Compliance was good for all CFTR modulators, azithromycin, aztreonam, and tobramycin in solution for inhalation. The patients with the best compliance were older; they had exacerbations and the greatest deterioration in lung function during this period. The three variables with the highest importance for the compliance of the generated Random Forest (RF) models were age, FEV1%, and use of Ivacaftor/Tezacaftor. This is one of the few studies to assess adherence to CFTR modulators and symptomatic treatment longitudinally. CF patient therapy is expensive, and the assessment of variables with the highest importance for a high MPR, helped by new Machine learning tools, can contribute to defining new efficient TA strategies with higher benefits.Cystic fibrosis (CF) is a genetic and multisystemic disease that requires a high therapeutic demand for its control. The aim of this study was to assess therapeutic adherence (TA) to different treatments to study possible clinical consequences and clinical factors influencing adherence. This is an ambispective observational study of 57 patients aged over 18 years with a diagnosis of CF. The assessment of TA was calculated using the Medication Possession Ratio (MPR) index. These data were related to exacerbations and the rate of decline in FEV Cystic fibrosis (CF) is a genetic and multisystemic disease that requires significant therapeutic demands for its control, both in terms of nutritional recommendations and physical activity, as well as compliance with multiple treatments, with an estimated daily average of up to seven . The plaUntil a few years ago, the existing therapies were only aimed at alleviating the symptoms caused by the disease: physical exercise, physiotherapy, bronchodilators, hypertonic or mucolytic substances to improve mucociliary clearance, immunomodulators, and antibiotics to control inflammation and bronchial infection, dietary recommendations, pancreatic enzymes, and vitamin supplements to maintain a good nutritional status . In receIn CF, therapeutic adherence (TA) is essential for controlling the disease, preventing exacerbations, and slowing progression . TA is tAdherence questionnaires are generally unreliable in CF compared to other methods because they overestimate adherence ,15. The Due to the high availability of medical and health information provided by portable networked devices, the use of health data for research purposes is becoming increasingly common . In mediThe study’s hypothesis is that CF is a multisystem disease requiring complex, time-consuming treatments for patients, so a low TA is assumed. Poor TA will have consequences on the clinical course. The aim of this study was to evaluate longitudinally, in a population of adult patients with CF, how adherence to the different treatments has been over the last 2.5 years, to study, in relation to the degree of TA the clinical consequences in terms of lung function and the number of respiratory exacerbations, and to assess the possible predictors of adherence.n = 30; 49.2%), moderate or MPR50–80%  and low or MPR < 50%  , resulting in a total of 57 patients to evaluate. Patients evaluated in this study were analyzed according to the mean compliance for all treatments followed throughout the study, classified into patients with high or MPR > 80%  .1%) and the mean percentage of Forced Vital Capacity (FVC%), was 71.0 ± 20.3 and 82.7 ± 15.6, respectively. Chronic bronchial infection by Pseudomonas aeruginosa was presented in 22 patients (38.6%), and 16 (28.1%) and 10 (17.5%) for methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA), respectively , mainly for the age range 20–30 years (p = 0.017). Gender, Body Mass Index (BMI), and genotype type were similar in the three groups, being the most compliant group (MPR > 80%), the group with the highest proportion for females (63.3%), and F508del homozygotes (46.7%). In addition, the most compliant group had a higher proportion of patients with pancreatic insufficiency , allergic bronchopulmonary aspergillosis , and massive hemoptysis , although this difference was non-statistically significant. Lung function showed a trend of improvement for all three groups during the study, although a non-statistically significant differences were observed between them. The group with the highest compliance had the lowest FEV1% and FVC values each year and overall. For chronic bronchial infection, a non-statistically significant difference was observed except for Haemophilus influenzae (p = 0.004), which was higher for patients with low compliance (4 patients: 50.0%) (Patients with low compliance (MPR < 50%) had a statistically significantly lower mean age (: 50.0%) .The mean number of exacerbations and number of days with exacerbations were assessed for both oral and intravenous cycles of antibiotic treatment. During the study period (2.5 years), for oral cycles, there was a mean total number of exacerbations of 1.5 ± 1.1, and for intravenous cycles, it was 0.2 ± 0.3 . The meap > 0.05).Regarding the statistical evaluation of the resulting change, only mean values for full years  were considered. Therefore, months in the year 2021 were excluded to avoid potential biases. The decreasing trend was only statistically significant for the reduction in the number of days assessed per intravenous cycle between 2019 and 2020 (Mean compliance was assessed for each type of treatment . All treThe mean compliance for each treatment type was assessed each year . All treχ2(1) = 3.133, p =0.077). This trend continued throughout all years of the study , the lung function of MPR <50% group showed a positive percentage change between the first and last year of the study for FEV1% and FVC% . The MPR 50–80% group had an increase in percentage change . However, the group MPR > 80% decreased its percentage change for FEV1% (−0.25%) and improved for FVC% (+6.0%). The three groups’ percentage change was not statistically significantly different for FEV1 (p = 0.625) but for FVC% (p = 0.029).When we analyzed which group, based on their MPR% , had better lung function , we founIn order to assess which variables have the most predictive power to the response variable and due to the RF method can handle a large number of predictor variables with a small size of observations , the preValidation was estimated internally during the algorithm’s execution using the out-of-bag (OOB) method, which measures the prediction error of random forests using Bootstrap aggregation . The assThe RF analysis provides the Mean Decrease Accuracy (MDA) and Mean Decrease Gini (MDG) indices of the predictors evaluated in each of the models . We main1% (MDG: 3.3), use of Ivacaftor/Tezacaftor treatment (3.0), age (MDG: 3.0), total days of oral exacerbations (3.0), presence of methicillin-sensitive Staphylococcus aureus (MSSA) (2.5). Similarly, for the model using non-compliance (MPR < 50%) as a response variable, the main predictors were mean FEV1% (MDG: 1.4), mean FVC% (1.4), BMI (1.4), presence of Haemophilus influenzae (1.4), BMI (1.5), age (1.3).For the model that uses high compliance (MPR > 80%) as a response variable, the top five relevant predictors were mean FEVy-axis) for a data point of the characteristics of the individual variables. The marks on the x-axis reflect the distribution of the assessed feature, showing how relevant a region is for interpretation (few or no points mean that we should not over-interpret this region).Besides analyzing which characteristics were most important for explaining the response variables in each generated model , we are interested in how the characteristics of these variables influence the predicted outcome. Partial dependence plots (PDP) were constructed for the factor variables with the highest relevance to characterize the relationship between the characteristics of the factors with the highest importance and the %MPR . Each PD1%) or the mean number of days with oral exacerbations and MPR > 80% (Staphylococcus aureus (MSSA).Our results showed that most quantitative variable factors assessed in both models were not linearly associated . For exaPR > 80% A–D. FurtHaemophilus influenzae, was also positively related to low compliance (For low compliance (MPR < 50%), there was a positive non-linear effect for lung function A,B and ampliance A,E.This is one of the first studies to longitudinally assess TA in CF, including CFTR modulator therapy among the different treatments evaluated. In addition, this study is also novel in terms of the application of new Machine Learning tools, specifically the creation of RF models, to assess which variables are most important in terms of compliance.Overall, compliance was good for all CFTR modulators , azithromycin, as well as aztreonam and tobramycin in inhalation solution. Compliance was moderate for the remaining treatments, and close to low compliance was observed with rhDNAsa. Among the inhaled antibiotics, colistin for inhalation and dry powder had the poorest MPR.In our study, we observed a decrease in respiratory exacerbations, both oral and intravenous, during the study period, favored by isolation and prevention measures, hand washing and widespread use of masks during the pandemic ,23.1% mean, use of Ivacaftor/Tezacaftor, Age, mean number of days with oral exacerbations, and presence of methicillin-sensitive Staphylococcus aureus. On the other hand, for low compliance, the top predictors in the model were FEV1% mean, FVC% mean, presence of Haemophilus influenzae, BMI, and age.We identified relevant predictors of high (MPR > 80%) and low compliance (MPR < 50%) from the generated RF model. Overall, we found that the top 5 variables contributing to high compliance were FEVA recent study on compliance over one year with the CFTR modulators Ivacaftor, Lumacaftor-Ivacaftor, and Tezacaftor-Ivacaftor analyzed a total of 2548 patients, 1289 (50.6%) patients with Lumacaftor/Ivacaftor, 784 (30.8%) with Ivacaftor and 475 (18.6%) with Tezacaftor/Ivacaftor. The mean MPR value for all CFTR modulators was higher than 80; they concluded the importance of monitoring TA due to the high cost of these treatments .Haemophilus influenzae chronic infections were associated with TA, and in CF, this pathogen is often associated with milder disease, perhaps not surprisingly associated with lower MPR  . Data we1% in each year of the study and the number of respiratory exacerbations per year, an exacerbation is considered the need to prescribe additional antibiotic treatment in the presence of clinical changes, with the number of courses of oral antibiotics in the case of mild or moderate exacerbations or intravenous antibiotics in the case of severe exacerbations being collected.The following clinical variables were collected: age and BMI at baseline, sex, CFTR gene mutation, exocrine pancreatic insufficiency, CF-related diabetes , chronic bronchial infection (defined by the presence of the same microorganism in 50% of sputum samples collected in 1 year), history of allergic bronchopulmonary aspergillosis and massive hemoptysis (more than 240 mL in 24 h or 100 mL in 1 h), the best FEV1% decline rate, and a number of exacerbations, whether treated with oral or intravenous antibiotics.Low, moderate, or high MPR was related to age, sex, chronic bronchial infection, lung function, 2.5-year FEVt-test) was performed. When one of these two assumptions was not met, a non-parametric test  was performed. In the case of qualitative variables, the comparison of proportions was tested using the chi-square test or Fisher’s exact test whenever necessary.First, a descriptive analysis of the characteristics of the total patients and their total compliance during the study was performed, calculating the mean and standard deviation of the quantitative variables. The Shapiro-W Kolmogorov-Smirnov test was performed to check the normality of continuous variables. Homoscedasticity was tested using Levene’s test. When the distributions were normal and homoscedastic, a parametric test  and non-compliant (MPR < 50) patient profiles. The importance of the variables was assessed through the Mean Decline in Accuracy (MDA) and Mean Decline in Gini (MDG) indicators, although to assess the importance of the variables, we focused mainly on the use of the MDG parameter since the classifications produced by this index provide more robust results . The hig


