Obesity is a highly prevalent chronic disease that is associated with the development of other metabolic comorbidities. Its etiology is complex and multiple risk factors have been reported. In women, weight gain during pregnancy and the effect of pregnancy on subsequent weight gain are important events in women’s history. Both pregnancy and postpartum are critical periods for the development of obesity.To identify sociodemographic and reproductive risk factors associated with obesity in women in their fourth decade of life.Cohort study conducted on women born from June 1978 to May 1979 in Ribeirão Preto, Brazil. Sociodemographic, clinical, and obstetric data were collected by interview and clinical evaluation. Univariable and multivariable binomial logistic regression models were constructed to identify the risk factors of obesity and the adjusted relative risk (RR) was calculated.The cohort included 916 women and 309 (33.7%) of them were obese. Obesity was associated with low educational level  and teenage pregnancy . There was no association of obesity with the other covariates studied.Obesity is associated with years of schooling and teenage pregnancy. In adul30 kg/m2 , 2.It has been increasing in epidemic proportions in both adults and children , 4. The Many chronic conditions that reduce longevity and quality of life are caused or affected by obesity, including diabetes mellitus, systemic arterial hypertension, dyslipidemia, metabolic syndrome (association of these previous conditions), and cardiovascular diseases , 7. The Different scholars mention a lot of predisposing factors which vary depending on geography, social conditions, political and economic factors, and human genetics. In aggregate, the commonest factors were sociodemographic, behavioral, genetic, and living in obesogenic environment. Many of the risk factors are known; however, a major gap in the scientific literature is how these factors are interrelated . KnowingThere are multiple factors and processes that lead to obesity. In fact, the traditional view is usually that the main cause is the excess energy stored in fat cells than the energy the body used. In addition, more and more etiologies or defects that lead to obesity can be identified under the lights of social, genetic, epigenetic, environmental and microenvironment issues .While there has been a plethora of articles about obesity published worldwide , 2, 7, 8Women tend to be more overweight and obese than males around the world and studies have shown that some NCDs have a predilection for women . This woThe primary objective of the present study was to identify sociodemographic and reproductive risk factors associated with obesity in women in their fourth decade of life, from a birth cohort that has been analyzed since 1978/79. The secondary objectives were to identify reproductive risk factors of obesity in women with previous pregnancies, as well as to describe the frequency of obesity and other chronic diseases  in women in their fourth decade of life.This is an analytical, observational cross-sectional study (nested in a cohort study). The sample of the present study consisted of women from the 1978/79 birth cohort conducted in the city of Ribeirão Preto, State of São Paulo, Brazil . This isRibeirão Preto is a city in the State of São Paulo, a rich and industrialized region with a Human Development Index (HDI) of 0.800 in 2010. Its population was 604,682 inhabitants in 2010 . It is oFor the cohort study, the records and charts of the eight maternity hospitals (three public and five private) that attended 98% of all deliveries in the municipality  from JunThis research has been performed in accordance with the Declaration of Helsinki and has been approved by the Research Ethics Committee of the School of Medicine of Ribeirão Preto, University of São Paulo. All stages of this cohort were submitted to the Research Ethics Committee. Before the review of records and charts of the eight maternity hospitals in 1978/79, permission was obtained from all clinical directors and the registry offices. The interviewed mothers from whom data and data of their children were collected provided oral consent and were released by their responsible physicians . The lasWomen who participated in the 2016/17 assessment were included in the present study (n = 929). The participants were invited to come to the research center on a scheduled day and time. Eligible participants received information about the objectives of the study and were invited to sign the free informed consent form. Data collection was started only after the participants had signed the form.2 may not reflect the diagnosis of obesity depending on the gestational age.Women who were pregnant at the time of assessment (n = 2), women who did not undergo collection of the anthropometric data (n = 6), and women who did not answer the questionnaire about obstetric history (n = 5) were excluded. Pregnant women were excluded because the BMI cut of 30 kg/mQuestionnaires including demographic, social, clinical, and reproductive data were applied during the interviews. Sociodemographic and reproductive health-related variables were included in this study. The following sociodemographic variables were collected: race/ethnicity (white and others), paid work (to be engaged in a paid activity at the time of application of the questionnaire), socioeconomic class, stable marital status , educational level , smoking , alcohol misuse , and ilSocioeconomic status was defined according to the ABEP categories . The estThe participants underwent body composition assessment, anthropometry, blood collection, and blood pressure measurement. Trained health professionals collected the data. Information on chronic diseases was obtained by interview. An existing condition was defined when the participant had a diagnosis or was being treated for systemic arterial hypertension, diabetes mellitus, and dyslipidemia. The criteria defined by the National Cholesterol Education Program Adult Treatment Panel III (NCEP-ATP III), revised by the American Heart Association and the National Heart, Lung and Blood Institute (AHA/NHLBI) , were coHeight was measured using a stadiometer graduated in centimeters and fixed to a smooth wall, with the patient standing erect, barefoot, with arms extended along the body, head positioned in the Frankfurt plane  with eyes fixed on a point at eye level, legs parallel and heels, calves, buttocks, shoulder blades and the back of the head  against the stadiometer or wall. Weight was measured using a Welmy ® digital anthropometric scale, with a capacity of 200 kg and accuracy of 100 g. The patient was positioned in the center of the scale, barefoot, erect, arms extended along the body, with feet together and in such a way that the weight was distributed symmetrically, avoiding the support more firmly on one of the legs and with the gaze fixed. at a point on the straight line.2) and classified according to the World Health Organization [Based on measured weight and height measurements, BMI was calculated according to the