OBJECTIVE To provide a scientific basis for the quality evaluation and clinical application of Qingwen hufei granules. METHODS Fourteen batches of Qingwen hufei granules were used as samples to establish high-performance liquid chromatography （HPLC） fingerprints using the Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine （2012 Edition）. The chromatographic peaks were identified and the similarity was evaluated. Cluster analysis （CA）， principal component analysis （PCA）， and orthogonal partial least squares-discriminant analysis （OPLS-DA） were used to conduct chemical pattern recognition analysis on the 14 batches of samples. Meanwhile， the contents of neochlorogenic acid （NGA）， chlorogenic acid （CHA）， cryptochlorogenic acid （CGA）， forsythoside A （FTA）， 3，5-O-dicaffeoylquinic acid （3，5-O- DA）， 4，5-O-dicaffeoylquinic acid （4，5-O-DA）， and angoroside C （AGC） in the samples were determined by HPLC. RESULTS The methodological investigation results of both the fingerprint and the content determination complied with the relevant requirements. Fourteen common peaks were indicated in the HPLC fingerprints of the 14 batches of samples， and 7 of them were identified [NGA （peak 2）， CHA （peak 3）， CGA （peak 5）， FTA （peak 11）， 3，5-O-DA （peak 12）， 4，5-O-DA （peak 13）， and AGC （peak 14）]； the similarity of each sample was greater than 0.94. The results of CA and PCA showed that the samples could be classified into 3 categories； the results of OPLS-DA indicated that peak 4 （unknown）， peak 11 （FTA）， peak 8 （unknown）， peak 9 （unknown）， and peak 1 （unknown） were the differential components. The content ranges of NGA， CHA， CGA， 3，5-O-DA， FTA， 4，5-O-DA and AGC in the 14 batches of samples were 0.210 4-0.458 7， 0.269 1-0.506 3， 0.228 1-0.461 1， 0.443 9-1.044 6， 0.066 7-0.155 7， 0.062 8-0.143 8， and 0.057 4-0.105 7 mg/g， respectively. CONCLUSIONS The HPLC fingerprint and multi-component content determination methods established in this study are efficient and reliable， and can be used for the quality evaluation of Qingwen hufei granules.

Objective To investigate the regulatory pathway of lipid peroxidation in ferroptosis and the pathogenesis of Parkinson's disease(PD)at animal level.Methods Zebrafish PD model was established by exposure to 200 μmol/L 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).Zebrafish embryo in 48 h after fertilisation were grouped into a control group(n=100),a MPTP group(n=100),and a MPTP+Nomi group(1.5 μg/ml nomifensine,n=100).Behavioral analysis was performed on zebrafish in 5 d after fertilization for movement ability,rate and distance.The expression of α-synuclein and tyrosine hydroxylase in the zebrafish brain was detected by immu-nofluorescence assay to evaluate the activity of dopaminergic neurons(DA).The levels of iron,glutathione(GSH)and lipid peroxidation,which includes catalase(CAT),glutathione peroxidase 4(GPX4),lipid peroxide(LPO),malondialdehyde(MDA)and reactive oxygen species(ROS),were measured to verify whether iron deposition may cause damage to DA through the metabolic regulation mechanism of lipid peroxidation in iron death.Results Compared with the control group,the movement rate and distance were significantly reduced in the MPTP group(P＜0.01),while Nomi treatment obviously increased the movement rate and distance when compared with the MPTP group(P＜0.01).The MPTP group exhibited obvious DA damage in comparison with the control group,and Nomi treatment reversed the damage caused by MPTP.There was no sta-tistical difference in the CAT level in the three group(P＞0.05).The MPTP group had signifi-cantly increased levels of iron,LPO and MDA and decreased levels of GSH and GPX4 when com-pared with the control group(838.18±143.42 vs 478.53±112.58,P＜0.05;1.71±0.11 vs 1.48±0.14,P＜0.05;4.50±0.64 vs 4.23±0.13,P＜0.05;38.93±1.72 vs 45.97±2.32,P＜0.05;0.17±0.03 vs 0.39±0.04,P＜0.05).The MPTP+Nomi group had significantly increased levels of GSH and GPX4 and decreased levels of iron and LPO when compared with the MPTP group(40.79±1.02 vs 38.93±1.72,P＜0.05;0.26±0.05 vs 0.17±0.03,P＜0.05;326.75±110.95 vs 838.18±143.42,P＜0.05;1.01±0.27 vs 1.71±0.11,P＜0.05).The results of the ROS flow cytometry analysis showed that the percentages of ROS positive cells in the control group,MPTP group,and Nomi group were as follows:Histogram:31.6％,46.2％and 31.3％,Scatter plot:62.4％,73.0％and 65.7％,respectively.Conclusion Ferroptosis is found in PD zebrafish induced by MPTP exposure.Our study has clarified at animal level that the damage of DA caused by iron deposition is related to the metabolic mechanism of lipid peroxidation caused by iron death.