Salvia miltiorrhiza are lacking. In this study, metabonomic and transcriptomic analyses of different S. miltiorrhiza phenotypes were conducted to explore pathways of synthesis, catalysis, accumulation, and transport of the main flavonoid secondary metabolites regulating pigment accumulation. Tissue localization and quantitative analysis of flavonoid secondary metabolites were conducted by laser scanning confocal microscopy (LSCM). A total 3090 differentially expressed genes were obtained from 114,431 full-length unigenes in purple and green phenotypes, and 108 functional genes were involved in flavonoid biosynthesis. Five key phenylpropane structural genes  were highly differentially expressed, and four transcription factor genes  were identified. In addition, six GST genes, nine ABC transporters, 22 MATE genes, and three SNARE genes were detected with key roles in flavonoid transport. According to LSCM, flavonoids were mainly distributed in epidermis, cortex, and collenchyma. Thus, comprehensive and systematic analyses were used to determine biosynthesis, accumulation, and transport of flavonoids in stems and leaves of different S. miltiorrhiza phenotypes. The findings will provide a reference for flavonoid production and cultivar selection.Flavonoid secondary metabolites can treat and prevent many diseases, but systematic studies on regulation of the biosynthesis of such metabolites in aboveground parts of  Salvia miltiorrhiza Bunge is an important herb in Chinese medicine that was first recorded in Shennong’s herbal classics and is currently widely used to prevent and treat cardiovascular and cerebrovascular diseases2. Tanshinones and phenolic acids are two major groups with significant pharmacological constituents obtained from S. miltiorrhiza, which have important roles in traditional Chinese clinical practice and Western medicine4. Flavonoids are another active ingredient that are widespread in aboveground parts of S. miltiorrhiza7. Because aerial parts of S. miltiorrhiza, are nonmedicinal, aboveground parts are always discarded during harvesting, resulting in huge waste of resources and increased environmental pressure. However, owing to the high flavonoid content and associated antioxidant characteristics, aerial parts of S. miltiorrhiza can be developed into natural antioxidants and used in foods.9. Flavonoids are important secondary metabolites involved in plant growth and development but have also been studied in many plants because of excellent antioxidant and anticancer effects7. Flavonoids primarily include anthocyanins (red to purple pigments), flavonols (colorless to light yellow pigments), flavanols (colorless pigments that turn brown after oxidation), and procyanidins (PAS)8. In addition, flavonoid metabolite distribution and accumulation vary with plant species, tissues, organs, development stages, and growth conditions11.Flavonoids are a group of bioactive compounds found extensively in foodstuffs of plant origin, and regular consumption of flavonoids is associated with reduced risk of several chronic diseases11. Diets that include flavonoid containing products are currently recommended12, further studies are necessary for confirmation of the beneficial effects, such as determine their mechanisms of action at the molecular level, to lay a foundation for the rational promotion of diet rich in flavonoids. Biosynthesis of flavonoids shares the same upstream pathway as lignin and phenolic acid biosynthesis, which is derived from the phenylpropane pathway13. In addition, biosynthesis and accumulation of flavonoids in S. miltiorrhiza are influenced by a series of structural genes, including PAL, C4H, 4CL, CHS, CHI, FLS, FNS, DFR, F3H, F3′H, F3′5′H, ANS, 3GT, and 3AT, and regulatory transcription factors, including MYB, bHLH, bZiP, WD40, and WRKY, or transcriptional complexes (MYB-bHLH-WD) in the flavonoid metabolic pathway. Genes  from other pathways can also be involved. Although expression of those genes affects the formation of color in plant organs15, biological and abiotic factors, especially hormones and environmental factors, also regulate flavonoid biosynthesis16.The growing evidence of the versatile health benefits of flavonoids including anti-inflammatory, antioxidant, antiproliferative and anticancer activity18. For example, a decrease in flavonoids/anthocyanins was the primary reason leaf color changed in purple-leaf tea plant20. In addition, the purple inner leaves of purple ornamental cabbage result from a high level of anthocyanin biosynthesis, a high level of chlorophyll degradation, and an extremely low level of chlorophyll biosynthesis18. In Suaeda salsa, two anthocyanins are responsible for red in leaves19, and in Ziziphus jujuba, flavonoids are the major differential metabolites determining leaf color20.Color phenotypes of plants organs are associated with the biosynthesis and accumulation of flavonoidsS. miltiorrhiza purple and green phenotypes were investigated in this work. Differentially accumulated metabolites (DAMs), including flavonoids and phenolic acids, and differentially expressed genes (DEGs) in flavonoid biosynthesis pathways were determined. To further explore the biosynthesis, accumulation, and transport of flavonoids in aboveground tissues of S. miltiorrhiza, laser scanning confocal microscopy (LSCM) with 2-Aminoethyl diphenylborinate (NA) taining-induced fluorescence was performed. On the basis of the analyses, the biosynthesis, accumulation, and transport of flavonoids in stems and leaves of different phenotypes were determined. The work revealed dynamic changes in metabolites and molecular mechanisms regulating underlying color changes in S. miltiorrhiza stem and leaf.To better understand the mechanism of color change and the dynamic changes in related metabolites and genes expression levels, metabolic and transcriptional profiles of stems and leaves of S. miltiorrhiza were significantly different. Purple plants had a better agronomic phenotype than that of green plants, with tall plants, thick stems, and purple stem skin with more villi. In addition, the color of buds and leaves on purple plants was different from that of old leaves . The clean data of each sample reached 6.3 GB; the Q20 value of obtained sequences was greater than 96%; the percentage of Q30 bases was ≥ 90%; and the GC content was more than 43%  ≥ 1, 1299 DEGs were identified in S1–S2, including 666 up-regulated genes and 633 down-regulated genes, and 1091 DEGs were identified in L1–L2, including 393 up-regulated genes and 698 down-regulated genes  21. In the S1–S2 group  was performed on DEGs with a pheatmap package , including members of S. miltiorrhiza is shown in Fig. S. miltiorrhiza, metabolite separation and detection were conducted. After data preprocessing, in leaves, 10,636 metabolites were identified in positive ion mode, and 5231 metabolites were identified in negative ion mode. In stems, 6264 metabolites were identified in positive ion mode, and 3503 metabolites were identified in negative ion mode  was selected from each group  of samples to display the base peak ion Base Peak Chromatogram (BPC) chromatogram. The total ion flow chromatogram of different phenotypes of stems and leaves of , L1, L2 S. miltiorrhiza leaves were classified in the Beijing Genomics Institute (BGI) library. Of the metabolites, 44 were flavonoids, 50 were terpenoids, 37 were phenylpropanoids, 14 were phenolic acids, 15 were phenols, 13 were classified as miscellaneous, and 3 were steroids. Eleven metabolites were alkaloids , and two were quinones (quinones and glycosides). Metabolites identified in S. miltiorrhiza stems were also classified in the BGI library. Of the metabolites, 45 were flavonoids and glycosides, 42 were terpenoids and glycosides, 35 were phenylpropanoids and glycosides, 18 were phenolic acids and glycosides, 14 were phenols and glycosides, 12 were in other classes, and one was in the steroids and glycosides group. Eleven metabolites were alkaloids and glycosides, and two metabolites were quinones and glycosides. Thus, flavonoids and glycosides, terpenoids and glycosides, and phenylpropanoids and glycosides were the main metabolites identified in all samples of aboveground parts of S. miltiorrhiza  and signal transduction (22.22%). Genetic information processing also had two branches: translation (80%) and folding, sorting, and degradation (20%). All metabolites accounted for the largest proportion (65.93%) in global and overview maps. In stem tissue of S. miltiorrhiza, two branches under environmental information processing included membrane transport (77.78%) and signal transduction (22.22%). Under genetic information processing, there was a branch of translation. All metabolites accounted for the largest proportion (65.32%) in global and overview maps  and the other the metabolites after the color change   were selected. In stems, seven DAMs were annotated in the metabolic pathway , and four DAMs were annotated in the secondary metabolite biosynthesis pathway  (Table P < 0.05) in 14 pathways. The largest proportion of DAMs was annotated to the pentose phosphate pathway. The largest number of DAMs was annotated to ABC transporters, accounting for 17.65%. The metabolites (d-(+)-glucose, biotin, d-(+)-xylose), d-(+)-glucose, and d-(+)-xylose were involved in the pentose phosphate pathway and antibiotic biosynthesis. The differential metabolite d-(−)-quinic acid was involved in the metabolic pathway of phenylalanine, tyrosine and tryptophan biosynthesis, and d-(+)-glucose was involved in almost all other metabolic pathways  was used to quantify gene expression. Reverse-transcription quantitative PCR (RT-qPCR) indicated that six genes had higher expression levels and four had lower expression levels in the purple phenotype than in the green phenotype Fig. . The RT-S. miltiorrhiza, frozen sections were prepared and observed. At the excitation wavelength of 488 nm, samples showed chloroplast autofluorescence before staining. The interference was eliminated by subtracting the background signal. After staining with NA solution, epidermis, glandular hairs, and thick horn tissue cells of stems in S. miltiorrhiza showed strong fluorescence. In addition, a small amount of fluorescence also appeared in the phloem. The observations suggested that flavonoids were biosynthesized and accumulated in epidermis, villi, collenchyma, phloem, and fascicle of S. miltiorrhiza  had higher expression in purple than in green samples, and two key anthocyanin structural genes  had higher expression in green than in purple samples. As key upstream enzyme genes of the phenylpropane pathway, PAL, 4CL, and CYP73A affect proportions in the metabolic flow of flavonoids, phenolic acids, and lignin1. The key enzymes in anthocyanins biosynthesis are DFR, ANS, and 3AT6. Overexpression of SmANS also increases anthocyanin accumulation and