ObjectiveHeadspace solid-phase microextraction-gas chromatography-mass spectrometry（HS-SPME-GC-MS） and GC-triple quadrupole MS（GC-QqQ-MS） in combination with non-targeted and targeted metabolomics were employed to systematically analyze the chemical composition differences of Xihuangwan prepared with natural musk and artificial musk， and establish an identification system for them. MethodsThe volatile components of 9 batches of Xihuangwan samples from 8 manufacturers were analyzed by HS-SPME-GC-MS non-targeted metabolomics， and identified by comparing their MS data with the National Institute of Standards and Technology（NIST） spectral library. Orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to identify differential volatile components of Xihuangwan prepared with natural musk and artificial musk. Additionally， GC-QqQ-MS targeted metabolomics was applied to quantify the levels of α-pinene， β-elemene， muscone， dehydroepiandrosterone， bornyl acetate， and octyl acetate in 27 batches of samples from 9 manufacturers. Cluster analysis， principal component analysis（PCA）， and partial least squares-discriminant analysis（PLS-DA） were conducted to further explore the differences in volatile components between Xihuangwan samples prepared with natural musk and artificial musk. ResultsNon-targeted metabolomics identified 291 volatile compounds in Xihuangwan， including alkanes， esters， alkanes， alcohols， ketones， naphthalenes and others. OPLS-DA analysis revealed distinct separation between Xihuangwan samples containing artificial musk（A1， C1， D1， E1， F1， G1， I1） and those containing natural musk（H1， H3）. A total of 30 differential metabolites were identified. The relative contents of these 30 differential metabolites were visualized using a radar chart， revealing significant differences in the levels of octanol， borneol acetate and muscone. Cluster analysis and PCA results from targeted metabolomics indicated that Xihuangwan could be classified into two distinct groups：one composed of natural musk（H1， H3） and the other of artificial musk， sample H2. PLS-DA identified muscone， octyl acetate， and dehydroepiandrosterone as key differential volatile components. Although no significant difference was observed in the content of octyl acetate between the two groups， statistically significant differences were found for muscone and dehydroepiandrosterone（P<0.05）. ConclusionMuscone and dehydroepiandrosterone can be used for the differentiation of Xihuangwan samples containing natural musk from those containing artificial musk. This study systematically and comprehensively analyzed the differences in the types and contents of major volatile components in Xihuangwan prepared with natural musk and artificial musk， providing a scientific basis for quality evaluation and control of Xihuangwan.

BackgroundAnxiety disorder is a common mental disorder， with its prevalence showing a continuous upward trend， significantly affecting the quality of life and social function of patients. Due to the lack of objective and reliable biomarkers in clinical practice， the early identification and treatment of anxiety disorder have been somewhat limited. Plasma proteins have the potential to serve as biomarkers for mental diseases， however， the causal relationship between them and anxiety disorder remains unclear. ObjectiveTo identify the plasma proteins that have a causal relationship with anxiety disorders， and to elucidate the associated biological pathways， in order to provide references for the search for biomarkers of anxiety disorders and the exploration of potential therapeutic targets. MethodsBased on the protein quantitative trait locus （pQTL） data of 4 907 plasma proteins covering 35 559 Icelandic individuals from the deCODE database， and the genome-wide association studies （GWAS） data of 50 486 patients with anxiety disorders and 330 460 healthy controls， the inverse-variance weighted （IVW） method was used as the main analysis method， supplemented by MR-Egger method， weighted median method， simple model method， and weighted model method for bidirectional Mendelian randomization analysis. Enrichment analysis of gene ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathways was conducted for the related proteins. Sensitivity analysis was performed