Objective To investigate whether Yiqicongming Decoction(YCD)can alleviate dry eye syndrome in rats.Methods Thirty-six male SD rats at 6 weeks of age were divided into blank group,model group,positive group,YCD high-dose group,YCD medium-dose group,and YCD low-dose group,with six rats in each group.The rats underwent iris,tear secretion volume,and corneal epithelial damage scoring assessments.Conjunctival tissue was stained with PAS,and the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)were detected using the en-zyme-linked immunosorbent assay(ELISA).The level of reactive oxygen species(ROS)was measured by flow cytometry.Western blot was used to detect the protein expression of nuclear factor erythroid 2-related factor 2(Nrf2)and hymeoxyge-nase-1(HO-1)in corneal tissues.Results Compared with the blank group,the number of goblet cells in the conjuncti-va,corneal epithelial injury score,MDA content and ROS level in the model group were significantly increased at all time points(all P＜0.05).The tear secretion volume of rats decreased on days 3,7,and 14,and the number of conjunctival goblet cells and SOD activity also decreased(all P＜0.05).Compared with the model group,the conjunctival score,corne-al epithelial injury score,MDA content,and ROS level in the positive control group and high-,medium-,and low-dose YCD groups were significantly reduced(all P＜0.05),while SOD activity and protein expression levels of Nrf2 and HO-1 in rats on day 14 were significantly increased(all P＜0.05).Compared with the high-dose YCD group,the conjunctival score,corneal epithelial injury score,and ROS level in the low-and medium-dose YCD groups were significantly increased(all P＜0.05),while SOD activity and protein expression levels of Nrf2 and HO-1 on day 14 were significantly decreased(all P＜0.05).On day 14,the tear secretion volume and the number of goblet cells in the conjunctiva of rats administered a low dose of Yiqi Congming Decoction both decreased(all P＜0.05).Compared with the medium-dose YCD group,the conjunc-tival score,corneal epithelial injury score and ROS level in the low-dose YCD group were significantly increased(all P＜0.05),while the tear secretion volume and protein expression levels of Nrf2 and HO-1 on day 14 were significantly de-creased(all P＜0.05).Conclusion YCD can increase tear secretion,alleviate iris and corneal epithelial damage,acti-vate Nrf2/HO-1 pathway,regulate oxidative stress,and alleviate dry eye syndrome.

This study aims to establish BHK-21 stable cell lines expressing APN from four species(human,pig,dog,and cat),the APN fragments were amplified from pEGFP-C1-APN plasmids of the four species stored in the laboratory to generate the recombinant plasmids pcDNA4.0-APN.Af-ter the recombinant plasmids were transfected into BHK-21 cells,the stable BHK-21 cell lines ex-pressing the APNs were selected by two rounds of limited dilution.The constructed BHK-21 cell lines were identified by indirect immunofluorescence assay(IFA),and their susceptibility to PD-CoV and TGEV was tested for these four cell lines.Virus infection experiments revealed that PD-CoV infected cells expressing human,pig,and dog APNs,while it did not infect cells expressing cat APN.Simultaneously,TGEV infected cells expressing pig,dog,and cat APNs,but did not infect cells expressing human APN.The results suggest that the risk of cross-species infection for different coronaviruses and the established cell line can be used effectively to evaluate the virus in-fection.The findings also revealed that PDCoV has the potential risk of cross-species infection of human and dog,and TGEV has the potential risk of cross-species infection of dog and cat.These results provide a basis for the prevention and control strategy of coronaviruses.

BACKGROUND:In the research and application of neural stem cells,cell culture and passage are key links,which directly affect the quality of cells and experimental results.It is of great significance to find the most suitable digestive enzymes that can maintain the biological characteristics of embryonic mouse spinal cord neural stem cells and enhance their passage efficiency.OBJECTIVE:To explore the most suitable digestive enzyme for passage of neural stem cells from the spinal cord of embryonic mice.METHODS:Microscopic dissection was used to isolate and extract spinal cord tissue from E14 d embryonic mice,which was cultured in DMEM/F12 serum-free medium containing epidermal growth factor,basic fibroblast growth factor,and B27.After spherulation,Nestin and Sox2 immunofluorescence identification was performed.During neural stem cell passage and culture,single-cell suspensions were prepared using trypsin,papain,and TrypLETM Express enzyme digestion combined with blow molding.The cell dispersion and spheroidization were observed,and passage 3 cells were stained with propidium iodide to detect cell death.Cell proliferation was detected by counting the total number of cells.Immunofluorescence staining,western blot assay and RT-PCR were used to detect the expression of Olig2,Tuj1,GFAP,and NeuN at the protein and mRNA levels and to identify cell differentiation.RESULTS AND CONCLUSION:After 72 hours of culture,E14 d embryonic mouse spinal cord tissue cells could form suspended neurospheres,which could be passaged after 5-7 days.Compared with trypsin and papain,TrypLETM Express enzyme combined with blow beating method was used for passage.The cell dispersion rate was high,the activity was good,and more NeuN-and Tuj1-positive neurons differentiated.This study optimized the culture and passaging process of neural stem cells,laying a foundation for further research on stem cell transplantation therapy for spinal cord diseases.