alters phenolic acids content in S. miltiorrhiza17. The genes ANS, DFR, and F3′5′H also have key roles in regulating the biosynthesis of anthocyanins in purple stems of Astragalus membranaceus13. The 3AT from Perilla frutescens can be used as a new molecular tool to modify anthocyanin structure and further increase flower color by recombinant DNA technology27. The gene HCT mainly controls the metabolic flow between lignin and the flavonoid branches. In Arabidopsis, HCT can inhibit the synthesis of lignin, which redirects metabolic flux to flavonoids via chalcone synthase activity28. In S. miltiorrhiza leaves in this study, expression level of HCT and content of flavonoids  were significantly higher in the purple phenotype than in the green phenotype. Therefore, HCT might be a positive regulator of flavonoid biosynthesis and thereby affect the color of organs and tissues in S. miltiorrhiza.The color of plant tissues and organs is determined primarily by the accumulation of flavonoids. Flavonoids, phenolic acids, and lignin are all in downstream branches of the phenylpropane metabolic pathwayS. miltiorrhiza, are regulated by single or multiple TFs as well as complexes formed between TFs14. Transcription factors MYB, AP2/ERFs, WRKY, bHLH, MADS, WD40, zinc finger, NACs, bZiP, and LBD may be involved in the formation of white and purple flowers in S. miltiorrhiza7. In this work, MYB, WRKY, bHLH, and bZiP also had important roles in pigment formation of S. miltiorrhiza stems and leaves. The regulatory network of MYB in S. miltiorrhiza is important and complex and therefore can provide useful information to improve the growth and defense ability of S. miltiorrhiza as well as the production of active compounds. Transcription factors MYB58 and MYB63 can activate PAL, C4H, 4CL, and other genes involved in lignin biosynthesis in Arabidopsis29. In S. miltiorrhiza, MYB37 is in the same subgroup as MYB58 and MYB63. Therefore, it was inferred that MYB37 had a similar role in lignin biosynthesis30. Arabidopsis thalianaMYB4 can interfere with the transcriptional activity of MBW complexes or inhibit biosynthesis of phenylalanine by repressing expression of the gene encoding arogenate dehydratase 631, which catalyzes the last step in phenylalanine biosynthesis that produces the precursor of flavonoid biosynthesis. In the current study, the green phenotype had high content of MYB4e, indicating that the gene had an obvious inhibitory effect on phenylalanine and thereby affected biosynthesis of flavonoids. The color variation in sweet gum young and senescent leaves is attributed to the composition of anthocyanidins produced by transcriptional regulation of F3′H and F3′5′H by MYB5, MYB113, and MYB12332. Therefore, because the MYB3 and MYB5 in S. miltiorrhiza are in the same group, the two TFs are also likely involved in anthocyanin biosynthesis28. The TFs MYB5-like and bHLH have roles in the first step of flavonoid production in flowers and immature fruits, and expression patterns of those TFs may be the key to determine differences in flavonoids in pomegranate flowers and fruits33. In addition, MYB37 has an important and positive role in plant response to abscisic acid (ABA) and drought stress34. The interaction of TFs MYB21 and MYB24 affects the regulation of jasmonic acid35, and SmMYB39 participates in regulation of the rosemary acid pathway by inhibiting the transcription of key enzyme factors36.The structural genes that catalyze the formation of flavonoids in WRKY family is one of the largest TF families in plants, and it regulates the growth and development of plants37. The TF WRKY55 positively regulates leaf senescence and defense by regulating the transcription of reactive oxygen species (ROS) and salicylic acid (SA) biosynthesis-related genes in Arabidopsis38. The TF WRKY34 interacts with the VQ20 protein to regulate pollen development and function39. The bHLH family TF PIF4 is necessary for plants to adapt to light and high ambient temperature, and interaction between PIF4 and PIF4 interacting proteins establishes a strong network for plants to respond to changes in the environment40. In addition, phytochrome interaction factors PIF4 and PIF5 negatively regulate anthocyanin synthesis in Arabidopsis seedlings under red light41. In plant response to jasmonic acid, MYC2 is the core TF, and overexpression of MYC2 can promote the production of phenolic acids in S. miltiorrhiza42. In this study, the differential expression of those genes in S. miltiorrhiza purple and green phenotypes was also significant. However, the complexity and integrity of biological processes, the complex regulatory network of plant anthocyanin metabolism, and various genetic and environmental factors affect the whole biosynthesis process.The d-glucose, l-rhamnose, d-galactose, d-xylose, and l-arabinose. Glycosylated forms of flavonoids and anthocyanins in flavonoids appear most often and are highly soluble in water. Therefore, hydrolysis may occur and other substances may form, resulting in a decrease in anthocyanin content. Moreover, the high expression of the glycosyltransferase 3AT could explain how glucose and flavonoids form anthocyanins through glycosylation, which eventually led to the difference in anthocyanin components between the two S. miltiorrhiza phenotypes. The DEGs and DAMs were more abundant in stems than in leaves, and expression in purple stems was relatively high. Flavonoids can be selectively transported from one organ to another in plants and from sites of synthesis to other parts43. Flavonoid distribution may be mediated by the action of MRP/ABC transporters rather than passive diffusion44. In this study, GST, ABC transporter, MATE, and SNARE genes were detected, which may have important roles in transporting anthocyanins to plant vacuoles. Therefore, some flavonoids and anthocyanins were likely transported to leaves by ABC transporters and plant hormone signal transduction, resulting in purple characters on the edges of some leaves. However, some anthocyanins might have been hydrolyzed during transport, which could also explain the accumulation of many of differential metabolites in the ABC transporter pathway and the variety of differential carbohydrate metabolites.Among the different metabolites, multiple carbohydrates were highly enriched, including Salvia miltiorrhiza seedlings were provided by Shaanxi Tasly Plant Medicine Co., Ltd.  and were identified by Prof. Yuan Chen . Seedlings were planted in the herbal garden of Gansu Agricultural University . The collection of plant materials complied with relevant institutional, national, and international guidelines and legislation. Completely green stems (S1), purple stems (S2), completely green leaves (L1), and leaves with purple edges (L2) were collected in a full-blossom period, frozen with liquid nitrogen, and stored at − 80 °C. Samples from six individual plants were pooled as one biological replicate, The voucher specimens are deposited in the herbarium of medicinal plants of Gansu Agricultural University . Three biological replicates were used in transcriptome and metabolome analyses conducted at the Shenzhen Genomics Institute (BGI) Co., Ltd.45 was used to filter out low-quality, joint pollution, and reads with high content of unknown base N (> 5%). The filtered data were considered “clean reads”46. The clean reads were assembled to obtain unigenes, and the unigenes were analyzed for function annotation, predicted coding area, and SSRs. The expression amount of each sample was calculated on the basis of all unigenes. The DEGs were screened according to q-value  ≤ 0.05. Based on the principle of negative binomial distribution, DEseq2 software was used for differential analysis47. The screening threshold was q-value ≤ 0.05 and log2 |(fold-change)|≥ 148.Because original sequence data obtained by sequencing could not be directly used for subsequent information analysis, the filtering software SOA Pnuke49. The metabolomics software package metaX and metabolomic information analysis processing were used for data preprocessing, statistical analysis, metabolite classification, and functional annotation50. This work used VIP values of the first two principal components in a multivariate Partial Least Squares Discriminant Analysis (PLS-DA) model51, combined with fold-change and t-test of univariate analysis to identify DAMs according to fold-change ≥ 1.2 or ≤ 0.83 and P < 0.0552.Untargeted metabolomics analysis was performed using LC–MS/MS. The data obtained were processed by Compound Discoverer 3.1 http://www.plabipd.de/portal/mercator-sequence-annotation). The provided online annotation tool performed functional annotation classification, obtained a mapping file with similar structure, imported log2 fold-change data of DEGs, and then used MAPMAN software for joint analysis of transcriptional and metabolomic data53. Last, a working model was proposed to explain the internal mechanism of purple pigmentation in aboveground parts of S. miltiorrhiza, and a schematic diagram of flavonoid biosynthesis and regulation in S. miltiorrhiza was constructed.Each metabolite may be regulated by multiple genes, or multiple metabolites may be regulated by one gene. The differential genes obtained by sequencing the transcriptome were targeted at the utilization site and reverse-transcribed according to a PrimeScriptTM RT Reagent Kit with gDNA Eraser . The cDNA was used as the template to measure gene expression. The specific primers for genes involved in flavonoid biosynthesis and the S. miltiorrhiza actin gene  are listed in Supplementary Table (test) = CT − CT, ΔCT = CT − CT. Secondly, the ΔCT value is the same as that of the test sample ΔCT value: ΔΔCT = ΔCT(test) − ΔCT. Finally, the comparative CT method (2−ΔΔCT method) was used to quantify gene expression. Statistical analysis was performed using Excel 2020 software .Total RNA of S. miltiorrhiza phenotypes were evaluated by an LSCM method54. Flavonoids react with NA reagent (0.1% 2-aminoethyl diphenylborinate (w/v) in 80% methanol) and produce fluorescence55. The middle part of a stem or leave was cut into small pieces (width of 3 mm), which were then cut into 30-μm sections with a freezing microtome . Sections required treatment to induce fluorescence. A drop of self-made phosphoric acid buffer solution (with the addition of 1% NaCl (w/v), pH 6.5) was added to the sections, followed by a drop of NA solution. After incubation for 5 min, sections were analyzed with LSCM .Microstructure and distribution of flavonoids in aboveground parts of different S. miltiorrhiza and thus can also provide valuable theoretical support for further exploration of new medicines and health foods with flavonoids.Contents of flavonoids and anthocyanins in the purple phenotype depended on the up-regulation of genes associated with biosynthesis of flavonoids and anthocyanins. The difference in color between purple and green phenotypes was the result of differential anthocyanin accumulation and differential gene expression. Those results and differences in morphological phenotypes suggested that regulation of structural genes in the flavonoid biosynthesis pathway promoted anthocyanin accumulation and contributed to the formation of the purple phenotype. The results clarify the important role of flavonoids in developmental processes and provide a basis for further understanding of organ coloring and the networks regulating flavonoid metabolism. Overall, the work revealed the dynamic changes in metabolites and the molecular regulation mechanisms underlying the color change in stem and leaf in Supplementary Information.