using Cochran's Q test， MR-Egger intercept test， MR-PRESSO test， and leave-one-out analysis to evaluate the robustness of the results. ResultsA total of 10 plasma proteins were identified as significantly associated with anxiety disorders. Among these， SPATA9 （OR=0.856， 95% CI： 0.784–0.934， P<0.01） and PDE5A （OR=0.911， 95% CI： 0.864–0.961， P<0.01） were identified as protective factors， while CRYGD （OR=1.209， 95% CI： 1.095–1.334， P<0.01）， BTN3A3 （OR=1.045， 95% CI： 1.018–1.073， P<0.01）， SERPINB13 （OR=1.102， 95% CI： 1.040–1.168， P<0.01）， ERBB4 （OR=1.283， 95% CI： 1.109–1.484， P<0.01）， LSAMP （OR=1.096， 95% CI： 1.037–1.158， P<0.01）， ICOSLG （OR=1.283， 95% CI： 1.104–1.490， P<0.01）， DNAJB11 （OR=1.172， 95% CI： 1.076–1.277， P<0.01）， and TREML1 （OR=1.115， 95% CI： 1.054–1.179， P<0.01） were identified as risk factors. The sensitivity analysis showed that the results were robust， with no heterogeneity （Cochran's Q test P>0.05） or pleiotropy （MR-Egger intercept test P>0.05）. Enrichment analysis indicated that these plasma proteins were enriched in biological processes such as T-cell signal transduction， lymphocyte proliferation， cell membrane structure and synaptic function， as well as the intestinal immune network that produces IgA and the ErbB signaling pathway. ConclusionThis study identified 10 plasma proteins associated with anxiety disorders. The functions of these plasma proteins involve multiple biological processes such as neural development and immune regulation.

ObjectiveHeadspace solid-phase microextraction-gas chromatography-mass spectrometry（HS-SPME-GC-MS） and GC-triple quadrupole MS（GC-QqQ-MS） in combination with non-targeted and targeted metabolomics were employed to systematically analyze the chemical composition differences of Xihuangwan prepared with natural musk and artificial musk， and establish an identification system for them. MethodsThe volatile components of 9 batches of Xihuangwan samples from 8 manufacturers were analyzed by HS-SPME-GC-MS non-targeted metabolomics， and identified by comparing their MS data with the National Institute of Standards and Technology（NIST） spectral library. Orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to identify differential volatile components of Xihuangwan prepared with natural musk and artificial musk. Additionally， GC-QqQ-MS targeted metabolomics was applied to quantify the levels of α-pinene， β-elemene， muscone， dehydroepiandrosterone， bornyl acetate， and octyl acetate in 27 batches of samples from 9 manufacturers. Cluster analysis， principal component analysis（PCA）， and partial least squares-discriminant analysis（PLS-DA） were conducted to further explore the differences in volatile components between Xihuangwan samples prepared with natural musk and artificial musk. ResultsNon-targeted metabolomics identified 291 volatile compounds in Xihuangwan， including alkanes， esters， alkanes， alcohols， ketones， naphthalenes and others. OPLS-DA analysis revealed distinct separation between Xihuangwan samples containing artificial musk（A1， C1， D1， E1， F1， G1， I1） and those containing natural musk（H1， H3）. A total of 30 differential metabolites were identified. The relative contents of these 30 differential metabolites were visualized using a radar chart， revealing significant differences in the levels of octanol， borneol acetate and muscone. Cluster analysis and PCA results from targeted metabolomics indicated that Xihuangwan could be classified into two distinct groups：one composed of natural musk（H1， H3） and the other of artificial musk， sample H2. PLS-DA identified muscone， octyl acetate， and dehydroepiandrosterone as key differential volatile components. Although no significant difference was observed in the content of octyl acetate between the two groups， statistically significant differences were found for muscone and dehydroepiandrosterone（P<0.05）. ConclusionMuscone and dehydroepiandrosterone can be used for the differentiation of Xihuangwan samples containing natural musk from those containing artificial musk. This study systematically and comprehensively analyzed the differences in the types and contents of major volatile components in Xihuangwan prepared with natural musk and artificial musk， providing a scientific basis for quality evaluation and control of Xihuangwan.