Objectives:To investigate the mechanism underlying the protective effects of polydatin(PD)a-gainst oxygen-glucose deprivation(OGD)-induced neuronal injury and acute spinal cord injury(SCI).Methods:The mouse hippocampal neuronal cell line HT22 was used in in vitro experiments.The cytotoxicity of PD was assessed using the CCK-8 assay:cells were treated with 0,1,3,10,30 and 100μM PD for 24h to de-termine the safe concentration range.After OGD modeling(24h),cells were treated with different concentrations of PD(0,3,10,and 30μM),and cell viability was measured via CCK-8 to identify the optimal protective concentration.Autophagy markers(LC3 and P62)were detected by immunofluorescence and Western blot(WB);Autophagosome formation was observed using transmission electron microscopy;And the apoptosis rate was e valuated by TUNEL staining.For in vivo studies,an acute SCI model was established in C57BL/6 mice using Allen's impact method.Animals were divided into sham,SCI model,and PD treatment(20mg/kg)groups,with n=10 per group.Tissues were collected on 7d and 14d post-injury.Spinal cord pathology was examined by HE staining on 7d and 14d.Immunofluorescence was performed on 14d to evaluate glial scar formation(GFAP+),neuronal survival(MAP2+),cell proliferation(BrdU+),and autophagy level(LC3 Ⅱ).Results:In vitro results showed that 1-100μM PD did not exhibit significant cytotoxicity toward HT22 cells.The optimal concentration of PD was determined to be 10μ M after OGD induction.Compared with the OGD group,10μM PD significantly enhanced cell viability after OGD[OGD group:(54.63±3.90)％vs OGD+PD(1 0μ M)group:(84.35±1.38)％,P＜0.05]and effectively attenuated autophagy activation,as evidenced by a decreased LC3Ⅱ/Ⅰratio(OGD group:11.0±0.57 vs OGD+PD group:3.50±0.28,P＜0.05),increased P62 accumulation(OGD group:0.55±0.04 vs OGD+PD group:0.93±0.06,P＜0.05),reduced number of autolysosomes,and significantly lower apoptosis rate[OGD group:(35.33±2.6)％vs OGD+PD group:(19.67±1.76)％,P＜0.05].In vivo,HE staining confirmed that pathological damage in spinal cord tissue was markedly alleviated in the PD-treated group compared with the SCI model group.Immunofluorescence results indicated that PD treatment inhibited fibrous scar formation(SCI group:2.32±0.19mm2 vs PD group:1.07±0.24mm2,P＜0.05),reduced neuronal damage(SCI group:1.72±0.28mm vs PD group:0.93±0.12mm,P＜0.05)and promoted cell proliferation[SCI group:(16.14±2.24)％vs PD group:(39.09±3.16)％,P＜0.05],and downregulated LC3 Ⅱ expression(SCI group:62.81±5.25 vs PD group:34.09±3.98,P＜0.05).Conclusions:PD ameliorates neural damage by concurrently suppressing autophagy and apoptosis,providing a dual-pathway therapeutic strategy for SCI.