formula BMI = weight (kg)/height  was used for all statistical analyses. A level of significance of 5% (p < 0.05) was adopted. Missing data were excluded from the analysis.The stage used in this study had 1759 individuals who attended for data collection. Of these individuals, those of the male gender were excluded (n = 846), allowing the analysis of a total of 929 women. Women who were pregnant at the time of assessment (n = 2), women who did not undergo collection of the anthropometric data (n = 6), and women who did not answer the questionnaire about obstetric history (n = 5) were excluded. Then, 916 women were analyzed.Most of them (80.2%) were white, had paid work (90%), lived with a companion (67.2%), had more than 12 years of schooling (45.9%), belonged to socioeconomic class A/B (68%), had a previous pregnancy (77.7%), were non-smokers (88.8%), were not abusive alcohol drinkers (78.4%), and did not use illicit drugs (96.6%) (Table 2) and obesity was 34.7% and 33.7%, respectively. The prevalence of clinical comorbidities was 11.2% for diabetes mellitus, 19.5% for systemic arterial hypertension, 18.3% for dyslipidemia, and 31.2% for metabolic syndrome  continued to be associated with obesity in women with a previous pregnancy when compared to age at first pregnancy over 30 years . The other covariates were not associated with obesity. Table The population consisted of women aged 37 to 39 years from a cohort study conducted in the city of Ribeirão Preto. Participants were recruited by telephone, advertisements through media and on social networks, and searches in a digital environment, strategies that may have been unable to reach marginalized groups. Thus, only 1,775 of 6,973 women were available for data collection. This sample corresponds to 25% of the initial sample, which means a loss of 75% of individuals. This loss can bring an important bias to the results of this study.A significant proportion of the population was overweight (68.4%) and approximately one-third was obese (33.7%). Although different methods were used for data collection, these percentages are much higher than the Brazilian estimates obtained in the same year (2016) by the VIGITEL survey (Surveillance System for Risk and Protective Factors for Chronic Diseases by Telephone Survey), in which the prevalence of overweight was 53.7%, with 17.7% of obese adults and 19% of obese women aged 35 to 44 years .The high prevalence of obesity found here agrees with North American estimates, which were slightly higher than the Brazilian rates. Data from the National Health and Nutrition Examination Survey (NHANES) showed a prevalence of obesity of 40% among adults aged 20 to 39 years in the United States; when stratified by age, there were 40.3% of obese men in this age group and 39.7% of obese women . The higThe difference between the national prevalence of obesity and the prevalence found in this study may be due to some limitations of this cohort follow-up, including the recruitment process, the number of women from the initial cohort who attended the data collection, and the fact that the data were from a single city.This percentage of women who were not followed may have brought some bias to the study, since it may determine a more frequent profile in the study. For example, women with more comorbidities may be more interested in health studies than women without chronic diseases, which would increase the prevalence of obesity in the sample.The characteristics of the women participating in the 2016/17 assessment rendered the population more homogeneous. The majority of these women were white, had more than 8 years of schooling, lived with a companion, had paid work, and belonged to higher socioeconomic classes; these conditions may also have brought some bias to the study. The fact that the study gathered a more homogeneous and biased population, perhaps due to the greater demand for participation in the study by women with chronic diseases, such as obesity, the primary outcome of the study, may have hindered the statistical analysis. For example, this difficulty may have limited the association of obesity with previous pregnancy. Besides, the question that the data were from a single Brazilian city can limit the generalizability of the present findings.The women were divided into two groups according to the presence or absence of obesity to identify factors associated with this disorder. The only associated sociodemographic factor was a low educational level (less than 8 years of schooling compared to more than 12 years) and the only reproductive health-related factor was teenage pregnancy (< 20 years). In the case of women with higher educational level, possible explanations are greater social pressure and better access to weight control and weight loss programs, regardless of whether or not they are healthy , 25. On In contrast to expectations, this study did not find an association between other reproductive factors and obesity. Pregnancy and postpartum are critical periods for the development of obesity; however, although the relationship between maternal pregnancy weight and the risk of becoming obese has been the focus of studies in recent years , 30, theSeveral Brazilian studies aimed to estimate the prevalence of obesity and to identify associated factors. One study reported a prevalence of obesity of 16.8% among men and of 24.4% among women in 2013/2014. Advanced age (over 50 years), low educational level (no schooling or incomplete elementary school), African descent, and living with a partner were risk factors for obesity. Leisure-time physical activity and the habit of watching more than 4 h of television per day had significant effects in both sexes. Regarding morbidity, obese people were more likely to have a diagnosis of hypertension, diabetes, or non-communicable chronic diseases .A study conducted in 2011 in Rio de Janeiro revealed a higher prevalence of abdominal obesity (waist circumference > 80 cm) among women older than 35 years with two or more children. When the analyses were stratified by BMI category, parity and age were no longer significantly associated with abdominal obesity . AnotherOne major strength of this study is its cohort design. Birth cohort studies have been a top priority on the research and technology agenda of developed countries . The assIn summary, the present results are in line with the global scenario of obesity being a highly prevalent disease in adults. In women, obesity is associated with a low educational level and teenage pregnancy. Primary prevention strategies are necessary, and attention must be paid to women with low educational level and to pregnant adolescents. During prenatal care of teenagers, interventions that promote appropriate weight gain are vital to prevent postpartum weight retention because excess gestational weight gain is a strong predictor of maternal overweight and obesity after pregnancy .


