after purification by Ni-NTA column.The optimal reaction con-ditions for the indirect ELISA of SFTSV antibody were as follows:coating antigen concentration(5 μg/mL),primary antibody(incubation at 37 ℃ for 1.5 h),and secondary antibody(diluted 1:10 000 and incubated at 37 ℃ for 1 h).The established method had no cross-reactivity with Rift Valley fever virus(RVFV),Ebola virus(EBOV),and tick-borne encephalitis virus(TBEV)posi-tive sera.The method had a high sensitivity,with P/N＞2.1 for SFTSV-positive sera diluted to 81920.Coefficients of variation for intra-and inter-batch reactions were less than 10％.Detection of four SFTSV-infected human clinical serum samples showed the serum samples from patients in re-mission were tested as positive(P/N＞2.1),while serum samples from patients with multiple or-gan failure were detected as negative(P/N＜2.1).The results indicated that the SFTSV Gn-D Ⅲ-Ⅲ protein was successfully expressed and purified,and it was used as the coating protein to estab-lish an indirect ELISA assay for SFTSV antibody,which possesses good specificity,sensitivity and reproducibility.This method might be applied to detect human SFTSV clinical serum samples.