Objective To investigate whether Yiqicongming Decoction(YCD)can alleviate dry eye syndrome in rats.Methods Thirty-six male SD rats at 6 weeks of age were divided into blank group,model group,positive group,YCD high-dose group,YCD medium-dose group,and YCD low-dose group,with six rats in each group.The rats underwent iris,tear secretion volume,and corneal epithelial damage scoring assessments.Conjunctival tissue was stained with PAS,and the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)were detected using the en-zyme-linked immunosorbent assay(ELISA).The level of reactive oxygen species(ROS)was measured by flow cytometry.Western blot was used to detect the protein expression of nuclear factor erythroid 2-related factor 2(Nrf2)and hymeoxyge-nase-1(HO-1)in corneal tissues.Results Compared with the blank group,the number of goblet cells in the conjuncti-va,corneal epithelial injury score,MDA content and ROS level in the model group were significantly increased at all time points(all P＜0.05).The tear secretion volume of rats decreased on days 3,7,and 14,and the number of conjunctival goblet cells and SOD activity also decreased(all P＜0.05).Compared with the model group,the conjunctival score,corne-al epithelial injury score,MDA content,and ROS level in the positive control group and high-,medium-,and low-dose YCD groups were significantly reduced(all P＜0.05),while SOD activity and protein expression levels of Nrf2 and HO-1 in rats on day 14 were significantly increased(all P＜0.05).Compared with the high-dose YCD group,the conjunctival score,corneal epithelial injury score,and ROS level in the low-and medium-dose YCD groups were significantly increased(all P＜0.05),while SOD activity and protein expression levels of Nrf2 and HO-1 on day 14 were significantly decreased(all P＜0.05).On day 14,the tear secretion volume and the number of goblet cells in the conjunctiva of rats administered a low dose of Yiqi Congming Decoction both decreased(all P＜0.05).Compared with the medium-dose YCD group,the conjunc-tival score,corneal epithelial injury score and ROS level in the low-dose YCD group were significantly increased(all P＜0.05),while the tear secretion volume and protein expression levels of Nrf2 and HO-1 on day 14 were significantly de-creased(all P＜0.05).Conclusion YCD can increase tear secretion,alleviate iris and corneal epithelial damage,acti-vate Nrf2/HO-1 pathway,regulate oxidative stress,and alleviate dry eye syndrome.

Objectives:To investigate the mechanism underlying the protective effects of polydatin(PD)a-gainst oxygen-glucose deprivation(OGD)-induced neuronal injury and acute spinal cord injury(SCI).Methods:The mouse hippocampal neuronal cell line HT22 was used in in vitro experiments.The cytotoxicity of PD was assessed using the CCK-8 assay:cells were treated with 0,1,3,10,30 and 100μM PD for 24h to de-termine the safe concentration range.After OGD modeling(24h),cells were treated with different concentrations of PD(0,3,10,and 30μM),and cell viability was measured via CCK-8 to identify the optimal protective concentration.Autophagy markers(LC3 and P62)were detected by immunofluorescence and Western blot(WB);Autophagosome formation was observed using transmission electron microscopy;And the apoptosis rate was e valuated by TUNEL staining.For in vivo studies,an acute SCI model was established in C57BL/6 mice using Allen's impact method.Animals were divided into sham,SCI model,and PD treatment(20mg/kg)groups,with n=10 per group.Tissues were collected on 7d and 14d post-injury.Spinal cord pathology was examined by HE staining on 7d and 14d.Immunofluorescence was performed on 14d to evaluate glial scar formation(GFAP+),neuronal survival(MAP2+),cell proliferation(BrdU+),and autophagy level(LC3 Ⅱ).Results:In vitro results showed that 1-100μM PD did not exhibit significant cytotoxicity toward HT22 cells.The optimal concentration of PD was determined to be 10μ M after OGD induction.Compared with the OGD group,10μM PD significantly enhanced cell viability after OGD[OGD group:(54.63±3.90)％vs OGD+PD(1 0μ M)group:(84.35±1.38)％,P＜0.05]and effectively attenuated autophagy activation,as evidenced by a decreased LC3Ⅱ/Ⅰratio(OGD group:11.0±0.57 vs OGD+PD group:3.50±0.28,P＜0.05),increased P62 accumulation(OGD group:0.55±0.04 vs OGD+PD group:0.93±0.06,P＜0.05),reduced number of autolysosomes,and significantly lower apoptosis rate[OGD group:(35.33±2.6)％vs OGD+PD group:(19.67±1.76)％,P＜0.05].In vivo,HE staining confirmed that pathological damage in spinal cord tissue was markedly alleviated in the PD-treated group compared with the SCI model group.Immunofluorescence results indicated that PD treatment inhibited fibrous scar formation(SCI group:2.32±0.19mm2 vs PD group:1.07±0.24mm2,P＜0.05),reduced neuronal damage(SCI group:1.72±0.28mm vs PD group:0.93±0.12mm,P＜0.05)and promoted cell proliferation[SCI group:(16.14±2.24)％vs PD group:(39.09±3.16)％,P＜0.05],and downregulated LC3 Ⅱ expression(SCI group:62.81±5.25 vs PD group:34.09±3.98,P＜0.05).Conclusions:PD ameliorates neural damage by concurrently suppressing autophagy and apoptosis,providing a dual-pathway therapeutic strategy for SCI.