Yinlan Tiaozhi capsule (YLTZC) has been widely used to treat hyperlipidemia (HLP). However, its material basis and underlying pharmacological effects remain unclean. The current study aimed to explore the mechanisms involved in the treatment of YLTZC on HLP based on network pharmacology, molecular docking, and experimental verification. Firstly, UPLC-Q-TOF–MS/MS was used to comprehensively analyze and identify the chemical constituents in YLTZC. A total of 66 compounds, mainly including flavonoids, saponins, coumarins, lactones, organic acids, and limonin were characterized and classified. Simultaneously, the mass fragmentation pattern of different types of representative compounds was further explored. By network pharmacology analysis, naringenin and ferulic acid may be the core constituents. The 52 potential targets of YLTZC, including ALB, IL-6, TNF, and VEGFA, were considered potential therapeutic targets. Molecular docking results showed that the core active constituents of YLTZC (naringenin and ferulic acid) have a strong affinity with the core targets of HLP. Lastly, animal experiments confirmed that naringenin and ferulic acid significantly upregulated the mRNA expression of ALB and downregulated the mRNA expression of IL-6, TNF, and VEGFA. In sum, the constituents of YLTZC, such as naringenin and ferulic acid, might treat HLP by regulating the mechanism of angiogenesis and inhibiting inflammatory responses. Furthermore, our data fills the gap in the material basis of YLTZC. The main drugs currently used in the treatment of HLP include statins, fibrates, and cholesterol absorption inhibitors, which have caused many side effects for patients during treatment, such as gastrointestinal tract issues, myopathy, and rhabdomyolysis3. Therefore, it is necessary to find alternative medicines for the treatment of HLP.Hyperlipidemia (HLP) is one of the most common disorders of lipid metabolism, which is closely related to metabolic syndrome, diabetes, obesity, and arteriosclerosis, and is a major risk factor for cardiovascular and cerebrovascular diseasesCitri Grandis Exocarpium, Ginkgo Folium, Gynostemma pentaphyllum, and propolis7. The clinical trials of new drug approval (2012L01011) have been successfully admitted by National Medical Products Administration (NMPA), and is currently in phase II clinical stage. Data from previous studies revealed that the YLTZC showed obvious anti-HLP activity and could regulate lipid levels in various animal models of experimental HLP, including mice, rats, and New Zealand rabbits. The experimental results showed that YLTZC remarkably lowered the levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein cholesterol (LDL-c) in HLP rabbits, mice, and rats, while increasing the levels of high-density lipoprotein cholesterol (HDL-c), and the anti-HLP effect of YLTZC might be related to inhibiting PXR expression, promoting bile acid excretion and RCT processes, and enhancing TG hydrolysis7. In addition, in the treatment of type 2 diabetes mellitus, YLTZC was performed to prohibit the FA β-oxidation, synthesis of cholesterol and phospholipids, gluconeogenesis, and inflammation level, as well as promote TG hydrolysis, glycolysis, and blood circulation8. However, there are still some problems in the related research of YLTZC. On the one hand, its pharmacodynamic substance basis and quality control system are still unclear. On the other hand, most of the current reports are limited to a single constituent and single target, while for the treatment of HLP, YLTZC plays a role primarily by acting on multiple ingredients, targets, and pathways, which means that its potential pharmacological effects are complex. The above-mentioned problems limited the systematic understanding of the mechanism of action of YLTZC. Therefore, it is necessary to establish a rapid and effective method to investigate the material basis in YLTZC, as well as the relationship and mechanism of action between the core active constituents and the core targets.Yinlan Tiaozhi capsule (YLTZC) is a classic traditional Chinese medicine (TCM) prescription and patent formula. It has been clinically used for treating HLP and consists of 9. With the rapid and continuous development of bioinformatics, network pharmacology has become an important approach in the study of TCM, which provides a basis for systematically understanding the complexity between drug action and disease and elucidating potential therapeutic mechanisms11.Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF–MS/MS), with high resolution, high sensitivity, and short analysis time, has become a powerful tool for the qualitative analysis in complex systems of TCMIn this research, we revealed the effective ingredients and predicted the potential targets and signaling pathways of YLTZC for the treatment of HLP by integrating the chemical profile, network pharmacology, molecular docking, and experiment verification. The detailed flowchart is shown in Fig. Methanol, formic acid, and acetonitrile of MS grade were purchased from Thermo Fisher Scientific (United States), and laboratory-deionized water generated by a Milli-Q system (United States) was used in all experiments.YLTZC (Batch No. 20211101) was provided by Guangdong Efong Pharmaceutical Co. Ltd. . Reference substances including vicenin-2, luteolin, naringenin, isomeranzin, ferulic acid and pinocembrin were all purchased from Chengdu Herbpurify Co., Ltd . Gallic acid, protocatechuic acid, rutin, naringin, ginkgolide A, ginkgolide B, limonin, and chrysin were all purchased from National Institutes for Food and Drug Control . Triton WR-1339 was purchased from Sigma-Aldrich .YLTZC was ground into powder, 0.25 g of powder was accurately weighed and extracted with 50 ml 50% aqueous ethanol under ultrasonication for 30 min. The extract solution was centrifuged at 12,000 rpm for 10 min at 25 °C. And then, the supernatant was filtrated through 0.22 μm filter membrane and 1.0 μl of filtrate was injected into UPLC-Q-TOF–MS/MS of analysis. 14 reference standards  were weighed in appropriate amounts, dissolved in methanol and filtered by 0.22 μm filter membrane to prepare 0.3 mg/ml stock solution, respectively.18 column  at 30 °C, with mobile phases A (0.1% formic acid) and B (acetonitrile). The flow rate was 0.3 ml/min, and the gradient profile was showed as follows: 0–3 min, 8–25% B; 3–12 min, 25–47% B; 12–25 min, 47–79% B; 25–32 min, 79–95% B; 32–35 min, 95% B; 35–36 min, 95–8% B. The sample injection volume was 1 μl.Liquid chromatography analysis was conducted by using a SHIMADZU ExionLC system (Japan). Chromatographic separation was used Waters ACQUITY BEH Cm/z 50–1000. Data were collected in information-dependent acquisition mode, and the instrument was recalibrated every four hours in order to exclude dynamic background. All data collected and analyzed by SCIEX OS v2.1 software .The MS analysis was acquired using an AB SCIEX X500R Q-TOF–MS/MS system (United States) with an electrospray ionization (ESI). The results of the mass spectrometry optimization conditions are as follows: ion voltage: − 4.0 kV and + 5.5 kV, Gas1 (nebulizer gas): 55 psi; Gas2 (heater gas): 55psi; curtain gas: 35 psi; declustering potential voltage: 80 V; ion source temperature: 500 °C; collision energy: 60 V; collision energy spread: 15 V; scan range: https://www.cnki.net/), SciFinder (https://scifinder.cas.org/) and literature searches, etc. Then, a self-built database of YLTZC chemical constituents containing component names and molecular formula information was established  was used for analyzing active constituents with absorption, distribution, metabolism, and excretion (ADME) properties and druglikeness evaluation to screen for active constituents with potential therapeutic effects. We applied in this study screening criteria of (1) pharmacokinetics “high” and (2) druglikeness (DL) with more than two “yes”12.The SwissADME tool (http://www.swisstargetprediction.ch/), and targets with probability greater than 0 were selected. The HLP-related targets were selected from CTD (http://ctdbase.org/) and GeneCards (https://www.genecards.org/) database using the keywords “hyperlipidemias” and “hyperlipidemia”. Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/) was used to identify the overlapped targets between YLTZC-related targets and HLP-related targets. The common targets were entered into the STRING database (https://string-db.org), and biological species was set to Homo sapiens and the confidence > 0.413. Cytoscape 3.7.1 was used to construct the component-target-disease (C-T-D) network and Protein–Protein Interaction (PPI) Network14.In this research, the targets of active constituents in YLTZC were searched by Swiss Target Prediction database (https://david.ncifcrf.gov/), and were selected for “OFFICE_GENE_SYMBOL” and “Homo sapiens”17. The GO enrichment analysis included the following 3 categories: biological process (BP), cellular component (CC), and molecular function (MF). The top 10 enriched GO entries and the top 20 enriched KEGG pathways were selected, and uploaded to the Bioinformatics (http://www.bioinformatics.com.cn/) cloud platform for data visualization. Cytoscape 3.7.1 was used to construct the component-target-pathway (C-T-P) network, and the core active constituents were obtained through the C-T-P network.The GO and KEGG pathway enrichment analysis of the common targets involved in the PPI network were performed using the DAVID 6.8 database (https://www.rcsb.org/) and the 3D structure of compounds were acquired from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/)18, and docking validation after hydrogenation and dehydration was carried out by AutoDockTools (version 1.5.6) and AutoDock Vina (version 1.1.2). Proteins should be selected from human proteins with one or more cocrystallized ligands and crystal structures with small “resolution” value19. Due to the still unclean functional binding sites of HLP-related targets to ingredients of YLTZC, all binding sites were restricted to the docking pockets region predicted by DeepSite software (https://www.playmolecule.com)20. Lastly, the molecular docking results were visualized into Pymol (2.5.0) software.The core targets and the core active constituents were obtained from the PPI and C-T-P network for molecular Docking. The protein PDB files of the core targets were obtained from the RSCB PDB online platform , and all animal experiments were performed in accordance with relevant ARRIVE guidelines. The 30 male KM mice weighing between 18 and 22 g were obtained from the Guangdong Medical Laboratory Animal Center . All animals were housed in barrier system at standard room temperature and a 12 h light/dark cycle conditions and fed normal food and water. The food was supported by Guangdong Medical Laboratory Animal Center . The experiment mice were divided into the following five groups with six mice in each: control group, model group, fenofibrate group (26 mg/kg), ferulic acid group (50 mg/kg), and naringenin group (50 mg/kg). The administration groups were given corresponding drugs by gavage, once a day, for 5 days. On the third day of administration, triton WR-1339 (480 mg/kg) was administered intramuscularly to all groups except the normal control group to establish a model of acute HLP. On the fifth day, one hour after gavage administration, all mice were anesthetized with isoflurane, sacrificed by inner canthus artery exsanguination, and the organs were retained for analysis.The serum sample was obtained by centrifugation of blood at 3000 rpm at 4 °C for 10 min and preserved at − 80 °C until analysis. TC, TG, HDL-c and LDL-c levels of serum were measured by Microplate Reader  with assay kits were purchased from Nanjing Jiancheng Bioengineering Institute .−ΔΔCt method, and Gene-expression data were normalized to that of the internal control GAPDH. The primer sequences are shown in Table About 50 mg of liver was weighed and transferred into a 1.5 ml grinding tube. After adding 500 µl Trizol reagent  and 2 grinding beads to the grinding tube, the liver was crashed by a freezing grinder . The grinding liquid was centrifuged  and the supernatant (about 400 µl) was transferred into a 1.5 mL centrifuge tube. 100 µl chloroform was added to the centrifuge tube and they were fully mixed by sharking. After centrifuging at 12,000 rpm at 4 °C for 10 min, the supernatant (about 200 µl) and the equivalent isopropyl alcohol were added into a 1.5 mL centrifuge tube and stored overnight in a − 20 °C refrigerator. The crushed total RNA was settled at the bottom of the centrifuge tube after centrifugation at 12,000 rpm at 4 °C for 10 min. After washing with 75% ethanol 2 times, the pure total RNA was gained and dissolved in 50 µl RNase-free water. The content of total RNA was measured by UV and 500 ng total RNA was reverse transcribed by using the Evo M-MLV RT Premix for qPCR . RT-qPCR reactions were performed on iQ5 Multicolor Real-Time PCR detection system  with SYBR Green Dye detection. The relative gene expression was determined by the 2p < 0.05 was considered statistically significant.Results were expressed as the mean ± standard deviation (SD), and analyzed by SPSS 20.0 software. Statistical significance was assessed using one-way analysis of variance (ANOVA). The value of 32. The main chemical constituent cluster was composed of 42 flavonoids, 6 saponins, 4 coumarins, 4 lactones, 9 organic acids and 1 limonin. Meanwhile, the identified constituents were attributed and classified based on the sources of their medicinal materials. Among them, 14 constituents  were determined by comparing with the reference standards. An additional 52 constituents were identified by comparing relevant literatures and mass fragmentation patterns.The mass spectrum data of YLTZC were gained by UPLC-Q-TOF–MS/MS. As shown in Table Citri Grandis Exocarpium and Ginkgo Folium, in the form of free or glycoside, and etc. The identification process was analyzed by taking peak 5, peak 12, and 55 as examples. In negative ion mode, peak 5 had [M − H]− ions at m/z 593.148 9, its MS/MS fragment ions at m/z 473.105 3 [M − H-C4H8O4]−, m/z 383.073 8 [M − H-C4H8O4-C3H6O3]−, m/z 353.063 4 [M − H-2C4H8O4]−, and m/z 353.063 4 [M − H-C4H8O4-C3H6O3-CH2O]− were further pointed out by comparing with the reference standard and