Objective:To investigate the situation of mild cognitive impairment (MCI) in elderly people in fluorosis areas, and analyze its potential influencing factors.Methods:From April to October 2020, a cluster random sampling method was used to select local elderly people aged 60 and older in areas of drinking-water-borne endemic fluorosis in Tianjin, and their general information was collected through face-to-face surveys. Terminal drinking water samples were collected from survey subjects. Water fluoride concentration was measured using ion-selective electrode method. Cognitive function was evaluated using the Mini-Mental State Examination (MMSE), and survey subjects were categorized into MCI group and control group based on MMSE scores. Logistic regression and Chi-squared automatic interaction detection classification tree models were constructed to analyze the influencing factors of MCI.Results:A total of 677 subjects were included, including 160 cases in the MCI group and 517 cases in the control group, with a MCI detection rate of 23.63%. The detection rates of MCI in the high fluoride exposure group and non high fluoride exposure group were 30.82% (94/305) and 17.74% (66/372), respectively, with a statistically significant difference between the groups (χ 2 = 15.88, P < 0.001). The results of univariate analysis showed that MCI was associated with high fluoride exposure, age, per capita annual household income, sleep duration, personality, and interpersonal relationships (χ 2 = 15.88, 16.17, 12.32, 8.91, 14.03, 9.81, P < 0.05). The results of multiple logistic regression analysis showed that moderate interpersonal relationships ( OR = 2.13, 95% CI: 1.08, 4.20) and high fluoride exposure ( OR = 1.91, 95% CI: 1.03, 3.54) were both risk factors for MCI ( P < 0.05). The classification tree model analysis result showed that high fluoride exposure was the primary factor affecting MCI in elderly people in fluorosis areas, followed by per capita annual household income, personality, and age. Conclusions:The detection rate of MCI in the high fluoride exposure group of elderly people in fluorosis areas is higher than that in the non high fluoride exposure group. High fluoride exposure, interpersonal relationships, per capita annual household income, personality, and age are all influencing factors of MCI in elderly people in fluorosis areas.