Objective: To determine the association between exposure to entertainment screen content on mobile phones and symptoms of anxiety-depression co-morbidity among college students,so as to provide evidence for mental health interventions. Methods: A baseline survey was conducted from April to May 2019. A total of 1 135 college students were selected from one university each in Shangrao City, Jiangxi Province and Hefei City, Anhui Province using cluster random sampling method. A follow up study was conducted in November 2019, resulting in 1 110 matched valid responses. Self rating questionnaires were used to assess the exposure of entertainment screen content. The Depression Anxiety Stress Scale-21(DASS-21) and the Patient Health Questionnaire-9 (PHQ-9) were used to evaluate the anxiety symptoms, depressive symptoms, and symptoms of anxiety-depression co-morbidity among college students. A multivariate binary Logistic regression model was constructed following initial intergroup comparisons with Chi-square test to determine the association between baseline exposure to mobile entertainment screen content and the risk of symptoms of anxiety depression co-morbidity at baseline and the 6 month follow up. Results: The prevalence rates of symptoms of anxiety-depression co-morbidity among college students were 25.4% and 20.6% at baseline and follow up, respectively.After adjusting for confounding factors such as gender, self rated family economic status and self rated health status, the results of multivariate binary Logistic regression analysis showed that compared with the appropriate exposure level group, the exposure of entertainment screen content on mobile phones at baseline, including frequent exposure to reading( OR =1.65,95% CI =1.14-2.39), occasional exposure to other entertainment screen content ( OR =1.46,95% CI =1.01-2.10)and frequent exposure to other entertainment screen content( OR =1.76,95% CI =1.20-2.60), increased the co-occurrence risk of symptoms of anxiety-depression co-morbidity among college students during the follow up period (all P <0.05). Conclusion Occasional or frequert exposure to mobile entertainment screen content can increase the risk of symptoms of anxiety depression co-morbidity among college students.

Objective: To systematically analyze the confirmatory positivity of different combinations of HBsAg screening results in blood testing, providing data to support the optimization of blood donor eligibility management. Methods: A retrospective analysis was conducted on blood screening data from 174 266 voluntary blood donor samples at the Chongqing Blood Center between October 2021 and September 2022. Samples with inconsistent results between the two HBsAg enzymelinked immunosorbent assays (ELISA) and individual donor nucleic acid testing (NAT) were confirmed using an electrochemiluminescence immunoassay (ECLIA) and a neutralization test. The detection efficacy of four different HBsAg ELISA reagents was compared using the HBsAg-confirmed positive samples. Results: A total of 767(0.44%) HBV-reactive (HB-sAg and/or HBV DNA reactive) samples were detected. Among them, 344 samples with discordant serological and NAT results were collected, of which 64(18.6%) were confirmed positive by neutralization test. Additionally, 5 samples that were neutralization-negative but double-reactive for HBsAg and HBV DNA were confirmed as positive according to FDA guidance, resulting in a total of 69(20.1%) confirmed HBsAg-positive samples. There were significant differences in the neutralization test confirmation rates among different screening result categories (P<0.05): The group with dual HBsAg reagent reactivity (double reactive) & NAT-negative had the highest confirmation rate (96.9%, 31/32); the group reactive to only reagent 2 (single reactive) had a rate of 25.7% (29/113); while the confirmation rates for samples reactive to only reagent 1 and samples with isolated HBV DNA positivity were extremely low [0(0/34) and 2.4%(4/165), respectively]. The four commercial reagents showed significant differences in their ability to detect confirmed positive samples that were initially single reactive (P<0.05). Conclusion: Given the performance variations among HBsAg screening reagents, thorough performance verification is essential before implementation. When NAT is negative, dual HBsAg reactivity in screening can serve as a basis for confirming infection and directly deferring blood donors. However, confirming infection in donors with single HBsAg reactivity is more challenging, necessitating supplementary tests to rule out infection risk.