literature21. Thus, peak 5 was deduced as vicenin-2, the detailed fragmentation pathway of peak 5 is shown in Fig. m/z 327.085 9 [M − H]−, with m/z 253.047 9 [M-C3H5O-H2O]−, m/z 209.060 2 [M-C3H5O-H2O-CO2]−, m/z 181.065 8 [M-C3H5O-H2O-CO2-CO]−, m/z 185.060 5 [M-C3H5O-H2O-C3O2]−, m/z 165.070 9 [M-C3H5O-H2O-2CO2]− being the main fragment ions in negative ion mode. Based on the reports in literature24, peak 55 was deduced as pinobanksin-3-O-propionate, the detailed fragmentation pathway of peak 55 is shown in Fig. A total of 42 flavonoids  were identified from YLTZC, and the flavonoids mainly came from propolis, Gynostemma pentaphyllum. These compounds mainly exist in the form of anion [M + HCOO]−, and the cracking law is mainly manifested as loss of –C2H2O, –C4H4O and glycosidic. For instance, the mass loss of 162 Da, 146 Da and 132 Da represents the loss of glucose (Glc), rhamnose (Rha), and xylose (Xyl), respectively. Peak 53 had [M − H]− and [M + HCOO]− ion at m/z 799.482 6 and m/z 845.489 4 in negative ion mode. The fragment ions at m/z 637.431 1 [M − H-Glc]− and m/z 475.379 9 [M − H-2Glc]− were formed by the [M − H]− ion following succession to lose of Glc residue. Based on the reports in literature, it was tentatively characterized as gypenoside LXXIV, the detailed fragmentation pathway of peak 53 is shown in Fig. − and [M + HCOO]− ion at m/z 783.488 2 and m/z 829.492 8 in negative ion mode. The fragment ions at m/z 621.438 0 [M − H-Glc]− and m/z 459.382 0 [M − H-2Glc]− were formed by the [M − H]− ion following succession to eliminate Glc residue. Hence, it was tentatively characterized as ginsenoside-F2, the detailed fragmentation pattern of ginsenoside-F2 is shown in Fig. 22.Six saponins  were detected from YLTZC, all from Citri Grandis Exocarpium, and most coumarins have stronger response in the positive ion mode. Peak 38 exhibited [M + H]+ ion at m/z 261.111 3M + H]+, with m/z 189.053 9 [M + H-C4H8O]+, m/z 159.044 8 [M + H-C4H8O-CH2O]+, m/z 131.049 1 [M + H-C4H8O-CH2O-CO]+ as the fragment ions in positive mode, by comparing with the reference standard, peak 38 was deduced as isomeranzin. Peak 20, and 38 are a group of isomers, based on the reports in literature, peak 20 was deduced as meranzin. In positive ion mode, peak 66 had [M + H]+ ions at m/z 299.164 7, and the main fragment ions at m/z 163.040 0 [M + H-C10H16]+, m/z 145.103 8 [M + H-C10H16-H2O]+, m/z 135.042 6 [M + H-C10H16-CO]+, m/z 107.050 0 [M + H-C10H16-2CO]+, by based on the reports in literature33, peak 66 was inferred as auraptene. The detailed mass fragment pathway of peak 66 is shown in Fig. Four coumarins  were identified from YLTZC, all of which were derived from 9 M + H-CH8O+, m/zGinkgo Folium. Ginkgolides have similar cleavage pathways, and the typical cleavage pathways is the opening of the lactone ring, with continuous loss of CO, CO2, and H2O. Peak 22, and peak 23 were identified as ginkgolide A, and ginkgolide B by comparing with reference standards. Taking peak 22 as an example in detail to illuminate the MS fragmentation pattern of these diterpenoid lactones23. Peak 22 had [M − H]− ions at m/z 407.134 1 in negative ion mode. And then [M − H]− ion continuous loss of CO, CO2, and loss of H2O to form m/z 351.143 6 [M − H-2CO]−, m/z 333.132 1 [M − H-2CO-H2O]−, m/z 319.144 0 [M − H-2CO2]−, m/z 307.160 3 [M − H-2CO-CO2]−, m/z 289.143 3 [M − H-2CO-CO2-H2O]−, m/z 273.148 1 [M − H-2CO2-CO-H2O]−, m/z 245.154 0 [M − H-2CO-H2O-2CO2]−. Therefore, the compound was identified as ginkgolide A. The detailed mass fragment pathway of peak 22 is shown in Fig. Four lactones  were detected from YLTZC, all of which were derived from − ion at m/z 153.018 9 [M − H]−, with m/z 109.028 5 [M − H-CO2]−, m/z 91.018 9 [M − H-CO2-H2O]− as the fragment ions in negative mode, by comparing with the reference standard24, peak 2 was deduced as protocatechuic acid, the detailed mass fragment pathway of peak 2 is shown in Fig. A total of nine organic acids  were identified. Taking peak 2 as an example, peak 2 exhibited [M − H]We performed an in-depth assessment of the absorption, distribution, metabolism, and excretion-related properties of 66 constituents in YLTZC using the online tool SwissADME. A total of 38 constituents in YLTZC were screened from the SwissADME tool  KEGG pathways were shown in Fig. Gene Ontology (GO) and KEGG pathway enrichment analysis were undertaken on the 52 common targets mentioned above using the DAVID 6.8 database. The top 10 GO enrichment analysis results listed in BP, MF and CC are shown in Fig. 34. Naringenin and ferulic acid had good affinity with the core targets, which was consistent with the results of literature reports, indicating that naringenin and ferulic acid have good anti-HLP effects. Based on these data, we suggested that the core active constituents have a good affinity for the core targets, which also demonstrated that YLTZC exerted its efficacy through multi-target combination.Based on the PPI and C–T–P network, we selected molecular docking between the 2 core active constituents and the top 5 core targets. The docking score and local structure of the results are presented in Table p < 0.01) were significantly increased in model group compared with the control group, and the levels of HDL-C (p < 0.05) was decreased. Compared with the model group, the levels of serum TC, TG, and LDL-c  were reduced in the naringenin and ferulic acid group, and the levels of serum HDL-C (p < 0.01) was significantly increased. These results suggested that naringenin and ferulic acid from YLTZC have good effect on improving blood lipid levels.Firstly, we investigate ferulic acid and naringenin on serum lipid levels in acute hyperlipidemia in triton WR-1339-induced mice. As shown in Fig. p < 0.01), while naringenin and ferulic acid treatment markedly reversed these key targets. As expected, naringenin and ferulic acid treatment significantly increased ALB mRNA expression and decreased IL6, TNF and VEGFA mRNA expression compared to the model group.To clarify the molecular mechanism of YLTZC treatment for HLP, the mRNA expressions of the targets predicted above were measured by RT-PCR. As shown in Fig. In conclusion, these results demonstrated that naringenin and ferulic acid from YLTZC may modulate the mechanism of angiogenesis and inhibiting inflammatory responses by regulating the expression of ALB, IL-6, TNF-α, and VEGFA to alleviate HLP.1. In our previous study, YLTZC showed a strong hypolipidemic effect, the results showed that YLTZC significantly decreased the levels of serum TC, TG, and LDL-c, and enhanced the level of serum HDL-c in HLP mice7. However, previous studies provided clues for the current study, but were not comprehensive, and relevant studies on the material basis and related mechanisms of action of YLTZC in the treatment of HLP are still lacking. This limits the further clinical studies and quality control and evaluation of YLTZC. Therefore, this study fills these shortages by developing a comprehensive research method combining chemical profile with network pharmacology, molecular docking, and experimental