Objective To establish the reference interval of urine Alzheimer-associated neuronal thread protein(AD7c-NTP)for healthy adults in Mianyang area using the indirect method.Methods The detection results of urine AD7c-NTP from 5 093 healthy in-dividuals were collected from the information management database of Medical Laboratory Department of Sichuan Science City Hospital from March 2017 to March 2022.Skewness-kurtosis and Kolmogorov-Smirnov tests were used to determine whether the data followed a normal distribution.After removing outliers using the Box Plots method,the enrolled subjects were grouped by gender and age.The Mann-Whitney U or Kruska-Wallis H tests were used to analyze the between-group differences of urine AD7c-NTP in healthy individu-als with different genders and ages.The adjacent age groups without statistically significant difference(P＞0.05)were combined,and the indirect method(non-parametric test method)was used to calculate the reference intervals for different gender and age groups.Results Skewness-kurtosis and Kolmogorov-Smirnov tests showed that the data followed a non-normal distribution.After removing 293 outliers using the Box Plots method,a total of 4 800 subjects,including 3 199 males and 1 601 females,were enrolled.The enrolled subjects were grouped by gender and age,and the non-parametric test method were used to establish the reference intervals of urine AD7c-NTP in healthy populations with different genders.The Mann-Whitney U test confirmed that urine AD7c-NTP levels existed gen-der differences(Z=14.09,P＜0.01),and the reference intervals for males and females were≤1.10 ng/mL and≤1.40 ng/mL,re-spectively.There were also statistical differences in urine AD7c-NTP levels among different age groups of the same gender.After combi-ning adjacent age groups without statistically significant difference(P＞0.05),the reference intervals of urine AD7c-NTP in healthy populations with different genders and ages were established by the non-parametric test method,which were≤1.00 ng/mL for male 20-39 years old group,≤1.10 ng/mL for male 40-79 years old group,≤1.60 ng/mL for male≥80 years old group,≤1.30 ng/mL for female 20-69 years old group,and≤1.60 ng/mL for female≥70 years old group,respectively.The established reference intervals of urine AD7c-NTP were further verified by healthy individuals,and the results met the standards.Conclusion The reference intervals of urine AD7c-NTP in healthy populations with different genders and ages in Mianyang area are established successfully using the indi-rect method,which may help to predict the risk of Alzheimer's disease in clinical practice and provide support for the diagnosis and treatment of the disease.

OBJECTIVE To investigate the positive test of high-risk human papillomavirus(HR-HPV)among the female population undergoing physical examination in Shenyang,analyze the influencing factors and establish and validate the risk prediction model.METHODS The data were collected from the female population who received HPV test in the physical examination center of a three-A hospital in the whole year of 2023.The prevalence rates of HR-HPV infections and subtypes were described,the influencing factors for the infections were identified.Uni-variate analysis and multivariate logistic regression analysis were performed for the influencing factors for positive test of HR-HPV,and the prediction model was established and validated.RESULTS Totally 6 130 out of 7 759 fe-male population who received HPV test were from Shenyang,the total positive rate of HR-HPV was 10.72％a-mong the population from Shenyang,11.11％among the population from other areas,and there was no signifi-cant difference.The population from Shenyang aged between 21 and 84 years old,with the mean age(48.58±11.64)years old.Among the local population who had the infections,80.21％were the single HPV infection,and 19.79％were multiple infections;HPV52 was the predominant subtype of HPV causing the infections,followed by HPV58 and HPV 16.The result of multivariate analysis showed age,smoking history,gynecological surgery history,allergic history,family annual income and sleep condition were the influencing factors for the positive HR-HPV.The prediction model was established based on the result of the multivariate analysis,the internal vali-dation of the model was carried out by modeling data and receiver operating characteristic(ROC)curves,the area under the curve(AUC)of the prediction model was 0.919,and 95％CI was 0.878 to 0.960,indicating that the prediction model had a high efficiency.CONCLUSIONS The positive rate of HR-HPV test is not relatively high among the physical examination female population in Shenyang,and the positive result is affected by a variety of factors.The population can be vaccinated for prevention and control based on the prediction model targeting to the non-variable factors such as age,meanwhile,the measures such as enhancement of health education,adjustment of health polies and interven-tion to health behaviors should be taken for other controllable factors.