Background: Bone metastasis (BM) is one of the typical metastatic types of head and neck cancer (HNC). The occurrence of BM prevents the HNC patients from obtaining a long survival period. Early assessment of the possibility of BM could bring more therapy options for HNC patients, as well as a longer overall survival time. This study aims to identify independent BM risk factors and develop a diagnostic nomogram to predict BM risk in HNC patients.Methods: Patients diagnosed with HNC between 2010 and 2015 were retrospectively evaluated in the Surveillance, Epidemiology, and End Results (SEER) database, and then eligible patients were enrolled in our study. First, those patients were randomly assigned to training and validation sets in a 7:3 ratio. Second, univariate and multivariate logistic regression analyses were used to determine the HNC patients’ independent BM risk factors. Finally, the diagnostic nomogram’s risk prediction capacity and clinical application value were assessed using calibration curves, receiver operating characteristic (ROC), and decision curve analysis (DCA) curves.Results: 39,561 HNC patients were enrolled in the study, and they were randomly divided into two sets: training  and validation . According to multivariate logistic regression analysis, race, primary site, tumor grade, T stage, N stage, and distant metastases  were all independent risk predictors of BM in HNC patients. The diagnostic nomogram was created using the above independent risk factors and had a high predictive capacity. The training and validation sets’ area under the curves (AUC) were 0.893 and 0.850, respectively. The AUC values of independent risk predictors were all smaller than that of the constructed diagnostic nomogram. Meanwhile, the calibration curve and DCA also proved the reliability and accuracy of the diagnostic nomogram.Conclusion: The diagnostic nomogram can quickly assess the probability of BM in HNC patients, help doctors allocate medical resources more reasonably, and achieve personalized management, especially for HNC patients with a potentially high BM risk, thus acquiring better early education, early detection, and early diagnosis and treatment to maximize the benefits of patients. According to the latest GLOBOCAN 2020 compiled by the International Agency for Research on Cancer, head and neck cancer (HNC) is the seventh most prevalent malignant tumor globally. There were approximately 932,000 new HNC patients worldwide in 2020, of which 467,000 patients had died . AlthougCurrently, the prognosis of HNC patients with BM and the OS prediction of HNC patients have been described . HoweverThe SEER database is a cancer database based on nearly 30% of the population of the United States of America. It includes demographic and clinical pathology information on cancer incidence and survival rates from 18 cancer registries . After oThe following were the criteria for inclusion: (1): HNC was the first or primary tumor; (2); the patient’s ICD-O-3 histological type was clear; (3); distant metastases  was evident, especially bone; (4); complete follow-up data. Meanwhile, the following were the exclusion criteria: (1): HNC was not the first or primary tumor; (2); unclear histological classification; (3); information about distant metastases , race, tumor grade, T stage, N stage, insurance status, and marital status was unknown; (4); the survival time was less than 1 month. Finally, a total of 39,561 HNC patients were enrolled in this study.From the SEER database, we retrieved 13 characteristics that may be related to the development of BM in HNC patients. Age, sex, race, insurance status, and marital status were among the demographic characteristics studied. Age was divided into ≥60 and <60 , sex wasp-value <0.05 was considered to be statistically different. These included 39561 patients were randomly separated into a training set  and a validation set  in a 7:3 ratio using R software. We used the training set to evaluated the independent risk predictors of BM in HNC patients, constructed a diagnostic nomogram model, and verified the constructed nomogram using the validation set. Specifically, we used the training set to perform univariate logistic analysis in SPSS to determine the risk factors related to BM. The variables with a p-value <0.05 in the univariate logistic regression analysis were further incorporated into the multivariate logistic regression analysis to determine the independent risk predictors of BM in HNC patients. Then, those independent risk predictors were used to construct a diagnostic nomogram model using R software, and the corresponding score assignment of independent risk predictors was obtained (SPSS (version 22.0) and R software (version 4.0.3) were used to conduct all statistical analyses in this study. Furthermore, a obtained . After tAccording to our inclusion and exclusion criteria, 39,561 HNC patients from 2010 to 2015 were finally included in the study, with 27,693 patients in the training set and the remaining 11,868 patients in the validation set. The goal of the study was to explore and verify independent risk predictors of BM in HNC patients. Patients in the training set showed male and white bias characteristics, accounting for 73.3% and 82.9% of the total patients, respectively. The majority of patients were insured (95.5%), while age and marital status had no significant difference. The primary common histological type of HNC patients was SCC (90.1%), and the top three primary sites were oral cavity (38.0%), larynx (24.0%), and oropharynx (18.1%). The most prevalent tumor grade, T stage, and N stage were grade II (48.8%), T2 (29.2%), and N0 (50.7%), respectively. At the same time, patients with distant metastases accounted for a small number of patients. The baseline information for all patients is shown in p < 0.05)  patients were diagnosed with BM, while 39,229 (99.2%) HNC patients were diagnosed without BM. Univariate logistic analysis was performed by incorporating the 13 variables. The results showed that race, insurance status, histological type, primary site, tumor grade, T stage, N stage, and distant metastases  were associated with the development of BM in HNC patients ( < 0.05) . Then, b < 0.05) . In otheThe above eight independent risk variables obtained from multivariate logistic regression analysis were used to create a diagnostic nomogram. . By assiet al., the average time from the diagnosis of any part of the distant metastases to the death of the patient was 4.3–7.3 months, and 86.7% of those patients would die within 12 months  and concluded that patients with bone exclusive and single BM had a considerably higher median survival time than multiple organs and polyostotic metastases significantly  as independent risk predictors of BM development by analyzing 39,561 HNC patients from the SEER database. Furthermore, based on these eight risk factors obtained, we constructed a diagnostic nomogram to predict BM’s risk in HNC patients. According to reports, only a limited percentage of patients were diagnosed with BM simultaneously as the initial diagnosis of HNC, and most patients were found BM only when there were more obvious skeletal-related events in the subsequent course of the disease. The median time between confirmed BM and the initial diagnosis of HNC was approximately 11.5 months, leading to the progression of the distant metastases and the loss of treatment opportunities . With thet al. performed autopsies on 832 HNC patients and found the distant metastases’ prevalence in the hypopharynx HNC were as high as 60%  may cause potential errors. Secondly, it is a retrospective study, and selection bias is inevitable. Then, although it contains several of the most common metastatic sites in HNC patients, it lacks information on the order and severity of metastases and other critical locations of potential metastases, such as skin and pleura, which are also common metastatic sites for HNC. Finally, more data from other research centers for external verification will improve the applicability and accuracy of our diagnostic nomogram.However, our research inevitably has several limitations. First, the limited number of HNC-BM patients  were independent risk factors for BM in HNC patients. The diagnostic nomogram constructed using the above risk factors could quickly determine the probability of BM in newly diagnosed HNC patients, assist doctors in providing personalized management of HNC patients, especially in HNC patients with potentially high-risk BM, and conduct better early education, early detection, and early diagnosis and treatment, to maximize the benefits of patients.


To evaluate the efficacy and safety of dexamethasone (DEXA) combined with tranexamic acid (TXA) in the perioperative period of total hip arthroplasty.A total of 100 cases were randomly divided into 2 groups (50 cases per group). All patients were given 15 mg/kg TXA before skin incision and 3 hours later. Patients in the intervention group (TXA + DEXA group) were given 20 mg dexamethasone intravenously after the onset of anesthesia, and the same dose of DEXA was administered again 24 hours later. Patients in the placebo group (TXA group) were only given the same dose of normal saline. Postoperative c-reactive protein and interleukin-6, postoperative nausea and vomiting, fatigue visual analogue scale score, postoperative length of stay, range of motion, and consumption of analgesic and antiemetics were statistically analyzed in the 2 groups.P < .001). Walking pain scores in the TXA + DEXA group were also significantly lower than those in the TXA group at 24 and 48 hours (P < .001); rest pain scores were lower at 24 hours (P < .001). Compared with the TXA group, the incidence of nausea VAS, postoperative nausea and vomiting, fatigue, analgesia and antiemetics consumption, postoperative length of stay, and range of motion were lower in the TXA + DEXA group , while there were no significant differences in postoperative hematocrit, total blood loss, and complications (P > .05).The levels of c-reactive protein and interleukin-6 in the TXA + DEXA group were lower than those in the TXA group at 24, 48, 72 hours post-operatively  and DEX  is an effective and safe strategy for patients undergoing total hip arthroplasty. The management of postoperative inflammation is of great benefit to the enhanced recovery of THA.,8Total hip arthroplasty (THA) is 1 of the most cost-effective treatments used for end-stage hip disease. It can significantly improve the function of the hip joint and patients’ quality of life, Numerous studies have suggested that TXA can significantly reduce blood loss and blood transfusion requirements, thus it become part of blood protection management during perioperative joint replacement,,11which is the guideline for blood protection during the perioperative period of joint replacement. In addition, some studies pointed out that TXA can effectively inhibit postoperative inflammatory response and reduce postoperative pain by inhibiting the activation of plasminase.,13 Nowadays, joint surgery is vigorously promoting the construction of accelerated rehabilitation wards, and multimodal analgesia is the core component, which also includes the suppression of postoperative inflammation is.,15As an analog of lysine, tranexamic acid (TXA) can competitively inhibit the activation of plasminogen and the binding of plasminogen to fibrin, thereby inhibiting fibrinolysis.,17 It has been widely used in perioperative management of various surgeries, including THA. Previous studies have shown that DEXA can reduce inflammatory responses, prevent PONV, and relieve postoperative pain and fatigue in patients with THA.,19 However, at present, the optimal dose and administration time of DEXA before THA remain unclear.,21 In addition, only a few studies reported on the application of DEXA combined with TXA in THA.Dexamethasone (DEXA) is a medium and long-acting glucocorticoid with strong anti-inflammatory effects. DEXA inhibits prostaglandin production by reducing the expression of cyclooxygenase 2. In this way, the expression of cell adhesion factors and the transcription of related cytokine genes can be inhibited. Neutrophils and macrophages can be inhibited to exudate to the inflammatory site and the activity of these cells can be inhibited, and their adhesion and aggregation on vascular endothelial cells in the inflammatory area can be reduced.Herein, we conducted a randomized controlled trial to compare the efficacy and safety of TXA + DEXA in THA.This study was approved by the Institutional review Committee of The People’s Hospital of Guangxi Zhuang Autonomous Region and registered in the International Clinical Trial Registry (Study Registry ChiCTR2000039801). Inclusion criteria were: unilateral THA; sign the informed consent; age > 18 years old or < 75 years old. Exclusion criteria were: allergy to DEXA; age ≤ 18 years old or ≥ 75 years old; use of any glucocorticoids within 3 months or any strong opioids within 1 week before surgery; history of severe heart disease (NYHA > 2), liver and kidney failure, systemic rheumatic diseases ; ipsilateral hip surgery history; lack of cognitive function or normal sensation; loss to follow-up.A total of 100 patients were randomly divided into the intervention group (TXA + DEXA group) and the placebo group (TXA group), with 50 patients in each group. According to the order of patients’ admission, 100 random numbers were generated on the computer by SPSS 24.0 software from 1 to 100. The random numbers were sorted from small to large, and the rank of random numbers was 1 to 50 as the control group (TXA group) and 51 to 100 as the intervention group (TXA + DEXA group).A random allocation sequence was concealed in opaque sealed envelopes that were only opened before surgery. All patients were treated with 15 mg/kg IV-TXA before skin incision and 3 hours later. All patients in the intervention group intravenously received 20 mg DEXA after anesthesia, and the same dose was administered again 24 hours after the operation. Patients in the placebo group were intravenously given the same dose of normal saline immediately after anesthesia, and the injection was repeated 24 hours after the operation. During this