verification.HLP is one of the leading risk factors for the development and progression of cardiovascular diseases, characterized by elevated TC, TG, LDL-c, and decreased HDL-cIn recent years, along with the generalization of systems biology, network pharmacology has become an important method to clarify the potential mechanisms of multiple constituents, targets, and pathways of TCM. In this study, the chemical constituent of YLTZC was comprehensively characterized by UPLC-Q-TOF–MS, and a total of 66 constituents, including naringenin, and ferulic acid, were identified. The results of the study provide more information on the chemical substance basis for further studies of YLTZC. According to the C-T-P and PPI network analysis, naringenin and ferulic acid were discovered to be the core active constituents associated with the most targets, and the HLP-related core targets of YLTZC were ALB, TNF, IL6, and VEGFA. In this study, enrichment analysis of GO and KEGG pathways was performed on 52 common targets. GO function enrichment analysis results showed that YLTZC treatment of HLP may involve the following BP: lipid metabolic process, positive regulation of smooth muscle cell proliferation, positive regulation of MAPK cascade, and positive regulation of inflammatory response. We hypothesized that the response to lipid metabolic process and inflammatory may be the most important BP in the treatment of HLP by YLTZC. KEGG pathway enrichment analysis results demonstrated that HIF-1 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, PPAR signaling pathway, PI3K-Akt signaling pathway, IR, and TNF signaling pathway were highly involved in YLTZC treatment of HLP. These pathways are highly associated with the regulation of angiogenesis and anti-inflammation systems, which may be significant pathways for the alleviation of the symptoms of HLP. Consequently, our findings suggest that the modulating the mechanism of angiogenesis and inhibiting inflammatory are potential therapeutic strategies of YLTZC for the treatment of HLP.36. It has been shown that naringenin prevents HLP, IR, and atherosclerosis by decreasing TC, TG, and LDL and increasing HDL37. In addition, naringenin inhibited fibroblast activation and inflammatory cell recruitment. The mRNA and protein expression levels of TNF‑α, IL‑1β, IL‑6, and TGF‑β1 were downregulated following naringenin treatment38. Meanwhile, some studies have found that the effects of naringenin are related to the activation of PPARs, which can regulate hepatic lipid metabolism at the transcriptional level in human and rat by activating PPARα, PPARβ, or PPARγ, respectively, and reduces serum lipids in HLP rats40. Ferulic acid has been shown to have anti-HLP, antioxidant, and anti-inflammatory effects. Compared with the placebo, the ferulic acid supplementation showed a statistically significant decrease in TC, LDL-c, and TG, and increased HDL-c42. In addition, ferulic acid could inhibit the expression of several pro-inflammatory cytokines including IL-1β, TNF-α, IL-10, and IL-6, and also inhibit angiogenesis by reducing the expression of VEGFA mRNA45. In our study, we established a triton WR-1339-induced HLP mice model, supplied with core active constituents of YLTZC for confirming its hypolipidemic effect. The results showed that naringenin and ferulic acid treatment significantly decreased the levels of serum TC, TG, and LDL-c, and enhanced the level of serum HDL-c in HLP mice. Therefore, naringenin and ferulic acid as the two core active constituents may be the potential material basis for YLTZC to alleviate HLP.Naringenin has an antioxidative activity to relieve oxidative stress, alleviate IR, and inhibit the production of inflammatory mediators46.On the one hand, YLTZC may treat HLP by regulating the mechanism of angiogenesis. VEGFA was found to be a significant factor in the regulation of vascular endothelial cells. Vascular remodeling during atherosclerosis was also associated with the expression of VEGFA, which has the effect of inducing angiogenesis, promoting their survival, and enhancing vascular permeability47. IL6 is also a pro-inflammatory cytokine that plays a crucial role in inflammation, atherosclerosis, and thrombosis, and can influence the rate of lipid metabolism, specifically the metabolism of TCs and TGs48. ALB is an important substance for maintaining plasma colloid osmotic pressure, and studies have found that ALB levels are closely related to cardiac function, and play a critical role in regulating the osmotic pressure and metabolic processes49. Meanwhile, studies have also shown that ALB is the most important carrier/transporter protein in vivo and plays an essential role in plasma antioxidant activity. In addition, in the HLP model, the decreased levels of ALB may be closely associated with the decline of liver function leading to reduced liver production51. Therefore, we speculate that ALB is mainly used as a drug transport platform to treat HLP by YLTZC. PPARs play a vital role in regulating the systemic inflammatory response, and they also modulate several biological processes that perturbed obesity, including inflammation, lipid and glucose metabolisms52.On the other hand, YLTZC can alleviate HLP by regulating inflammatory and oxidative stress targets. For example, TNF-α is one of the most significant pro-inflammatory mediators and a critical factor in IR, which is involved in the pathophysiology of various CVDs53. Activation of the AGE-RAGE signaling pathway can trigger the production of tissue factors, and inflammatory factors54. Moreover, the PPAR signaling pathway plays an essential part in cholesterol metabolism and cholesterol efflux55. IR is associated with hyperinsulinemia and HLP56. These pathways are highly involved in YLTZC treatment of HLP, among which the PI3K-Akt signaling pathway, PPAR signaling pathway, and IR play critical roles in insulin secretion and lipid metabolism. Furthermore, fluid shear stress and atherosclerosis, the HIF-1 signaling pathway, and the TNF signaling pathway are highly participated in angiogenesis, pro-inflammatory factor secretion, and vascular tone regulation48.The HIF-1 signaling pathway is primarily involved in maintaining the steady state of oxygen in the body, and is also engaged in regulating angiogenesis and inflammationp < 0.01), and decreased the mRNA expression of ALB (p < 0.01) in the model group. However, compared with the model group, the naringenin and ferulic acid groups inhibited the mRNA expression of IL-6, VEGFA, and TNF-α (p < 0.01), and promoted the mRNA expression of ALB . Therefore, we reasoned that YLTZC can effectively alleviate HLP by modulating the mechanism of angiogenesis and inhibiting inflammatory responses. The results of molecular docking and experimental verification were consistent with the predicted results of network pharmacology, indicating the accuracy of this method in screening the active constituents and action targets of YLTZC.In summary, we identified 2 core active constituents in YLTZC and the potential targets and pathways underlying the effects of YLTZC in HLP using the network pharmacology method. The results identified some pathways and biological processes that could be related to the lipid-lowering effects of YLTZC. To further validate the feasibility of network pharmacology analysis, IL-6, TNF-α, VEGFA, and ALB were selected as candidate targets of YLTZC against HLP. The molecular docking verified that the core active constituents have good binding properties with the core targets. In vivo experiment, compared with the control group, the mRNA expression of IL-6, VEGFA, and TNF-α mRNA were significantly increased (In this study, we revealed the therapeutic effect and underlying mechanism of YLTZC against HLP based on network pharmacology, molecular docking, and experimental validation. 