Objective: To evaluate repeated testing on blood screening platforms in confirmation of non-discriminatory reactive (NDR) samples in nucleic acid testing (NAT). Methods: A total of 102 HBsAg-negative/NAT NDR samples were collected from voluntary blood donors at Dalian Blood Center between January 2021 and December 2023. Repeated testing was performed using two NAT platforms (Cobas s201 and Panther). For the first round of repeated testing, all samples were tested 12 times on each system; for the second round, the samples which were non-reactive or only reactive once in the first round were tested an additional 8 times. Anti-HBc and anti-HBs was detected using electrochemiluminescence assay (ECA). Meanwhile, blood donors were followed up. Results: The proportion of anti-HBc+ in 102 NDR samples was 88.2%. Forty-one samples (40.2%, 41/102) and 7 samples were confirmed HBV DNA+ in first-round and second-round repeated testing, respectively. The cumulative confirmation rate of HBV DNA+ was 47.1% (48/102) after repeated testing. Extra five blood donors detected HBV DNA+ in follow-up were identified as anti-HBc+ occult hepatitis B virus infection (OBI), while no window period infection was observed. Ultimately, there were 53 HBV infected donors confirmed, 46 HBV infection-unconfirmed, and 3 HBV uninfected. No significant difference was observed between the confirmation rate of the first-round testing and the cumulative confirmation rate after the second-round testing (P>0.05). The proportion of anti-HBc+ donors was quite high in both HBV infection-confirmed (98.1%) and unconfirmed group (82.6%), and donors with seronegative and anti-HBs-only occupied a high proportion in the latter (P<0.05). Conclusion: Numerous repeated testing of NDR samples using NAT platforms cannot achieve complete confirmation of HBV infection. Supplementary anti-HBc testing can minimize potential OBI risk among NDR donors, and is low-cost and efficient.

ObjectiveThis study aimed to investigate whether Bushen Tongluo prescription inhibits macrophage polarization by regulating the Wnt3a/β-catenin signaling pathway， thereby reducing epithelial-mesenchymal transition and excessive extracellular matrix deposition， in order to elucidate the anti-pulmonary fibrosis mechanisms of Bushen Tongluo prescription and provide a new theoretical basis for the clinical treatment of pulmonary fibrosis. MethodsFifty male Sprague-Dawley （SD） rats were randomly divided into a blank group， model group， pirfenidone group， and high- and low-dose Bushen Tongluo prescription groups. Except for the blank group， the pulmonary fibrosis model was established by intratracheal instillation of bleomycin. Intervention was initiated on day 28 after modeling. The high- and low-dose Bushen Tongluo prescription groups were administered Bushen Tongluo prescription at doses of 30.88， 15.44 g·kg^-1， respectively， by intragastric gavage. The pirfenidone group was administered pirfenidone capsules at 110 mg·kg^-1 by intragastric gavage. The blank and model groups were given an equal volume of normal saline by gavage， once daily for 90 days. After treatment， the level of transforming growth factor-β₁ （TGF-β₁） in bronchoalveolar lavage fluid （BALF） was detected by enzyme-linked immunosorbent assay （ELISA）. Morphological changes in lung tissue and the collagen volume fraction were compared. The protein distribution and expression of E-cadherin， cytokeratin 19， α-smooth muscle actin （α-SMA）， vimentin， collagen type Ⅰ （Col Ⅰ）， and collagen type Ⅲ （Col Ⅲ） in lung tissue were detected by immunohistochemistry. The protein distribution and expression of CD68， arginase-1 （Arg-1）， inducible nitric oxide synthase （iNOS）， Wnt3a， and β-catenin in lung tissue were detected by immunofluorescence. The