Objective:To learn about the iodine nutrition status of pregnant women in Tianjin and analyze the influencing factors of moderate to severe iodine deficiency in pregnant women.Methods:From January 2022 to December 2024, a stratified random sampling method was adopted. Each year, five sampling areas were divided into the east, west, south, north and central directions in 16 districts of Tianjin. Ten pregnant women from one township/street in each area were selected for questionnaire surveys. Household salt samples and random urine samples were collected to measure salt iodine and urinary iodine levels. Multivariate logistic regression was used to analyze the influencing factors of moderate to severe iodine deficiency in pregnant women, and a restricted cubic spline model was used to analyze the dose-response relationship between the risk of moderate to severe iodine deficiency in pregnant women and salt iodine level.Results:A total of 2 532 pregnant women in Tianjin were surveyed, with an age of (30.70 ± 4.44) years. Among them, 53.20% (1 347/2 532) had received health education, and 52.45% (1 328/2 532) actively supplemented iodine. A total of 2 532 household salt samples of pregnant women were tested, with a median salt iodine level of 23.66 mg/kg. The coverage rate of iodized salt was 74.45% (1 885/2 532), the qualified rate of iodized salt was 87.59% (1 651/1 885), and the consumption rate of qualified iodized salt was 65.21% (1 651/2 532). A total of 2 532 urine samples of pregnant women were tested, with a median urinary iodine level of 151.68 μg/L. Among them, 652, 1 348 and 532 urine samples were collected from pregnant women in the early, middle, and late stages of pregnancy, respectively, with median urinary iodine levels of 150.80, 153.00 and 143.68 μg/L, respectively. Multivariate logistic regression analysis showed that consuming iodized salt was a protective factor for the moderate to severe iodine deficiency in pregnant women ( OR = 0.70, 95% CI: 0.58 - 0.86, P < 0.001). Restricted cubic spline analysis showed that there was a linear dose-response relationship between the risk of moderate to severe iodine deficiency in pregnant women and salt iodine level ( Poverall < 0.001, Pnonlinear = 0.065). Further stratified analysis revealed that there was a linear dose-response relationship between the risk of moderate to severe iodine deficiency in pregnant women who had received iodine supplementation and salt iodine level ( Poverall = 0.018, Pnonlinear = 0.554), while there was a nonlinear dose-response relationship between the risk of moderate to severe iodine deficiency in pregnant women who had not received iodine supplementation and salt iodine level ( Poverall < 0.001, Pnonlinear = 0.029). Conclusions:From 2022 to 2024, the overall iodine level of pregnant women in Tianjin is appropriate, but some pregnant women are at risk of iodine deficiency. Consuming iodized salt is a protective factor against the moderate to severe iodine deficiency in pregnant women. It is still necessary to further implement comprehensive prevention and control measures mainly based on iodized salt.

Objective:To investigate the prevalence of dental fluorosis in children in Tianjin and study its influencing factors.Methods:From January to December 2023, 5 affected villages were selected from each of the drinking water-borne endemic fluorosis (drinking water-borne fluorosis for short) areas of 10 agricultural areas in Tianjin, and 50 children aged 8 - 12 years (gender and age balanced) were selected from each affected village for questionnaire survey and dental fluorosis examination. At the same time, water samples from affected villages and children's one random urine sample were collected to test for fluoride levels in water and urine. Multivariate logistic regression was used to analyze the influencing factors of dental fluorosis prevalence in children, and a restricted cubic spline model was used to analyze the dose-response relationship between the years of water improvement and dental fluorosis prevalence.Results:A total of 50 water samples were collected, with water fluoride levels ranging from 0.05 to 0.85 mg/L. All affected villages had completed the water improvement and the water fluoride levels were qualified. A total of 2 439 urine samples were collected from children, with urinary fluoride levels ranging from 0.05 to 12.56 