study, neither the researcher nor the patient knew which group each subject was divided into, nor which group received the trial treatment.All surgeries were performed by the same group of senior joint surgeons, with an anterolateral incision approach and biotype prosthesis. All operations were evaluated by an anesthesiologist and performed under general anesthesia. In order to control the variable factors, patients were not treated with nerve block or patient-controlled intravenous analgesia during the perioperative period and were subcutaneously injected with low molecular weight heparin 6 hours after the operation. Deep vein thrombosis was detected by Doppler ultrasound 1 month after discharge. The walking and rest pain was assessed using a visual analogue scale : if VAS levels were between 4 and 6, oxycodone was given orally at 10 mg, Q8h; if the pain was greater than 6, 100 mg of tramadol was injected intramuscularly. The VAS  was used to assess the degree of nausea. If the VAS score exceeded 6, 10 mg of metoclopramide was given as a first-line antiemetic rescue; if nausea persisted within 30 minutes, 5 mg of ondasetron was used as second-line antiemesis aid.The total blood loss was calculated according to the general formula, and the blood transfusion indications were strictly controlled only when the hemoglobin level was < 70 g/L or 70 to 00 g/L, accompanied by anemia symptoms such as mental state, dizziness, etc. Activity (ROM) was assessed by the nurse with a goniometer before surgery and at discharge, and postoperative length of stay (p-LOS) and complications were carefully recorded.CRP, IL-6, VAS pain score, nausea VAS score, and POVN incidence were recorded at 24, 48, and 72 hours postoperatively. The primary outcome variables of this study were VAS, CRP and IL-6 after operation, and other outcome variables were secondary observation indexes. The total number and dose of postoperative analgesics  and antiemetic salvage drugs  were recorded. Fatigue was assessed preoperatively and at discharge using the 10-point numerical scoring scale .https://www.ncss.com/software/pass/) software based on a 2-sample t test. All data were analyzed by SPSS 22.0 . T test or Wilcoxon Mann-Whiney U test was used to analyze quantitative data, and Pearson Chi-square test or Fisher exact test was used to analyze qualitative comparative data. P < .05 was considered to be statistically significant. With the power of 0.90 and the significant level of 0.05, 43 patients per arm were required in the study. Considering the dropping out, 10% to 15% of the sample size should be increased at least. In this study, we calculated that at 15%. Therefore, the sample size of 50 cases each group was required for this trial, so the total sample size was 100 cases.Sample size calculations were analyzed by PASS 2011  than that in the TXA group  at 24 hours, 48 hours (P < .001), and 72 hours (P < .001) after surgery , and postoperative pain during walking at 24 hours (P < .001) and 48 hours (P < .001) were significantly lower in the DEXA + TXA group than in the TXA group. However, VAS scores for rest at 48 hours after surgery and VAS scores for rest and walking at 72 hours after surgery revealed no significant difference between the 2 groups (P > .05) . The cumulative consumption of oxycodone was 530 mg in the TXA + DEXA group and 740 mg in the TXA group, with no significant difference (P = .08). For tramadol hydrochloride, the number of patients required in the TXA + DEXA group was significantly different compared with the TXA group (P < .001), and the cumulative consumption of the TXA + DEXA group was also lower (P = .004)(Table 2).Thirty patients in the TXA + DEXA group and less than 37 patients in the TXA group needed oxycodone; however, the difference was not statistically significant (P = .006). The cumulative consumption of metoplatin in the TXA + DEXA group was 40 mg, and that in the TXA group was 180 mg; the observed difference was significantly different (P = .009). Moreover, in the TXA + DEXA group, 1 patient required ondansetron compared to 4 patients in the TXA group, but the difference was not statistically significant (P = .17). Meanwhile, there was no significant difference in the cumulative consumption of ondansetron between the 2 groups (P = .17) . In terms of PONV incidence, DEXA + TXA was 6% (3/50) and 24% (12/50) in the TXA group; the observed difference was statistically significant (P = .011) . The ROM of patients in the TXA + DEXA group was greater than that of the TXA group at discharge, and the comparison between the 2 groups was statistically significant (P = .002). The mean p-LOS of the TXA + DEXA and TXA groups was (5.02 ± 0.52) days and (5.56 ± 0.81) respectively . There was no statistical difference between the 2 groups in terms of total blood loss and transfusion rate   have demonstrated the efficacy of DEXA in preventing inflammatory stress without causing a wound and gastrointestinal bleeding complications during THA.,21 However, so far, no optimal dose and optimal time of DEXA injection before THA has been proposed. Moreover, only a few studies reported on the application of DEXA combined with TXA in THA. An et al evaluated the efficacy and safety of combined use of TXA and DEX for anti-inflammatory and clinical outcomes after THA. Patients who underwent THA were given 20 mg IV-DEXA only when anesthesia was completed in the intervention group, and the same dose of normal saline was applied in the placebo group. All patients were given 15 mg/kg TXA before skin incision and 3 hours later, which confirmed that DEXA + TXA could relieve postoperative pain, PONV, and fatigue and did not increase the risk of postoperative complications. In our study, patients received 15 mg/kg TXA before skin incision and 3 hours later. Patients in the intervention group were given 20 mg IV-DEXA when the anesthesia was completed and 24 hours later, which was different from An et al The control group was given the same dose of normal saline. We discovered that TXA + DEXA could significantly reduce postoperative CRP and the level of IL-6, reduce PONV, postoperative pain, and fatigue, increase the effect of analgesia and anti-nausea without affecting the hemostatic effect of TXA or increasing the risk of complications such as wound infection and gastrointestinal bleeding.DEXA is a medium long-acting glucocorticoid with strong anti-inflammatory properties, widely used to reduce perioperative inflammatory response, prevent PONV, and relieve perioperative and postoperative pain and fatigue.,28 In our study, 20 mg DEXA was intravenously administered before surgery and 24 hours later, and the postoperative CRP and IL-6 levels were significantly reduced, which was consistent with previous studies.,17,20,21 Nevertheless, the optimal dose, duration of treatment, and route of administration of DEXA remain controversial. In our study, DEXA combined with TXA further significantly reduced the levels of CRP and IL-6 , alleviated postoperative pain, and accelerated rapid recovery. In our study, unlike most studies using TXA or DEXA alone, we hypothesized that DEXA combined with TXA had synergistic and additional benefits in reducing postoperative inflammatory responses compared with TXA alone. Moreover, our data confirmed the hypothesis that combined strategies could reduce CRP and IL-6 levels.As markers of acute inflammation, CRP and IL-6 levels show similar dynamic changes during the inflammatory response. Previous studies have confirmed that local and systemic inflammatory responses are closely related to early postoperative rehabilitation and complications.,13 It is worthy to note that some patients still had pain symptoms after surgery. Therefore, we considered whether DEXA combined with TXA could potentially compensate for the lack of anti-inflammatory effects of TXA, further reducing pain and accelerating the recovery of patients. The analgesic effect of DEXA is to block the pathway of cyclooxygenase and lipase in the inflammatory chain reaction by inhibiting the phospholipase. In addition, DEXA can also inhibit the level of 35-bradykinin in tissue, release neuropeptides from the nerve endings, and reduce the inflammation and pain in the tissue. Previous studies have confirmed the good analgesic effect of DEXA in the perioperative period of THA. Lei et al examined 210 patients who underwent THA and were randomly divided into 3 groups: Group A was the blank group; group B was given 10 mg IV-DEXA after anesthesia and then again after 3 hours; Group C was given 10 mg IV-DEXA after anesthesia and then again after 3 and 24 hours. Our results showed that the additional DEXA could further reduce the pain and reduce the application of analgesic drugs. Based on the previous study of analgesic drug consumption, we considered that a single application of 20 mg IV-DEXA might not be enough for analgesic effect, so we injected a second dose again after 24 hours. We hypothesized that increasing the dose of DEXA might provide additional analgesic effects. In our study, pain scores were lower at 24 and 48 hours of walking and 24 hours of rest after surgery. In addition, the consumption of oxycodone and tramadol hydrochloride was lower, which further confirmed that the short-term efficacy of increasing DEXA dose could further reduce pain and also compensate for the deficiency of TXA as an anti-inflammatory. It is important to note that the long-term effects of DEXA on relieving pain have not yet been proved.Previous studies have confirmed the anti-inflammatory and analgesic effects of TXA.,31 In the current study, using a single dose of a glucocorticoid to reduce the incidence of PONV was supported and recommended.,21 We also hypothesized that patients would receive PONV remission 24 hours after surgery if the additional dose was supplied compared with single-dose treatment. In our study, the incidence of nausea VAS score and PONV in the TXA + DEXA group was significantly lower than that in the TXA group (P < .05), and the number of metoclopramide and total cumulative consumption were also significantly lower than that in the TXA group (P < .05). This data suggests that 1 additional dose of DEXA 24 hours after surgery can effectively reduce the incidence of PONV.The antiemetic effect of DEXA has been confirmed, mainly by regulating prostaglandin synthesis or inhibiting endogenous opioid release.,21,32 In their meta-analysis of the relationship among pain, sleep, and fatigue, Whibley et al pointed out that these 3 factors influence each other and interact. Only when each factor is positively controlled, this virtuous circle can be established to promote early recovery of patients, shorten the average length of stay in the hospital, and improve patients’ satisfaction. In our study, Identity-consequence-fatigue-scale was used to assess fatigue, and the average p-LOS was calculated. The results showed that postoperative Identity-consequence-fatigue-scale scores and p-LOS in the TXA + DEXA group were significantly reduced compared with the TXA group .Postoperative fatigue is a common clinical symptom, which often leads to delay in early recovery and affects patients’ initiative rehabilitation. Yet, patients given low doses of DEXA may still suffer from pain, fatigue, and PONV. Therefore, this study was based on the assumption that the effect of low-dose DEXA still did not meet the anti-inflammatory requirements, and its half-life was fully considered to be about 24 hours. Accordingly, it is reasonable to administer 20 mg DEXA again 24 hours after surgery. As for the dose of IV-TXA, the guidelines recommend that 15 to 20 mg/kg can be given, depending on the patients. In our previous studies, the dose of 15 mg/kg of TXA achieved satisfactory clinical effects. Our study showed that DEXA combined with TXA could effectively reduce postoperative inflammation and pain, without affecting the hemostatic effect of TXA.The optimal approach and dose of DEXA before THA are still controversial. Some studies have identified a dose range of 10 to 40 mg for primary THA.,17,29 DEXA has been applied again before and 24 hours after surgery in our study. One patient had a thrum reaction in the TXA + DEXA group, and in the TXA group, 1 patient had incision fat liquefaction, and the other had a thrum reaction. During observation, no incision infection or gastrointestinal bleeding and no thrombosis was found in any of patients. However, it should be pointed out that large-scale prospective studies are still needed to assess the safety of DEXA in perioperative application of THA and clinical application.Although DEXA is widely used in the perioperative period of THA, it is still controversial whether DEXA might increase the risk of adverse reactions.This study has some limitations. First, the follow-up time was too short. Three-month follow-up was insufficient to fully evaluate the efficacy and safety of TXA + DEXA. Second, this study has a relatively small sample size. Third, the optimal combination dose and timing of TXA + DEXA remain unclear and need to be further studied.Our data suggest that DEXA combined with TXA can significantly reduce postoperative CRP and IL-6 levels, reduce postoperative pain, improve the incidence of POVN, increase the analgesic and antiemetic effects, reduce postoperative fatigue, shorten the average p-LOS, and improve ROM without affecting the hemostatic effect of TXA or increasing the risk of perioperative complications of THA.Fulin-Li performed the data collection and analysis and participated in manuscript writing. Xiao Huang, Wenhui-Liu, Wenwen-Huang and Chaoqun-Wang performed the database setup and statistical analysis. Dong Yin performed the operations, participated in the study design and coordination, and helped to draft the manuscript. All of the authors have read and approved the final manuscript.Data curation: Fulin-Li, Xiao Huang, Wenwen Huang, Chaoqun-Wang, Dong Yin.Formal analysis: Xiao Huang, Chaoqun-Wang, Dong Yin.Funding acquisition: Fulin-Li.Investigation: Fulin-Li, Wenwen Huang.Methodology: Fulin-Li, Wenwen Huang, Dong Yin.Resources: Wenhui Liu.Software: Fulin-Li, Wenhui Liu.Supervision: Wenhui Liu.Writing – original draft: Fulin-Li, Dong Yin.Writing – review & editing: Fulin-Li, Dong Yin.