protein expression of Wnt3a and β-catenin in lung tissue was detected by Western blot， and the mRNA expression of Wnt3a and β-catenin was detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR）. ResultsCompared with the blank group， a large number of inflammatory cells infiltrated the airway walls， alveolar spaces， and interstitial tissue in the model group， with obvious fibrous tissue hyperplasia. The level of TGF-β₁ in BALF was significantly increased. The protein expression of E-cadherin and cytokeratin 19 in lung tissue was decreased， whereas the protein expression of α-SMA， Vimentin， Wnt3a， β-catenin， Col Ⅰ， and Col Ⅲ was increased. The fluorescence-positive area ratios of CD68， Arg-1， iNOS， Wnt3a， and β-catenin in lung tissue were increased. The protein and mRNA expression levels of Wnt3a and β-catenin in lung tissue were significantly increased （P<0.01）. Compared with the model group， all treatment groups showed varying degrees of improvement in inflammatory cell infiltration and fibrous tissue hyperplasia in the airway walls， alveolar spaces， and interstitial tissue， decreased TGF-β₁ levels in BALF， increased protein expression of E-cadherin and cytokeratin 19 in lung tissue， decreased protein expression of α-SMA， Vimentin， Col Ⅰ， and Col Ⅲ， decreased fluorescence-positive area ratios of CD68， Arg-1， iNOS， Wnt3a， and β-catenin in lung tissue， and decreased protein and mRNA expression levels of Wnt3a and β-catenin in lung tissue （P<0.05， P<0.01）. ConclusionBushen Tongluo prescription can improve bleomycin-induced pulmonary fibrosis in rats by inhibiting epithelial-mesenchymal transition and reducing excessive extracellular matrix deposition. The mechanism may be related to inhibition of the Wnt3a/β-catenin signaling pathway and the macrophage polarization mediated by this pathway.

Background: Adrenocortical hormones, particularly 11-oxygenated androgens, are pivotal in female reproductive health and fertility. Standardized detection kits and population-specific reference intervals are lacking in China, hindering related clinical applications. Methods: A HPLC-tandem mass spectrometry (HPLC-MS/MS) pipeline was developed, rigorously validated, and applied to simultaneously quantify corticosterone, cortisone, cortisol, 18-OH cortisol, androstenedione (A4), 11β-hydroxyandrostenedione (11-OH A4), dehydroepiandrosterone, and dehydroepiandrosterone sulfate in serum samples from 455 reproductive-aged women (18–45 yrs) in Guangxi, China. Age-dependent concentration trends were analyzed, and reference intervals stratified by age (2.5th to 97.5th percentiles) were established. Correlations with body-composition metrics, ethnicity, and the menstrual cycle were investigated. Results: The HPLC-MS/MS method demonstrated high precision (intra- and inter-assay CVs < 15%), accuracy, and sensitivity. All eight hormones exhibited significant age-related declines (P < 0.001 for seven hormones; P = 0.001 for 11-OH A4). Notably, 11-OH A4 levels were significantly lower in the 35–45-yr (3.05 nmol/L) and 25–34-yr (3.09 nmol/L) age groups than in the 18–24-yr (3.57 nmol/L) age group, whereas no significant difference was observed between the 35–45-yr and 25–34-yr age groups. Weak negative correlations were observed between the body mass index and corticosterone and cortisone levels, whereas ethnicity and the menstrual cycle showed no significant associations with hormone levels. Conclusions We developed an HPLC-MS/MS-based method for simultaneously quantifying eight adrenocortical hormones, including 11-OH A4, and defined age-specific reference intervals for reproductive-aged Chinese women. These findings advance the clinical utility of adrenocortical hormones in diagnosing and managing reproductive disorders.