mg/L and a geometric mean of 0.82 mg/L. A total of 2 439 children were examined for dental fluorosis, with a detection rate of 22.67% (553/2 439). The results of multivariate logistic regression analysis showed that age ( OR = 1.26, 95% CI: 1.15 - 1.37, P < 0.001), father's education level of junior high school or below ( OR = 1.57, 95% CI: 1.04 - 2.38, P = 0.033), and high urinary fluoride (0.74 - 1.58 mg/L: OR = 1.59, 95% CI: 1.19 - 2.13, P = 0.002; 1.59 - 12.56 mg/L: OR = 2.00, 95% CI: 1.48 - 2.70, P < 0.001) were risk factors for dental fluorosis prevalence in children. The total annual household income with 40 000 to 80 000 yuan ( OR = 0.76, 95% CI: 0.58 - 0.99, P = 0.041), father's occupation was self-employed and other occupation ( OR = 0.69, 95% CI: 0.51 - 0.92, P = 0.013), the years of water improvement in affected villages ≥10 years ( OR = 0.39, 95% CI: 0.30 - 0.50, P < 0.001), and the material of the drinking water container at home was stainless steel products ( OR = 0.58, 95% CI: 0.40 - 0.85, P = 0.005), ceramic or glass products ( OR = 0.66, 95% CI: 0.48 - 0.90, P = 0.010) were protective factors for dental fluorosis prevalence in children. By constructing a restricted cubic spline model, it was found that there was a linear dose-response relationship between the prevalence of dental fluorosis in children and the years of water improvement in affected villages ( Poverall < 0.001, Pnonlinear = 0.059). The longer the years of water improvement, the lower the risk of dental fluorosis prevalence. Conclusions:The detection rate of dental fluorosis in children in Tianjin is relatively high. The fluoride reduction and water improvement measures implemented in drinking water-borne fluorosis areas can effectively reduce the risk of dental fluorosis prevalence. Factors such as age, urinary fluoride, economic conditions, and lifestyle also have important impacts on the prevalence of dental fluorosis.

Objective:To investigate the awareness of prevention and treatment knowledge of drinking water-borne endemic fluorosis among children in Tianjin, explore its influencing factors, analyze the needs of children's health education methods, and provide a basis for carrying out health education in school.Methods:From January to December in 2024, a stratified sampling method was employed to select two endemic villages from each of the 10 areas with drinking water-borne endemic fluorosis in Tianjin as survey sites. In each village, no fewer than 50 children aged 8 - 12 (gender and age balanced) were recruited to conduct a questionnaire survey. Multivariate logistic regression analysis was used to analyze the influencing factors of children's awareness of prevention and treatment knowledge.Results:A total of 1 678 valid questionnaires were collected. The overall awareness rate of prevention and control knowledge of drinking water-borne endemic fluorosis among children in Tianjin was 67.62% (11 346/16 780). Children had the highest awareness of the hazards of drinking water-borne endemic fluorosis (83.61%, 1 403/1 678), while their awareness of the clinical manifestations of dental fluorosis was the lowest (44.87%, 753/1 678). Multivariate logistic regression analysis showed that children in higher grades [grades 5 to 6, OR (95% CI) = 1.66 (1.36, 2.03), P < 0.001], with a larger number of permanent residents in the family [≥6 people, OR (95% CI) = 1.58 (1.13, 2.23), P = 0.008], whose mothers had a college education or above [ OR (95% CI) = 1.45 (1.08, 1.95), P = 0.014], and who had received health education [ OR (95% CI) = 1.46 (1.19, 1.78), P < 0.001] had a higher awareness rate of the prevention and control knowledge of drinking water-borne endemic fluorosis. There were statistically significant differences in the demand rates for access to prevention and control knowledge via school teachers, promotional videos, and online/WeChat official accounts among children of different grades ( P < 0.05). Conclusions:The awareness rate of prevention and treatment knowledge of drinking water-borne endemic fluorosis among children in Tianjin is relatively low. Special attention should be paid to children with a small number of permanent residents in the family, mothers with low educational levels, and who have not received health education. At the same time, detailed health publicity services should be carried out for children of different grades.