Background: Accelerated atherosclerosis in patients with endometriosis has been hypothesised, and lifestyle improvement might control cardiovascular risk. We explored cardiometabolic markers and oxidative stress and evaluated the effects of the Mediterranean Diet (MD) in modulating these markers. Methods: In this prospective study, we included 35 women with endometriosis. At baseline (T0) and after 3 (T1) and 6 (T2) months from the start of the diet, we investigated cardiometabolic parameters, lifestyle and oxidative stress. Results: After a 3-month intervention with MD, we observed a significant reduction in total cholesterol (p = 0.01) and LDL-c (p = 0.003). We observed at T1 an increase in B12 and E vitamins, folate and zinc. After 6 months, zinc (p = 0.04) and folate (p = 0.08) increased in comparison to T0. A reduction in homocysteine from T0 to T1 (p = 0.01) was found. After 3 months, an increase in Rapid Assessment of Physical Activity tool 1 (RAPA) (p < 0.001) and RAPA 2 was observed (p = 0.009). We observed high levels of oxidative stress markers at baseline. After 6 months of MD, a significant improvement in lymphocyte Reactive Oxygen Species (ROS) (p < 0.001) and total antioxidant capacity was observed (p = 0.02). Conclusions: The improvement of lifestyle, and in particular the Mediterranean dietary intervention, allowed the improvement of the metabolic and oxidative profile and overall health-related quality of life. Atherosclerosis represents a multifocal and progressive systemic disease of the arterial wall. The pathogenesis of atherosclerosis is linked to the interaction of known and emerging risk factors . The keyEndometriosis is a chronic, debilitating disease affecting roughly 10% (190 million) of reproductive-age women globally . It is aEndometriosis and atherosclerosis are traditionally viewed as distinct entities, with endometriosis affecting young reproductive-age women, while atherosclerosis is an ageing-related process. The link between endometriosis and cardiovascular diseases is increasingly being studied and has been partly explained by systemic inflammation, with potentially underlying genetic similarities that, in turn, increase the risk of atherosclerosis, coronary artery disease and cardiovascular morbidity and mortality . ChronicOxidative stress, increased inflammatory cytokines and mast cell activation are considered relevant steps in the pathophysiology and progression of the disease . RetrogrEndometriosis has also been associated with an atherogenic lipid profile, notable for increased LDL, non-high-density lipoprotein and triglycerides, and decreased HDL levels ,13. The The role of diet in endometriosis has gained more attention since it has been observed that diet can affect several processes that are involved in endometriosis, including inflammation, prostaglandin metabolism, and oestrogen activity . To dateLifestyle improvement by adopting Mediterranean dietary patterns, an evidence-based nutritional model for the prevention of cardiovascular disease, might also represent a future non-pharmacological therapeutic intervention for controlling cardiovascular risk in women with endometriosis. No data are available concerning the role of the Mediterranean Diet in influencing subclinical atherosclerosis, and in turn cardiovascular risk, in endometriosis. Based on the above-mentioned observations, the aim of this study is to explore cardio-metabolic, endothelial, oxidative stress and inflammatory markers correlated with the atherosclerotic process, and to evaluate the effects of Mediterranean Diet intervention in modulating these markers.In this prospective study, we investigated 90 Caucasian women with endometriosis referred to the Internal Medicine Clinic at the Centre for Assisted Reproductive Technology, Division of Obstetrics and Gynaecology of Careggi University Hospital, Florence, from March 2020 to May 2022. All women were referred to the Endometriosis Centre of Careggi University Hospital, Florence, a third-level centre for endometriosis treatment. In all patients, the diagnosis of endometriosis was confirmed by diagnostic imaging (US or MRI) and/or laparoscopy performed by gynaecologists expert in this field. Pre-existing atherothrombotic disorders, hypertension, diabetes, autoimmune diseases, renal failure, obesity, pregnancy and chronic illnesses that are known to affect gastrointestinal absorption of nutrients  represented exclusion criteria. Women participating or who had participated in a weight loss treatment and nutrition programme in the last 3 months were excluded. Therefore, we included 35 women with endometriosis of reproductive age in the study. At the first evaluation, all women were in therapy with oestrogen-progestins or progestins. The baseline assessment included demographic information and cardiovascular risk factors. Moreover, at the first visit, we focused on the current nutritional habits of endometriosis patients before the start of the diet. We administered a validated questionnaire to evaluate accurate data about dietary intake and adherence to the Mediterranean Diet ,19.During the subsequent 10 days after the first visit, the participants completed a food diary giving a detailed description of each food consumed, the time of consumption, and amount, using household measures, in order to capture their habitual diet and stool habit assessment. After the evaluation of the food diary, we developed a personalised tailored nutrition Mediterranean Diet plan agreed upon with the patient to achieve the best compliance. The energy requirements were calculated for each woman. The diet consisted of 50–55% carbohydrate, 25–30% total fat (≤10% saturated fat) and 15–20% protein. Each woman was provided with a 1-week menu plan as well as information on the food groups that could be included and those that should be avoided.-Coagulative profile, including VIII Factor (FVIII), von Willebrand Factor (vWF), vitamin profile  and zinc, endothelial markers ), lipid and glucose profile, liver panel enzymes, hs-CRP;-Blood redox status .During the study, 3 clinical evaluations were performed: at the baseline, before the start of dietary treatment (T0), 3 months after the start of the dietary intervention (T1), and after a further 3 months (6 months from the beginning) (T2). At baseline (T0) and after 3 (T1) and 6 (T2) months from the start of the diet, we evaluated:At each clinical evaluation, anthropometric parameters  were measured. Compliance with the Mediterranean Diet was evaluated during follow-up visits using a validated adherence score .The investigation conformed to the principles outlined in the Declaration of Helsinki. The Local Ethics Committee  approved the original study (Reference: 21140).2, normal weight when the BMI was between 18.5 and 24.99 Kg/m2, and overweight when the BMI was 25 Kg/m2. In order to evaluate abdominal fat, anthropometric parameters were also measured. Hip circumference was measured at the widest point over the buttocks, and waist circumference was measured midway between the inferior margin of the lowest rib and the iliac crest in the horizontal plane at the end of normal expiration. A waist circumference of 80 cm was considered a marker of increased CV risk according to Alberti et al. [We assess women’s vascular profiles by collecting information about the family history of CVDs, defined as the history of CVDs in first-degree relatives <55 years of age in men and <65 years in women . During i et al. ; waist ti et al. . To limii et al. . HyperhoLipid profile (lipoprotein (a), total cholesterol, HDL-c, LDL-c, triglycerides), homocysteine and vitamins , zinc, hs-CRP, glucose and coagulative profiles, including FVIII and vWF, were determined at the University Careggi Hospital Laboratory.g for 15 min at 4 °C), plasma was collected and used for experiments or stored at −80 °C for further analysis. Oxidative Stress markers were measured in the Laboratory of Oxidative Medicine of the Department of Clinical and Experimental Biomedical Sciences “Mario Serio” University of Florence, Italy.Blood samples were collected in Vacutainer tubes containing 0.109 mol/L buffered trisodium citrate (1:10) or EDTA (0.17 mol/L). After centrifugation  was used for intracellular ROS evaluation, as previously reported ,27.Plasma lipid peroxide levels were estimated using an ALDetect Lipid Peroxidation assay (BML-AK170-Enzo Life) as previously reported . The resThe ORAC  assay was performed as previously reported . Plasma The sample size was calculated starting from historical data  and estip < 0.05. Statistical analysis was performed using IBM SPSS Statistics 28 for Windows .The results were expressed as median (range) or mean, as appropriate. Categorical variables were presented in terms of frequencies and percentages. Statistical analysis was performed using McNemar to analyse dichotomous variables and the Wilcoxon rank-sum test for continuous variables for paired data. Unpaired Mann–Whitney U-tests were performed to compare the independent groups. Repeated measures ANOVAs were used to examine the mean differences in the subgroups of women who completed three evaluations. The Spearman (rho) test was used to estimate the correlation between the variables. Differences were considered statistically significant if A total of 35 participants completed the first phase of the intervention. Sixteen women completed the entire study, and 19 women did not complete phase 2 (T2): two of them became pregnant in the meantime, while for what concerns, the remaining 17, less than three months have passed from T1.About 37% of the study population had a high level of education (>13 years of education).From gynaecologists’ medical reports, we obtained information about the phenotype of endometriosis; in particular, 17.1% of women had Deep Infiltrating Endometriosis (DIE) and 17.1% had a mixed phenotype (DIE + Ovarian Endometrioma).2 while about 23% of women were underweight. In about 26% of women, we evidenced values of waist circumference above 80 cm.The baseline demographic characteristics of the population studied are shown in Dyslipidaemia was present in more than 50%, in particular, in 40% of women with high levels of LDL-c.In framing women’s risk profiles, we also considered family history of CVD, and we observed that more than 25% had a family history of CVD. As concerns migraine, which is known as a stronger and more prevalent risk factor in women, we observed a high prevalence of migraine with aura (11.4%).Finally, by analysing the composition of the diet through a food diary at T0, we observed a daily intake of macronutrients, with carbohydrates accounting for 48% of dietary energy. Carbohydrate intake was lower than desirable for the Mediterranean dietary pattern (50–55%), while sugar consumption was high, if