Objective: To investigate the longitudinal association between biological rhythm trajectories and the risk of anxiety, depression, and comorbid symptoms among college students, so as to provide a cumulative empirical basis for early psychiatric tracking and mental health warning systems. Methods: Using random cluster sampling method, a baseline survey was conducted from April to May 2019. One comprehensive university and one medical university were selected in Shangrao City, Jiangxi Province, and Hefei City, Anhui Province, respectively. A total of 1 029 first year college students were recruited as study participants, and four waves of longitudinal follow up were carried out over a period of two years. The Self rating of Biological Rhythm Disorder for Adolescents was used to assess college students biological rhythms, and using the dynamic data from the first two follow ups to construct the trajectory of biological rhythm changes. Mental health outcomes (anxiety and depressive symptoms) at the end of the follow up period were assessed using Generalized Anxiety Disorder-7 and Patient Health Questionnaire-9. Multivariable Logistic regression models were employed to evaluate the independent predictive value of distinct lifestyle rhythm trajectories on the detection risk of long term anxiety, depression, and their comorbid symptoms. Results: The prevalence rates of anxiety, depression, and comorbid symptoms among participants were 23.5%, 22.6%, and 16.7%, respectively. Multivariable Logistic regression analysis showed that, after adjusting for confounding factors such as sex, major, smoking, and alcohol consumption, compared with the persistent low risk reference group of overall biological rhythm, the persistent high risk trajectory group of overall assessment had an increased risk of anxiety symptoms( OR=3.87, 95%CI =2.45-6.11), depressive symptoms( OR=3.18, 95%CI =1.98-5.10), and comorbid symptoms( OR=2.43, 95%CI =1.46-4.04)(all P ＜0.05).Among college students in the elevated overall assessment risk group, the risks of anxiety symptoms ( OR=1.97, 95%CI =1.22-3.17), depressive symptoms ( OR=2.13, 95%CI =1.33-3.42), and comorbid symptoms ( OR=1.81, 95%CI =1.07-3.06) were also increased (all P < 0. 05), and the various risks in the group with increased risks were also higher than those in the group with consistently low risks. Conclusions The dynamic deterioration and persistent disruption of college students lifestyle and behavioral rhythms serve as strong early warning indicators for long term anxiety and depressive symptoms. Mental health screening in colleges and universities should incorporate multi timepoint dynamic tracking of behavioral rhythms, and circadian rhythm regulation should be regarded as a core target for group level mental health interventions.

Objective To explore curative effect of two thrombolytic regimens in the treatment of ultra-early acute cerebral infarction(ACI)of different age groups and different thrombolytic time windows.Methods A total of 166 patients with ACI were enrolled,with interval from onset to treatment≤4.5 h.According to different thrombolytic regiments,patients were divided into the recombinant tissue plasminogen activator(rt-PA)group(104 cases,intravenous thrombolysis with 0.9 mg/kg rt-PA)and the tenecteplase(TNK)group(62 cases,intravenous thrombolysis with 0.25 mg/kg TNK).According to different age groups and different thrombolytic time windows,patients were divided into the high age group(≥70 years),the low age group(＜70 years),the short time window group(＜3 h)and the long time window group(3-4.5 h).After thrombolysis,laboratory indexes[neutrophil(NEU),lymphocyte(LYM)],scores of National Institutes of Health Stroke Scale(NIHSS),modified Rankin scale(mRS)and incidence of adverse events were compared in different groups by factorial analysis method.Results There was no significant difference in any index between the rt-PA group and the TNK group(P＞0.05).Compared with the high age group at 7 d after thrombolysis,NIHSS score,NEU,neutrophil to lymphocyte ratio(NLR)and high sensitive C-reactive protein(hs-CRP)were lower,while LYM and albumin(ALB)were higher in the low age group(P＜0.05).Compared with the long time window group,NEU and NLR were lower,while LYM was higher in the short time window group(P＜0.05).There was no significant difference in total incidence of adverse events within 14 d of thrombolysis between the different age groups and the different thrombolytic time window groups(P＞0.05).Conclusion For patients with ultra-early ACI,0.9 mg/kg rt-PA and 0.25 mg/kg TNK have comparable efficacy in intravenous thrombolysis,and both have better effects when patient age is＜70 years old and the thrombolysis time window is＜3 hours.