Objective To investigate the regulatory pathway of lipid peroxidation in ferroptosis and the pathogenesis of Parkinson's disease(PD)at animal level.Methods Zebrafish PD model was established by exposure to 200 μmol/L 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).Zebrafish embryo in 48 h after fertilisation were grouped into a control group(n=100),a MPTP group(n=100),and a MPTP+Nomi group(1.5 μg/ml nomifensine,n=100).Behavioral analysis was performed on zebrafish in 5 d after fertilization for movement ability,rate and distance.The expression of α-synuclein and tyrosine hydroxylase in the zebrafish brain was detected by immu-nofluorescence assay to evaluate the activity of dopaminergic neurons(DA).The levels of iron,glutathione(GSH)and lipid peroxidation,which includes catalase(CAT),glutathione peroxidase 4(GPX4),lipid peroxide(LPO),malondialdehyde(MDA)and reactive oxygen species(ROS),were measured to verify whether iron deposition may cause damage to DA through the metabolic regulation mechanism of lipid peroxidation in iron death.Results Compared with the control group,the movement rate and distance were significantly reduced in the MPTP group(P＜0.01),while Nomi treatment obviously increased the movement rate and distance when compared with the MPTP group(P＜0.01).The MPTP group exhibited obvious DA damage in comparison with the control group,and Nomi treatment reversed the damage caused by MPTP.There was no sta-tistical difference in the CAT level in the three group(P＞0.05).The MPTP group had signifi-cantly increased levels of iron,LPO and MDA and decreased levels of GSH and GPX4 when com-pared with the control group(838.18±143.42 vs 478.53±112.58,P＜0.05;1.71±0.11 vs 1.48±0.14,P＜0.05;4.50±0.64 vs 4.23±0.13,P＜0.05;38.93±1.72 vs 45.97±2.32,P＜0.05;0.17±0.03 vs 0.39±0.04,P＜0.05).The MPTP+Nomi group had significantly increased levels of GSH and GPX4 and decreased levels of iron and LPO when compared with the MPTP group(40.79±1.02 vs 38.93±1.72,P＜0.05;0.26±0.05 vs 0.17±0.03,P＜0.05;326.75±110.95 vs 838.18±143.42,P＜0.05;1.01±0.27 vs 1.71±0.11,P＜0.05).The results of the ROS flow cytometry analysis showed that the percentages of ROS positive cells in the control group,MPTP group,and Nomi group were as follows:Histogram:31.6％,46.2％and 31.3％,Scatter plot:62.4％,73.0％and 65.7％,respectively.Conclusion Ferroptosis is found in PD zebrafish induced by MPTP exposure.Our study has clarified at animal level that the damage of DA caused by iron deposition is related to the metabolic mechanism of lipid peroxidation caused by iron death.

Objective To explore the association between different levels of fluoride exposure and dyslipidemia in elderly people, and to analyze the influencing factors and their interactions. Methods A total of 1 143 elderly people over 60 years old were randomly selected from historical high water fluorosis areas and control areas in Tianjin. Logistic regression model and classification tree model were used to analyze the influencing factors of dyslipidemia, and to analyze the interaction between high fluoride exposure and relevant influencing factors on dyslipidemia. Results The prevalence of elevated low density lipoprotein cholesterol (LDL-C) was 5.69% (65/1 143). There was a significant difference in the prevalence of high LDL-C in different fluoride-exposed areas (2 = 0.092，P = 0.762). Multivariate logistic analysis showed that high fluoride exposure (OR=2.306，95%CI：1.185-4.491) and abdominal obesity (OR=2.274，95%CI：1.299-3.978) were risk factors for high LDL-C, while type B personality (OR=0.529，95%CI：0.308-0.908) was a protective factor for high LDL-C. The results of classification tree model showed that abdominal obesity contributed the most to the prevalence of high LDL-C in the elderly, followed by high fluoride exposure and hyperglycemia. There was a significant multiplicative interaction between high fluoride exposure and abdominal obesity on dyslipidemia (OR=5.191,95%CI：1.609-16.745，P=0.006). Conclusion High fluoride exposure may increase the risk of high LDL-C, and there is a multiplicative interaction between high fluoride exposure and abdominal obesity on dyslipidemia.