we consider that the baseline evaluation showed that only 14.3% of the patients assumed 2–3 portions of fruit a day. Indeed, we assumed that the quantity of total sugar intake was mainly due to the consumption of added sugars. The consumption of fibre was lower when compared to the 15 g/1000 Kcal/day recommended by the LARN , and thiFat consumption exceeded 30% of energy intake and, specifically, the median intake of saturated fatty acids was 10.5%, with values ranging from 6.6% up to 16.6%.Biochemical parameters at T0, including coagulative profile, vitamins, homocysteine, liver and renal function, and fasting glucose, are shown in The overall frequency of folate and vitamin B12 deficiencies was estimated to be 26.5% and 20%, respectively . The freWe observed no vitamin E deficiency and only 2.9% of women had reduced levels of vitamin B6 and zinc.We evaluated anthropometric parameters in women from T0 to T2 and we evidenced no significant differences in the BMI, waist circumference and WHR between T0 vs. T1 and T0 vs. T2.p = 0.04), while in overweight women (p = 0.06). Of relevance, 3 women underweight at T0 had normal BMI at T1 and T2.We analysed changes in anthropometric parameters according to BMI at baseline; therefore, we considered changes in women that at T0 were underweight, women with normal BMI and women overweight. ht women , we evidp = 0.02; Waist rho = −0.52, p = 0.001; WHR rho = −0.44, p = 0.009), indicating an inverse association more evident when adherence to the Mediterranean Diet increased.At T0, all the anthropometric parameters were inversely related to the Mediterranean Diet adherence score, albeit without statistical significance, whereas this correlation was significant at T1  who completed three evaluations , and in particular in LDL-c levels (p = 0.003). This trend of reduction in LDL-c seems to also be present at T2, even if not significant, probably due to the small sample size . After six months, in the 16 patients who completed the clinical evaluation at T2, zinc (T0 vs. T2 p = 0.04) and folate (T0 vs. T2 p = 0.08) levels increased in comparison to T0. Finally, at T1, we observed a reduction in the percentage of patients with B12 vitamin deficit (20% at T0 vs. 5.9% at T1), whereas all women with folate deficit had already corrected this condition.Moreover, we analysed the vitamin profile, and we observed an increase in the levels of B12 and E vitamins, folate and zinc . In partp = 0.01), probably due to an improvement in vitamins regulating its metabolism , was found . At T2, in the sample that completed the study, we observed a median value of hs-CRP of 0.6 (0.01–3.4).p < 0.001) (p < 0.001).Through the analysis of the Mediterranean Diet adherence score at the end of the first phase, we found a statistically significant increase in the median adherence score  , which wp = 0.07). This improvement may be due to the season in which each woman did their T1 visit .The nutrition evaluation took into account the daily water intake that resulted very low in some patients, with a median value of 1.5 L (0.25–3). At T1, we found an important increase to 1.75 L (0.25–4), although not statistically significant (p < 0.001) and RAPA 2 (p = 0.009). Regarding the patients who concluded the study, RAPA 1 and RAPA 2 significantly increased from T0 to T2   tool. At baseline, the median levels were very low for both RAPA 1 (Aerobic) 2.4 (1–6) and RAPA 2 (Strength and Flexibility) 0.3 (0–2). After the first 3 months from the beginning of the intervention, the increase was statistically significant for both RAPA 1 (ctively) . In genectively) .We evaluated blood global redox status by assessing leukocyte intracellular ROS production, plasma lipid peroxidation, and plasma total antioxidant capacity. We observed significant alterations compared to the normal range observed in a population of healthy age-matched women (as previously published) , in partp < 0.001) and an increase in total antioxidant capacity (p = 0.02). We also observed this result by analysing ROS production and plasma antioxidant capacity from T0 to T2 in 16 women who concluded the study .We did not observe significant changes between T0 and T1, but there was an improvement between T0 and T2 . In parthe study . MoreoveThis study aimed to evaluate the role of the Mediterranean Diet and lifestyle in improving the cardiovascular risk profile of women with endometriosis and investigated common mechanisms shared by atherosclerosis and endometriosis. The aetiopathogenesis of endometriosis is a multifactorial process that determines the development of an extremely heterogeneous disease. Several hypotheses have been suggested; nevertheless, none is able to completely explain its pathogenesis and all its different clinical features. Evidence suggests that immune cells, adhesion molecules, extracellular matrix metalloproteinase and pro-inflammatory cytokines activate the peritoneal microenvironment, leading to differentiation, adhesion, proliferation and survival of ectopic endometrial cells . SystemiData from the Nurses’ Health Study II  suggesteConcerning the link between endometriosis and dyslipidaemia, few studies have investigated the lipid profile in women with endometriosis . In our These data reinforce the previous evidence regarding the healthy effects of the Mediterranean Diet since the development of a less atherogenic LDL phenotype could be a possible explanation for some of the cardioprotective benefits of this dietary pattern . Data frIn the evaluation of biohumoral parameters, besides the lipid profile, we evaluated blood levels of vitamin E and zinc, beyond B6, B12 vitamins and folates that are known to be linked to homocysteine levels. Our endometriosis patients showed significantly lower levels of vitamin B12 and folate at baseline, just as higher levels of homocysteine were found in about 9% of women. Hyperhomocysteinemia is most commonly caused by B-vitamins’ deficiency, especially folate, B6 and B12. Elevated homocysteine promotes atherosclerosis through increased oxidative stress, impaired endothelial function, and thrombosis induction . Since vWith respect to antioxidant vitamins, the mean vitamin E level in our study population fell within the normal range. The main function of vitamin E is to act as a structural antioxidant, in particular at the membrane level. In general, a deficiency of this vitamin exacerbates the inflammatory response and impairs both cellular and humoral immunity. Vitamin E, a lipid-soluble antioxidant, can also delay or prevent oxidative stress-induced diseases. Indeed, it may be considered a neutralising agent against endometriotic cell-derived ROS ,48. Our An increasing body of evidence has suggested that oxidative stress is closely associated with atherosclerotic progression and plaque instability . OxidatiOther dietary interventions  evaluated the correlation between adherence to diet and improvement of symptoms in endometriosis, but not the effect on oxidative stress . MoreoveThere is growing evidence of the involvement of oxidative stress in female reproduction, particularly in endometriosis. The studies on this topic are highly heterogeneous and aimed to evaluate the effects of dietary supplementation on oxidative stress in endometriosis . Data frIn this study, there are some limits to consider together with the very promising data generated so far. First, the limited duration of the study and the limited number of participants who completed the whole intervention, as enrolment was performed during the COVID-19 pandemic, induced the Italian government to enforce restrictions on outdoor activities and collectively quarantined the population. Second, we are aware that 6 month intervention represents a limited period that permits only to suggest a possible interpretation of the given results. Studies with a larger population and a longer duration are necessary to confirm these intriguing results. Third, in our study, all participants were Caucasian; thus, the findings may not be generalisable to other racial and ethnic groups. Finally, no information concerning intima media thickness (IMT), a structural parameter of subclinical atherosclerosis, was provided. Nevertheless, IMT reflects structural vascular damage that takes a longer time to realise . NotwithOur findings provide evidence of an unfavourable cardiovascular profile, as well as of unhealthy lifestyle habits in women with endometriosis, with results appearing of clinical relevance due to the mean age of the study population. Lifestyle improvement, and in particular the Mediterranean dietary intervention, allowed to ameliorate the metabolic and oxidative profile and provided a substantial improvement in the overall health-related quality of life.Comprehensive and interdisciplinary approaches to managing endometriosis and interventions aiming to increase the education and disease awareness of patients are mandatory in order to provide prompt and accurate diagnosis and treatment and to allow progress in the discovery of possible effective pharmacological and non-pharmacological interventions. In the field of gender medicine, the evaluation of the cardiovascular risk profile cannot neglect a gender-specific approach, since women’s health is burdened by exclusive risk factors, such as endometriosis, currently the object of attention by experts in several disciplines. To date, the innovative contribution of this study is represented by the use of a non-pharmacological approach, such as the Mediterranean Diet. This dietary pattern is known to be a cornerstone in the prevention of cardiovascular risk but has limited clinical evidence in the gynaecological field, and specifically in the modulation of common pathogenetic mechanisms correlating endometriosis with subclinical atherosclerosis. This study, by promoting research, prevention, and treatment of this high social impact disease, could help improve the quality of life and long-term cardiovascular health of young women suffering from endometriosis.The results of this study will help to identify useful indicators in order to define the emerging role of endometriosis in the development of the early atherosclerotic process that exposes women of reproductive age to higher cardiovascular risk. Our results can contribute not only to scientific research, providing further evidence to the ongoing discussion about the correlation between atherosclerosis and endometriosis and the role of the latter as a gender-specific cardiovascular risk factor, but also to suggest intervention programmes aimed at promoting a healthy lifestyle in women with endometriosis.Further studies are needed to confirm our results and to deeply explore specific aspects related to different hormonal therapies, adherence to the Mediterranean Diet and endometriosis phenotype.