66 chemical constituents of YLTZC were rapidly characterized by UPLC-Q-TOF–MS/MS. Based on the network pharmacology approach, the core active constituents including naringenin and ferulic acid, as well as core targets such as ALB, TNF, IL6, and VEGFA were screened for YLTZC for the treatment of HLP. Functional enrichment analysis through GO and KEGG pathways demonstrated the regulation of lipid metabolism, angiogenesis, and anti-inflammation systems, which may be important pathways in alleviating HLP. Molecular docking verified the possibility of the core active constituents binding to the core targets. In vivo experiments further showed that the hypolipidemic mechanisms of YLTZC were associated with the down-regulation of TNF-α, IL6, and VEGFA mRNA expression levels, and the up-regulation of ALB mRNA expression levels. Collectively, this work demonstrated that YLTZC may act against HLP by modulating the mechanism of angiogenesis and inhibiting inflammatory responses, and provided an efficient way to understand the active constituents and underlying mechanisms of YLTZC.Supplementary Figure S1.Supplementary Table S1.


Background: Thyroid metastases (TMs) are a rare entity, ranging between 0 and 24% in the autopsy series. In the assessment of the best management, the discrimination between a primary and a metastatic thyroid lesion is crucial. In this regard, fine needle aspiration cytology (FNAC) is likely to play a crucial role especially when ancillary techniques  and molecular testing) are carried out. Methods: We searched for all the TMs diagnosed using FNAC and analyzed between 2014 and 2023. The cases were processed with liquid-based (LBC) and ICC and molecular testing performed on LBC-stored material. Results: We reported 2.2% (19 cases) of TMs out of 1022 malignancies. TMs included: 1 larynx carcinoma (LX-Ca), 1 melanoma, 2 breast carcinomas (B-Ca), 3 lung carcinomas (LG-Ca), 4 gastro-intestinal carcinomas (GI-Ca), and 8 clear cell renal carcinomas (CCRC). All patients had a previous cancer history, between 300 and 2 months from the primary cancers. The morphological features were supported by ICC, which were contributive in 100% of cases. All TMs cases were characterized by multiple thyroid nodules except the melanoma case. Four cases underwent total thyroidectomy  whilst 15 TMs were treated with radio-chemotherapy. Conclusions: FNAC empowered the diagnostic workup of patients with TMs avoiding useless surgery. The low sensitivity of cytology might be reinforced by the application of ancillary techniques. We found a predominant rate of kidney metastatic carcinomas, followed by lung and breast. TMs are frequently multifocal and in a context of a systemic disease so a tailored therapy seems to be the best treatment. In these last decades, fine needle aspiration cytology (FNAC) has clearly shown its crucial diagnostic role in the evaluation of thyroid nodules ,2,3,4,5.In the spectrum of cytological diagnoses, the majority of thyroid nodules are diagnosed as primary thyroid entities using FNAC even though a possible metastatic localization to the thyroid gland should be always kept in mind especially when the morphological features are not univocally in favor of a primary thyroid entity. Not only should the lesions be associated to a previous or current history of malignancy, but they are likely to represent the first evidence of the disease ,8,9,10. Some papers, especially those related to autopsy series, documented that the estimated incidence of thyroid metastatic lesions ranges between 0 and 24%. Furthermore, as emerged from a multi-institutional study of 62 secondary neoplasms to the thyroid gland, the most frequent metastatic diseases seem to be colon, kidney, breast, and lung cancers ,14,15,16Despite the fact that thyroid metastases (TMs) may represent an unexpected finding, either cytological or histological series have assessed that TMs account for 1.4% to 2.5% of all thyroid cancers ,19,20,21In 2014, our group published the results of a TMs series, diagnosed in the period between 2000 and 2013 [TM . Each FNAC was performed with two passes for each lesion, using 25 to 27 G needles without any rapid adequacy assessment of the material.We conducted a retrospective, computerized search of all metastatic thyroid cytological cases recorded in the files of the Division of Anatomic Pathology and Histology of the Catholic University, “Agostino Gemelli” Hospital of Rome  between January 2014 and February 2023. A global cohort of 19 (1.8%) cases out of 1022 total thyroid malignancies were diagnosed as positive for malignancy (PM), favoring a metastatic localization, using liquid-based cytology (LBC). We searched for cases with a known history of malignancies or primary suggested diagnoses of TMs with FNAC. In our institution, all the nodules were evaluated under sonographic guidance (US) by pathologists, surgeons, and endocrinologists. All the cases were processed with the LBC method Thin Prep 2000The size of nodules ranged from 0.7 cm to 7.0 cm and they were all found during routine US thyroid check-ups performed in the Centre for Thyroid Diseases of the Departments of Endocrinology and Endocrine Surgery of the Catholic University. Regarding the use of the LBC method, all the patients had been appropriately informed for processing their aspiration samples and a written informed consent was signed by each of them. Our study followed the tenets of the Declaration of Helsinki and we received the internal ethics approval for the study.TM solution at room temperature for 3 to 4 months to be used for eventual additional investigations or preparation of additional slides. These ancillary techniques can be performed when the remaining material is at about 2 mL eluted in 5 mL of PreservCyt solution. The adequacy was defined, according to the British RCPath classification, by six groups of epithelial cells within the submitted slides, each of them with at least 10 well-visualized epithelial cells [Concerning the LBC method, the aspirated material was processed according to the manufacturer’s suggestions and following the standard method that we have been using since the introduction of LBC in our institution in 2000. The resulting slide was fixed in 95% ethanol and stained with Papanicolaou while the remaining material was stored in the PreservCytal