Objective To explore the value of diffusion kurtosis imaging(DKI)in the diagnosis of Parkinson's disease with mild cognitive impairment(PD-MCI)patients.Methods A total of 18 patients with Parkinson's disease cognitive normal(PDN),22 patients with PD-MCI,and 24 healthy controls(HC)were prospectively included.All participants underwent DKI,and regions of interest(ROI)were selected in the substantia nigra,red nucleus,striatum,and posterior cingulate gyrus for post-processing.The diagnostic efficacy of DKI parameters on PD patients' cognitive status was analyzed by the receiver operating characteristic(ROC)curve.Results Compared with the PDN group,the PD-MCI group had a longer disease duration and a higher H-Y stage.Compared with the HC group,the PD-MCI group showed significantly lower mean kurtosis(MK),radial kurtosis(RK),axial kurtosis(AK),and fractional anisotropy(FA)values in the substantia nigra and posterior cingulate gyrus.In the PDN group,FA and MK values in the substantia nigra were significantly decreased,while FA values in the striatum and posterior cingulate gyrus were significantly increased(P＜0.05).Compared with the PDN group,the PD-MCI group showed significantly decreased DKI parameter values in the substantia nigra and posterior cingulate gyrus,and significantly decreased RK,AK,and FA values in the striatum(P＜0.05).The FA values of striatum,posterior cingulate gyrus and joint predictors were the most effective in the diagnosis of PD-MCI and the area under the curve(AUC)were 0.826,0.853 and 0.960,respectively.Conclusion DKI can detect microstructural changes in PD patients.Microstructural alterations in the striatum and posterior cingulate gyrus have an impact on early cognitive function changes in PD patients.FA demonstrate high sensitivity and specificity in the diagnosis of PD-MCI,and the combined diagnostic efficacy across multiple regions is even higher.

Objective: To explore the relationship between school sports environment and physical activity levels of middle school students, so as to provide theoretical and empirical support for optimizing school sports environment and enhance adolescent physical activity. Methods: Using multi-stage random cluster sampling, from September to December 2023, 1 329 junior and senior high school students from Xuancheng City of Anhui Province, Lianyungang City of Jiangsu Province, Wuhan City of Hubei Province, Qiqihar City and Suihua City of Heilongjiang Province, and Shenzhen City of Guangdong Province were selected. The International Physical Activity Questionnaire-Short Form (IPAQ-SF) assessed students physical activity levels, and the questionnaire on the characteristics of school sports environment was developed to evaluate the factors of school sports environment. Multivariate ordered Logistic regression was performed to analyze the correlation between school sports environment factors and physical activity levels, and the analytic hierarchy process determined the weight of key influencing factors. Results: The results showed that weekly vigorous physical activity time was [60 (25, 90)] minutes, moderate physical activity time was [60 (30, 90)] minutes, light physical activity time was [105 (40, 200)] minutes, and sedentary behavior time was [ 3 300 (2 100, 4 500)] minutes, only 10.53% of the students met World Health Organization physical activity recommendations, and 89.69% of the students averaged >8 h daily sedentary time. Multivariate ordered Logistic regression showed that adequate sports equipment significantly promoted physical activity across all intensities and reduced sedentary time ( OR = 4.97, 11.54, 4.03, 0.11); diverse sports activities improved vigorous and moderate physical activity while reducing sedentary time ( OR =4.20, 14.06, 0.17); and peer encouragement was associated with increased low-intensity physical activities and decreased sedentary time ( OR =10.40, 0.15)( P <0.05). The analytic hierarchy process weighting analysis identified the top three influential factors related to physical activity among middle school students: sufficient sports equipment, varied physical education activities, frequent peer encouragement, the influence weight accounts for 23.55% , 14.18% and 11.77% of the total, respectively. Conclusion Key school sports environmental factors for adolescent physical activity level include ensuring adequate sports equipment and class availability, diversifying activity content, fostering peer support, and cultivating an active sports culture and a comprehensive approach encourage students participation in extracurricular physical activities.