We studied the effects of silicon carbide (SiC) and SiC hybrid systems with different conventional fillers  on the rheometric measurements, crosslink density, mechanical performance, aging stability, morphology, thermal behaviour, and flammability of ethylene-propylene-diene (EPDM) rubber composites. The hybrid filler systems showed technically promising synergetic effects on the performance of the EPDM composites. A pronounced reinforcing effect in EPDM composites filled with hybrid SiC filler systems was noted. Tensile strength increased in the systems with carbon black, silica, and graphene nanoplatelets, by 21%, 37%, and 68%, respectively, compared to the neat EPDM. Dynamic-mechanical analysis (DMA) revealed a shift of the glass transition temperature (Tg) of EPDM composites towards higher values following the incorporation of hybrid SiC fillers, indicating that the mobility of the macromolecule chains was restricted by the presence of filler particles. Importantly, the application of SiC as a filler in EPDM rubber composites contributed to a considerable reduction in flammability, as demonstrated by microscale combustion calorimetry (MCC). The most promising results were obtained for HAL/SiC and LDH/SiC hybrid systems, which produced final composites with high flame retardancy and good mechanical performance. The study highlights the significant potential of SiC and SiC hybrid systems as effective fillers improving the properties of elastomer composites. Flame retardants must be incombustible, or at least difficult to ignite and burn ,2,3. Theaterials . Variousaterials ,9,10.In recent years, inorganic and synthetic nanomaterials, such as carbon nanotubes (CNTs) ,12, carbBecause of its excellent high tension, high tensile strength, toughness, and resistance to aging properties, EPDM (ethylene-propylene-diene rubber) is widely used in many fields ,23. HoweTo further enhance the flammability and mechanical properties of EPDM, a filler such as SiC could be added to the mixture. Silicon carbide powder is characterized by very favorable properties, such as high thermal conductivity, high thermal stability, high strength and hardness, and good resistance to oxidation ,30,31. FHere, we present the novel use of raw SiC and SiC hybrid fillers with carbon black, silica, halloysite, layered double hydroxide, inorganic pigment, or graphene as flame retardants in EPDM composites. It was expected that the use of a combination of different fillers would be a good way to obtain a balance between the properties and cost of the rubber materials. We investigated the impact of the SiC hybrid systems on the rheological, mechanical, and thermal properties of the elastomer materials, as well as on their aging behavior. In particular, we investigated the influence of SiC and SiC hybrid systems on the combustion properties of the composites.2/g; Fast Extrusion Furnace Black -Carbon black Evonik Degussa GmbH , particle size of 39–55 nm; graphene nanoplatelets, XGnP XG Science Inc. , surface area 500 m2/g, particle size, average particle thickness around 2 nm, average particle diameter >2 μm; hydrotalcite LDH, synthetic Sigma Aldrich, particle size 1 μm; halloysite nanoclay Sigma Aldrich, particle diameter 30–70 nm, length 1–3 μm; Pigment Yellow 119 Kremer Pigmente , chemical formula Fe2O3·ZnO. Eleven rubber composites were prepared, based on the formulations presented in 2; 7-EPDM/10SiC/5CB; 8-EPDM/10SiC/5XGnP; 9-EPDM/10SiC/5LDH; 10-EPDM/10SiC/5HAL; 11-EPDM/10SiC/5PY119.The list of components and their sources were as follows: ethylene-propylene-diene rubber (EPDM) Keltan 4450S, containing 4.3% ethylidene-norbornene (ENB) and 52% ethylene, from Brenntag Polska ; sulfur Siarkopol ; N-cyclohexyl-2-benzothiazolesulfenamide-CBS Alfa Aesar ; stearin POCH S.A. ; microsized zinc oxide ZnO Huta Bedzin ; silicon carbide SiC Sigma Aldrich, particle size 200–450 mesh; silica Aerosil 380 Evonik Degussa GmbH , surface area 350 m02; optimum cure time-t90; maximum elastic torque-Mmax; minimum elastic torque-Mmin; increment of elastic torque ΔM = Mmax − Mmin. Based on the results, the blends were vulcanized on a hydraulic press for the curing time (t90) determined in rheometric measurements. The equilibrium swelling method was employed to evaluate the crosslink density of the EPDM vulcanizates, according to the ISO 1817 standard procedure. Four differently shaped samples, weighing in the range of 20–30 mg, were used for swelling experiments in toluene solvent. The crosslink density was calculated based on the Flory-Rehner equation [s is the molar volume of the solvent, Vr is the volume fraction of the rubber in the swollen specimen, dr is the density of the rubber, and The ethylene-propylene-diene rubber composites were blended in an open laboratory two-roll mill. The length of the rolling mill was 450 mm and the diameter of 200 mm. The compounding temperature was kept at around 40 °C and each composite was mixed for about 20 min. The curing parameters of the elastomer compounds were determined using a MonTech D-RPA 3000 Moving Die Rheometer . For each composite the following were measured: scorch time-tequation ,37:(1)υetion (1) :(2)Agingmin value, which was related to the change in viscosity. This effect is typical for elastomer blends filled with rigid filler particles [min values were observed for uncured EPDM/10SiC/5SiO2 sample containing silica (0.76 dNm) and for EPDM/10SiC/5XGnP filled with graphene (0.72 dNm). This could be related to the particle size of the material. Furthermore, it can be seen that incorporation of the SiC filler into EPDM mixes did not contribute to the changes in the increment of torque (ΔM). This can most likely be explained by the relatively low specific surface area of the microsized SiC filler and its poor dispersion in the elastomer matrix, leading to low adhesion with EPDM. On the other hand, the presence of active fillers in the SiC hybrid filler systems produced EPDM composites with higher ΔM parameters. For instance, the ΔM parameter values for EPDM/10SiC/5SiO2, EPDM/10SiC/5CB, and EPDM/10SiC/5HAL were about 10%, 12%, and 16% higher, respectively, than the value for unfilled EPDM. This is a common effect observed in rubber compounds filled with reinforcing fillers [05). Increasing the concentration of SiC in the EPDM compounds caused a progressive reduction in the t05. The samples with graphene EPDM/10SiC/5XGnP and the sample with carbon black EPDM/10SiC/5LDH showed the shortest t05 of the composites with mixed additives. The scorch time of the uncured compound with graphene EPDM/10SiC/5XGnP fell from 4.66 min (for the reference EPDM sample) to 1.91 min. These decreases were caused by the particular properties of the additives: both graphene and carbon black exhibit thermal conductivity, which results in enhanced heat transfer inside the tested sample, leading to a decrease in t05 [The effect of the SiC filler and its hybrid systems used on the curing behavior of the prepared EPDM compounds was investigated in rheometric measurements. The curing curves and corresponding rhemoetric results are presented on articles ,40. The  fillers ,42. It se in t05 ,44.90. Based on our previous study [2. This may have been caused by the formation of larger agglomerates inside the vulcanizate. The sulfur crosslinking system could also partially be adsorbed onto the outer surface of the silica, which resulted in a slower curing process. Moreover, silica is characterized by low thermal conductivity, which may lead to deterioration of heat transfer in the sample and prolongation of curing time. On the other hand, the shortest vulcanization time was observed for the compounds containing SiC and CB fillers, which has favorable thermal conductivity.One of the crucial parameters determining the profitability of the vulcanization process, especially on an industrial scale, is the vulcanization time tus study , in whicEPDM rubber can be effectively cured using both the conventional crosslinking system and peroxides due to the presence of unsaturated bonds in its structure. In our study, sulfur was used for crosslinking, as well as CBS, which is a sulfenamide characterized as an accelerator with relatively fast curing speed. The similar crosslinking density for EPDM cured by peroxides to the reference sample EPDM in our study has been previously reported in other work . The comThe static mechanical properties (tensile strength and elongation at break) of the EPDM-filled composites are presented in 2, 17% for CB, 59% for xGnP compared to neat EPDM). This can be clearly seen on stress-strain curves of the studied composites presented in Increasing the concentration of SiC in the rubber also caused a reduction in elongation at break (EB), due to the increased viscosity and stiffness of the filled composites. For example, EPDM composite filled with 20 phr of SiC reached an EB of 210%, whereas for neat EPDM EB was 230%. The hybrid SiC filler systems with silica, carbon black, and graphene significantly improved the mechanical properties of the EPDM composites. All these systems strengthened the EPDM rubber, as shown by the increases in tensile strength . The resulting curves are presented in δ values  and in tδ values  of the sδ values  to higheThe flammability of the EPDM-filled composites was investigated using the microscale combustion calorimetry test (MCC), which is known to be suitable for assessing the combustion behavior of elastomer composites. The most important parameter monitored during this test is the heat release rate (HRR). 600) could also affect the flammability of the composites. Interestingly, the sample containing carbon black showed higher char residue than the sample containing graphene. This was most likely related to the formation of so-called “bound rubber” in the EPDM/10SiC/5CB composite. In contrast, graphene flakes form a layered material comprising sp2-bonded carbon atoms in a planar arrangement, separated by an interlayer distance about 0.335. The limited d-spacing in graphite makes it difficult for macromolecules to enter gallery spaces, preventing the formation of bound rubber.The thermal stability of the EPDM composites filled with different SiC systems was analyzed by thermogravimetric analysis (TGA). The TGA thermograms of the studied composites are presented in In this study, a series of EPDM composites filled with SiC and SiC hybrid filler systems were fabricated. The properties of the EPDM/SiC composites were tuned by adjusting the contents of selected conventional fillers used in polymer technology, such as silica, graphene, and carbon black. The use of these reinforcing fillers in the hybrid systems resulted in EPDM composites with improved static and dynamic mechanical properties. The EPDM/SiC composites were also modified by the addition of halloysite nanotubes. The use of SiC in filler systems with halloysite nanotubes resulted in a considerable improvement in the flame retardancy of the EPDM composites. The incorporation of 15 phr of SiC into EPDM caused a reduction in the heat release rate parameter from 1460 W/g for neat EPDM to 1167 W/g for the EPDM/15SiC composite, as measured by MCC tests. The results of this study show that SiC can be successfully used as filler for reducing the flammability of elastomer composites. Elastomer composites filled with SiC hybrid filler systems with different conventional fillers offer an attractive rubber material resistant to fire, thermo-oxidative aging, and mechanical stress. The low concentration of hybrid filler systems in the polymer matrix allows for relatively low production costs, which is often a determining factor in the selection of flame-retardant elastomer products. Future work should focus on improving the dispersion of SiC and its compatibility with the rubber matrix.