cells .At the beginning, the cytological cases were classified according to the Italian Working Group SIAPEC-IAP classification and then reclassified for scientific purposes, according to the Thyroid Bethesda System. Albeit the differences in classification systems, the selection of metastatic lesions did not incur any discrepancies, and they all belonged to the malignant category regardless of the different classification systems ,18.Patients with lymphomas and malignant lesions that invade the thyroid to contiguity were excluded from our series. We excluded any thyroid lymphoma because, as for the previous paper, we wanted to compare the epithelial metastases. Furthermore, we did not include any thyroid infiltration from a contiguous malignancy because we wanted to analyze the thyroid metastatic lesions mimicking a thyroid primary neoplasm instead of analyzing an infiltrative process to the thyroid. In our institution, all the cytological and histological sections were reviewed by two expert cytopathologists and those cases whose interpretation was equivocal were submitted to diagnostic judgment until a final agreement was achieved. In this specific series, there was not any disagreement that needed to be re-evaluated. The follow-up included a period between 2 and 120 months.Immunocytochemical stainings were carried out with the avidin-biotin peroxidase complex using a selection of specific antibodies mostly based on the morphological diagnostic hypotheses. The immunocytochemical method was standardized according to the manufacturer’s protocol adopted in our institution and previously published ,22. PosiThe surgical specimens were fixed in 10% buffered formaldehyde, embedded in paraffin and the 5 micron-thick sections were stained with haematoxylin-eosin. The concordance of immunohistochemistry between the primary surgical carcinomas and TM cytological samples was 100%. All the fibro-adipose tissue close to the thyroid gland was included for lymph node research and evaluation.p value less than 0.05 were considered significant.Statistical analysis was performed by using a commercially available statistical software package  for iOS . The comparison of categorical variables was performed by Fisher’s exact test when appropriate. A During the reference period, between January 2014 and February 2023, we found 19 metastatic thyroid FNACs out of 30,006 thyroid FNACs (0.06%). Those metastatic lesions represented 1.8% of 1022 thyroid malignant diagnoses. The series comprised 13 (68.4%) female and 6 (31.6%) male patients with a median age of 65 years old . No signIn In p for the CCRC. In fact, the current series is characterized by a high number of CCRCs, followed by colon adenocarcinoma. Notably, the gap between the primary RCC and the metastatic localization ranged from 20 to 30 years.The conclusive diagnosis resulted from a combination of morphological features, IIC yields, and clinical history in all our 19 cases. Notably, none of the cases was positive for Thyroglobulin , confirmThe main purpose of the current paper is to evaluate our institutional experience in the diagnosis of TMs, with reference to the period between 2014 and 2023. Furthermore, we correlate the yields with our previous results referring to a published series of TMs in the period between 2000 and 2013 [Unexpectedly, in the first series, we found a predominant number of gastrointestinal metastatic adenocarcinomas and only one clear cell renal carcinoma. Based on those data, we decided to compare them with our further cases from 2014 to 2023 and to discuss similarities and/or differences between the two series.Data from the autopsy series have documented that TMs range between 0 and 24% of thyroid nodules, whilst according to the clinical series, mostly published by Cheung et al. and Montero et al. ,8, the rAccording to our results, we found 2.2% TMs in the first series versus 1.8% in the second reference period. Apart from slight differences, these two figures showed an overlap in the numbers of TMs, with quite stable figures in the different decades. In fact, these percentages, belonging to two consecutive decades, have confirmed the trend that the thyroid gland represents a rare metastatic site for many different primary carcinomas despite its rich vascular structure and supply ,29,30,31Few papers have discussed the role of FNAC in the diagnosis of TM, mostly suggesting that the crucial step is represented by the ability to obtain the adequate amount of diagnostic neoplastic cells ,9. Both p < 0.0001) [In a series by Wysocka et al., the authors found TMs and nodal metastases (NM) in 57 patients . The authors assessed that their frequency was higher in the group of patients with a known history of primary cancer compared with the group of unknown primary lesions  .A revision of literature that referred to the years before 2014, reported a medium number of TMs ranging from 14 and 22 cases over a period between 55 years and 7 years . Both ouDespite the fact that different papers highlighted an equal gender distribution of TMs, we have to underline a slightly higher incidence in the female population. In agreement with the majority of the series, we have also documented a presentation in the sixth or seventh decade of life. Nonetheless, concerning the TMs from the current series, we found some significant discrepancies with the results obtained in the first period. In detail, the current series supported the evidence that CCRC is the most frequent thyroid metastasis 42.1%) representing a cause of common pitfalls. In a previous multicenter study, including 7 United States and European medical centers, also involving our institution, 3 out of 9 CCRC cases were misdiagnosed as follicular neoplasms [% represeHence, in our series, as also documented in other publications, only one case diagnosed as a metastatic melanoma was defined by a single solitary nodule, characterized by single-patterned and pleomorphic cells, which might lead to several differential diagnoses, including anaplastic thyroid carcinoma. Despite the fact that 100% of our cases were diagnosed as malignant-favoring a metastatic localization, the diagnosis of the primary carcinoma/adenocarcinoma cannot be further classified by the morphological evaluation alone. Regardless of the previous clinical history, the support of ancillary techniques, especially ICC, performed on LBC-stored material, is useful for making the definitive diagnosis ,26,27,28The last point is dedicated to the management of thyroid metastases, which is, however, not univocally defined by specific surgical guidelines. Whilst some papers suggest a lobectomy in cases of solitary thyroid metastasis, others propose total thyroidectomy in a multifocal metastatic process ,32. NotwIn conclusion, for those cases with a primary known cancer, FNAC shows high accuracy in the diagnosis of TM, especially when it is combined with the application of ancillary techniques which may facilitate to tailor the best and appropriate management (including chemotherapy and radiotherapy) for a metastatic patient ,34,35,36