BACKGROUND:Fibromyalgia syndrome,as a common rheumatic disease,is related to central sensitization and immune abnormalities.However,the specific mechanism has not been elucidated,and there is a lack of specific diagnostic markers.Exploring the possible pathogenesis of this disease has important clinical significance. OBJECTIVE:To screen the potential diagnostic marker genes of fibromyalgia syndrome and analyze the possible immune infiltration characteristics based on bioinformatics methods,such as weighted gene co-expression network analysis(WGCNA),and machine learning. METHODS:Gene expression profiles in peripheral serum of fibromyalgia syndrome patients and healthy controls were obtained from the gene expression omnibus(GEO)database.The differentially co-expressed genes were screened in the expression profile by differential analysis and WGCNA analysis.Least absolute shrinkage and selection operator(LASSO)and support vector machine-recursive feature elimination(SVM-RFE)machine learning algorithm were further used to identify hub biomarkers,and draw receiver operating characteristic curve(ROC)to evaluate the accuracy of diagnosing fibromyalgia syndrome.Finally,single sample gene set enrichment analysis(ssGSEA)and gene set enrichment analysis(GSEA)were used to evaluate the immune cell infiltration and pathway enrichment in patients with fibromyalgia syndrome. RESULTS AND CONCLUSION:Eight down-regulated differentially expressed genes(DEGs)were obtained after differential analysis of the GSE67311 dataset according to the conditions of log2|(FC)|>0 and P<0.05.After WGCNA analysis,497 genes were included in the module(MEdarkviolet)with the highest positive correlation(r=0.22,P=0.04),and 19 genes were included in the module(MEsalmon2)with the highest negative correlation(r=-0.41,P=6×10-5).After intersecting DEGs and the module genes of WGCNA,seven genes were obtained.Four genes were screened out by LASSO regression algorithm and five genes were screened out by SVM-RFE machine learning algorithm.After the intersection of the two,three core genes were identified,which were germinal center associated signaling and motility like,integrin beta-8,and carboxypeptidase A3.The areas under the ROC curve of the three core genes were 0.744,0.739,and 0.734,respectively,indicating that they have good diagnostic value and can be used as biomarkers for fibromyalgia syndrome.The results of immune infiltration analysis showed that memory B cells,CD56 bright NK cells,and mast cells were significantly down-regulated in patients with fibromyalgia syndrome compared with the control group(P<0.05),and were significantly positively correlated with the above three biomarkers(P<0.05).The enrichment analysis suggested that there were nine fibromyalgia syndrome enrichment pathways,mainly related to olfactory transduction pathway,neuroactive ligand-receptor interaction,and infection pathway.The above results showed that the occurrence and development of fibromyalgia syndrome are related to the involvement of multiple genes,abnormal immune regulation,and multiple pathways imbalance.However,the interactions between these genes and immune cells,as well as their relationships with various pathways need to be further investigated.

Objective:To establish a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for accurate determination of plasma Lyso-GL-3 concentration.Method:Solid phase extraction technology was used to process plasma samples, and under positive ion mode and multiple reaction monitoring (MRM) conditions, LC-MS/MS was used to determine the concentration of Lyso-GL-3. The linear range, detection and quantification limits, accuracy, precision, matrix effect, carrier effect of the method, and plasma sample stability were validated. And the accuracy of Lyso-GL-3 positive patients was compared by combining enzymatic and genetic testing results.Result:Lyso-GL-3 had good linearity in the range of 1.25-400 nmol/L. The detection limit and quantification limit were 0.15 nmol/L and 0.50 nmol/L, respectively. The spiked recovery rate was 88.78%-108.96%. The coefficient of variation ( CV) for intra batch precision, inter batch precision, and matrix effect were all less than 15%, the result of carrier effect was 0.55%. Plasma samples could be stably stored for 30 days under refrigeration conditions. The clinical conformity of the patient was 100%. Conclusion:The established LC-MS/MS detection method for plasma Lyso-GL-3 concentration takes 2.5 minutes, which is simple, fast, accurate, and reliable.